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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. 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.

  7. 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

  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. 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.

  5. 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

  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. 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

  5. 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

  6. 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

  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. 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

  6. 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

  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. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  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. 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

  16. 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

  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. 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

  20. 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

  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 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

  4. 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

  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. 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.

  8. 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.

  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. 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

  16. 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

  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. 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

  2. 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

  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. 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

  9. 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

  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

    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 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

  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. 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

  17. 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...

  18. 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.

  19. 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

  20. 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

  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. 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

  10. 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

  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. 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

  19. 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

  20. 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

  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. 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

  3. 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

  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. 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

  10. 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