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Sample records for egfr fish assay

  1. EGFR fluorescence in situ hybridisation assay: guidelines for application to non-small-cell lung cancer.

    PubMed

    Varella-Garcia, M; Diebold, J; Eberhard, D A; Geenen, K; Hirschmann, A; Kockx, M; Nagelmeier, I; Rüschoff, J; Schmitt, M; Arbogast, S; Cappuzzo, F

    2009-11-01

    There is a need for predictive biomarkers that identify non-small-cell lung cancer (NSCLC) patients most likely to respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment. There are numerous potential candidates, although none has been proven in prospective clinical trials. The EGFR gene copy number evaluated by fluorescence in situ hybridisation (FISH) has been highlighted as one of the most effective markers for sensitivity to EGFR TKIs in large phase III, randomised placebo-controlled trials and has been used in clinical settings to assist physicians in defining the therapeutic regimen. The EGFR FISH assay has technical challenges and it is critical that detailed guidelines are provided to help clinical laboratories in performing and interpreting the test. Excellent assay reproducibility and portability rates among laboratories are crucial to guarantee that accurate clinical decisions can be made for patients with NSCLC. This article discusses the consensus outcomes of a global workshop convened to discuss key technical issues and standardise reading strategies for the EGFR FISH assay of NSCLC tumour tissue. PMID:19861557

  2. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

    PubMed Central

    Smith, Matthew A.; Hall, Richard; Fisher, Kate; Haake, Scott M.; Khalil, Farah; Schabath, Matthew B.; Vuaroqueaux, Vincent; Fiebig, Heinz-Herbert; Altiok, Soner; Chen, Y. Ann; Haura, Eric B.

    2015-01-01

    Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. Here, we used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules. PMID:25587191

  3. Validation of a Fish Short-term Reproduction Assay

    EPA Science Inventory

    The Fish Short-term Reproduction Assay is an in vivo assay conducted with fathead minnows and is designed to detect changes in spawning, gross morphology, histopathology, and specific biochemical endpoints that reflect disturbances in the hypothalamic-pituitary-gonadal (HPG) axis...

  4. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

    PubMed Central

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Chicchinelli, Nicoletta; Mancini, Rita; Pisconti, Salvatore; Botti, Gerardo; Morabito, Alessandro

    2013-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples. PMID:24364033

  5. PNA-FISH assays for early targeted bacteraemia treatment.

    PubMed

    Parcell, B J; Orange, G V

    2013-11-01

    PNA-FISH S. aureus/CNS and GNR Traffic Light assays were compared with standard culture methods for identifying bacteraemia in 156 blood cultures from 131 patients. Results correlated with final culture results in 153 cultures. Retrospective case note review revealed that earlier targeted treatment would have occurred in 10.7% of cases. PMID:24055387

  6. Behavioral assay for assessing effects of pollutants on fish chemoreception

    SciTech Connect

    Lemly, A.D.; Smith, R.J.

    1986-04-01

    Behavioral assays are sensitive to sublethal levels of pollution but they usually require highly trained personnel and long observation periods. We describe a system that combines the sensitivity of a behavioral assay with commercially available automated monitoring equipment. The observation system consists of a special aquarium coupled to a recirculating water system, and an Opto-Varimex-Aqua activity tracking meter (Columbus Instruments, Columbus, Ohio) interfaced to a microcomputer. The tracking meter forms an intersecting, planar grid of light beams which, when interrupted by fish movements, is translated into a digitized signal and fed to the computer. The assay is based on the response of fish to natural chemical stimuli such as food odors or pheromones. When these stimulus solutions are injected into the water circulation the response of the fish is monitored by the computer system, which is capable of discriminating and quantifying changes in eight parameters. Normal responses to stimuli are compared with the response of fish that have been exposed to pollutants. We have successfully used this technique to examine effects of reduced pH on the response of fathead minnows, Pimephales promelas, to chemical feeding stimuli. The system should be easily adapted to any laboratory concerned with testing for effects of toxic substances, and will identify effects of pollution that have thus far been difficult or impossible to assess.

  7. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    PubMed Central

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Flores Quintana, Harold A.; Morris, James A.; Litaker, R. Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  8. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    PubMed

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  9. Pyrosequencing for EGFR mutation detection: diagnostic accuracy and clinical implications.

    PubMed

    Sahnane, Nora; Gueli, Rossana; Tibiletti, Maria G; Bernasconi, Barbara; Stefanoli, Michele; Franzi, Francesca; Pinotti, Graziella; Capella, Carlo; Furlan, Daniela

    2013-12-01

    EGFR-activating mutations predict responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients. Mutation screening is crucial to support therapeutic decisions and is commonly conducted using dideoxy sequencing, although its sensitivity is suboptimal in clinical settings. To evaluate the diagnostic performance of pyrosequencing and dideoxy sequencing, we examined EGFR mutation status in a retrospective cohort of 53 patients with NSCLCs clinically selected for TKI therapy and whose clinical outcome was available. Moreover, pyrosequencing quantitative results were compared with EGFR amplification data. EGFR mutations were investigated by pyrosequencing and by dideoxy sequencing. Detection rates of both methods were determined by titration assays using NCI-H1975 and HCC-827 cell lines. Increased EGFR copy number was assessed by fluorescence in situ hybridization (FISH). Pyrosequencing showed a higher detection rate than dideoxy sequencing. Tumor control rate of cases with mutant and wild-type EGFR was 86% and 29%, respectively. EGFR amplification was significantly associated with EGFR mutation and a positive correlation between high percentages of mutant alleles and clinical response to TKI was observed. We concluded that pyrosequencing is more sensitive than dideoxy sequencing in mutation screening for EGFR mutations. Detection rate of dideoxy sequencing was suboptimal when low frequencies of mutant alleles or low tumor cell contents were observed. Pyrosequencing enables quantification of mutant alleles that correlates well with increased EGFR copy number assessed by FISH. Pyrosequencing should be used in molecular diagnostic of NSCLC to appropriately select patients who are likely to benefit from TKI therapy. PMID:24193003

  10. A microscaled mercury saturation assay for metallothionein in fish.

    PubMed

    Shaw-Allen, Patricia; Elliott, Muriel; Jagoe, Charles H

    2003-09-01

    A mercury (Hg) saturation assay for measuring metallothionein (MT) in fish liver was modified by optimizing binding conditions to minimize the mercury and tissue consumed. The revised method uses stable Hg at low concentrations instead of 203Hg. At the reduced Hg concentrations used, MT concentrations in livers homogenized in saline appeared to increase systematically with dilution in both bluegill sunfish (Lepomis macrochirus) and largemouth bass (Micropterus salmoides). This error suggested a binding limitation due to sulfhydryl oxidation or competition for and removal of mercury by non-MT proteins. Homogenizing tissues in trichloroacetic acid (TCA) eliminated the interference. To further evaluate the method, the protocol was tested in the laboratory and field. Metallothionein in bluegill injected with 0.6 mg/kg zinc chloride increased at a rate of 0.03 nmole MT/g liver/h (r2 = 0.53, p = 0.001). Linearity improved when data were corrected for protein content (r2 = 0.74, p < 0.0001). Metallothionein levels in bluegill from a coal ash-contaminated environment were significantly increased over that of hatchery-reared sunfish (F = 20.17, p = 0.0003). The microscaled procedure minimizes concerns related to radioisotope use and waste generation while retaining the high sensitivity of the 203Hg assay. PMID:12959524

  11. Molecular Assays for Detecting Aphanomyces invadans in Ulcerative Mycotic Fish Lesions

    PubMed Central

    Vandersea, Mark W.; Litaker, R. Wayne; Yonnish, Bryan; Sosa, Emilio; Landsberg, Jan H.; Pullinger, Chris; Moon-Butzin, Paula; Green, Jason; Morris, James A.; Kator, Howard; Noga, Edward J.; Tester, Patricia A.

    2006-01-01

    The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans. PMID:16461710

  12. Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

    PubMed Central

    Shah, Jyotsna; Mark, Olivia; Weltman, Helena; Barcelo, Nicolas; Lo, Wai; Wronska, Danuta; Kakkilaya, Srinivas; Rao, Aravinda; Bhat, Shalia T.; Sinha, Ruchi; Omar, Sabah; Moro, Manuel; Gilman, Robert H.; Harris, Nick

    2015-01-01

    Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively. PMID:26333092

  13. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

    PubMed

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen's' reports and fish community monitorings. PMID:26814998

  14. Analysis of three marine fish cell lines by rapd assay.

    PubMed

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  15. DEVELOPMENT OF A CARCINOGEN ASSAY SYSTEM UTILIZING ESTUARINE FISHES

    EPA Science Inventory

    The objective of this project was the development of systems to assay the effects of chemical carcinogens on marine teleosts. It was determined that the LC-50 for benzidine with respect to Cyprinodon variegatus was ca. 64 ppm. Weekly contaminations of 1 ppm benzidine caused some ...

  16. Rapid facile solid-phase immunobead assay for screening ciguatoxic fish in the market place.

    PubMed

    Park, D L; Gamboa, P M; Goldsmith, C H

    1992-01-01

    The precision of the solid-phase immunobead assay (Ciguatect) to detect toxins associated with ciguatera poisoning have been evaluated through analysis of toxic and non-toxic fish obtained from fishing areas around the Hawaiian Islands. The Ciguatect test kit has been optimized for application to field/marketplace screening of ciguatoxic fish. Twelve parrot, surgeon, and amberjack fish fillet and fish extract test portions containing various concentrations of toxins were distributed to participating laboratories for analysis. The presence or absence of ciguatera-related toxins is determined by binding the toxins to a membrane attached to a plastic strip and exposing the toxin ladened membrane to a monoclonal antibody-colored latex bead complex which has a high specificity for ciguatera-related toxins. The intensity of the color on the membrane denotes the presence of the toxins in the fish or fish extract. Toxic components in the fish were confirmed by extraction, column purification, and toxicity testing using the brine shrimp (Artemia sp.) assay. Okadaic acid was used to standardize both the S-PIA and brine shrimp assays. For determination of ciguatoxin and related polyether toxins in parrot, surgeon, and amberjack fish fillets, the relative standard deviations for repeatability (RSDR) were 13.5, 9.0 and 4.3%, respectively, and the relative standard deviations for reproducibility (RSDR) were 44.4, 29.7 and 14.3%, respectively, for concentrations ranging from 1-4 ng/test strip. For determination of ciguatoxin and related polyether toxins in parrot, surgeon, and amberjack fish extracts, the RSDR were 5.8, 4.8, and 3.7%, respectively, and the RSDR were 11.9, 9.9, and 7.6%, respectively, for concentrations ranging from 3-5 ng/test strip.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1340354

  17. A mercury saturation assay for measuring metallothionein in fish

    SciTech Connect

    Dutton, M.D. . Dept. of Zoology); Stephenson, M. . Environmental Science Branch); Klaverkamp, J.F. )

    1993-07-01

    An accurate, rapid, sensitive, and simple method using mercury saturation for quantifying metallothionein (MT) is described. A complex solution of enzymatic and nonenzymatic thiols, including rabbit liver MT-2, and supernatants from homogenized samples of rainbow trout liver were incubated in the presence of [sup 203]Hg in 10% trichloroacetic acid. Excess Hg was bound to an removed by chicken egg albumin, which denatured on contact with the acidic assay medium. After centrifugation, MT labeled with [sup 203]Hg remained in the TCA supernatant and was estimated using known stoichiometry for Hg-MT binding. A dilution series was used to establish that nonspecific metal binding, a common problem with other metal saturation assays, is negligible. Analysis of hepatic MT with high Cu content from rainbow trout demonstrated virtually complete displacement of Cu, Cd, and Zn by Hg. When compared to other metal-saturation assays developed for vertebrates, this method requires the least number of technical steps, and one-third or less of total preparatory and analytical time.

  18. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater

    PubMed Central

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s’ reports and fish community monitorings. PMID:26814998

  19. CELL-FREE NEUROCHEMICAL SCREENING ASSAYS TO PREDICT ADVERSE EFFECTS IN MAMMALS, FISH, AND BIRDS

    EPA Science Inventory

    This work will result in the establishment of a high-throughput screening assay that can be used to predict reproductive impairment across multiple ecologically relevant species (birds, fish, mammals). Resources exist to adapt this platform to screen 1,000s of toxicants. It...

  20. Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

    PubMed

    Altinok, Ilhan; Capkin, Erol; Kayis, Sevki

    2008-10-15

    A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method. PMID:18499358

  1. Immunochemical determination of oxytetracycline in fish: comparison between enzymatic and time-resolved fluorometric assays.

    PubMed

    Cháfer-Pericás, Consuelo; Maquieira, Angel; Puchades, Rosa; Miralles, Javier; Moreno, Amelia; Pastor-Navarro, Nuria; Espinós, Francisco

    2010-03-10

    An indirect competitive enzyme-linked immunosorbent assay (ELISA) with photometric detection of horseradish peroxidase (HRP) activity, was developed in plate to detect oxytetracycline (OTC) in Gilthead sea bream (Sparus aurata) samples. The results were compared to those obtained by time-resolved fluoroimmunoassay (TR-FIA) using a secondary antibody with coproporphyrin of platinum (II) (PtCP) as marker. The limits of detection obtained in fish extract were 16 and 0.08 microg kg(-1) for photometric and fluorometric detections, respectively; therefore, they were suitable for fish quality control according to the maximum residue level established by the European Union. An extraction procedure using methanol:water 70:30 (v/v)+1 mL EDTA 0.1 M, and different clean-up procedures based on solid-phase extraction (C(18), polymeric reversed phase, SCX, Si) was assayed. The matrix effects were overcome by means of an average tetracycline-free fish extract calibration curve used for quantification. The OTC optimized ELISA can also be applied to determine tetracycline and chlortetracycline residues with good results. Thus, the developed immunoassay could be considered as a generic assay for the most used tetracyclines in aquaculture antibiotic treatments. In order to confirm the utility of the developed immunoassay as a semi-quantitative methodology, fish samples obtained from different supermarkets were analyzed. Results correlate well with those obtained with a reference HPLC method. PMID:20171317

  2. Alkaline comet assay for genotoxic effect detection in neotropical fish Prochilodus lineatus (Pisces, Curimatidae).

    PubMed

    Simoniello, M F; Gigena, F; Poletta, G; Loteste, A; Kleinsorge, E; Campana, M; Scagnetti, J; Parma, M J

    2009-08-01

    Toxicants on fish may induce genetic alterations that can be used as genotoxic markers. We evaluated DNA damage using alkaline comet assay applied on erythrocytes after in vivo exposure of Prochilodus lineatus to different concentrations of Cypermethrin (0.300, 0.150, 0.075 and 0.000 microg/L) as a probable chemical mutagen. The results revealed a significantly higher level of DNA damage at all concentrations of Cypermethrin tested compared to control and background level (p < 0.05). We have standardized the technique for one of the most common native fish species that will be useful for biomonitoring genotoxicity in polluted waters of the region. PMID:19466374

  3. Important Considerations for Methemoglobin Measurement in Fish Blood: Assay Choice and Storage Conditions

    SciTech Connect

    Kuntz, Mel Anton; Rodnick, K. J.; J. A. Lacey

    2002-05-01

    Spectrophotometric assays of methaemoglobin (metHb) in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, tilapias Tilapia niloticus and Tilapia zillii and white sturgeon Acipenser transmontanus, under baseline conditions, were low (<4%) for each species, and yet higher than human values (<1%). MetHb results for a given fish species varied significantly between assays and two assays were deemed unacceptable for particular animals. For rainbow trout, white sturgeon, and the two species of tilapia, the Dubowski method gave uncharacteristically high estimates of metHb. MetHb could not measured in tilapia blood using the Evelyn & Malloy method due to spectral interference. Only the Horecker & Brackett assay worked well for all species. Storage conditions were extremely important in the quantification of metHb in rainbow trout blood. For consistent values, samples can be stored up to 4 h on ice (0 degrees C) or at least 20 days under liquid nitrogen (-196 degrees C). Auto-oxidation, however, elevates rainbow trout metHb at -20 and -80 degrees C. It should not be assumed that the blood of fishes and humans perform similarly during assays of metHb.

  4. Determination of PCBs in fish using enzyme-linked immunosorbent assay (ELISA)

    USGS Publications Warehouse

    Lasrado, J.A.; Santerre, C.R.; Zajicek, J.L.; Stahl, J.R.; Tillitt, D.E.; Deardorff, D.

    2003-01-01

    Polychlorinated biphenyls (PCBs) were determined in fish tissue using an enzyme-linked immunosorbent assay (ELISA). Standard curves for Aroclor 1248, 1254, and 1260 in catfish tissue were developed with ranges from 0.05 to 0.5 ppm and 0.5 to 5.0 ppm. Wild fish were initially analyzed using gas chromatography/electron-capture detection (GC/ECD) and those having residues within the standard curve ranges were analyzed with ELISA. Results obtained using ELISA and GC/ECD were not significantly different (p < 0.05) from 0.05 to 0.5 ppm. From 0.5 to 5.0 ppm, the standard curve for Aroclor 1254 was the best predictor of total PCB in wild fish samples.

  5. In vivo virus growth competition assays demonstrate equal fitness of fish rhabdovirus strains that co-circulate in aquaculture

    USGS Publications Warehouse

    Troyer, R.M.; Garver, K.A.; Ranson, J.C.; Wargo, A.R.; Kurath, G.

    2008-01-01

    A novel virus growth competition assay for determining relative fitness of RNA virus variants in vivo has been developed using the fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), in juvenile rainbow trout (Oncorhynchus mykiss). We have conducted assays with IHNV isolates designated B, C, and D, representing the three most common genetic subtypes that co-circulate in Idaho trout farm aquaculture. In each assay, groups of 30 fish were immersed in a 1:1 mixture of two genotypes of IHNV, and then held in individual beakers for a 72 h period of in vivo competitive virus replication. Progeny virus populations in each fish were analyzed for the presence and proportion of each viral genotype. In two independent assays of the B:C isolate pair, and two assays of the B:D pair, all fish were co-infected and there was a high level of fish-to-fish variation in the ratio of the two competing genotypes. However, in each assay the average ratio in the 30-fish group was not significantly different from the input ratio of 1:1, indicating equal or nearly equal viral fitness on a host population basis, under the conditions tested. ?? 2008.

  6. Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors

    PubMed Central

    Brevet, Marie; Johnson, Melissa L.; Azzoli, Christopher G.; Ladanyi, Marc

    2012-01-01

    Aims EGFR mutations now guide the clinical use of EGFR-targeted therapy in lung cancer. However, standard EGFR mutation analysis requires a minimum amount of tumor tissue, which may not be available in certain situations. In this study, we combined a mass spectrometry genotyping assay (Sequenom) with a mutant-enriched PCR (ME-PCR) to detect EGFR mutations in free plasma DNA from patients with lung cancer. Method DNAs were extracted from 31 plasma samples from 31 patients and analyzed by both methods for EGFR exon 19 deletion and EGFR L858R mutation. Results in plasma DNA samples were compared with EGFR mutation status obtained in tumor DNA (18/31 EGFR mutant). The relationship of EGFR mutation status in tumor and/or plasma samples to overall survival was assessed. Results The EGFR mutation status in plasma DNA was identical to the primary tumor in 61% of patients (19/31). By mass spectrometry genotyping, the plasma samples contained mutant DNA corresponding to 5/14 EGFR exon 19 deletions and 3/4 EGFR L858R mutations previously diagnosed in the matched tumors. Two samples were positive in plasma DNA but negative in primary tumor tissue. Results were similar for ME-PCR. For patients treated with erlotinib, overall survival was correlated with the presence of EGFR mutation in plasma and/or tumor tissue (p=0.002), with the two patients positive only in plasma DNA showing responses and favorable outcomes. Conclusion The detection of EGFR mutations in plasma DNA samples by mass spectrometry genotyping and ME-PCR is feasible. A positive EGFR result in plasma DNA has a high predictive value for tumor EGFR status and for favorable clinical course on EGFR-targeted therapy and could therefore be useful in guiding clinical decisions in patients with insufficient or unavailable tumor specimens. PMID:21130517

  7. Application of a fish DNA damage assay as a biological toxicity screening tool for metal plating wastewater

    SciTech Connect

    Choi, K.; Zong, M.; Meier, P.G.

    2000-01-01

    The utility of a fish DNA damage assay as a rapid monitoring tool was investigated. Metal plating wastewater was chosen as a sample because it contains various genotoxic metal species. Fish DNA damage assay results were compared to data generated from the conventional whole effluent toxicity (WET) test procedure. The Microtox{reg_sign} assay (Azur Environmental, Carlsbad, CA, USA) using Vibrio fischeri was also employed. Eleven samples from two metal plating companies were collected for this evaluation. For the fish DNA damage assay, 7-d-old fathead minnow larvae, Pimephales promelas, were utilized. They were exposed to a series of dilutions at 20 C for 2 h. Whole effluent toxicity tests conducted in this study included two acute toxicity tests with Daphnia magna and fathead minnows and two chronic toxicity tests with Ceriodaphnia dubia and fathead minnows. The fish DNA damage assay showed good correlations with both the acute and chronic WET test results, especially with those obtained with fathead minnows. The kappa values, an index of agreement, between the fish DNA damage assay and WET tests were shown to be acceptable. These findings imply that this novel fish DNA damage assay has use as an expedient toxicity screening procedure since it produces comparable results to those of the acute and chronic fathead minnow toxicity tests.

  8. Verification by the FISH translocation assay of historic doses to Mayak workers from external gamma radiation.

    PubMed

    Sotnik, Natalia V; Azizova, Tamara V; Darroudi, Firouz; Ainsbury, Elizabeth A; Moquet, Jayne E; Fomina, Janna; Lloyd, David C; Hone, Pat A; Edwards, Alan A

    2015-11-01

    The aim of this study was to apply the fluorescence in situ hybridization (FISH) translocation assay in combination with chromosome painting of peripheral blood lymphocytes for retrospective biological dosimetry of Mayak nuclear power plant workers exposed chronically to external gamma radiation. These data were compared with physical dose estimates based on monitoring with badge dosimeters throughout each person's working life. Chromosome translocation yields for 94 workers of the Mayak production association were measured in three laboratories: Southern Urals Biophysics Institute, Leiden University Medical Center and the former Health Protection Agency of the UK (hereinafter Public Health England). The results of the study demonstrated that the FISH-based translocation assay in workers with prolonged (chronic) occupational gamma-ray exposure was a reliable biological dosimeter even many years after radiation exposure. Cytogenetic estimates of red bone marrow doses from external gamma rays were reasonably consistent with dose measurements based on film badge readings successfully validated in dosimetry system "Doses-2005" by FISH, within the bounds of the associated uncertainties. PMID:26319788

  9. Monitoring Conformational Changes in the Receptor Tyrosine Kinase EGFR.

    PubMed

    Becker, Christian; Öcal, Sinan; Nguyen, Hoang D; Phan, Trang; Keul, Marina; Simard, Jeffrey R; Rauh, Daniel

    2016-06-01

    The receptor tyrosine kinase EGFR is regulated by complex conformational changes, and this conformational control is disturbed in certain types of cancer. Many ligands are known to bind EGFR in its active conformation, thereby preventing ATP from binding. Only a few ligands are known to stabilize EGFR in its inactive conformation, thus providing novel strategies for perturbing EGFR activity. We report a direct binding assay that enables the identification of novel ligands that bind to and stabilize the inactive conformation of EGFR. PMID:26991964

  10. Targeting EGFR in bladder cancer.

    PubMed

    Villares, G J; Zigler, M; Blehm, K; Bogdan, C; McConkey, D; Colin, D; Bar-Eli, Menashe

    2007-12-01

    Expression and overexpression of the epidermal growth factor receptor (EGFR) have been described in several solid tumors including bladder, breast, colorectal, NSCLC, prostate, and ovarian cancers. In addition to gene amplification, point mutations within the kinase domain also occur. Previous reports indicate that the patient's response to gefitinib depends on either the presence of mutations within the kinase domain of EGFR or the expression of the most frequent alteration, the truncated EGFR variant III (EGFRvIII). Therefore, it is important to determine if these EGFR alterations are present in urothelial carcinoma. The kinase domain of EGFR (exons 18-21) from 11 bladder cancer cell lines as well as from 75 patient tumors was analyzed by automated sequencing. No mutations were detected in all samples tested. Furthermore, analysis of EGFRvIII by immunohistochemistry revealed that almost half of all the patient samples expressed this truncation in a urothelial carcinoma tissue microarray. However, there have been previous reports of inconsistencies in detecting EGFRvIII by immunohistochemistry owing to the specificity of the antibodies and the methodologies utilized. Therefore, these results were validated by reverse transcription PCR, real-time PCR and western blot analysis. In these assays, none of the samples tested positive for EGFRvIII. Taken together, these results indicate that mutations within the tyrosine kinase domain of EGFR and expression of EGFRvIII are rare events in bladder cancer and therefore do not contribute to the malignant phenotype of this tumor. These results have clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma. PMID:17690890

  11. Developing immune function assays to monitor fish health in field studies

    SciTech Connect

    Rice, C.D.; Kergosien, D.H.; Adams, S.M. |

    1995-12-31

    The East Fork Poplar Creek system, a 24km long stream in TN that receives point source discharges of contaminants near its headwaters, was chosen to evaluate a field approach to fish immunotoxicology. Previous studies in this stream have shown that cytochrome P4501A activity, liver somatic indices, macrophage aggregates, and parasitic liver lesions are significantly elevated in sunfish with the degree of impact decreasing with distance from the contaminant source. Red-breasted sunfish were collected between May 23 and June 3 of 1994. Captured fish were anesthetized in MS-222 and processed by two different methods. One group was sacrificed at each sampling station, weights and lengths recorded, and the spleen and anterior kidney tissues removed and placed in buffer on ice. The other group was kept in MS-222 for 2 hr and transported to the laboratory. The spleen and anterior kidney from each fish were then prepared as a single cell suspension and shipped overnight to Mississippi State University. Cells were then washed by centrifugation and resuspended in appropriate media to evaluate PMA-stimulated phagocyte oxidative burst and non-specific cytotoxic cell (NCC) activity against K562 tumor targets. Oxidative burst responses were dramatically suppressed in both groups at stations near the headwaters but returned to reference levels further downstream. There were no differences between treatment groups at each station. NCC activities did not follow gradient-response patterns observed with phagocyte oxidative burst data and there were inconsistent differences between treatment groups at each station. These data show that simple immune function assays, such as phagocyte oxidative burst responses, can be used as an ancillary biomarker in fish health monitoring.

  12. eGFR

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? Estimated Glomerular Filtration Rate (eGFR) Share this page: Was ... page helpful? Also known as: Glomerular Filtration Rate, Estimated; GFR; eGFR; Calculated Glomerular Filtration Rate; cGFR Formal ...

  13. Studies on the genotoxicity of endosulfan in different tissues of fresh water fish Mystus vittatus using the comet assay.

    PubMed

    Sharma, Shilpi; Nagpure, N S; Kumar, Ravindra; Pandey, Sanjay; Srivastava, Satish K; Singh, Poonam J; Mathur, P K

    2007-11-01

    Endosulfan, a widely used organochlorine pesticide, is readily bio-accumulative in fishes and can be indirectly harmful to human populations. Limited efforts have been made to study long-term genotoxic effects of endosulfan in different tissues of fish using gentoxicity biomarkers. Therefore, the current investigation was undertaken to detect single-cell DNA strand breaks induced by endosulfan in the fresh water teleost fish Mystus vittatus using the comet assay. The LC(50) value of technical grade endosulfan was first determined for the fish species in a semistatic system, and on the basis of the LC(50) value, the sublethal and nonlethal concentrations were determined. The DNA damage was measured in gill, kidney, and erythrocytes as the percentage of DNA in comet tails of fish specimens exposed to the sublethal and nonlethal concentrations of endosulfan. In general, significant effects (p < 0.01) from both concentration and time of exposure were observed in exposed fishes. It was found that all the tissues at all concentrations exhibited the highest DNA damage on day 1, after which there was a nonlinear decline in the percentage of tail DNA. The comparison of DNA damage among the tissues at different concentrations could not show the sensitivity of particular tissue to endosulfan. The current study explored the utility of the comet assay for in vivo laboratory studies using fish species to screen the genotoxic potential of chemical agents. PMID:17713809

  14. Inhibition of DYRK1A destabilizes EGFR and reduces EGFR-dependent glioblastoma growth

    PubMed Central

    Pozo, Natividad; Zahonero, Cristina; Fernández, Paloma; Liñares, Jose M.; Ayuso, Angel; Hagiwara, Masatoshi; Pérez, Angel; Ricoy, Jose R.; Hernández-Laín, Aurelio; Sepúlveda, Juan M.; Sánchez-Gómez, Pilar

    2013-01-01

    Glioblastomas (GBMs) are very aggressive tumors that are resistant to conventional chemo- and radiotherapy. New molecular therapeutic strategies are required to effectively eliminate the subpopulation of GBM tumor–initiating cells that are responsible for relapse. Since EGFR is altered in 50% of GBMs, it represents one of the most promising targets; however, EGFR kinase inhibitors have produced poor results in clinical assays, with no clear explanation for the observed resistance. We uncovered a fundamental role for the dual-specificity tyrosine phosphorylation–regulated kinase, DYRK1A, in regulating EGFR in GBMs. We found that DYRK1A was highly expressed in these tumors and that its expression was correlated with that of EGFR. Moreover, DYRK1A inhibition promoted EGFR degradation in primary GBM cell lines and neural progenitor cells, sharply reducing the self-renewal capacity of normal and tumorigenic cells. Most importantly, our data suggest that a subset of GBMs depends on high surface EGFR levels, as DYRK1A inhibition compromised their survival and produced a profound decrease in tumor burden. We propose that the recovery of EGFR stability is a key oncogenic event in a large proportion of gliomas and that pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for EGFR-dependent GBMs. PMID:23635774

  15. Are fish and standardized FETAX assays protective enough for amphibians? A case study on Xenopus laevis larvae assay with biologically active substances present in livestock wastes.

    PubMed

    Martini, Federica; Tarazona, José V; Pablos, M Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC(50)) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  16. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    PubMed Central

    Martini, Federica; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  17. Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

    PubMed Central

    Ishii, Hidenobu; Azuma, Koichi; Sakai, Kazuko; Kawahara, Akihiko; Yamada, Kazuhiko; Tokito, Takaaki; Okamoto, Isamu; Nishio, Kazuto; Hoshino, Tomoaki

    2015-01-01

    As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs. PMID:26334838

  18. Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

    PubMed

    Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2016-09-15

    A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes. PMID:27469903

  19. Chromogenic in situ hybridization to detect EGFR gene copy number in cell blocks from fine-needle aspirates of non small cell lung carcinomas and lung metastases from colo-rectal cancer

    PubMed Central

    2010-01-01

    Background Several studies demonstrated that epidermal growth factor receptor (EGFR) gene copy number (GCN) correlates to the response to tyrosine kinase inhibitors in non small cell lung cancer (NSCLC) and to anti-EGFR monoclonal antibodies (MoAbs) in metastatic colorectal cancer (CRC). In the presence of lung nodules, cytology is often the only possible diagnostic approach. Chromogenic in situ hybridization (CISH) is an alternative technique to fluorescence in situ hybridization (FISH), but its feasibility in detecting EGFR GCN in cell blocks from fine-needle aspiration cytology (FNAC) of lung nodules has not yet been established. Methods We evaluated the feasibility of CISH on 33 FNAC from 20 primary NSCLC (5 squamous carcinomas, 8 large cell carcinomas and 7 adenocarcinomas) and 13 lung metastases from CRC. Results Of the 33 FNAC analyzed by CISH, 27 (82%) presented a balanced increase in EGFR gene and chromosome 7 number: 10 cases (30%) showed a low polysomy, 15 (45%) a high polysomy and 2 (6%) NSCLC were amplified. No significant differences between NSCLC and CRC lung metastases were found in relation to disomic or polysomic status. In addition, no correlation between EGFR GCN and EGFR immunohistochemical overexpression was found. Furthermore, we compared CISH results with those obtained by FISH on the same samples and we found 97% overall agreement between the two assays (k = 0.78, p < 0.0001). Two cases were amplified with both assays, whereas 1 case of NSCLC was amplified by FISH only. CISH sensitivity was 67%, the specificity and positive predictive value (PPV) was 100%, and the negative predictive value (NPV) was 97%. Conclusions Our study shows that CISH is a valid method to detect EGFR GCN in cell blocks from FNAC of primary NSCLC or metastatic CRC to the lung. PMID:20843314

  20. EGFR-tyrosine kinase inhibitor treatment in a patient with advanced non-small cell lung cancer and concurrent exon 19 and 21 EGFR mutations: A case report and review of the literature

    PubMed Central

    YANG, YANG; ZHANG, BIAO; LI, RUTIAN; LIU, BAORUI; WANG, LIFENG

    2016-01-01

    Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are considered to be effective treatments for advanced non-small cell lung cancer (NSCLC) patients with sensitizing EGFR mutations, including exon 19 deletion and exon 21 L858R mutations. However, with the development of EGFR mutation detection assays, patients with complex EGFR mutations are emerging, and their response to EGFR-TKIs remains unclear. The present study reports a case of a 62-year-old, non-smoking female patient with advanced NSCLC, presenting with concurrent EGFR 19+21 sensitizing mutations, who had a poor response to the first-line EGFR-TKI erlotinib and succumbed 5 months subsequent to diagnosis. Furthermore, the present study performed a literature review, and 18 patients with complex EGFR 19+21 mutations that had received EGFR-TKIs were identified. The majority of these patients responded well to EGFR-TKIs. To the best of our knowledge, the present case is the first to report a patient with lung adenocarcinoma with complex EGFR 19+21 sensitizing mutations that had a poor clinical response to a first-line EGFR-TKI. Despite the 70% response rate of sensitizing EGFR-mutant NSCLCs to EGFR-TKIs, there is still a proportion of patients that experience de novo resistance, and heterogeneity is likely to be important in this resistance mechanism. Therefore, comprehensive genomic detection assays and multi-targeted therapies for patients with NSCLC with complex EGFR mutations require additional investigation. PMID:27123149

  1. Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish.

    PubMed

    Nakajima, Nao; Kawanishi, Michiko; Imamura, Saiki; Hirano, Fumiya; Uchiyama, Mariko; Yamamoto, Kinya; Nagai, Hidetaka; Futami, Kunihiko; Katagiri, Takayuki; Maita, Masashi; Kijima, Mayumi

    2014-05-01

    Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations. PMID:24657319

  2. Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae.

    PubMed

    López, Jose R; Hamman-Khalifa, Abdel M; Navas, José I; de la Herran, Roberto

    2011-11-01

    The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen. PMID:22092820

  3. A short-term sublethal toxicity assay with zebra fish based on preying rate and its integration with mortality.

    PubMed

    Abdel-moneim, Ahmed; Moreira-Santos, Matilde; Ribeiro, Rui

    2015-02-01

    Contaminant-induced feeding inhibition has direct and immediate consequences at higher levels of biological organization, by depressing the population consumption and thus hampering ecosystem functioning (e.g. grazing, organic matter decomposition). Thus, similarly to lethality and avoidance, feeding is mechanistically linked to ecosystem processes and is therefore an unequivocal ecologically meaningful response. The objective of the present study was to develop a short-term assay with the small freshwater fish Danio rerio, based on feeding. For this, a methodology to easily and precisely quantify feeding was first optimized: each fish was allowed to prey on ten live Daphnia magna juveniles, for 1h, just before the end of a 48-h exposure test period. Secondly, copper sensitivity of feeding relatively to survival and growth was evaluated. At the growth EC20 (40 μg L(-1)), feeding was inhibited by 53%, and at the feeding EC50 (36 μg L(-1)), mortality was negligible (1.3%). Integrating feeding and survival revealed a 97% depression in the population consumption at the LC50 (61 μg L(-1)). Thirdly, the influence of pH, conductivity and hardness on the feeding background variability was assessed by assaying waters collected at eight reference sites and was found to be negligible, within tested ranges. Fourthly, feeding assays with natural waters contaminated with acid mine drainage confirmed the integration of lethality and feeding to be pertinent at estimating contaminant effects at higher levels of biological organization. PMID:25462299

  4. Development of a polymerase chain reaction diagnostic assay for Ceratomyxa shasta, a myxosporean parasite of salmonid fish.

    PubMed

    Palenzuela, O; Trobridge, G; Bartholomew, J L

    1999-04-15

    A diagnostic procedure based on the polymerase chain reaction (PCR) was developed for the myxosporean parasite Ceratomyxa shasta. Three sets of oligonucleotide primers were designed to specifically amplify C. shasta ribosomal RNA genes and several parameters of the assay were tested and optimised. A simple protocol for the processing of fish tissue samples was also developed. In a single round, 20 microliters volume reaction the optimised procedure allows the detection of 50 fg of purified C. shasta genomic DNA, or 0.01 spore from a seeded fish intestine sample. This protocol is considerably faster, cheaper and more reliable than any previous diagnostic procedure for a myxosporean parasite, and can be an invaluable tool for the monitoring of early and/or subclinical C. shasta infections in wild and cultured salmon populations. PMID:10349552

  5. Molecular genetic alterations in egfr CA-SSR-1 microsatellite and egfr copy number changes are associated with aggressiveness in thymoma

    PubMed Central

    Conti, Salvatore; Gallo, Enzo; Sioletic, Stefano; Facciolo, Francesco; Palmieri, Giovannella; Lauriola, Libero; Evoli, Amelia; Martucci, Robert; Di Benedetto, Anna; Novelli, Flavia; Giannarelli, Diana; Deriu, Gloria; Granone, Pierluigi; Ottaviano, Margaret; Muti, Paola; Pescarmona, Edoardo

    2016-01-01

    Background The key role of egfr in thymoma pathogenesis has been questioned following the failure in identifying recurrent genetic alterations of egfr coding sequences and relevant egfr amplification rate. We investigated the role of the non-coding egfr CA simple sequence repeat 1 (CA-SSR-1) in a thymoma case series. Methods We used sequencing and egfr-fluorescence in situ hybridization (FISH) to genotype 43 thymomas; (I) for polymorphisms and somatic loss of heterozygosity of the non-coding egfr CA-SSR-1 microsatellite and (II) for egfr gene copy number changes. Results We found two prevalent CA-SSR-1 genotypes: a homozygous 16 CA repeat and a heterozygous genotype, bearing alleles with 16 and 20 CA repeats. The average combined allele length was correlated with tumor subtype: shorter sequences were significantly associated with the more aggressive WHO thymoma subtype group including B2/B3, B3 and B3/C histotypes. Four out of 29 informative cases analysed for somatic CA-SSR-1 loss of heterozygosity showed allelic imbalance (AI), 3/4 with loss of the longer allele. By egfr-FISH analysis, 9 out of 33 cases were FISH positive. Moreover, the two integrated techniques demonstrated that 3 out of 4 CA-SSR-1-AI positive cases with short allele relative prevalence showed significantly low or high chromosome 7 “polysomy”/increased gene copy number by egfr-FISH. Conclusions Our molecular and genetic and follow up data indicated that CA-SSR-1-allelic imbalance with short allele relative prevalence significantly correlated with EGFR 3+ immunohistochemical score, increased egfr Gene Copy Number, advanced stage and with relapsing/metastatic behaviour in thymomas. PMID:27076933

  6. Development of critical life stage assays: Teratogenic effects of SRS effluent components on freshwater fish, gambusia

    SciTech Connect

    Guram, M.S.

    1990-11-01

    The final report of the research carried out at Voorhees College contains a composite compilation of the last two years work. The data note variation in the number of young fish delivered per female vary markedly between several ponds on the SRS and of SRS ponds. The reasons for this are unknown at present. Initial research was carried out on the effects on the developing fish fetus of various substances that may have produced these variations. Further study is necessary to identify the factors that produce the observed alterations. 7 figs., 5 tabs.

  7. EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance.

    PubMed

    Dua, Rajiv; Zhang, Jianhuan; Nhonthachit, Phets; Penuel, Elicia; Petropoulos, Chris; Parry, Gordon

    2010-08-01

    HER2 is gene amplified or over-expressed in 20-25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909-4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype. PMID:19859802

  8. Protein digestibility evaluations of meat and fish substrates using laboratory, avian and ileally cannulated dog assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish and meat protein serves as important protein sources in the human and companion animal diets: however, limited information is available on differences in protein quality. Pollock fillet, and salmon fillet, beef loin, pork loin and chicken breast, were evaluated for protein quality and amino aci...

  9. Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

    SciTech Connect

    Yu, Zhiwei; Cui, Binbin; Jin, Yinghu; Chen, Haipeng; Wang, Xishan

    2011-08-12

    Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

  10. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors.

    PubMed

    Jia, Yong; Yun, Cai-Hong; Park, Eunyoung; Ercan, Dalia; Manuia, Mari; Juarez, Jose; Xu, Chunxiao; Rhee, Kevin; Chen, Ting; Zhang, Haikuo; Palakurthi, Sangeetha; Jang, Jaebong; Lelais, Gerald; DiDonato, Michael; Bursulaya, Badry; Michellys, Pierre-Yves; Epple, Robert; Marsilje, Thomas H; McNeill, Matthew; Lu, Wenshuo; Harris, Jennifer; Bender, Steven; Wong, Kwok-Kin; Jänne, Pasi A; Eck, Michael J

    2016-06-01

    The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors. PMID:27251290

  11. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  12. Bridging the Gap From Screening Assays to Estrogenic Effects in Fish: Potential Roles of Multiple Estrogen Receptor Subtypes

    PubMed Central

    2015-01-01

    This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hERα. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hERα and hERβ, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ERβ subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ERβ subtypes are critically involved in the teleost estrogenic response, with the ERα:ERβ ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures. PMID:24422420

  13. A single ligand is sufficient to activate EGFR dimers

    PubMed Central

    Liu, Ping; Cleveland, Thomas E.; Bouyain, Samuel; Byrne, Patrick O.; Longo, Patti A.; Leahy, Daniel J.

    2012-01-01

    Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1β that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions. PMID:22699492

  14. A PCR-based assay for the identification of the fish pathogen Renibacterium salmoninarum.

    PubMed

    León, G; Maulén, N; Figueroa, J; Villanueva, J; Rodríguez, C; Vera, M I; Krauskopf, M

    1994-01-15

    By means of a one-step one-tube extraction from less than 1 mg of tissue it is possible to identify, via the polymerase chain reaction, Renibacterium salmoninarum in salmon with bacterial kidney disease. A 149-bp DNA sequence unique to R. salmoninarum was specifically amplified and its nature confirmed by Southern hybridization using a non-isotopically labelled probe. The sensitivity of the approach allowed the detection of 22 R. salmoninarum cells. The procedure was successfully applied in the identification of the causative agent of bacterial kidney disease in kidney tissue from infected fishes. PMID:8138127

  15. Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish.

    PubMed

    Bjornsdottir-Butler, Kristin; Jones, Jessica L; Benner, Ronald; Burkhardt, William

    2011-05-01

    Prompt detection of bacteria that contribute to scombrotoxin (histamine) fish poisoning can aid in the detection of potentially toxic fish products and prevent the occurrence of illness. We report development of the first real-time PCR method for rapid detection of Gram-negative histamine-producing bacteria (HPB) in fish. The real-time PCR assay was 100% inclusive for detecting high-histamine producing isolates and did not detect any of the low- or non-histamine producing isolates. The efficiency of the assay with/without internal amplification control ranged from 96-104% and in the presence of background flora and inhibitory matrices was 92/100% and 73-96%, respectively. This assay was used to detect HPB from naturally contaminated yellowfin tuna, bluefish, and false albacore samples. Photobacterium damselae (8), Plesiomonas shigelloides (2), Shewanella sp. (1), and Morganella morganii (1) were subsequently isolated from the real-time PCR positive fish samples. These results indicate that the real-time PCR assay developed in this study is a rapid and sensitive method for detecting high-HPB. The assay may be adapted for quantification of HPB, either directly or with an MPN-PCR method. PMID:21356438

  16. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma

    PubMed Central

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

  17. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

    PubMed

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

  18. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

    PubMed

    Yamada, T; Takeuchi, S; Fujita, N; Nakamura, A; Wang, W; Li, Q; Oda, M; Mitsudomi, T; Yatabe, Y; Sekido, Y; Yoshida, J; Higashiyama, M; Noguchi, M; Uehara, H; Nishioka, Y; Sone, S; Yano, S

    2013-09-12

    Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3K (phosphoinositide 3-kinase)/PDK1 (3-phosphoinositide-dependent protein kinase)/Akt that determines receptor signal selectivity for non-mutated EGFR, and assessed its role in EGFR mutant lung cancer with or without gatekeeper T790M mutation. Cell line-based assays showed that Aki1 constitutively associates with mutant EGFR in lung cancer cells with (H1975) or without (PC-9 and HCC827) T790M gatekeeper mutation. Silencing of Aki1 induced apoptosis of EGFR mutant lung cancer cells. Treatment with Aki1 siRNA dramatically inhibited growth of H1975 cells in a xenograft model. Moreover, silencing of Aki1 further potentiated growth inhibitory effect of new generation EGFR-TKIs against H1975 cells in vitro. Aki1 was frequently expressed in tumor cells of EGFR mutant lung cancer patients (53/56 cases), including those with acquired resistance to EGFR-TKI treatment (7/7 cases). Our data suggest that Aki1 may be a critical mediator of survival signaling from mutant EGFR to Akt, and may therefore be an ideal target for EGFR mutant lung cancer patients, especially those with acquired EGFR-TKI resistance due to EGFR T790M gatekeeper mutation. PMID:23045273

  19. Diagnosis of adults Xp11.2 translocation renal cell carcinoma by immunohistochemistry and FISH assays: clinicopathological data from ethnic Chinese population

    PubMed Central

    Qu, Yuanyuan; Gu, Chengyuan; Wang, Hongkai; Chang, Kun; Yang, Xiaoqun; Zhou, Xiaoyan; Dai, Bo; Zhu, Yao; Shi, Guohai; Zhang, Hailiang; Ye, Dingwei

    2016-01-01

    This study aimed to assess the utility of transcription factor E3 (TFE3) break-apart fluorescence in situ hybridization (FISH) assay in diagnosis of Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) and to compare the clinicopathological features between adult Xp11.2 RCC and non-Xp11.2 RCC. 76 pathologically suspected Xp11.2 RCCs were recruited from our institution. Both TFE3 immunohistochemistry (IHC) and TFE3 FISH assay were performed for the entire cohort. The progression-free survival (PFS) and overall survival (OS) curves were estimated using the Kaplan-Meier method. FISH analysis confirmed 30 Xp11.2 RCCs, including 28 cases with positive TFE3 immunostaining and 2 cases with negative immunostaining. The false-positive and false-negative rates were 6.7% (2/30) and 4.3% (2/46), respectively, for TFE3 IHC compared with FISH assay. Xp11.2 RCC was significantly associated with higher pathological stage and Fuhrman nuclear grade compared with non-Xp11.2 RCC (P < 0.05). The median PFS and OS for TFE3 FISH-positive group were 13.0 months (95% CI, 8.4–17.6 months) and 50.0 months (95% CI, 27.6–72.4 months), respectively, while the median PFS and OS had not been reached for TFE3 FISH-negative group. In conclusion, TFE3 break-apart FISH assay is a highly useful and standard diagnostic method for Xp11.2 RCC. Adult Xp11.2 RCC is clinically aggressive and often presents at advanced stage with poor prognosis. PMID:26880493

  20. TELEOST FISH (Solea solea): A NOVEL MODEL FOR ECOTOXICOLOGICAL ASSAY OF CONTAMINATED SEDIMENTS

    PubMed Central

    Ribecco, C; Hardiman, G; Sásik, R; Vittori, S; Carnevali, O

    2012-01-01

    Chemical analysis of sediment is not indicative of the downstream biological effects on aquatic organisms. In this study, the biological effects of sediment were examined using: Teleost fish, (Solea solea), Artemia and rotifers. Although chemicals levels were below the limits permissible by Italian law, S. solea juveniles exposed to sediment (0.3% w/v) for 96 hours, revealed significant induction in the expression levels of HSP70, ERα, TRα, RXRα, PPARα, β, CYP4501A1 and CYP3A mRNAs, suggesting the utility of this species as a novel biosensor. The bio-toxicity of the sediment was further validated by exposing Artemia and rotifers to concentrations of elutriate (derived from the sediment) from 10 to 100 % v/v (with a 50% mortality rate). These results suggest that sediment defined as moderately contaminated, solely on the basis of the chemical profile, may in fact cause harmful effects to aquatic organisms. This study highlights the need for biological approaches in the establishment of sediment toxicity levels. PMID:22217502

  1. A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity.

    PubMed

    Castoldi, R; Ecker, V; Wiehle, L; Majety, M; Busl-Schuller, R; Asmussen, M; Nopora, A; Jucknischke, U; Osl, F; Kobold, S; Scheuer, W; Venturi, M; Klein, C; Niederfellner, G; Sustmann, C

    2013-12-12

    Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling. PMID:23812422

  2. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrined-disrupting chemicals

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely to...

  3. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely t...

  4. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals (poster)

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely to...

  5. EGFR Signaling in Liver Diseases

    PubMed Central

    Komposch, Karin; Sibilia, Maria

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that is activated by several ligands leading to the activation of diverse signaling pathways controlling mainly proliferation, differentiation, and survival. The EGFR signaling axis has been shown to play a key role during liver regeneration following acute and chronic liver damage, as well as in cirrhosis and hepatocellular carcinoma (HCC) highlighting the importance of the EGFR in the development of liver diseases. Despite the frequent overexpression of EGFR in human HCC, clinical studies with EGFR inhibitors have so far shown only modest results. Interestingly, a recent study has shown that in human HCC and in mouse HCC models the EGFR is upregulated in liver macrophages where it plays a tumor-promoting function. Thus, the role of EGFR in liver diseases appears to be more complex than what anticipated. Further studies are needed to improve the molecular understanding of the cell-specific signaling pathways that control disease development and progression to be able to develop better therapies targeting major components of the EGFR signaling network in selected cell types. In this review, we compiled the current knowledge of EGFR signaling in different models of liver damage and diseases, mainly derived from the analysis of HCC cell lines and genetically engineered mouse models (GEMMs). PMID:26729094

  6. Monitoring of gefitinib sensitivity with radioiodinated PHY based on EGFR expression.

    PubMed

    Yoshimoto, Mitsuyoshi; Hirata, Masahiko; Kanai, Yasukazu; Naka, Sadahiro; Nishii, Ryuichi; Kagawa, Shinya; Kawai, Keiichi; Ohmomo, Yoshiro

    2014-01-01

    Epidermal growth factor receptor (EGFR) is attractive target for tumor diagnosis and therapy, as it is specifically and abundantly expressed in tumor cells. EGFR-tyrosine kinase (TK) inhibitors such as gefitinib and erlotinib are widely used in the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated whether radioiodinated 4-(3-iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), which is a candidate EGFR-TK imaging agent for single photon emission computed tomography (SPECT) is able to predict gefitinib sensitivity. We used four NSCLC cell lines-A549 (wild-type EGFR), H1650 (mutant EGFR; del E746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR; L858R)-and one epidermoid carcinoma cell line, A431 (wild-type EGFR). Cell proliferation assay and Western blotting revealed that A431 and H3255 with high EGFR expression showed high sensitivity to gefitinib. On the other hand, A549, H1650 and H1975 showed much lower sensitivity to gefitinib. The blocking study revealed that gefitinib decreased tumor uptake in (125)I-PHY in A431-bearing mice. Moreover, in vivo tumor uptake of (125)I-PHY was correlated with the IC50 of gefitinib for cell proliferation. In the present study, tumor uptake of (125)I-PHY was correlated with the gefitinib sensitivity and this uptake was based on expression levels of EGFR, but not on mutation status. Although the mutation status is the most important factor for predicting gefitinib sensitivity, the abundant expression of EGFR is essential for therapy with EGFR-TK inhibitors. Therefore, radioiodinated PHY is a potential imaging agent to predict gefitinib sensitivity based on EGFR expression levels though further modifications of the imaging agent is needed to accurately estimate the mutation status. PMID:24583857

  7. Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands.

    PubMed

    Caillaud, A; Eixarch, H; de la Iglesia, P; Rodriguez, M; Dominguez, L; Andree, K B; Diogène, J

    2012-01-01

    The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands. PMID:22394180

  8. A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum.

    PubMed

    Zhang, Yang; Xiao, Jingfan; Wang, Qiyao; Zhang, Yuanxing

    2016-08-28

    Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method. PMID:27116991

  9. Serological evidence of infectious salmon anaemia virus (ISAV) infection in farmed fishes, using an indirect enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Kibenge, Molly T; Opazo, Beatriz; Rojas, Alejandro H; Kibenge, Frederick S B

    2002-08-15

    Antibody detection tests are rarely used for diagnostic purposes in fish diseases. Infectious salmon anaemia (ISA) caused by ISA virus (ISAV) is an emerging disease of Atlantic salmon Salmo salar L. The virus has also been isolated from diseased coho salmon Oncorhynchus kisutch in Chile. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate serodiagnosis of ISAV infection, the study of epidemiology, and the control of ISA in farmed fishes has been developed using purified ISAV as the coating antigen, and monoclonal antibodies that detect fish immunoglobulins bound to the antigen on the plate. Application of the test to a random sample of farmed Atlantic salmon from the Bay of Fundy, New Brunswick, Canada, positively identified 5 of the 7 ISAV RT-PCR-positive fish, and all 10 RT-PCR-negative fish were also negative in the ELISA. Some RT-PCR-negative fish had an elevated non-specific antibody reactivity suggestive of chronic infection or resistance to ISAV. This test was also able to detect 11 of the 14 coho salmon pooled serum samples from a clinically affected farm in Chile that were positive by the virus neutralization (VN) test, and 2 of the 4 VN-negative samples. We conclude that this ELISA would be suitable as a routine test for ISAV infection or for assessing ISAV vaccine efficacy before placing smolts in sea cages, and for testing fishes in sea cages to detect level of resistance to ISA. The assay enables vaccination in combination with depopulation control methods. PMID:12240966

  10. Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2

    PubMed Central

    Wu, Yifen; Li, Rong; Zhang, Junyi; Wang, Gang; Liu, Bin; Huang, Xiaofang; Zhang, Tao; Luo, Rongcheng

    2015-01-01

    Background Trastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2. Methods Trastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model. Results Trastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are

  11. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR)

    PubMed Central

    Trussoni, Christy E.; Tabibian, James H.; Splinter, Patrick L.; O’Hara, Steven P.

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes. PMID:25915403

  12. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes. PMID:25915403

  13. Assessment of EGFR Mutation Status in Lung Adenocarcinoma by Immunohistochemistry Using Antibodies Specific to the Two Major Forms of Mutant EGFR

    PubMed Central

    Brevet, Marie; Arcila, Maria; Ladanyi, Marc

    2010-01-01

    EGFR mutations are the best predictors of response to EGFR kinase inhibitors in lung adenocarcinoma. We evaluated two mutation-specific monoclonal antibodies for the detection of EGFR mutations by immunohistochemistry (IHC), generated respectively against the L858R mutant and the exon 19 mutant with the common 15bp/5AA deletion. These two mutations account for approximately 90% of all EGFR mutations. IHC staining performed on 218 paraffin-embedded lung adenocarcinomas was assessed on a 0 to 3+ scale, and positivity cutoffs of 1+ and 2+ were compared. All cases were studied by standard molecular methods for these two mutations, and selected cases were also studied using higher sensitivity molecular assays. The EGFR L858R mutant antibody showed a sensitivity of 95% and a positive predictive value (PPV) of 99% with a positivity cutoff of 1+ and a sensitivity of 76% and a PPV of 100% with a positivity cutoff of 2+. The EGFR exon 19 mutant–specific antibody showed reduced sensitivity for exon 19 deletions other than 15bp. A positivity cutoff of 1+ resulted in a sensitivity of 85% and a PPV of 99%, whereas a 2+ cutoff gave a sensitivity of 67% and a PPV of 100%. IHC with EGFR mutant–specific antibodies could be used as a screen to identify most candidates for EGFR inhibitors. PMID:20093391

  14. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR.

    PubMed

    Brevet, Marie; Arcila, Maria; Ladanyi, Marc

    2010-03-01

    EGFR mutations are the best predictors of response to EGFR kinase inhibitors in lung adenocarcinoma. We evaluated two mutation-specific monoclonal antibodies for the detection of EGFR mutations by immunohistochemistry (IHC), generated respectively against the L858R mutant and the exon 19 mutant with the common 15bp/5AA deletion. These two mutations account for approximately 90% of all EGFR mutations. IHC staining performed on 218 paraffin-embedded lung adenocarcinomas was assessed on a 0 to 3+ scale, and positivity cutoffs of 1+ and 2+ were compared. All cases were studied by standard molecular methods for these two mutations, and selected cases were also studied using higher sensitivity molecular assays. The EGFR L858R mutant antibody showed a sensitivity of 95% and a positive predictive value (PPV) of 99% with a positivity cutoff of 1+ and a sensitivity of 76% and a PPV of 100% with a positivity cutoff of 2+. The EGFR exon 19 mutant-specific antibody showed reduced sensitivity for exon 19 deletions other than 15bp. A positivity cutoff of 1+ resulted in a sensitivity of 85% and a PPV of 99%, whereas a 2+ cutoff gave a sensitivity of 67% and a PPV of 100%. IHC with EGFR mutant-specific antibodies could be used as a screen to identify most candidates for EGFR inhibitors. PMID:20093391

  15. Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.

    2006-01-01

    The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.

  16. Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

    PubMed Central

    Brinkmann, Markus; Blenkle, Henning; Salowsky, Helena; Bluhm, Kerstin; Schiwy, Sabrina; Tiehm, Andreas; Hollert, Henner

    2014-01-01

    Heterocyclic aromatic hydrocarbons are, together with their un-substituted analogues, widely distributed throughout all environmental compartments. While fate and effects of homocyclic PAHs are well-understood, there are still data gaps concerning the ecotoxicology of heterocyclic PAHs: Only few publications are available investigating these substances using in vitro bioassays. Here, we present a study focusing on the identification and quantification of clastogenic and aneugenic effects in the micronucleus assay with the fish liver cell line RTL-W1 that was originally derived from rainbow trout (Oncorhynchus mykiss). Real concentrations of the test items after incubation without cells were determined to assess chemical losses due to, e.g., sorption or volatilization, by means of gas chromatography-mass spectrometry. We were able to show genotoxic effects for six compounds that have not been reported in vertebrate systems before. Out of the tested substances, 2,3-dimethylbenzofuran, benzothiophene, quinoline and 6-methylquinoline did not cause substantial induction of micronuclei in the cell line. Acridine caused the highest absolute induction. Carbazole, acridine and dibenzothiophene were the most potent substances compared with 4-nitroquinoline oxide, a well characterized genotoxicant with high potency used as standard. Dibenzofuran was positive in our investigation and tested negative before in a mammalian system. Chemical losses during incubation ranged from 29.3% (acridine) to 91.7% (benzofuran) and may be a confounding factor in studies without chemical analyses, leading to an underestimation of the real potency. The relative potency of the investigated substances was high compared with their un-substituted PAH analogues, only the latter being typically monitored as priority or indicator pollutants. Hetero-PAHs are widely distributed in the environment and even more mobile, e.g. in ground water, than homocyclic PAHs due to the higher water solubility. We

  17. Development of a loop-mediated isothermal amplification assay for rapid detection of Nocardia salmonicida, the causative agent of nocardiosis in fish.

    PubMed

    Xia, Liqun; Zhang, Honglian; Lu, Yishan; Cai, Jia; Wang, Bei; Jian, Jichang

    2015-03-01

    Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10(3) CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis. PMID:25262681

  18. Validation of an accelerated solvent extraction liquid chromatography-tandem mass spectrometry method for Pacific ciguatoxin-1 in fish flesh and comparison with the mouse neuroblastoma assay.

    PubMed

    Wu, Jia Jun; Mak, Yim Ling; Murphy, Margaret B; Lam, James C W; Chan, Wing Hei; Wang, Mingfu; Chan, Leo L; Lam, Paul K S

    2011-07-01

    Ciguatera fish poisoning (CFP) is a global foodborne illness caused by consumption of seafood containing ciguatoxins (CTXs) originating from dinoflagellates such as Gambierdiscus toxicus. P-CTX-1 has been suggested to be the most toxic CTX, causing ciguatera at 0.1 μg/kg in the flesh of carnivorous fish. CTXs are structurally complex and difficult to quantify, but there is a need for analytical methods for CFP toxins in coral reef fishes to protect human health. In this paper, we describe a sensitive and rapid extraction method using accelerated solvent extraction combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the detection and quantification of P-CTX-1 in fish flesh. By the use of a more sensitive MS system (5500 QTRAP), the validated method has a limit of quantification (LOQ) of 0.01 μg/kg, linearity correlation coefficients above 0.99 for both solvent- and matrix-based standard solutions as well as matrix spike recoveries ranging from 49% to 85% in 17 coral reef fish species. Compared with previous methods, this method has better overall recovery, extraction efficiency and LOQ. Fish flesh from 12 blue-spotted groupers (Cephalopholis argus) was assessed for the presence of CTXs using HPLC-MS/MS analysis and the commonly used mouse neuroblastoma assay, and the results of the two methods were strongly correlated. This method is capable of detecting low concentrations of P-CTX-1 in fish at levels that are relevant to human health, making it suitable for monitoring of suspected ciguateric fish both in the environment and in the marketplace. PMID:21505950

  19. Accurate Detection and Quantification of the Fish Viral Hemorrhagic Septicemia virus (VHSv) with a Two-Color Fluorometric Real-Time PCR Assay

    PubMed Central

    Palsule, Vrushalee V.; Yeo, Jiyoun; Shepherd, Brian S.; Crawford, Erin L.; Stepien, Carol A.

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb – appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106 actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics. PMID:23977162

  20. Dual MET-EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer.

    PubMed

    Tang, Z; Du, R; Jiang, S; Wu, C; Barkauskas, D S; Richey, J; Molter, J; Lam, M; Flask, C; Gerson, S; Dowlati, A; Liu, L; Lee, Z; Halmos, B; Wang, Y; Kern, J A; Ma, P C

    2008-09-16

    Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV = 1.1). At 2 microM, SU 11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P = 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET-EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T

  1. Differential Sensitivity of Glioma- versus Lung Cancer-specific EGFR mutations to EGFR Kinase Inhibitors

    PubMed Central

    Vivanco, Igor; Robins, H. Ian; Rohle, Daniel; Campos, Carl; Grommes, Christian; Nghiemphu, Phioanh Leia; Kubek, Sara; Oldrini, Barbara; Chheda, Milan G.; Yannuzzi, Nicolas; Tao, Hui; Zhu, Shaojun; Iwanami, Akio; Kuga, Daisuke; Dang, Julie; Pedraza, Alicia; Brennan, Cameron W.; Heguy, Adriana; Liau, Linda M.; Lieberman, Frank; Yung, W.K. Alfred; Gilbert, Mark R.; Reardon, David A.; Drappatz, Jan; Wen, Patrick Y.; Lamborn, Kathleen R.; Chang, Susan M.; Prados, Michael D.; Fine, Howard A.; Horvath, Steve; Wu, Nian; Lassman, Andrew B.; DeAngelis, Lisa M.; Yong, William H.; Kuhn, John G.; Mischel, Paul S.; Mehta, Minesh P.; Cloughesy, Timothy F.; Mellinghoff, Ingo K.

    2012-01-01

    Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors which bind to the inactive EGFR conformation, on the other hand, potently inhibit EGFR EC mutants and induce cell death in EGFR mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM, and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types. PMID:22588883

  2. The Use of a Two-Tiered Testing Strategy for the Simultaneous Detection of Small EGFR Mutations and EGFR Amplification in Lung Cancer

    PubMed Central

    Lewandowska, Marzena Anna; Czubak, Karol; Klonowska, Katarzyna; Jozwicki, Wojciech; Kowalewski, Janusz; Kozlowski, Piotr

    2015-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy. PMID:25719557

  3. The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

    PubMed

    Lewandowska, Marzena Anna; Czubak, Karol; Klonowska, Katarzyna; Jozwicki, Wojciech; Kowalewski, Janusz; Kozlowski, Piotr

    2015-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy. PMID:25719557

  4. A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

    PubMed

    Cook, M; Lynch, W H

    1999-07-01

    A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission. PMID:10388701

  5. Differences in detection of Aeromonas salmonicida in covertly infected salmonid fishes by the stress-inducible furunculosis test and culture-based assays

    USGS Publications Warehouse

    Cipriano, R.C.; Ford, L.A.; Smith, D.R.; Schachte, J.H.; Petrie, C.J.

    1997-01-01

    Accurate detection of Aeromonas salmonicida subsp. salmonicida (the cause of furunculosis disease) in covertly infected salmonids is difficult and is a cause of concern for those involved in fish health inspection and resource management programs. In this study, we examined populations of brook trout Salvelinus fontinalis, Atlantic salmon Salmo salar, and lake trout Salvelinus namaycush that previously sustained natural episodes of furunculosis. Consequently, the sampled fish were presumed to harbor latent infections. Mucus, gill, liver, kidney, heart, spleen, and intestine samples (N = 100 fish per group sampled) were processed and examined by (1) direct dilution counts and (2) quadrant streaking after a 48-h pre-enrichment in trypticase soy broth (TSB). Another subsample of fish from each group was then subjected to stress-inducible furunculosis tests. Stress tests detected A. salmonicida in three of four groups of fish that were examined whereas the pathogen was detected in only two of the groups analyzed with culture-based assays. Although pre-enrichment in TSB enhanced detection within internal sampling sites including the liver, heart, spleen, and kidney, enrichment did not enhance detection from mucus, gill, or intestinal samples.

  6. 2,3,7,8 tetrachlorodibenzodioxin in fish from the Pigeon river of Eastern Tennessee: Its toxicity and mutagenicity as revealed by the Ames Salmonella Assay

    SciTech Connect

    Blevins, R.D. )

    1990-04-01

    Levels of 2,3,7,8 tetrachlorodibenzodioxin (TCDD)were determined in both striated muscle (fillets) and whole body extracts of fish specimens harvested during a two year period (1987-1989) from the Pigeon River (between Hartford and Newport) of Eastern Tennessee. Whole body (wet weight) fish extract levels as high as 117 {mu}g/kg body weight and composite fish fillet (wet weight) extract levels as high 87 {mu}g/kg fillet weight were observed. Pure TCDD was found to be highly toxic to the Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA1535 at TCDD dosages which exceeded 825 ng/ml in the top agar of the Ames Salmonella assay. An 825 ng/ml TCDD dosage was not mutagenic to any of the tested Salmonella strains, (both with and without metabolic activation (S9) mix). However, when both acidic and alcohol fish extracts from the Pigeon River were tested for mutagenicity, several of the fish extracts were found to be mutagenic to Salmonella strains TA97, TA98, and TA100 (having mutagenic ratios which greatly exceeded the 2.5 {times} spontaneous ratio). These mutagenic extracts also demonstrated mutagenic dose-response curves. Other chemicals within the extracts as well as synergistic effects may account for the mutagenicity.

  7. A Sensitive Nested Reverse Transcriptase PCR Assay To Detect Viable Cells of the Fish Pathogen Renibacterium salmoninarum in Atlantic Salmon (Salmo salar L.)

    PubMed Central

    Cook, Marcia; Lynch, William H.

    1999-01-01

    A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission. PMID:10388701

  8. Rapid detection of Flavobacterium columnare in fish using TaqMan real-time polymerase chain reaction assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease caused by Flavobacterium columnare, a ubiquitous Gram-negative organism prevalent among both warm and cold water reared fish species is important from an economical perspective to the aquaculture industry, worldwide. Because, in commercial aquaculture large numbers of fish are rea...

  9. The antitumorigenic function of EGFR in metastatic breast cancer is regulated by expression of Mig6.

    PubMed

    Wendt, Michael K; Williams, Whitney K; Pascuzzi, Pete E; Balanis, Nikolas G; Schiemann, Barbara J; Carlin, Cathleen R; Schiemann, William P

    2015-01-01

    Numerous studies by our lab and others demonstrate that epidermal growth factor receptor (EGFR) plays critical roles in primary breast cancer (BC) initiation, growth and dissemination. However, clinical trials targeting EGFR function in BC have lead to disappointing results. In the current study we sought to identify the mechanisms responsible for this disparity by investigating the function of EGFR across the continuum of the metastatic cascade. We previously established that overexpression of EGFR is sufficient for formation of in situ primary tumors by otherwise nontransformed murine mammary gland cells. Induction of epithelial-mesenchymal transition (EMT) is sufficient to drive the metastasis of these EGFR-transformed tumors. Examining growth factor receptor expression across this and other models revealed a potent downregulation of EGFR through metastatic progression. Consistent with diminution of EGFR following EMT and metastasis EGF stimulation changes from a proliferative to an apoptotic response in in situ versus metastatic tumor cells, respectively. Furthermore, overexpression of EGFR in metastatic MDA-MB-231 BC cells promoted their antitumorigenic response to EGF in three dimensional (3D) metastatic outgrowth assays. In line with the paradoxical function of EGFR through EMT and metastasis we demonstrate that the EGFR inhibitory molecule, Mitogen Induced Gene-6 (Mig6), is tumor suppressive in in situ tumor cells. However, Mig6 expression is absolutely required for prevention of apoptosis and ultimate metastasis of MDA-MB-231 cells. Further understanding of the paradoxical function of EGFR between primary and metastatic tumors will be essential for application of its targeted molecular therapies in BC. PMID:25622905

  10. Decreased HCRP1 promotes breast cancer metastasis by enhancing EGFR phosphorylation.

    PubMed

    Yang, Wenlin; Wang, Ji-Gang; Wang, Qiangxiu; Qin, Yejun; Lin, Xiaoyan; Zhou, Danmei; Ren, Kehan; Hou, Chenjian; Xu, Jiawen; Liu, Xiuping

    2016-08-19

    Previous study showed that hepatocellular carcinoma related protein 1 (HCRP1) is decreased in breast cancer. HCRP1 expression is inversely related to epithelial growth factor receptor (EGFR) in breast cancer tissues, and patients with breast cancer expressing lower HCRP1 tended to suffer a shorter life expectancy. However, the detailed biological functions of HCRP1 in breast cancer as well as the interaction between HCRP1 and EGFR remain unexplored. In this study, we examined HCRP1 expression in breast cancer tissues and cell lines by western blot. Thereafter, we performed transwell migration and matrigel invasion assays after siRNA interference and lentiviral vector of HCRP1 infection. To further investigate the interaction between HCRP1 downregulation and EGFR signaling pathway, we evaluated the phosphorylation status of EGFR, Erk1/2 and Akt by western blot following HCRP1-siRNA transfection. Moreover, we investigated the in vivo functions of HCRP1 using a breast cancer xenograft model. We found that HCRP1 depletion significantly promoted breast cancer migration and invasion while HCRP1 overexpression produced an opposite effect. In addition, HCRP1 depletion decreased EGFR degradation and enhanced phosphorylation of EGFR. Interestingly, HCRP1 depletion also led to insensitivity to EGFR inhibitors treatment. The in vivo experiment confirmed the metastasis inhibition function of HCRP1. The present data indicate that HCRP1 inhibits breast cancer metastasis through downregulating EGFR phosphorylation. PMID:27311861

  11. Utilization of Structure-Based Design to Identify Novel, Irreversible Inhibitors of EGFR Harboring the T790M Mutation.

    PubMed

    Hennessy, Edward J; Chuaqui, Claudio; Ashton, Susan; Colclough, Nicola; Cross, Darren A E; Debreczeni, Judit É; Eberlein, Cath; Gingipalli, Lakshmaiah; Klinowska, Teresa C M; Orme, Jonathan P; Sha, Li; Wu, Xiaoyun

    2016-05-12

    A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers. PMID:27190603

  12. Src inhibitors act through different mechanisms in Non-Small Cell Lung Cancer models depending on EGFR and RAS mutational status

    PubMed Central

    Formisano, Luigi; D'Amato, Valentina; Servetto, Alberto; Brillante, Simona; Raimondo, Lucia; Di Mauro, Concetta; Marciano, Roberta; Orsini, Roberta Clara; Cosconati, Sandro; Randazzo, Antonio; Parsons, Sarah J.; Montuori, Nunzia; Veneziani, Bianca Maria; De Placido, Sabino

    2015-01-01

    Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non-Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs. PMID:26325669

  13. Study of 30 patients with unexplained developmental delay and dysmorphic features or congenital abnormalities using conventional cytogenetics and multiplex FISH telomere (M-TEL) integrity assay.

    PubMed

    Popp, Susanne; Schulze, Birgit; Granzow, Martin; Keller, Monika; Holtgreve-Grez, Heidi; Schoell, Brigitte; Brough, Michaela; Hager, Hans-Dieter; Tariverdian, Gholamali; Brown, Jill; Kearney, Lyndal; Jauch, Anna

    2002-07-01

    Cryptic subtelomeric chromosome rearrangements are a major cause of mild to severe mental retardation pointing out the necessity of sensitive screening techniques to detect such aberrations among affected patients. In this prospective study a group of 30 patients with unexplained developmental retardation and dysmorphic features or congenital abnormalities were analysed using the recently published multiplex FISH telomere (M-TEL) integrity assay in combination with conventional G-banding analysis. The patients were selected by one or more of the following criteria defined by de Vries et al.: (a) family history with two or more affected individuals, (b) prenatal onset growth retardation, (c) postnatal growth abnormalities, (d) facial dysmorphic features, (e) non-facial dysmorphism and congenital abnormalities. In addition, we included two patients who met these criteria and revealed questionable chromosome regions requiring further clarification. In four patients (13.3%) cryptic chromosome aberrations were successfully determined by the M-TEL integrity assay and in two patients with abnormal chromosome regions intrachromosomal aberrations were characterized by targetted FISH experiments. Our results accentuate the requirement of strict selection criteria prior to patient testing with the M-TEL integrity assay. Another essential precondition is high-quality banding analysis to identify structural abnormal chromosomes. The detection of familial balanced translocation carriers in 50% of the cases emphasizes the significance of such an integrated approach for genetic counselling and prenatal diagnosis. PMID:12136233

  14. Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer

    PubMed Central

    Lococo, Filippo; Paci, Massimiliano; Rapicetta, Cristian; Rossi, Teresa; Sancisi, Valentina; Braglia, Luca; Cavuto, Silvio; Bisagni, Alessandra; Bongarzone, Italia; Noonan, Douglas M.; Albini, Adriana; Maramotti, Sally

    2015-01-01

    Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies. PMID:26295387

  15. Studies for development of novel quinazolinones: new biomarker for EGFR.

    PubMed

    Aggarwal, Swati; Sinha, Deepa; Tiwari, Anjani Kumar; Pooja, Pooja; Kaul, Ankur; Singh, Gurmeet; Mishra, Anil Kumar

    2015-05-15

    The binding capabilities of a series of novel quinazolinone molecules were established and stated in a comprehensive computational methodology as well as by in vitro analysis. The main focus of this work was to achieve more insight of the interactions with crystal structure of PDB ID: 1M17 and predict their binding mode to EGFR. Three molecules were screened for further examination, which were synthesized and characterized using spectroscopic techniques. The persuasive affinity of these molecules towards EGFR inhibition (IC50 for QT=45nM) was established and validated from specific kinase assay including the cell viability spectrophotometric assay (QT=12nM). Drug likeliness property were also considered by analysing, the ADME of these molecules by using scintigraphic techniques. The result showed antitumour activity of QT (4.17 tumour/muscle at 4h). Further photo physical properties were also analysed to see in vitro HSA binding to QT. PMID:25766241

  16. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

    PubMed Central

    2010-01-01

    Background Immunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients. PMID:21167064

  17. Selective and sensitive determination of cypermethrin in fish via enzyme-linked immunosorbent assay-like method based on molecularly imprinted artificial antibody-quantum dot optosensing materials.

    PubMed

    Xiao, Ting-Ting; Shi, Xi-Zhi; Jiao, Hai-Feng; Sun, Ai-Li; Ding, Hao; Zhang, Rong-Rrong; Pan, Dao-Dong; Li, De-Xiang; Chen, Jiong

    2016-01-15

    Molecularly imprinted silica layers appended to quantum dots (MIP-QDs) with customized selective artificial recognition sites were fabricated in this study by optimizing the ratio of the functional monomer to the template. Scanning electron microscopy, transmission electron microscopy, Brunauer–emmett–teller, Fourier transform infrared spectroscopy, and selectivity assay analyses were also performed. Results demonstrated that the selective fluorescence quenching properties of MIP-QDs toward cypermethrin (CYP) are due to strong interactions between these molecules. An enzyme-linked immunosorbent assay (ELISA)-like method based on the MIP-QDs was established under optimal conditions. The fluorescence quenching observed from this method showed a linear relationship with CYP concentration over the range of 0.05–60.0 mg/kg with a correlation coefficient of 0.9838. Good recovery (82.7–92.4%) and a relative standard deviation of less than 10.1% were obtained from fish samples spiked with three levels of CYP. This method also demonstrated a low detection limit of 1.2 μg/kg. The ELISA-like method based on MIP-QDs can be successfully employed to detect residual of CYP in fish samples. PMID:26283587

  18. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

    PubMed Central

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

    2015-01-01

    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

  19. Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

    PubMed

    Zheng, Junfang; Dai, Yuanping; Yang, Zhiyu; Yang, Longyan; Peng, Zhiqiang; Meng, Ran; Xiong, Ying; He, Junqi

    2016-01-01

    Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular

  20. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

    SciTech Connect

    Zhou, Wenjun; Ercan, Dalia; Chen, Liang; Yun, Cai-Hong; Li, Danan; Capelletti, Marzia; Cortot, Alexis B.; Chirieac, Lucian; Iacob, Roxana E.; Padera, Robert; Engen, John R.; Wong, Kwok-Kin; Eck, Michael J.; Gray, Nathanael S.; Jänne, Pasi A.

    2010-01-12

    The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

  1. Cell surface protein C23 affects EGF-EGFR induced activation of ERK and PI3K-AKT pathways.

    PubMed

    Lv, Shunzeng; Dai, Congxin; Liu, Yuting; Sun, Bowen; Shi, Ranran; Han, Mingzhi; Bian, Ruixiang; Wang, Renzhi

    2015-02-01

    The epidermal growth factor (EGF) pathway has been reported as canonical causes in cancer development. Meanwhile, the involvement of C23 in multiple signaling pathways has been also investigated (Lv et al., 2014). However, the effect of C23 on EGF pathway in glioblastoma is not fully characterized. In the present study, C23 and the epidermal growth factor receptor (EGFR) of U251 cell line were inhibited by C23 and EGFR antibodies, respectively; and then C23 and EGFR siRNAs were used to knock down endogenous C23 and EGFR, respectively. In addition, soft-agar and MTT assay were also introduced. Compared with control, either C23 or EGFR antibodies efficiently repressed the phosphorylation levels of ERK1/2 (p<0.000) and AKT (p<0.000). Similarly, either C23 or EGFR siRNAs indeed resulted in C23 and EGFR knockdown, and further suppressed the expression of p-ERK1/2 and p-AKT. Most importantly, immunoprecipitation revealed C23 interacted with EGFR once U251 was exposed to EGF treatment. In addition, the MTT and soft-agar assay also identified that C23 or EGFR siRNAs could obviously affected cell growth (p=0.004) and invasiveness, as cell viability and colony formation decreased markedly. Our results suggest that C23 plays a crucial role in activation of EGF-induced ERK and PI3K-AKT pathways via interacting with EGFR; furthermore, C23 could be indicative of an important factor in glioblastoma development and a useful target for glioblastoma treatment. PMID:25015231

  2. Development of sensitive, high-throughput one-tube RT-PCR-enzyme hybridisation assay to detect selected bacterial fish pathogens.

    PubMed

    Wilson, T; Carson, J

    2003-03-31

    Bacterial monitoring and surveillance is critical for the early detection of pathogens to avoid the spread of disease. To facilitate this, an efficient, high-performance and high-throughput method to detect the presence of femotgram amounts of ribosomal RNA from 4 bacterial fish pathogens: Aeromonas salmonicida; Tenacibaculum maritimum (formerly Flexibacter maritimus); Lactococcus garvieae; and Yersinia ruckeri was developed. The system uses NucleoLink strips for liquid- and solid-phase PCR in 1 tube, to perform RT-PCR-enzyme hybridisation assays (RT-PCR-EHA) detecting 4 fg or less of rRNA from pure cultures and between 1 and 9 CFU per 200 microl sample volume from selective-enrichment culture media. The liquid-phase amplicons were visualised by gel electrophoresis and the solid-phase amplicons detected using internal probes and visualised using colorimetric detection and p-nitrophenylphosphate. PMID:12747638

  3. Antioxidant activity of Cod (Gadus morhua) protein hydrolysates: in vitro assays and evaluation in 5% fish oil-in-water emulsion.

    PubMed

    Farvin, K H Sabeena; Lystbæk Andersen, Lisa; Hauch Nielsen, Henrik; Jacobsen, Charlotte; Jakobsen, Greta; Johansson, Inez; Jessen, Flemming

    2014-04-15

    Cod protein hydrolysates were fractionated according to the molecular mass into three fractions of >5 kDa, 3-5 kDa and <3 kDa using an ultrafiltration membrane system. The antioxidative activity of the crude hydrolysates and the fractions were investigated, both in in vitro assays (DPPH radical scavenging activity, reducing power, Fe²⁺ chelating activity and inhibition of lipid oxidation in liposome model system), and in 5% fish oil-in-water emulsions. The <3 kDa fractions had very good radical scavenging activity, Fe²⁺ chelating activity and reducing power while the fraction 3-5 kDa resulted in higher protection against oxidation in the liposome model system. When tested in 5% oil-in-water emulsions, all the fractions, including the crude protein hydrolysate, were able to protect fish oil against oxidation in an iron induced oxidation system. However, none of the peptide fractions were effective in preventing tocopherol loss and showed no tocopherol sparing property. Volatile oxidation products showed an interaction between the aldehydes and the protein/peptides added in the emulsions, and this needs further investigation. PMID:24295714

  4. CRIPTO1 expression in EGFR-mutant NSCLC elicits intrinsic EGFR-inhibitor resistance.

    PubMed

    Park, Kang-Seo; Raffeld, Mark; Moon, Yong Wha; Xi, Liqiang; Bianco, Caterina; Pham, Trung; Lee, Liam C; Mitsudomi, Tetsuya; Yatabe, Yasushi; Okamoto, Isamu; Subramaniam, Deepa; Mok, Tony; Rosell, Rafael; Luo, Ji; Salomon, David S; Wang, Yisong; Giaccone, Giuseppe

    2014-07-01

    The majority of non-small cell lung cancer (NSCLC) patients harbor EGFR-activating mutations that can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib and gefitinib. Unfortunately, a subset of patients with EGFR mutations are refractory to EGFR-TKIs. Resistance to EGFR inhibitors reportedly involves SRC activation and induction of epithelial-to-mesenchymal transition (EMT). Here, we have demonstrated that overexpression of CRIPTO1, an EGF-CFC protein family member, renders EGFR-TKI-sensitive and EGFR-mutated NSCLC cells resistant to erlotinib in culture and in murine xenograft models. Furthermore, tumors from NSCLC patients with EGFR-activating mutations that were intrinsically resistant to EGFR-TKIs expressed higher levels of CRIPTO1 compared with tumors from patients that were sensitive to EGFR-TKIs. Primary NSCLC cells derived from a patient with EGFR-mutated NSCLC that was intrinsically erlotinib resistant were CRIPTO1 positive, but gained erlotinib sensitivity upon loss of CRIPTO1 expression during culture. CRIPTO1 activated SRC and ZEB1 to promote EMT via microRNA-205 (miR-205) downregulation. While miR-205 depletion induced erlotinib resistance, miR-205 overexpression inhibited CRIPTO1-dependent ZEB1 and SRC activation, restoring erlotinib sensitivity. CRIPTO1-induced erlotinib resistance was directly mediated through SRC but not ZEB1; therefore, cotargeting EGFR and SRC synergistically attenuated growth of erlotinib-resistant, CRIPTO1-positive, EGFR-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome intrinsic EGFR-inhibitor resistance in patients with CRIPTO1-positive, EGFR-mutated NSCLC. PMID:24911146

  5. CRIPTO1 expression in EGFR-mutant NSCLC elicits intrinsic EGFR-inhibitor resistance

    PubMed Central

    Park, Kang-Seo; Raffeld, Mark; Moon, Yong Wha; Xi, Liqiang; Bianco, Caterina; Pham, Trung; Lee, Liam C.; Mitsudomi, Tetsuya; Yatabe, Yasushi; Okamoto, Isamu; Subramaniam, Deepa; Mok, Tony; Rosell, Rafael; Luo, Ji; Salomon, David S.; Wang, Yisong; Giaccone, Giuseppe

    2014-01-01

    The majority of non–small cell lung cancer (NSCLC) patients harbor EGFR-activating mutations that can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib and gefitinib. Unfortunately, a subset of patients with EGFR mutations are refractory to EGFR-TKIs. Resistance to EGFR inhibitors reportedly involves SRC activation and induction of epithelial-to-mesenchymal transition (EMT). Here, we have demonstrated that overexpression of CRIPTO1, an EGF-CFC protein family member, renders EGFR-TKI–sensitive and EGFR-mutated NSCLC cells resistant to erlotinib in culture and in murine xenograft models. Furthermore, tumors from NSCLC patients with EGFR-activating mutations that were intrinsically resistant to EGFR-TKIs expressed higher levels of CRIPTO1 compared with tumors from patients that were sensitive to EGFR-TKIs. Primary NSCLC cells derived from a patient with EGFR-mutated NSCLC that was intrinsically erlotinib resistant were CRIPTO1 positive, but gained erlotinib sensitivity upon loss of CRIPTO1 expression during culture. CRIPTO1 activated SRC and ZEB1 to promote EMT via microRNA-205 (miR-205) downregulation. While miR-205 depletion induced erlotinib resistance, miR-205 overexpression inhibited CRIPTO1-dependent ZEB1 and SRC activation, restoring erlotinib sensitivity. CRIPTO1-induced erlotinib resistance was directly mediated through SRC but not ZEB1; therefore, cotargeting EGFR and SRC synergistically attenuated growth of erlotinib-resistant, CRIPTO1-positive, EGFR-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome intrinsic EGFR-inhibitor resistance in patients with CRIPTO1-positive, EGFR-mutated NSCLC. PMID:24911146

  6. Genotoxicity evaluation of the herbicide Garlon(®) and its active ingredient (triclopyr) in fish (Anguilla anguilla L.) using the comet assay.

    PubMed

    Guilherme, Sofia; Santos, Maria A; Gaivão, Isabel; Pacheco, Mário

    2015-09-01

    Triclopyr-based herbicides are broadly used worldwide for site preparation and forest vegetation management. Thus, following application, these agrochemicals can inadvertently reach the aquatic ecosystems. Garlon(®) is one of the most popular commercial denominations of this group of herbicides, considered as highly toxic to fish, even by its manufacturer. Although DNA is frequently regarded as a target of pesticide toxicity, the genotoxic potential of Garlon(®) to fish remains completely unknown. Hence, the main goal of this study was to evaluate the genotoxicity of Garlon(®) and its active ingredient (triclopyr), clarifying the underlying mechanisms. Therefore, the comet assay, implemented as the standard procedure, with an extra step involving DNA lesion-specific repair enzymes (formamidopyrimidine DNA glycosylase and endonuclease III), was used to identify DNA damage in blood cells of Anguilla anguilla L. Short-term exposures (1 and 3 days) to Garlon(®) and triclopyr were carried out, adopting environmentally realistic concentrations (67.6 and 270.5 µg L(-1) Garlon(®) and 30 and 120 µg L(-1) triclopyr). The results concerning the nonspecific DNA damage proved the risk of the herbicide Garlon(®) and its active ingredient triclopyr in both tested concentrations and exposure lengths. In addition, the higher genotoxic potential of the formulation, in comparison with the active ingredient, was demonstrated. When the additional breaks corresponding to net enzyme-sensitive sites were considered, none of the conditions revealed significant levels of oxidative damage. This identification of the genotoxic properties of triclopyr-based herbicides to fish highlights the need to develop less hazardous formulations, as well as the adoption of mitigation measures related to the application of these agrochemicals in the framework of forestry and agriculture sustainable management. PMID:24623388

  7. ReadMax-a novel reading and scoring approach for EGFR gene copy number to predict therapeutic benefit of erlotinib treatment in EGFR wild-type non-small-cell lung cancer.

    PubMed

    Moecks, Joachim; Soulières, Denis; Klughammer, Barbara

    2015-07-01

    EGFR mutation testing is now well established as a means of selecting the optimal first-line therapy for patients with advanced non-small-cell lung cancer (NSCLC). However, deciding on the correct treatment for EGFR wild-type NSCLC remains a challenge. EGFR fluorescence in-situ hybridization (FISH) testing of gene copy number has been a promising marker, but has provided mixed results despite attempts to standardize the reading and scoring process. The novel ReadMax reading and scoring system focuses on the most aberrant cells, to identify oncogene addiction, rather than taking a representative reading as in the Colorado method. The methodology was developed using historical samples from the TRUST and MERIT studies, followed by re-reading of the samples from the SATURN trial. Analysis of samples using the ReadMax methodology revealed that progression-free survival (PFS) and overall survival (OS) were improved in patients with ReadMax FISH-positive (RM FISH+) tumours, compared with those whose tumours were not RM FISH+: PFS hazard ratios (HRs) were 0.52 for RM FISH+ versus 0.93 for not RM FISH+; OS HRs were 0.69 and 0.92, respectively. For PFS, HR for RM FISH+ versus not RM FISH+ in the SATURN erlotinib group was 0.53 (p = 0.003). The PFS and OS results were also similar in the EGFR wild-type population (PFS HRs were 0.63 and 0.96; OS HRs were 0.61 and 0.84, respectively), although amplification of the EGFR gene in patients with EGFR wild-type disease was not found to be predictive of treatment outcomes, which was unexpected but not unprecedented. KRAS status was not found to affect outcomes. Further experience is required to refine the ReadMax methodology and fully establish its validity and robustness. In conclusion, the ReadMax scoring system to identify patients with EGFR FISH-positive NSCLC is a promising technique, which could improve treatment options and outcomes for patients with advanced NSCLC, in particular for EGFR wild-type tumours. PMID:27499899

  8. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals.

    PubMed

    Ankley, Gerald T; Jensen, Kathleen M

    2014-11-01

    The fish short-term reproduction assay (FSTRA) is a key component of the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), which uses a weight-of-evidence analysis based on data from several assays to identify the potential for chemicals to act as agonists or antagonists of the estrogen or androgen receptors (ER and AR), or inhibitors of steroidogenic enzymes. The FSTRA considers a variety of mechanistic and apical responses in 21-d exposures with the fathead minnow (Pimephales promelas), including plasma steroid and vitellogenin (VTG; egg yolk protein) concentrations, secondary sex characteristics, gonad size and histopathology, and egg production. Although the FSTRA initially was described several years ago, recent data generation associated with implementation of the EDSP highlighted the need for more formal guidance regarding evaluation of information from the assay. The authors describe a framework for interpretation of FSTRA data relative to perturbation of endocrine pathways of concern to the EDSP. The framework considers end points individually and as suites of physiologically related responses relative to pathway identification. Sometimes changes in single end points can be highly diagnostic (e.g., induction of VTG in males by ER agonists, production of male secondary sex characteristics in females by AR agonists); in other instances, however, multiple, related end points are needed to reliably assess pathway perturbation (e.g., AR antagonism, steroid synthesis inhibition). In addition to describing an interpretive framework, the authors demonstrate its practical utility using publicly available FSTRA data for a wide range of known and hypothesized endocrine-disrupting chemicals. Environ Toxicol Chem 2014;33:2529-2540. Published 2014 Wiley Periodicals Inc., on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America. PMID:25098918

  9. A quantitative assay for reductive metabolism of a pesticide in fish using electrochemistry coupled with liquid chromatography tandem mass spectrometry.

    PubMed

    Bussy, Ugo; Chung-Davidson, Yu-Wen; Li, Ke; Li, Weiming

    2015-04-01

    This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography-mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography-high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than those of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide. PMID:25730707

  10. Pan-HER-An antibody mixture targeting EGFR, HER2 and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers.

    PubMed

    Ellebaek, Sofie; Brix, Susanne; Grandal, Michael; Lantto, Johan; Horak, Ivan D; Kragh, Michael; Poulsen, Thomas Tuxen

    2016-11-01

    The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings. PMID:27342948

  11. Development of in vivo and in vitro assays to evaluate the physiological effects of environmental estrogens in fish

    SciTech Connect

    Tremblay, L.; Yao, Z.; Kraak, G. Van Der

    1995-12-31

    There are many reports of environmental chemicals that may act as estrogens by binding to the nuclear 17-{beta} estradiol (E{sub 2}) receptor. Experiments were conducted to evaluate whether the plant sterol {beta}-sitosterol and the detergent nonylphenol interact with hepatic estrogen receptors in fish. These compounds are estrogenic in mammals and are found in treated industrial and municipal sewage waters and in pulp and paper mill effluents. Specific high affinity binding sites were characterized in rainbow trout. Nonylphenol and sitosterol were found to have relative affinities of 0.009 and 0.0001 compared to E{sub 2}. To determine if these compounds act as E{sub 2} agonists, their ability to induce estrogen dependent processes was monitored. Induction of estrogen receptors is a common E{sub 2} dependent effect. While other groups have shown in other systems that induction of hepatic E2 receptor levels was estrogen dependent, the authors found that E{sub 2} did not increase E{sub 2} binding in goldfish. However, using isolated goldfish hepatocytes cells, E{sub 2}, sitosterol and nonylphenol induced vitellogenin production. Current studies are aimed at evaluating the structure and activity relationships of these compounds responsible for causing E{sub 2} binding and vitellogenin inductions. Other means to evaluate E{sub 2} binding in goldfish liver are also being investigated.

  12. Specific PCR assays to determine bovine, porcine, fish and plant origin of gelatin capsules of dietary supplements.

    PubMed

    Lee, Jae-Hwang; Kim, Mi-Ra; Jo, Cheon-Ho; Jung, Yoo-Kyung; Kwon, Kisung; Kang, Tae Sun

    2016-11-15

    Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules. PMID:27283629

  13. Heterogeneous EGFR Gene Copy Number Increase Is Common in Colorectal Cancer and Defines Response to Anti-EGFR Therapy

    PubMed Central

    Ålgars, Annika; Lintunen, Minnamaija; Sundström, Jari; Jokilehto, Terhi; Ristimäki, Ari; Ristamäki, Raija; Carpén, Olli

    2014-01-01

    Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12–0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC. PMID:24940619

  14. Development of an upconverting chelate assay

    NASA Astrophysics Data System (ADS)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  15. Identification of novel drug-resistant EGFR mutant inhibitors by in silico screening using comprehensive assessments of protein structures.

    PubMed

    Sato, Tomohiro; Watanabe, Hisami; Tsuganezawa, Keiko; Yuki, Hitomi; Mikuni, Junko; Yoshikawa, Seiko; Kukimoto-Niino, Mutsuko; Fujimoto, Takako; Terazawa, Yumiko; Wakiyama, Motoaki; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Shirouzu, Mikako; Yokoyama, Shigeyuki; Tanaka, Akiko; Honma, Teruki

    2012-06-15

    EGFR is a target protein for the treatment of non small cell lung cancer (NSCLC). The mutations associated with the activation of EGFR kinase activity, such as L858R and G719S, destabilize the inactive conformation of EGFR and are closely linked with the development of NSCLC. The additional T790M mutation reportedly causes drug resistance against the commercially available EGFR inhibitors, gefitinib and erlotinib. In this study, we searched for novel G719S/T790M EGFR inhibitors by a new in silico screening strategy, using two datasets. The results of in silico screening using protein-ligand docking are affected by the selection of 3D structure of the target protein. As the first strategy, we chose the 3D structures for in silico screening by test dockings using the G719S/T790M crystal structure, its molecular dynamics snapshots, and known inhibitors of the drug-resistant EGFR. In the second strategy, we selected the 3D structures by test dockings using all of the EGFR structures, regardless of the mutations, and all of the known EGFR inhibitors. Using each of the 3D structures selected by the strategies, 1000 compounds were chosen from the 71,588 compounds. Kinase assays identified 15 G719S/T790M EGFR inhibitors, including two compounds with novel scaffolds. Analyses of their structure-activity relationships revealed that interactions with the mutated Met790 residue specifically increase the inhibitory activity against G719S/T790M EGFR. PMID:22607878

  16. Decreased autocrine EGFR signaling in metastatic breast cancer cells inhibits tumor growth in bone and mammary fat pad.

    PubMed

    Nickerson, Nicole K; Mohammad, Khalid S; Gilmore, Jennifer L; Crismore, Erin; Bruzzaniti, Angela; Guise, Theresa A; Foley, John

    2012-01-01

    Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland. PMID:22276166

  17. Decreased Autocrine EGFR Signaling in Metastatic Breast Cancer Cells Inhibits Tumor Growth in Bone and Mammary Fat Pad

    PubMed Central

    Nickerson, Nicole K.; Mohammad, Khalid S.; Gilmore, Jennifer L.; Crismore, Erin; Bruzzaniti, Angela; Guise, Theresa A.; Foley, John

    2012-01-01

    Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland. PMID:22276166

  18. ReadMax—a novel reading and scoring approach for EGFR gene copy number to predict therapeutic benefit of erlotinib treatment in EGFR wild‐type non‐small‐cell lung cancer

    PubMed Central

    Soulières, Denis; Klughammer, Barbara

    2015-01-01

    Abstract EGFR mutation testing is now well established as a means of selecting the optimal first‐line therapy for patients with advanced non‐small‐cell lung cancer (NSCLC). However, deciding on the correct treatment for EGFR wild‐type NSCLC remains a challenge. EGFR fluorescence in‐situ hybridization (FISH) testing of gene copy number has been a promising marker, but has provided mixed results despite attempts to standardize the reading and scoring process. The novel ReadMax reading and scoring system focuses on the most aberrant cells, to identify oncogene addiction, rather than taking a representative reading as in the Colorado method. The methodology was developed using historical samples from the TRUST and MERIT studies, followed by re‐reading of the samples from the SATURN trial. Analysis of samples using the ReadMax methodology revealed that progression‐free survival (PFS) and overall survival (OS) were improved in patients with ReadMax FISH‐positive (RM FISH+) tumours, compared with those whose tumours were not RM FISH+: PFS hazard ratios (HRs) were 0.52 for RM FISH+ versus 0.93 for not RM FISH+; OS HRs were 0.69 and 0.92, respectively. For PFS, HR for RM FISH+ versus not RM FISH+ in the SATURN erlotinib group was 0.53 (p = 0.003). The PFS and OS results were also similar in the EGFR wild‐type population (PFS HRs were 0.63 and 0.96; OS HRs were 0.61 and 0.84, respectively), although amplification of the EGFR gene in patients with EGFR wild‐type disease was not found to be predictive of treatment outcomes, which was unexpected but not unprecedented. KRAS status was not found to affect outcomes. Further experience is required to refine the ReadMax methodology and fully establish its validity and robustness. In conclusion, the ReadMax scoring system to identify patients with EGFR FISH‐positive NSCLC is a promising technique, which could improve treatment options and outcomes for patients with advanced NSCLC, in particular for EGFR

  19. Cytosolic phosphorylated EGFR is predictive of recurrence in early stage penile cancer patients: a retropective study

    PubMed Central

    2013-01-01

    Background Penile cancer (PC) is a rare tumor, and therapeutic options are limited for this disease, with an overall 5-year overall survival around 65-70%. Adjuvant therapy is not recommended for patients with N0-1 disease, despite up to 60% of these patients will die within 5 years from diagnosis. Methods Medical records of all patients who underwent radical surgery at University Federico II of Naples and at National Tumor Institute “Pascale” of Naples for early squamous cell carcinoma of the penis from January, 2000 to December, 2011 were retrieved. Paraffin wax embedded tissue specimens were retrieved from the pathology archives of the participating Institutions for all patients. Expression of p-EGFR, EGFR and positivity to HPV were evaluated along with other histological variables of interest. Demographic data of eligible patients were retrieved along with clinical characteristics such as type of surgical operation, time of follow up, time of recurrence, overall survival. A multivariable model was constructed using a forward stepwise selection procedure. Results Thirty eligible patients were identified. All patients were positive for EGFR by immunohistochemistry, while 13 and 16 were respectively positive for nuclear and cytosolic p-EGFR. No EGFR amplification was detected by FISH. Eight patients were positive for high-risk HPV by ISH. On univariable analysis, corpora cavernosa infiltration (OR 7.8; 95% CI = 0,8 to 75,6; P = 0,039) and positivity for cytosolic p-EGFR (OR 7.6; 95% CI =1.49 to 50; P = 0.009) were predictive for recurrence, while only positivity for cytosolic p-EGFR (HR =9.0; 95% CI 1.0-100; P = 0,0116) was prognostic for poor survival. Conclusion It is of primary importance to identify patients with N0-1 disease who are at increased risk of recurrence, as they do not normally receive any adjuvant therapy. Expression of p-EGFR was found in this series to be strongly related to increase risk of recurrence and shorter overall

  20. Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

    PubMed Central

    2014-01-01

    Background Small liver and minute intestinal flukes are highly prevalent in Southeast Asia. Definitive diagnosis of parasite infection is usually achieved parasitologically by finding the fluke eggs in feces. However, their eggs are difficult to differentiate morphologically in fecal samples, even for experienced technicians. The present study developed a PCR assay coupled with DNA pyrosequencing for identification of the fish-borne trematodes (FBT), Opisthorchis viverrini, Clonorchis sinensis, Haplorchis taichui, H. pumilio and Stellantchasmus falcatus, and to evaluate potential detection in fecal specimens, and identification and differentiation of cercarial and metacercarial stages. Methods Primers targeting the partial 28S large subunit ribosomal RNA gene were designed and about 46–47 nucleotides were selected as the target region for species identification by a PCR assay coupled with a pyrosequencing technique. Results The nucleotide variations at 24 positions, which is sufficient for the identification of the five species of FBT were selected. The method could identify O. viverrini and C. sinensis eggs in feces, cercarial and metacercarial stages of O. viverrini, and metacercarial stage of H. pumilio and H. taichui. The detection limit was as little as a single O. viverrini or C. sinensis egg artificially inoculated in 100 mg of non-infected fecal sample (equivalent to 10 eggs per gram), indicating highly sensitivity. The method was found to be superior to the traditional microscopy method and was more rapid than Sanger DNA sequencing. Conclusions DNA pyrosequencing-based identification is a valuable tool for differentiating O. viverrini and other Opisthorchis-like eggs, and can be applied to epidemiological studies and for molecular taxonomic investigation of FBT in endemic areas. PMID:24589167

  1. Dominant Enhancers of Egfr in Drosophila Melanogaster: Genetic Links between the Notch and Egfr Signaling Pathways

    PubMed Central

    Price, J. V.; Savenye, E. D.; Lum, D.; Breitkreutz, A.

    1997-01-01

    The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F(1) screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context. PMID:9383058

  2. Oncogenic KRAS Impairs EGFR Antibodies' Efficiency by C/EBPβ-Dependent Suppression of EGFR Expression12

    PubMed Central

    Derer, Stefanie; Berger, Sven; Schlaeth, Martin; Schneider-Merck, Tanja; Klausz, Katja; Lohse, Stefan; Overdijk, Marije B; Dechant, Michael; Kellner, Christian; Nagelmeier, Iris; Scheel, Andreas H; Lammerts van Bueren, Jeroen J; van de Winkel, Jan GJ; Parren, Paul WHI; Peipp, Matthias; Valerius, Thomas

    2012-01-01

    Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRASG12V) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRASG12V on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRASG12V impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRASG12V also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs—such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRASG12V downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPβ-LIP. Thus, siRNA-mediated knockdown of C/EBPβ led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRASG12V signaling induced C/EBPβ-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents. PMID

  3. Evaluation of EGFR as a prognostic and diagnostic marker for head and neck squamous cell carcinoma patients

    PubMed Central

    Polanska, Hana; Raudenska, Martina; Hudcová, Kristyna; Gumulec, Jaromir; Svobodova, Marketa; Heger, Zbynek; Fojtu, Michaela; Binkova, Hana; Horakova, Zuzana; Kostrica, Rom; Adam, Vojtech; Kizek, Rene; Masarik, Michal

    2016-01-01

    Approximately 90% of all head and neck tumors are squamous cell carcinomas. The overall survival of patients with head and neck squamous cell carcinoma (HNSCC) is low (≤50%). A non-invasive marker of disease progression is sorely required. The present study focused on the plasmatic levels of epidermal growth factor receptor (EGFR) in HNSCC patients (N=92) compared with healthy (N=29) and diabetic [type 2 diabetes mellitus (T2DM); N=26] controls. Enzyme-linked immunosorbent assay using antibodies against the extracellular region of EGFR (L25-S645) was performed. No significant changes were observed between diabetic and healthy controls. However, there were significantly higher EGFR plasma levels in HNSCC patients compared with both control groups (P=0.001 and 0.005, respectively). Receiver operating characteristic curve analysis identified a sensitivity of 76.09%, a specificity of 67.27% and an area under curve of 0.727 for this comparison. No significant association was observed between EGFR plasma levels and tumor stage, tumor grade, lymph node or distant metastasis occurrence, smoking habit or hypertension. However, the presence of human papillomavirus infection and T2DM in HNSCC patients had borderline effect on the plasma EGFR levels. Survival analysis revealed no significant influence of plasmatic EGFR levels on the overall and disease-specific survival of HNSCC patients. In conclusion, EGFR plasma levels appear to be a relatively promising diagnostic, but poor prognostic, HNSCC marker. PMID:27602151

  4. A comparative study of affibody, panitumumab, and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors.

    PubMed

    Gong, Haibiao; Kovar, Joy L; Cheung, Lael; Rosenthal, Eben L; Olive, D Michael

    2014-02-01

    Aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers. EGFR variant III (EGFRvIII) is a common in-frame deletion mutant, which lacks a large part of the extracellular portion (exons 2-7), including components of the ligand-binding domain. Although EGFR has been extensively studied as a molecular imaging target, information about EGFRvIII-targeted molecular imaging is lacking. In this study, the EGFR-specific affibody, therapeutic antibody panitumumab, and ligand EGF were labeled with IRDye 800CW (Ex/Em: 774/789 nm), yielding Aff800, Pan800, and EGF800, respectively. The binding affinities of the labeled agents were compared in cell-based assays using a rat glioma cell line F98 parental (F98-p) lacking EGFR expression, and 2 F98-derived transgenic cell lines expressing EGFR or EGFRvIII (designated as F98-EGFR and F98-vIII, respectively). Results showed that all agents could bind to F98-EGFR, with Pan800 having the highest binding affinity, followed by Aff800 and EGF800. Pan800 and Aff800, but not EGF800, also bound to F98-vIII. In vivo animal imaging demonstrated that compared with F98-p tumors, F98-EGFR tumors generated higher signals with all three agents. However, in the case of F98-vIII, only Pan800 and Aff800 signals were higher. Analysis of tissue lysates showed that a large portion of Pan800 was degraded into small fragments in F98-EGFR and F98-vIII tumors, possibly due to proteolytic digestion after its specific binding and internalization. In conclusion, Pan800 and Aff800 could be used as imaging agents for both wild-type EGFR and EGFRvIII, whereas EGF800 only targets wild-type EGFR. PMID:24100437

  5. The Cobas® EGFR Mutation Test v2 assay.

    PubMed

    Brown, Paul

    2016-02-01

    Paul Brown speaks to Gemma Westcott, Commissioning Editor: Paul Brown has served as the Head of Roche Molecular Diagnostics at Roche Diagnostics Corporation since February 2010 having previously held a variety of positions within Roche Pharma. After completing his doctorate in organic chemistry he was awarded a post-doctoral fellowship at the California Institute of Technology in Pasadena, CA, USA, but soon returned to his native UK to join Roche Pharma Research. Paul's first post within Roche was as group leader, doing drug discovery and making new small molecule drugs, but later moved into the business part of the company. Since then, he has enjoyed roles such as lifecycle leader for brands such as Tamiflu(®) and Xenical, vice president of sales and marketing of the pharmaceutical division and most recently general manager of Roche Pharma, Sweden. PMID:26838018

  6. A real-time PCR assay for differential expression of vitellogenin I and II genes in the liver of the sentinel fish species Lipophrys pholis.

    PubMed

    Ferreira, F; Monteiro, N M; Vieira, M N; Reis-Henriques, M A; Castro, L Filipe C; Santos, M M

    2013-10-01

    Abstract The recent advances in molecular biology techniques have prompted the use of vitellogenin (VTG) gene expression as a sensitive and reliable indicator of estrogenic chemicals (EC) exposure. However, data on the dynamic response of the different VTGs genes upon EC exposure is still poorly understood, particularly in sentinel fish species used in field monitoring studies. Hence, the present study aimed at developing a sensitive real-time PCR assay for determining the response of VTG I and II in the recently proposed marine sentinel species Lipophrys pholis upon exposure to the model EC 17α-ethinylestradiol (EE2). The findings of the laboratory study indicate that L. pholis VTG I proved to be not only more inducible but also more sensitive to EE2 exposure than VTG II, for the same range of concentrations. In fact, VTG I gene induction was 475-fold higher than VTG II at 15 ng/L EE2, and 13-fold at 5 ng/L EE2. Overall, the findings of the present study indicate that in the field, expression of VTG I in L. pholis should be preferentially used in the screening of EC exposure because of its higher sensitivity. Furthermore, the present study favors L. pholis integration in monitoring programs associated with EC's pollution within the European water policy legislation. PMID:23718563

  7. Investigational EGFR-targeted therapies in HNSCC

    PubMed Central

    Cassell, Andre; Grandis, Jennifer R.

    2010-01-01

    Importance of the Field The epidermal growth factor receptor (EGFR) is an established therapeutic target in head and neck squamous cell carcinoma (HNSCC). The EGFR-targeting monoclonal antibody cetuximab (™Erbitux) was FDA-approved for use in HNSCC in 2006. The molecular basis for the efficacy of an antibody approach compared with inhibition of EGFR tyrosine kinase function using small molecule inhibitors, or downregulation of protein expression via antisense strategies remains incompletely understood. Areas covered in this review A literature search was performed to identify studies elucidating mechanisms of action of several approaches to targeting EGFR in HNSCC (monoclonal antibodies, tyrosine kinase inhibitors, antisense approaches, and ligand toxin conjugates). What the reader will gain Monoclonal antibodies decrease tumor growth via receptor endocytosis and recruitment of host immune defenses. Tyrosine kinase inhibitors bind to the ATP binding pocket of the tyrosine kinase domain, inhibiting signaling. Antisense approaches decrease EGFR expression with high specificity although drug delivery remains problematic. Ligand-toxin conjugates facilitate the entry of toxin and the ADP-ribosylation of the ribosome, thereby inhibiting translation. Take home message Elucidation mechanisms by which these different strategies inhibit EGFR function may enhance the development of more effective treatments for HNSCC and enable prospective identification of individuals who will benefit from EGFR inhibition. PMID:20415598

  8. EGFR/HER2 inhibitors effectively reduce the malignant potential of MDR breast cancer evoked by P-gp substrates in vitro and in vivo.

    PubMed

    Jin, Yiting; Zhang, Wei; Wang, Hongying; Zhang, Zijing; Chu, Chengyu; Liu, Xiuping; Zou, Qiang

    2016-02-01

    Multidrug resistance (MDR) induced by chemotherapy in breast cancer frequently leads to tumor invasion, metastasis and poor clinical outcome. We preliminarily found that the epidermal growth factor receptor (EGFR) is involved in enhancing the malignant potential of MDR breast cancer cells, but the mechanism remains unclear. In the present study, we demonstrated in vitro and in vivo that EGFR/HER2 promote the invasive and metastatic abilities of MDR breast cancer. More importantly, a new function of EGFR/HER2 inhibitors was revealed for the first time, which could improve the treatment efficacy of breast cancer by reversing the MDR process rather than by inhibiting tumor growth. Firstly, using quantitative real‑time PCR and western blot analysis, we found that overexpression of EGFR/HER2 in MCF7/Adr cells upregulated CD147 and MMP2/9 at both the transcription and protein expression levels, which promoted tumor cell migration, as determined using an in vitro invasion assay. Secondly, the upregulated levels of CD147 and MMP2/9 were decreased when EGFR/HER2 activity was inhibited, and therefore tumor invasion was also significantly inhibited. These phenomena were also demonstrated in nude mouse assays. Additionally, in MDR breast cancer patients, we found that overexpression of EGFR and P‑gp levels led to shorter overall survival (OS) and disease‑free survival (DFS) by IHC assays and Kaplan‑Meier survival analysis. In conclusion, EGFR/HER2 play a crucial role in enhancing CD147 and MMP expression to establish favorable conditions for invasion/metastasis in MDR breast cancer. The scope of application of EGFR/HER2 inhibitors may be expanded in EGFR/HER2‑positive patients. We suggest that MDR breast cancer patients may benefit from novel therapies targeting EGFR/HER2. PMID:26718028

  9. Whacking a mole-cule: clinical activity and mechanisms of resistance to third generation EGFR inhibitors in EGFR mutated lung cancers with EGFR-T790M

    PubMed Central

    Costa, Daniel B.

    2015-01-01

    Epidermal growth factor receptor (EGFR) mutations, especially EGFR-exon 19 deletions and EGFR-L858R, are the most frequent actionable genomic events in lung adenocarcinomas. Tumors arise due to constitutively activated EGFR signaling and are susceptible to EGFR tyrosine kinase inhibitors (TKIs). First generation EGFR TKIs (gefitinib and erlotinib) and the second generation EGFR TKI afatinib are approved worldwide. Although targeted therapies against EGFR mutants induce dramatic initial responses, acquired resistance (through multiple biological mechanisms) to erlotinib, gefitinib and afatinib emerges within the first 1-2 years of continued monotherapy. EGFR-T790M accounts for more than half of acquired resistance to first or second generation EGFR TKIs by modifying ATP affinity and drug binding kinetics. Two new studies have shown that two covalent pyrimidine inhibitors—AZD9291 and rociletinib of EGFR-T790M (i.e., third generation EGFR TKIs) shown remarkable clinical activity in patients with acquired resistance to erlotinib, gefitinib and afatinib when the tumor carries EGFR-T790M in conjunction with an activating mutation. However, and regrettably, acquired resistance to these third generation EGFR TKIs has already been reported in preclinical models and clinical specimens; such as a tertiary mutation at EGFR-C797S that prevents covalent binding of EGFR TKIs. The experience with sequential EGFR TKI monotherapy highlights tumor heterogeneity and adaptability (i.e., relentless game of whack-a-mole played between TKIs and cancer), and will help shape future clinical development of novel combinatory approaches to manage EGFR mutated lung adenocarcinomas. PMID:26798593

  10. Whacking a mole-cule: clinical activity and mechanisms of resistance to third generation EGFR inhibitors in EGFR mutated lung cancers with EGFR-T790M.

    PubMed

    Costa, Daniel B; Kobayashi, Susumu S

    2015-12-01

    Epidermal growth factor receptor (EGFR) mutations, especially EGFR-exon 19 deletions and EGFR-L858R, are the most frequent actionable genomic events in lung adenocarcinomas. Tumors arise due to constitutively activated EGFR signaling and are susceptible to EGFR tyrosine kinase inhibitors (TKIs). First generation EGFR TKIs (gefitinib and erlotinib) and the second generation EGFR TKI afatinib are approved worldwide. Although targeted therapies against EGFR mutants induce dramatic initial responses, acquired resistance (through multiple biological mechanisms) to erlotinib, gefitinib and afatinib emerges within the first 1-2 years of continued monotherapy. EGFR-T790M accounts for more than half of acquired resistance to first or second generation EGFR TKIs by modifying ATP affinity and drug binding kinetics. Two new studies have shown that two covalent pyrimidine inhibitors-AZD9291 and rociletinib of EGFR-T790M (i.e., third generation EGFR TKIs) shown remarkable clinical activity in patients with acquired resistance to erlotinib, gefitinib and afatinib when the tumor carries EGFR-T790M in conjunction with an activating mutation. However, and regrettably, acquired resistance to these third generation EGFR TKIs has already been reported in preclinical models and clinical specimens; such as a tertiary mutation at EGFR-C797S that prevents covalent binding of EGFR TKIs. The experience with sequential EGFR TKI monotherapy highlights tumor heterogeneity and adaptability (i.e., relentless game of whack-a-mole played between TKIs and cancer), and will help shape future clinical development of novel combinatory approaches to manage EGFR mutated lung adenocarcinomas. PMID:26798593

  11. Simultaneous Inhibition of EGFR and PI3K Enhances Radiosensitivity in Human Breast Cancer

    SciTech Connect

    Li Ping; Zhang Qing; Torossian, Artour; Li Zhaobin; Xu Wencai; Lu Bo; Fu Shen

    2012-07-01

    Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used to investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF{sub SF2}) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF{sub SF2} at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may have

  12. The receptor AXL diversifies EGFR signaling and limits the response to EGFR-targeted inhibitors in triple-negative breast cancer cells.

    PubMed

    Meyer, Aaron S; Miller, Miles A; Gertler, Frank B; Lauffenburger, Douglas A

    2013-08-01

    The relationship between drug resistance, changes in signaling, and emergence of an invasive phenotype is well appreciated, but the underlying mechanisms are not well understood. Using machine learning analysis applied to the Cancer Cell Line Encyclopedia database, we identified expression of AXL, the gene that encodes the epithelial-to-mesenchymal transition (EMT)-associated receptor tyrosine kinase (RTK) AXL, as exceptionally predictive of lack of response to ErbB family receptor-targeted inhibitors. Activation of EGFR (epidermal growth factor receptor) transactivated AXL, and this ligand-independent AXL activity diversified EGFR-induced signaling into additional downstream pathways beyond those triggered by EGFR alone. AXL-mediated signaling diversification was required for EGF (epidermal growth factor)-elicited motility responses in AXL-positive TNBC (triple-negative breast cancer) cells. Using cross-linking coimmunoprecipitation assays, we determined that AXL associated with EGFR, other ErbB receptor family members, MET (hepatocyte growth factor receptor), and PDGFR (platelet-derived growth factor receptor) but not IGF1R (insulin-like growth factor 1 receptor) or INSR (insulin receptor). From these AXL interaction data, we predicted AXL-mediated signaling synergy for additional RTKs and validated these predictions in cells. This alternative mechanism of receptor activation limits the use of ligand-blocking therapies and indicates against therapy withdrawal after acquired resistance. Further, subadditive interaction between EGFR- and AXL-targeted inhibitors across all AXL-positive TNBC cell lines may indicate that increased abundance of EGFR is principally a means to transactivation-mediated signaling. PMID:23921085

  13. Accurate detection and quantification of the fish viral hemorrhagic septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting > 80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, killed many game fish species...

  14. The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

    PubMed Central

    Chang, Xiaofei; Izumchenko, Eugene; Solis, Luisa M.; Kim, Myoung Sook; Chatterjee, Aditi; Ling, Shizhang; Monitto, Constance L.; Harari, Paul M.; Hidalgo, Manuel; Goodman, Steve N.; Wistuba, Ignacio I.; Bedi, Atul; Sidransky, David

    2013-01-01

    The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01). PMID:23935914

  15. Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR).

    PubMed

    Akbari, Bahman; Farajnia, Safar; Zarghami, Nosratollah; Mahdieh, Nejat; Rahmati, Mohammad; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2016-11-01

    Various strategies have been attempted for targeting of epidermal growth factor receptor (EGFR), as an essential biomarker in a variety of cancers. Several anti-EGFR antibodies including cetuximab are used in clinics for treatment of EGFR-overexpressing colorectal and head and neck cancers but the efficiency of these antibodies is threatened by their large size and chimeric nature. Humanized single chains antibodies (huscFv) are smaller generation of antibodies with lower immunogenicity may overcome these limitations. This article reports production and evaluation of a novel humanized anti-EGFR scFv. The CDRs of cetuximab heavy and light chains were grafted onto human antibody frameworks as framework donors. To maintain the antigen binding affinity of murine antibody, the murine vernier zone residues were retained in framework regions of huscFv. Additionally, two point mutations in CDR-L1 and CDR-L3 and three point mutations in CDR-H2 and CDR-H3 loops of the humanized scFv (huscFv) were introduced to increase affinity of the huscFv to EGFR. Analysis of results demonstrated that the humanness degree of resultant huscFv was increased as 19%. HuscFv was expressed in BL21 (DE3) and affinity purified via Ni-NTA column. The reactivity of huscFv with EGFR was evaluated by ELISA and dot blot techniques. Analysis by ELISA and dot blot showed that the huscFv was able to recognize and react with EGFR. Toxicity analysis by MTT assay indicated an inhibitory effect on growth of EGFR-overexpressing A431 cells. In conclusion, the huscFv produced in this study revealed decreased immunogenicity while retained growth inhibitory effect on EGFR-overexpressing tumor cells. PMID:27298212

  16. Histone Deacetylase-3/CAGE Axis Targets EGFR Signaling and Regulates the Response to Anti-Cancer Drugs.

    PubMed

    Kim, Hyuna; Kim, Youngmi; Goh, Hyeonjung; Jeoung, Dooil

    2016-03-31

    We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of pEGFR(Y845). HDAC3 was found to negatively regulate the expression of pEGFR(Y845). CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, pEGFR(Y845), and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant Malme3M(R) cells, decreasing the tumorigenic potential of Malme3M(R) cells in a manner associated with its effect on the expression of HDAC3, CAGE and pEGFR(Y845). The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that PKCδ was responsible for the increased expression of pEGFR(Y845) and CAGE in Malme3M(R) cells. CAGE showed an interaction with PKCδ in Malme3M(R) cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors. PMID:26883907

  17. A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lung cancer

    PubMed Central

    OFUJI, KAZUYA; TADA, YOSHITAKA; YOSHIKAWA, TOSHIAKI; SHIMOMURA, MANAMI; YOSHIMURA, MAYUKO; SAITO, KEIGO; NAKAMOTO, YASUNARI; NAKATSURA, TETSUYA

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have demonstrated marked clinical activity against non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutations. However, in most cases, patients develop acquired resistance to EGFR-TKI therapy. The threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation is the most common acquired resistance mutation, and is present in ~50% cases of TKI resistance. New treatment strategies for NSCLC patients harboring the EGFR T790M mutation are required. We evaluated the immunogenicity of an antigen derived from EGFR with the T790M mutation. Using BIMAS we selected several EGFR T790M-derived peptides bound to human leukocyte antigen (HLA)-A*02:01. T790M-A peptide (789–797) (IMQLMPFGC)-specific cytotoxic T lymphocytes (CTLs) were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors. An established T790M-A-specific CTL line showed reactivity against the NCSLC cell line, H1975-A2 (HLA-A2+, T790M+), but not H1975 (HLA-A2−, T790M+), and the corresponding wild-type peptide (ITQLMPFGC)-pulsed T2 cells using an interferon-γ (IFN-γ) enzyme-linked immuno spot (ELISPOT) assay. This CTL line also demonstrated peptide-specific cytotoxicity against H1975-A2 cells. This finding suggests that the EGFR T790M mutation-derived antigen could be a new target for cancer immunotherapy. PMID:25532027

  18. Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling.

    PubMed

    Merkulova, Yulia; Shen, Yue; Parkinson, Leigh G; Raithatha, Sheetal A; Zhao, Hongyan; Westendorf, Kathryn; Sharma, Mehul; Bleackley, Robert Chris; Granville, David J

    2016-09-01

    Chronic non-healing wounds including diabetic, venous, and decubitus skin ulcers are currently lacking effective therapies. Non-healing diabetic ulcers can lead to amputations as progress into a highly chronic state before detection and existing treatments for these wounds often fail. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during excessive or chronic inflammation, GzmB can accumulate in the extracellular milieu, retain its activity, and cleave a number of important extracellular proteins. Epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in cellular processes such as proliferation and migration. EGFR signaling is integral to the wound healing process. The present study investigated the effects of GzmB on keratinocyte cell migration using HaCaT cell line. Using electric cell-substrate impedance sensing and scratch assays, the present study demonstrates that GzmB inhibits keratinocyte migration by interfering with the EGFR pathway. GzmB limited cell transition into a migratory morphology and was found to reduce ligand-induced EGFR phosphorylation. Inhibition of GzmB reversed the aforementioned effects. In summary, data from the present study suggest key role for GzmB in the pathogenesis of impaired wound healing through the impairment of EGFR signaling and cell migration. PMID:27060743

  19. The Emergent Landscape of Detecting EGFR Mutations Using Circulating Tumor DNA in Lung Cancer.

    PubMed

    Huang, Wei-Lun; Wei, Fang; Wong, David T; Lin, Chien-Chung; Su, Wu-Chou

    2015-01-01

    The advances in targeted therapies for lung cancer are based on the evaluation of specific gene mutations especially the epidermal growth factor receptor (EGFR). The assays largely depend on the acquisition of tumor tissue via biopsy before the initiation of therapy or after the onset of acquired resistance. However, the limitations of tissue biopsy including tumor heterogeneity and insufficient tissues for molecular testing are impotent clinical obstacles for mutation analysis and lung cancer treatment. Due to the invasive procedure of tissue biopsy and the progressive development of drug-resistant EGFR mutations, the effective initial detection and continuous monitoring of EGFR mutations are still unmet requirements. Circulating tumor DNA (ctDNA) detection is a promising biomarker for noninvasive assessment of cancer burden. Recent advancement of sensitive techniques in detecting EGFR mutations using ctDNA enables a broad range of clinical applications, including early detection of disease, prediction of treatment responses, and disease progression. This review not only introduces the biology and clinical implementations of ctDNA but also includes the updating information of recent advancement of techniques for detecting EGFR mutation using ctDNA in lung cancer. PMID:26448936

  20. EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

    PubMed Central

    Arasada, Rajeswara Rao; Amann, Joseph M.; Rahman, Mohammad A; Huppert, Stacey S.; Carbone, David P.

    2014-01-01

    Mutations in the epidermal growth factor receptor (EGFR) are the most common actionable genetic abnormalities yet discovered in lung cancer. However, targeting these mutations with kinase inhibitors is not curative in advanced disease and has yet to demonstrate an impact on potentially curable, early-stage disease, with some data suggesting adverse outcomes. Here, we report that treatment of EGFR-mutated lung cancer cell lines with erlotinib, while showing robust cell death, enriches the ALDH+ stem-like cells through EGFR-dependent activation of Notch3. Additionally, we demonstrate that erlotinib treatment increases the clonogenicity of lung cancer cells in a sphere-forming assay, suggesting increased stem-like cell potential. We demonstrate that inhibition of EGFR kinase activity leads to activation of Notch transcriptional targets in a gamma secretase inhibitor sensitive manner and causes Notch activation. leading to an increase in ALDH high+ cells. We also find a kinase-dependent physical association between the Notch3 and EGFR receptors and tyrosine phosphorylation of Notch3. This could explain the worsened survival observed in some studies of erlotinib treatment at early-stage disease, and suggests that specific dual targeting might overcome this adverse effect. PMID:25125655

  1. The Emergent Landscape of Detecting EGFR Mutations Using Circulating Tumor DNA in Lung Cancer

    PubMed Central

    Huang, Wei-Lun; Wei, Fang; Wong, David T.; Lin, Chien-Chung; Su, Wu-Chou

    2015-01-01

    The advances in targeted therapies for lung cancer are based on the evaluation of specific gene mutations especially the epidermal growth factor receptor (EGFR). The assays largely depend on the acquisition of tumor tissue via biopsy before the initiation of therapy or after the onset of acquired resistance. However, the limitations of tissue biopsy including tumor heterogeneity and insufficient tissues for molecular testing are impotent clinical obstacles for mutation analysis and lung cancer treatment. Due to the invasive procedure of tissue biopsy and the progressive development of drug-resistant EGFR mutations, the effective initial detection and continuous monitoring of EGFR mutations are still unmet requirements. Circulating tumor DNA (ctDNA) detection is a promising biomarker for noninvasive assessment of cancer burden. Recent advancement of sensitive techniques in detecting EGFR mutations using ctDNA enables a broad range of clinical applications, including early detection of disease, prediction of treatment responses, and disease progression. This review not only introduces the biology and clinical implementations of ctDNA but also includes the updating information of recent advancement of techniques for detecting EGFR mutation using ctDNA in lung cancer. PMID:26448936

  2. PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum.

    PubMed Central

    Miriam, A; Griffiths, S G; Lovely, J E; Lynch, W H

    1997-01-01

    A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells. PMID:9163437

  3. PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum.

    PubMed

    Miriam, A; Griffiths, S G; Lovely, J E; Lynch, W H

    1997-06-01

    A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells. PMID:9163437

  4. Intratumoral distribution of EGFR-amplified and EGFR-mutated cells in pulmonary adenocarcinoma.

    PubMed

    Soma, Shingo; Tsuta, Koji; Takano, Toshimi; Hatanaka, Yutaka; Yoshida, Akihiko; Suzuki, Kenji; Asamura, Hisao; Tsuda, Hitoshi

    2014-03-01

    Alterations in the epidermal growth factor receptor (EGFR) gene are associated with carcinogenesis in non-small cell lung cancer. However, the intratumoral distribution of these abnormalities has not been elucidated. This study included patients with surgically resected lung adenocarcinoma. The predominant histological growth pattern was determined. Chromogenic in situ hybridization (CISH) and EGFR-mutation specific-antibodies were used for analysis of changes in gene copy number and EGFR mutations, respectively. EGFR mutation detected immunohistochemistry (IHC) and amplification were identified in 31 (53%) and 30 (52%) cases, respectively. The predominant growth patterns in the 58 tumors evaluated were papillary (28, 48%), lepidic (8, 14%), acinar (15, 26%), and solid (7, 12%). EGFR mutations were the least common in cases with a solid predominant pattern. The incidence of EGFR amplification did not differ among predominant patterns. Analyzing each histological subtype, no differences were noted between the prevalence of EGFR-IHC positive and CISH-positive rates. In the analysis of EGFR amplification, CISH-positive status was more prevalent in IHC-positive cases than in IHC-negative cases. All 19 cases that were both IHC and CISH positive were analyzed. In 17 cases (90%), the IHC-positive area was equal to or larger than the CISH-positive area. Among the histological subtypes of lung adenocarcinoma, the solid predominant subtype was distinguishable by its infrequent EGFR mutations. EGFR gene mutations preceded changes in oncogenic drive, more so than did EGFR gene number alterations during the developmental process of lung adenocarcinoma. PMID:24355440

  5. Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)

    PubMed Central

    Nitta, Hiroaki; Hauss-Wegrzyniak, Beatrice; Lehrkamp, Megan; Murillo, Adrian E; Gaire, Fabien; Farrell, Michael; Walk, Eric; Penault-Llorca, Frederique; Kurosumi, Masafumi; Dietel, Manfred; Wang, Lin; Loftus, Margaret; Pettay, James; Tubbs, Raymond R; Grogan, Thomas M

    2008-01-01

    Background Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. Methods The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Results Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO

  6. Combined EGFR/MEK Inhibition Prevents the Emergence of Resistance in EGFR mutant Lung Cancer

    PubMed Central

    Uddin, Sharmeen; Capelletti, Marzia; Ercan, Dalia; Ogino, Atsuko; Pratilas, Christine A.; Rosen, Neal; Gray, Nathanael S.; Wong, Kwok-Kin; Jänne, Pasi A.

    2016-01-01

    Irreversible pyrimidine based EGFR inhibitors, including WZ4002, selectively inhibit both EGFR activating and EGFR inhibitor resistant T790M mutations more potently than wild type EGFR. While this class of mutant selective EGFR inhibitors is effective clinically in lung cancer patients harboring EGFR T790M, prior preclinical studies demonstrate that acquired resistance can occur through genomic alterations that activate ERK1/2 signaling. Here we find that ERK1/2 reactivation occurs rapidly following WZ4002 treatment. Concomitant inhibition of ERK1/2 by the MEK inhibitor trametinib prevents ERK1/2 reactivation, enhances WZ4002 induced apoptosis and inhibits the emergence of resistance in WZ4002 sensitive models known to acquire resistance via both T790M dependent and independent mechanisms. Resistance to WZ4002 in combination with trametinib eventually emerges due to AKT/mTOR reactivation. These data suggest that initial co-targeting of EGFR and MEK could significantly impede the development of acquired resistance in mutant EGFR lung cancer. PMID:26036643

  7. Electrochemical aptamer/antibody based sandwich immunosensor for the detection of EGFR, a cancer biomarker, using gold nanoparticles as a signaling probe.

    PubMed

    Ilkhani, Hoda; Sarparast, Morteza; Noori, Abolhassan; Zahra Bathaie, S; Mousavi, Mir F

    2015-12-15

    Detection of epidermal growth factor receptor (EGFR) in biological fluids is of paramount importance, since it has significant application in cancer diagnosis, drug development, and therapy monitoring. EGFR is a cancer biomarker, and its overexpression is associated with the development of some types of cancer. Herein, we report on the development of a sensitive and selective electrochemical aptamer/antibody (Apt/Ab) sandwich immunosensor for detection of EGFR. In this study, a biotinylated anti-human EGFR Apt was immobilized on streptavidin-coated magnetic beads (MB) and served as a capture probe. A polyclonal anti-human EGFR Ab was conjugated to citrate-coated gold nanoparticles (AuNPs) and used as a signaling probe. In the presence of EGFR, an Apt-EGFR-Ab sandwich was formed on the MB surface. The extent of the complexation was evaluated by differential pulse voltammetry of AuNPs after their dissolution in HCl. Under optimal conditions, the dynamic concentration range of the immunosensor for EGFR spanned from 1 to 40 ng/mL, with a low detection limit of 50 pg/mL, and RSD percent of less than 4.2%. The proposed approach takes advantage of sandwich assay for high specificity, MBs for fast separation, and electrochemical method for cost-effective and sensitive detection. In this proof-of-principle study, we demonstrate the potential clinical efficacy of the immunosensor for monitoring of chemotherapy effectiveness in breast cancer samples. PMID:26176209

  8. Disappearance of an activated EGFR mutation after treatment with EGFR tyrosine kinase inhibitors.

    PubMed

    Honda, Yoshihiro; Takigawa, Nagio; Fushimi, Soichiro; Ochi, Nobuaki; Kubo, Toshio; Ozaki, Saeko; Tanimoto, Mitsune; Kiura, Katsuyuki

    2012-10-01

    A 34-year-old Japanese woman presented with left supraclavicular lymph node swelling. Computed tomography scans revealed a mass on the left lower lobe, pulmonary nodules, and pleural effusion. A lymph node biopsy revealed large-cell carcinoma with an epidermal growth factor receptor (EGFR) deletion mutation, L747-T751 in exon 19. Although malignant pleural effusions carried the same EGFR mutation, progressive pleural effusions after treatment with chemotherapy, gefitinib, and erlotinib did not show any EGFR mutation. A cell line established from the pleural effusion 3 days before the patient expired also did not harbor the EGFR mutation. Histological sections of the lymph node of the patient were similar to those of the xenograft tumor of the cell line. There may be genetic heterogeneity in EGFR mutant tumors. PMID:22835516

  9. EGFR-TKI down-regulates PD-L1 in EGFR mutant NSCLC through inhibiting NF-κB.

    PubMed

    Lin, Kailong; Cheng, Jianan; Yang, Tao; Li, Yongsheng; Zhu, Bo

    Non-small-cell lung cancer (NSCLC) is a severe disease threatening human health. Targeted therapy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has obtained potent efficacy in the treatment of NSCLC patients. However, the effects of EGFR-TKIs on tumor immune microenvironment are unclear. In this study, we show that NSCLCs with EGFR mutation express higher programmed cell death ligand 1 (PD-L1) than NSCLCs with wild type EGFR. The EGFR activation is also associated with high expression of PD-L1. The EGFR-TKI gefitinib can reduce PD-L1 expression, via inhibiting NF-κB, in EGFR mutant NSCLC in vitro and in vivo. These findings elucidate a novel anti-tumor mechanism of EGFR-TKI and provide the possibility of combined strategy of targeted therapy and immunotherapy for EGFR mutant NSCLC patients. PMID:25998384

  10. Rapid and label-free amplification and detection assay for genotyping of cancer biomarker.

    PubMed

    Shin, Yong; Soo, Ross A; Yoon, Jaeyun; Perera, Agampodi Promoda; Yoon, Yong-Jin; Park, Mi Kyoung

    2015-06-15

    As understanding of the molecular pathways that drive malignancy in human cancer improves, personalized genotype-based therapy in combination with the predictive biomarker for the efficacy of targeted therapy is becoming more popular in cancer management. Sanger sequencing, that has been the gold standard for mutation analysis in cancer since the 1970s, suffers from low sensitivity, complexity, and time-consuming and labor-intensive procedure. Although several PCR based molecular testing methods are being emerged, there is no universal assay available for genotyping of cancer biomarkers. Here we present a rapid, simple and sensitive assay for the detection of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancers (NSCLCs). The assay employs a novel double mis-matched primer (DMP) set to improve the detection ability of isothermal solid-phase amplification/detection (ISAD) based on silicon microring biosensor. We show that the EGFR-DMP can detect EGFR gene mutations within 20 min in a label-free and real-time manner. The EGFR-DMP was able to detect a mutation in a sample containing only 1% of the mutant cells in a mixture of wild-type cells. Furthermore, to validate the proposed assay for potential applications in clinical diagnostics, we examined paraffin-embedded tissue samples from 10 NSCLC patients for the presence of EGFR mutations by performing EGFR-DMP and direct sequencing. The EGFR-DMP assay was able to rapidly detect the mutation, with high sensitivity and specificity. The EGFR-DMP assay offers a robust and sensitive approach for the rapid identification of the EGFR mutation. The high sensitivity and specificity and rapidity of this approach may make it useful for predicting the clinical response to targeted EGFR TKIs as a companion diagnostic. PMID:25569872

  11. MITF Modulates Therapeutic Resistance through EGFR Signaling.

    PubMed

    Ji, Zhenyu; Erin Chen, Yiyin; Kumar, Raj; Taylor, Michael; Jenny Njauw, Ching-Ni; Miao, Benchun; Frederick, Dennie T; Wargo, Jennifer A; Flaherty, Keith T; Jönsson, Göran; Tsao, Hensin

    2015-07-01

    Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF (V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs), although the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR-induced vemurafenib resistance is ligand dependent. We employed whole-genome expression analysis and discovered that vemurafenib resistance correlated with the loss of microphthalmia-associated transcription factor (MITF), along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance, and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients' tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an "autocrine drug resistance loop" is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition. PMID:25789707

  12. MITF Modulates Therapeutic Resistance through EGFR Signaling

    PubMed Central

    Ji, Zhenyu; Chen, Yiyin Erin; Kumar, Raj; Taylor, Michael; Njauw, Ching-Ni Jenny; Miao, Benchun; Frederick, Dennie T.; Wargo, Jennifer A.; Flaherty, Keith T.; Jönsson, Göran; Tsao, Hensin

    2015-01-01

    Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF(V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs) though the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR induced vemurafenib resistance is ligand dependent. We then employed whole-genome expression analysis and discovererd that vemurafenib resistance correlated with the loss of MITF, along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients’ tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an “autocrine drug resistance loop” is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition. PMID:25789707

  13. Aberrant large tumor suppressor 2 (LATS2) gene expression correlates with EGFR mutation and survival in lung adenocarcinomas

    PubMed Central

    Luo, Susan Y.; Sit, Ko-Yung; Sihoe, Alan D.L.; Suen, Wai-Sing; Au, Wing-Kuk; Tang, Ximing; Ma, Edmond S.K.; Chan, Wai-Kong; Wistuba, Ignacio I.; Minna, John D.; Tsao, George S.W.; Lam, David C.L.

    2015-01-01

    Background Large tumor suppressor 2 (LATS2) gene is a putative tumor suppressor gene with potential roles in regulation of cell proliferation and apoptosis in lung cancer. The aim of this study is to explore the association of aberrant LATS2 expression with EGFR mutation and survival in lung adenocarcinoma (AD), and the effects of LATS2 silencing in both lung AD cell lines. Methods LATS2 mRNA and protein expression in resected lung AD were correlated with demographic characteristics, EGFR mutation and survival. LATS2-specific siRNA was transfected into four EGFR wild-type (WT) and three EGFR mutant AD cell lines and the changes in LATS2 expression and relevant signaling molecules before and after LATS2 knockdown were assayed. Results Fifty resected lung AD were included (M:F = 23:27, smokers:non-smokers = 19:31, EGFR mutant:wild-type = 21:29) with LATS2 mRNA levels showed no significant difference between gender, age, smoking and pathological stages while LATS2 immunohistochemical staining on an independent set of 79 lung AD showed similar trend. LATS2 mRNA level was found to be a significant independent predictor for survival status (disease-free survival RR = 0.217; p = 0.003; Overall survival RR = 0.238; p = 0.036). siRNA-mediated suppression of LATS2 expression resulted in augmentation of ERK phosphorylation in EGFR wild-type AD cell lines with high basal LATS2 expression, discriminatory modulation of Akt signaling between EGFR wild-type and mutant cells, and induction of p53 accumulation in AD cell lines with low baseline p53 levels. Conclusions LATS2 expression level is predictive of survival in patients with resected lung AD. LATS2 may modulate and contribute to tumor growth via different signaling pathways in EGFR mutant and wild-type tumors. PMID:24976335

  14. Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment.

    PubMed

    Chia, Puey Ling; Do, Hongdo; Morey, Adrienne; Mitchell, Paul; Dobrovic, Alexander; John, Thomas

    2016-08-01

    AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms. Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA. In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse. ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression. PMID:27393503

  15. Dual inhibition of MEK1/2 and EGFR synergistically induces caspase-3-dependent apoptosis in EGFR inhibitor-resistant lung cancer cells via BIM upregulation.

    PubMed

    Song, Ji-Young; Kim, Choung-Soo; Lee, Je-Hwan; Jang, Se Jin; Lee, Sang-wook; Hwang, Jung Jin; Lim, Chulsoo; Lee, Gilnam; Seo, Jeongbeob; Cho, Suk Young; Choi, Jene

    2013-12-01

    Epidermal growth factor receptor (EGFR) gene mutations activate the KRAS-RAF-MEK-ERK pathway in lung cancer cells. EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib induce apoptosis of cancer cells, but prolonged treatment is often associated with acquired resistance. Here, we identified a novel MEK1/2 inhibitor, CZ0775, and compared its cytotoxic effects to those of AZD6244 (selumetinib) in non-small cell lung cancer (NSCLC) cell lines harboring EGFR mutations. The lapatinib-sensitive HCC827 and PC9 and lapatinib-resistant H1650 and H1975 cell lines showed poor responses to CZ0775 and AZD6244 monotherapy with an IC50 > 10 μM. By contrast, combination treatment with lapatinib and CZ0775 inhibited cell proliferation and produced a 2-fold higher number of annexin V-labeled cells than lapatinib alone in H1975 cells. Furthermore, combination treatment decreased phosphorylated extracellular signal related kinase (p-ERK) and survivin levels and upregulated the expression of the pro-apoptotic protein BIM. siRNA-mediated BIM depletion reduced caspase-3 activity (~40%) in lapatinib and CZ0775 treated H1975 cells. An in vitro ERK activity assay showed that p-ERK levels were approximately a 3-fold lower in H1975 cells treated with CZ0775 and lapatinib combination than in cells treated with lapatinib alone. CZ0775 was more cytotoxic than AZD6244 when used in combination with lapatinib. Our results suggest that combination treatment with CZ0774 and EGFR inhibitors is a promising therapeutic approach for the treatment of EGFR-TKI-resistant lung cancers and its effect is mediated by the inhibition of ERK and the induction of BIM. PMID:24068620

  16. Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

    PubMed Central

    2013-01-01

    Background Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting. Results A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants. Conclusions Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome. PMID:23806066

  17. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations

    PubMed Central

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-01-01

    Abstract This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer. This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated. Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23–52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2–26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7–14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856). Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation. PMID:27368002

  18. A synonymous EGFR polymorphism predicting responsiveness to anti-EGFR therapy in metastatic colorectal cancer patients.

    PubMed

    Bonin, Serena; Donada, Marisa; Bussolati, Gianni; Nardon, Ermanno; Annaratone, Laura; Pichler, Martin; Chiaravalli, Anna Maria; Capella, Carlo; Hoefler, Gerald; Stanta, Giorgio

    2016-06-01

    Genetic factors are known to affect the efficiency of therapy with monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer (mCRC). At present, the only accepted molecular marker predictive of the response to anti-EGFR mAbs is the somatic mutation of KRAS and NRAS as a marker of resistance to anti-EGFR. However, only a fraction of KRAS wild-type patients benefit from that treatment. In this study, we show that the EGFR gene polymorphism rs1050171 defines, independently of RAS mutational status, a sub-population of 11 % of patients with a better clinical outcome after anti-EGFR treatment. Median PFS for patients with the GG genotype was 10.17 months compared to 5.37 of those with AG + AA genotypes. Taken together, our findings could be used to better define CRC populations responding to anti-EGFR therapy. Further studies in larger independent cohorts are necessary to validate the present observation that a synonymous polymorphism in EGFR gene impacts on clinical responsiveness. PMID:26666825

  19. Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

    PubMed Central

    Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

    2016-01-01

    Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

  20. Ability of the Met Kinase Inhibitor Crizotinib and New Generation EGFR Inhibitors to Overcome Resistance to EGFR Inhibitors

    PubMed Central

    Nanjo, Shigeki; Yamada, Tadaaki; Nishihara, Hiroshi; Takeuchi, Shinji; Sano, Takako; Nakagawa, Takayuki; Ishikawa, Daisuke; Zhao, Lu; Ebi, Hiromichi; Yasumoto, Kazuo; Matsumoto, Kunio; Yano, Seiji

    2013-01-01

    Purpose Although EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have shown dramatic effects against EGFR mutant lung cancer, patients ultimately develop resistance by multiple mechanisms. We therefore assessed the ability of combined treatment with the Met inhibitor crizotinib and new generation EGFR-TKIs to overcome resistance to first-generation EGFR-TKIs. Experimental Design Lung cancer cell lines made resistant to EGFR-TKIs by the gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression and mice with tumors induced by these cells were treated with crizotinib and a new generation EGFR-TKI. Results The new generation EGFR-TKI inhibited the growth of lung cancer cells containing the gatekeeper EGFR-T790M mutation, but did not inhibit the growth of cells with Met amplification or HGF overexpression. In contrast, combined therapy with crizotinib plus afatinib or WZ4002 was effective against all three types of cells, inhibiting EGFR and Met phosphorylation and their downstream molecules. Crizotinib combined with afatinib or WZ4002 potently inhibited the growth of mouse tumors induced by these lung cancer cell lines. However, the combination of high dose crizotinib and afatinib, but not WZ4002, triggered severe adverse events. Conclusions Our results suggest that the dual blockade of mutant EGFR and Met by crizotinib and a new generation EGFR-TKI may be promising for overcoming resistance to reversible EGFR-TKIs but careful assessment is warranted clinically. PMID:24386407

  1. Mechanisms of tumor resistance to EGFR-targeted therapies

    PubMed Central

    Hopper-Borge, Elizabeth A; Nasto, Rochelle E; Ratushny, Vladimir; Weiner, Louis M; Golemis, Erica A

    2009-01-01

    Background Much effort has been devoted to development of cancer therapies targeting EGFR, based on its role in regulating cell growth. Small-molecule and antibody EGFR inhibitors have clinical roles based on their efficacy in a subset of cancers, generally as components of combination therapies. Many cancers are either initially resistant to EGFR inhibitors or become resistant during treatment, limiting the efficacy of these reagents. Objective/Methods To review cellular resistance mechanisms to EGFR-targeted therapies. Results/Conclusions The best validated of these mechanisms include activation of classic ATP-binding casette (ABC) multidrug transporters; activation or mutation of EGFR; and overexpression or activation of signaling proteins operating in relation to EGFR. We discuss current efforts and potential strategies to override these sources of resistance. We describe emerging systems-biology-based concepts of alternative resistance to EGFR-targeted therapies, and discuss their implications for use of EGFR-targeted and other targeted therapies. PMID:19236156

  2. EGFR-mediated Intracellular Delivery of Pc 4 Nanoformulation for Targeted Photodynamic Therapy of Cancer: In Vitro Studies

    PubMed Central

    Master, Alyssa M.; Qi, Yizhi; Oleinick, Nancy L.; Gupta, Anirban Sen

    2011-01-01

    In photodynamic therapy (PDT), the light-activation of a photosensitizer leads to the generation of reactive oxygen species that can trigger various mechanisms of cell death. Harnessing this process within cancer cells enables minimally invasive yet targeted cancer treatment. With this rationale, here we demonstrate tumor-targeted delivery of a highly hydrophobic photosensitizer Pc 4 loaded within biocompatible PEG-PCL block-copolymer micelles. The micelles were surface-modified with EGFR-targeting GE11-peptides for active targeting of EGFR-overexpressing cancer cells, in vitro. Pc 4-loaded EGFR-targeted micelles were incubated with EGFR-overexpressing A431 epidermoid carcinoma cells for various time periods, to determine Pc 4 uptake by epifluorescence microscopy. The cells were subsequently photoirradiated and PDT-induced cell death for various incubation periods was determined by MTT assay and fluorescence Live/Dead assay. Our results indicate that active EGFR-targeting of the Pc 4-loaded micelles accelerates intracellular uptake of the drug. Consequently this enhances the PDT-induced cytotoxicity within shorter time periods. PMID:22024195

  3. Optimizing the sequence of anti-EGFR-targeted therapy in EGFR-mutant lung cancer.

    PubMed

    Meador, Catherine B; Jin, Hailing; de Stanchina, Elisa; Nebhan, Caroline A; Pirazzoli, Valentina; Wang, Lu; Lu, Pengcheng; Vuong, Huy; Hutchinson, Katherine E; Jia, Peilin; Chen, Xi; Eisenberg, Rosana; Ladanyi, Marc; Politi, Katerina; Zhao, Zhongming; Lovly, Christine M; Cross, Darren A E; Pao, William

    2015-02-01

    Metastatic EGFR-mutant lung cancers are sensitive to the first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and afatinib, but resistance develops. Acquired resistance to gefitinib or erlotinib occurs most commonly (>50%) via the emergence of a second-site EGFR mutation, T790M. Two strategies to overcome T790M-mediated resistance are dual inhibition of EGFR with afatinib plus the anti-EGFR antibody cetuximab (A+C), or mutant-specific EGFR inhibition with AZD9291. A+C and AZD9291 are now also being tested as first-line therapies, but whether these therapies will extend progression-free survival or induce more aggressive forms of resistance in this setting remains unknown. We modeled resistance to multiple generations of anti-EGFR therapies preclinically to understand the effects of sequential treatment with anti-EGFR agents on drug resistance and determine the optimal order of treatment. Using a panel of erlotinib/afatinib-resistant cells, including a novel patient-derived cell line (VP-2), we found that AZD9291 was more potent than A+C at inhibiting cell growth and EGFR signaling in this setting. Four of four xenograft-derived A+C-resistant cell lines displayed in vitro and in vivo sensitivity to AZD9291, but four of four AZD9291-resistant cell lines demonstrated cross-resistance to A+C. Addition of cetuximab to AZD9291 did not confer additive benefit in any preclinical disease setting. This work, emphasizing a mechanistic understanding of the effects of therapies on tumor evolution, provides a framework for future clinical trials testing different treatment sequences. This paradigm is applicable to other tumor types in which multiple generations of inhibitors are now available. PMID:25477325

  4. Optimizing the sequence of anti-EGFR targeted therapy in EGFR-mutant lung cancer

    PubMed Central

    Meador, Catherine B.; Jin, Hailing; de Stanchina, Elisa; Nebhan, Caroline A.; Pirazzoli, Valentina; Wang, Lu; Lu, Pengcheng; Vuong, Huy; Hutchinson, Katherine E.; Jia, Peilin; Chen, Xi; Eisenberg, Rosana; Ladanyi, Marc; Politi, Katerina; Zhao, Zhongming; Lovly, Christine M.; Cross, Darren A. E.; Pao, William

    2015-01-01

    Metastatic EGFR-mutant lung cancers are sensitive to the first- and second- generation EGFR tyrosine kinase inhibitors (TKIs), gefitinib, erlotinib, and afatinib, but resistance develops. Acquired resistance (AR) to gefitinib or erlotinib occurs most commonly (>50%) via the emergence of a second-site EGFR mutation, T790M. Two strategies to overcome T790M-mediated resistance are dual inhibition of EGFR with afatinib plus the anti-EGFR antibody, cetuximab (A+C), or mutant-specific EGFR inhibition with AZD9291. A+C and AZD9291 are now also being tested as first-line therapies, but whether these therapies will extend progression-free survival or induce more aggressive forms of resistance in this setting remains unknown. We modeled resistance to multiple generations of anti-EGFR therapies preclinically in order to understand the effects of sequential treatment with anti-EGFR agents on drug resistance and determine the optimal order of treatment. Using a panel of erlotinib/afatinib-resistant cells including a novel patient-derived cell line (VP-2), we found that AZD9291 was more potent than A+C at inhibiting cell growth and EGFR signaling in this setting. 4 of 4 xenograft-derived A+C-resistant cell lines displayed in vitro and in vivo sensitivity to AZD9291, but 4 of 4 AZD9291-resistant cell lines demonstrated cross-resistance to A+C. Addition of cetuximab to AZD9291 did not confer additive benefit in any preclinical disease setting. This work, emphasizing a mechanistic understanding of the effects of therapies on tumor evolution, provides a framework for future clinical trials testing different treatment sequences. This paradigm is applicable to other tumor types in which multiple generations of inhibitors are now available. PMID:25477325

  5. Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

    PubMed Central

    2015-01-01

    Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit. PMID:24697349

  6. Evaluation of therapeutic effectiveness of 131I-antiEGFR-BSA-PCL in a mouse model of colorectal cancer

    PubMed Central

    Li, Wei; Ji, Yan-Hui; Li, Cheng-Xia; Liu, Zhong-Yun; Li, Ning; Fang, Lei; Chang, Jin; Tan, Jian

    2016-01-01

    AIM: To investigate the biological effects of internal irradiation, and the therapeutic effectiveness was assessed of 131I-labeled anti-epidermal growth factor receptor (EGFR) liposomes, derived from cetuximab, when used as a tumor-targeting carrier in a colorectal cancer mouse model. METHODS: We described the liposomes and characterized their EGFR-targeted binding and cellular uptake in EGFR-overexpressing LS180 colorectal cancer cells. After intra-tumor injections of 74 MBq (740 MBq/mL) 131I-antiEGFR-BSA-PCL, we investigated the biological effects of internal irradiation and the therapeutic efficacy of 131I-antiEGFR-BSA-PCL on colorectal cancer in a male BALB/c mouse model. Tumor size, body weight, histopathology, and SPECT imaging were monitored for 33 d post-therapy. RESULTS: The rapid radioiodine uptake of 131I-antiEGFR-BSA-PCL and 131I-BSA-PCL reached maximum levels at 4 h after incubation, and the 131I uptake of 131I-antiEGFR-BSA-PCL was higher than that of 131I-BSA-PCL in vitro. The 131I tissue distribution assay revealed that 131I-antiEGFR-BSA-PCL was markedly taken up by the tumor. Furthermore, a tissue distribution assay revealed that 131I-antiEGFR-BSA-PCL was markedly taken up by the tumor and reached its maximal uptake value of 21.0 ± 1.01 %ID/g (%ID/g is the percentage injected dose per gram of tissue) at 72 h following therapy; the drug concentration in the tumor was higher than that in the liver, heart, colon, or spleen. Tumor size measurements showed that tumor development was significantly inhibited by treatments with 131I-antiEGFR-BSA-PCL and 131I-BSA-PCL. The volume of tumor increased, and treatment rate with 131I-antiEGFR-BSA-PCL was 124% ± 7%, lower than that with 131I-BSA-PCL (127% ± 9%), 131I (143% ± 7%), and normal saline (146% ± 10%). The percentage losses in original body weights were 39% ± 3%, 41% ± 4%, 49% ± 5%, and 55% ± 13%, respectively. The best survival and cure rates were obtained in the group treated with 131I-antiEGFR

  7. Comparison of an enzyme-linked immunosorbent assay (ELISA) to gas chromatography (GC) - measurement of polychlorinated biphenyls (PCBs) in selected US fish extracts

    USGS Publications Warehouse

    Zajicek, J.L.; Tillitt, D.E.; Schwartz, T.R.; Schmitt, C.J.; Harrison, R.O.

    2000-01-01

    The analysis of PCBs in fish tissues by immunoassay methods was evaluated using fish collected from a US monitoring program, the National Contaminant Biomonitoring Program of the US Department of Interior, Fish and Wildlife Service. Selected composite whole fish samples, which represented widely varying concentrations and sources of PCBs, were extracted and subjected to congener PCB analysis by gas chromatography (GC) and total PCB analysis using an ELISA (ePCBs) calibrated against technical Aroclor 1248. PCB congener patterns in these fishes were different from the patterns found in commercial Aroclors or their combinations as demonstrated by principal component analysis of normalized GC congener data. The sum of the PCB congeners measured by GC (total-PCBs) ranged from 37 to 4600 ng/g (wet weight). Concentrations of PCBs as determined by the ELISA method were positively correlated with total-PCBs and the ePCBs/total-PCBs ratios for individual samples ranged from 1 to 6. Ratios of ePCBs/total-PCBs for dilutions of Aroclors 1242, 1254, and 1260 and for matrix spikes range from 0.6 for 1242 to 2.5 for 1254 and 1260. These results suggest that higher chlorinated PCB congeners have higher affinity for the anti-PCB antibodies. Partial least squares with latent variable analysis of GC and ELISA data of selected Aroclors and fish samples also support the conclusion that ELISA derived PCB concentrations are dependent on the degree on chlorination.

  8. Development of critical life stage assays: Teratogenic effects of ash basin effluent components on freshwater fish, Gambusia affinis and Daphnia: Progress report, May 21, 1987-- June 1, 1988

    SciTech Connect

    Guram, M.S.; Boatwright, B.

    1988-04-06

    The Voorhees College research project directed toward describing and evaluating the reproductive activity of a representative fish species (Gambusia) in several ponds on the SRP property has established several important points during the first phase (year I) of its operation. It has been demonstrated that significant numbers of the selected fish can be obtained from each of the test ponds, and can be returned to the Voorhees College laboratories in good physiological condition. This is shown by very minimal loss of fish during and following capture, transportation, and subsequent placement in individual tanks for rearing young when delivered during subsequent weeks. Forty individual fry rearing tanks are now functioning with capture of fry being carried out as they are delivered by each female fish. Preservation of fry is made in Bouins solution for detailed analysis of any abnormalities. The goal of the first phase is to establish a year long (several seasons) profile of the number of fry delivered per female fish, presence or absence of abnormal fry at birth, variation in fry between successive deliveries of fry, variations throughout the year, and variations of fry characteristics between the several ponds from which the adult female fish were obtained. Excellent technical help has been obtained from Voorhees College senior biology major students. These students have maintained the health of the rearing ranks throughout the study period, have collected the fry as they are born, and analyzed data obtained in observations on those fry.

  9. Aeromonas salmonicida infection levels in pre- and post-stocked cleaner fish assessed by culture and an amended qPCR assay.

    PubMed

    Gulla, S; Duodu, S; Nilsen, A; Fossen, I; Colquhoun, D J

    2016-07-01

    Due to increasing resistance to chemical therapeutants, the use of 'cleaner fish' (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de-licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A-layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real-time PCR (qPCR), targeting the A. salmonicida A-layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A-layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild-caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre- and post-stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded. PMID:26514414

  10. Cathepsin S attenuates endosomal EGFR signalling: A mechanical rationale for the combination of cathepsin S and EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Chien-Chang; Lee, Cheng-Che; Lin, Hsiao-Han; Chang, Jang-Yang

    2016-01-01

    EGF-mediated EGFR endocytosis plays a crucial role in the attenuation of EGFR activation by sorting from early endosomes to late endosomes and transporting them into lysosomes for the final proteolytic degradation. We previously observed that cathepsin S (CTSS) inhibition induces tumour cell autophagy through the EGFR-mediated signalling pathway. In this study, we further clarified the relationship between CTSS activities and EGFR signalling regulation. Our results revealed that CTSS can regulate EGFR signalling by facilitating EGF-mediated EGFR degradation. CTSS inhibition delayed the EGFR degradation process and caused EGFR accumulation in the late endosomes at the perinuclear region, which provides spatial compartments for prolonged EGFR and sustained downstream signal transducer and activator of transcription 3 and AKT signalling. Notably, cellular apoptosis was markedly enhanced by combining treatment with the EGFR inhibitor Iressa and CTSS inhibitor 6r. The data not only reveal a biological role of CTSS in EGFR signalling regulation but also evidence a rationale for its clinical evaluation in the combination of CTSS and EGFR tyrosine kinase inhibitors. PMID:27387133

  11. Immunohistochemical assay for epidermal growth factor receptor on paraffin-embedded sections: validation against ligand-binding assay and clinical relevance in breast cancer.

    PubMed Central

    Newby, J. C.; A'Hern, R. P.; Leek, R. D.; Smith, I. E.; Harris, A. L.; Dowsett, M.

    1995-01-01

    Epidermal growth factor receptor (EGFR) has been the subject of much research since it was first described as a prognostic factor in breast cancer. The assay methods used and results obtained vary widely between studies. In this study 88 primary breast cancers were assayed for EGFR using a novel immunohistochemical assay performed on paraffin-embedded sections. The monoclonal antibody used was raised against purified, denatured EGFR, reacts with an epitope on the external domain and does not interfere with ligand binding. Twenty-two per cent of the tumours were EGFR positive using this assay. The results obtained were significantly correlated with those obtained by ligand-binding assay (r = 0.621, P = 0.011). The concordance rate was 82% (P < 0.001). The majority of discordant results could be explained by the presence of benign breast tissue and other non-malignant elements which could be seen to express EGFR on the immunohistochemical assay and were excluded from the score for this, but would be incorporated into ligand-binding assay results. The well-established inverse relationship between EGFR (as measured by this assay) and oestrogen receptor (ER) was seen (chi 2 = 24.9, P < 0.0001). In addition, in this exploratory study on a limited tumour set, EGFR was a significant adverse prognostic factor (on univariate but not multivariate analysis) for both relapse-free survival (P = 0.02) and overall survival (P = 0.03) when measured by this immunohistochemical assay, but was not significant when measured by ligand-binding assay. Images Figure 1 Figure 2 PMID:7779717

  12. Intratumor Heterogeneity of ALK-Rearrangements and Homogeneity of EGFR-Mutations in Mixed Lung Adenocarcinoma

    PubMed Central

    Marino, Federica Zito; Liguori, Giuseppina; Aquino, Gabriella; La Mantia, Elvira; Bosari, Silvano; Ferrero, Stefano; Rosso, Lorenzo; Gaudioso, Gabriella; De Rosa, Nicla; Scrima, Marianna; Martucci, Nicola; La Rocca, Antonello; Normanno, Nicola; Morabito, Alessandro; Rocco, Gaetano; Botti, Gerardo; Franco, Renato

    2015-01-01

    Background Non Small Cell Lung Cancer is a highly heterogeneous tumor. Histologic intratumor heterogeneity could be ‘major’, characterized by a single tumor showing two different histologic types, and ‘minor’, due to at least 2 different growth patterns in the same tumor. Therefore, a morphological heterogeneity could reflect an intratumor molecular heterogeneity. To date, few data are reported in literature about molecular features of the mixed adenocarcinoma. The aim of our study was to assess EGFR-mutations and ALK-rearrangements in different intratumor subtypes and/or growth patterns in a series of mixed adenocarcinomas and adenosquamous carcinomas. Methods 590 Non Small Cell Lung Carcinomas tumor samples were revised in order to select mixed adenocarcinomas with available tumor components. Finally, only 105 mixed adenocarcinomas and 17 adenosquamous carcinomas were included in the study for further analyses. Two TMAs were built selecting the different intratumor histotypes. ALK-rearrangements were detected through FISH and IHC, and EGFR-mutations were detected through IHC and confirmed by RT-PCR. Results 10/122 cases were ALK-rearranged and 7 from those 10 showing an intratumor heterogeneity of the rearrangements. 12/122 cases were EGFR-mutated, uniformly expressing the EGFR-mutated protein in all histologic components. Conclusion Our data suggests that EGFR-mutations is generally homogeneously expressed. On the contrary, ALK-rearrangement showed an intratumor heterogeneity in both mixed adenocarcinomas and adenosquamous carcinomas. The intratumor heterogeneity of ALK-rearrangements could lead to a possible impact on the therapeutic responses and the disease outcomes. PMID:26422230

  13. Novel EGFR mutation specific antibodies for NSCLC: Immunohistochemistry as a possible screening method for EGFR mutations

    PubMed Central

    Kato, Yasufumi; Peled, Nir; Wynes, Murry W.; Yoshida, Koichi; Pardo, Marta; Mascaux, Celine; Ohira, Tatsuo; Tsuboi, Masahiro; Matsubayashi, Jun; Nagao, Toshitaka; Ikeda, Norihiko; Hirsch, Fred R.

    2010-01-01

    Background Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict better outcome to EGFR tyrosine kinase inhibitors (TKIs). The most common mutations are exon 19 deletions (most frequently E746-A750) and L858R point mutation in exon 21. Here, we evaluated the accuracy of novel EGFR mutation specific antibodies in a Japanese cohort with NSCLC and compared to direct DNA sequencing and clinical outcome. Materials and methods Immunohistochemistry (IHC) using antibodies specific for the E746-A750 and L858R mutations in EGFR was performed on tissue microarrays of tumors from 70 gefitinib treated NSCLC patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21. Results DNA sequencing showed EGFR mutations in 41 patients (58.6%), and exon 19 deletions in 18 patients (25.7%), 61% (11/18) had a deletion in the range of E746-A750) and 12 (17.1%) had exon 21 mutations (L858R). IHC showed, for the E746-A750 and L858R mutations, sensitivity (81.8% and 75%), specificity (100%, 96.6%), PPV (100%, 81.8%) and NPV (96.7%, 94.9%). Analysis for objective response rates (ORR) and survival were not correlated to IHC staining, although the combined staining showed non-significant trends towards better overall survival for patients with EGFR mutations. Conclusions The mutation specific IHC antibodies have high sensitivity and specificity for pre-defined EFGR mutations and may be suitable for screening for these pre-defined mutations. However, negative IHC results require further mutation analyses prior to excluding EGFR-targeted therapy. PMID:20697298

  14. Polymorphisms in epidermal growth factor receptor (EGFR) and AKT1 as possible predictors of clinical outcome in advanced non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors.

    PubMed

    Zhang, Xiaoqing; Fan, Junwei; Li, Yuping; Lin, Shengtao; Shu, Ping; Ni, Jian; Qin, Shengying; Zhang, Zhemin

    2016-01-01

    This study aimed to investigate the association of epidermal growth factor receptor (EGFR) gene polymorphism and AKT1 polymorphism with the clinical outcomes in advanced non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs). The clinical outcome and the survival of NSCLC of 230 patients after treatment with EGFR-TKIs were measured. The rs712829, rs1468727 of the EGFR gene and rs1130214 of the AKT1 gene from peripheral blood cell were detected by a multiplexed single nucleotide polymorphism (SNP) MassEXTEND assay. The relationship between genetic polymorphisms and clinical outcomes of treatment with EGFR-TKIs was analyzed. The response rates and the disease control rate of patients with genotype GG, GT, and TT in EGFR rs712829 were statistically very significant difference(19.7 vs 36.1 vs 50.0 %, P = 0.016 and 57.7 vs 77.8 vs 83.3 %, P = 0.026, respectively). Better disease control was also achieved in patients with the GG genotype of AKT1 rs1130214 than those with the GT and TT genotypes (65.6 vs. 48.7 %, P = 0.043). Patients carrying the EGFR rs712829 TT genotype had significantly longer PFS and OS than those with the GT or GG genotypes (9.0 vs. 7.0 vs. 5.0 months, P = 0.001 and 13.1 vs. 14.6 vs. 18.8 months, P = 0.008, respectively). In addition, patients carrying the AKT1 rs1130214 GG genotype also had significantly longer PFS than those with the GT and TT genotypes (5.5 vs. 4.5 months, P = 0.008). EGFR rs712829 polymorphism and AKT1 rs1130214 could influence the response to EGFR-TKIs therapy in patients with advanced NSCLC. PMID:26269114

  15. Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition.

    PubMed

    Soucheray, Margaret; Capelletti, Marzia; Pulido, Inés; Kuang, Yanan; Paweletz, Cloud P; Becker, Jeffrey H; Kikuchi, Eiki; Xu, Chunxiao; Patel, Tarun B; Al-Shahrour, Fatima; Carretero, Julián; Wong, Kwok-Kin; Jänne, Pasi A; Shapiro, Geoffrey I; Shimamura, Takeshi

    2015-10-15

    Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. PMID:26282169

  16. Lupeol evokes anticancer effects in oral squamous cell carcinoma by inhibiting oncogenic EGFR pathway.

    PubMed

    Rauth, Sanchita; Ray, Sudipta; Bhattacharyya, Sayantan; Mehrotra, Debapriya Ghosh; Alam, Neyaz; Mondal, Goutam; Nath, Partha; Roy, Asoke; Biswas, Jaydip; Murmu, Nabendu

    2016-06-01

    Epidermal growth factor receptor (EGFR) pathway is overexpressed in head and neck cancer (HNC). Lupeol, a natural triterpene (phytosterol found in fruits, vegetables, etc.), has been reported to be effective against multiple cancer indications. Here we investigate the antitumor effects of Lupeol and underlying mechanism in oral cancer. Lupeol-induced antitumor response was evaluated in two oral squamous cell carcinoma (OSCC) cell lines (UPCI:SCC131 and UPCI:SCC084) by viability (MTT), proliferation, and colony formation assays. Lupeol-mediated induction of apoptosis was examined by caspase 3/7 assay and flow cytometry. Effect of Lupeol on EGFR in the presence or absence of EGF was delineated by Western blot. The mRNA stability assay was performed to check the role of Lupeol on COX-2 mRNA regulation. Lupeol inhibited proliferation of OSCC cells in vitro by inducing apoptosis 48 h post treatment. Ligand-induced phosphorylation of EGFR and subsequent activation of its downstream molecules such as protein kinase B (PKB or AKT), I kappa B (IκB), and nuclear factor kappa B (NF-κB) was also found to be, in part, suppressed. Interestingly, Lupeol suppressed expression of COX-2 at mRNA and protein level in a time-dependent manner. Primary explants from oral squamous cell carcinoma tissues further confirmed significant inhibition of proliferation (Ki67) in Lupeol-treated explants as compared to untreated control at 48 h. Together these data suggest that Lupeol may act as a potent inhibitor of the EGFR signaling in OSCC and therefore imply its role in triggering antitumor efficacy. PMID:27206736

  17. Copper improves the anti-angiogenic activity of disulfiram through the EGFR/Src/VEGF pathway in gliomas.

    PubMed

    Li, Yi; Fu, Shi-Yuan; Wang, Li-Hui; Wang, Fang-Yang; Wang, Nan-Nan; Cao, Qi; Wang, Ya-Ting; Yang, Jing-Yu; Wu, Chun-Fu

    2015-12-01

    Disulfiram (DSF) possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. DSF also potently inhibits angiogenesis, but the effect of Cu on this anti-angiogenic activity is unknown. Here we show that DSF inhibits the proliferation, migration, invasion, adhesion and complex tube formation of human umbilical vascular endothelial cells (HUVECs). Aortic ring assays and Matrigel plug assays revealed that DSF significantly inhibited the formation of microvessels. Importantly, Cu improved the anti-angiogenic activity of DSF in all these assays, while copper alone had no effect. DSF/Cu treatment of U87 human glioblastoma cells resulted in suppression of VEGF secretion through the EGFR/c-Src/VEGF pathway. Reduction of EGFR phosphorylation disables recruitment of multiple Src homology 2 (SH2) domains, resulting in transcriptional down-regulation of VEGF. The role of EGFR/c-Src/VEGF pathway was further confirmed by using specific inhibitor, which significantly improved the anti-angiogenic activity of DSF/Cu. DSF/Cu also exerted increased anti-tumor effects on subcutaneous and intracerebral U87 xenograft models by reducing microvessel density (MVD) and VEGF expression. These results indicate that Cu improves the anti-angiogenic activity of DSF by targeting the EGFR/Src/VEGF signaling pathway, thus providing a rationale for the use of DSF/Cu rather than DSF alone as an angiogenesis inhibitor in clinical applications. PMID:26254539

  18. Synergistic inhibitory effect of cetuximab and tectochrysin on human colon cancer cell growth via inhibition of EGFR signal.

    PubMed

    Park, Mi Hee; Hong, Ji Eun; Hwang, Chul Ju; Choi, Mingi; Choi, Jeong Soon; An, Young Jin; Son, Dong Ju; Hong, Jin Tae

    2016-05-01

    The purpose of this study was to evaluate the enhancing potency of tectochrysin, a flavonoid isolated from Alpinia oxyphylla Miquel by combining cetuximab, an anti-EGFR monoclonal antibody, on human colon cancer cell growth through further inhibition of EGFR pathway. HCT116 and SW480 colon cancer cells were treated with cetuximab (30 μg/mL, 1/10 of IC50), tectochrysin (5 μg/mL, 1/3 of IC50), or the combination of both agents. The growth inhibitory effect was examined using the MTT assay while apoptotic cell death was performed using TUNEL staining assays. The DNA binding activity of NF-kappa B and AP-1 was investigated by electrophoretic mobility shift assay. Protein expression was determined by Western blot. Cell proliferation was significantly inhibited by the combination of cetuximab and tectochrysin than treatment with cetuximab or tectochrysin alone (combination index: 0.572 and 0.533, respectively). Combination treatment of cells with cetuximab and tectochrysin significantly reduced the expressions of p-EGFR and COX-2 in both cell lines. Combination treatment also significantly inhibited activities of NF-kB and AP-1 compared to the single agent treatment. Our results indicate that combined therapy with lower concentration of cetuximab and tectochrysin could significantly enhance the cancer cell growth inhibitory effect through the inhibition of EGFR signaling. PMID:27025376

  19. EGFR-mutated lung cancer: a paradigm of molecular oncology

    PubMed Central

    Zhang, Zhenfeng; Stiegler, Amy L.; Boggon, Titus J.; Kobayashi, Susumu; Halmos, Balazs

    2010-01-01

    The development of EGFR tyrosine kinase inhibitors for clinical use in non-small cell lung cancer and the subsequent discovery of activating EGFR mutations have led to an explosion of knowledge in the fields of EGFR biology, targeted therapeutics and lung cancer research. EGFR-mutated adenocarcinoma of the lung has clearly emerged as a unique clinical entity necessitating the routine introduction of molecular diagnostics into our current diagnostic algorithms and leading to the evidence-based preferential usage of EGFR-targeted agents for patients with EGFR-mutant lung cancers. This review will summarize our current understanding of the functional role of activating mutations, key downstream signaling pathways and regulatory mechanisms, pivotal primary and acquired resistance mechanisms, structure-function relationships and ultimately the incorporation of molecular diagnostics and small molecule EGFR tyrosine kinase inhibitors into our current treatment paradigms. PMID:21165163

  20. Interaction of EGFR to δ-catenin leads to δ-catenin phosphorylation and enhances EGFR signaling

    PubMed Central

    He, Yongfeng; Ryu, Taeyong; Shrestha, Nensi; Yuan, Tingting; Kim, Hangun; Shrestha, Hridaya; Cho, Young-Chang; Seo, Young-Woo; Song, Woo Keun; Kim, Kwonseop

    2016-01-01

    Expression of δ-catenin reportedly increases during late stage prostate cancer. Furthermore, it has been demonstrated that expression of EGFR is enhanced in hormone refractory prostate cancer. In this study, we investigated the possible correlation between EGFR and δ-catenin in prostate cancer cells. We found that EGFR interacted with δ-catenin and the interaction decreased in the presence of EGF. We also demonstrated that, on one hand, EGFR phosphorylated δ-catenin in a Src independent manner in the presence of EGF and on the other hand, δ-catenin enhanced protein stability of EGFR and strengthened the EGFR/Erk1/2 signaling pathway. Our findings added a new perspective to the interaction of EGFR to the E-cadherin complex. They also provided novel insights to the roles of δ-catenin in prostate cancer cells. PMID:26883159

  1. Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2

    PubMed Central

    Rexer, Brent N.; Ghosh, Ritwik; Narasanna, Archana; Estrada, Mónica Valeria; Chakrabarty, Anindita; Song, Youngchul; Engelman, Jeffrey A.; Arteaga, Carlos L.

    2013-01-01

    Purpose Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. Experimental Design HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR-HER2-HER3-PI3K axis. Results A low 3% allelic frequency of the T798M mutant shifted10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting increased EGFR ligand production was causally associated with drug resistance. Conclusions Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles. PMID:23948973

  2. Novel Quinazoline Derivatives Bearing Various 4-Aniline Moieties as Potent EGFR Inhibitors with Enhanced Activity Against NSCLC Cell Lines.

    PubMed

    Wang, Changyan; Sun, Yajun; Zhu, Xingqi; Wu, Bin; Wang, Qiao; Zhen, Yuhong; Shu, Xiaohong; Liu, Kexin; Zhou, Youwen; Ma, Xiaodong

    2016-04-01

    A class of novel quinazoline derivatives bearing various C-4 aniline moieties was synthesized and biologically evaluated as potent epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Most of these inhibitors are comparable to gefitinib in inhibiting these cancer cell lines, and several of them even displayed superior inhibitory activity. In particular, analogue 5b with an IC50 of 0.10 μm against the EGFR wild-type A431 cells and 5c with an IC50 of 0.001 μm against the gefitinib-sensitive HCC827 cells (EGFR del E746-A750) was identified as highly active EGFR inhibitors. It was also significant that the discovered analogue 2f, not only has high potency against the gefitinib-sensitive cells (IC50 = 0.031 μm), but also possesses remarkably improved activity against the gefitinib-resistant cells. In addition, the enzymatic assays and the Western blot analysis for evaluating the effects of the typical inhibitors indicated that these molecules strongly interfere with the EGFR target. PMID:26613384

  3. Lower serum soluble-EGFR is a potential biomarker for metastasis of HCC demonstrated by N-glycoproteomic analysis.

    PubMed

    Hu, Heng; Gao, Lingling; Wang, Cun; Li, Yan; Ma, Huiying; Chen, Long; Qin, Jie; Liu, Binbin; Liu, Yinkun; Liang, Chunmin

    2015-05-01

    Hepatocellular carcinoma (HCC) is one of the most deadly cancers in the world due to its high metastatic potential. By using the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative N-glycoproteomic analysis, 26 differentially expressed serum glycoproteins derived from defined stages in orthotopic xenograft tumor model were identified. Among them, expression level of soluble EGFR (sEGFR) was verified in HCC cell lines. We found that non-metastasis HCC cell lines express significantly more sEGFR than HCC cell lines with metastasis potential both in cell lysates and culture media. Serum samples from 28 non-metastatic HCC patients and 28 metastatic HCC patients were assayed. Compared with the non-metastatic HCC group, serum level of sEGFR in metastatic HCC group was statistically lower (p<0.01). All these results provide evidence that sEGFR is a potential candidate for metastasis-associated biomarkers of HCC. The related molecular mechanism deserves to be further explored. PMID:26105696

  4. Anti-EGFR-Conjugated Hollow Gold Nanospheres Enhance Radiocytotoxic Targeting of Cervical Cancer at Megavoltage Radiation Energies

    NASA Astrophysics Data System (ADS)

    Liu, Jiao; Liang, Ying; Liu, Ting; Li, Dengke; Yang, Xingsheng

    2015-05-01

    The study aimed to confirm that anti-epidermal growth factor receptor (EGFR) monoclonal antibody-conjugated hollow gold nanospheres (anti-EGFR/HGNs) can be selectively uptaken by cervical cancer cells and induce its apoptosis when combined with radiotherapy, as a result enhancing radiosensitivity of cervical cancer cells. HGNs with a mean diameter of 54.6 ± 7.11 nm and wall thickness of 5.01 ± 2.23 nm were viewed by transmission electron microscopy (TEM). Cell uptake was assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The cytotoxicity on HeLa cells, which were used in our experiment, was assessed by CCK-8 assay. Cell cycle and apoptosis were examined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry (FCM). The expression of several critical apoptosis-related proteins, including Bcl-2, Bax, Bad, and active caspase 3, was tested by western blot analysis. Cells treated by anti-EGFR/HGNs showed an obvious increase in nanoparticle uptake compared to naked HGNs. Anti-EGFR/HGNs combined with radiation resulted in a significant growth inhibition, compared with radiation combined with naked HGNs. Anti-EGFR/HGNs remarkably increased the ratio of HeLa cells in the G2/M phase and induced more apoptosis by an obvious deregulation of Bcl-2 and upregulation of Bax, Bad, and caspase 3 when combined with radiation. Therefore, anti-EGFR/HGNs can increase the targeted uptake of HGNs by HeLa cells and enhance radiocytotoxic targeting of cervical cancer at megavoltage radiation energies.

  5. Heptameric targeting ligands against EGFR and HER2 with high stability and avidity.

    PubMed

    Kim, Dongwook; Yan, Yitang; Valencia, C Alexander; Liu, Rihe

    2012-01-01

    Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, Z(EGFR) and Z(HER2), were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric Z(EGFR) and Z(HER2) ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents.. PMID:22912791

  6. Cloning and expression of a surface immunogenic protein in Streptococcus dysgalactiae isolated from fish and its application in enzyme-linked immunosorbent assays to diagnose S. dysgalactiae infections in fish.

    PubMed

    Nishiki, I; Minami, T; Itami, T; Yoshida, T

    2014-12-01

    Lancefield group C Streptococcus dysgalactiae (GCSD) causes severe necrotic lesions in the caudal peduncle in the genus Seriola farmed in Japan. To develop a sero-diagnostic method for GCSD infection in farmed fish, we attempted to identify a surface immunogenic protein that induces an antibody after infection with GCSD by immunoblot analysis using sera collected from infected fish. A protein obtained from sodium dodecyl sulfate (SDS) extracts of GCSD was identified as S. dysgalactiae surface immunogenic protein (Sd-Sip). Sd-Sip exhibited more than 94% homology with a surface antigen or a hypothetical protein from S. dysgalactiae mammalian isolates at the nucleotide sequence level. Expression of the recombinant Sd-Sip (rSd-Sip) was confirmed by immunoblot analysis, that is, its reactivity to GCSD-infected sera. Antibody detection ELISA using rSd-Sip and their usefulness for diagnosis of GCSD infection were examined. GCSD-infected sera collected from farmed amberjack, Seriola dumerili (Risso), showed strong reaction with immobilized rSd-Sip. Meanwhile, sera immunized by other pathogenic bacteria of fish were showed ELISA values similar to those of non-infected sera. These results of this study suggest that the antibody detection ELISA using rSd-Sip is an effective diagnostic method for GCSD infection in fish. PMID:24131210

  7. The Pitfalls of Companion Diagnostics: Evaluation of Discordant EGFR Mutation Results from a Clinical Laboratory and a Central Laboratory.

    PubMed

    Turner, Scott A; Peterson, Jason D; Pettus, Jason R; de Abreu, Francine B; Amos, Christopher I; Dragnev, Konstantin H; Tsongalis, Gregory J

    2016-05-01

    Accurate identification of somatic mutations in formalin-fixed, paraffin-embedded tumor tissue is required for enrollment into clinical trials for many novel targeted therapeutics, including trials requiring EGFR mutation status in non-small-cell lung carcinomas. Central clinical trial laboratories contracted to perform this analysis typically rely on US Food and Drug Administration-approved targeted assays to identify these mutations. We present two cases in which central laboratories inaccurately reported EGFR mutation status because of improper identification and isolation of tumor material and failure to accurately report assay limitations, resulting in enrollment denial. Such cases highlight the need for increased awareness by clinical trials of the limitation of these US Food and Drug Administration-approved assays and the necessity for a mechanism to reevaluate discordant results by alternative laboratory-developed procedures, including clinical next-generation sequencing. PMID:26923179

  8. Application of endocrine disruptor screening program fish short-term reproduction assay: Reproduction and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus) exposed to Bermuda pond sediment.

    PubMed

    Fort, Douglas J; Mathis, Michael; Fort, Chelsea E; Fort, Hayley M; Bacon, Jamie P

    2015-06-01

    A modified tier 1 Endocrine Disruptor Screening Program (EDSP) 21-d fish short-term reproduction assay (FSTRA) was used to evaluate the effects of sediment exposure from freshwater and brackish ponds in Bermuda on reproductive fecundity and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus). Reproductively active male and female fish were exposed to control sediment and sediment from 2 freshwater ponds (fathead minnow) and 2 marine ponds (killifish) contaminated with polyaromatic hydrocarbons and metals via flow-through exposure for 21 d. Reproductive fecundity was monitored daily. At termination, the status of the reproductive endocrine system was assessed by the gonadosomatic index, gonadal histology, plasma steroids (estrogen [E2], testosterone [T], and 11-ketotestosterone [11-KT]), steroidogenic enzymes (aromatase and combined 3β/17β -hydroxysteroid dehydrogenase [3β/17β-HSD]), and plasma vitellogenin (VTG). Decreased reproductive fecundity, lower male body weight, and altered endocrinological measures of reproductive status were observed in both species. Higher plasma T levels in female minnows and 11-KT levels in both male and female minnows and female killifish exposed to freshwater and brackish sediments, respectively. Decreased female E2 and VTG levels and gonadal cytochrome P19 (aromatase) activity were also found in sediment exposed females from both species. No effect on female 3β/17β-HSD activity was found in either species. The FSTRA provided a robust model capable of modification to evaluate reproductive effects of sediment exposure in fish. PMID:25565366

  9. Targeting the EGFR signaling pathway in cancer therapy

    PubMed Central

    Seshacharyulu, Parthasarathy; Ponnusamy, Moorthy P.; Haridas, Dhanya; Jain, Maneesh; Ganti, AparK.; Batra, Surinder K.

    2012-01-01

    Introduction Cancer is a devastating disease; however, several therapeutic advances have recently been made, wherein EGFR and its family members have emerged as useful biomarkers and therapeutic targets. EGFR, a transmembrane glycoprotein is a member of the ERBB receptor tyrosine kinase superfamily. EGFR binds to its cognate ligand EGF, which further induces tyrosine phosphorylation and receptor dimerization with other family members leading to enhanced uncontrolled proliferation. Several anti-EGFR therapies such as monoclonal antibodies and tyrosine kinase inhibitors have been developed, which has enabled clinicians to identify and treat specific patient cohorts. Areas covered In this review, the basic mechanism of EGFR activation and the role of EGFR signaling in cancer progression, has been covered. Furthermore, current developments made towards targeting the EGFR signaling pathway for the treatment of epithelial cancers and a summary of the various anti-EGFR therapeutic agents that are currently in use, has also been made. Expert opinion EGFR signaling is a part of a complex network that has been the target of effective cancer therapies. However, further understanding of the system is required to develop an effective anticancer regiment. A combination therapy comprising of an anti-EGFR and a chemotherapeutic/chemopreventive agent will exhibit a multi-pronged approach that can be developed into a highly attractive and specific molecular oriented remedy. PMID:22239438

  10. NSCLC harboring EGFR exon-20 insertions after the regulatory C-helix of kinase domain responds poorly to known EGFR inhibitors.

    PubMed

    Yang, Mengmeng; Xu, Xiaoxi; Cai, Jie; Ning, Jinying; Wery, Jean Pierre; Li, Qi-Xiang

    2016-07-01

    Anecdote clinical observations hint that non-small cell lung cancer (NSCLC) with exon-20 insertions might respond poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), contrasting to those with classic mutations. Lack of patient-derived experimental models has been a major hurdle for the discovery of new treatment for the diseases. We established two NSCLC-PDXs harboring two different exon-20 insertions, LU0387-adenocarcinoma (ADC) with a nine-base insertion at 2319 (H773-V774insNPH) and LU3075-squamous cell carcinoma (SCC) with a nine-base insertion at 2316 (P772-H773insDNP). Both insertions immediately follow the regulatory C-helix of the kinase domain. Contrary to the generally good responses to EGFR inhibitors observed in PDXs with classic mutations, both exon-20 insertions are largely resistant to cetuximab and TKIs in vivo, suggesting fundamental difference from the classic EGFR mutations, consistent with the poor response rate to TKI seen in anecdotal clinic reports. It is worth noting that although responses are generally poor, they differ between the two exon-20 mutants depending on the type of TKI. In vitro drug sensitivity assays using established primary cell lines from our two PDXs largely confirmed the in vivo data. Our data from patient-derived experimental models confirmed that exon-20 insertions in domain immediately following the C-helix confer poor response to all known EGFR inhibitors, and suggested that these models can be utilized to facilitate the discovery of new therapies targeting NSCLC harboring exon-20 insertions. PMID:26891175

  11. Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish

    PubMed Central

    2013-01-01

    Background The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters. Results The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus. Conclusions Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile. PMID:24040749

  12. Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease.

    PubMed

    Zhou, Shun; Gao, Zhi-Xin; Zhang, Min; Liu, Dan-Yang; Zhao, Xin-Peng; Liu, Yong

    2016-01-01

    A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four Vibrio species, including Vibrio ichthyoenteri, Vibrio parahaemolyticus, Vibrio scophthalmi, and Vibrio vulnificus, simultaneously. Four sets of species-specific primers were designed with different restriction sites contained in the inner primers. The quadruplex LAMP method could distinguish four Vibrio species via the subsequent restriction enzyme analysis. The sensitivity of the quadruplex LAMP method were 10(2)-10(3) times higher than the sensitivity of conventional PCR. V. scophthalmi, V. vulnificus, V. parahaemolyticus and V. ichthyoenteri could be detected in the different tissues of the infected fish by the quadruplex LAMP method simply and conveniently through using SYBR Green I to facilitate visual inspection of the LAMP products. The method we developed in this study could be a simple and convenient diagnostic tool for field detection of Vibrio infection in fish. PMID:27468405

  13. Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining (1)H NMR metabolomics and conventional biochemical assays.

    PubMed

    Cappello, Tiziana; Brandão, Fátima; Guilherme, Sofia; Santos, Maria Ana; Maisano, Maria; Mauceri, Angela; Canário, João; Pacheco, Mário; Pereira, Patrícia

    2016-04-01

    Oxidative stress has been described as a key pathway to initiate mercury (Hg) toxicity in fish. However, the mechanisms underlying Hg-induced oxidative stress in fish still need to be clarified. To this aim, environmental metabolomics in combination with a battery of conventional oxidative stress biomarkers were applied to the gills of golden grey mullet (Liza aurata) collected from Largo do Laranjo (LAR), a confined Hg contaminated area, and São Jacinto (SJ), selected as reference site (Aveiro Lagoon, Portugal). Higher accumulation of inorganic Hg and methylmercury was found in gills of fish from LAR relative to SJ. Nuclear magnetic resonance (NMR)-based metabolomics revealed changes in metabolites related to antioxidant protection, namely depletion of reduced glutathione (GSH) and its constituent amino acids, glutamate and glycine. The interference of Hg with the antioxidant protection of gills was corroborated through oxidative stress endpoints, namely the depletion of glutathione peroxidase and superoxide dismutase activities at LAR. The increase of total glutathione content (reduced glutathione+oxidized glutathione) at LAR, in parallel with GSH depletion aforementioned, indicates the occurrence of massive GSH oxidation under Hg stress, and an inability to carry out its regeneration (glutathione reductase activity was unaltered) or de novo synthesis. Nevertheless, the results suggest the occurrence of alternative mechanisms for preventing lipid peroxidative damage, which may be associated with the enhancement of membrane stabilization/repair processes resulting from depletion in the precursors of phosphatidylcholine (phosphocholine and glycerophosphocholine), as highlighted by NMR spectroscopy. However, the observed decrease in taurine may be attributable to alterations in the structure of cell membranes or interference in osmoregulatory processes. Overall, the novel concurrent use of metabolomics and conventional oxidative stress endpoints demonstrated to be

  14. siRNA delivered by EGFR-specific scFv sensitizes EGFR-TKI-resistant human lung cancer cells.

    PubMed

    Lu, Yuan; Liu, Li; Wang, Yuan; Li, Fakai; Zhang, Jian; Ye, Mingxiang; Zhao, Hu; Zhang, Xiang; Zhang, Mi; Zhao, Jing; Yan, Bo; Yang, Angang; Feng, Huasong; Zhang, Rui; Ren, Xinling

    2016-01-01

    The overexpression of epidermal growth factor receptor (EGFR) is closely associated with a poor outcome in non-small cell lung cancer (NSCLC), and EGFR is an ideal biomarker for the targeted therapy of NSCLC. Although patients with EGFR-activating mutations respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs), they eventually acquire resistance, which typically results from a secondary EGFR mutation or the activation of other signaling pathways. Novel approaches to overcome or prevent EGFR-TKI resistance are clinically important. In this study, we developed an EGFR-scFv-arginine nonamer peptide fusion protein, s-9R, as an siRNA carrier. Here, we show that s-9R effectively and specifically delivers EGFR-siRNAs, KRAS-siRNA and MET-siRNA into NSCLC cells and silences the expression of target genes. The sensitivity of NSCLC cells to gefitinib was restored after treatment with the s-9R/siRNA complex, and the apoptosis rates of the treated cells were significantly higher than those of the control groups. Furthermore, the co-administration of s-9R/siRNA and gefitinib successfully suppressed the progression of H1975 xenograft tumors and extended the life span of tumor-bearing nude mice. Collectively, the results of this study provide not only a new scFv derivative for delivering siRNA into EGFR-overexpressing, TKI-resistant NSCLC cells but also a novel method for overcoming TKI resistance. PMID:26524539

  15. TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells.

    PubMed

    Otani, Hiroki; Yamamoto, Hiromasa; Takaoka, Munenori; Sakaguchi, Masakiyo; Soh, Junichi; Jida, Masaru; Ueno, Tsuyoshi; Kubo, Takafumi; Asano, Hiroaki; Tsukuda, Kazunori; Kiura, Katsuyuki; Hatakeyama, Shinji; Kawahara, Eiji; Naomoto, Yoshio; Miyoshi, Shinichiro; Toyooka, Shinichi

    2015-01-01

    TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation. PMID:26090892

  16. TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

    PubMed Central

    Otani, Hiroki; Yamamoto, Hiromasa; Takaoka, Munenori; Sakaguchi, Masakiyo; Soh, Junichi; Jida, Masaru; Ueno, Tsuyoshi; Kubo, Takafumi; Asano, Hiroaki; Tsukuda, Kazunori; Kiura, Katsuyuki; Hatakeyama, Shinji; Kawahara, Eiji; Naomoto, Yoshio; Miyoshi, Shinichiro; Toyooka, Shinichi

    2015-01-01

    TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation. PMID:26090892

  17. Utility of Gene Expression and Ex vivo Steroid Production in a 96 h Assay for Predicting Impacts of Endocrine Active Chemicals on Fish Reproduction.

    EPA Science Inventory

    Development of efficient test methods that can generate reliable data to inform risk assessment is an on-going challenge in the field of ecotoxicology. In the present study we evaluated whether a 96 h in vivo assay focused on a small number of quantitative real-time polymerase ch...

  18. Stress-Induced EGFR Trafficking: Mechanisms, Functions, and Therapeutic Implications.

    PubMed

    Tan, Xiaojun; Lambert, Paul F; Rapraeger, Alan C; Anderson, Richard A

    2016-05-01

    Epidermal growth factor receptor (EGFR) has fundamental roles in normal physiology and cancer, making it a rational target for cancer therapy. Surprisingly, however, inhibitors that target canonical, ligand-stimulated EGFR signaling have proven to be largely ineffective in treating many EGFR-dependent cancers. Recent evidence indicates that both intrinsic and therapy-induced cellular stress triggers robust, noncanonical pathways of ligand-independent EGFR trafficking and signaling, which provides cancer cells with a survival advantage and resistance to therapeutics. Here, we review the mechanistic regulation of noncanonical EGFR trafficking and signaling, and the pathological and therapeutic stresses that activate it. We also discuss the implications of this pathway in clinical treatment of EGFR-overexpressing cancers. PMID:26827089

  19. Inhibiting EGFR Dimerization Using Triazolyl-Bridged Dimerization Arm Mimics

    PubMed Central

    Hanold, Laura E.; Oruganty, Krishnadev; Ton, Norman T.; Beedle, Aaron M.; Kannan, Natarajan; Kennedy, Eileen J.

    2015-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed in multiple carcinomas and is the focus of a variety of targeted therapies. Here we report the design of peptide-based compounds that mimic the EGFR dimerization arm and inhibit allosteric activation of EGFR. These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop. In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge. We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability. This is the first example of a biologically active triazolyl-bridged peptide targeting the EGFR dimerization interface that effectively downregulates EGFR activation. PMID:25790232

  20. Mechanisms of resistance to EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Lihua; Fu, Liwu

    2015-01-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies. PMID:26579470

  1. EGFR-Targeted Hybrid Plasmonic Magnetic Nanoparticles Synergistically Induce Autophagy and Apoptosis in Non-Small Cell Lung Cancer Cells

    PubMed Central

    Kuroda, Shinji; Scott, Ailing W.; Aaron, Jesse; Larson, Tim; Shanker, Manish; Correa, Arlene M.; Kondo, Seiji; Roth, Jack A.; Sokolov, Konstantin; Ramesh, Rajagopal

    2011-01-01

    Background The epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival. In recent years, EGFR-targeted inhibitors have been tested in the clinic for NSCLC. Despite the emergence of novel therapeutics and their application in cancer therapy, the overall survival rate of lung cancer patients remains 15%. To develop more effective therapies for lung cancer we have combined the anti-EGFR antibody (Clone 225) as a molecular therapeutic with hybrid plasmonic magnetic nanoparticles (NP) and tested on non-small cell lung cancer (NSCLC) cells. Methodology/Principal Findings Cell viability was determined by trypan-blue assay. Cellular protein expression was determined by Western blotting. C225-NPs were detected by electron microscopy and confocal microscopy, and EGFR expression using immunocytochemistry. C225-NP exhibited a strong and selective antitumor effect on EGFR-expressing NSCLC cells by inhibiting EGFR-mediated signal transduction and induced autophagy and apoptosis in tumor cells. Optical images showed specificity of interactions between C225-NP and EGFR-expressing NSCLC cells. No binding of C225-NP was observed for EGFR-null NSCLC cells. C225-NP exhibited higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody in tumor cells with different levels of EGFR expression. Furthermore, in contrast to C225-NP, free C225 antibody did not induce autophagy in cells. However, the therapeutic efficacy of C225-NP gradually approached the level of free antibodies as the amount of C225 antibody conjugated per nanoparticle was decreased. Finally, attaching C225 to NP was important for producing the enhanced tumor cell killing as addition of mixture of free C225 and NP did not demonstrate the same degree of cell killing activity. Conclusions/Significance We demonstrated for the first time the molecular mechanism of C225-NP induced cytotoxic effects in

  2. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    SciTech Connect

    Ebi, Masahide; Kataoka, Hiromi; Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  3. EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT

    PubMed Central

    Choudhary, Kumari Sonal; Rohatgi, Neha; Briem, Eirikur; Gudjonsson, Thorarinn; Gudmundsson, Steinn; Rolfsson, Ottar

    2016-01-01

    Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend. PMID:27253373

  4. EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT.

    PubMed

    Choudhary, Kumari Sonal; Rohatgi, Neha; Halldorsson, Skarphedinn; Briem, Eirikur; Gudjonsson, Thorarinn; Gudmundsson, Steinn; Rolfsson, Ottar

    2016-06-01

    Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend. PMID:27253373

  5. Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria, after incorporation into biopolymer edible films.

    PubMed

    Iturriaga, L; Olabarrieta, I; de Marañón, I Martínez

    2012-08-01

    The antimicrobial activity of twelve natural extracts was tested against two fish spoilage bacteria (Pseudomonas fluorescens and Aeromonas hydrophila/caviae) and Listeria innocua, in order to assess their potential utilization in the preservation and safety of minimally processed fish products. After a screening of the active extracts by agar diffusion and vapour diffusion methods, oregano and thyme essential oils and citrus extract were selected. The minimum inhibitory concentration (MIC) of the selected extracts was determined by disc diffusion method against target bacteria and at two temperatures: bacteria's optimal growth temperature (30 °C or 37 °C) and refrigeration temperature (4 °C). Due to its better solubility, lack of odour and greater inhibitory effect obtained against L. innocua at refrigerated temperature, citrus extract was selected and incorporated at 1% (v/v) into different biopolymer film forming solutions (gelatin, methyl cellulose and their blend 50:50 w/w). The antimicrobial activity of the developed films was then evaluated, just after preparation of the films and after one month of storage at 43±3% relative humidity and 24±3 °C. Regardless of the biopolymer matrix, all the developed films showed antimicrobial activity against the target bacteria. The most sensitive bacterium towards active films was L. innocua while P. fluorescens appeared as the most resistant one, in accordance with the previously performed antimicrobial tests for pure extracts. The differences in activity of the films between the tested two temperatures were not significant except for L. innocua, for which three times higher inhibition diameters were observed at refrigerated temperature. The inhibitory effectiveness of the films against the tested strains was maintained regardless of the biopolymer matrix for at least one month. Therefore, these edible films show potential for their future use in fresh fish fillets preservation. PMID:22824340

  6. EGFR Mutation Testing Practices within the Asia Pacific Region

    PubMed Central

    Kerr, Keith M.; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma. Luisa D.; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony

    2015-01-01

    Introduction: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non–small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. Methods: A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Results: Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%–32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. Conclusions: In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia. PMID:25376513

  7. Design, synthesis and biological activities of novel oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as EGFR inhibitors.

    PubMed

    Yin, Siyuan; Zhou, Liliang; Lin, Jinsheng; Xue, Lingjing; Zhang, Can

    2015-08-28

    A series of oxazolo[4,5-g]quinazolin-2(1H)-one derivatives employing Erlotinib as lead compound were synthesized and evaluated for their EGFR inhibition activity. These compounds having variation at the 1 and 8-position, included ether and esters hydrophilic side-chain and aromatic head fragment, respectively. All these compounds were evaluated by EGFR inhibition and two anti-proliferation assays in vitro. Four compounds were found more potent than Erlotinib in EGFR-TK assay. Furthermore, compounds 18, 42 and 50 also had good to excellent anti-proliferation activity against human epidermoid cancer cell line (KB) and renal cell carcinoma cell line (A498). Finally, compound 50 presented remarkably higher inhibition efficacy towards tumor growth than Erlotinib in a mouse lewis lung cancer (LLC) xenograft model. Furthermore, compound 50 displayed the most distinguished effect on extending the survival period of the tumor-bearing mice. PMID:26188620

  8. A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.

    PubMed

    Moores, Sheri L; Chiu, Mark L; Bushey, Barbara S; Chevalier, Kristen; Luistro, Leopoldo; Dorn, Keri; Brezski, Randall J; Haytko, Peter; Kelly, Thomas; Wu, Sheng-Jiun; Martin, Pauline L; Neijssen, Joost; Parren, Paul W H I; Schuurman, Janine; Attar, Ricardo M; Laquerre, Sylvie; Lorenzi, Matthew V; Anderson, G Mark

    2016-07-01

    Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR. PMID:27216193

  9. Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer

    PubMed Central

    Regales, Lucia; Gong, Yixuan; Shen, Ronglai; de Stanchina, Elisa; Vivanco, Igor; Goel, Aviva; Koutcher, Jason A.; Spassova, Maria; Ouerfelli, Ouathek; Mellinghoff, Ingo K.; Zakowski, Maureen F.; Politi, Katerina A.; Pao, William

    2009-01-01

    EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers. PMID:19759520

  10. Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

    PubMed Central

    2011-01-01

    Background Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment. The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness. Results In this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that the metabolism of gefitinib in the sensitive cells is a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation. Conclusion Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, might represent an early assessment of gefitinib responsiveness in NSCLC

  11. ERβ localization influenced outcomes of EGFR-TKI treatment in NSCLC patients with EGFR mutations

    PubMed Central

    Wang, Zhijie; Li, Zhenxiang; Ding, Xiaosheng; Shen, Zhirong; Liu, Zhentao; An, Tongtong; Duan, Jianchun; Zhong, Jia; Wu, Meina; Zhao, Jun; Zhuo, Minglei; Wang, Yuyan; Wang, Shuhang; Sun, Yu; Bai, Hua; Wang, Jie

    2015-01-01

    Effects of estrogen receptorβ (ERβ) localization on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC) are unknown. First, we analyzed the relationship between ERβ localization determined by immunohistochemistry and EGFR-TKI outcomes in 184 patients with advanced NSCLC and found that ERβ expression localized in the cytoplasm and/or nucleus. The frequency of cytoplasmic ERβ (c-ERβ) and nuclear ERβ (n-ERβ) co-expression was 12% (22/184). C-ERβ and n-ERβ co-expression was correlated with poor median progression-free survival compared to patients without co-expression. In subsequent in vitro experiments, PC9 cells transfected with ERβ isoform1 (ERβ1, strong expression of both c-ERβ and n-ERβ) were more resistant to gefitinib than PC9 cells transfected with ERβ isoform2 or 5 (ERβ2 or ERβ5, strong expression of ERβ in cytoplasm but not nucleus). Resistance was identified due to interactions between ERβ1 and other isoforms, and mediated by activation of non-genomic pathways. Moreover, gefitinib resistance was reversed by a combination treatment with gefitinib and fulvestrant, both in cell lines and in one NSCLC patient. These results suggested that c-ERβ and n-ERβ co-expression was a potential molecular indicator of EGFR-TKI resistance, which might be overcome by combining EGFR-TKI and ER antagonist. PMID:26096604

  12. ERβ localization influenced outcomes of EGFR-TKI treatment in NSCLC patients with EGFR mutations.

    PubMed

    Wang, Zhijie; Li, Zhenxiang; Ding, Xiaosheng; Shen, Zhirong; Liu, Zhentao; An, Tongtong; Duan, Jianchun; Zhong, Jia; Wu, Meina; Zhao, Jun; Zhuo, Minglei; Wang, Yuyan; Wang, Shuhang; Sun, Yu; Bai, Hua; Wang, Jie

    2015-01-01

    Effects of estrogen receptorβ (ERβ) localization on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC) are unknown. First, we analyzed the relationship between ERβ localization determined by immunohistochemistry and EGFR-TKI outcomes in 184 patients with advanced NSCLC and found that ERβ expression localized in the cytoplasm and/or nucleus. The frequency of cytoplasmic ERβ (c-ERβ) and nuclear ERβ (n-ERβ) co-expression was 12% (22/184). C-ERβ and n-ERβ co-expression was correlated with poor median progression-free survival compared to patients without co-expression. In subsequent in vitro experiments, PC9 cells transfected with ERβ isoform1 (ERβ1, strong expression of both c-ERβ and n-ERβ) were more resistant to gefitinib than PC9 cells transfected with ERβ isoform2 or 5 (ERβ2 or ERβ5, strong expression of ERβ in cytoplasm but not nucleus). Resistance was identified due to interactions between ERβ1 and other isoforms, and mediated by activation of non-genomic pathways. Moreover, gefitinib resistance was reversed by a combination treatment with gefitinib and fulvestrant, both in cell lines and in one NSCLC patient. These results suggested that c-ERβ and n-ERβ co-expression was a potential molecular indicator of EGFR-TKI resistance, which might be overcome by combining EGFR-TKI and ER antagonist. PMID:26096604

  13. Facile and efficient synthesis and biological evaluation of 4-anilinoquinazoline derivatives as EGFR inhibitors.

    PubMed

    Wang, Zheng; Wu, Xue; Wang, Li; Zhang, Jiao; Liu, Jianli; Song, Zhongxing; Tang, Zhishu

    2016-06-01

    Series of 4-anilinoquinazoline derivatives were conveniently and efficiently synthesized and their antitumor activities were evaluated by MTT assay in three human cancer cell lines: H1975, HepG2 and SMMC-7721. New compounds 19a-19h were designed and synthesized to seek for powerful EGFR inhibitors and to explore whether methyl group at C-2 position of quinazoline ring has a positive effect on EGFR inhibition. All the compounds of 19a-19h were found potent against all three cell lines and five compounds (19c, 19d, and 19f-19h) were found more potent against H1975 than gefitinib. SAR studies revealed that methyl group at C-2 position of quinazoline ring could significantly improve the antitumor potency of 4-anilinoquinazolines. The same conclusion was also drawn according to the results of Western blotting analysis. Among all the tested compounds, 19g exhibited extremely potent against H1975 with an IC50 value of 0.11μM, remarkably lower than that of gefitinib (1.23μM). The results of western blotting analysis showed that compounds 19c and 19g could notably inhibit the expression of phosphorylated EGFR, especially 19g, almost inhibited completely. PMID:27118497

  14. Five-year survival in EGFR-mutant metastatic lung adenocarcinoma treated with EGFR-TKIs

    PubMed Central

    Lin, Jessica J.; Cardarella, Stephanie; Lydon, Christine A.; Dahlberg, Suzanne E.; Jackman, David M.; Jänne, Pasi A.; Johnson, Bruce E.

    2016-01-01

    Introduction Activating mutations in the epidermal growth factor receptor (EGFR) predict for prolonged progression-free survival in patients with advanced non-small cell lung cancer (NSCLC) treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) versus chemotherapy. Long-term survival outcomes, however, remain undefined. The objective of this study was to determine the 5-year survival in these patients, and identify clinical factors associated with overall survival (OS). Methods Patients with EGFR-mutant metastatic lung adenocarcinoma treated with erlotinib or gefitinib at Dana-Farber Cancer Institute between 2002 and 2009 were included. OS was analyzed. Results Among 137 patients, median PFS and OS were 12.1 months (95% CI, 10.2-13.5 months) and 30.9 months (95% CI, 28.2-35.7 months), respectively. Twenty patients (14.6%) were 5-year survivors. In multivariate analysis, exon 19 deletions (hazard ratio [HR], 0.63; 95% CI, 0.44-0.91; P = 0.01), absence of extrathoracic (HR 0.62; 95% CI, 0.41-0.93; P = 0.02) or brain metastasis (HR 0.48; 95% CI, 0.30-0.77, P = 0.002), and not a current smoker (HR 0.23; 95% CI, 0.09-0.59; P = 0.002) were associated with prolonged OS. Age, gender, stage at diagnosis, liver or bone or adrenal metastasis, specific TKI, and line of TKI therapy were not associated with OS. Conclusions Our data suggest that the prevalence of 5-year survival among EGFR-mutant metastatic lung adenocarcinoma patients treated with erlotinib or gefitinib is 14.6%. Exon 19 deletions and absence of extrathoracic or brain metastasis are associated with prolonged survival. Based on our findings, clinicians can gain an enhanced estimation of long-term outcomes in this population. PMID:26724471

  15. MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

    PubMed Central

    Kubo, Takafumi; Yamamoto, Hiromasa; Lockwood, William W.; Valencia, Ilse; Soh, Junichi; Peyton, Michael; Jida, Masaru; Otani, Hiroki; Fujii, Tetsuya; Ouchida, Mamoru; Takigawa, Nagio; Kiura, Katsuyuki; Shimizu, Kenji; Date, Hiroshi; Minna, John D.; Varella-Garcia, Marileila; Lam, Wan L.; Gazdar, Adi F.; Toyooka, Shinichi

    2009-01-01

    We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. PMID:19117057

  16. Detecting and treating breast cancer resistance to EGFR inhibitors

    DOEpatents

    Moonlee, Sun-Young; Bissell, Mina J.; Furuta, Saori; Meier, Roland; Kenny, Paraic A.

    2016-04-05

    The application describes therapeutic compositions and methods for treating cancer. For example, therapeutic compositions and methods related to inhibition of FAM83A (family with sequence similarity 83) are provided. The application also describes methods for diagnosing cancer resistance to EGFR inhibitors. For example, a method of diagnosing cancer resistance to EGFR inhibitors by detecting increased FAM83A levels is described.

  17. Aspirin Prevents Colorectal Cancer by Normalizing EGFR Expression

    PubMed Central

    Li, Haitao; Zhu, Feng; Boardman, Lisa A.; Wang, Lei; Oi, Naomi; Liu, Kangdong; Li, Xiang; Fu, Yang; Limburg, Paul J.; Bode, Ann M.; Dong, Zigang

    2015-01-01

    Background Aspirin intake reduces the risk of colorectal cancer (CRC), but the molecular underpinnings remain elusive. Epidermal growth factor receptor (EGFR), which is overexpressed in about 80% of CRC cases, is implicated in the etiology of CRC. Here, we investigated whether aspirin can prevent CRC by normalizing EGFR expression. Methods Immunohistochemistry staining was performed on paraffin-embedded tissue sections from normal colon mucosa, adenomatous polyps from FAP patients who were classified as regular aspirin users or nonusers. The interplay between cyclooxygenase-2 (COX-2) and EGFR was studied in primary intestinal epithelial cells isolated from ApcMin mice, immortalized normal human colon epithelial cells (HCECs) as well as murine embryonic fibroblasts (MEFs). Results Immunohistochemistry staining results established that EGFR overexpression is an early event in colorectal tumorigenesis, which can be greatly attenuated by regular use of aspirin. Importantly, EGFR and COX-2 were co-overexpressed and co-localized with each other in FAP patients. Further mechanistic studies revealed that COX-2 overexpression triggers the activation of the c-Jun-dependent transcription factor, activator protein-1 (AP-1), which binds to the Egfr promoter. Binding facilitates the cellular accumulation of EGFR and lowers the threshold required for pre-neoplastic cells to undergo transformation. Conclusion Aspirin might exert its chemopreventive activity against CRC, at least partially, by normalizing EGFR expression in gastrointestinal precancerous lesions. PMID:26097892

  18. DNA methylation down-regulates EGFR expression in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The epidermal growth factor receptor (EGFR), a growth-factor-receptor tyrosine kinase, was found up-regulated in numerous tumors, which provides a good target for cancer therapy. Although it was documented that oncoviruses are responsible for the activation of EGFR in tumors, the impact of Marek’s d...

  19. EGFRs mediate chemotactic migration in the developing telencephalon.

    PubMed

    Caric, D; Raphael, H; Viti, J; Feathers, A; Wancio, D; Lillien, L

    2001-11-01

    Epidermal growth factor receptors (EGFRs) have been implicated in the control of migration in the telencephalon, but the mechanism underlying their contribution is unclear. We show that expression of a threshold level of EGFRs confers chemotactic competence in stem cells, neurons and astrocytes in cortical explants. This level of receptor expression is normally achieved by a subpopulation of cells during mid-embryonic development. Cells that express high levels of EGFR are located in migration pathways, including the tangential pathway to the olfactory bulb via the rostral migratory stream (RMS), the lateral cortical stream (LCS) leading to ventrolateral cortex and the radial pathway from proliferative zones to cortical plate. The targets of these pathways express the ligands HB-EGF and/or TGFalpha. To test the idea that EGFRs mediate chemotactic migration these pathways, we increased the size of the population of cells expressing threshold levels of EGFRs in vivo by viral transduction. Our results suggest that EGFRs mediate migration radially to the cortical plate and ventrolaterally in the LCS, but not tangentially in the RMS. Within the bulb, however, EGFRs also mediate radial migration. Our findings suggest that developmental changes in EGFR expression, together with changes in ligand expression regulate the migration of specific populations of cells in the telencephalon by a chemoattractive mechanism. PMID:11684657

  20. A Novel Technique to Detect EGFR Mutations in Lung Cancer

    PubMed Central

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-01-01

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples. PMID:27223277

  1. A Novel Technique to Detect EGFR Mutations in Lung Cancer.

    PubMed

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-01-01

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples. PMID:27223277

  2. Dual-targeting organometallic ruthenium(II) anticancer complexes bearing EGFR-inhibiting 4-anilinoquinazoline ligands.

    PubMed

    Zhang, Yang; Zheng, Wei; Luo, Qun; Zhao, Yao; Zhang, Erlong; Liu, Suyan; Wang, Fuyi

    2015-08-01

    We have recently demonstrated that complexation with (η(6)-arene)Ru(II) fragments confers 4-anilinoquinazoline pharmacophores a higher potential for inducing cellular apoptosis while preserving the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine (Chem. Commun., 2013, 49, 10224-10226). Reported herein are the synthesis, characterisation and evaluation of the biological activity of a new series of ruthenium(ii) complexes of the type [(η(6)-arene)Ru(N,N-L)Cl]PF6 (arene = p-cymene, benzene, 2-phenylethanol or indane, L = 4-anilinoquinazolines). These organometallic ruthenium complexes undergo fast hydrolysis in aqueous solution. Intriguingly, the ligation of (arene)Ru(II) fragments with 4-anilinoquinazolines not only makes the target complexes excellent EGFR inhibitors, but also confers the complexes high affinity to bind to DNA minor grooves while maintaining their reactivity towards DNA bases, characterising them with dual-targeting properties. Molecular modelling studies reveal that the hydrolysis of these complexes is a favourable process which increases the affinity of the target complexes to bind to EGFR and DNA. In vitro biological activity assays show that most of this group of ruthenium complexes are selectively active inhibiting the EGF-stimulated growth of the HeLa cervical cancer cell line, and the most active complex [(η(6)-arene)Ru(N,N-L13)Cl]PF6 (, IC50 = 1.36 μM, = 4-(3'-chloro-4'-fluoroanilino)-6-(2-(2-aminoethyl)aminoethoxy)-7-methoxyquinazoline) is 29-fold more active than its analogue, [(η(6)-arene)Ru(N,N-ethylenediamine)Cl]PF6, and 21-fold more active than gefitinib, a well-known EGFR inhibitor in use clinically. These results highlight the strong promise to develop highly active ruthenium anticancer complexes by ligation of cytotoxic ruthenium pharmacophores with bioactive organic molecules. PMID:26106875

  3. Everolimus prolonged survival in transgenic mice with EGFR-driven lung tumors.

    PubMed

    Yasugi, Masayuki; Takigawa, Nagio; Ochi, Nobuaki; Ohashi, Kadoaki; Harada, Daijiro; Ninomiya, Takashi; Murakami, Toshi; Honda, Yoshihiro; Ichihara, Eiki; Tanimoto, Mitsune; Kiura, Katsuyuki

    2014-08-15

    Everolimus is an orally administered mTOR inhibitor. The effect, and mechanism of action, of everolimus on lung cancers with an epidermal growth factor receptor (EGFR) mutation remain unclear. Four gefitinib-sensitive and -resistant cell lines were used in the present work. Growth inhibition was determined using the MTT assay. Transgenic mice carrying the EGFR L858R mutation were treated with everolimus (10 mg/kg/day), or vehicle alone, from 5 to 20 weeks of age, and were then sacrificed. To evaluate the efficacy of everolimus in prolonging survival, everolimus (10 mg/kg/day) or vehicle was administered from 5 weeks of age. The four cell lines were similarly sensitive to everolimus. Expression of phosphorylated (p) mTOR and pS6 were suppressed upon treatment with everolimus in vitro, whereas the pAKT level increased. The numbers of lung tumors with a long axis exceeding 1mm in the everolimus-treated and control groups were 1.9 ± 0.9 and 9.4 ± 3.2 (t-test, p<0.001), respectively. pS6 was suppressed during eve r olimus treatment. Although apoptosis and autophagy were not induced in everolimus-treated EGFR transgenic mice, angiogenesis was suppressed. The median survival time in the everolimus-treated group (58.0 weeks) was significantly longer than that in the control group (31.2 weeks) (logrank test, p<0.001). These findings suggest that everolimus had an indirect effect on tumor formation by inhibiting angiogenesis and might be effective to treat lung tumors induced by an activating EGFR gene mutation. PMID:24768699

  4. Bisphosphonates inactivate human EGFRs to exert antitumor actions.

    PubMed

    Yuen, Tony; Stachnik, Agnes; Iqbal, Jameel; Sgobba, Miriam; Gupta, Yogesh; Lu, Ping; Colaianni, Graziana; Ji, Yaoting; Zhu, Ling-Ling; Kim, Se-Min; Li, Jianhua; Liu, Peng; Izadmehr, Sudeh; Sangodkar, Jaya; Bailey, Jack; Latif, Yathin; Mujtaba, Shiraz; Epstein, Solomon; Davies, Terry F; Bian, Zhuan; Zallone, Alberta; Aggarwal, Aneel K; Haider, Shozeb; New, Maria I; Sun, Li; Narla, Goutham; Zaidi, Mone

    2014-12-16

    Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers. PMID:25453081

  5. A semi-automated FISH-based micronucleus-centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation

    PubMed Central

    VRAL, ANNE; DECORTE, VEERLE; DEPUYDT, JULIE; WAMBERSIE, ANDRÉ; THIERENS, HUBERT

    2016-01-01

    The aim of the present study was to perform cytogenetic analysis by means of a semi-automated micro-nucleus-centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere-negative micronucleus (MNCM−) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low-dose' (0–100 mGy) MNCM− dose-response curve to evaluate the sensitivity and suitability of the micronucleus-centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi-automated scoring of MNCM− enables the detection of a dose of 50 mGy. The comparison of MNCM− yields in medical radiation workers and control individuals showed enhanced MNCM− scores in the medical radiation workers group (P=0.15). The highest MNCM− scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM− scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the

  6. EGFR and colon cancer: a clinical view.

    PubMed

    de Castro-Carpeño, J; Belda-Iniesta, C; Casado Sáenz, E; Hernández Agudo, E; Feliu Batlle, J; González Barón, M

    2008-01-01

    Signalling pathways that emerge from EGFR activation are critical in colon cancer (CC) biology. Its targeting with specific drugs has opened a new window in the treatment of this disease. In this regard, monoclonal antibodies (mAb) have evidenced a high degree of efficiency opposed to the uselessness of tyrosine-kinase inhibitors. Cetuximab is the mAb that has evidenced most activity in CC. After its initial approval as an irinotecan-resistance reversal agent, cetuximab has demonstrated its efficiency from the first line to heavily pretreated patients. In the first line, its addition may increase response rate to chemotherapy, improving liver metastases resection rate. Another promising approach has been suggested from combination schedules with bevacizumab. Panitumumab has been recently approved for CC. Although there is limited clinical experience, the latest data have confirmed its activity in heavily pretreated patients resulting in a clinical benefit vs. best support care. In spite of the clinical benefits, adverse events and the high sanitary cost derived from these drugs force the selection of patients with the highest probability of benefit. At the moment, when EGFR expression evidenced by immunohistochemistry has no value, skin toxicity and, fundamentally, K-Ras mutations may hint at critical information for confirmatory prospective studies. PMID:18208787

  7. Characterization of a complex chromosomal rearrangement using chromosome, FISH, and microarray assays in a girl with multiple congenital abnormalities and developmental delay

    PubMed Central

    2014-01-01

    Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomal pairs. The phenotypic anomalies in such cases are attributed to gene disruption, superimposed cryptic imbalances in the genome, and/or position effects. We report a 14-year-old girl who presented with multiple congenital anomalies and developmental delay. Chromosome and FISH analysis indicated a highly complex chromosomal rearrangement involving three chromosomes (3, 7 and 12), seven breakpoints as a result of one inversion, two insertions, and two translocations forming three derivative chromosomes. Additionally, chromosomal microarray study (CMA) revealed two submicroscopic deletions at 3p12.3 (467 kb) and 12q13.12 (442 kb). We postulate that microdeletion within the ROBO1 gene at 3p12.3 may have played a role in the patient’s developmental delay, since it has potential activity-dependent role in neurons. Additionally, factors other than genomic deletions such as loss of function or position effects may also contribute to the abnormal phenotype in our patient. PMID:25478007

  8. Prognostic implications of immunohistochemistry markers for EGFR-TKI therapy in Chinese patients with advanced lung adenocarcinoma harboring EGFR mutations

    PubMed Central

    Zhang, Ruiguang; Li, Yan; Nie, Xiu; Dong, Xiaorong; Wu, Gang

    2016-01-01

    Background Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer predict dramatic clinical responses to tyrosine kinase inhibitor (TKI) treatment. The conclusion on EGFR mutation-specific antibodies by immunohistochemistry (IHC) is not consistent. We evaluated the clinical availability of EGFR mutation-specific antibodies, investigating the prediction role of mutant EGFRs and other IHC markers in TKI therapy in patients with advanced lung adenocarcinoma. Materials and methods We analyzed 637 primary lung adenocarcinomas from an unselected Chinese population. For IHC, antibodies against EGFR exon 19 E746_A750 deletions, exon 21 L858R mutations, thyroid transcription factor-1 (TTF-1), and Napsin-A were applied. Positivity was defined as staining score >0. Results Specificities of E746_A750 and L858R antibodies were 99.6% and 99.3%, while sensitivities were 86.0% and 82.7%, respectively. Tumors with Napsin-A positivity, TTF-1 positivity, EGFR mutations, and lepidic pattern showed a lower marker of proliferation index (Ki67). Higher expression scores of mutant EGFR protein, TTF-1 positivity, lower Ki67 proliferation index, and lepidic pattern were associated with longer progression-free survival. Conclusion High scores of mutant EGFR, Napsin-A positivity, TTF-1 positivity, lower Ki67 index, and lepidic pattern were favorable predictors for TKI therapy in patients with advanced lung adenocarcinoma. PMID:26848271

  9. A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): Old successes and future perspectives

    PubMed Central

    Russo, Alessandro; Franchina, Tindara; Rita Ricciardi, Giuseppina Rosaria; Picone, Antonio; Ferraro, Giuseppa; Zanghì, Mariangela; Toscano, Giuseppe; Giordano, Antonio; Adamo, Vincenzo

    2015-01-01

    The discovery of Epidermal Growth Factor Receptor (EGFR) mutations in Non Small Cell Lung Cancer (NSCLC) launched the era of personalized medicine in advanced NSCLC, leading to a dramatic shift in the therapeutic landscape of this disease. After ten years from the individuation of activating mutations in the tyrosine kinase domain of the EGFR in NSCLC patients responding to the EGFR tyrosine kinase inhibitor (TKI) Gefitinib, several progresses have been done and first line treatment with EGFR TKIs is a firmly established option in advanced EGFR-mutated NSCLC patients. During the last decade, different EGFR TKIs have been developed and three inhibitors have been approved so far in these selected patients. However, despite great breakthroughs have been made, treatment of these molecularly selected patients poses novel therapeutic challenges, such as emerging of acquired resistance, brain metastases development or the need to translate these treatments in earlier clinical settings, such as adjuvant therapy. The aim of this paper is to provide a comprehensive review of the major progresses reported so far in the EGFR inhibition in this molecularly-selected subgroup of NSCLC patients, from the early successes with first generation EGFR TKIs, Erlotinib and Gefitinib, to the novel irreversible and mutant-selective inhibitors and ultimately the emerging challenges that we, in the next future, are called to deal with. PMID:26308162

  10. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

    PubMed Central

    Cross, Darren A. E.; Ashton, Susan E.; Ghiorghiu, Serban; Eberlein, Cath; Nebhan, Caroline A.; Spitzler, Paula J.; Orme, Jonathon P.; Finlay, M. Raymond V.; Ward, Richard A.; Mellor, Martine J.; Hughes, Gareth; Rahi, Amar; Jacobs, Vivien N.; Brewer, Monica Red; Ichihara, Eiki; Sun, Jing; Jin, Hailing; Ballard, Peter; Al-Kadhimi, Katherine; Rowlinson, Rachel; Klinowska, Teresa; Richmond, Graham H. P.; Cantarini, Mireille; Kim, Dong-Wan; Ranson, Malcolm R.; Pao, William

    2014-01-01

    First generation EGF receptor tyrosine kinase inhibitors (EGFR TKIs) provide significant clinical benefit in patients with advanced EGFR mutant (EGFRm+) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent and selective third generation irreversible inhibitor of both EGFRm+ sensitizing and T790M resistance mutants that spares wild-type EGFR. This monoanilino-pyrimidine compound is structurally distinct from other third generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Pre-clinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm+ and EGFRm+/T790M mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR mutant tumor xenograft and transgenic models. The treatment of two patients with advanced EGFRm T790M+ NSCLC is described as proof of principle. PMID:24893891

  11. A Novel Mechanism of PPAR Gamma Induction via EGFR Signalling Constitutes Rational for Combination Therapy in Bladder Cancer

    PubMed Central

    Mansure, Jose Joao; Nassim, Roland; Chevalier, Simone; Szymanski, Konrad; Rocha, Joice; Aldousari, Saad; Kassouf, Wassim

    2013-01-01

    Background Two signalling molecules that are attractive for targeted therapy are the epidermal growth factor receptor (EGFR) and the peroxisome proliferator-activated receptor gamma (PPARγ). We investigated possible crosstalk between these 2 pathways, particularly in light of the recent evidence implicating PPARγ for anticancer therapy. Principal Findings As evaluated by MTT assays, gefitinib (EGFR inhibitor) and DIM-C (PPARγ agonist) inhibited growth of 9 bladder cancer cell lines in a dose-dependent manner but with variable sensitivity. In addition, combination of gefitinib and DIM-C demonstrated maximal inhibition of cell proliferation compared to each drug alone. These findings were confirmed in vivo, where combination therapy maximally inhibited tumor growth in contrast to each treatment alone when compared to control (p<0.04). Induction of PPARγ expression along with nuclear accumulation was observed in response to increasing concentrations of gefitinib via activation of the transcription factor CCAT/enhancer-binding protein-β (CEBP-β). In these cell lines, DIM-C significantly sensitized bladder cancer cell lines that were resistant to EGFR inhibition in a schedule-specific manner. Conclusion These results suggest that PPARγ agonist DIM-C can be an excellent alternative to bladder tumors resistant to EGFR inhibition and combination efficacy might be achieved in a schedule-specific manner. PMID:23409107

  12. Combined targeting of EGFR/HER promotes anti-tumor efficacy in subsets of KRAS mutant lung cancer resistant to single EGFR blockade

    PubMed Central

    Umelo, Ijeoma Adaku; De Wever, Olivier; Kronenberger, Peter; Van Deun, Jan; Noor, Alfiah; Singh, Kshitiz; Teugels, Erik; Chen, Gang; Bracke, Marc; De Grève, Jacques

    2015-01-01

    KRAS is a frequently mutated oncogene in lung cancer and among the most refractory to EGFR targeted therapy. Recently, preclinical evidence in pancreatic cancer has demonstrated that mutant KRAS can be regulated by EGFR. However, the distinct correlation between the EGFR/HER family members and mutant KRAS has not been investigated. Here, we show that non-small cell lung cancer cell lines harboring differing isoforms of mutant KRAS, can be broadly divided into EGFR/HER dependent and EGFR/HER independent groups. Combined therapeutic targeting of EGFR, HER2 and HER3 in isoforms regulated by extracellular growth signals promotes in vitro and in vivo efficacy. We also provide evidence that depletion of EGFR via RNA interference specifically abolishes the EGFR/KRAS interaction in the dependent subset. Taken together, these findings suggest that upstream inhibition of the EGFR/HER receptors may be effective in treating a subset of KRAS mutant lung cancers. PMID:25992771

  13. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    PubMed

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types. PMID:22623992

  14. Fish Allergy

    MedlinePlus

    ... Story" 5 Things to Know About Zika & Pregnancy Fish Allergy KidsHealth > For Parents > Fish Allergy Print A ... From Home en español Alergia al pescado About Fish Allergy A fish allergy is not exactly the ...

  15. EGFR-TKI resistance in NSCLC patients: mechanisms and strategies

    PubMed Central

    Lin, Yuxin; Wang, Xian; Jin, Hongchuan

    2014-01-01

    The epidermal growth factor receptor (EGFR) is a kind of receptor tyrosine kinase (RTK) that plays a critical role in the initiation and development of malignant tumors via modulating downstream signaling pathways. In non-small cell lung cancer (NSCLC), the activating mutations located in the tyrosine kinase domains of EGFR have been demonstrated in multiple researches as the “Achilles’ heel” of this deadly disease since they could be well-targeted by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). However, it’s still too early to celebrate since the first-generation EGFR-TKIs such as gefitinib and erlotinib have only achieved limited clinical benefits and acquired resistance to this kind of drugs occurred inevitably in almost all the NSCLC patients. In order to make the most of EGFR-TKIs and develop more effective regimens for the NSCLC patients, researchers majoring in different aspects start a battle against EGFR-TKI resistance. Challenging as it is, we still progress stably and step firmly toward the final victory. This review will summarize the major mechanisms of acquired resistance to EGFR-TKIs, and then discuss the development of rationally designed molecular target drugs in accordance with each mechanism, in the hope of shedding light on the great achievements we have obtained and tough obstacles we have to overcome in the battle against this deadly disease. PMID:25232485

  16. Genetic modifiers of EGFR dependence in non-small cell lung cancer

    PubMed Central

    Sharifnia, Tanaz; Rusu, Victor; Piccioni, Federica; Bagul, Mukta; Imielinski, Marcin; Cherniack, Andrew D.; Pedamallu, Chandra Sekhar; Wong, Bang; Wilson, Frederick H.; Garraway, Levi A.; Altshuler, David; Golub, Todd R.; Root, David E.; Subramanian, Aravind; Meyerson, Matthew

    2014-01-01

    Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival. PMID:25512530

  17. Alpha-Particle Emitting 213Bi-Anti-EGFR Immunoconjugates Eradicate Tumor Cells Independent of Oxygenation

    PubMed Central

    Gaertner, Florian C.; Bruchertseifer, Frank; Morgenstern, Alfred; Essler, Markus; Senekowitsch-Schmidtke, Reingard

    2013-01-01

    Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that cell-bound α-particle emitting 213Bi immunoconjugates kill hypoxic and normoxic CAL33 tumor cells with identical efficiency. For that purpose CAL33 cells were incubated with 213Bi-anti-EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing 213Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml–1.48 MBq/ml) and irradiation with increasing doses of photons (0.5–12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by 213Bi-anti-EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate that α-particle emitting 213Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, 213Bi-radioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors. PMID:23724085

  18. EGFR mutation and lobar location of lung adenocarcinoma.

    PubMed

    Tseng, Chien-Hua; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Ho, Chao-Chi; Hsia, Te-Chun; Su, Kang-Yi; Wu, Ming-Fang; Chiu, Kuo-Liang; Liu, Chien-Ming; Wu, Tzu-Chin; Chen, Hung-Jen; Chen, Hsuan-Yu; Chang, Chi-Sheng; Hsu, Chung-Ping; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Lin, Chin-Hung; Chen, Jeremy J W; Chen, Kuan-Yu; Liao, Wei-Yu; Shih, Jin-Yuan; Yu, Sung-Liang; Yu, Chong-Jen; Yang, Pan-Chyr; Yang, Tsung-Ying; Chang, Gee-Chen

    2016-02-01

    The objective of this study was to investigate the associations among lung cancer location, and epidermal growth factor receptor (EGFR) mutation status. Treatment-naive, pathologically confirmed lung adenocarcinomas with tumor specimens available for genetic analysis were included from 2011 through 2014. Overall, 1771 patients with lung adenocarcinoma were included for analysis, after excluding those with carcinoma not otherwise specified, or synchronous multiple primary lung cancers. The median age was 64 years, and the female:male and never smoker:ever smoker ratios were 930:855 (52:48%) and 1167:604 (65:35%), respectively. The EGFR mutation rate was 56%. Among patients, 1093 (62%) had primary tumors in the upper lobes. Compared with the characteristics of the EGFR wild-type, tumors with EGFR activating mutations were more common in women (P < 0.001), never smokers (P < 0.001), and in the upper lobes (P = 0.004). Among EGFR activating mutations, compared with the EGFR exon 19 deletion, L858R mutation were more common in women (P = 0.002), never smokers (P = 0.038), and the upper lobes P < 0.0005). The present study is the first to address that different pulmonary lobar locations might harbor different EGFR mutation subtypes. We demonstrated that adenocarcinomas with L858R mutation, rather than exon 19 deletion or wild-type EGFR gene, prefer to locate over the upper lungs. This phenomenon was more significant in females and never-smokers, implying the result of complex interactions between genetic susceptibility and environmental factors. Therefore, EGFR L858R mutation and exon 19 deletion may not be identical disease entity from the point of carcinogenesis. PMID:26645716

  19. Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

    PubMed Central

    Li, Chunrong; Iida, Mari; Dunn, Emily F.; Ghia, Amol J.; Wheeler, Deric L.

    2010-01-01

    Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR blocking-antibody that has been approved for the treatment of patients with cancers of the head and neck (HNSCC) and metastatic colorectal cancer (mCRC). Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin (AR), heparin-binding EGF (HB-EGF) and β-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and re-sensitization to cetuximab. In addition, expression of a nuclear localization sequence tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab. PMID:19684613

  20. Nuclear EGFR contributes to acquired resistance to cetuximab.

    PubMed

    Li, C; Iida, M; Dunn, E F; Ghia, A J; Wheeler, D L

    2009-10-29

    Epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase involved in the etiology of several human cancers. Cetuximab is an EGFR-blocking antibody that has been approved for the treatment of patients with head and neck squamous cell carcinoma and metastatic colorectal cancer. Previous reports have shown that EGFR translocation to the nucleus is associated with cell proliferation. Here we investigated mechanisms of acquired resistance to cetuximab using a model derived from the non-small cell lung cancer line H226. We demonstrated that cetuximab-resistant cells overexpress HER family ligands including epidermal growth factor (EGF), amphiregulin, heparin-binding EGF and beta-cellulin. Overexpression of these ligands is associated with the nuclear translocation of the EGFR and this process was mediated by the Src family kinases (SFK). Treatment of cetuximab-resistant cells with the SFK inhibitor, dasatinib, resulted in loss of nuclear EGFR, increased membrane expression of the EGFR and resensitization to cetuximab. In addition, expression of a nuclear localization sequence-tagged EGFR in cetuximab-sensitive cells increased resistance to cetuximab both in vitro and in mouse xenografts. Collectively, these data suggest that nuclear expression of EGFR may be an important molecular determinant of resistance to cetuximab therapy and provides a rationale for investigating nuclear EGFR as a biomarker for cetuximab response. Further, these data suggest a rationale for the design of clinical trials that examine the value of treating patients with cetuximab-resistant tumors with inhibitors of SFKs in combination with cetuximab. PMID:19684613

  1. One Fish Two Fish.

    ERIC Educational Resources Information Center

    Hoffman, Michele

    1998-01-01

    This activity explains fisheries resource management to seven-year olds. First-grade students learn concepts such as offspring viability, life expectancy, and distribution of species, which help to determine when, where, and how people fish and the importance of fishing responsibly. Lists materials, procedures, and extensions. (SJR)

  2. miR-217 and CAGE form feedback loop and regulates the response to anti-cancer drugs through EGFR and HER2

    PubMed Central

    Han, Minho; Lee, Hansoo; Lee, Yun Sil; Choe, Jongseon; Kim, Young Myeong; Jeoung, Dooil

    2016-01-01

    MicroRNA array analysis revealed that miR-217 expression was decreased in anti-cancer drug-resistant Malme3MR cancer cells. CAGE, a cancer/testis antigen, was predicted as a target of miR-217. Luciferase activity and ChIP assays revealed a negative feedback relationship between CAGE and miR-217. miR-217 and CAGE oppositely regulated the response to anti-cancer drugs such as taxol, gefitinib and trastuzumab, an inhibitor of HER2. miR-217 negatively regulated the tumorigenic, metastatic, angiogenic, migration and invasion potential of cancer cells. The xenograft of Malme3MR cells showed an increased expression of pEGFRY845. CAGE and miR-217 inhibitor regulated the expression of pEGFRY845. CAGE showed interactions with EGFR and HER2 and regulated the in vivo sensitivity to trastuzumab. The down-regulation of EGFR or HER2 enhanced the sensitivity to anti-cancer drugs. CAGE showed direct regulation of HER2 and was necessary for the interaction between EGFR and HER2 in Malme3MR cells. miR-217 inhibitor induced interactions of CAGE with EGFR and HER2 in Malme3M cells. The inhibition of EGFR by CAGE-binding GTGKT peptide enhanced the sensitivity to gefitinib and trastuzumab and prevented interactions of EGFR with CAGE and HER2. Our results show that miR-217-CAGE feedback loop serves as a target for overcoming resistance to various anti-cancer drugs, including EGFR and HER2 inhibitors. PMID:26863629

  3. STAT1-Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients

    PubMed Central

    Srivastava, Raghvendra M.; Trivedi, Sumita; Concha-Benavente, Fernando; Hyun-bae, Jie; Wang, Lin; Seethala, Raja R.; Branstetter, Barton F.; Ferrone, Soldano; Ferris, Robert L.

    2015-01-01

    The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was utilized to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1−/− cells. Cetuximab enhanced HNC cell recognition by EGFR853–861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271–279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. PMID:25972070

  4. Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR*

    PubMed Central

    Jung, Sung Keun; Lee, Mee-Hyun; Lim, Do Young; Kim, Jong Eun; Singh, Puja; Lee, Sung-Young; Jeong, Chul-Ho; Lim, Tae-Gyu; Chen, Hanyong; Chi, Young-In; Kundu, Joydeb Kumar; Lee, Nam Hyouck; Lee, Charles C.; Cho, Yong-Yeon; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

    2014-01-01

    Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR. PMID:25368326

  5. In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

    PubMed Central

    Yasuda, Hiroyuki; Hamamoto, Junko; Oashi, Ayano; Ishioka, Kota; Arai, Daisuke; Nukaga, Shigenari; Miyawaki, Masayoshi; Kawada, Ichiro; Naoki, Katsuhiko; Costa, Daniel B.; Kobayashi, Susumu S.; Betsuyaku, Tomoko; Soejima, Kenzo

    2015-01-01

    EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations. PMID:26515464

  6. Anti-EGFR Antibody Reduces Lung Nodules by Inhibition of EGFR-Pathway in a Model of Lymphangioleiomyomatosis

    PubMed Central

    Ancona, Silvia; Orpianesi, Emanuela; Di Giulio, Anna Maria; Gorio, Alfredo

    2015-01-01

    EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cells has been linked with increased proliferation, angiogenesis, and metastasis. Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that occurs sporadically or in association with tuberous sclerosis complex (TSC), a genetic, multisystem disorder characterized by hamartomas in several organs. From chylous of a LAM/TSC patient, we previously isolated smooth muscle-like LAM/TSC cells whose proliferation depends on EGF and monoclonal anti-EGFR antibodies reduced proliferation and caused cell death. We demonstrated that the dependency from EGF was caused by the absence of tuberin. To study the role of EGFR pathway in vivo, we developed a mouse model by administration of LAM/TSC cells to female nude mice. LAM/TSC cells caused pulmonary airspace enlargement and, after 30 weeks, nodule formation which express EGFR. Anti-EGFR antibody decreased the number and dimension of lung nodules likely for the inhibition of Erk and S6 signaling, reversed the pulmonary alterations, and reduced lymphatic and blood vessels. Moreover, in pulmonary nodules anti-EGFR antibody reduced the positivity to estrogen and progesterone receptors which enhance survival of LAM cells and Snail expression. These results suggest that the inhibition of EGFR signalling has a potential in treatment of LAM/TSC lung alterations. PMID:25699271

  7. IKBKE Is a Substrate of EGFR and a Therapeutic Target in Non-Small Cell Lung Cancer with Activating Mutations of EGFR.

    PubMed

    Challa, Sridevi; Guo, Jian-Ping; Ding, Xiaowen; Xu, Cheng-Xiong; Li, Yajuan; Kim, Donghwa; Smith, Matthew A; Cress, Douglas W; Coppola, Domenico; Haura, Eric B; Cheng, Jin Q

    2016-08-01

    Non-small cell lung cancers (NSCLC) marked by EGFR mutations tend to develop resistance to therapeutic EGFR inhibitors, often due to secondary mutation EGFR(T790M) but also other mechanisms. Here we report support for a rationale to target IKBKE, an IκB kinase family member that activates the AKT and NF-κB pathways, as one strategy to address NSCLC resistant to EGFR inhibitors. While wild-type and mutant EGFR directly interacted with IKBKE, only mutant EGFR phosphorylated IKBKE on residues Y153 and Y179. The unphosphorylatable mutant IKBKE-Y153F/Y179-F that lost kinase activity failed to activate AKT and inhibited EGFR signaling. In clinical specimens of NSCLC with activating mutations of EGFR, we observed elevated levels of phospho-Y153 IKBKE. IKBKE ablation with shRNA or small-molecule inhibitor amlexanox selectively inhibited the viability of NSCLC cells with EGFR mutations in vitro In parallel, we found that these treatments activated the MAPK pathway due to attenuation of an IKBKE feedback mechanism. In vivo studies revealed that combining amlexanox with MEK inhibitor AZD6244 significantly inhibited the xenograft tumor growth of NSCLC cells harboring activating EGFR mutations, including EGFR(T790M) Overall, our findings define IKBKE as a direct effector target of EGFR and provide a therapeutic rationale to target IKBKE as a strategy to eradicate EGFR-TKI-resistant NSCLC cells. Cancer Res; 76(15); 4418-29. ©2016 AACR. PMID:27287717

  8. Synthesis of new 4-anilinoquinazoline analogues and evaluation of their EGFR inhibitor activity.

    PubMed

    Wang, Zheng; WANG, Cui-Ling; Li, Jun-lin; Zhang, Ning; Sun, Yan-ni; Liu, Zhu-lan; Tang, Zhi-shu; Liu, Jian-li

    2015-12-01

    Thirteen of 4-anilinoquinazoline derivatives with imine groups at position 6 of quinazoline ring were synthesized and their antitumor activities were evaluated by MTT assay and Western blotting analysis. Among these compounds, 13a-131 were reported first time. The MTT assay was carried out on three human cancer cell lines (A549, HepG2 and SMMC7721) with EGFR highly expressed. Among the tested compounds, 13i and 13j exhibited notable inhibition potency and their IC50 values on three cell lines were equivalent to or less than those of gefitinib. Compound 14, without imine group substituted, displayed excellent inhibitor potency only on A549 cell line. Compounds 14 and 13j were chosen to perform Western blotting analysis on A549. The results showed that both of the compounds could inhibit the expression level of phosphorylated EGFR remarkably. It was concluded that the inhibitor potency of compound 14 was almost equivalent to that of gefitinib and the inhibitor potency of 13j was better than that of gefitinib. PMID:27169285

  9. Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.

    PubMed

    Singer, Josef; Fazekas, Judit; Wang, Wei; Weichselbaumer, Marlene; Matz, Miroslawa; Mader, Alexander; Steinfellner, Willibald; Meitz, Sarah; Mechtcheriakova, Diana; Sobanov, Yuri; Willmann, Michael; Stockner, Thomas; Spillner, Edzard; Kunert, Renate; Jensen-Jarolim, Erika

    2014-07-01

    Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. PMID:24755200

  10. Assessment of DDR2, BRAF, EGFR and KRAS mutations as therapeutic targets in non-adenocarcinoma lung cancer patients

    PubMed Central

    YASHIMA, HIDEAKI; SHIMIZU, KIMIHIRO; ARAKI, TAKUYA; AOMORI, TOHRU; OHTAKI, YOICHI; NAGASHIMA, TOSHITERU; ENOKIDA, YASUAKI; ATSUMI, JUN; NAKAMURA, TOMONORI; TAKEYOSHI, IZUMI; YAMAMOTO, KOUJIROU

    2014-01-01

    Molecular-targeted therapy has not been established in non-adenocarcinoma lung cancer (non-AdLC), as no targets that affect the clinical efficacy of molecular-targeted drugs have yet been identified. In this study, we investigated the frequency of genetic variations in discoidin domain receptor 2 (DDR2), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-AdLC patients, in order to evaluate the possibility of genetic mutations in these genes being used as therapeutic targets for the treatment of patients with non-AdLC. For this purpose, we enrolled 150 non-AdLC patients who had undergone surgery at the Gunma University Hospital between December, 2003 and December, 2012. Genetic mutations in the EGFR, KRAS, DDR2 and BRAF genes were detected by a sequencing method or probe assay using DNA derived from cancer tissues. No somatic mutations in DDR2 or BRAF were detected in non-AdLC patients. Conversely, genetic mutations in EGFR exon 19 were found in 3 squamous cell carcinoma (SCC) and 3 adenosquamous carcinoma patients, whereas KRAS codon 12 mutations were also found in 3 SCC patients and 1 large-cell neuroendocrine carcinoma patient. EGFR and KRAS mutations were mutually exclusive. This study indicated that, although DDR2 and BRAF mutations may only rarely be used as therapeutic targets, EGFR and KRAS mutations may represent candidate therapeutic targets, at least in the non-AdLC patients investigated. PMID:25054035

  11. Integrative Genomics Implicates EGFR as a Downstream Mediator in NKX2-1 Amplified Non-Small Cell Lung Cancer

    PubMed Central

    Clarke, Nicole; Biscocho, Jewison; Kwei, Kevin A.; Davidson, Jean M.; Sridhar, Sushmita; Gong, Xue; Pollack, Jonathan R.

    2015-01-01

    NKX2-1, encoding a homeobox transcription factor, is amplified in approximately 15% of non-small cell lung cancers (NSCLC), where it is thought to drive cancer cell proliferation and survival. However, its mechanism of action remains largely unknown. To identify relevant downstream transcriptional targets, here we carried out a combined NKX2-1 transcriptome (NKX2-1 knockdown followed by RNAseq) and cistrome (NKX2-1 binding sites by ChIPseq) analysis in four NKX2-1-amplified human NSCLC cell lines. While NKX2-1 regulated genes differed among the four cell lines assayed, cell proliferation emerged as a common theme. Moreover, in 3 of the 4 cell lines, epidermal growth factor receptor (EGFR) was among the top NKX2-1 upregulated targets, which we confirmed at the protein level by western blot. Interestingly, EGFR knockdown led to upregulation of NKX2-1, suggesting a negative feedback loop. Consistent with this finding, combined knockdown of NKX2-1 and EGFR in NCI-H1819 lung cancer cells reduced cell proliferation (as well as MAP-kinase and PI3-kinase signaling) more than knockdown of either alone. Likewise, NKX2-1 knockdown enhanced the growth-inhibitory effect of the EGFR-inhibitor erlotinib. Taken together, our findings implicate EGFR as a downstream effector of NKX2-1 in NKX2-1 amplified NSCLC, with possible clinical implications, and provide a rich dataset for investigating additional mediators of NKX2-1 driven oncogenesis. PMID:26556242

  12. Generation of a Canine Anti-EGFR (ErbB-1) Antibody for Passive Immunotherapy in Dog Cancer Patients

    PubMed Central

    Wang, Wei; Weichselbaumer, Marlene; Matz, Miroslawa; Mader, Alexander; Steinfellner, Willibald; Meitz, Sarah; Mechtcheriakova, Diana; Sobanov, Yuri; Willmann, Michael; Stockner, Thomas; Spillner, Edzard; Kunert, Renate; Jensen-Jarolim, Erika

    2014-01-01

    Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a “caninized” version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. PMID:24755200

  13. Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy.

    PubMed

    Jing, Hui; Cheng, Wen; Li, Shouqiang; Wu, Bolin; Leng, Xiaoping; Xu, Shouping; Tian, Jiawei

    2016-10-01

    The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy. PMID:27388967

  14. 2-Triazenoazaindoles: A novel class of triazenes inducing transcriptional down-regulation of EGFR and HER-2 in human pancreatic cancer cells

    PubMed Central

    KREUTZER, JAN N.; SALVADOR, ALESSIA; DIANA, PATRIZIA; CIRRINCIONE, GIROLAMO; VEDALDI, DANIELA; LITCHFIELD, DAVID W.; ISSINGER, OLAF-GEORG; GUERRA, BARBARA

    2012-01-01

    Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases. PMID:22134789

  15. Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

    PubMed Central

    Ma, Jian Jun; Xu, Peng; Zhang, Wei; Li, Yu Mei; Fu, Qiang; Zhu, Guang Feng; Xue, Wei; Lei, Yong Hua; Gao, Jing Yu; Wang, Juan Ying; Shao, Chen; Yi, Cheng Gang; Wang, He

    2013-01-01

    Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment. PMID:23950876

  16. Differential Efficacy of Combined Therapy With Radiation and AEE788 in High and Low EGFR-Expressing Androgen-Independent Prostate Tumor Models

    SciTech Connect

    Huamani, Jessica; Willey, Christopher; Thotala, Dinesh; Niermann, Kenneth J.; Reyzer, Michelle; Leavitt, Lauren; Jones, Cameron; Fleishcher, Arthur; Caprioli, Richard; Hallahan, Dennis E.; Kim, Dong Wook Nathan

    2008-05-01

    Purpose: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. Methods and Materials: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT {+-} AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flow and growth measurements were performed using immunohistochemistry and imaging. Results: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. Conclusions: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell

  17. Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6

    PubMed Central

    YI, YONG WEON; YOU, KYUSIC; BAE, EDWARD JEONG; KWAK, SAHNG-JUNE; SEONG, YEON-SUN; BAE, INSOO

    2015-01-01

    Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB-231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB-231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB-231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through down-regulation of RPS6. PMID:25955731

  18. Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6.

    PubMed

    Yi, Yong Weon; You, Kyusic; Bae, Edward Jeong; Kwak, Sahng-June; Seong, Yeon-Sun; Bae, Insoo

    2015-07-01

    Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB‑231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB‑231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB‑231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through downregulation of RPS6. PMID:25955731

  19. Design, synthesis and antitumor activity of novel pyrazolo[3,4-d]pyrimidine derivatives as EGFR-TK inhibitors.

    PubMed

    Abdelgawad, Mohamed A; Bakr, Rania B; Alkhoja, Olla A; Mohamed, Wafaa R

    2016-06-01

    A novel series of 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-substitutedbenzylidene)acetohydrazide (12a-g) was prepared and their structures were confirmed by spectral and elemental analyses. The cytotoxic activity of the newly synthesized compounds was evaluated against breast carcinoma (MCF-7), non-small cell lung cancer (A549) and human colorectal adenocarcinoma (HT-29) cell lines using MTT and colony formation assays. The tested compounds showed a marked anticancer activity against all the tested cell lines, especially compound 12g, which was the most potent anticancer agent with half maximal inhibitory concentrations (IC50) between 5.36 and 9.09μM. Docking studies into ATP binding site of EGFR protein tyrosine kinase were performed to predict their scores and mode of binding to amino acids, In addition, the inhibitory activity of the target compounds against epidermal growth factor receptor tyrosine kinase (EGFR-TK) was evaluated. Results indicated the ability of the target compounds to inhibit EGFR-TK with half maximal inhibitory concentrations (IC50) in the range of 4.18-35.88μM. Furthermore, The most active compounds 12g, 12c and 12d were assayed against Fibroblast Growth Factor Receptor (FGFR), Insulin Receptor (IR) and Vascular Endothelial Growth Factor Receptor (VEGFR). The activity of the reported compounds warrants further optimization as novel members in cancer treatment protocols. PMID:27043178

  20. EGFR and EGFRvIII interact with PUMA to inhibit mitochondrial translocalization of PUMA and PUMA-mediated apoptosis independent of EGFR kinase activity

    PubMed Central

    Zhu, Hu; Cao, Xinyu; Ali-Osman, Francis; Keir, Stephen; Lo, Hui-Wen

    2010-01-01

    EGFR and its constitutively activated variant EGFRvIII are linked to glioblastoma resistance to therapy, the mechanisms underlying this association, however, are still unclear. We report that in glioblastoma, EGFR/EGFRvIII paradoxically co-expresses with p53-upregulated modulator of apoptosis (PUMA), a proapoptotic member of the Bcl-2 family of proteins primarily located on the mitochondria. EGFR/EGFRvIII binds to PUMA constitutively and under apoptotic stress, and subsequently sequesters PUMA in the cytoplasm. The EGFR-PUMA interaction is independent of EGFR activation and is sustained under EGFR inhibition. A Bcl-2/Bcl-xL inhibitor that mimics PUMA activity sensitizes EGFR/EGFRvIII-expressing glioblastoma cells to Iressa. Collectively, we uncovered a novel kinase-independent function of EGFR/EGFRvIII that leads to tumor drug resistance. PMID:20153921

  1. Management of egfr tki–induced dermatologic adverse events

    PubMed Central

    Melosky, B.; Leighl, N.B.; Rothenstein, J.; Sangha, R.; Stewart, D.; Papp, K.

    2015-01-01

    Targeting the epidermal growth factor receptor (egfr) pathway has become standard practice for the treatment of advanced non-small-cell lung cancer. Compared with chemotherapy, egfr tyrosine kinase inhibitors (tkis) have been associated with improved efficacy in patients with an EGFR mutation. Together with the increase in efficacy comes an adverse event (ae) profile different from that of chemotherapy. That profile includes three of the most commonly occurring dermatologic aes: acneiform rash, stomatitis, and paronychia. Currently, no randomized clinical trials have evaluated the treatments for the dermatologic aes that patients experience when taking egfr tkis. Based on the expert opinion of the authors, some basic strategies have been developed to manage those key dermatologic aes. Those strategies have the potential to improve patient quality of life and compliance and to prevent inappropriate dose reductions. PMID:25908911

  2. Imaging EGFR distribution using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Lucas, L.; Chen, X. K.; Smith, A.; Korbelik, M.; Zeng, H.; Lee, P. W. K.; Hewitt, K. C.

    2009-02-01

    The purpose of this study is to explore the feasibility of using Surface Enhanced Raman Spectroscopy (SERS) to image the distribution of Epidermal Growth Factor Receptor (EGFR) in cells. To accomplish this task, 30 nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (1012 per ml) are incubated with cells (106 per ml) of the A431 human epidermoid carcinoma cell line and normal human bronchial epithelial (NHBE) cells. Using the 632.8 nm excitation line of a He-Ne laser, Raman spectroscopy measurements are performed using a point mapping scheme. SERS signals are observed with an overall enhancement of 4-7 orders of magnitude. Raman intensity maps of the 1480 and 1583 cm-1 peaks correlate well with the expected distribution of AuNPs and EGFR. Normal cells show little to no enhancement. The results therefore present a simple yet effective means to image EGFR over-expression.

  3. Regulatory mechanisms of EGFR signalling during Drosophila eye development.

    PubMed

    Malartre, Marianne

    2016-05-01

    EGFR signalling is a well-conserved signalling pathway playing major roles during development and cancers. This review explores what studying the EGFR pathway during Drosophila eye development has taught us in terms of the diversity of its regulatory mechanisms. This model system has allowed the identification of numerous positive and negative regulators acting at specific time and place, thus participating to the tight control of signalling. EGFR signalling regulation is achieved by a variety of mechanisms, including the control of ligand processing, the availability of the receptor itself and the transduction of the cascade in the cytoplasm. Ultimately, the transcriptional responses contribute to the establishment of positive and negative feedback loops. The combination of these multiple mechanisms employed to regulate the EGFR pathway leads to specific cellular outcomes involved in functions as diverse as the acquisition of cell fate, proliferation, survival, adherens junction remodelling and morphogenesis. PMID:26935860

  4. IgA EGFR antibodies mediate tumour killing in vivo

    PubMed Central

    Boross, Peter; Lohse, Stefan; Nederend, Maaike; Jansen, Johannes Hendrik Marco; van Tetering, Geert; Dechant, Michael; Peipp, Matthias; Royle, Louise; Liew, Li Phing; Boon, Louis; van Rooijen, Nico; Bleeker, Wim K; Parren, Paul W H I; van de Winkel, Jan G J; Valerius, Thomas; Leusen, Jeanette H W

    2013-01-01

    Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer. The study reveals that IgA antibodies directed against EGFR and engaging Fcalpha receptor (FcαRI) on effector cells, have in vivo anti-cancer activity. These data support the development of novel immunotherapeutic strategies based on targeting FcαRI. PMID:23918228

  5. EGFR signaling defines Mcl-1 survival dependency in neuroblastoma

    PubMed Central

    Nalluri, Srilatha; Peirce, Susan K; Tanos, Rachel; Abdella, Haneen A; Karmali, Dipan; Hogarty, Michael D; Goldsmith, Kelly C

    2015-01-01

    The pediatric solid tumor neuroblastoma (NB) often depends on the anti-apoptotic protein, Mcl-1, for survival through Mcl-1 sequestration of pro-apoptotic Bim. High affinity Mcl-1 inhibitors currently do not exist such that novel methods to inhibit Mcl-1 clinically are in high demand. Receptor tyrosine kinases (RTK) regulate Mcl-1 in many cancers and play a role in NB survival, yet how they regulate Bcl-2 family interactions in NB is unknown. We found that NB cell lines derived to resist the Bcl-2/-xl/-w antagonist, ABT-737, acquire a dependence on Mcl-1 and show increased expression and activation of the RTK, EGFR. Mcl-1 dependent NB cell lines derived at diagnosis and from the same tumor following relapse also have increased EGFR expression compared to those dependent on Bcl-2. Inhibition of EGFR by shRNA or erlotinib in Mcl-1 dependent NBs disrupts Bim binding to Mcl-1 and enhances its affinity for Bcl-2, restoring sensitivity to ABT-737 as well as cytotoxics in vitro. Mechanistically treatment of NBs with small molecule inhibitors of EGFR (erlotinib, cetuximab) and ERK (U0126) increases Noxa expression and dephosphorylates Bim to promote Bim binding to Bcl-2. Thus, EGFR regulates Mcl-1 dependence in high-risk NB via ERK-mediated phosphorylation of Bim such that EGFR/ERK inhibition renders Mcl-1 dependent tumors now reliant on Bcl-2. Clinically, EGFR inhibitors are ineffective as single agent compounds in patients with recurrent NB, likely due to this transferred survival dependence to Bcl-2. Likewise, EGFR or ERK inhibitors warrant further testing in combination with Bcl-2 antagonists in vivo as a novel future combination to overcome therapy resistance in the clinic. PMID:25756510

  6. EGFR and microvessel density in canine malignant mammary tumours.

    PubMed

    Carvalho, Maria Isabel; Guimarães, Maria João; Pires, Isabel; Prada, Justina; Silva-Carvalho, Ricardo; Lopes, Carlos; Queiroga, Felisbina L

    2013-12-01

    The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor which has been shown to have an important role in human breast cancer. Its role appears to be associated with increased angiogenesis and metastasis. In order to clarify its role in canine mammary tumours (CMT), 61 malignant neoplasms were studied by using immunohistochemistry, comparing expression of EGFR, microvessel density (MVD) by CD31 immunolabelling and characteristics of tumour aggressiveness. High EGFR immunoexpression was statistically significantly associated with tumour size, tumour necrosis, mitotic grade, histological grade of malignancy and clinical stage. High CD31 immunoreactivity was statistically significantly associated with tubule formation, histological grade of malignancy and clinical stage. A positive correlation between EGFR and CD31 immunoexpression (r = 0.843; P < 0.001) was also observed. Results suggest that an over-expression of EGFR may contribute to increased angiogenesis and aggression in malignant CMT, presenting the possibility of using EGFR inhibitors in the context of metastatic disease treatment. PMID:24091029

  7. Mechanisms underlying skin disorders induced by EGFR inhibitors

    PubMed Central

    Holcmann, Martin; Sibilia, Maria

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is frequently mutated or overexpressed in a large number of tumors such as carcinomas or glioblastoma. Inhibitors of EGFR activation have been successfully established for the therapy of some cancers and are more and more frequently being used as first or later line therapies. Although the side effects induced by inhibitors of EGFR are less severe than those observed with classic cytotoxic chemotherapy and can usually be handled by out-patient care, they may still be a cause for dose reduction or discontinuation of treatment that can reduce the effectiveness of antitumor therapy. The mechanisms underlying these cutaneous side effects are only partly understood. Important questions, such as the reasons for the correlation between the intensity of the side effects and the efficiency of treatment with EGFR inhibitors, remain to be answered. Optimized adjuvant strategies to accompany anti-EGFR therapy need to be found for optimal therapeutic application and improved quality of life of patients. Here, we summarize current literature on the molecular and cellular mechanisms underlying the cutaneous side effects induced by EGFR inhibitors and provide evidence that keratinocytes are probably the optimal targets for adjuvant therapy aimed at alleviating skin toxicities. PMID:27308503

  8. Inequalities in lung cancer: a world of EGFR.

    PubMed

    Carbonnaux, Mélodie; Souquet, Pierre-Jean; Meert, Anne-Pascale; Scherpereel, Arnaud; Peters, Matthew; Couraud, Sébastien

    2016-05-01

    Epidermal growth factor receptor gene (EGFR) mutation status has emerged as a crucial issue in lung cancer management. Availability and cost of tests and tyrosine kinase inhibitors (TKIs) may vary as a function of country development.We conducted a prospective specialist opinion survey to map EGFR test and EGFR-TKI availability and detect associations with the Human Development Index (HDI). A questionnaire was sent to specialists in thoracic oncology in all United Nations Member States.We obtained responses from 74 countries, comprising 78% of the worldwide population. Nonresponding countries had significantly lower HDI rank than responding countries. EGFR mutation analysis was routinely available in 57 countries (70% of the worldwide population). The cost of the test was EGFR mutation testing and EGFR-TKIs are widely accessible in routine practice worldwide. However, there are large discrepancies in access to this innovative treatment path and in its cost for patients as a function of country development. PMID:27030679

  9. Management of EGFR mutated nonsmall cell lung carcinoma patients.

    PubMed

    Grigoriu, Bogdan; Berghmans, Thierry; Meert, Anne-Pascale

    2015-04-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) are common in the therapeutic armentarium of lung cancer today. Initially tested in an unselected population, they have been of limited usefulness until the identification EGFR gene mutations. Activating mutations generate conformational changes that result in a shift toward an active state of the catalytic domain and are associated with sensitivity to first generation EGFR TKI. Other mutations have been associated with resistance to these drugs, but for rare mutations there is limited data concerning their role in predicting response to EGFR TKI. To date, four molecules have been approved for the treatment of EGFR mutated lung cancer. Gefitinib and/or erlotinib are available in almost all countries. Afatinib has been approved by the US Food and Drug Administration and by the European Medicines Agency, and icotinib has been approved only in China. Other, more active, third generation agents with a higher binding affinity for the receptor, or that are directed against specific mutations, are under development. EGFR TKIs have a favourable impact on progression-free survival when given as first line treatment in mutated patients, but may also have a moderate effect as a salvage therapy and in maintenance in an unselected population. PMID:25700389

  10. The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph; Ferguson, Kathryn M.

    2014-01-01

    The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface. PMID:24691965

  11. Hippo pathway effector YAP inhibition restores the sensitivity of EGFR-TKI in lung adenocarcinoma having primary or acquired EGFR-TKI resistance.

    PubMed

    Lee, Jeong Eun; Park, Hee Sun; Lee, Dahye; Yoo, Geon; Kim, Tackhoon; Jeon, Haeyon; Yeo, Min-Kyung; Lee, Choong-Sik; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Kim, Sun Young; Park, Dong Il; Park, Yeon Hee; Lee, Jae Cheol; Oh, In-Jae; Lim, Dae Sik; Chung, Chaeuk

    2016-05-20

    The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is significantly limited by various resistance mechanisms to those drugs. The resistance to EGFR-TKI is largely divided by two classes; acquired resistance after EGFR-TKI treatment, and primary resistance marked by cancer cell's dependence on other oncogene, such as KRAS. YAP has emerged as critical oncogene in conferring drug resistance against targeted therapy. In this study, we evaluated the role of YAP in primary and acquired EGFR-TKI resistance using gefitinib-resistant A549 and PC9 cells and their parental cell lines. Our study revealed that EGFR-TKI resistance is associated with enhanced YAP activity. Notably, YAP activation was independent of the Hippo pathway. We confirmed that AXL is a downstream target of YAP that confers EGFR-TKI resistance. And our results showed that YAP can induce ERK activation in lung adenocarcinoma. The combination of YAP inhibition with EGFR-TKI overcomes primary and acquired EGFR-TKI resistance. We also found increased YAP expression in human lung cancer after acquiring EGFR-TKI resistance. Collectively, we suggest a novel EGFR-TKI resistance mechanism involving YAP activation and suggest targeting YAP and EGFR simultaneously may be a breakthrough treatment of primary and acquired EGFR-TKI resistant lung cancer. PMID:27105908

  12. EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib

    PubMed Central

    Gallant, Jean-Nicolas; Sheehan, Jonathan H.; Shaver, Timothy M.; Bailey, Mark; Lipson, Doron; Chandramohan, Raghu; Brewer, Monica Red; York, Sally J.; Kris, Mark G.; Pietenpol, Jennifer A.; Ladanyi, Marc; Miller, Vincent A.; Ali, Siraj M.; Meiler, Jens; Lovly, Christine M.

    2015-01-01

    Oncogenic EGFR mutations are found in 10-35% of lung adenocarcinomas. Such mutations, which present most commonly as small in-frame deletions in exon 19 or point mutations in exon 21 (L858R), confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). In analyzing the tumor from a 33-year-old male never smoker, we identified a novel EGFR alteration in lung cancer: EGFR exon 18-25 kinase domain duplication (EGFR-KDD). Through analysis of a larger cohort of tumor samples, we detected additional cases of EGFR-KDD in lung, brain, and other cancers. In vitro, EGFR-KDD is constitutively active, and computational modeling provides potential mechanistic support for its auto-activation. EGFR-KDD-transformed cells are sensitive to EGFR TKIs and, consistent with these in vitro findings, the index patient had a partial response to the EGFR TKI, afatinib. The patient eventually progressed, at which time, re-sequencing revealed an EGFR-dependent mechanism of acquired resistance to afatinib, thereby validating EGFR-KDD as a driver alteration and therapeutic target. PMID:26286086

  13. Nanobiophotonics for molecular imaging of cancer: Au- and Ag-based Epidermal Growth Factor receptor (EGFR) specific nanoprobes

    NASA Astrophysics Data System (ADS)

    Lucas, Leanne J.; Hewitt, Kevin C.

    2012-03-01

    Our aim is to create and validate a novel SERS-based nanoprobe for optical imaging of the epidermal growth factor receptor (EGFR). Gold and silver nanoparticles (Au/AgNPs) of various sizes were synthesized and coupled to epidermal growth factor (EGF) via a short ligand, α-lipoic acid (206 g/mol), which binds strongly to both Au and Ag nanoparticles via its disulfide end group. We used carbodiimide chemistry to couple EGF to α-lipoic acid. These nanoprobes were tested for binding affinity using Enzyme Linked ImmunoSorbent Assay (ELISA) and, in-vitro, using EGFRoverexpressing A431 cells. The nanoprobes show excellent EGFR-specific binding. Time of Flight Mass Spectrometry demonstrate the carbodiimide based linking of the carboxylic acid end-group of α-lipoic acid to one or more of the three (terminal, or 2 lysine) amine groups on EGF. ELISA confirms that the linked EGF is active by itself, and following conjugation with gold or silver nanoparticles. Compared with bare nanoparticles, UV-Vis spectroscopy of Ag-based nanoprobes exhibit significant plasmon red-shift, while there was no discernable shift for Au-based ones. Dark field microscopy shows abundant uptake by EGFR overexpressing A431 cells, and serves to further confirm the excellent binding affinity. Nanoprobe internalization and consequent aggregation is thought to be the basis of enhanced light scattering in the dark field images, supporting the notion that these nanoprobes should provide excellent SERS signals at all nanoprobe sizes. In summary, novel EGFR-specific nanoprobes have been synthesized and validated by standard assay and in cell culture for use as SERS optical imaging probes.

  14. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  15. Silibinin suppresses EGFR ligand-induced CD44 expression through inhibition of EGFR activity in breast cancer cells.

    PubMed

    Kim, Sangmin; Han, Jeonghun; Kim, Jee Soo; Kim, Jung-Han; Choe, Jun-Ho; Yang, Jung-Hyun; Nam, Seok Jin; Lee, Jeong Eon

    2011-11-01

    CD44, the transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. The expression of CD44 and its variants is associated with poor prognosis in breast cancer. Here, we investigated the effect of silibinin (a polyphenolic flavonolignan of the herbal plant of Silybum marianum, milk thistle) on the epidermal growth factor (EGF) ligand-induced CD44 expression in human breast cancer cells. The levels of CD44 mRNA and protein expression were greatly increased by EGF and by TGF-α in SKBR3 and BT474 breast cancer cells. In contrast, EGFR ligand-induced CD44 expression was reduced by EGFR inhibitors, AG1478 and lapatinib, respectively. Interestingly, we observed that EGFR ligand-induced CD44 and matrix metalloproteinase-9 (MMP-9) expression was reduced by silibinin treatment in a dose-dependent manner. In addition, silibinin suppressed the EGF-induced phosphorylation of EGFR and extracellular signal-regulated kinase1/2 (ERK1/2), a downstream signaling molecule of EGFR. Therefore, we suggest that silibinin prevents the EGFR signaling pathway and may be used as an effective drug for the inhibition of metastasis of human breast cancer. PMID:22110198

  16. Fish Hearing.

    ERIC Educational Resources Information Center

    Blaxter, J. H. S.

    1980-01-01

    Provides related information about hearing in fish, including the sensory stimulus of sound in the underwater environment, mechanoreceptors in fish, pressure perception and the swimbladder, specializations in sound conduction peculiar to certain fish families. Includes numerous figures. (CS)

  17. City Fishing.

    ERIC Educational Resources Information Center

    Lange, Robert E.

    1979-01-01

    A program of supplying opportunities for fishing at locations within and near urban areas was developed. This effort included stocking, management of bodies of water for fishing, and presentation of fishing clinics for urban fishermen. (RE)

  18. Modulation of Morphogenesis by Egfr during Dorsal Closure in Drosophila

    PubMed Central

    Cormier, Olga; Cheng, David Chung-Pei; Reed, Bruce; Harden, Nicholas

    2013-01-01

    During Drosophila embryogenesis the process of dorsal closure (DC) results in continuity of the embryonic epidermis, and DC is well recognized as a model system for the analysis of epithelial morphogenesis as well as wound healing. During DC the flanking lateral epidermal sheets stretch, align, and fuse along the dorsal midline, thereby sealing a hole in the epidermis occupied by an extra-embryonic tissue known as the amnioserosa (AS). Successful DC requires the regulation of cell shape change via actomyosin contractility in both the epidermis and the AS, and this involves bidirectional communication between these two tissues. We previously demonstrated that transcriptional regulation of myosin from the zipper (zip) locus in both the epidermis and the AS involves the expression of Ack family tyrosine kinases in the AS in conjunction with Dpp secreted from the epidermis. A major function of Ack in other species, however, involves the negative regulation of Egfr. We have, therefore, asked what role Egfr might play in the regulation of DC. Our studies demonstrate that Egfr is required to negatively regulate epidermal expression of dpp during DC. Interestingly, we also find that Egfr signaling in the AS is required to repress zip expression in both the AS and the epidermis, and this may be generally restrictive to the progression of morphogenesis in these tissues. Consistent with this theme of restricting morphogenesis, it has previously been shown that programmed cell death of the AS is essential for proper DC, and we show that Egfr signaling also functions to inhibit or delay AS programmed cell death. Finally, we present evidence that Ack regulates zip expression by promoting the endocytosis of Egfr in the AS. We propose that the general role of Egfr signaling during DC is that of a braking mechanism on the overall progression of DC. PMID:23579691

  19. EGFR: The Paradigm of an Oncogene-Driven Lung Cancer

    PubMed Central

    Riely, Gregory J.; Yu, Helena A.

    2015-01-01

    Somatic, activating mutations in Epidermal Growth Factor Receptor (EGFR) identify a significant minority of patients with non-small cell lung cancer (NSCLC). While these mutations are associated with an ~70% response rate to some EGFR tyrosine kinase inhibitors (gefitinib, erlotinib, and afatinib), patients develop resistance (i.e. “acquired resistance”) after a median of 9–12 months. In patients with clinical acquired resistance, repeat biopsy of tumors has identified a number of relevant mechanisms of resistance, but by far the most frequent event is the acquisition of EGFR T790M, a mutation in the “gatekeeper” residue that confers resistance to gefitinib, erlotinib, and afatinib. This emphasizes the critical dependence upon EGFR signaling for some tumors, a property that has been exploited therapeutically. Dual EGFR blockade using afatinib and cetuximab led to a 29% radiographic response rate. More recently, drugs which target EGFR T790M (e.g. rociletinib, AZD9291, and others) have entered clinical trials, with impressive results observed in phase 1 clinical trials. The development of these newer drugs, with efficacy after resistance to first line EGFR TKI, has led to exploration of these strategies in multiple disease settings: at resistance, in the first line, and adjuvant treatment of those with completely resected early stage disease who would otherwise die of recurrent/metastatic disease. This example of translational research that identifies mechanisms of resistance to first generation drugs, and then targets those mechanisms yielding clinical benefit is a paradigm for how targeted therapies can be developed. PMID:25979928

  20. EGFR: The Paradigm of an Oncogene-Driven Lung Cancer.

    PubMed

    Riely, Gregory J; Yu, Helena A

    2015-05-15

    Somatic, activating mutations in EGFR identify a significant minority of patients with non-small cell lung cancer (NSCLC). Although these mutations are associated with an approximately 70% response rate to some EGFR tyrosine kinase inhibitors (gefitinib, erlotinib, and afatinib), patients develop resistance (i.e., "acquired resistance") after a median of 9 to 12 months. In patients with clinical acquired resistance, repeat biopsy of tumors has identified a number of relevant mechanisms of resistance, but by far the most frequent event is the acquisition of EGFR T790M, a mutation in the "gatekeeper" residue that confers resistance to gefitinib, erlotinib, and afatinib. This emphasizes the critical dependence upon EGFR signaling for some tumors, a property that has been exploited therapeutically. Dual EGFR blockade using afatinib and cetuximab led to a 29% radiographic response rate. More recently, drugs that target EGFR T790M (e.g., rociletinib, AZD9291, and others) have entered clinical trials, with impressive results observed in phase I clinical trials. The development of these newer drugs, with efficacy after resistance to first-line EGFR tyrosine kinase inhibitor, has led to exploration of these strategies in multiple disease settings: at resistance, in the first line, and in adjuvant treatment of those with completely resected early-stage disease who would otherwise die of recurrent/metastatic disease. This example of translational research that identifies mechanisms of resistance to first-generation drugs, and then targets those mechanisms yielding clinical benefit, is a paradigm for how targeted therapies can be developed. PMID:25979928

  1. Biomarkers that currently affect clinical practice: EGFR, ALK, MET, KRAS

    PubMed Central

    Vincent, M.D.; Kuruvilla, M.S.; Leighl, N.B.; Kamel–Reid, S.

    2012-01-01

    New drugs such as pemetrexed, the epidermal growth factor receptor (egfr) tyrosine kinase inhibitors, and the Alk inhibitor crizotinib have recently enabled progress in the management of advanced non-small-cell lung cancer (nsclc). More drugs, especially Met inhibitors, will follow. However, the benefits of these agents are not uniform across the spectrum of nsclc, and optimizing their utility requires some degree of subgrouping of nsclc by the presence or absence of certain biomarkers. The biomarkers of current or imminent value are EGFR and KRAS mutational status, ALK rearrangements, and MET immunohistochemistry. As a predictor of benefit for anti-egfr monoclonal antibodies, EGFR immunohistochemistry is also of potential interest. Some of the foregoing biomarkers (EGFR, ALK, MET) are direct drivers of the malignant phenotype. As such, they are, quite rationally, the direct targets of inhibitory drugs. However, KRAS, while definitely a driver, has resisted attempts at direct pharmacologic manipulation, and its main value might lie in its role as part of an efficient testing algorithm, because KRAS mutations appear to exclude EGFR and ALK mutations. The indirect value of KRAS in determining sensitivity to other targeted agents or to pemetrexed remains controversial. The other biomarkers (EGFR, ALK, MET) may also have indirect value as predictors of sensitivity to chemotherapy in general, to pemetrexed specifically, and to radiotherapy and molecularly targeted agents. These biomarkers have all enabled the co-development of new drugs with companion diagnostics, and they illustrate the paradigm that will govern progress in oncology in the immediate future. However, in nsclc, the acquisition of sufficient biopsy material remains a stubborn obstacle to the evolution of novel targeted therapies. PMID:22787409

  2. EGFR biomarkers predict benefit from vandetanib in combination with docetaxel in a randomized phase III study of second-line treatment of patients with advanced non-small cell lung cancer

    PubMed Central

    Heymach, J. V.; Lockwood, S. J.; Herbst, R. S.; Johnson, B. E.; Ryan, A. J.

    2014-01-01

    Background ZODIAC was a randomized phase III study of second-line treatment in patients with advanced non-small cell lung cancer (NSCLC) that evaluated the addition of vandetanib to docetaxel. The study showed a statistically significant improvement in progression-free survival and objective response rate, but not in overall survival for unselected patients. This study evaluated epidermal growth factor receptor (EGFR) gene mutation, copy number gain, and protein expression, and KRAS gene mutation, in pretreatment tumor samples as potential biomarkers predicting benefit from vandetanib as second-line treatment of NSCLC. Patients and methods After progression following first-line chemotherapy, 1391 patients with locally advanced or metastatic (stage IIIB/IV) NSCLC were randomized 1 : 1 to receive vandetanib (100 mg/day) plus docetaxel (75 mg/m2 every 21 days) or placebo plus docetaxel in the ZODIAC study. Archival tumor samples (n = 570) were collected from consenting patients (n = 958) for predefined, prospective biomarker analyses. Results Of evaluable samples, 14% were EGFR mutation positive, 35% were EGFR FISH positive, 88% were EGFR protein expression positive, and 13% were KRAS mutation positive. Compared with the overall study population, in which progression-free survival (PFS) [hazard ratio (HR) = 0.79] but not OS (HR = 0.91) were significantly improved with vandetanib, there was greater relative clinical benefit for patients with EGFR mutation-positive tumors [PFS HR 0.51, confidence interval (CI) 0.25–1.06 and OS HR 0.46, CI 0.14–1.57] and EGFR FISH-positive tumors (PFS HR 0.61, CI 0.39–0.94 and OS HR 0.48, CI 0.28–0.84). Similarly, patients with EGFR mutation or FISH-positive tumor samples who received vandetanib had an increased chance of objective tumor response (odds ratios 3.34, CI 0.8–13.89, and 3.90, CI 1.02–14.82, respectively). There did not appear to be benefit for vandetanib in patients with KRAS mutation-positive tumors. Conclusions

  3. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  4. Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

    PubMed Central

    Barber, Paul R.; Tullis, Iain D.C.; Vojnovic, Borivoj; Kong, Anthony

    2011-01-01

    The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers. PMID:21908901

  5. Neoadjuvant and adjuvant epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy for lung cancer

    PubMed Central

    Zhai, Haoran; Zhong, Wenzhao; Yang, Xuening

    2015-01-01

    The Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis and the meta-analysis of individual participant data reported by non-small cell lung cancer (NSCLC) Meta-analysis Collaborative Group in neo-adjuvant setting validated respectively that adjuvant and neoadjuvant chemotherapy would significantly improve overall survival (OS) and recurrence-free survival for resectable NSCLC. However, chemotherapy has reached a therapeutic plateau. It has been confirmed that epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeting therapy provides a dramatic response to patients with advanced EGFR-mutation positive NSCLC. Researchers have paid more attention to exploring applications of TKIs to early resectable NSCLCs. Several studies on adjuvant TKI treatment concluded its safety and feasibility. But there existed certain limitations of these studies as inference factors to interpret data accurately: the BR19 study recruited patients among which almost 52% had stage IB and only 15 (3.0%, 15/503) had been confirmed with EGFR-mutant type; retrospective studies performed at Memorial Sloan Kettering Cancer Center (MSKCC) selected EGFR mutant-type NSCLC patients but couldn’t avoid inherent defects inside retrospective researches; the RADIANT study revised endpoints from targeting at EGFR immunohistochemistry (IHC)+ and/or fluorescence in situ hybridization (FISH)+ mutation to only EGFR IHC+ mutation, leading to selective bias; despite that the SELECT study validated efficacy of adjuvant TKI and second round of TKI after resistance occurred, a single-arm clinical trial is not that persuasive in the absence of comparison with chemotherapy. Taking all these limitations into account, CTONG1104 in China and IMPACT in Japan have been conducted and recruiting patients to offer higher level of evidences to explore efficacy of preoperative TKI therapy for early resectable EGFR mutation positive NSCLC patients (confirmed by pathological results of tumor tissue or

  6. ASSAYS OF TOXIC POLLUTANTS BY FISH BLOOD

    EPA Science Inventory

    The authors have developed a biological multichannel analyzer which, using a sensor that operates on the Coulter Principle, measures and distributes mixed cell populations by cell size. It provides an analog distribution and digital printed readout for future analysis. Although p...

  7. MiR-200c overexpression is associated with better efficacy of EGFR-TKIs in non-small cell lung cancer patients with EGFR wild-type

    PubMed Central

    Li, Jiayu; Li, Xuefei; Ren, Shengxiang; Chen, Xiaoxia; Zhang, Yishi; Zhou, Fei; Zhao, Mingchuan; Zhao, Chao; Chen, Xiu; Cheng, Ningning; Zhao, Yinmin; Zhou, Caicun; Hirsch, Fred R.

    2014-01-01

    Several randomized trials have demonstrated non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations can achieve favorable clinical outcomes on treatment with EGFR tyrosine kinase inhibitors (TKIs). EGFR mutation is considered as a predictive marker for efficacy of EGFR-TKIs in NSCLC. Here we show miR-200c overexpression was correlated with the epithelial phenotype and sensitivity to gefitinib in EGFR wild-type NSCLC cell lines. Up-regulated miR-200c could regain the sensitivity to gefitinib in the EGFR wild-type cell lines and miR-200c could regulate epithelial to mesenchymal transition through PI3K/AKT and MEK/ERK pathways. NSCLC patients at advanced stage (N=150) who received EGFR-TKIs (gefitinib or erlotinib) as second- or third-line therapy from September 2008 to December 2012 were included in the study. In 66 NSCLC patients with wild-type EGFR, high levels of miR-200c expression was associated with higher disease control rate (DCR), longer progression-free survival (PFS) and longer overall survival (OS) compared with low miR-200c expression subgroup. In the subgroup with EGFR mutation, the trend remained the same but not statistically significant. Overall, these findings indicated that miR-200c might be a predictive biomarker for sensitivity to EGFR-TKIs in advanced NSCLC patients with wild-type EGFR. PMID:25277203

  8. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

    SciTech Connect

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-07-15

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  9. Design, synthesis and biological evaluation of novel EGFR/HER2 dual inhibitors bearing a oxazolo[4,5-g]quinazolin-2(1H)-one scaffold.

    PubMed

    Yin, Siyuan; Tang, Chunming; Wang, Bin; Zhang, Ying; Zhou, Liliang; Xue, Lingjing; Zhang, Can

    2016-09-14

    For the purpose of developing novel EGFR/HER2 tyrosine kinases inhibitors with high inhibition activity and low toxicity, two novel series of oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as EGFR/HER2 dual inhibitors introducing two electrophiles 2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl group as side-chain at 1-position respectively and evaluated their EGFR and HER2 inhibition activity and toxicity comparing with Lapatinib. All these compounds were evaluated by EGFR and HER2 kinase inhibition and two anti-proliferation assays in vitro. Most of the designed compounds exhibited moderate to high inhibition activity against EGFR and HER2. Especially, compounds 11o, 11p, 12e and 12f presented high inhibition against EGFR and HER2. Furthermore, compounds 11p and 12f also had well exhibition to excellent anti-proliferation activity against human lung adenocarcinoma cell line (A549) and human breast cancer cell line (SK-Br3), and 12f also exhibited the lowest toxicity against human embryonic lung fibroblast cell line (HELF) cell. Finally, compound 12f presented remarkably higher inhibition efficacy towards tumour growth than Lapatinib in a mouse lewis lung cancer (LLC) xenograft model. PMID:27187856

  10. Epidermal growth factor receptor (EGFR) antisense transfection reduces the expression of EGFR and suppresses the malignant phenotype of a human ovarian cancer cell line.

    PubMed

    Brader, K R; Wolf, J K; Chakrabarty, S; Price, J E

    1998-01-01

    An EGFR-expressing clone of the human ovarian cancer line 2774 was transfected with an antisense construct of EGFR to test how suppression of this gene modulates the malignant phenotype. Transfected clones were screened for EGFR expression by Western blot and FACS analysis. Anchorage-independent growth was used to assess the effect of reduced EGFR on the malignant behavior of the cells. Several transfected clones with decreased EGFR (40-50% reduction) were identified. A correlation was noted between reduced EGFR and decreased anchorage-independent growth, with the transfected clones losing the ability to grow in agarose and responsiveness to exogenous EGF. These results suggest that EGFR may be an important factor in the malignant behavior of this ovarian cancer cell line. PMID:9683849

  11. Driver mutations among never smoking female lung cancer tissues in China identify unique EGFR and KRAS mutation pattern associated with household coal burning

    PubMed Central

    Hosgood, H. Dean; Pao, William; Rothman, Nathaniel; Hu, Wei; Pan, Yumei Helen; Kuchinsky, Kyle; Jones, Kirk D.; Xu, Jun; Vermeulen, Roel; Simko, Jeff; Lan, Qing

    2013-01-01

    Lung cancer in never smokers, which has been partially attributed to household solid fuel use (i.e coal), is etiologically and clinically different from lung cancer attributed to tobacco smoking. To explore the spectrum of driver mutations among lung cancer tissues from never smokers, specifically in a population where high lung cancer rates have been attributed to indoor air pollution from domestic coal use, multiplexed assays were used to detect >40 point mutations, insertions, and deletions (EGFR, KRAS, BRAF, HER2, NRAS, PIK3CA, MEK1, AKT1, and PTEN) among the lung tumors of confirmed never smoking females from Xuanwei, China [32 adenocarcinomas (ADCs), 7 squamous cell carcinomas (SCCs), 1 adenosquamous carcinoma (ADSC)]. EGFR mutations were detected in 35% of tumors. 46% of these involved EGFR exon 18 G719X, while 14% were exon 21 L858R mutations. KRAS mutations, all of which were G12C_34G>T, were observed in 15% of tumors. EGFR and KRAS mutations were mutually exclusive, and no mutations were observed in the other tested genes. Most point mutations were transversions and were also found in tumors from patients who used coal in their homes. Our high mutation frequencies in EGFR exon 18 and KRAS and low mutation frequency in EGFR exon 21 are strikingly divergent from those in other smoking and never smoking populations from Asia. Given that our subjects live in a region where coal is typically burned indoors, our findings provide new insights into the pathogenesis of lung cancer among never smoking females exposed to indoor air pollution from coal. PMID:24055406

  12. Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: Involvement of Src/EGFR/PI3-K/Akt pathway

    SciTech Connect

    Lin, W.-N.; Luo, S.-F.; Wu, C.-B.; Lin, C.-C.; Yang, C.-M.

    2008-04-15

    In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-{kappa}B in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS.

  13. Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1.

    PubMed

    Lo, Hui-Wen; Ali-Seyed, Mohamed; Wu, Yadi; Bartholomeusz, Geoffrey; Hsu, Sheng-Chieh; Hung, Mien-Chie

    2006-08-15

    Many receptor tyrosine kinases (RTKs) can be detected in the cell nucleus, such as EGFR, HER-2, HER-3, HER-4, and fibroblast growth factor receptor. EGFR, HER-2 and HER-4 contain transactivational activity and function as transcription co-factors to activate gene promoters. High EGFR in tumor nuclei correlates with increased tumor proliferation and poor survival in cancer patients. However, the mechanism by which cell-surface EGFR translocates into the cell nucleus remains largely unknown. Here, we found that EGFR co-localizes and interacts with importins alpha1/beta1, carriers that are critical for macromolecules nuclear import. EGFR variant mutated at the nuclear localization signal (NLS) is defective in associating with importins and in entering the nuclei indicating that EGFR's NLS is critical for EGFR/importins interaction and EGFR nuclear import. Moreover, disruption of receptor internalization process using chemicals and forced expression of dominant-negative Dynamin II mutant suppressed nuclear entry of EGFR. Additional evidences suggest an involvement of endosomal sorting machinery in EGFR nuclear translocalization. Finally, we found that nuclear export of EGFR may involve CRM1 exportin as we detected EGFR/CRM1 interaction and markedly increased nuclear EGFR following exposure to leptomycin B, a CRM1 inhibitor. Collectively, these data suggest the importance of receptor endocytosis, endosomal sorting machinery, interaction with importins alpha1/beta1, and exportin CRM1 in EGFR nuclear-cytoplasmic trafficking. Together, our work sheds light into the nature and regulation of the nuclear EGFR pathway and provides a plausible mechanism by which cells shuttle cell-surface EGFR and potentially other RTKs through the nuclear pore complex and into the nuclear compartment. PMID:16552725

  14. Design, Synthesis, and Biological Evaluation of Novel Conformationally Constrained Inhibitors Targeting EGFR

    PubMed Central

    2013-01-01

    This letter describes the construction of conformationally constrained quinazoline analogues. Structure–activity relationship studies led to the identification of the lead compound 9n. Compound 9n exhibits effective in vitro activity against A431WT,overexpression and H1975[L858R/T790M] cancer cell lines but is significantly less effective against EGFR negative cancer cell lines (SW620, A549, and K562). Compound 9n was also assessed for potency in enzymatic assays and in vivo antitumor studies. The results indicated that 9n is a potent kinase inhibitor against both wild-type and T790M mutant EGFR kinase. Meanwhile, an oral administration of 9n at a dose of 200 mg/kg produced a considerable antitumor effect in a A431 xenograft model, as compared to gefitinib. A preliminary pharmacokinetic study of 9n also indicates it has good pharmacokinetic properties, and therefore, it is a good starting point for further development. PMID:24900594

  15. Generation and evaluation of bispecific affibody molecules for simultaneous targeting of EGFR and HER2.

    PubMed

    Ekerljung, Lina; Wållberg, Helena; Sohrabian, Azita; Andersson, Karl; Friedman, Mikaela; Frejd, Fredrik Y; Ståhl, Stefan; Gedda, Lars

    2012-09-19

    Coexpression of several ErbB receptors has been found in many cancers and has been linked with increased aggressiveness of tumors and a worse patient prognosis. This makes the simultaneous targeting of two surface receptors by using bispecific constructs an increasingly appreciated strategy. Here, we have generated six such bispecific targeting proteins, each comprising two monomeric affibody molecules with specific binding to either of the two human epidermal growth factor receptors, EGFR and HER2, respectively. The bispecific constructs were designed with (i) alternative positioning (N- or C-terminal) of the different affibody molecules, (ii) two alternative peptide linkers (Gly(4)Ser)(3) or (Ser(4)Gly)(3), and (iii) affibody molecules with different affinity (nanomolar or picomolar) for HER2. Using both Biacore technology and cell binding assays, it was demonstrated that all six constructs could bind simultaneously to both their target proteins. N-terminal positioning of the inherent monomeric affibody molecules was favorable to promote the binding to the respective target. Interestingly, bispecific constructs containing the novel (Ser(4)Gly)(3) linker displayed a higher affinity in cell binding, as compared to constructs containing the more conventional linker, (Gly(4)Ser)(3). It could further be concluded that bispecific constructs (but not the monomeric affibody molecules) induced dimer formation and phosphorylation of EGFR in SKBR3 cells, which express fairly high levels of both receptors. It was also investigated whether the bispecific binding would influence cell growth or sensitize cells for ionizing radiation, but no such effects were observed. PMID:22882002

  16. Potent anti-tumor effects of EGFR-targeted hybrid peptide on mice bearing liver metastases.

    PubMed

    Gaowa, Arong; Horibe, Tomohisa; Kohno, Masayuki; Harada, Hiroshi; Hiraoka, Masahiro; Kawakami, Koji

    2016-01-01

    In this study, we investigated the therapeutic efficacy of EGFR2R-lytic hybrid peptide for the treatment of liver metastasis from colon carcinoma. The cytotoxic activity of the hybrid peptide against luciferase-expressing human colon cancer (HCT-116-luc) cells was determined by the WST-8 assay. The experimental mouse model of liver metastases was generated by splenic injection of HCT-116-luc cells. The hybrid peptide was intravenously injected into mice the day after cell implantation at a dose of 5 mg/kg and this was repeated on alternate days for a total of 7 doses. Saline-treated mice were used as controls. Tumor growth and therapeutic responses were monitored by an IVIS imaging system. It was shown that the hybrid peptide exhibited potent cytotoxic activity against HCT-116-luc cells and the liver metastases were significantly reduced after intravenous injections of hybrid peptide compared with controls. Furthermore, Kaplan–Meier analysis showed that hybrid peptide-treated mice had significantly longer survival than controls. In addition, bright-field and ex vivo imaging of liver tissue revealed that mice treated with the hybrid peptide had significantly fewer tumors compared with controls. These results demonstrated that the EGFR2R-lytic hybrid peptide is a potential treatment option for patients with colorectal cancer metastases in the liver. PMID:26467564

  17. Design, Synthesis, and Biological Evaluation of Novel Conformationally Constrained Inhibitors Targeting EGFR.

    PubMed

    Wu, Jianwei; Chen, Wenteng; Xia, Guangxin; Zhang, Jing; Shao, Jiaan; Tan, Biqin; Zhang, Chunchun; Yu, Wanwan; Weng, Qinjie; Liu, Haiyan; Hu, Miao; Deng, Hailin; Hao, Yu; Shen, Jingkang; Yu, Yongping

    2013-10-10

    This letter describes the construction of conformationally constrained quinazoline analogues. Structure-activity relationship studies led to the identification of the lead compound 9n . Compound 9n exhibits effective in vitro activity against A431(WT,overexpression) and H1975([L858R/T790M]) cancer cell lines but is significantly less effective against EGFR negative cancer cell lines (SW620, A549, and K562). Compound 9n was also assessed for potency in enzymatic assays and in vivo antitumor studies. The results indicated that 9n is a potent kinase inhibitor against both wild-type and T790M mutant EGFR kinase. Meanwhile, an oral administration of 9n at a dose of 200 mg/kg produced a considerable antitumor effect in a A431 xenograft model, as compared to gefitinib. A preliminary pharmacokinetic study of 9n also indicates it has good pharmacokinetic properties, and therefore, it is a good starting point for further development. PMID:24900594

  18. Nuclear trafficking of EGFR by Vps34 represses Arf expression to promote lung tumor cell survival.

    PubMed

    Dayde, D; Guerard, M; Perron, P; Hatat, A-S; Barrial, C; Eymin, B; Gazzeri, S

    2016-07-28

    Epidermal growth factor receptor (EGFR) is a cell surface receptor that has an essential role in cell proliferation and survival, and overexpression of EGFR is a common feature of human cancers. In Non-small-cell lung cancer (NSCLC), activating mutations of EGFR have also been described. We recently showed that mutant EGFR-L858R inhibits the expression of the p14ARF tumor-suppressor protein to promote cell survival. In this study, we defined the molecular bases by which EGFR controls Arf expression. Using various lung tumor models, we showed that EGF stimulation inhibits Arf transcription by a mechanism involving the nuclear transport and recruitment of EGFR to the Arf promoter. We unraveled the vesicular trafficking protein Vps34 as a mediator of EGFR nuclear trafficking and showed that its neutralization prevents the accumulation of EGFR to the Arf promoter in response to ligand activation. Finally, in lung tumor cells that carry mutant EGFR-L858R, we demonstrated that inhibition of Vps34 using small interfering RNA restrains nuclear EGFR location and restores Arf expression leading to apoptosis. These findings identify the Arf tumor suppressor as a new transcriptional target of nuclear EGFR and highlight Vps34 as an important regulator of the nuclear EGFR/Arf survival pathway. As a whole, they provide a mechanistic explanation to the inverse correlation between nuclear expression of EGFR and overall survival in NSCLC patients. PMID:26686095

  19. Novel EGFR-targeted strategy with hybrid peptide against oesophageal squamous cell carcinoma

    PubMed Central

    Kikuchi, Osamu; Ohashi, Shinya; Horibe, Tomohisa; Kohno, Masayuki; Nakai, Yukie; Miyamoto, Shin’ichi; Chiba, Tsutomu; Muto, Manabu; Kawakami, Koji

    2016-01-01

    Epidermal growth factor receptor (EGFR) is a key molecule in the pathophysiology of oesophageal squamous cell carcinoma (OSCC). However, EGFR-targeted agents such as anti-EGFR antibody or tyrosine kinase inhibitors for OSCC have not demonstrated any clinical benefits. Recently, a novel chemotherapeutic agent, EGFR(2R)-lytic hybrid peptide, a composite of EGFR-binding peptide and lytic peptide fragments, has been shown to exhibit a potent anti-tumour effect against cancers that express high EGFR levels. In this study, we investigated the validity of employing EGFR(2R)-lytic hybrid peptide against OSCC cells both in vitro and in vivo. Additionally, the toxicity of this peptide was assessed in mice. We found high EGFR expression levels on the cell surface of OSCC cells, and the EGFR-binding peptide fragment showed high affinity for OSCC cells. A potent cytotoxic effect was induced within 30 minutes by the exposure of OSCC cells to EGFR(2R)-lytic hybrid peptide. Furthermore, EGFR(2R)-lytic hybrid peptide markedly suppressed the tumour growth of OSCC cells in a xenograft model. Moreover, it did not cause any identifiable adverse effects in mice. Taken together, EGFR(2R)-lytic hybrid peptide was shown to be a valid therapeutic agent against OSCC, providing a crucial rationale regarding novel EGFR-targeted therapies against OSCC. PMID:26956916

  20. Integrating anti-EGFR therapies in metastatic colorectal cancer

    PubMed Central

    Haraldsdottir, Sigurdis

    2013-01-01

    Colorectal cancer remains one of the most common causes of cancer diagnoses and mortality in the United States. The treatment of metastatic colorectal cancer has evolved significantly over the last decade with near-tripling of patient survival rate. A significant contribution to this outcome was the advent of novel targeted agents, such as the epidermal growth factor (EGFR) inhibitors. In an era of emphasis on refining therapy, the presence of KRAS mutation will predict for resistance and limit exposure to patients who are more likely to benefit. In contrast, the presence of BRAF mutations does not seem to have a predictive value. Agents that are thought to reverse resistance to EGFR inhibitors such as those targeting PI3K, c-MET or IGF-1R are currently under study. EGFR inhibitors have exhibited single agent activity, and seem to synergize very well with standard chemotherapy except for cetuximab and 5-fluorouracil, leucovorin, oxaliplatin (FOLFOX). Preliminary data suggests that EGFR inhibitors have similar effectiveness to vascular endothelial growth factor (VEGF) inhibitors in the first line setting. Skin toxicity remains the main limiting factor for the utilization of EGFR inhibitors, but strategies including the use of agents such as minocycline or doxycycline added to topical care seem to limit the severity of the rash. PMID:23997940

  1. Kinase-mediated quasi-dimers of EGFR

    PubMed Central

    Bublil, Erez M.; Pines, Gur; Patel, Gargi; Fruhwirth, Gilbert; Ng, Tony; Yarden, Yosef

    2010-01-01

    Ligand-induced dimerization of the epidermal growth factor receptor (ErbB-1/EGFR) involves conformational changes that expose an extracellular dimerization interface. Subsequent alterations within the cytoplasmic kinase domain, which culminate in tyrosine phosphorylation, are less understood. Our study addressed this question by using two strategies: a chimeric receptor approach employed ErbB-3, whose defective kinase domain was replaced by the respective part of EGFR. The implanted full-length kinase, unlike its subdomains, conferred dimerization and catalysis. The data infer that the kinase function of EGFR is restrained by the carboxyl tail; once grafted distally to the ectopic tail of ErbB-3, the kinase domain acquires quasi-dimerization and activation. In an attempt to alternatively refold the cytoplasmic tail, our other approach employed kinase inhibitors. Biophysical measurements and covalent cross-linking analyses showed that inhibitors targeting the active conformation of EGFR, in contrast to a compound recognizing the inactive conformation, induce quasi-dimers in a manner similar to the chimeric ErbB-3 molecule. Collectively, these observations unveil kinase domain-mediated quasi-dimers, which are regulated by an autoinhibitory carboxyl tail. On the basis of these observations, we propose that quasi-dimers precede formation of ligand-induced, fully active dimers, which are stabilized by both extracellular and intracellular receptor-receptor interactions.—Bublil, E. M., Pines, G., Patel, G., Fruhwirth, G., Ng, T., Yosef Yarden. Kinase-mediated quasi-dimers of EGFR. PMID:20682838

  2. Molecular Pathways: Sensitivity and Resistance to Anti-EGFR Antibodies.

    PubMed

    Bertotti, Andrea; Sassi, Francesco

    2015-08-01

    Monoclonal antibodies targeting the EGF receptor (EGFR) tyrosine kinase, such as cetuximab and panitumumab, achieve clinically meaningful responses in patients affected by head and neck and colorectal cancers. Despite this evidence of efficacy, no genomic abnormalities that robustly predict sensitivity to EGFR blockade have been yet identified. This suggests that, in some tumor contexts, EGFR dependency is not acquired during neoplastic transformation and rather reflects an aberrant declination of physiologic traits typical of normal tissue counterparts. Indeed, EGFR signals are crucial for the reconstitution of damaged mucosa in the context of acute inflammation, and their sustained activation is likely to turn into a pro-oncogenic cue during chronic inflammation. Although positive predictors of response to anti-EGFR antibodies remain unknown, multiple determinants of resistance have been described, including alterations interfering with antibody-receptor interaction, deregulation of parallel signaling pathways, and mutations in downstream transducers. These findings provide new opportunities for the optimization of therapeutic strategies based on drug combinations. However, the emerging notion that genetic interactions and compensatory mechanisms may affect-both positively and negatively-the efficacy of targeted therapies complicates the rational design of combinatorial approaches and implies a rethinking of the criteria required to prioritize laboratory findings for clinical validation in investigational trials. PMID:26071484

  3. EGFR and HER2 signaling in breast cancer brain metastasis

    PubMed Central

    Sirkisoon, Sherona R.; Carpenter, Richard L.; Rimkus, Tadas; Miller, Lance; Metheny-Barlow, Linda; Lo, Hui-Wen

    2016-01-01

    Breast cancer occurs in approximately 1 in 8 women and 1 in 37 women with breast cancer succumbed to the disease. Over the past decades, new diagnostic tools and treatments have substantially improved the prognosis of women with local diseases. However, women with metastatic disease still have a dismal prognosis without effective treatments. Among different molecular subtypes of breast cancer, the HER2-enriched and basal-like subtypes typically have higher rates of metastasis to the brain. Basal-like metastatic breast tumors frequently express EGFR. Consequently, HER2- and EGFR-targeted therapies are being used in the clinic and/or evaluated in clinical trials for treating breast cancer patients with brain metastases. In this review, we will first provide an overview of the HER2 and EGFR signaling pathways. The roles that EGFR and HER2 play in breast cancer metastasis to the brain will then be discussed. Finally, we will summarize the preclinical and clinical effects of EGFR- and HER2-targeted therapies on breast cancer metastasis. PMID:26709660

  4. Structural basis for EGFR ligand sequestration by Argos

    SciTech Connect

    Klein, Daryl E.; Stayrook, Steven E.; Shi, Fumin; Narayan, Kartik; Lemmon, Mark A.

    2008-06-26

    Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR5. Here we describe the 1.6-{angstrom} resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-{beta} family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.

  5. Inhibition of Cell Proliferation by an Anti-EGFR Aptamer

    PubMed Central

    Li, Na; Nguyen, Hong Hanh; Byrom, Michelle; Ellington, Andrew D.

    2011-01-01

    Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (Kd = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents. PMID:21687663

  6. Covalent EGFR Inhibitors: Binding Mechanisms, Synthetic Approaches, and Clinical Profiles.

    PubMed

    Hossam, Monia; Lasheen, Deena S; Abouzid, Khaled A M

    2016-08-01

    Being overexpressed in several types of cancer, the epidermal growth factor receptor (EGFR) is considered one of the key therapeutic targets in oncology. Although many first-generation EGFR inhibitors had been FDA approved for the treatment of certain types of cancer, patients soon developed resistance to these reversible ATP competitive inhibitors via mutations in the kinase domain of EGFR. A new trend was adopted to design covalent irreversible inhibitors, that is, second- and third-generation inhibitors. Second-generation inhibitors can inhibit the mutant forms but, unfortunately, they had dose limiting side effects due to wild-type EGFR inhibition. Third-generation inhibitors emerged shortly, which were capable of inhibiting the mutant forms exclusively while sparing the wild type. Many other strategies have also been developed to reduce the risk of covalent interactions with off-targets, thus improving the pharmacokinetic and/or pharmacodynamic profile of the antiproliferative agents. In this review, we focused mainly on second- and third-generation EGFR inhibitors, their binding mechanisms (either docking studies or co-crystallized structures), their synthetic approaches, clinical profiles, and limitations. PMID:27258393

  7. Transgenic Fish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish into which foreign DNA is artificially introduced and integrated into their genome are called transgenic fish. Since the development of the first transgenic fish in 1985, techniques to produce transgenic fish have improved tremendously, resulting in the production of genetically modified (GM) ...

  8. Anti-EGFR-iRGD recombinant protein conjugated silk fibroin nanoparticles for enhanced tumor targeting and antitumor efficiency

    PubMed Central

    Bian, Xinyu; Wu, Puyuan; Sha, Huizi; Qian, Hanqing; Wang, Qing; Cheng, Lei; Yang, Yang; Yang, Mi; Liu, Baorui

    2016-01-01

    In this study, we report a novel kind of targeting with paclitaxel (PTX)-loaded silk fibroin nanoparticles conjugated with iRGD–EGFR nanobody recombinant protein (anti-EGFR-iRGD). The new nanoparticles (called A-PTX-SF-NPs) were prepared using the carbodiimide-mediated coupling procedure and their characteristics were evaluated. The cellular cytotoxicity and cellular uptake of A-PTX-SF-NPs were also investigated. The results in vivo suggested that NPs conjugated with the recombinant protein exhibited more targeting and anti-neoplastic property in cells with high EGFR expression. In the in vivo antitumor efficacy assay, the A-PTX-SF-NPs group showed slower tumor growth and smaller tumor volumes than PTX-SF-NPs in a HeLa xenograft mouse model. A real-time near-infrared fluorescence imaging study showed that A-PTX-SF-NPs could target the tumor more effectively. These results suggest that the anticancer activity and tumor targeting of A-PTX-SF-NPs were superior to those of PTX-SF-NPs and may have the potential to be used for targeted delivery for tumor therapies. PMID:27313461

  9. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    SciTech Connect

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  10. EGFR signaling and autophagy dependence for growth, survival, and therapy resistance

    PubMed Central

    Jutten, Barry; Rouschop, Kasper MA

    2014-01-01

    The epidermal growth factor receptor (EGFR) is amplified or mutated in various human epithelial tumors. Its expression and activation leads to cell proliferation, differentiation, and survival. Consistently, EGFR amplification or expression of EGFR variant 3 (EGFRvIII) is associated with resistance to conventional cancer therapy through activation of pro-survival signaling and DNA-repair mechanisms. EGFR targeting has successfully been exploited as strategy to increase treatment efficacy. Nevertheless, these targeting strategies have only been proven effective in a limited percentage of human tumors.   Recent knowledge indicates that EGFR deregulated tumors display differences in autophagy and dependence on autophagy for growth and survival and the use of autophagy to increase resistance to EGFR-targeting drugs. In this review the dependency on autophagy and its role in mediating resistance to EGFR-targeting agents will be discussed. Considering the current knowledge, autophagy inhibition could provide a novel strategy to enhance therapy efficacy in treatment of EGFR deregulated tumors. PMID:24335351

  11. ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinase activity in combination with radiotherapy

    SciTech Connect

    Frederick, Barbara; Gustafson, Dan; Bianco, Cataldo; Ciardiello, Fortunato; Dimery, Isaiah; Raben, David . E-mail: david.raben@uchsc.edu

    2006-01-01

    Radiation enhances both epithelial growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) expression, which are a part of key pathways for tumor progression. Some tumors may not respond well to EGFR inhibitors alone or may develop resistance to EGFR inhibitors. Therefore, drug therapy targeted to VEGF receptors and EGFRs, when combined with radiotherapy (RT), may improve tumor control and provide wider applicability. This article focuses on ZD6474, an inhibitor of EGFR and VEGF receptor signaling in combination with RT. We discuss preclinical and clinical studies with RT and inhibitors of VEGF or EGFR signaling first. We then address issues associated with ZD6474 pharmacokinetic dosing, and scheduling when combined with RT. We also discuss ZD6474 in the context of anti-EGFR therapy resistance. Dual inhibition of EGFR and VEGF receptor signaling pathways shows promise in enhancing RT efficacy.

  12. KRAS, HRAS and EGFR Mutations in Sporadic Sebaceous Gland Hyperplasia.

    PubMed

    Groesser, Leopold; Singer, Sebastian; Peterhof, Eva; Landthaler, Michael; Heigl, Ulrike; Schneider-Brachert, Wulf; Berneburg, Mark; Hafner, Christian

    2016-08-23

    Sporadic sebaceous gland hyperplasia (SGH) is a benign skin lesion, with a high prevalence in the general population. Although SGH has been attributed to both extrinsic and intrinsic factors, the underlying genetic changes have not yet been characterized. Recently, HRAS and KRAS mutations have been identified in sebaceous naevus, a hamartoma sharing histological characteristics with SGH. Therefore we screened 43 SGH for activating mutations in RAS genes and other oncogenes. We identified a wide spectrum of mutually exclusive activating HRAS (8/43), KRAS (11/43) and EGFR mutations (7/31) in altogether 60% of the lesions investigated. A RAS and EGFR wildtype status was found in 15 normal sebaceous glands in the head and neck area. Our findings indicate that activating HRAS, KRAS and EGFR mutations play a major role in the pathogenesis of sporadic SGH. These results support the concept that SGH is a true benign neoplasm rather than a reactive hyperplasia. PMID:26804118

  13. Renal toxicity of anticancer agents targeting HER2 and EGFR.

    PubMed

    Cosmai, Laura; Gallieni, Maurizio; Porta, Camillo

    2015-12-01

    EGFR and HER2 are found overexpressed and/or activated in many different human malignancies (e.g. breast and colon cancer), and a number of drugs specifically targeting these two tyrosine kinases have been developed over the years as anticancer agents. In the present review, the renal safety profile of presently available agents targeting either HER2 or EGFR will be discussed, together with the peculiarities related to their clinical use in patients with impaired renal function, or even in dialysis. Indeed, even though renal toxicity is not so common with these agents, it may nevertheless happen, especially when these agents are combined with traditional chemotherapeutic agents. As a whole, kidney impairment or dialysis should not be regarded per se as reasons not to administer or to stop an active anti-HER or anti-EGFR anticancer treatment, especially given the possibility of significantly improving the life expectancy of many cancer patients with the use of these agents. PMID:26341657

  14. Histone Deacetylase-3/CAGE Axis Targets EGFR Signaling and Regulates the Response to Anti-Cancer Drugs

    PubMed Central

    Kim, Hyuna; Kim, Youngmi; Goh, Hyeonjung; Jeoung, Dooil

    2016-01-01

    We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of pEGFRY845. HDAC3 was found to negatively regulate the expression of pEGFRY845. CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, pEGFRY845, and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant Malme3MR cells, decreasing the tumorigenic potential of Malme3MR cells in a manner associated with its effect on the expression of HDAC3, CAGE and pEGFRY845. The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that PKCδ was responsible for the increased expression of pEGFRY845 and CAGE in Malme3MR cells. CAGE showed an interaction with PKCδ in Malme3MR cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors. PMID:26883907

  15. Implementation and Quality Control of Lung Cancer EGFR Genetic Testing by MALDI-TOF Mass Spectrometry in Taiwan Clinical Practice

    PubMed Central

    Su, Kang-Yi; Kao, Jau-Tsuen; Ho, Bing-Ching; Chen, Hsuan-Yu; Chang, Gee-Cheng; Ho, Chao-Chi; Yu, Sung-Liang

    2016-01-01

    Molecular diagnostics in cancer pharmacogenomics is indispensable for making targeted therapy decisions especially in lung cancer. For routine clinical practice, the flexible testing platform and implemented quality system are important for failure rate and turnaround time (TAT) reduction. We established and validated the multiplex EGFR testing by MALDI-TOF MS according to ISO15189 regulation and CLIA recommendation in Taiwan. Totally 8,147 cases from Aug-2011 to Jul-2015 were assayed and statistical characteristics were reported. The intra-run precision of EGFR mutation frequency was CV 2.15% (L858R) and 2.77% (T790M); the inter-run precision was CV 3.50% (L858R) and 2.84% (T790M). Accuracy tests by consensus reference biomaterials showed 100% consistence with datasheet (public database). Both analytical sensitivity and specificity were 100% while taking Sanger sequencing as the gold-standard method for comparison. EGFR mutation frequency of peripheral blood mononuclear cell for reference range determination was 0.002 ± 0.016% (95% CI: 0.000–0.036) (L858R) and 0.292 ± 0.289% (95% CI: 0.000–0.871) (T790M). The average TAT was 4.5 working days and the failure rate was less than 0.1%. In conclusion, this study provides a comprehensive report of lung cancer EGFR mutation detection from platform establishment, method validation to clinical routine practice. It may be a reference model for molecular diagnostics in cancer pharmacogenomics. PMID:27480787

  16. High efficacy of third generation EGFR inhibitor AZD9291 in a leptomeningeal carcinomatosis model with EGFR-mutant lung cancer cells.

    PubMed

    Nanjo, Shigeki; Ebi, Hiromichi; Arai, Sachiko; Takeuchi, Shinji; Yamada, Tadaaki; Mochizuki, Satsuki; Okada, Yasunori; Nakada, Mitsutoshi; Murakami, Takashi; Yano, Seiji

    2016-01-26

    Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer. PMID:26716903

  17. High efficacy of third generation EGFR inhibitor AZD9291 in a leptomeningeal carcinomatosis model with EGFR-mutant lung cancer cells

    PubMed Central

    Nanjo, Shigeki; Ebi, Hiromichi; Arai, Sachiko; Takeuchi, Shinji; Yamada, Tadaaki; Mochizuki, Satsuki; Okada, Yasunori; Nakada, Mitsutoshi; Murakami, Takashi; Yano, Seiji

    2016-01-01

    Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer. PMID:26716903

  18. Direct interaction between surface β1,4-galactosyltransferase 1 and epidermal growth factor receptor (EGFR) inhibits EGFR activation in hepatocellular carcinoma

    SciTech Connect

    Tang, Wenqing; Weng, Shuqiang; Zhang, Si; Wu, Weibing; Dong, Ling; Shen, Xizhong; Zhang, Songwen; Gu, Jianxin; Xue, Ruyi

    2013-05-10

    Highlights: •β1,4GT1 interacts with EGFR both in vitro and in vivo. •β1,4GT1 co-localizes with EGFR on the cell surface. •β1,4GT1 inhibits {sup 125}I-EGF binding to EGFR. •β1,4GT1 inhibits EGF induced EGFR dimerization and phosphorylation. -- Abstract: Our previous studies showed that cell surface β1,4-galactosyltransferase 1 (β1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that β1,4-galactosyltransferase 1 (β1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that β1,4GT1 bound to EGFR in vivo by co-immunoprecipitation and determined the co-localization of β1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using {sup 125}I-EGF binding experiments and Western blot analysis, we found that overexpression of β1,4GT1 inhibited {sup 125}I-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface β1,4GT1 interacts with EGFR and inhibits EGFR activation.

  19. Radiation Nanomedicine for EGFR-Positive Breast Cancer: Panitumumab-Modified Gold Nanoparticles Complexed to the β-Particle-Emitter, (177)Lu.

    PubMed

    Yook, Simmyung; Cai, Zhongli; Lu, Yijie; Winnik, Mitchell A; Pignol, Jean-Philippe; Reilly, Raymond M

    2015-11-01

    Our objective was to construct a novel radiation nanomedicine for treatment of breast cancer (BC) expressing epidermal growth factor receptors (EGFR), particularly triple-negative tumors (TNBC). Gold nanoparticles (AuNP; 30 nm) were modified with polyethylene glycol (PEG) chains (4 kDa) derivatized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators for complexing the β-emitter, (177)Lu and with PEG chains (5 kDa) linked to panitumumab for targeting BC cells expressing EGFR. The AuNP were further coated with PEG chains (2 kDa) to stabilize the particles to aggregation. The binding and internalization of EGFR-targeted AuNP ((177)Lu-T-AuNP) into BC cells was studied and compared to nontargeted (177)Lu-NT-AuNP. The cytotoxicity of (177)Lu-T-AuNP and (177)Lu-NT-AuNP was measured in clonogenic assays using BC cells with widely different EGFR densities: MDA-MB-468 (10(6) receptors/cell), MDA-MB-231 (10(5) receptors/cell), and MCF-7 cells (10(4) receptors/cell). Radiation absorbed doses to the cell nucleus of MDA-MB-468 cells were estimated based on subcellular distribution. Darkfield and fluorescence microscopy as well as radioligand binding assays revealed that (177)Lu-T-AuNP were specifically bound by BC cells dependent on their EGFR density whereas the binding and internalization of (177)Lu-NT-AuNP was significantly lower. The affinity of binding of (177)Lu-T-AuNP to MDA-MB-468 cells was reduced by 2-fold compared to (123)I-labeled panitumumab (KD = 1.3 ± 0.2 nM vs 0.7 ± 0.4 nM, respectively). The cytotoxicity of (177)Lu-T-AuNP was dependent on the amount of radioactivity incubated with BC cells, their EGFR density and the radiosensitivity of the cells. The clonogenic survival (CS) of MDA-MB-468 cells overexpressing EGFR was reduced to <0.001% at the highest amount of (177)Lu-T-AuNP tested (4.5 MBq; 6 × 10(11) AuNP per 2.5 × 10(4)-1.2 × 10(5) cells). (177)Lu-T-AuNP were less effective for killing MDA-MB-231 cells or MCF-7 cells with

  20. A Mutation-Sensitive Switch Assay to Detect Five Clinically Significant Epidermal Growth Factor Receptor Mutations

    PubMed Central

    Liu, Bin; Zhou, Lin; Wang, Qian

    2015-01-01

    Epidermal growth factor receptor (EGFR) mutations can affect the therapeutic efficacy of drugs used to treat nonsmall-cell lung cancer (NSCLC). We aimed to develop methods to detect five common EGFR somatic mutations in tumor tissues from NSCLC patients by using a nanoscale mutation-sensitive switch consisting of a high-fidelity polymerase and phosphorothioate-modified allele-specific primers. The five clinically significant EGFR mutations examined here are S768I, T790M, L858R, and 15- and 18-bp deletion mutations in exon 19. Our assays showed sensitivities of 100 copies and specificities of more than three log scales for matched templates relative to mismatched templates by routine polymerase chain reaction (PCR), real-time PCR, and multiplex PCR. This assay would be superior to DNA sequencing in situations where mutant DNA is not abundant. PMID:25918867

  1. Modulating the Structure of EGFR with UV Light: New Possibilities in Cancer Therapy

    PubMed Central

    Thiagarajan, Viruthachalam; Coutinho, Isabel; Gajula, Gnana Prakash; Petersen, Steffen B.

    2014-01-01

    The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies. PMID:25386651

  2. JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

    PubMed Central

    Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.

    2016-01-01

    Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

  3. Emerging Roles of MicroRNAs in EGFR-Targeted Therapies for Lung Cancer

    PubMed Central

    Han, Fei; He, Jinxi; Li, Feng; Yang, Jiali; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung cancer is a leading cause of cancer mortality worldwide. Several molecular pathways underlying mechanisms of this disease have been partly elucidated, among which the epidermal growth factor receptor (EGFR) pathway is one of the well-known signaling cascades that plays a critical role in tumorigenesis. Dysregulation of the EGFR signaling is frequently found in lung cancer. The strategies to effectively inhibit EGFR signaling pathway have been mounted for developing anticancer therapeutic agents. However, most anti-EGFR-targeted agents fail to repress cancer progression because of developing drug-resistance. Therefore, studies of the mechanisms underpinning the resistance toward anti-EGFR agents may provide important findings for lung cancer treatment using anti-EGFR therapies. Recently, increasing numbers of miRNAs are correlated with the drug resistance of lung cancer cells to anti-EGFR agents, indicating that miRNAs may serve as novel targets and/or promising predictive biomarkers for anti-EGFR therapy. In this paper, we summarize the emerging role of miRNAs as regulators to modulate the EGFR signaling and the resistance of lung cancer cells to anti-EGFR therapy. We also highlight the evidence supporting the use of miRNAs as biomarkers for response to anti-EGFR agents and as novel therapeutic targets to circumvent the resistance of lung cancer cells to EGFR inhibitors. PMID:26273639

  4. Modulating the structure of EGFR with UV light: new possibilities in cancer therapy.

    PubMed

    Correia, Manuel; Thiagarajan, Viruthachalam; Coutinho, Isabel; Gajula, Gnana Prakash; Petersen, Steffen B; Neves-Petersen, Maria Teresa

    2014-01-01

    The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies. PMID:25386651

  5. JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors.

    PubMed

    Gao, Sizhi P; Chang, Qing; Mao, Ninghui; Daly, Laura A; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Red Brewer, Monica; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F

    2016-01-01

    Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells' dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

  6. Involvement of EGFR in the promotion of malignant properties in multidrug resistant breast cancer cells.

    PubMed

    Xu, Jia-Wen; Li, Qing-Quan; Tao, Li-Li; Cheng, Yuan-Yuan; Yu, Juan; Chen, Qi; Liu, Xiu-Ping; Xu, Zu-De

    2011-12-01

    Multidrug resistance is the most predominant phenomenon leading to chemotherapy treatment failure in breast cancer patients. Despite many studies having suggested that overexpression of epidermal growth factor receptor (EGFR) is a potent predictor of malignancy in cancers, systematic research of EGFR in multidrug resistant (MDR) breast cancer cells is lacking. In order to clarify the role of EGFR in MDR breast cancer cells, MCF7/Adr expressing relatively higher EGFR, and its parental cell line MCF7 expressing relatively lower EGFR, were chosen for this study. Knockdown of EGFR by siRNA in MCF7/Adr cells showed that EGFR siRNA inhibits cell migration, invasion and proliferation in vitro; converse effects were observed in MCF7 cells transfected with pcDNA3.0-EGFR plasmid. Moreover, we found that EGFR upregulated migration and invasion via EMMPRIN, MMP2 and MMP9 in addition to promoting cell cycle passage via elevation of cyclin D1 and CDK4 in MDR breast cancer cells. Interestingly, MCF7/Adr cells not expressing EGFR showed significant decrease of P-glycoprotein (P-gp) and ABCG2 expression levels, and became more sensitive to treatment of adriamycin (ADR) and paclitaxel (Taxol); the above results indicated that MDR of cancer cells is related to S-phase arrest. In conclusion, EGFR is an important factor enhancing the malignancy of MDR breast cancer cells, partially, inducing MDR. Anti-EGFR therapy may improve outcome in chemorefractory breast cancer patients. PMID:21805028

  7. EGFR Signaling in the Brain Is Necessary for Olfactory Learning in "Drosophila" Larvae

    ERIC Educational Resources Information Center

    Rahn, Tasja; Leippe, Matthias; Roeder, Thomas; Fedders, Henning

    2013-01-01

    Signaling via the epidermal growth factor receptor (EGFR) pathway has emerged as one of the key mechanisms in the development of the central nervous system in "Drosophila melanogaster." By contrast, little is known about the functions of EGFR signaling in the differentiated larval brain. Here, promoter-reporter lines of EGFR and its most prominent…

  8. Potential roles of Centipede Scolopendra extracts as a strategy against EGFR-dependent cancers.

    PubMed

    Ma, Weina; Zhang, Dongdong; Zheng, Lei; Zhan, Yingzhuan; Zhang, Yanmin

    2015-01-01

    Centipede Scolopendra, a commonly used traditional Chinese medicine, has been shown to have anti-cancer effects. In this study, the inhibitory effect of alcohol extracts of Centipede Scolopendra (AECS) was more prominent when treating cells highly expressing epidermal growth factor receptor (EGFR) (A431 and HEK293/EGFR cells versus HEK293 cells). The elution profiles of AECS on cell membrane chromatography (CMC) column showed that AECS could bind to EGFR, and competition studies indicated that AECS and gefitinib may have direct competition at a single common binding site on EGFR. SiRNA knockdown of EGFR in A431 cells attenuated AECS effects, suggesting that EGFR was a target mediated by AECS. In a cell culture system, AECS dramatically induced apoptosis of A431 and HEK293/EGFR cells, which was associated with the effects on Bcl-2 family. Furthermore, AECS could alter EGFR kinase activity and reduce phosphorylation of EGFR and downstream signaling players AKT and Erk1/2. The mechanism of AECS to inhibit high-EGFR expression cell proliferation is due to its ability to induce apoptosis and modulate the EGFR pathway. This study might provide a novel therapy for cancer with high-EGFR expression. PMID:25755827

  9. Pooled analysis of clinical outcome for EGFR TKI-treated patients with EGFR mutation-positive NSCLC

    PubMed Central

    Paz-Ares, Luis; Soulières, Denis; Moecks, Joachim; Bara, Ilze; Mok, Tony; Klughammer, Barbara

    2014-01-01

    Patients with non-small-cell lung cancer (NSCLC) appear to gain particular benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKI) if their disease tests positive for EGFR activating mutations. Recently, several large, controlled, phase III studies have been published in NSCLC patients with EGFR mutation-positive tumours. Given the increased patient dataset now available, a comprehensive literature search for EGFR TKIs or chemotherapy in EGFR mutation-positive NSCLC was undertaken to update the results of a previously published pooled analysis. Pooling eligible progression-free survival (PFS) data from 27 erlotinib studies (n = 731), 54 gefitinib studies (n = 1802) and 20 chemotherapy studies (n = 984) provided median PFS values for each treatment. The pooled median PFS was: 12.4 months (95% accuracy intervals [AI] 11.6–13.4) for erlotinib-treated patients; 9.4 months (95% AI 9.0–9.8) for gefitinib-treated patients; and 5.6 months (95% AI 5.3–6.0) for chemotherapy. Both erlotinib and gefitinib resulted in significantly longer PFS than chemotherapy (permutation testing; P = 0.000 and P = 0.000, respectively). Data on more recent TKIs (afatinib, dacomitinib and icotinib) were insufficient at this time-point to carry out a pooled PFS analysis on these compounds. The results of this updated pooled analysis suggest a substantial clear PFS benefit of treating patients with EGFR mutation-positive NSCLC with erlotinib or gefitinib compared with chemotherapy. PMID:25100284

  10. Ibrutinib selectively and irreversibly targets EGFR (L858R, Del19) mutant but is moderately resistant to EGFR (T790M) mutant NSCLC Cells

    PubMed Central

    Wang, Wenchao; Hu, Chen; Ye, Zi; Zhao, Zheng; Wang, Li; Li, Xixiang; Yu, Kailin; Liu, Juan; Wu, Jiaxin; Yan, Xiao-E; Zhao, Peng; Wang, Jinhua; Wang, Chu; Weisberg, Ellen L.; Gray, Nathanael S.; Yun, Cai-Hong; Liu, Jing; Chen, Liang; Liu, Qingsong

    2015-01-01

    Through comprehensive comparison study, we found that ibrutinib, a clinically approved covalent BTK kinase inhibitor, was highly active against EGFR (L858R, del19) mutant driven NSCLC cells, but moderately active to the T790M ‘gatekeeper’ mutant cells and not active to wild-type EGFR NSCLC cells. Ibrutinib strongly affected EGFR mediated signaling pathways and induced apoptosis and cell cycle arrest (G0/G1) in mutant EGFR but not wt EGFR cells. However, ibrutinib only slowed down tumor progression in PC-9 and H1975 xenograft models. MEK kinase inhibitor, GSK1120212, could potentiate ibrutinib's effect against the EGFR (L858R/T790M) mutation in vitro but not in vivo. These results suggest that special drug administration might be required to achieve best clinical response in the ongoing phase I/II clinical trial with ibrutinib for NSCLC. PMID:26375053

  11. Detection of rearrangement of anaplastic lymphoma kinase (ALK) and mutation of epidermal growth factor receptor (EGFR) in primary pulmonary lymphoepithelioma-like carcinoma

    PubMed Central

    Wang, Liang; Lin, Yongbin; Cai, Qingqing; Long, Hao; Zhang, Yu; Rong, Tiehua

    2015-01-01

    Background Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a distinct rare subtype of lung cancer. The prevalence of anaplastic lymphoma kinase (ALK) rearrangement and epidermal growth factor receptor (EGFR) mutation in primary pulmonary LELC had not been thoroughly investigated. Methods We investigated a cohort of 42 patients with primary pulmonary LELC and genotyped for ALK rearrangement and EGFR mutation. ALK rearrangement was detected by fluorescence in situ hybridization (FISH). EGFR mutational analysis of exons 18 through 21 was analyzed by TaqMan real-time polymerase chain reaction (PCR). Results Epstein-Barr virus-encoded RNAs (EBERs) showed positive signals in all 42 patients. By immunohistochemistry staining, all patients demonstrated positive expression of CK5/6 and P63, but almost all patients were negative for TTF-1 (34/34, 100%) or CK7 (34/35, 97.1%). None of the 42 patients had ALK rearrangement. Of 42 patients tested, only one patient (2.4%) harbored L858R mutation and gefitinib was applied to this case, however no objective response was observed and the progression free survival (PFS) time was only 1 month. Conclusions Primary pulmonary LELC is a unique histological subtype of lung cancer. ALK rearrangement and EGFR mutation are lack and they may not be the oncogenic driver gene in pulmonary LELC. Future efforts should be made to explore other oncogenic driver gene to guide targeted therapy in this rare disease to determine the optimal treatment. Keywords Pulmonary lymphoepithelioma-like carcinoma (LELC); anaplastic lymphoma kinase (ALK); epidermal growth factor receptor (EGFR); targeted therapy; Epstein-Barr virus (EBV) PMID:26543602

  12. Investigating molecular dynamics-guided lead optimization of EGFR inhibitors.

    PubMed

    Lavecchia, Martin J; Puig de la Bellacasa, Raimon; Borrell, José I; Cavasotto, Claudio N

    2016-02-15

    The epidermal growth factor receptor (EGFR) is part of an extended family of proteins that together control aspects of cell growth and development, and thus a validated target for drug discovery. We explore in this work the suitability of a molecular dynamics-based end-point binding free energy protocol to estimate the relative affinities of a virtual combinatorial library designed around the EGFR model inhibitor 6{1} as a tool to guide chemical synthesis toward the most promising compounds. To investigate the validity of this approach, selected analogs including some with better and worse predicted affinities relative to 6{1} were synthesized, and their biological activity determined. To understand the binding determinants of the different analogs, hydrogen bonding and van der Waals contributions, and water molecule bridging in the EGFR-analog complexes were analyzed. The experimental validation was in good qualitative agreement with our theoretical calculations, while also a 6-dibromophenyl-substituted compound with enhanced inhibitory effect on EGFR compared to the reference ligand was obtained. PMID:26810832

  13. EGFR-mutated lung cancer in Li-Fraumeni syndrome.

    PubMed

    Michalarea, Vasiliki; Calcasola, Matthew; Cane, Paul; Tobal, Khalid; Izatt, Louise; Spicer, James

    2014-09-01

    This is a revised case report of a 52 year old Caucasian female with Li-Fraumeni syndrome with a rare TP53 mutation, who was treated for breast cancer and later developed epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer. PMID:25047674

  14. Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging

    NASA Astrophysics Data System (ADS)

    Gao, Jing; Wang, Ye; Cai, Mingjun; Pan, Yangang; Xu, Haijiao; Jiang, Junguang; Ji, Hongbin; Wang, Hongda

    2015-01-01

    The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR activation state, whereas suspended COS-7 cells exhibit a moderate clustering state and a homogeneous distribution pattern, indicating that the external environment surrounding the cell membrane is the decisive factor in the EGFR clustering pattern. A dual-color dSTORM image reveals the significant colocalization of EGFR and lipid rafts; interestingly MβCD treatment leads to a dramatic decrease of the amount and size of EGFR clusters on both apical and basal surfaces, highlighting a key role of lipid rafts in EGFR cluster formation. Altogether, our results illustrate the distribution pattern of EGFR in polarized cells and uncover the essential role of lipid rafts in EGFR cluster maintenance.The clustering of membrane receptors such as EGFR is critical for various biological processes, for example cell signaling and tumorigenesis. However, the mechanism involved remains poorly understood. Here, we used a super resolution imaging technique, which has shattered the longstanding resolution barrier of light diffraction, to investigate the distribution of membrane EGFR on apical or basal surfaces of COS-7 cells and on the surface of suspended COS-7 cells. Our data show that more and larger EGFR clusters are detected on the apical surface in comparison with those on the basal surface and this difference is not affected by the EGFR

  15. Fish meals, fish components, and fish protein hydrolysates as potential ingredients in pet foods.

    PubMed

    Folador, J F; Karr-Lilienthal, L K; Parsons, C M; Bauer, L L; Utterback, P L; Schasteen, C S; Bechtel, P J; Fahey, G C

    2006-10-01

    An experiment to determine the chemical composition and protein quality of 13 fish substrates (pollock by-products, n = 5; fish protein hydrolysates, n = 5; and fish meals, n = 3) was conducted. Two of these substrates, salmon protein hydrolysate (SPH) and salmon meal with crushed bones (SMB), were used to determine their palatability as components of dog diets. Pollock by-products differed in concentrations of CP, crude fat, and total AA by 71, 79, and 71%, respectively, and GE by 4.1 kcal/g. Fish protein hydrolysates and fish meals were less variable (approximately 18, 14, and 17%, and 1.4 kcal/g, respectively). Biogenic amine concentrations were much higher in fish protein hydrolysates as compared with pollock by-products and fish meals. Pollock liver and viscera had the highest total fatty acid concentrations; however, red salmon hydrolysate and SMB had the highest total PUFA concentrations (49.63 and 48.60 mg/g, respectively). Salmon protein hydrolysate had the highest protein solubility in 0.2% KOH. Based on calculations using immobilized digestive enzyme assay values, lysine digestibility of fish meal substrates was comparable to in vivo cecectomized rooster assay values and averaged approximately 90.3%. Also, pollock milt, pollock viscera, red salmon hydrolysate, and sole hydrolysate had comparable values as assessed by immobilized digestive enzyme assay and rooster assays. A chick protein efficiency ratio (PER) assay compared SMB and SPH to a whole egg meal control and showed that SMB had high protein quality (PER = 3.5), whereas SPH had poor protein quality (PER value less than 1.5). However, using whole egg meal as the reference protein, both fish substrates were found to be good protein sources with an essential AA index of 1.0 and 0.9 for SMB and SPH, respectively. In the dog palatability experiments, a chicken-based control diet and 2 diets containing 10% of either SPH or SMB were tested. Dogs consumed more of the SPH diet compared with the control

  16. The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling

    PubMed Central

    Asbagh, Layka Abbasi; Vazquez, Iria; Vecchione, Loredana; Budinska, Eva; De Vriendt, Veerle; Baietti, Maria Francesca; Steklov, Mikhail; Jacobs, Bart; Hoe, Nicholas; Singh, Sharat; Imjeti, Naga-Sailaja; Zimmermann, Pascale

    2014-01-01

    Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies. PMID:25301722

  17. Clinical activity of the mutant-selective EGFR inhibitor AZD9291 in patients with EGFR inhibitor-resistant non-small cell lung cancer.

    PubMed

    Jiang, Tao; Zhou, Caicun

    2014-12-01

    The first generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in advanced non-small cell lung cancer (NSCLC) with EGFR mutations. Unfortunately, disease progression generally occurs after 9 to 14 months of targeted therapy. The substitution of threonine with methionine at amino acid position 790 (T790M), as the second mutation in EGFR, is the most common resistance mechanism and is detected in tumor cells from more than 50-60% of patients after disease progression. However, current targeted therapeutic strategies for patients with acquired resistance are limited. This has led to the development of "third generation" EGFR-TKIs that are designed to target T790M and EGFR-TKI sensitizing mutations more selectively than wild-type. AZD9291, as a mono-anilino-pyrimidine compound, is a novel, irreversible EGFR-TKI, has proved to be more effective against both EGFR-TKI sensitizing and resistance T790M mutations in preclinical models. This phase I clinical study showed that AZD9291 has robust efficacy and is well tolerated in EGFR mutant NSCLC patients with acquired resistance to EGFR-TKIs. PMID:25806323

  18. Fish Dishes.

    ERIC Educational Resources Information Center

    Derby, Marie

    2003-01-01

    Describes an art project that was inspired by Greek pottery, specifically dishes shaped as fish. Explains that fourth-grade students drew a fish shape that was later used to create their clay version of the fish. Discusses how the students examined the pottery to make decisions about color and design. (CMK)

  19. ADAM-mediated amphiregulin shedding and EGFR transactivation

    PubMed Central

    Kasina, S.; Scherle, P. A.; Hall, C. L.; Macoska, J. A.

    2011-01-01

    Introduction The ectodomain shedding of epidermal growth factor receptor (EGFR) ligands, such as amphiregulin (AREG), by ADAMs (A Disintegrin And Metalloproteases) can be stimulated by G protein-coupled receptor (GPCR) agonists. Interactions between the CXCR4 GPCR and the CXCL12 chemokine have been shown to mediate gene transcription and cellular proliferation in non-transformed and transformed prostate epithelial cells, as well as motility/invasiveness in transformed cells. Objectives In this report, we investigated the ability of CXCL12 to stimulate amphiregulin ectodomain shedding in non-transformed and transformed prostate epithelial cells that respond proliferatively to sub-nanomolar levels of CXCL12 and amphiregulin. Materials and Methods Non-transformed N15C6 and transformed PC3 prostate epithelial cells were assessed for amphiregulin shedding, ADAM activation, Src phosphorylation and EGFR activation using ELISA, immunoblot, and immunoprecipitation techniques, and for proliferation using cell counting after stimulation with CXCL12 or vehicle. Results The results of these studies identify CXCL12 as a novel inducer of amphiregulin ectodomain shedding and show that both basal and CXCL12-mediated amphiregulin shedding are ADAM10- and Src kinase-dependent in non-transformed N15C6 cells. In contrast, amphiregulin shedding is not amplified subsequent to stimulation with exogenous CXCL12, and is not reduced subsequent to metalloprotease- or Src kinase-inhibition, in highly aggressive PC3 prostate cancer cells. These data also show that CXCL12-mediated cellular proliferation requires EGFR transactivation in a Src-and ADAM-dependent manner in non-transformed prostate epithelial cells. However, these same mechanisms are dysfunctional in highly transformed prostate cancer cells, which secrete amphiregulin in an autocrine manner that cannot be repressed through metalloprotease- or Src kinase inhibition. Conclusion These findings show that non-transformed and transformed

  20. Epidermal EGFR controls cutaneous host defense and prevents inflammation.

    PubMed

    Lichtenberger, Beate M; Gerber, Peter A; Holcmann, Martin; Buhren, Bettina A; Amberg, Nicole; Smolle, Viktoria; Schrumpf, Holger; Boelke, Edwin; Ansari, Parinaz; Mackenzie, Colin; Wollenberg, Andreas; Kislat, Andreas; Fischer, Jens W; Röck, Katharina; Harder, Jürgen; Schröder, Jens M; Homey, Bernhard; Sibilia, Maria

    2013-08-21

    The epidermal growth factor receptor (EGFR) plays an important role in tissue homeostasis and tumor progression. However, cancer patients treated with EGFR inhibitors (EGFRIs) frequently develop acneiform skin toxicities, which are a strong predictor of a patient's treatment response. We show that the early inflammatory infiltrate of the skin rash induced by EGFRI is dominated by dendritic cells, macrophages, granulocytes, mast cells, and T cells. EGFRIs induce the expression of chemokines (CCL2, CCL5, CCL27, and CXCL14) in epidermal keratinocytes and impair the production of antimicrobial peptides and skin barrier proteins. Correspondingly, EGFRI-treated keratinocytes facilitate lymphocyte recruitment but show a considerably reduced cytotoxic activity against Staphylococcus aureus. Mice lacking epidermal EGFR (EGFR(Δep)) show a similar phenotype, which is accompanied by chemokine-driven skin inflammation, hair follicle degeneration, decreased host defense, and deficient skin barrier function, as well as early lethality. Skin toxicities were not ameliorated in a Rag2-, MyD88-, and CCL2-deficient background or in mice lacking epidermal Langerhans cells. The skin phenotype was also not rescued in a hairless (hr/hr) background, demonstrating that skin inflammation is not induced by hair follicle degeneration. Treatment with mast cell inhibitors reduced the immigration of T cells, suggesting that mast cells play a role in the EGFRI-mediated skin pathology. Our findings demonstrate that EGFR signaling in keratinocytes regulates key factors involved in skin inflammation, barrier function, and innate host defense, providing insights into the mechanisms underlying EGFRI-induced skin pathologies. PMID:23966300

  1. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

    PubMed Central

    George, Amee J.; Purdue, Brooke W.; Gould, Cathryn M.; Thomas, Daniel W.; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A.; Morgan, Kylie A.; Simpson, Kaylene J.; Thomas, Walter G.; Hannan, Ross D.

    2013-01-01

    Summary The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R–EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R–EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R–EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR–EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  2. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation.

    PubMed

    George, Amee J; Purdue, Brooke W; Gould, Cathryn M; Thomas, Daniel W; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A; Morgan, Kylie A; Simpson, Kaylene J; Thomas, Walter G; Hannan, Ross D

    2013-12-01

    The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  3. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation.

    PubMed

    Faria, Jerusa A Q A; de Andrade, Carolina; Goes, Alfredo M; Rodrigues, Michele A; Gomes, Dawidson A

    2016-09-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. PMID:27462018

  4. Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive downstream signaling networks

    PubMed Central

    Chakraborty, Sharmistha; Li, Li; Puliyappadamba, VineshkumarThidil; Guo, Gao; Hatanpaa, Kimmo J.; Mickey, Bruce; Souza, Rhonda F.; Vo, Peggy; Herz, Joachim; Chen, Mei-Ru; Boothman, David A.; Pandita, Tej K.; Wang, David H.; Sen, Ganes C.; Habib, Amyn A.

    2014-01-01

    EGFR overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling. Constitutive or non-canonical EGFR signaling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1, and TRAIL. Ligand-mediated activation of EGFR switches off IRF3 dependent transcription, activates canonical ERK and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of EGF dissociates TBK1, IRF3, and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signaling in glioblastoma. PMID:25503978

  5. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway

    PubMed Central

    AMONYINGCHAROEN, SUMET; SURIYO, TAWIT; THIANTANAWAT, APINYA; WATCHARASIT, PIYAJIT; SATAYAVIVAD, JUTAMAAD

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1–40 μM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth. PMID:25815516

  6. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

    PubMed Central

    Jiang, Huaqi; Grenley, Marc O.; Bravo, Maria-Jose; Blumhagen, Rachel Z.; Edgar, Bruce A.

    2010-01-01

    Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thus maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration following enteric infection by the bacterium, Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis. PMID:21167805

  7. Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping

    PubMed Central

    2013-01-01

    Background Epidermal growth factor receptor (EGFR)-activating mutations are major determinants in predicting the tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). Noninvasive test for the detection of EGFR mutations is required, especially in NSCLC patients from whom tissue is not available. In this study, we assessed the feasibility of detection of EGFR mutations in free DNA circulating in plasma. Methods Plasma samples of 60 patients with partial response to gefitinib were analyzed to detect EGFR-activating mutations in exons 19 and 21. Forty (66.7%) of patients had tumor EGFR mutation results. EGFR mutations in plasma were detected using the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method. All clinical data and plasma samples were obtained from 11 centers of the Korean Molecular Lung Cancer Group (KMLCG). Results Of the 60 patients, 39 were female and the median age was 62.5 years. Forty-three patients never smoked, 53 had adenocarcinomas, and seven had other histologic types. EGFR-activating mutation was detected in plasma of 10 cases (exon 19 deletion in seven and exon 21 L858R point mutation in three). It could not be found in plasma after treatment for 2 months. When only patients with confirmed EGFR mutation in tumor were analyzed, 17% (6 of 35) of them showed positive plasma EGFR mutation and the mutation type was completely matched with that in tumor. There was no statistically significant difference in clinical parameters between patients with EGFR mutations in plasma and those without EGFR mutations. Conclusions The detection rate of EGFR mutations from plasma was not so high despite highly sensitive EGFR mutation test suggesting that more advances in detection methods and further exploration of characteristics of circulating free DNA are required. PMID:23927790

  8. Antibodies specifically targeting a locally misfolded region of tumor associated EGFR.

    PubMed

    Garrett, Thomas P J; Burgess, Antony W; Gan, Hui K; Luwor, Rod B; Cartwright, Glenn; Walker, Francesca; Orchard, Suzanne G; Clayton, Andrew H A; Nice, Edouard C; Rothacker, Julie; Catimel, Bruno; Cavenee, Webster K; Old, Lloyd J; Stockert, Elisabeth; Ritter, Gerd; Adams, Timothy E; Hoyne, Peter A; Wittrup, Dane; Chao, Ginger; Cochran, Jennifer R; Luo, Cindy; Lou, Mezhen; Huyton, Trevor; Xu, Yibin; Fairlie, W Douglas; Yao, Shenggen; Scott, Andrew M; Johns, Terrance G

    2009-03-31

    Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells. PMID:19289842

  9. Targeted Therapy Against VEGFR and EGFR With ZD6474 Enhances the Therapeutic Efficacy of Irradiation in an Orthotopic Model of Human Non-Small-Cell Lung Cancer

    SciTech Connect

    Shibuya, Keiko; Komaki, Ritsuko; Shintani, Tomoaki; Itasaka, Satoshi; Ryan, Anderson; Juergensmeier, Juliane M.; Milas, Luka; Ang, Kian; Herbst, Roy S.; O'Reilly, Michael S.

    2007-12-01

    Purpose: Conventional therapies for patients with lung cancer have reached a therapeutic plateau. We therefore evaluated the feasibility of combined vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and epidermal growth factor (EGF) receptor (EGFR) targeting with radiation therapy in an orthotopic model that closely recapitulates the clinical presentation of human lung cancer. Methods and Materials: Effects of irradiation and/or ZD6474, a small-molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were studied in vitro for human lung adenocarcinoma cells by using proliferation and clonogenic assays. The feasibility of combining ZD6474 with radiation therapy was then evaluated in an orthotopic model of human lung adenocarcinoma. Lung tumor burden and spread within the thorax were assessed, and tumor and adjacent tissues were analyzed by means of immunohistochemical staining for multiple parameters, including CD31, VEGF, VEGFR2, EGF, EGFR, matrix metalloproteinase-2 and -9, and basic fibroblast growth factor. Results: ZD6474 enhanced the radioresponse of NCI-H441 human lung adenocarcinoma cells by a factor of 1.37 and markedly inhibited sublethal damage repair. In vivo, the combined blockade of VEGFR2 and EGFR by ZD6474 blocked pleural effusion formation and angiogenesis and enhanced the antivascular and antitumor effects of radiation therapy in the orthotopic human lung cancer model and was superior to chemoradiotherapy. Conclusions: When radiation therapy is combined with VEGFR2 and EGFR blockade, significant enhancement of antiangiogenic, antivascular, and antitumor effects are seen in an orthotopic model of lung cancer. These data provide support for clinical trials of biologically targeted and conventional therapies for human lung cancer.

  10. Toward discovery of mutant EGFR inhibitors; Design, synthesis and in vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives.

    PubMed

    Mowafy, Samar; Galanis, A; Doctor, Zainab M; Paranal, Raymond M; Lasheen, Deena S; Farag, Nahla A; Jänne, Pasi A; Abouzid, Khaled A M

    2016-08-15

    A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type (WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type (EGFR WT) with IC50 values in the low nanomolar range (<0.495-9.05nM) and displayed more potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed. Compounds 4d, 6f, 7e showed significant inhibition (IC50=1.76-2.38μM) in these mutant lines and significant Her2 enzyme inhibition (IC50=19.2-40.6nM) compared to lapatinib (60.1nM). The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested, compounds 6f and 7e showed about eight and three folds respectively greater potency than gefitinib. Our structure-activity relationships (SAR) studies suggested that presence of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for developing inhibitors against wild type and mutant EGFR. PMID:27288180

  11. Longitudinal monitoring of EGFR mutations in plasma predicts outcomes of NSCLC patients treated with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02).

    PubMed

    Lee, Ji Yun; Qing, Xu; Xiumin, Wei; Yali, Bai; Chi, Sangah; Bak, So Hyeon; Lee, Ho Yun; Sun, Jong-Mu; Lee, Se-Hoon; Ahn, Jin Seok; Cho, Eun Kyung; Kim, Dong-Wan; Kim, Hye Ryun; Min, Young Joo; Jung, Sin-Ho; Park, Keunchil; Mao, Mao; Ahn, Myung-Ju

    2016-02-01

    We hypothesized that plasma-based EGFR mutation analysis for NSCLC may be feasible for monitoring treatment response to EGFR TKIs and also predict drug resistance.Clinically relevant mutations including exon 19 deletion (ex19del), L858R and T790M were analyzed using droplet digital PCR (ddPCR) in longitudinally collected plasma samples (n = 367) from 81 NSCLC patients treated with EGFR TKI. Of a total 58 baseline cell-free DNA (cfDNA) samples available for ddPCR analysis, 43 (74.1%) had the same mutation in the matched tumors (clinical sensitivity: 70.8% [17/24] for L858R and 76.5% [26/34] for ex19del). The concordance rates of plasma with tissue-based results of EGFR mutations were 87.9% for L858R and 86.2% for ex19del. All 40 patients who were detected EGFR mutations at baseline showed a dramatic decrease of mutant copies (>50%) in plasma during the first two months after treatment. Median progression-free survival (PFS) was 10.1 months for patients with undetectable EGFR v 6.3 months for detectable EGFR mutations in blood after two-month treatment (HR 3.88, 95% CI 1.48-10.19, P = 0.006). We observed emerging resistance with early detection of T790M as a secondary mutation in 14 (28.6%) of 49 patients. Plasma-based EGFR mutation analysis using ddPCR can monitor treatment response to EGFR TKIs and can lead to early detection of EGFR TKIs resistance. Further studies confirming clinical implications of EGFR mutation in plasma are warranted to guide optimal therapeutic strategies upon knowledge of treatment response and resistance. PMID:26755650

  12. EGFR protein expression using a specific intracellular domain antibody and PTEN and clinical outcomes in squamous cell lung cancer patients with EGFR-tyrosine kinase inhibitor therapy

    PubMed Central

    Chang, Hyun; Oh, Jisu; Zhang, Xianglan; Kim, Yu Jung; Lee, Jae Ho; Lee, Choon-Taek; Chung, Jin-haeng; Lee, Jong-Seok

    2016-01-01

    Purpose The aim of this research was to examine the molecular and clinical features that are related with EGFR-tyrosine kinase inhibitor (EGFR-TKI) efficacy in previously treated patients with squamous cell carcinoma of the lung (SCCL). Materials and methods This retrospective study included 67 SCCL patients with obtainable lung cancer tissue and records on EGFR-TKI treatment response and survival. EGFR protein expression in lung cancer tissue was measured by immunohistochemistry with a specific antibody that recognizes the intracellular domain (ID) of EGFR. PTEN expression in lung cancer tissue was also evaluated with immunohistochemistry. PI3KCA gene amplification was detected by quantitative real-time polymerase chain reaction, and FGFR1 amplification was assessed by fluorescent in situ hybridization. Results EGFR ID expression (hazard ratio [HR] 0.53, P=0.022) and Eastern Cooperative Oncology Group (ECOG) performance status (PS) (HR 0.43, P=0.022) were significantly related with progression-free survival following EGFR-TKIs treatment. PTEN expression (HR 0.52, P=0.025) was significantly related to overall survival. The group of EGFR-positive or PTEN-positive patients with ECOG PS of 0 or 1 had better clinical outcomes than patients who were EGFR-negative and PTEN-negative or who had poor ECOG PS with longer median progression-free survival (2.1 vs 1.0 months, P=0.05) and overall survival (6.2 vs 2.1 months, P=0.05). Conclusion EGFR expression using an ID-specific antibody and PTEN protein expression may be used to identify SCCL patients who might benefit from EGFR-TKI treatment. PMID:27578983

  13. Longitudinal monitoring of EGFR mutations in plasma predicts outcomes of NSCLC patients treated with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02)

    PubMed Central

    Xiumin, Wei; Yali, Bai; Chi, Sangah; Bak, So Hyeon; Lee, Ho Yun; Sun, Jong-Mu; Lee, Se-Hoon; Ahn, Jin Seok; Cho, Eun Kyung; Kim, Dong-Wan; Kim, Hye Ryun; Min, Young Joo; Jung, Sin-Ho; Park, Keunchil; Mao, Mao; Ahn, Myung-Ju

    2016-01-01

    We hypothesized that plasma-based EGFR mutation analysis for NSCLC may be feasible for monitoring treatment response to EGFR TKIs and also predict drug resistance. Clinically relevant mutations including exon 19 deletion (ex19del), L858R and T790M were analyzed using droplet digital PCR (ddPCR) in longitudinally collected plasma samples (n = 367) from 81 NSCLC patients treated with EGFR TKI. Of a total 58 baseline cell-free DNA (cfDNA) samples available for ddPCR analysis, 43 (74.1%) had the same mutation in the matched tumors (clinical sensitivity: 70.8% [17/24] for L858R and 76.5% [26/34] for ex19del). The concordance rates of plasma with tissue-based results of EGFR mutations were 87.9% for L858R and 86.2% for ex19del. All 40 patients who were detected EGFR mutations at baseline showed a dramatic decrease of mutant copies (>50%) in plasma during the first two months after treatment. Median progression-free survival (PFS) was 10.1 months for patients with undetectable EGFR v 6.3 months for detectable EGFR mutations in blood after two-month treatment (HR 3.88, 95% CI 1.48-10.19, P = 0.006). We observed emerging resistance with early detection of T790M as a secondary mutation in 14 (28.6%) of 49 patients. Plasma-based EGFR mutation analysis using ddPCR can monitor treatment response to EGFR TKIs and can lead to early detection of EGFR TKIs resistance. Further studies confirming clinical implications of EGFR mutation in plasma are warranted to guide optimal therapeutic strategies upon knowledge of treatment response and resistance. PMID:26755650

  14. Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell-Free DNA from Patients with Advanced Non-Small Cell Lung Cancer.

    PubMed

    Zhu, Guanshan; Ye, Xin; Dong, Zhengwei; Lu, Ya Chao; Sun, Yun; Liu, Yi; McCormack, Rose; Gu, Yi; Liu, Xiaoqing

    2015-05-01

    Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing. PMID:25769900

  15. Identification of Potent EGFR Inhibitors from TCM Database@Taiwan

    PubMed Central

    Yang, Shun-Chieh; Chang, Su-Sen; Chen, Hsin-Yi; Chen, Calvin Yu-Chian

    2011-01-01

    Overexpression of epidermal growth factor receptor (EGFR) has been associated with cancer. Targeted inhibition of the EGFR pathway has been shown to limit proliferation of cancerous cells. Hence, we employed Traditional Chinese Medicine Database (TCM Database@Taiwan) (http://tcm.cmu.edu.tw) to identify potential EGFR inhibitor. Multiple Linear Regression (MLR), Support Vector Machine (SVM), Comparative Molecular Field Analysis (CoMFA), and Comparative Molecular Similarities Indices Analysis (CoMSIA) models were generated using a training set of EGFR ligands of known inhibitory activities. The top four TCM candidates based on DockScore were 2-O-caffeoyl tartaric acid, Emitine, Rosmaricine, and 2-O-feruloyl tartaric acid, and all had higher binding affinities than the control Iressa®. The TCM candidates had interactions with Asp855, Lys716, and Lys728, all which are residues of the protein kinase binding site. Validated MLR (r2 = 0.7858) and SVM (r2 = 0.8754) models predicted good bioactivity for the TCM candidates. In addition, the TCM candidates contoured well to the 3D-Quantitative Structure-Activity Relationship (3D-QSAR) map derived from the CoMFA (q2 = 0.721, r2 = 0.986) and CoMSIA (q2 = 0.662, r2 = 0.988) models. The steric field, hydrophobic field, and H-bond of the 3D-QSAR map were well matched by each TCM candidate. Molecular docking indicated that all TCM candidates formed H-bonds within the EGFR protein kinase domain. Based on the different structures, H-bonds were formed at either Asp855 or Lys716/Lys728. The compounds remained stable throughout molecular dynamics (MD) simulation. Based on the results of this study, 2-O-caffeoyl tartaric acid, Emitine, Rosmaricine, and 2-O-feruloyl tartaric acid are suggested to be potential EGFR inhibitors. PMID:22022246

  16. Design and Synthesis of Novel Schiff Base-Benzothiazole Hybrids as Potential Epidermal Growth Factor Receptor (EGFR) Inhibitors.

    PubMed

    Singh, Meenakshi; Singh, Sudhir Kumar; Thakur, Bhushan; Ray, Pritha; Singh, Sushil K

    2016-01-01

    A series of novel Schiff bases -benzothiazole hybrids was designed, synthesized and evaluated for their anticancer activity by MTT assay and western blot method. Antiproliferative screening indicated that compound containing dihydroxy substituents had potent inhibitory activity with IC50 value 34µg/ml against SKOV3, A2780-S and A2780-CR cell lines. It showed more potent cytotoxicity in combination with cisplatin and paclitaxel than alone in the selected cell lines (SKOV3, A2780 and A2780-CR models). The in vitro cytotoxicity of the compounds on IOSE 364 cell line was evaluated to establish the selectivity. Molecular docking study exhibited good binding against epidermal growth factor receptor, which was further ascertained by immunoblot assay using specific antibody against phosphorylated EGFR, and thus unravelling the targeted anticancer mechanism. PMID:26443027

  17. Fish Rhabdoviruses

    USGS Publications Warehouse

    Kurath, G.; Winton, J.

    2008-01-01

    Many important viral pathogens of fish are members of the family Rhabdoviridae. The viruses in this large group cause significant losses in populations of wild fish as well as among fish reared in aquaculture. Fish rhabdoviruses often have a wide host and geographic range, and infect aquatic animals in both freshwater and seawater. The fish rhabdoviruses comprise a diverse collection of isolates that can be placed in one of two quite different groups: isolates that are members of the established genusNovirhabdovirus, and those that are most similar to members of the genus Vesiculovirus. Because the diseases caused by fish rhabdoviruses are important to aquaculture, diagnostic methods for their detection and identification are well established. In addition to regulations designed to reduce the spread of fish viruses, a significant body of research has addressed methods for the control or prevention of diseases caused by fish rhabdoviruses, including vaccination. The number of reported fish rhabdoviruses continues to grow as a result of the expansion of aquaculture, the increase in global trade, the development of improved diagnostic methods, and heightened surveillance activities. Fish rhabdoviruses serve as useful components of model systems to study vertebrate virus disease, epidemiology, and immunology.

  18. Fish flavor.

    PubMed

    Kawai, T

    1996-02-01

    This article reviews features of flavor in three groups of fishes and summarizes them as follows: (1) fresh saltwater fish are nearly odorless because they contain a small quantity of volatiles; (2 freshwater fish give off pyrrolidine and earthy-odor compounds, which are responsible for their maturity and surrounding water pollution, and (3) euryhaline fish exhibit a variety of unsaturated carbonyls and alcohols derived from enzymatic and nonenzymatic oxidation of polyunsaturated fatty acids (PAs). These features are discussed, as are the effects of different enzymatic activities on PA oxidation and the effects of pH on mechanisms of formation of the volatiles. The monotonous volatile constitution of saltwater fish is likely caused by an unknown antioxidation system restraining the fish from oxidizing. The variety of constitution of euryhaline fish, especially that of anadromous fish under spawning conditions, could result from the loss of that system. The thermal environments of heated foods are also reviewed. The basic environment of fish, which allows the formation of flavor compounds, is discussed to confirm the volatiles found in unheated fish. PMID:8744606

  19. Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR

    PubMed Central

    Campbell, Patrick; Morton, Penny; Takeichi, Takuya; Salam, Amr; Roberts, Nerys; Proudfoot, Laura E.; Mellerio, Jemima E.; Aminu, Kingi; Wellington, Cheryl; Patil, Sachin N.; Akiyama, Masashi; Liu, Lu; McMillan, James R.; Aristodemou, Sophia; Ishida-Yamamoto, Akemi; Abdul-Wahab, Alya; Petrof, Gabriela; Fong, Kenneth; Harnchoowong, Sarawin; Stone, Kristina; Harper, John I.; McLean, W. H. Irwin; Simpson, Michael A.; Parsons, Maddy; McGrath, John A.

    2014-01-01

    Epidermal growth factor receptor (EGFR) signaling is fundamentally important for tissue homeostasis through EGFR/ligand interactions that stimulate numerous signal transduction pathways. Aberrant EGFR signaling has been reported in inflammatory and malignant diseases but thus far no primary inherited defects in EGFR have been recorded. Using whole-exome sequencing, we identified a homozygous loss-of-function missense mutation in EGFR (c.1283G>A; p.Gly428Asp) in a male infant with life-long inflammation affecting the skin, bowel and lungs. During the first year of life, his skin showed erosions, dry scale, and alopecia. Subsequently, there were numerous papules and pustules – similar to the rash seen in patients receiving EGFR inhibitor drugs. Skin biopsy demonstrated an altered cellular distribution of EGFR in the epidermis with reduced cell membrane labeling, and in vitro analysis of the mutant receptor revealed abrogated EGFR phosphorylation and EGF-stimulated downstream signaling. Microarray analysis on the patient’s skin highlighted disturbed differentiation/premature terminal differentiation of keratinocytes and upregulation of several inflammatory/innate immune response networks. The boy died aged 2.5 years from extensive skin and chest infections as well as electrolyte imbalance. This case highlights the major mechanism of epithelial dysfunction following EGFR signaling ablation and illustrates the broader impact of EGFR inhibition on other tissues. PMID:24691054

  20. Endophilin B1 regulates EGFR endocytic degradation in prostate cancer cell.

    PubMed

    Zhu, J-Y; Xiong, Y; Zhang, W; Wan, J; Wan, J

    2016-01-01

    Prostate cancer (Pca) is one of the most common types of cancer for elder men. Aberrant expression of epidermal growth factor receptor (EGFR) and EGFR downstream signaling have been known to contribute to disease progression in prostate cancer. EGF-stimulated EGFR is internalized and the process of endocytic degradation of EGFR mediates its signaling which is frequently dysregulated in many kinds of cancer. In the present study, we demonstrated that endophilin B1 expression was inhibited and EGFR expression was significantly increased in prostate cancer cell lines. We demonstrated that suppression of endophilin B1 increased EGFR levels via delaying EGFR internalization triggered by EGF and its intracellular degradation. Endophilin B1 decreased also sustained EGFR downstream signaling such as Erk1/2 phosphorylation in response to EGF stimulation and promoted prostate cancer cell proliferation which is EGF independent. Our data indicated that endophilin B1 mediated the biological function of EGFR in cancer cell proliferation through regulating the EGFR endocytic trafficking and downstream signaling. PMID:27609472

  1. An Unbiased Screen Identifies the DEP-1 Tumour Suppressor as a Phosphatase Controlling EGFR Endocytosis

    PubMed Central

    Tarcic, Gabi; Boguslavsky, Shlomit K.; Wakim, Jean; Kiuchi, Tai; Liu, Angela; Reinitz, Felicia; Nathanson, David; Takahashi, Takamune; Mischel, Paul S.; Ng, Tony; Yarden, Yosef

    2009-01-01

    Background The epidermal growth factor (EGF) stimulates rapid tyrosine phosphorylation of EGF-receptor (EGFR). This event precedes signalling from both the plasma membrane and from endosomes, and it is essential for recruitment of an ubiquitin ligase, CBL, that sorts activated receptors to endosomes and degradation. Because hyper-phosphorylation of EGFR is involved in oncogenic pathways, we performed an unbiased screen of siRNA oilgonucleotides targeting all human tyrosine phosphatases. Results We report the identification of PTPRK and PTPRJ (DEP-1) as EGFR-targeting phosphatases. DEP-1 is a tumour suppressor that dephosphorylates, thereby stabilizes EGFR by hampering its ability to associate with the CBL-GRB2 ubiquitin ligase complex. DEP-1 silencing enhanced tyrosine phosphorylation of endosomal EGFRs and, accordingly, increased cell proliferation. In line with functional interactions, EGFR and DEP-1 form physical associations, and EGFR phosphorylates a substrtae trapping mutant of DEP-1. Interestingly, the interactions of DEP-1 and EGFR are followed by physical segregation: whereas EGFR undergoes endocytosis, DEP-1 remains confined to the cell surface. Conclusions EGFR and DEP-1 physically interact at the cell surface and maitain bidirectional enzyme-substrate interactions, which are relevant to their respective oncogenic and tumor suppressive functions. These observations highlight the emerging roles of vesicular trafficking in malignant processes. PMID:19836242

  2. Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR.

    PubMed

    Campbell, Patrick; Morton, Penny E; Takeichi, Takuya; Salam, Amr; Roberts, Nerys; Proudfoot, Laura E; Mellerio, Jemima E; Aminu, Kingi; Wellington, Cheryl; Patil, Sachin N; Akiyama, Masashi; Liu, Lu; McMillan, James R; Aristodemou, Sophia; Ishida-Yamamoto, Akemi; Abdul-Wahab, Alya; Petrof, Gabriela; Fong, Kenneth; Harnchoowong, Sarawin; Stone, Kristina L; Harper, John I; McLean, W H Irwin; Simpson, Michael A; Parsons, Maddy; McGrath, John A

    2014-10-01

    Epidermal growth factor receptor (EGFR) signaling is fundamentally important for tissue homeostasis through EGFR/ligand interactions that stimulate numerous signal transduction pathways. Aberrant EGFR signaling has been reported in inflammatory and malignant diseases, but thus far no primary inherited defects in EGFR have been recorded. Using whole-exome sequencing, we identified a homozygous loss-of-function missense mutation in EGFR (c.1283 G>A; p.Gly428Asp) in a male infant with lifelong inflammation affecting the skin, bowel, and lungs. During the first year of life, his skin showed erosions, dry scale, and alopecia. Subsequently, there were numerous papules and pustules--similar to the rash seen in patients receiving EGFR inhibitor drugs. Skin biopsy demonstrated an altered cellular distribution of EGFR in the epidermis with reduced cell membrane labeling, and in vitro analysis of the mutant receptor revealed abrogated EGFR phosphorylation and EGF-stimulated downstream signaling. Microarray analysis on the patient's skin highlighted disturbed differentiation/premature terminal differentiation of keratinocytes and upregulation of several inflammatory/innate immune response networks. The boy died at the age of 2.5 years from extensive skin and chest infections as well as electrolyte imbalance. This case highlights the major mechanism of epithelial dysfunction following EGFR signaling ablation and illustrates the broader impact of EGFR inhibition on other tissues. PMID:24691054

  3. Differential Contribution to Neuroendocrine Tumorigenesis of Parallel Egfr Signaling in Cancer Cells and Pericytes

    PubMed Central

    Nolan-Stevaux, Olivier; Truitt, Morgan C.; Pahler, Jessica C.; Olson, Peter; Guinto, Cristina; Lee, David C.; Hanahan, Douglas

    2010-01-01

    Factors associated with tumor sensitivity to epidermal growth factor receptor (EGFR) inhibitors in the context of wild-type EGFR remain elusive. This study investigates the mechanistic basis of responsiveness to EGFR inhibitors in the RIP1-Tag2 (RT2) mouse model of pancreatic neuroendocrine tumorigenesis (PNET). Upon treatment of RT2 mice with EGFR inhibitors, PNET tumors harboring wild-type, nonamplified alleles of Egfr grow at a markedly reduced rate and display a significant increase in tumor cell apoptosis, as well as reduced neovascularization. The authors identify Tgf-α and Hb-egf as key limiting mediators of separable pathological functions of Egfr in neuroendocrine tumor progression: Tgf-α mutant tumors present with an elevated apoptotic index, whereas Hb-egf mutant lesions exhibit decreased angiogenic switching and neovascularization. This study not only associates Tgf-α and Hb-egf expression with wild-type Egfr oncogenicity but also ascribes the proangiogenic activity of Egfr in this tumor model to a novel mesenchymal Hb-egf/Egfr signaling axis, whereby endothelial and pericyte-derived Hb-egf activates Egfr specifically in tumor-associated perivascular cells, leading to increased pericyte coverage of the tumor endothelium and enhanced angiogenesis. PMID:20975924

  4. Metastasis-associated PRL-3 induces EGFR activation and addiction in cancer cells

    PubMed Central

    Al-aidaroos, Abdul Qader Omer; Yuen, Hiu Fung; Guo, Ke; Zhang, Shu Dong; Chung, Tae-Hoon; Chng, Wee Joo; Zeng, Qi

    2013-01-01

    Metastasis-associated phosphatase of regenerating liver-3 (PRL-3) has pleiotropic effects in driving cancer progression, yet the signaling mechanisms of PRL-3 are still not fully understood. Here, we provide evidence for PRL-3–induced hyperactivation of EGFR and its downstream signaling cascades in multiple human cancer cell lines. Mechanistically, PRL-3–induced activation of EGFR was attributed primarily to transcriptional downregulation of protein tyrosine phosphatase 1B (PTP1B), an inhibitory phosphatase for EGFR. Functionally, PRL-3–induced hyperactivation of EGFR correlated with increased cell growth, promigratory characteristics, and tumorigenicity. Moreover, PRL-3 induced cellular addiction to EGFR signaling, as evidenced by the pronounced reversion of these oncogenic attributes upon EGFR-specific inhibition. Of clinical significance, we verified elevated PRL-3 expression as a predictive marker for favorable therapeutic response in a heterogeneous colorectal cancer (CRC) patient cohort treated with the clinically approved anti-EGFR antibody cetuximab. The identification of PRL-3–driven EGFR hyperactivation and consequential addiction to EGFR signaling opens new avenues for inhibiting PRL-3–driven cancer progression. We propose that elevated PRL-3 expression is an important clinical predictive biomarker for favorable anti-EGFR cancer therapy. PMID:23867504

  5. EGFR Mutation Analysis of Circulating Tumor DNA Using an Improved PNA-LNA PCR Clamp Method

    PubMed Central

    Watanabe, Kana; Fukuhara, Tatsuro; Tsukita, Yoko; Morita, Mami; Suzuki, Aya; Tanaka, Nobuyuki; Terasaki, Hiroshi; Nukiwa, Toshihiro

    2016-01-01

    Introduction. Rebiopsies have become more crucial in non-small cell lung cancer (NSCLC). Instead of invasive biopsies, development of collecting biological data of the tumor from blood samples is expected. We conducted a prospective study to assess the feasibility of detection of epidermal growth factor receptor (EGFR) mutation in plasma samples. Method. NSCLC patients harboring EGFR activating mutations, who were going to receive EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatment, were enrolled in this study. Plasma EGFR activating mutations and the T790M resistance mutation were analyzed by an improved PNA-LNA PCR clamp method, characterized by a 10-fold or more sensitivity compared with the original methods. Result. Six patients with wild-type EGFR and 24 patients with EGFR mutations were enrolled in this study. Pretreatment plasma samples achieved sensitivity of 79%. The 6 patients with wild-type EGFR were all negative for plasma EGFR mutations. At the time of disease progression, plasma T790M mutation was detected in 8 of 16 cases. Absence of T790M before and during TKI treatment and disappearance of activating mutations during TKI treatment were considered as predictors of EGFR-TKIs efficacy. Conclusion. We were able to detect EGFR mutations in plasma samples by using an improved PNA-LNA PCR clamp method. PMID:27478396

  6. Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data.

    PubMed

    Jorge, S E D C; Kobayashi, S S; Costa, D B

    2014-09-01

    Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC. PMID:25211582

  7. Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data.

    PubMed

    Jorge, S E D C; Kobayashi, S S; Costa, D B

    2014-11-01

    Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC. PMID:25296354

  8. Role of EGFR mutations in lung cancers: prognosis and tumor chemosensitivity.

    PubMed

    Suda, Kenichi; Mitsudomi, Tetsuya

    2015-08-01

    Lung cancers with an epidermal growth factor receptor (EGFR) gene mutation account for ~40 % of adenocarcinomas in East Asians and ~15 % of those in Caucasians and African Americans, which makes them one of the most common molecularly defined lung cancer subsets. The discriminative clinical and pathological features of lung cancers with EGFR mutations have been intensively studied, and the predictive role of an EGFR mutation for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) is well established. However, controversial issues remain regarding the clinical and therapeutic implications of EGFR mutations in lung cancers. These include the prognostic impact of the EGFR mutation, its predictive implication for successful treatment with anticancer agents other than EGFR-TKIs, appropriate cytotoxic agents for lung cancers with this mutation, and the chemosensitivity of EGFR-mutation-positive lung cancers after acquisition of resistance to EGFR-TKIs. In this review, we discuss these unanswered but important questions, referring to in vitro studies, basic research, retrospective analyses, and the results of phase III clinical trials. PMID:25983263

  9. EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing

    PubMed Central

    Francis, Joshua M.; Zhang, Cheng-Zhong; Maire, Cecile L.; Jung, Joonil; Manzo, Veronica E.; Adalsteinsson, Viktor A.; Homer, Heather; Haidar, Sam; Blumenstiel, Brendan; Pedamallu, Chandra Sekhar; Ligon, Azra H.; Love, J. Christopher; Meyerson, Matthew; Ligon, Keith L.

    2014-01-01

    Glioblastomas with EGFR amplification represent approximately 50% of newly diagnosed cases and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same tumor with implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here we applied a population-based single-cell whole genome sequencing methodology to characterize genomic heterogeneity in EGFR amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal loss-of-heterozygosity and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR Carboxyl-terminal deletions) identified in the bulk tumor segregate into non-overlapping subclonal populations. In vitro and in vivo functional studies show EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment. PMID:24893890

  10. Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

    PubMed

    Yen, Hsin-Yung; Liu, Ying-Chih; Chen, Nai-Yu; Tsai, Chia-Feng; Wang, Yi-Ting; Chen, Yu-Ju; Hsu, Tsui-Ling; Yang, Pan-Chyr; Wong, Chi-Huey

    2015-06-01

    Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation. PMID:25971727

  11. Treatment approaches for EGFR-inhibitor-resistant patients with non-small-cell lung cancer.

    PubMed

    Tan, Chee-Seng; Gilligan, David; Pacey, Simon

    2015-09-01

    Discovery of activating mutations in EGFR and their use as predictive biomarkers to tailor patient therapy with EGFR tyrosine kinase inhibitors (TKIs) has revolutionised treatment of patients with advanced EGFR-mutant non-small-cell lung cancer (NSCLC). At present, first-line treatment with EGFR TKIs (gefitinib, erlotinib, and afatinib) has been approved for patients harbouring exon 19 deletions or exon 21 (Leu858Arg) substitution EGFR mutations. These agents improve response rates, time to progression, and overall survival. Unfortunately, patients develop resistance, limiting patient benefit and posing a challenge to oncologists. Optimum treatment after progression is not clearly defined. A more detailed understanding of the biology of EGFR-mutant NSCLC and the mechanisms of resistance to targeted therapy mean that an era of treatment approaches based on rationally developed drugs or therapeutic strategies has begun. Combination approaches-eg, dual EGFR blockade-to overcome resistance have been trialled and seem to be promising but are potentially limited by toxicity. Third-generation EGFR-mutant-selective TKIs, such as AZD9291 or rociletininb, which target Thr790Met-mutant tumours, the most common mechanism of EGFR TKI resistance, have entered clinical trials, and exciting, albeit preliminary, efficacy data have been reported. In this Review, we summarise the scientific literature and evidence on therapy options after EGFR TKI treatment for patients with NSCLC, aiming to provide a guide to oncologists, and consider how to maximise therapeutic advances in outcomes in this rapidly advancing area. PMID:26370354

  12. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  13. Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule.

    PubMed

    Friedman, Mikaela; Lindström, Sara; Ekerljung, Lina; Andersson-Svahn, Helene; Carlsson, Jörgen; Brismar, Hjalmar; Gedda, Lars; Frejd, Fredrik Y; Ståhl, Stefan

    2009-10-01

    HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G4S)3 [(Gly4-Ser)3]-encoding gene fragment. The encoded 30 kDa affibody construct (ZHER2)2-(G4S)3-(ZEGFR)2, with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed. PMID:19492986

  14. EGFR phosphorylates FAM129B to promote Ras activation

    PubMed Central

    Ji, Haitao; Lee, Jong-Ho; Wang, Yugang; Pang, Yilin; Zhang, Tao; Xia, Yan; Zhong, Lianjin; Lyu, Jianxin; Lu, Zhimin

    2016-01-01

    Ras GTPase-activating proteins (GAPs) are important regulators for Ras activation, which is instrumental in tumor development. However, the mechanism underlying this regulation remains elusive. We demonstrate here that activated EGFR phosphorylates the Y593 residue of the protein known as family with sequence similarity 129, member B (FAM129B), which is overexpressed in many types of human cancer. FAM129B phosphorylation increased the interaction between FAM129B and Ras, resulting in reduced binding of p120-RasGAP to Ras. FAM129B phosphorylation promoted Ras activation, increasing ERK1/2- and PKM2-dependent β-catenin transactivation and leading to the enhanced glycolytic gene expression and the Warburg effect; promoting tumor cell proliferation and invasion; and supporting brain tumorigenesis. Our studies unearthed a novel and important mechanism underlying EGFR-mediated Ras activation in tumor development. PMID:26721396

  15. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  16. Combination of afatinib with cetuximab in patients with EGFR-mutant non-small-cell lung cancer resistant to EGFR inhibitors.

    PubMed

    Ribeiro Gomes, Jéssica; Cruz, Marcelo Rocha S

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown effectiveness for advanced non-small-cell lung cancer (NSCLC) with activating mutations in the EGFR gene. However, resistance to the EGFR TKIs develops mostly secondary to T790M mutation in exon 20. The use of afatinib associated with cetuximab represents a new possibility of therapy following progression on gefitinib or erlotinib. We present two patients who acquired resistance to first-generation TKI and who underwent combination treatment with afatinib plus cetuximab as third-line therapy. Both patients presented partial response, and the time duration of disease control was 8 months and 10 months. The combined use of afatinib plus cetuximab emerges as a new possibility for the treatment of patients with advanced NSCLC harboring mutated EGFR after progression on first-generation EGFR TKIs with consequently acquired resistance to TKIs. Further studies are necessary to consolidate the data. PMID:26056478

  17. Combination of afatinib with cetuximab in patients with EGFR-mutant non-small-cell lung cancer resistant to EGFR inhibitors

    PubMed Central

    Ribeiro Gomes, Jéssica; Cruz, Marcelo Rocha S

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown effectiveness for advanced non-small-cell lung cancer (NSCLC) with activating mutations in the EGFR gene. However, resistance to the EGFR TKIs develops mostly secondary to T790M mutation in exon 20. The use of afatinib associated with cetuximab represents a new possibility of therapy following progression on gefitinib or erlotinib. We present two patients who acquired resistance to first-generation TKI and who underwent combination treatment with afatinib plus cetuximab as third-line therapy. Both patients presented partial response, and the time duration of disease control was 8 months and 10 months. The combined use of afatinib plus cetuximab emerges as a new possibility for the treatment of patients with advanced NSCLC harboring mutated EGFR after progression on first-generation EGFR TKIs with consequently acquired resistance to TKIs. Further studies are necessary to consolidate the data. PMID:26056478

  18. Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways.

    PubMed

    Luo, Liang; Sun, Ying-Jian; Wu, Yi-Jun

    2013-08-01

    Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC(50) values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood-brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects. PMID:23648830

  19. Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

    PubMed Central

    Sette, G; Salvati, V; Mottolese, M; Visca, P; Gallo, E; Fecchi, K; Pilozzi, E; Duranti, E; Policicchio, E; Tartaglia, M; Milella, M; De Maria, R; Eramo, A

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFRtyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFRtyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFRtyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFRtyr1068 was associated with

  20. Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer.

    PubMed

    Sette, G; Salvati, V; Mottolese, M; Visca, P; Gallo, E; Fecchi, K; Pilozzi, E; Duranti, E; Policicchio, E; Tartaglia, M; Milella, M; De Maria, R; Eramo, A

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFRtyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFRtyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFRtyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFRtyr1068 was associated with

  1. ADAM17 promotes breast cancer cell malignant phenotype through EGFR-PI3K-AKT activation

    PubMed Central

    Zheng, Xuguang; Jiang, Feng; Katakowski, Mark; Zhang, Zheng Gang; Lu, Qing-e; Chopp, Michael

    2009-01-01

    A disintegrin and metalloproteinase-17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane-bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and glioma. ADAM17 promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis after stroke and brain tumor growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to breast cancer progression and its mechanisms. To this end, we examined the role of ADAM17 in the proliferation, invasion, and tube formation of MDA-MB-231 breast cancer cells in vitro. Stable transfection of the MDA-MB-231 cell line with either a plasmid for over-expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high or low ADAM17 expression in breast cancer cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI-2 and the PI3K-AKT inhibitor LY294002 were used to counteract high ADAM17 expression or the activated PI3K-AKT pathway. Proliferation of MDA-MB-231 breast cancer cells were tested by MTT, Bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of MDA-MB-231 cells to penetrate the Extra Cellular Matrix. A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR-PI3K-Akt pathway activation in MDA-MB-231 cells under different ADAM17 expression levels were tested by Western blot and ELISA. Our data show that ADAM17 promotes the MDA-MB-231 malignant phenotype by increased proliferation, invasion and angiogenesis. TGF-α, VEGF secretion and VEGF expression was increasing by ADAM17 and counteracted by ADAM17 siRNA, TAPI-2, and LY294002 in MDA-MB-231 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI-2, and LY294002 deactivated the EGFR-PI3K-AKT signal pathway, which correlated with MDA-MB-231 cell malignant phenotype

  2. An in vitro chemoresponse assay defines a subset of colorectal and lung carcinomas responsive to cetuximab.

    PubMed

    Rice, Shara D; Cassino, Theresa R; Sakhamuri, Lahari; Song, Nan; Williams, Karl E; Brower, Stacey L

    2011-01-15

    Cetuximab is a chimeric monoclonal antibody for the epidermal growth factor receptor (EGFR) that may provide benefit to select cancer patients; however, identification of the characteristics of those patients who may benefit from its use is not complete. The ChemoFx® drug response marker (DRM) is an in vitro assay that can provide drug response data on tumor specimens before any patient treatment is initiated. We determined the feasibility of using the ChemoFx DRM to test tumor samples for sensitivity to cetuximab. We exposed four non-small cell lung carcinoma (NSCLC) cell lines (H358, H520, HCC827, and H1666) to cetuximab and determined their sensitivity using the ChemoFx DRM and, in parallel, EGFR status using immunocytochemistry, Western blotting, and In-Cell Western (TM) analysis. We used the ChemoFx DRM to determine cetuximab sensitivity of primary NSCLC and colorectal tumor samples. The ChemoFx DRM distinguished between cetuximab-sensitive and -resistant cell lines. Cetuximab sensitivity was not dependent on EGFR mutational status; H358 cells were non-responsive to cetuximab yet contain wild-type EGFR, whereas H1666 cells were intermediately responsive to cetuximab and contain wild-type EGFR. HCC827 (EGFR-mutant) cells were intermediately responsive and, as expected, H520 cells (EGFR-null) were non-responsive to cetuximab. ChemoFx-determined cetuximab sensitivity of primary NSCLC and colorectal tumor samples was 9.0% and 7.5%, respectively. Use of the ChemoFx DRM is feasible for determining cetuximab sensitivity. The ChemoFx-determined cetuximab responses of primary NSCLC and colorectal tumor specimens were similar to published response rates of patients to treatment with cetuximab monotherapy. PMID:20980824

  3. Cell assay using a two-photon-excited europium chelate

    PubMed Central

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2011-01-01

    We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu3+ emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles. PMID:21833362

  4. Exon 19 L747P mutation presented as a primary resistance to EGFR-TKI: a case report

    PubMed Central

    Wang, Yu-Ting; Ning, Wei-Wei; Li, Jing

    2016-01-01

    Active mutations of the EGFR gene have been proved to predict the activity of EGFR-TKI. The most common mutations are the exon 19 deletion and exon 21 point mutation, both of which are sensitive to EGFR-TKI. However, rare EGFR mutations or complex mutations still exist, and data of which are scarce and controversial. Their response to EGFR-TKI remains uncertain. We presented a patient diagnosed with stage IV lung adenocarcinoma who was found to have the EGFR mutation in exon 19 (L747P) before any treatment. The disease progressed 2 months after the chemotherapy containing cisplatin and pemetrexed, and erlotinib was administered, but there was no response found. This EGFR-TKI naïve patient failed to achieve the desired effect with the therapy of EGFR-TKI. L747P may be associated with primary resistance to EGFR-TKI in this case. PMID:27499993

  5. Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment

    PubMed Central

    Lauriola, Mattia; Enuka, Yehoshua; Zeisel, Amit; D’Uva, Gabriele; Roth, Lee; Sharon-Sevilla, Michal; Lindzen, Moshit; Sharma, Kirti; Nevo, Nava; Feldman, Morris; Carvalho, Silvia; Cohen-Dvashi, Hadas; Kedmi, Merav; Ben-Chetrit, Nir; Chen, Alon; Solmi, Rossella; Wiemann, Stefan; Schmitt, Fernando; Domany, Eytan; Yarden, Yosef

    2014-01-01

    Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR’s positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR’s feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy. PMID:25278152

  6. EGFR-targeting therapy as an evolving concept: learning from nimotuzumab clinical development.

    PubMed

    Perez, Rolando; Moreno, Ernesto

    2014-03-01

    Epidermal growth factor receptor (EGFR)-targeted therapies have been extensively evaluated in the clinic for different tumor localizations and using different EGFR-targeting products, either registered or still in clinical development. Nonetheless, there still is a long way to go to optimize the clinical benefit from EGFR-targeted therapies. In this article we briefly discuss on current paradigms guiding the use of EGFR-targeting agents in the clinic, and on new emergent concepts. The discussion is largely based on experiences from the clinical development of the monoclonal antibody nimotuzumab, which has shown a quite particular clinical profile, characterized by a very low toxicity. In order to optimize the design of EGFR-targeting therapies, clinical researchers should take into account the interconnection between the EGFR pathway and other cellular pathways. Thus, clinical trials need to incorporate more translational research. PMID:25842083

  7. Adaptive and Acquired Resistance to EGFR Inhibitors Converge on the MAPK Pathway

    PubMed Central

    Ma, Pengfei; Fu, Yujie; Chen, Minjiang; Jing, Ying; Wu, Jie; Li, Ke; Shen, Ying; Gao, Jian-Xin; Wang, Mengzhao; Zhao, Xiaojing; Zhuang, Guanglei

    2016-01-01

    Both adaptive and acquired resistance significantly limits the efficacy of the epidermal growth factor receptor (EGFR) kinase inhibitors. However, the distinct or common mechanisms of adaptive and acquired resistance have not been fully characterized. Here, through systematic modeling of erlotinib resistance in lung cancer, we found that feedback reactivation of MAPK signaling following erlotinib treatment, which was dependent on the MET receptor, contributed to the adaptive resistance of EGFR inhibitors. Interestingly, acquired resistance to erlotinib was also associated with the MAPK pathway activation as a result of CRAF or NRAS amplification. Consequently, combined inhibition of EGFR and MAPK impeded the development of both adaptive and acquired resistance. These observations demonstrate that adaptive and acquired resistance to EGFR inhibitors can converge on the same pathway and credential cotargeting EGFR and MAPK as a promising therapeutic approach in EGFR mutant tumors. PMID:27279914

  8. Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

    PubMed

    Shimizu, Nobuyoshi; Maekawa, Masahiko; Asai, Satoko; Shimizu, Yoshiko

    2015-12-01

    We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed. PMID:25947045

  9. MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells.

    PubMed

    Presutti, Dario; Santini, Simonetta; Cardinali, Beatrice; Papoff, Giuliana; Lalli, Cristiana; Samperna, Simone; Fustaino, Valentina; Giannini, Giuseppe; Ruberti, Giovina

    2015-01-01

    Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30-40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation

  10. MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

    PubMed Central

    Presutti, Dario; Santini, Simonetta; Cardinali, Beatrice; Papoff, Giuliana; Lalli, Cristiana; Samperna, Simone; Fustaino, Valentina; Giannini, Giuseppe; Ruberti, Giovina

    2015-01-01

    Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation

  11. Aquaporin-8 mediates human esophageal cancer Eca-109 cell migration via the EGFR-Erk1/2 pathway

    PubMed Central

    Chang, Heng; Shi, Yong-Hua; Talaf, Tuo-Kan; Lin, Chen

    2014-01-01

    Abnormal expression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases1/2 (ERK1/2) are associated with tumorigenesis and cancer progression and may upregulate AQPs expression. In this study, we investigated acquaporin-8 expression and signaling via epidermal growth factor receptor-extracellular signal-regulated kinases1/2 in human esophageal cancer Eca-109 cells by western blot, immunofluorescence and wound healing (scratch) assays. Our results showed that epidermal growth factor (EGF) induced both Eca-109 migration and AQP8 expression. Wound healing results showed that cell migration was increased by 1.23-1.10-fold at 24 h and 48 h after EGF treatment. AQP8 expression was significantly increased (1.19-fold) at 48 h after EGF treatment in Eca-109. The EGFR kinase inhibitor, PD153035, blocked EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 3.65-fold (EGF-treated) to 0.55-fold (PD153035-treated) in Eca-109. Furthermore, the MEK [MAPK (mitogen-activated protein kinase)/Erk1/2]/Erk1/2 inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 3.92-fold (EGF-treated) to 1.38-fold (U0126-treated) in Eca-109. In conclusions, EGF induces AQP8 expression and cell migration in Eca-109 cells via the EGFR/Erk1/2 signal transduction pathway. PMID:25550802

  12. A view on EGFR-targeted therapies from the oncogene-addiction perspective

    PubMed Central

    Perez, Rolando; Crombet, Tania; de Leon, Joel; Moreno, Ernesto

    2013-01-01

    Tumor cell growth and survival can often be impaired by inactivating a single oncogen– a phenomenon that has been called as “oncogene addiction.” It is in such scenarios that molecular targeted therapies may succeed. among known oncogenes, the epidermal growth factor receptor (EGFR) has become the target of different cancer therapies. So far, however, the clinical benefit from EGFR-targeted therapies has been rather limited. a critical review of the large amount of clinical data obtained with anti-EGFR agents, carried out from the perspective of the oncogene addiction concept, may help to understand the causes of the unsatisfactory results. In this article we intend to do such an exercise taking as basis for the analysis a few case studies of anti-EGFR agents that are currently in the clinic. There, the “EGFR addiction” phenomenon becomes apparent in high-responder patients. We further discuss how the concept of oncogene addiction needs to be interpreted on the light of emerging experimental evidences and ideas; in particular, that EGFR addiction may reflect the interconnection of several cellular pathways. In this regard we set forth several hypotheses; namely, that requirement of higher glucose uptake by hypoxic tumor cells may reinforce EGFR addiction; and that chronic use of EGFR-targeted antibodies in EGFR-addicted tumors would induce stable disease by reversing the malignant phenotype of cancer stem cells and also by sustaining an anti-tumor T cell response. Finally, we discuss possible reasons for the failure of certain combinatorial therapies involving anti-EGFR agents, arguing that some of these agents might produce either a negative or a positive trans-modulation effect on other oncogenes. It becomes evident that we need operational definitions of EGFR addiction in order to determine which patient populations may benefit from treatment with anti-EGFR drugs, and to improve the design of these therapies. PMID:23637683

  13. Association between human papillomavirus and EGFR mutations in advanced lung adenocarcinoma

    PubMed Central

    Li, Ming; Deng, Fang; Qian, Li-Ting; Meng, Shui-Ping; Zhang, Yang; Shan, Wu-Lin; Zhang, Xiao-Lei; Wang, Bao-Long

    2016-01-01

    Previous studies have demonstrated an association between human papillomavirus (HPV) and mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer patients; however, few studies have investigated this association in advanced lung adenocarcinoma patients undergoing gefitinib treatment. The present study investigated the association between HPV and EGFR mutations in advanced lung adenocarcinoma patients. A total of 95 advanced lung adenocarcinoma patients were enrolled in the study. The HPV infection status and presence of EGFR mutations in tumor tissue was evaluated. Patient clinical characteristics were also determined and compared with HPV infection and EGFR mutation status to analyze their impact on progression-free survival. HPV DNA was identified in 27/95 (28.4%) lung adenocarcinoma tumors and was most common in patients with lymph node metastasis (P=0.016). A total of 44/95 (46.3%) cases exhibited EGFR mutations, which were predominantly observed in female patients and non-smokers. The presence of HPV DNA was significantly associated with EGFR mutations (P=0.012) and multivariate analysis also revealed that HPV DNA was significantly associated with EGFR mutations (odds ratio=3.971) in advanced lung adenocarcinoma. Patients with both HPV infections and EGFR mutations exhibit a marked decrease in the risk of lung cancer progression when compared with those without HPV infection or EGFR mutations (adjusted HR=0.640; 95% confidence interval: 0.488–0.840; P=0.001). HPV infection was significantly associated with EGFR mutations in advanced lung adenocarcinoma patients. Furthermore, patients with HPV infections exhibited the longest progression-free survival times, which may be due to good response to tyrosine kinase inhibitor- or platinum-based-adjuvant therapy in these patients. Patients with EGFR mutations exhibited a better prognosis when compared with those exhibiting wild-type EGFR, regardless of HPV status. PMID:27602120

  14. Known and putative mechanisms of resistance to EGFR targeted therapies in NSCLC patients with EGFR mutations—a review

    PubMed Central

    Stewart, Erin L.; Tan, Samuel Zhixing; Liu, Geoffrey

    2015-01-01

    Lung cancer is the leading cause of cancer related deaths in Canada with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Tumor characterization can identify cancer-driving mutations as treatment targets. One of the most successful examples of cancer targeted therapy is inhibition of mutated epidermal growth factor receptor (EGFR), which occurs in ~10-30% of NSCLC patients. While this treatment has benefited many patients with activating EGFR mutations, almost all who initially benefited will eventually acquire resistance. Approximately 50% of cases of acquired resistance (AR) are due to a secondary T790M mutation in exon 20 of the EGFR gene; however, many of the remaining mechanisms of resistance are still unknown. Much work has been done to elucidate the remaining mechanisms of resistance. This review aims to highlight both the mechanisms of resistance that have already been identified in patients and potential novel mechanisms identified in preclinical models which have yet to be validated in the patient settings. PMID:25806347

  15. Texture Fish

    ERIC Educational Resources Information Center

    Stone, Julie

    2007-01-01

    In an effort to provide an opportunity for her first graders to explore texture through an engaging subject, the author developed a three-part lesson that features fish in a mixed-media artwork: (1) Exploring Textured Paint; (2) Creating the Fish; and (3) Role Playing. In this lesson, students effectively explore texture through painting, drawing,…

  16. Development of an epidermal growth factor derivative with EGFR blocking activity.

    PubMed

    Panosa, Clara; Tebar, Francesc; Ferrer-Batallé, Montserrat; Fonge, Humphrey; Seno, Masaharu; Reilly, Raymond M; Massaguer, Anna; De Llorens, Rafael

    2013-01-01

    The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR. PMID:23935985

  17. Evolution of the EGFR pathway in Metazoa and its diversification in the planarian Schmidtea mediterranea.

    PubMed

    Barberán, Sara; Martín-Durán, José M; Cebrià, Francesc

    2016-01-01

    The EGFR pathway is an essential signaling system in animals, whose core components are the epidermal growth factors (EGF ligands) and their trans-membrane tyrosine kinase receptors (EGFRs). Despite extensive knowledge in classical model organisms, little is known of the composition and function of the EGFR pathway in most animal lineages. Here, we have performed an extensive search for the presence of EGFRs and EGF ligands in representative species of most major animal clades, with special focus on the planarian Schmidtea mediterranea. With the exception of placozoans and cnidarians, we found that the EGFR pathway is potentially present in all other analyzed animal groups, and has experienced frequent independent expansions. We further characterized the expression domains of the EGFR/EGF identified in S. mediterranea, revealing a wide variety of patterns and localization in almost all planarian tissues. Finally, functional experiments suggest an interaction between one of the previously described receptors, Smed-egfr-5, and the newly found ligand Smed-egf-6. Our findings provide the most comprehensive overview to date of the EGFR pathway, and indicate that the last common metazoan ancestor had an initial complement of one EGFR and one putative EGF ligand, which was often expanded or lost during animal evolution. PMID:27325311

  18. Evolution of the EGFR pathway in Metazoa and its diversification in the planarian Schmidtea mediterranea

    PubMed Central

    Barberán, Sara; Martín-Durán, José M.; Cebrià, Francesc

    2016-01-01

    The EGFR pathway is an essential signaling system in animals, whose core components are the epidermal growth factors (EGF ligands) and their trans-membrane tyrosine kinase receptors (EGFRs). Despite extensive knowledge in classical model organisms, little is known of the composition and function of the EGFR pathway in most animal lineages. Here, we have performed an extensive search for the presence of EGFRs and EGF ligands in representative species of most major animal clades, with special focus on the planarian Schmidtea mediterranea. With the exception of placozoans and cnidarians, we found that the EGFR pathway is potentially present in all other analyzed animal groups, and has experienced frequent independent expansions. We further characterized the expression domains of the EGFR/EGF identified in S. mediterranea, revealing a wide variety of patterns and localization in almost all planarian tissues. Finally, functional experiments suggest an interaction between one of the previously described receptors, Smed-egfr-5, and the newly found ligand Smed-egf-6. Our findings provide the most comprehensive overview to date of the EGFR pathway, and indicate that the last common metazoan ancestor had an initial complement of one EGFR and one putative EGF ligand, which was often expanded or lost during animal evolution. PMID:27325311

  19. β-catenin contributes to lung tumor development induced by EGFR mutations

    PubMed Central

    Nakayama, Sohei; Sng, Natasha; Carretero, Julian; Welner, Robert; Hayashi, Yuichiro; Yamamoto, Mihoko; Tan, Alistair J.; Yamaguchi, Norihiro; Yasuda, Hiroyuki; Li, Danan; Soejima, Kenzo; Soo, Ross A.; Costa, Daniel B.; Wong, Kwok-Kin; Kobayashi, Susumu S.

    2014-01-01

    The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and development of EGFR tyrosine kinase inhibitors (TKIs) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. β-catenin was upregulated and activated in EGFR mutated cells. Mutant EGFR preferentially bound to and tyrosine-phosphorylated β-catenin, leading to increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacological inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs. PMID:25164010

  20. Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells.

    PubMed

    Jackson, Nicole M; Ceresa, Brian P

    2016-08-15

    The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway. PMID:27381222

  1. NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor

    PubMed Central

    Galvani, Elena; Sun, Jing; Leon, Leticia G.; Sciarrillo, Rocco; Narayan, Ravi S.; Tjin Tham Sjin, Robert; Lee, Kwangho; Ohashi, Kadoaki; Heideman, Daniëlle A.M.; Alfieri, Roberta R.; Heynen, Guus J.; Bernards, René; Smit, Egbert F.; Pao, William; Peters, Godefridus J.; Giovannetti, Elisa

    2015-01-01

    The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs. PMID:26015408

  2. Characterization of EGFR and ErbB2 expression in atopic dermatitis patients.

    PubMed

    Sääf, Annika; Pivarcsi, Andor; Winge, Mårten C G; Wahlgren, Carl-Fredrik; Homey, Bernhard; Nordenskjöld, Magnus; Tengvall-Linder, Maria; Bradley, Maria

    2012-12-01

    Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases in industrialized countries. To identify candidate genes involved in the pathogenesis of AD, we previously undertook a genome-wide approach using DNA microarrays. A transcript encoding the epidermal growth factor receptor (EGFR) was found to be among the down-regulated transcripts in AD skin. Here, we further investigated the expression pattern of two EGFR family members (EGFR and ErbB2) in AD skin on a protein level. Immunohistochemical (IHC) analysis of EGFR and ErbB2 showed decreased expression of EGFR and ErbB2 proteins in AD lesional skin as compared to skin from healthy individuals. Interestingly, we found that EGFR and ErbB2 were reciprocally expressed in an in vitro model of keratinocyte proliferation and differentiation, paralleling the expression patterns found in epidermis of healthy skin. The highest levels of EGFR transcripts were found in proliferating cells, while ErbB2 was found in differentiated cells. We show that blocking EGFR activity combined with co-stimulation of the Th2-cytokine IL4 in keratinocytes leads to induction of the inflammatory chemokine CCL26/eotaxin-3 in vitro. Accordingly, increased CCL26 transcriptional levels were observed in AD lesional skin. Taken together, suppression of EGFR may contribute to the pathogenesis of AD via the regulation of inflammatory chemokines. PMID:22552355

  3. Managing acquired resistance in EGFR-mutated non-small cell lung cancer.

    PubMed

    Forde, Patrick M; Ettinger, David S

    2015-08-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) deliver high response rates with relatively modest toxicity in patients with advanced EGFR-mutated non-small cell lung cancer. Despite this, nearly all tumors eventually develop resistance to first-line therapy. At present, the only standard treatment option for patients with acquired resistance is cytotoxic chemotherapy. In this article, we review the latest research into methods of targeting acquired resistance to EGFR TKI therapy, including third-generation EGFR TKIs that target the T790M resistance mutation and other novel agents in development. PMID:26351816

  4. Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

    PubMed Central

    Wang, Ye; Gao, Jing; Guo, Xingdong; Tong, Ti; Shi, Xiaoshan; Li, Lunyi; Qi, Miao; Wang, Yajie; Cai, Mingjun; Jiang, Junguang; Xu, Chenqi; Ji, Hongbin; Wang, Hongda

    2014-01-01

    The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers. PMID:25001389

  5. Combined therapy with mutant-selective EGFR inhibitor and Met kinase inhibitor for overcoming erlotinib resistance in EGFR-mutant lung cancer.

    PubMed

    Nakagawa, Takayuki; Takeuchi, Shinji; Yamada, Tadaaki; Nanjo, Shigeki; Ishikawa, Daisuke; Sano, Takako; Kita, Kenji; Nakamura, Takahiro; Matsumoto, Kunio; Suda, Kenichi; Mitsudomi, Tetsuya; Sekido, Yoshitaka; Uenaka, Toshimitsu; Yano, Seiji

    2012-10-01

    Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression, thereafter relapsing. Thus, it is urgent to develop novel agents to overcome EGFR-TKI resistance. We have tested the effects of the mutant-selective EGFR-TKI WZ4002 and the mutant-selective Met-TKI E7050 on 3 EGFR mutant lung cancer cell lines resistant to erlotinib by different mechanisms: PC-9/HGF cells with an exon 19 deletion, H1975 with an L858R mutation, and HCC827ER with an exon 19 deletion, with acquired resistance to erlotinib because of HGF gene transfection, gatekeeper T790M mutation, and Met amplification, respectively. WZ4002 inhibited the growth of H1975 cells with a gatekeeper T790M mutation, but did not inhibit the growth of HCC827ER and PC-9/HGF cells. HGF triggered the resistance of H1975 cells to WZ4002, whereas E7050 sensitized HCC827ER, PC-9/HGF, and HGF-treated H1975 cells to WZ4002, inhibiting EGFR and Met phosphorylation and their downstream molecules. Combined treatment potently inhibited the growth of tumors induced in severe-combined immunodeficient mice by H1975, HCC827ER, and PC-9/HGF cells, without any marked adverse events. These therapeutic effects were associated with the inhibition of EGFR and Met phosphorylation in vivo. The combination of a mutant-selective EGFR-TKI and a Met-TKI was effective in suppressing the growth of erlotinib-resistant tumors caused by gatekeeper T790M mutation, Met amplification, and HGF overexpression. Further evaluations in clinical trials are warranted. PMID:22844075

  6. 177Lu-DO3A-HSA-Z EGFR:1907: characterization as a potential radiopharmaceutical for radionuclide therapy of EGFR-expressing head and neck carcinomas.

    PubMed

    Hoppmann, Susan; Qi, Shibo; Miao, Zheng; Liu, Hongguang; Jiang, Han; Cutler, Cathy S; Bao, Ande; Cheng, Zhen

    2012-06-01

    Epidermal growth factor receptor 1 (EGFR) is an attractive target for radionuclide therapy of head and neck carcinomas. Affibody molecules against EGFR (Z(EGFR)) show excellent tumor localizations in imaging studies. However, one major drawback is that radiometal-labeled Affibody molecules display extremely high uptakes in the radiosensitive kidneys which may impact their use as radiotherapeutic agents. The purpose of this study is to further explore whether radiometal-labeled human serum albumin (HSA)-Z(EFGR) bioconjugates display desirable profiles for the use in radionuclide therapy of EGFR-positive head and neck carcinomas. The Z(EFGR) analog, Ac-Cys-Z(EGFR:1907), was site-specifically conjugated with HSA. The resulting bioconjugate 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A)-HSA-Z(EGFR:1907) was then radiolabeled with either (64)Cu or (177)Lu and subjected to in vitro cell uptake and internalization studies using the human oral squamous carcinoma cell line SAS. Positron emission tomography (PET), single photon emission computed tomography (SPECT), and biodistribution studies were conducted using SAS-tumor-bearing mice. Cell studies revealed a high (8.43 ± 0.55 % at 4 h) and specific (0.95 ± 0.09 % at 4 h) uptake of (177)Lu-DO3A-HSA-Z(EGFR:1907) as determined by blocking with nonradioactive Z(EGFR:1907). The internalization of (177)Lu-DO3A-HSA-Z(EGFR:1907) was verified in vitro and found to be significantly higher than that of (177)Lu-labeled Z(EFGR) at 2-24 h of incubation. PET and SPECT studies showed good tumor imaging contrasts. The biodistribution of (177)Lu-DO3A-HSA-Z(EGFR:1907) in SAS-tumor-bearing mice displayed high tumor uptake (5.1 ± 0.44 % ID/g) and liver uptake (31.5 ± 7.66 % ID/g) and moderate kidney uptake (8.5 ± 1.08 % ID/g) at 72 h after injection. (177)Lu-DO3A-HSA-Z(EGFR:1907) shows promising in vivo profiles and may be a potential radiopharmaceutical for radionuclide therapy of EGFR-expressing head and neck carcinomas

  7. Large-Scale Computational Screening Identifies First in Class Multitarget Inhibitor of EGFR Kinase and BRD4

    PubMed Central

    Allen, Bryce K.; Mehta, Saurabh; Ember, Stewart W. J.; Schonbrunn, Ernst; Ayad, Nagi; Schürer, Stephan C.

    2015-01-01

    Inhibition of cancer-promoting kinases is an established therapeutic strategy for the treatment of many cancers, although resistance to kinase inhibitors is common. One way to overcome resistance is to target orthogonal cancer-promoting pathways. Bromo and Extra-Terminal (BET) domain proteins, which belong to the family of epigenetic readers, have recently emerged as promising therapeutic targets in multiple cancers. The development of multitarget drugs that inhibit kinase and BET proteins therefore may be a promising strategy to overcome tumor resistance and prolong therapeutic efficacy in the clinic. We developed a general computational screening approach to identify novel dual kinase/bromodomain inhibitors from millions of commercially available small molecules. Our method integrated machine learning using big datasets of kinase inhibitors and structure-based drug design. Here we describe the computational methodology, including validation and characterization of our models and their application and integration into a scalable virtual screening pipeline. We screened over 6 million commercially available compounds and selected 24 for testing in BRD4 and EGFR biochemical assays. We identified several novel BRD4 inhibitors, among them a first in class dual EGFR-BRD4 inhibitor. Our studies suggest that this computational screening approach may be broadly applicable for identifying dual kinase/BET inhibitors with potential for treating various cancers. PMID:26596901

  8. One Fish, Two Fish, Redfish, You Fish!

    ERIC Educational Resources Information Center

    White, Katherine; Timmons, Maryellen; Medders, Paul

    2011-01-01

    The recreational fishing activity presented in this article provides a hands-on, problem-based experience for students; it unites biology, math, economics, environmental policy, and population dynamics concepts. In addition, the activity allows students to shape environmental policy in a realistic setting and evaluate their peers' work. By…

  9. Fishing Forecasts

    NASA Technical Reports Server (NTRS)

    1988-01-01

    ROFFS stands for Roffer's Ocean Fishing Forecasting Service, Inc. Roffer combines satellite and computer technology with oceanographic information from several sources to produce frequently updated charts sometimes as often as 30 times a day showing clues to the location of marlin, sailfish, tuna, swordfish and a variety of other types. Also provides customized forecasts for racing boats and the shipping industry along with seasonal forecasts that allow the marine industry to formulate fishing strategies based on foreknowledge of the arrival and departure times of different fish. Roffs service exemplifies the potential for benefits to marine industries from satellite observations. Most notable results are reduced search time and substantial fuel savings.

  10. The Genomic Landscape of Response to EGFR Blockade in Colorectal Cancer

    PubMed Central

    Bertotti, Andrea; Papp, Eniko; Jones, Siân; Adleff, Vilmos; Anagnostou, Valsamo; Lupo, Barbara; Sausen, Mark; Phallen, Jillian; Hruban, Carolyn A.; Tokheim, Collin; Niknafs, Noushin; Nesselbush, Monica; Lytle, Karli; Sassi, Francesco; Cottino, Francesca; Migliardi, Giorgia; Zanella, Eugenia R.; Ribero, Dario; Russolillo, Nadia; Mellano, Alfredo; Muratore, Andrea; Paraluppi, Gianluca; Salizzoni, Mauro; Marsoni, Silvia; Kragh, Michael; Lantto, Johan; Cassingena, Andrea; Li, Qing Kay; Karchin, Rachel; Scharpf, Robert; Sartore-Bianchi, Andrea; Siena, Salvatore; Diaz, Luis A.; Trusolino, Livio; Velculescu, Victor E.

    2016-01-01

    Colorectal cancer (CRC) is the third most common cancer world-wide with 1.2 million patients diagnosed yearly. In late stage CRC, the most commonly used targeted therapies are monoclonal antibodies cetuximab and panitumumab, which inactivate EGFR1. Recent studies have identified alterations in KRAS2–4 and other genes5–13 as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in CRC and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in CRC on response to anti-EGFR antibody therapy, we performed complete exome sequence and copy number analyses of 129 patient-derived tumorgrafts and targeted genomic analyses of 55 patient tumors, all of which were KRAS wild-type. We analyzed the response of tumors to anti-EGFR antibody blockade in tumorgraft models or in clinical settings. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumors with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumorgraft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluate response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and provide new avenues for intervention in the management of CRC. PMID:26416732

  11. COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

    SciTech Connect

    Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito; Lee, Hong-Jen; Lee, Heng-Huan; Hung, Mien-Chie

    2010-09-03

    Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH{sub 2}-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with {gamma}-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.

  12. The genomic landscape of response to EGFR blockade in colorectal cancer.

    PubMed

    Bertotti, Andrea; Papp, Eniko; Jones, Siân; Adleff, Vilmos; Anagnostou, Valsamo; Lupo, Barbara; Sausen, Mark; Phallen, Jillian; Hruban, Carolyn A; Tokheim, Collin; Niknafs, Noushin; Nesselbush, Monica; Lytle, Karli; Sassi, Francesco; Cottino, Francesca; Migliardi, Giorgia; Zanella, Eugenia R; Ribero, Dario; Russolillo, Nadia; Mellano, Alfredo; Muratore, Andrea; Paraluppi, Gianluca; Salizzoni, Mauro; Marsoni, Silvia; Kragh, Michael; Lantto, Johan; Cassingena, Andrea; Li, Qing Kay; Karchin, Rachel; Scharpf, Robert; Sartore-Bianchi, Andrea; Siena, Salvatore; Diaz, Luis A; Trusolino, Livio; Velculescu, Victor E

    2015-10-01

    Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer. PMID:26416732

  13. Correlation between serum CEA levels and EGFR mutations in Chinese nonsmokers with lung adenocarcinoma

    PubMed Central

    Jin, Bo; Dong, Yu; Wang, Hui-min; Huang, Jin-su; Han, Bao-hui

    2014-01-01

    Aim: To evaluate the relationship between epidermal growth factor receptor (EGFR) mutations and serum carcinoembryonic antigen (CEA) levels in Chinese nonsmokers with pulmonary adenocarcinoma. Methods: We sequenced exons 18–21 of the EGFR gene in 98 cases. The patients were divided into two groups based on their pre-treatment serum CEA levels (below or above 5 ng/mL) for analyzing the correlations with EGFR mutations. Results: Sixty-seven cases harbored EGFR mutations. The rates of EGFR mutations and exon 19 mutations in the high-CEA group (78.2% and 49.1%, respectively) were significantly higher those in the low-CEA group (55.8% and 20.9%, respectively). Serum CEA levels were found to be the only independent predictor of EGFR mutation (OR 2.837; 95% CI: 1.178–6.829) and exon 19 mutation (OR 3.618; 95% CI: 1.319–9.918). Furthermore, a higher serum CEA level was associated with a higher EGFR mutation rate and a higher exon 19 mutation rate: patients with serum CEA levels <5 ng/mL, ≥5 and <20 ng/mL, ≥20 ng/mL showed the EGFR mutation rate of 55.8%, 74.1%, 82.1%, respectively, and the exon 19 mutation rate of 20.9%, 40.7%, 57.1%, respectively. Patients with EGFR mutations displayed a significantly higher incidence of abnormal serum CEA levels (>5 ng/mL) than patients without EGFR mutations (64.2% vs 38.7%). Conclusion: Elevated serum CEA levels predict the presence of EGFR gene mutations in Chinese nonsmokers with pulmonary adenocarcinoma. PMID:24487967

  14. Gallic acid abolishes the EGFR/Src/Akt/Erk-mediated expression of matrix metalloproteinase-9 in MCF-7 breast cancer cells.

    PubMed

    Chen, Ying-Jung; Lin, Ku-Nan; Jhang, Li-Mei; Huang, Chia-Hui; Lee, Yuan-Chin; Chang, Long-Sen

    2016-05-25

    Several studies have revealed that natural compounds are valuable resources to develop novel agents against dysregulation of the EGF/EGFR-mediated matrix metalloproteinase-9 (MMP-9) expression in cancer cells. In view of the findings that EGF/EGFR-mediated MMP-9 expression is closely related to invasion and metastasis of breast cancer. To determine the beneficial effects of gallic acid on the suppression of breast cancer metastasis, we explored the effect of gallic acid on MMP-9 expression in EGF-treated MCF-7 breast cancer cells. Treatment with EGF up-regulated MMP-9 mRNA and protein levels in MCF-7 cells. EGF treatment induced phosphorylation of EGFR and elicited Src activation, subsequently promoting Akt/NFκB (p65) and ERK/c-Jun phosphorylation in MCF-7 cells. Activation of Akt/p65 and ERK/c-Jun was responsible for the MMP-9 up-regulation in EGF-treated cells. Gallic acid repressed the EGF-induced activation of EGFR and Src; furthermore, inactivation of Akt/p65 and ERK/c-Jun was a result of the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. Over-expression of constitutively active Akt and MEK1 or over-expression of constitutively active Src eradicated the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. A chromosome conformation capture assay showed that EGF induced a chromosomal loop formation in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun activation. Treatment with gallic acid, EGFR inhibitor, or Src inhibitor reduced DNA looping. Taken together, our data suggest that gallic acid inhibits the activation of EGFR/Src-mediated Akt and ERK, leading to reduced levels of p65/c-Jun-mediated DNA looping and thus inhibiting MMP-9 expression in EGF-treated MCF-7 cells. PMID:27087131

  15. NSK-01105, a Novel Sorafenib Derivative, Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

    PubMed Central

    Yu, Pengfei; Ye, Liang; Wang, Hongbo; Du, Guangying; Zhang, Jianzhao; Zuo, Yanhua; Zhang, Jinghai; Tian, Jingwei

    2014-01-01

    The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities. PMID:25551444

  16. Fish Facts

    MedlinePlus

    ... not eat any fish because they worry about mercury in seafood. Mercury is a metal that, at high levels, can ... many types of seafood have little or no mercury at all. So your risk of mercury exposure ...

  17. Designer Fish.

    ERIC Educational Resources Information Center

    Hall, William R., Jr.

    1990-01-01

    Described is an activity in which students are asked to design a fish that would survive in a natural system. A project to computerize the activity is discussed. The development of this artificial intelligence software is detailed. (CW)

  18. Fish Allergy

    MedlinePlus

    ... specific fish used on the label. Read all product labels carefully before purchasing and consuming any item. Ingredients ... Getting Started Newly Diagnosed Emergency Care Plan Food Labels Mislabeled Products Tips for Managing Food Allergies Resources For... Most ...

  19. Ultrasound-guided photoacoustic imaging for the selective detection of EGFR-expressing breast cancer and lymph node metastases

    PubMed Central

    Zhang, Meihua; Kim, Hoe Suk; Jin, Tiefeng; Yi, Ann; Moon, Woo Kyung

    2016-01-01

    We assessed the use of ultrasound (US)-guided photoacoustic imaging (PAI) and anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs) to non-invasively detect EGFR-expressing primary tumor masses and regional lymph node (LN) metastases in breast tumor mice generated by injecting MCF-7 (EGFR-negative) or MDA-MB-231 (EGFR-positive) human breast cells using a preclinical Vevo 2100 LAZR Imaging system. Anti-EGFR-GNs provided a significant enhancement in the PA signal in MDA-MB-231 tumor and the axillary LN metastases relative to MCF-7 tumor and non-LN metastases. We demonstrated that US-guided PAI using anti-EGFR-GNs is highly sensitive for the selective visualization of EGFR-expressing breast primary tumors as well as LN micrometastases. PMID:27231631

  20. Ultrasound-guided photoacoustic imaging for the selective detection of EGFR-expressing breast cancer and lymph node metastases.

    PubMed

    Zhang, Meihua; Kim, Hoe Suk; Jin, Tiefeng; Yi, Ann; Moon, Woo Kyung

    2016-05-01

    We assessed the use of ultrasound (US)-guided photoacoustic imaging (PAI) and anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs) to non-invasively detect EGFR-expressing primary tumor masses and regional lymph node (LN) metastases in breast tumor mice generated by injecting MCF-7 (EGFR-negative) or MDA-MB-231 (EGFR-positive) human breast cells using a preclinical Vevo 2100 LAZR Imaging system. Anti-EGFR-GNs provided a significant enhancement in the PA signal in MDA-MB-231 tumor and the axillary LN metastases relative to MCF-7 tumor and non-LN metastases. We demonstrated that US-guided PAI using anti-EGFR-GNs is highly sensitive for the selective visualization of EGFR-expressing breast primary tumors as well as LN micrometastases. PMID:27231631

  1. EGFR-activating mutations, DNA copy number abundance of ErbB family, and prognosis in lung adenocarcinoma

    PubMed Central

    Chen, Hsuan-Yu; Liu, Chia-Hsin; Chang, Ya-Hsuan; Yu, Sung-Liang; Ho, Bing-Ching; Hsu, Chung-Ping; Yang, Tsung-Ying; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Chang, Chi-Sheng; Li, Yu-Cheng; Li, Ker-Chau; Chang, Gee-Chen; Yang, Pan-Chyr

    2016-01-01

    In this study, EGFR-activating mutation status and DNA copy number abundances of members of ErbB family were measured in 261 lung adenocarcinomas. The associations between DNA copy number abundances of ErbB family, EGFR-activating mutation status, and prognosis were explored. Results showed that DNA copy number abundances of EGFR, ERBB2, ERBB3, and ERBB4 had associations with overall survival in lung adenocarcinoma with EGFR-activating mutations. In the stratification analysis, only ERBB2 showed significant discrepancy in patients carrying wild type EGFR and other members of ErbB family in patients carrying EGFR-activating mutation. This indicated that CNAs of ErbB family had effect modifications of EGFR-activating mutation status. Findings of this study demonstrate potential molecular guidance of patient management of lung adenocarcinoma with or without EGFR-activating mutations. PMID:26824984

  2. The Microtubule-associated Histone Deacetylase 6 (HDAC6) Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation*

    PubMed Central

    Gao, Ya-sheng; Hubbert, Charlotte C.; Yao, Tso-Pang

    2010-01-01

    Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase with tubulin deacetylase activity, and it binds dynein motors. Recent studies revealed that microtubule acetylation affects the affinity and processivity of microtubule motors. These unique properties implicate a role for HDAC6 in intracellular organelle transport. Here, we show that HDAC6 associates with the endosomal compartments and controls epidermal growth factor receptor (EGFR) trafficking and degradation. We found that loss of HDAC6 promoted EGFR degradation. Mechanistically, HDAC6 deficiency did not cause aberrant EGFR internalization and recycling. Rather, it resulted in accelerated segregation of EGFR from early endosomes and premature delivery of EGFR to the late endosomal and lysosomal compartments. The deregulated EGFR endocytic trafficking was accompanied by an increase in microtubule-dependent movement of EGFR-bearing vesicles, revealing a novel regulation of EGFR vesicular trafficking and degradation by the microtubule deacetylase HDAC6. PMID:20133936

  3. EZH2 inhibition enhances the efficacy of an EGFR inhibitor in suppressing colon cancer cells

    PubMed Central

    Katona, Bryson W; Liu, Yuanyuan; Ma, Anqi; Jin, Jian; Hua, Xianxin

    2014-01-01

    Metastatic colon cancer has a 5-year survival of less than 10% despite the use of aggressive chemotherapeutic regimens. As signaling from epidermal growth factor receptor (EGFR) is often enhanced and epigenetic regulation is often altered in colon cancer, it is desirable to enhance the efficacy of EGFR-directed therapy by co-targeting an epigenetic pathway. We showed that the histone methyltransferase EZH2, which catalyzes methylation of histone H3 lysine 27 (H3K27), was upregulated in colon cancers in The Cancer Genome Atlas (TCGA) database. Since co-inhibition of both EGFR and EZH2 has not been studied in colon cancer, we examined the effects of co-inhibition of EGFR and EZH2 on 2 colon cancer cell lines, HT-29 and HCT-15. Co-inhibition of EZH2 and EGFR with the small molecules UNC1999 and gefitinib, led to a significant decrease in cell number and increased apoptosis compared to inhibition of either pathway alone, and similar results were noted after EZH2 shRNA knockdown. Moreover, co-inhibition of EZH2 and EGFR also significantly induced autophagy, indicating that autophagy may play a role in the observed synergy. Together, these findings suggest that inhibition of both EZH2 and EGFR serves as an effective method to increase the efficacy of EGFR inhibitors in suppressing colon cancer cells. PMID:25535899

  4. Effect of the BRCA1-SIRT1-EGFR axis on cisplatin sensitivity in ovarian cancer

    PubMed Central

    Li, Da; Wu, Qi-Jun; Bi, Fang-Fang; Chen, Si-Lei; Zhou, Yi-Ming; Zhao, Yue; Yang, Qing

    2016-01-01

    There is accumulating evidence that breast cancer 1 (BRCA1), sirtuin 1 (SIRT1), and epidermal growth factor receptor (EGFR) help to modulate cisplatin cytotoxicity. The role of dynamic crosstalk among BRCA1, SIRT1, and EGFR in cisplatin sensitivity remains largely unknown. We found that BRCA1, SIRT1, and EGFR levels were increased in cisplatin-resistant ovarian cancers compared with those in cisplatin-sensitive ovarian cancers. Hypomethylation in the BRCA1 promoter was associated with BRCA1 activation, significantly elevated SIRT1 levels, decreased nicotinamide adenine dinucleotide (NAD)-mediated SIRT1 activity, and decreased EGFR levels. Treatment with 5 and 10 μg/ml cisplatin induced a gradual increase in BRCA1 and SIRT1 levels and a gradual decrease in NAD levels and NAD-mediated SIRT1 activity, whereas EGFR levels were increased or decreased by treatment with 5 or 10 μg/ml cisplatin, respectively. The overexpression of SIRT1 or the enhancement of SIRT1 activity synergistically enhanced the BRCA1-mediated effects on EGFR transcription. In contrast, the knockdown of SIRT1 or the inhibition of SIRT1 activity inhibited the BRCA1-mediated effects on EGFR transcription. BRCA1 regulates EGFR through a BRCA1-mediated balance between SIRT1 expression and activity. Those results improve our understanding of the basic molecular mechanism underlying BRCA1-related cisplatin resistance in ovarian cancer. PMID:27186285

  5. Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits

    PubMed Central

    Ibach, Jenny; Radon, Yvonne; Gelléri, Márton; Sonntag, Michael H.; Brunsveld, Luc; Bastiaens, Philippe I. H.; Verveer, Peter J.

    2015-01-01

    Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane. PMID:26575183

  6. Micropapillary: A component more likely to harbour heterogeneous EGFR mutations in lung adenocarcinomas

    PubMed Central

    Cai, Yi-Ran; Dong, Yu-Jie; Wu, Hong-Bo; Liu, Zi-Chen; Zhou, Li-Juan; Su, Dan; Chen, Xue-Jing; Zhang, Li; Zhao, Ying-Li

    2016-01-01

    The micropapillary (MP) subtype has recently been established to be a distinct marker of poor prognosis in lung adenocarcinomas (LACs). According to the 2015 WHO classification system, LAC constituents are required to be precisely reported. T790M mutation and an insertion in exon 20 (E20ins) are associated with EGFR-TKI resistance. A total of 211 LAC patients were involved in this study, and EGFR mutations were determined using an amplification refractory mutation system (ARMS). Sex, smoking history, lymph node status, and clinical stage differed significantly between the EGFR wild type and mutant groups (p < 0.05). The EGFR mutation occurred more frequently in female, non-smokers, ACs with papillary (85.7%) or MP components (91.4%) (p < 0.001). Twenty ACs with naïve T790M or E20ins were microdissected. The AC constituents metastasizing to lymph nodes exhibited a phenotype and EGFR status that was consistent with the primary loci constituents. Glomerulus-like solid components exhibited the same EGFR status as the surrounding T790M-mutated MP components. The MP and glomerulus-like portions in AC tumours exhibited a congenial EGFR status, but the acinar cells with papillary cells were heterogeneous. The naïve T790M mutants, although minor in the MP component, dramatically increased after EGFR-TKI therapy and indicate that the MP components feature intrinsic heterogeneity. PMID:27046167

  7. Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system

    PubMed Central

    Fukuda, Shinji; Nishida-Fukuda, Hisayo; Nanba, Daisuke; Nakashiro, Koh-ichi; Nakayama, Hironao; Kubota, Hiroyuki; Higashiyama, Shigeki

    2016-01-01

    Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. Epidermal growth factor (EGF) and amphiregulin (AREG) are high- and low-affinity ligands for EGF receptor (EGFR), respectively. EGFR signaling is known to promote epithelial-mesenchymal transition (EMT) by the activation of ERK and the induction of an EMT transcription factor, ZEB1. Here, we demonstrate that ligand-switching between EGF and AREG at equivalent molarity reversibly interconverts epithelial and mesenchymal-like states of EGFR signal-dependent mammary epithelial cells. The EGF- and AREG-cultured cells also differ in their epithelial characteristics, including the expression of cell surface markers, the mode of migration and the ability for acinus-formation. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling. This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. Further, AREG-induced EGFR accumulation on the plasma membrane compensates for the weak association between AREG and EGFR. The EGFR dynamics enables AREG to support proliferation as efficiently as EGF at equivalent molarity and to maintain epithelial characteristics. Our findings reveal a role of EGFR ligands-generated signal strength in the regulation of mammary epithelial cell plasticity. PMID:26831618

  8. Role of EGFR expression levels in the regulation of integrin function by EGF.

    PubMed

    Vial, Daniel; McKeown-Longo, Paula J

    2016-06-01

    Activation of β1 integrins in dormant tumor cells has been linked to metastatic progression, suggesting that therapies designed to maintain β1 integrins in an inactive state may be useful in the prevention of metastatic disease. Our earlier studies have demonstrated that EGF regulates the activation state of the α5β1 integrin in EGFR overexpressing tumor cells through an ERK/p90RSK signaling pathway. Activation of this pathway by EGF resulted in the filamin A dependent inactivation of the α5β1 integrin receptor for fibronectin. The current study was designed to address the role of EGFR overexpression in the regulation of α5β1 integrin activation state by EGF. Lentiviral knockdown of EGFR coupled with limited dilution cloning was used to develop A431 squamous carcinoma cell lines expressing high, moderate, and low levels of EGFR. Inactivation of α5β1 integrin by EGF was shown to correlate with both the level of EGFR expression and the extent of p90RSK phosphorylation, but not with the level of ERK phosphorylation, suggesting that high levels of EGFR promote α5β1 integrin inactivation through sustained activation of p90RSK. Treatment of cells with EGFR kinase inhibitor resulted in a reactivation of the integrin which could be reversed with the phosphatase inhibitor, menadione. Taken together, these findings indicate that p90RSK may function to maintain dormancy in tumor cells expressing high levels of EGFR. © 2015 Wiley Periodicals, Inc. PMID:26053065

  9. EGFR-Mediated Chromatin Condensation Protects KRAS-Mutant Cancer Cells Against Ionizing Radiation

    PubMed Central

    Wang, Meng; Kern, Ashley M.; Hülskötter, Marieke; Greninger, Patricia; Singh, Anurag; Pan, Yunfeng; Chowdhury, Dipanjan; Krause, Mechthild; Baumann, Michael; Benes, Cyril H.; Efstathiou, Jason A.; Settleman, Jeff; Willers, Henning

    2014-01-01

    Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in-vitro and in-vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like co-localization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is co-dependent upon EGFR and PKCα. PKCα, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. PMID:24648348

  10. AZD9291 in EGFR-mutant advanced non-small-cell lung cancer patients.

    PubMed

    Remon, Jordi; Planchard, David

    2015-11-01

    Non-small-cell lung cancer (NSCLC) patients whose tumors have an EGFR-activating mutation develop acquired resistance after a median of 9-11 months from the beginning of treatment with erlotinib, gefitinib and afatinib. T790M mutation is the cause of this resistance in approximately 60% of cases. AZD9291 is an oral, irreversible, mutant-selective EGF receptor (EGFR) tyrosine kinase inhibitor (TKI) developed to have potency against EGFR mutations, including T790M mutation, while sparing wild-type EGFR. A Phase I trial of AZD9291 in EGFR-mutant NSCLC patients, demonstrated high activity, essentially among T790M-mutant tumors, with a manageable tolerability profile. Ongoing Phase III trials are evaluating AZD9291 in EGFR-mutant patients as first-line treatment compared with erlotinib and gefitinib; and as second-line treatment compared with chemotherapy after progression on EGFR TKI in T790M-mutant tumors. Better identification of T790M-mutant tumors post EGFR TKI relapse and mechanisms of resistance to AZD9291 are the future challenges. This article reviews the emerging data regarding AZD9291 in the treatment of patients with advanced NSCLC. PMID:26450446

  11. The Next Wave of EGFR Tyrosine Kinase Inhibitors Enter the Clinic.

    PubMed

    Politi, Katerina; Ayeni, Deborah; Lynch, Thomas

    2015-06-01

    The T790M mutation in EGFR accounts for approximately half of all lung cancer cases with acquired resistance to the current clinical EGFR tyrosine kinase inhibitors. In tyrosine kinase inhibitor-resistant lung tumors, rociletinib and AZD9291 are highly active when T790M is present and modestly active when T790M is absent. PMID:26058074

  12. EPHA2 Blockade Overcomes Acquired Resistance to EGFR Kinase Inhibitors in Lung Cancer.

    PubMed

    Amato, Katherine R; Wang, Shan; Tan, Li; Hastings, Andrew K; Song, Wenqiang; Lovly, Christine M; Meador, Catherine B; Ye, Fei; Lu, Pengcheng; Balko, Justin M; Colvin, Daniel C; Cates, Justin M; Pao, William; Gray, Nathanael S; Chen, Jin

    2016-01-15

    Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFR(T790M) mutations in vitro and inhibited tumor growth and progression in an inducible EGFR(L858R+T790M)-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors. PMID:26744526

  13. Inhibition of Receptor Dimerization as a Novel Negative Feedback Mechanism of EGFR Signaling

    PubMed Central

    Kluba, Malgorzata; Engelborghs, Yves; Hofkens, Johan; Mizuno, Hideaki

    2015-01-01

    Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca2+ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization. PMID:26465157

  14. Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms

    PubMed Central

    Li, Jiannong; Bennett, Keiryn; Stukalov, Alexey; Fang, Bin; Zhang, Guolin; Yoshida, Takeshi; Okamoto, Isamu; Kim, Jae-Young; Song, Lanxi; Bai, Yun; Qian, Xiaoning; Rawal, Bhupendra; Schell, Michael; Grebien, Florian; Winter, Georg; Rix, Uwe; Eschrich, Steven; Colinge, Jacques; Koomen, John; Superti-Furga, Giulio; Haura, Eric B

    2013-01-01

    We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance. PMID:24189400

  15. Bile acids regulate intestinal cell proliferation by modulating EGFR and FXR signaling.

    PubMed

    Dossa, Avafia Y; Escobar, Oswaldo; Golden, Jamie; Frey, Mark R; Ford, Henri R; Gayer, Christopher P

    2016-01-15

    Bile acids (BAs) are synthesized in the liver and secreted into the intestine. In the lumen, enteric bacteria metabolize BAs from conjugated, primary forms into more toxic unconjugated, secondary metabolites. Secondary BAs can be injurious to the intestine and may contribute to disease. The epidermal growth factor receptor (EGFR) and the nuclear farnesoid X receptor (FXR) are known to interact with BAs. In this study we examined the effects of BAs on intestinal epithelial cell proliferation and investigated the possible roles for EGFR and FXR in these effects. We report that taurine-conjugated cholic acid (TCA) induced proliferation, while its unconjugated secondary counterpart deoxycholic acid (DCA) inhibited proliferation. TCA stimulated phosphorylation of Src, EGFR, and ERK 1/2. Pharmacological blockade of any of these pathways or genetic ablation of EGFR abrogated TCA-stimulated proliferation. Interestingly, Src or EGFR inhibitors eliminated TCA-induced phosphorylation of both molecules, suggesting that their activation is interdependent. In contrast to TCA, DCA exposure diminished EGFR phosphorylation, and pharmacological or siRNA blockade of FXR abolished DCA-induced inhibition of proliferation. Taken together, these results suggest that TCA induces intestinal cell proliferation via Src, EGFR, and ERK activation. In contrast, DCA inhibits proliferation via an FXR-dependent mechanism that may include downstream inactivation of the EGFR/Src/ERK pathway. Since elevated secondary BA levels are the result of specific bacterial modification, this may provide a mechanism through which an altered microbiota contributes to normal or abnormal intestinal epithelial cell proliferation. PMID:26608185

  16. Multiplex FISH telomere integrity assay identifies an unbalanced cryptic translocation der(5)t(3;5)(q27;p15.3) in a family with three mentally retarded individuals.

    PubMed

    Granzow, M; Popp, S; Keller, M; Holtgreve-Grez, H; Brough, M; Schoell, B; Rauterberg-Ruland, I; Hager, H D; Tariverdian, G; Jauch, A

    2000-07-01

    Cryptic rearrangements involving the terminal regions of chromosomes are suspected to be the cause of idiopathic mental retardation in a significant number of cases. This finding highlights the necessity of a primary screening test for such chromosome aberrations. Here we present a multiplex fluorescence in situ hybridization telomere integrity assay which allows the detection of submicroscopic aberrations in the telomeric regions of all chromosomes. This novel approach identified an unbalanced cryptic translocation der(5)t(3;5)(q27;p15.3) in a family with three cases of unexplained mental retardation and dysmorphic features. The symptoms of the patients represent neither the classical dup(3q)- nor cri du chat syndrome, although all affected individuals demonstrate several features of both syndromes. The identification of two balanced translocation carriers emphasizes the significance of the telomere integrity assay for genetic counseling and prenatal diagnosis. PMID:10982035

  17. Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

    SciTech Connect

    Song, Xiulong Wei, Zhengxi; Shaikh, Zahir A.

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  18. MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands

    PubMed Central

    CHUNG, EUN JOO; URICK, MARY ELLEN; KURSHAN, NAAMIT; SHIELD, WILLIAM; ASANO, HIROAKI; SMITH, PAUL D.; SCROGGINS, BRADLEY S.; BURKEEN, JEFFREY; CITRIN, DEBORAH E.

    2013-01-01

    The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and check-point kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR. PMID:23588995

  19. DESC1, a novel tumor suppressor, sensitizes cells to apoptosis by downregulating the EGFR/AKT pathway in esophageal squamous cell carcinoma.

    PubMed

    Ng, Hoi Yan; Ko, Josephine Mun-Yee; Yu, Valen Zhuoyou; Ip, Joseph Chok Yan; Dai, Wei; Cal, Santiago; Lung, Maria Li

    2016-06-15

    Esophageal cancer is ranked as the eighth most common cancer and the sixth leading cause of cancer deaths worldwide. To identify candidate tumor suppressor genes related to esophageal squamous cell carcinoma (ESCC) development, a cDNA microarray analysis was performed using paired tumor and nontumor tissue samples from ESCC patients. Differentially expressed in squamous cell carcinoma 1 (DESC1), which belongs to the Type II transmembrane serine protease family, was frequently downregulated in ESCC. This study aims to elucidate the molecular mechanism for the tumor suppressive function of DESC1 in ESCC. We show that DESC1 reduced cell viability and sensitized cells to apoptosis, when cells were under apoptotic stimuli. The proapoptotic effect of DESC1 was mediated through downregulating AKT1 activation and the restoration of AKT activation by the introduction of the constitutively active AKT, myr-AKT, abolished the apoptosis-sensitizing effect of DESC1. DESC1 also reduced EGFR protein level, which was abrogated when the proteolytic function of DESC1 was lost, suggesting that DESC1 cleaved EGFR and downregulated the EGFR/AKT pathway to favor apoptosis. The transmembrane localization and the structural domains provide an opportunity for DESC1 to interact with the extracellular environment. The importance of such interaction was highlighted by the finding that DESC1 reduced cell colony formation ability in three-dimensional culture. In line with this, DESC1 reduced tumor growth kinetics in the in vivo orthotopic tumorigenesis assay. Taken together, our novel findings suggest how DESC1 may suppress ESCC development by sensitizing cells to apoptosis under an apoptotic stimulus through downregulating the EGFR/AKT signaling pathway. PMID:26856390

  20. MALT1 is required for EGFR-induced NF-κB activation and contributes to EGFR-driven lung cancer progression.

    PubMed

    Pan, D; Jiang, C; Ma, Z; Blonska, M; You, M J; Lin, X

    2016-02-18

    The transcription factor nuclear factor kappa B (NF-κB) has been implicated in having a crucial role in the tumorigenesis of many types of human cancers. Although epidermal growth factor receptor (EGFR) can directly activate NF-κB, the mechanism by which EGFR induces NF-κB activation and the role of NF-κB in EGFR-associated tumor progression is still not fully defined. Herein, we found that mucosa-associated lymphoid tissue 1 (MALT1) is involved in EGFR-induced NF-κB activation in cancer cells, and that MALT1 deficiency impaired EGFR-induced NF-κB activation. MALT1 mainly functions as a scaffold protein by recruiting E3 ligase TRAF6 to IKK complex to activate NF-κB in response to EGF stimulation. Functionally, MALT1 inhibition shows significant defects in EGFR-associated tumor malignancy, including cell migration, metastasis and anchorage-independent growth. To further access a physiological role of MALT1-dependent NF-κB activation in EGFR-driven tumor progression, we generated triple-transgenic mouse model (tetO-EGFR(L858R); CCSP-rtTA; Malt1(-/-)), in which mutant EGFR-driven lung cancer was developed in the absence of MALT1 expression. MALT1-deficient mice show significantly less lung tumor burden when compared with its heterozygous controls, suggesting that MALT1 is required for the progression of EGFR-induced lung cancer. Mechanistically, MALT1 deficiency abolished both NF-κB and STAT3 activation in vivo, which is a result of a defect of interleukin-6 production. In comparison, MALT1 deficiency does not affect tumor progression in a mouse model (LSL-K-ras(G12D); CCSP-Cre; Malt1(-/-)) in which lung cancer is induced by expressing a K-ras mutant. Thus, our study has provided the cellular and genetic evidence that suggests MALT1-dependent NF-κB activation is important in EGFR-associated solid-tumor progression. PMID:25982276

  1. Novel morpholin-3-one fused quinazoline derivatives as EGFR tyrosine kinase inhibitors.

    PubMed

    Qin, Xuemei; Lv, Yongjuan; Liu, Peng; Li, Zhipeng; Hu, Liming; Zeng, Chengchu; Yang, Leifu

    2016-03-15

    A series of novel morpholin-3-one-fused quinazoline derivatives were designed, synthesized and evaluated as EGFR tyrosine kinase inhibitors. Nineteen compounds showed significant inhibitory activities against EGFR(wt) kinase (IC50<1 μM). Compound a8 demonstrated the most potent inhibitory activity toward EGFR(wt) (IC50=53.1 nM). Compound a7 and a8 showed excellent inhibitory activities against mutant EGFR(T790M/L858R) and strong antiproliferative activity against H358 and A549 cell lines. Finally, molecular docking studies were performed to predict the possible binding mode of the target compounds. It is believed that this work would be very useful for designing a new series of tyrosine kinase inhibitors targeting EGFR. PMID:26879314