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Sample records for activating kras mutations

  1. KRAS Mutation

    PubMed Central

    Franklin, Wilbur A.; Haney, Jerry; Sugita, Michio; Bemis, Lynne; Jimeno, Antonio; Messersmith, Wells A.

    2010-01-01

    Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing. PMID:20007845

  2. The Differential Effects of Wild-Type and Mutated K-Ras on MST2 Signaling Are Determined by K-Ras Activation Kinetics

    PubMed Central

    Romano, David; Maccario, Helene; Doherty, Carolanne; Quinn, Niall P.

    2013-01-01

    K-Ras is frequently mutated in human cancers. Mutant (mt) K-Ras can stimulate both oncogenic transformation and apoptosis through activation of extracellular signal-regulated kinase (ERK) and AKT pathways and the MST2 pathway, respectively. The biological outcome is determined by the balance and cross talk between these pathways. In colorectal cancer (CRC), a K-Ras mutation is negatively correlated with MST2 expression, as mt K-Ras can induce apoptosis by activating the MST2 pathway. However, wild-type (wt) K-Ras can prevent the activation of the MST2 pathway upon growth factor stimulation and enable transformation by mt K-Ras in CRC cells that express MST2. Here we have investigated the mechanism by which wt and mt K-Ras differentially regulate the MST2 pathway and MST2-dependent apoptosis. The ability of K-Ras to activate MST2 and MST2-dependent apoptosis is determined by the differential activation kinetics of mt K-Ras and wt K-Ras. Chronic activation of K-Ras by mutation or overexpression of Ras exchange factors results in the activation of MST2 and LATS1, increased MST2-LATS1 complex formation, and apoptosis. In contrast, transient K-Ras activation upon epidermal growth factor (EGF) stimulation prevents the formation of the MST2-LATS1 complex in an AKT-dependent manner. Our data suggest that the close relationship between Ras prosurvival and proapoptotic signaling is coordinated via the differential regulation of the MST2-LATS1 interaction by transient and chronic stimuli. PMID:23459937

  3. Rosai-Dorfman Disease Harboring an Activating KRAS K117N Missense Mutation.

    PubMed

    Shanmugam, Vignesh; Margolskee, Elizabeth; Kluk, Michael; Giorgadze, Tamara; Orazi, Attilio

    2016-09-01

    Rosai-Dorfman disease (RDD) or sinus histiocytosis with massive lymphadenopathy is a rare histiocytic proliferation that is generally considered to be reactive with a benign clinical course. The etiology of RDD is very poorly understood. Recent studies have shown frequent BRAF, NRAS, KRAS, and PIK3CA activating mutations in several histiocytic neoplasms highlighting the emerging importance of the RAF/MEK/ERK pathway in the pathogenesis of these diseases. Here we report a case of Rosai-Dorfman disease involving the submandibular salivary gland with a KRAS K117N missense mutation discovered by next-generation sequencing. These results suggest that at least a subset of RDD cases may be clonal processes. Further mutational studies on this rare histiocytic disease should be undertaken to better characterize its pathogenesis as well as open up potential avenues for therapy. PMID:26922062

  4. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    PubMed

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  5. Anti-tumor activity of ESX1 on cancer cells harboring oncogenic K-ras mutation

    SciTech Connect

    Nakajima, Junta; Ishikawa, Susumu; Hamada, Jun-Ichi; Yanagihara, Masatomo; Koike, Takao; Hatakeyama, Masanori

    2008-05-23

    Human ESX1 is a 65-kilodalton (kDa) paired-like homeoprotein that is proteolytically processed into N-terminal 45-kDa and C-terminal 20-kDa fragments. The N-terminal ESX1 fragment, which contains the homeodomain, localizes to the nucleus and represses mRNA transcription from the K-ras gene. When we inoculated human colorectal carcinoma HCT116 constitutive expressing N-terminal region of ESX1 (N-ESX1) into nude mice, transfectant cells uniformly showed decreased tumor-forming activity compared with that of the parental cells. Furthermore, pretreatment of HCT116 carcinoma cells with a fusion protein consisting of N-ESX1 and the protein-transduction domain derived from the human immunodeficiency virus type-1 TAT protein gave rise to a dramatic reduction in the tumorigenicity of HCT116 cells in nude mice. Our results provide first in vivo evidence for the molecular targeting therapeutic application of the K-ras repressor ESX1, especially TAT-mediated transduction of N-ESX1, in the treatment of human cancers having oncogenic K-ras mutations.

  6. KRAS Mutations in Canine and Feline Pancreatic Acinar Cell Carcinoma.

    PubMed

    Crozier, C; Wood, G A; Foster, R A; Stasi, S; Liu, J H W; Bartlett, J M S; Coomber, B L; Sabine, V S

    2016-07-01

    Companion animals may serve as valuable models for studying human cancers. Although KRAS is the most commonly mutated gene in human ductal pancreatic cancers (57%), with mutations frequently occurring at codons 12, 13 and 61, human pancreatic acinar cell carcinomas (ACCs) lack activating KRAS mutations. In the present study, 32 pancreatic ACC samples obtained from 14 dogs and 18 cats, including seven metastases, were analyzed for six common activating KRAS mutations located in codons 12 (n = 5) and 13 (n = 1) using Sequenom MassARRAY. No KRAS mutations were found, suggesting that, similar to human pancreatic ACC, KRAS mutations do not play a critical role in feline or canine pancreatic ACC. Due to the similarity of the clinical disease in dogs and cats to that of man, this study confirms that companion animals offer potential as a suitable model for investigating this rare subtype of pancreatic carcinoma. PMID:27290644

  7. Diagnostic application of KRAS mutation testing in uterine microglandular proliferations.

    PubMed

    Hong, Wei; Abi-Raad, Rita; Alomari, Ahmed K; Hui, Pei; Buza, Natalia

    2015-07-01

    Microglandular proliferations often pose a diagnostic challenge in small endocervical and endometrial biopsies. Microglandular hyperplasia (MGH) is one of the most common pseudoneoplastic glandular proliferations of uterine cervix, which can closely mimic endometrial adenocarcinomas (EAC) with a microglandular pattern (microglandular EAC). Although MGH is typically characterized by relatively uniform nuclei and rare to absent mitoses, atypical forms with architectural and/or cytologic deviation from the usual morphology have been previously described. Recently, a series of MGH with high mitotic activity has also been documented. Although careful morphological assessment and immunohistochemical workup can resolve the diagnostic dilemma in some cases, additional differential diagnostic tools are needed to separate both the common and atypical variants of MGH from EAC with microglandular pattern. Frequent KRAS mutation has been previously reported in endometrial complex mucinous lesions and endometrial mucinous carcinomas. However, the diagnostic utility of KRAS mutation analysis has not yet been explored in the context of cervical and endometrial microglandular lesions. Twelve mitotically active MGH cases and 15 cases of EAC with microglandular growth pattern were selected for the study. KRAS mutation analysis was performed in all cases by highly sensitive single-strand conformation polymorphism analysis. Clinical history and follow-up data were retrieved from electronic medical records. KRAS mutation was absent in all MGH cases, whereas 9 (60%) of 15 microglandular EAC cases tested positive for KRAS mutation. Our data indicate that KRAS mutation analysis may offer additional discriminatory power in separating benign MGH from EAC with microglandular pattern. PMID:25997988

  8. Molecular spectrum of BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias: implication for MEK-ERK pathway activation

    PubMed Central

    Lionetti, Marta; Barbieri, Marzia; Todoerti, Katia; Agnelli, Luca; Marzorati, Simona; Fabris, Sonia; Ciceri, Gabriella; Galletti, Serena; Milesi, Giulia; Manzoni, Martina; Mazzoni, Mara; Greco, Angela; Tonon, Giovanni; Musto, Pellegrino; Baldini, Luca; Neri, Antonino

    2015-01-01

    Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell (PC) malignancy. Whole-exome sequencing has identified therapeutically targetable mutations such as those in the mitogen-activated protein kinase (MAPK) pathway, which are the most prevalent MM mutations. We used deep sequencing to screen 167 representative patients with PC dyscrasias [132 with MM, 24 with primary PC leukemia (pPCL) and 11 with secondary PC leukemia (sPCL)] for mutations in BRAF, NRAS and KRAS, which were respectively found in 12%, 23.9% and 29.3% of cases. Overall, the MAPK pathway was affected in 57.5% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). The majority of BRAF variants were comparably expressed at transcript level. Additionally, gene expression profiling indicated the MAPK pathway is activated in mutated patients. Finally, we found that vemurafenib inhibition of BRAF activation in mutated U266 cells affected the expression of genes known to be associated with MM. Our data confirm and extend previous published evidence that MAPK pathway activation is recurrent in myeloma; the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications. PMID:26090869

  9. Molecular spectrum of BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias: implication for MEK-ERK pathway activation.

    PubMed

    Lionetti, Marta; Barbieri, Marzia; Todoerti, Katia; Agnelli, Luca; Marzorati, Simona; Fabris, Sonia; Ciceri, Gabriella; Galletti, Serena; Milesi, Giulia; Manzoni, Martina; Mazzoni, Mara; Greco, Angela; Tonon, Giovanni; Musto, Pellegrino; Baldini, Luca; Neri, Antonino

    2015-09-15

    Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell (PC) malignancy. Whole-exome sequencing has identified therapeutically targetable mutations such as those in the mitogen-activated protein kinase (MAPK) pathway, which are the most prevalent MM mutations. We used deep sequencing to screen 167 representative patients with PC dyscrasias [132 with MM, 24 with primary PC leukemia (pPCL) and 11 with secondary PC leukemia (sPCL)] for mutations in BRAF, NRAS and KRAS, which were respectively found in 12%, 23.9% and 29.3% of cases. Overall, the MAPK pathway was affected in 57.5% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). The majority of BRAF variants were comparably expressed at transcript level. Additionally, gene expression profiling indicated the MAPK pathway is activated in mutated patients. Finally, we found that vemurafenib inhibition of BRAF activation in mutated U266 cells affected the expression of genes known to be associated with MM. Our data confirm and extend previous published evidence that MAPK pathway activation is recurrent in myeloma; the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications. PMID:26090869

  10. KRAS, HRAS and EGFR Mutations in Sporadic Sebaceous Gland Hyperplasia.

    PubMed

    Groesser, Leopold; Singer, Sebastian; Peterhof, Eva; Landthaler, Michael; Heigl, Ulrike; Schneider-Brachert, Wulf; Berneburg, Mark; Hafner, Christian

    2016-08-23

    Sporadic sebaceous gland hyperplasia (SGH) is a benign skin lesion, with a high prevalence in the general population. Although SGH has been attributed to both extrinsic and intrinsic factors, the underlying genetic changes have not yet been characterized. Recently, HRAS and KRAS mutations have been identified in sebaceous naevus, a hamartoma sharing histological characteristics with SGH. Therefore we screened 43 SGH for activating mutations in RAS genes and other oncogenes. We identified a wide spectrum of mutually exclusive activating HRAS (8/43), KRAS (11/43) and EGFR mutations (7/31) in altogether 60% of the lesions investigated. A RAS and EGFR wildtype status was found in 15 normal sebaceous glands in the head and neck area. Our findings indicate that activating HRAS, KRAS and EGFR mutations play a major role in the pathogenesis of sporadic SGH. These results support the concept that SGH is a true benign neoplasm rather than a reactive hyperplasia. PMID:26804118

  11. Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

    PubMed Central

    Sloan, S R; Newcomb, E W; Pellicer, A

    1990-01-01

    Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA. Images PMID:2403644

  12. KRAS insertion mutations are oncogenic and exhibit distinct functional properties

    PubMed Central

    White, Yasmine; Bagchi, Aditi; Van Ziffle, Jessica; Inguva, Anagha; Bollag, Gideon; Zhang, Chao; Carias, Heidi; Dickens, David; Loh, Mignon; Shannon, Kevin; Firestone, Ari J.

    2016-01-01

    Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-RasG60_A66dup and K-RasE62_A66dup proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications. PMID:26854029

  13. The Presence of Telomere Fusion in Sporadic Colon Cancer Independently of Disease Stage, TP53/KRAS Mutation Status, Mean Telomere Length, and Telomerase Activity

    PubMed Central

    Tanaka, Hiromi; Beam, Matthew J.; Caruana, Kevin

    2014-01-01

    Defects in telomere maintenance can result in telomere fusions that likely play a causative role in carcinogenesis by promoting genomic instability. However, this proposition remains to be fully understood in human colon carcinogenesis. In the present study, the temporal sequence of telomere dysfunction dynamics was delineated by analyzing telomere fusion, telomere length, telomerase activity, hotspot mutations in KRAS or BRAF, and TP53 of tissue samples obtained from 18 colon cancer patients. Our results revealed that both the deficiency of p53 and the shortening of mean telomere length were not necessary for producing telomere fusions in colon tissue. In five cases, telomere fusion was observed even in tissue adjacent to cancerous lesions, suggesting that genomic instability is initiated in pathologically non-cancerous lesions. The extent of mean telomere attrition increased with lymph node invasiveness of tumors, implying that mean telomere shortening correlates with colon cancer progression. Telomerase activity was relatively higher in most cancer tissues containing mutation(s) in KRAS or BRAF and/or TP53 compared to those without these hotspot mutations, suggesting that telomerase could become fully active at the late stage of colon cancer development. Interestingly, the majority of telomere fusion junctions in colon cancer appeared to be a chromatid-type containing chromosome 7q or 12q. In sum, this meticulous correlative study not only highlights the concept that telomere fusion is present in the early stages of cancer regardless of TP53/KRAS mutation status, mean telomere length, and telomerase activity, but also provides additional insights targeting key telomere fusion junctions which may have significant implications for colon cancer diagnoses. PMID:25379018

  14. Epidermal growth factor receptor and KRAS mutations in Brazilian lung cancer patients

    PubMed Central

    Bacchi, Carlos E.; Ciol, Heloísa; Queiroga, Eduardo M.; Benine, Lucimara C.; Silva, Luciana H.; Ojopi, Elida B.

    2012-01-01

    OBJECTIVE: Epidermal growth factor receptor is involved in the pathogenesis of non-small cell lung cancer and has recently emerged as an important target for molecular therapeutics. The KRAS oncogene also plays an important role in the development of lung cancer. The aim of this study was to evaluate the frequency of epidermal growth factor receptor and KRAS mutations in a population of Brazilian patients with non-small cell lung cancer. METHODS: A total of 207 specimens from Brazilian patients with non-small cell lung cancer were analyzed for activating epidermal growth factor receptor and KRAS somatic mutations, and their associations with clinicopathological characteristics (including age, gender, ethnicity, smoking habits, and histological subtype) were examined. RESULTS: We identified 63 cases (30.4%) with epidermal growth factor receptor mutations and 30 cases (14.6%) with KRAS mutations. The most frequent epidermal growth factor receptor mutation we detected was a deletion in exon 19 (60.3%, 38 patients), followed by an L858R amino acid substitution in exon 21 (27%, 17 patients). The most common types of KRAS mutations were found in codon 12. There were no significant differences in epidermal growth factor receptor or KRAS mutations by gender or primary versus metastatic lung cancer. There was a higher prevalence of KRAS mutations in the non-Asian patients. Epidermal growth factor receptor mutations were more prevalent in adenocarcinomas than in non-adenocarcinoma histological types. Being a non-smoker was significantly associated with the prevalence of epidermal growth factor receptor mutations, but the prevalence of KRAS mutations was significantly associated with smoking. CONCLUSIONS: This study is the first to examine the prevalence of epidermal growth factor receptor and KRAS mutations in a Brazilian population sample with non-small cell lung cancer. PMID:22666783

  15. p53 mutations cooperate with oncogenic Kras to promote adenocarcinoma from pancreatic ductal cells.

    PubMed

    Bailey, J M; Hendley, A M; Lafaro, K J; Pruski, M A; Jones, N C; Alsina, J; Younes, M; Maitra, A; McAllister, F; Iacobuzio-Donahue, C A; Leach, S D

    2016-08-11

    Pancreatic cancer is one of the most lethal malignancies, with virtually all patients eventually succumbing to their disease. Mutations in p53 have been documented in >50% of pancreatic cancers. Owing to the high incidence of p53 mutations in PanIN 3 lesions and pancreatic tumors, we interrogated the comparative ability of adult pancreatic acinar and ductal cells to respond to oncogenic Kras and mutant Tp53(R172H) using Hnf1b:CreER(T2) and Mist1:CreER(T2) mice. These studies involved co-activation of a membrane-tethered GFP lineage label, allowing for direct visualization and isolation of cells undergoing Kras and mutant p53 activation. Kras activation in Mist1(+) adult acinar cells resulted in brisk PanIN formation, whereas no evidence of pancreatic neoplasia was observed for up to 6 months following Kras activation in Hnf1beta(+) adult ductal cells. In contrast to the lack of response to oncogenic Kras alone, simultaneous activation of Kras and mutant p53 in adult ductal epithelium generated invasive PDAC in 75% of mice as early as 2.5 months after tamoxifen administration. These data demonstrate that pancreatic ductal cells, whereas exhibiting relative resistance to oncogenic Kras alone, can serve as an effective cell of origin for pancreatic ductal adenocarcinoma in the setting of gain-of-function mutations in p53. PMID:26592447

  16. Heterogeneity of KRAS Mutation Status in Rectal Cancer

    PubMed Central

    Jo, Peter; König, Alexander; Schirmer, Markus; Kitz, Julia; Conradi, Lena-Christin; Azizian, Azadeh; Bernhardt, Markus; Wolff, Hendrik A.; Grade, Marian; Ghadimi, Michael; Ströbel, Philipp; Schildhaus, Hans-Ulrich; Gaedcke, Jochen

    2016-01-01

    Introduction Anti-EGFR targeted therapy is of increasing importance in advanced colorectal cancer and prior KRAS mutation testing is mandatory for therapy. However, at which occasions this should be performed is still under debate. We aimed to assess in patients with locally advanced rectal cancer whether there is intra-specimen KRAS heterogeneity prior to and upon preoperative chemoradiotherapy (CRT), and if there are any changes in KRAS mutation status due to this intervention. Materials and Methods KRAS mutation status analyses were performed in 199 tumor samples from 47 patients with rectal cancer. To evaluate the heterogeneity between different tumor areas within the same tumor prior to preoperative CRT, 114 biopsies from 34 patients (mean 3 biopsies per patient) were analyzed (pre-therapeutic intratumoral heterogeneity). For the assessment of heterogeneity after CRT residual tumor tissue (85 samples) from 12 patients (mean 4.2 tissue samples per patient) were analyzed (post-therapeutic intratumoral heterogeneity) and assessment of heterogeneity before and after CRT was evaluated in corresponding patient samples (interventional heterogeneity). Primer extension method (SNaPshot™) was used for initial KRAS mutation status testing for Codon 12, 13, 61, and 146. Discordant results by this method were reevaluated by using the FDA-approved KRAS Pyro Kit 24, V1 and the RAS Extension Pyro Kit 24, V1 Kit (therascreen® KRAS test). Results For 20 (43%) out of the 47 patients, a KRAS mutation was detected. With 12 out of 20, the majority of these mutations affected codon 35. We did not obtained evidence that CRT results in changes of the KRAS mutation pattern. In addition, no intratumoral heterogeneity in the KRAS mutational status could be proven. This was true for both the biopsies prior to CRT and the resection specimens thereafter. The discrepancy observed in some samples when using the SNaPshot™ assay was due to insufficient sensitivity of this technique upon

  17. A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer.

    PubMed Central

    Taya, Y; Hosogai, K; Hirohashi, S; Shimosato, Y; Tsuchiya, R; Tsuchida, N; Fushimi, M; Sekiya, T; Nishimura, S

    1984-01-01

    Amplifications of two oncogenes, c-K-ras-2 and c-myc, were found in a human lung giant cell carcinoma (LGCC) Lu-65, which is maintained in nude mice. The extent of c-K-ras-2 and myc amplifications were estimated to be 10- and 8-fold, respectively, by means of the Southern hybridization procedure. In addition, NIH3T3 cells were transformed by transfection of Lu-65 DNA and the transforming gene was identified as c-K-ras-2. c-K-ras-2 genes were cloned from a gene library of Lu-65 and a single point mutation causing a substitution of cysteine for glycine in codon 12 was found by DNA sequencing. It was concluded that the amplification of the c-myc and c-K-ras-2 genes are accompanied by point mutational activation of c-K-ras-2 in the human LGCC Lu-65. This is the first report of multiple gene amplification accompanied by a point mutation of oncogenes in human cancer cells, providing further support for the idea that co-operation of at least two activated cellular oncogenes is required for carcinogenesis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:6098458

  18. Frequent KRAS mutation in complex mucinous epithelial lesions of the endometrium.

    PubMed

    Alomari, Ahmed; Abi-Raad, Rita; Buza, Natalia; Hui, Pei

    2014-05-01

    KRAS mutation correlates with mucinous differentiation in various human cancers, and recently, was found in a high proportion of a small cohort of papillary mucinous lesions of the endometrium. In this study, a large number of endometrial mucinous lesions were analyzed for the presence of KRAS mutation along with clinical progression. A total of 45 endometrial biopsy/curettage cases were included in the study and classified into the following categories: simple mucinous change (5 cases), complex mucinous change (33 cases) and mucinous adenocarcinoma (7 cases). Follow-up hysterectomy specimens were available in 14 of 33 patients (42%) with complex mucinous lesions, of which 9 cases (64%) showed atypical complex hyperplasia with an average interval of 21 weeks. None of the 5 cases of simple mucinous change showed KRAS mutation. KRAS mutation was observed in 18 of 33 patients with complex mucinous lesions (55%) and in 6 of 7 cases of mucinous adenocarcinoma (86%). Overall, KRAS mutation has a positive predictive value (PPV) of 88% (7/8 cases) for complex atypical hyperplasia or adenocarcinoma in the follow-up hysterectomy. In conclusion, the current data further emphasizes the architectural complexity as an important prognostic indicator for patients with mucinous endometrial lesions. The presence of KRAS mutation in both mucinous adenocarcinoma and complex mucinous changes indicates that KRAS mutational activation is implicated in the pathogenesis of a significant subset of endometrial mucinous carcinoma. With a high PPV, KRAS mutation analysis may offer an additional discriminatory power to refine risk stratification algorithm for patients with endometrial mucinous lesions. PMID:24186144

  19. Characterization of a novel oncogenic K-ras mutation in colon cancer

    SciTech Connect

    Akagi, Kiwamu . E-mail: akagi@cancer-c.pref.saitama.jp; Uchibori, Ryosuke; Yamaguchi, Kensei; Kurosawa, Keiko; Tanaka, Yoichiro; Kozu, Tomoko

    2007-01-19

    Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.

  20. MicroRNA Expression Signatures Associated With BRAF-Mutated Versus KRAS-Mutated Colorectal Cancers

    PubMed Central

    Choi, Yong Won; Song, Young Soo; Lee, Hyunwoo; Yi, Kijong; Kim, Young-Bae; Suh, Kwang Wook; Lee, Dakeun

    2016-01-01

    Abstract BRAF and KRAS genes are known to play a similar role in the activation of RAS-RAF-MEK-ERK signaling pathway in colorectal tumorigenesis. However, BRAF-mutated colorectal cancers (CRCs) have distinct clinicopathologic characteristics different from those of the KRAS mutated ones as in comparison the BRAF-mutated CRCs are associated with a much worse prognosis for the afflicted patients. This study aimed to determine the different miRNA expression signatures associated with BRAF-mutated CRCs in comparison to KRAS-mutated ones, and to identify the specific miRNAs possibly mediating the aggressive phenotype of the BRAF-mutated CRCs. We screened 535 formalin-fixed paraffin-embedded CRC tissue samples for the BRAF V600E mutation, and selected 7 BRAF-mutated and 7 KRAS-mutated CRCs that were tumor size, stage, and microsatellite status-matched. Affymetrix GeneChip® miRNA 4.0 Array was used for detection of miRNA expression differences in the selected samples. We validated the array results by quantitative reverse transcription polymerase chain reaction (qRT-PCR) for selected miRNAs. A total of 10 differentially expressed (DE) miRNAs associated with BRAF-mutated CRCs were obtained, including miR-31-5p, miR-877-5p, miR-362-5p, and miR-425-3p. miR-31-5p showed the highest fold change (8.3-fold) among all of the miRNAs analyzed. From the analyses of GO biological processes, the DE-miRNAs were functionally relevant to cellular proliferation such as positive regulation of gene expression (P = 1.26 × 10−10), transcription (P = 9.70 × 10−10), and RNA metabolic process (P = 1.97 × 10−9). Bioinformatics analysis showed that the DE-miRNAs were significantly enriched in cancer-associated pathways including neutrophin signaling (P = 6.84 × 10−5), pathways in cancer (P = 0.0016), Wnt signaling (P = 0.0027), and MAPK signaling pathway (P = 0.0036). Our results suggest that the DE-miRNAs in BRAF-mutated CRCs in comparison

  1. PIK3CA mutation uncouples tumor growth and Cyclin D1 regulation from MEK/ERK and mutant KRAS signaling

    PubMed Central

    Halilovic, Ensar; She, Qing-Bai; Ye, Qing; Pagliarini, Raymond; Sellers, William R.; Solit, David B.; Rosen, Neal

    2010-01-01

    Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small molecule MEK inhibitor, decreases MEK/ERK pathway signaling, and destabilizes Cyclin D1, resulting in significant anti-cancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores Cyclin D1 expression and G1/S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and PI3K mutations are unlikely to respond to inhibition of the MEK pathway alone but will require effective inhibition of both MEK and PI3K/AKT pathway signaling. PMID:20699365

  2. Analysis of KRAS and BRAF genes mutation in the central nervous system metastases of non-small cell lung cancer.

    PubMed

    Nicoś, Marcin; Krawczyk, Paweł; Jarosz, Bożena; Sawicki, Marek; Szumiłło, Justyna; Trojanowski, Tomasz; Milanowski, Janusz

    2016-05-01

    KRAS mutations are associated with tumor resistance to EGFR TKIs (erlotinib, gefitinib) and to monoclonal antibody against EGFR (cetuximab). Targeted treatment of mutated RAS patients is still considered as a challenge. Inhibitors of c-Met (onartuzumab or tiwantinib) and MEK (selumetinib-a dual inhibitor of MEK1 and MEK2) signaling pathways showed activity in patients with mutations in KRAS that can became an effective approach in carriers of such disorders. BRAF mutation is very rare in patients with NSCLC, and its presence is associated with sensitivity of tumor cells to BRAF inhibitors (vemurafenib, dabrafenib). In the present study, the frequency and type of KRAS and BRAF mutation were assessed in 145 FFPE tissue samples from CNS metastases of NSCLC. In 30 patients, material from the primary tumor was simultaneously available. Real-time PCR technique with allele-specific molecular probe (KRAS/BRAF Mutation Analysis Kit, Entrogen, USA) was used for molecular tests. KRAS mutations were detected in 21.4 % of CNS metastatic lesions and in 23.3 % of corresponding primary tumors. Five mutations were identified both in primary and in metastatic lesions, while one mutation only in primary tumor and one mutation only in the metastatic tumor. Most of mutations were observed in codon 12 of KRAS; however, an individual patient had diagnosed a rare G13D and Q61R substitutions. KRAS mutations were significantly more frequent in adenocarcinoma patients and smokers. Additional analysis indicated one patient with rare coexistence of KRAS and DDR2 mutations. BRAF mutation was not detected in the examined materials. KRAS frequency appears to be similar in primary and CNS. PMID:25902737

  3. The impact of KRAS mutations on VEGF-A production and tumour vascular network

    PubMed Central

    2013-01-01

    Background The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Methods Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Results Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Conclusion Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis. PMID:23506169

  4. An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations.

    PubMed

    Liu, Yuliang; Gudnason, Haukur; Li, Yi-Ping; Bang, Dang Duong; Wolff, Anders

    2014-10-01

    Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidity and mortality worldwide. CRC is curable if diagnosed at an early stage. Mutations in the oncogene KRAS play a critical role in early development of CRC. Detection of activated KRAS is of diagnostic and therapeutic importance. In this study, KRAS gene fragments containing mutations in codon 12 were amplified by multiplex PCR using a 5'-Cy5-labeled reverse primer in combination with 3'-mutation-specific forward primers that were linked with four unique nucleotide-sequence tags at the 5'-end. The Cy5-labeled reverse primer was extended under PCR amplification to the 5'-end of the mutation-specific forward primers and thus included the complimentary sequence of the tag. PCR products were hybridized to tag-probes immobilized on various substrates and detected by a scanner. Our results indicate that all mutations at codon 12 of KRAS derived from cancer cells and clinical samples could be unambiguously detected. KRAS mutations were accurately detected when the mutant DNA was present only in 10% of the starting mixed materials including wild-type genomic DNA, which was isolated from either cancer cells or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable to be used for detection of KRAS mutations and clinical diagnosis of CRC. PMID:25018048

  5. KRAS Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in Prediction of Lung Metastasis

    PubMed Central

    Cejas, Paloma; López-Gómez, Miriam; Aguayo, Cristina; Madero, Rosario; de Castro Carpeño, Javier; Belda-Iniesta, Cristóbal; Barriuso, Jorge; Moreno García, Víctor; Larrauri, Javier; López, Rocío; Casado, Enrique; Gonzalez-Barón, Manuel; Feliu, Jaime

    2009-01-01

    Background KRAS mutations in colorectal cancer primary tumors predict resistance to anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody therapy in patients with metastatic colorectal cancer, and thus represent a true indicator of EGFR pathway activation status. Methodology/Principal Findings KRAS mutations were retrospectively studied using polymerase chain reactions and subsequent sequencing of codons 12 and 13 (exon 2) in 110 patients with metastatic colorectal tumors. These studies were performed using tissue samples from both the primary tumor and their related metastases (93 liver, 84%; 17 lung, 16%). All patients received adjuvant 5-Fluorouracil-based polychemotherapy after resection of metastases. None received anti-EGFR therapy. Mutations in KRAS were observed in 37 (34%) of primary tumors and in 40 (36%) of related metastases, yielding a 94% level of concordance (kappa index 0.86). Patients with primary tumors possessing KRAS mutations had a shorter disease-free survival period after metastasis resection (12.0 vs 18.0 months; P = 0.035) than those who did not. A higher percentage of KRAS mutations was detected in primary tumors of patiens with lung metastases than in patients with liver metastases (59% vs 32%; p = 0.054). To further evaluate this finding we analyzed 120 additional patients with unresectable metastatic colorectal cancer who previously had their primary tumors evaluated for KRAS mutational status for clinical purposes. Separately, the analysis of these 120 patients showed a tendency towards a higher degree of KRAS mutations in primary tumors of patients with lung metastases, although it did not reach statistical significance. Taken together the group of 230 patients showed that KRAS was mutated significantly more often in the primary tumors of patients with lung metastases (57% vs 35%; P = 0.006). Conclusions/Significance Our results suggest a role for KRAS mutations in the propensity of primary colorectal tumors to

  6. KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer

    PubMed Central

    Niitsu, H; Hinoi, T; Kawaguchi, Y; Sentani, K; Yuge, R; Kitadai, Y; Sotomaru, Y; Adachi, T; Saito, Y; Miguchi, M; Kochi, M; Sada, H; Shimomura, M; Oue, N; Yasui, W; Ohdan, H

    2016-01-01

    KRAS mutations occur in 30–40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apcflox/flox; LSL-KrasG12D and CDX2P-G22Cre;Apcflox/flox mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription–PCR (qRT–PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT–PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin–nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin–NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC. PMID:27526107

  7. Picoliter droplet-based digital peptide nucleic acid clamp PCR and dielectric sorting for low abundant K-ras mutations

    NASA Astrophysics Data System (ADS)

    Zhang, Huidan; Sperling, Ralph; Rotem, Assaf; Shan, Lianfeng; Heyman, John; Zhang, Yizhe; Weitz, David

    2012-02-01

    Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality in the US, and the 5-year survival of metastatic CRC (mCRC) is less than 10%. Although monoclonal antibodies against epidermal growth factor receptor (EGFR) provide incremental improvements in survival, approximately 40% of mCRC patients with activating KRAS mutations won't benefit from this therapy. Peptide nucleic acid (PNA), a synthetic non-extendable oligonucleotides, can bind strongly to completely complementary wild-type KRAS by Watson-Crick base pairing and suppress its amplification during PCR, while any mutant allele will show unhindered amplification. The method is particularly suitable for the simultaneously detection of several adjoining mutant sites, just as mutations of codons 12 and 13 of KRAS gene where there are totally 12 possible mutation types. In this work, we describe the development and validation of this method, based on the droplet-based digital PCR. Using a microfluidic system, single target DNA molecule is compartmentalized in microdroplets together with PNA specific for wild-type KRAS, thermocycled and the fluorescence of each droplet was detected, followed by sorting and sequencing. It enables the precise determination of all possible mutant KRAS simultaneously, and the precise quantification of a single mutated KRAS in excess background unmutated KRAS.

  8. The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

    PubMed Central

    Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

    2016-01-01

    Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP–GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer. PMID:26902995

  9. KrasG12D and p53 mutation cause primary intra-hepatic cholangiocarcinoma

    PubMed Central

    O’Dell, Michael R.; Huang, Jing-Li; Whitney-Miller, Christa L.; Deshpande, Vikram; Rothberg, Paul; Grose, Valerie; Rossi, Randall M.; Zhu, Andrew X.; Land, Hartmut; Bardeesy, Nabeel; Hezel, Aram F.

    2012-01-01

    Intrahepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver with a rising incidence and poor prognosis. Preclinical studies of the etiology and treatment of this disease are hampered by the relatively small number of available IHCC cell lines or genetically faithful animal models. Here we report the development of a genetically engineered mouse model of IHCC that incorporates two of the most common mutations in human IHCC, activating mutations of Kras (KrasG12D) and deletion of p53. Tissue-specific activation of KrasG12D alone resulted in the development of invasive IHCC with low penetrance and long latency. Latency was shortened by combining KrasG12D activation with heterozygous or homozygous deletion of p53 (mean survival of 56 weeks versus 19 weeks, respectively), which also resulted in widespread local and distant metastasis. Serial analysis showed that the murine models closely recapitulated the multistage histopathologic progression of the human disease, including the development of stroma-rich tumors and the pre-malignant biliary lesions, intraductal papillary biliary neoplasms (IPBN) and Von Meyenburg complexes (VMC; also known as biliary hamartomas). These findings establish a new genetically and histopathologically faithful model of IHCC and lend experimental support to the hypothesis that IPBN and VMC are precursors to invasive cancers. PMID:22266220

  10. Monitoring KRAS mutations in circulating DNA and tumor cells using digital droplet PCR during treatment of KRAS-mutated lung adenocarcinoma.

    PubMed

    Guibert, Nicolas; Pradines, Anne; Farella, Magali; Casanova, Anne; Gouin, Sandrine; Keller, Laura; Favre, Gilles; Mazieres, Julien

    2016-10-01

    Liquid biopsies are a new non-invasive strategy to detect and monitor the biology of non-small-cell lung cancer (NSCLC) in the era of personalized medicine. KRAS is an oncogenic driver that is mutated in 30% of NSCLCs and is associated with a poor prognosis. 62 samples from 32 patients, treated for metastatic KRAS-mutated lung adenocarcinoma, had DNA extracted from plasma and circulating tumor cells (CTCs) prospectively tested for the presence of KRAS mutations using droplet digital PCR. A KRAS mutation was detected in 82% of patients. Sensitivity was 78% for circulating free DNA (cfDNA) and 34% for CTCs. The presence of a KRAS mutation in cfDNA was correlated with a poor response to chemotherapy or targeted therapy. When a KRAS-mutated-DNA was detected and then monitored in cfDNA, its variation during targeted or conventional therapy was correlated with response, according to RECIST criteria, in 87.5% of cases (n=14/16), whereas this correlation was observed in 37.5% of cases for CTCs (n=3/8). We report the usefulness of using digital droplet PCR on liquid biopsies to predict and monitor responses to treatment of KRAS-mutated lung adenocarcinoma. ctDNA was much more sensitive than CTCs in this context. PMID:27597273

  11. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer.

    PubMed

    Yamada, Takeshi; Iwai, Takuma; Takahashi, Goro; Kan, Hayato; Koizumi, Michihiro; Matsuda, Akihisa; Shinji, Seiichi; Yamagishi, Aya; Yokoyama, Yasuyuki; Tatsuguchi, Atsushi; Kawagoe, Tatsuro; Kitano, Shiro; Nakayama, Masato; Matsumoto, Satoshi; Uchida, Eiji

    2016-07-01

    In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. PMID:27116474

  12. Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.

    PubMed

    Sloane, Hillary S; Kelly, Kimberly A; Landers, James P

    2015-10-20

    Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis. PMID:26339780

  13. G48A, a New KRAS Mutation Found in Lung Adenocarcinoma.

    PubMed

    Marabese, Mirko; Caiola, Elisa; Garassino, Marina C; Rastelli, Giulio; Settanni, Giulio; Brugnara, Sonia; Broggini, Massimo; Ganzinelli, Monica

    2016-07-01

    A new somatic mutation in the coding region of Kirsten rat sarcoma viral oncogene homolog gene (KRAS), G48A, has been identified in a patient with non-small cell lung cancer (NSCLC). No other mutations were found by screening several genes known to be mutated in NSCLC. The patient responded to first-line therapy and is still under maintenance treatment 18 months after diagnosis. Normal and cancer cells were engineered to express the KRAS(G48A) mutation. KRAS(G48A) overexpression did not change the growth or the response to treatment compared with KRAS(wild type)-expressing cells. Analysis of the structure of the KRAS(G48A) mutant predicted altered interactions with other proteins. Analysis of KRAS binding to B-Raf proto-oncogene, serine/threonine kinase showed that the KRAS(G48A) mutant behaves more like a wild-type than a classical KRAS(G12) mutant. In conclusion, this new mutation in the coding region of KRAS, found in NSCLC, does not induce phenotypic changes similar to those induced by G12 mutants but presumably affects KRAS binding to proteins other than B-Raf proto-oncogene, serine/threonine kinase. PMID:27058911

  14. Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer.

    PubMed

    Saber, Ali; van der Wekken, Anthonie J; Kerner, Gerald S M A; van den Berge, Maarten; Timens, Wim; Schuuring, Ed; ter Elst, Arja; van den Berg, Anke; Hiltermann, T Jeroen N; Groen, Harry J M

    2016-01-01

    Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56-4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14-14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04-0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients. PMID:27008036

  15. Association of KRAS and EGFR Mutations with Survival in Patients with Advanced Lung Adenocarcinomas

    PubMed Central

    Johnson, Melissa L.; Sima, Camelia S.; Chaft, Jamie; Paik, Paul K.; Pao, William; Kris, Mark G.; Ladanyi, Marc; Riely, Gregory J.

    2014-01-01

    Background Lung adenocarcinomas can be distinguished by identifying mutated driver oncogenes including EGFR and KRAS. Mutations in EGFR are associated with both an improved survival as well as response to treatment with erlotinib and gefitinib. However, the prognostic significance of KRAS has not been evaluated in large numbers of patients and remains controversial. We examined the association of EGFR and KRAS mutations with survival among patients with advanced lung adenocarcinomas. Methods We analyzed data from patients with advanced lung adenocarcinomas and known EGFR and KRAS mutation status evaluated between 2002 and 2009. We collected clinical variables including age, gender, Karnofsky Performance Status, smoking history, and treatment history. Overall survival from diagnosis of advanced disease was analyzed using Kaplan-Meier and Cox proportional hazard methods. Results We evaluated 1036 patients, including 610 women (59%) and 344 never-smokers (33%). Patients had a median age of 65 (range, 25–92) and the majority (81%) had a KPS ≥80%. In multivariate analysis, EGFR mutations were associated with a longer overall survival (HR= 0.6, p<0.001) and KRAS mutations with a shorter survival (HR=1.21, p=0.048). Conclusions KRAS mutations predict shorter survival for patients with advanced lung adenocarcinomas. The presence of EGFR and KRAS mutations define distinct subsets of patients with lung adenocarcinomas, and should be determined in patients upon diagnosis of advanced disease. Clinical trial reports should include EGFR and KRAS mutation status along with other prognostic factors. PMID:22810899

  16. K-ras gene mutation in gall bladder carcinomas and dysplasia.

    PubMed Central

    Ajiki, T; Fujimori, T; Onoyama, H; Yamamoto, M; Kitazawa, S; Maeda, S; Saitoh, Y

    1996-01-01

    Epithelial dysplasia of gall bladder is an important precancerous lesion of gall bladder carcinogenesis. To investigate the frequency of K-ras gene mutation in gall bladder carcinoma and dysplasia, K-ras codon 12 mutations were investigated by the polymerase chain reaction/restriction enzyme based method following direct sequencing. Mutation was detected in 59% (30 of 51) of gall bladder carcinomas, in 73% (8 of 11) of gall bladder dysplasia in gall stone cases, and in 0% of the normal gall bladder epithelium. There was, however, no correlation between K-ras mutation and clinicopathological factors of gall bladder carcinoma. K-ras gene mutation occurs even in gall bladder dysplasia at an incidence similar to that in carcinomas, suggesting that testing for K-ras gene mutation may prove useful as an adjunct to bile cytological or biopsy analysis. Images Figure 1 Figure 2 Figure 3 PMID:8675098

  17. KRAS Mutation in Small Cell Lung Carcinoma and Extrapulmonary Small Cell Cancer

    PubMed Central

    Kodaz, Hilmi; Taştekin, Ebru; Erdoğan, Bülent; Hacıbekiroğlu, İlhan; Tozkır, Hilmi; Gürkan, Hakan; Türkmen, Esma; Demirkan, Bora; Uzunoğlu, Sernaz; Çiçin, İrfan

    2016-01-01

    Background: Lung cancer is one of the most lethal cancers. It is mainly classified into 2 groups: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Extrapulmonary small cell carcinomas (EPSCC) are very rare. The Ras oncogene controls most of the cellular functions in the cell. Overall, 21.6% of human cancers contain a Kirsten Ras (KRAS) mutation. SCLC and EPSCC have several similar features but their clinical course is different. Aims: We investigated the KRAS mutation status in SCLC and EPSCC. Study design: Mutation research. Methods: Thirty-seven SCLC and 15 EPSCC patients were included in the study. The pathological diagnoses were confirmed by a second pathologist. KRAS analysis was performed in our medical genetic department. DNA isolation was performed with primary tumor tissue using the QIAamp DNA FFPE Tissue kit (Qiagen; Hilden, Germany) in all patients. The therascreen KRAS Pyro Kit 24 V1 (Qiagen; Hilden, Germany) was used for KRAS analyses. Results: Thirty-four (91.9%) of the SCLC patients were male, while 11 (73.3%) of the EPSCC l patients were female. SCLC was more common in males, and EPSCC in females (p=0.001). A KRAS mutation was found in 6 (16.2%) if SCLC patients. The most common mutation was Q61R (CAA>CGA). Among the 15 EPSCC patients, 2 had a KRAS mutation (13.3%). When KRAS mutant and wild type patients were compared in the SCLC group, no difference was found for overall survival (p=0.6). Conclusion: In previous studies, the incidence of KRAS mutation in SCLC was 1–3%; however, it was 16.2% in our study. Therefore, there may be ethnic and geographical differences in the KRAS mutations of SCLC. As a result, KRAS mutation should not be excluded in SCLC.

  18. Comparison of KRAS mutation status between primary tumor and metastasis in Chinese colorectal cancer patients.

    PubMed

    Li, Zhe-Zhen; Bai, Long; Wang, Feng; Zhang, Zi-Chen; Wang, Fang; Zeng, Zhao-Lei; Zeng, Jun-Bo; Zhang, Dong-Sheng; Wang, Feng-Hua; Wang, Zhi-Qiang; Li, Yu-Hong; Shao, Jian-Yong; Xu, Rui-Hua

    2016-07-01

    Detection of KRAS mutation status is a routine clinical procedure for predicting response to anti-EGFR therapy in colorectal cancer (CRC) patients. Previous studies showed high concordance of KRAS mutation status in primary lesion and corresponding metastatic sites in CRC. However, the data were mostly from Caucasians. The aim of this study is to compare KRAS mutation and other molecules mutation status between primary tumor and corresponding metastatic lesion in Chinese patients with CRC. In this retrospective study, Chinese CRC patients with paired samples of primary tumor and metastatic site were detected for KRAS codon 12 and 13 with quantitative real-time PCR, or detected for OncoCarta™ panel of 19 genes with MassARRAY(®) technique, including KRAS, BRAF, NRAS and PIK3CA et al. Forty-eight paired CRC samples were analyzed for KRAS codon 12 and 13 using quantitative real-time PCR. Ten paired samples were analyzed by 19 genes OncoCarta™ Panel with MassARRAY(®) technique. KRAS mutation was found in 15 (25.9 %) primary tumors and 18 (31.0 %) metastases. The discordance of KRAS was observed in 11 (19.0 %) patients. Alteration of mutation points in primary site with mutant KRAS was not observed. In the 10 patients with multiple gene detection, PIK3CA mutation showed concordant mutation status in primary tumor and metastatic site, whereas discordance in BRAF, NRAS and AKT1 was detected. A concordance rate of 81.0 % was detected in KRAS mutation between primary tumor and metastatic lesion in Chinese patients with CRC. Discordance of BRAF, NRAS and AKT1 mutation status in primary tumor and metastases was observed. PMID:27270901

  19. Identification of a KRAS mutation in a patient with non-small cell lung cancer treated with chemoradiotherapy and panitumumab

    PubMed Central

    Zaorsky, Nicholas G; Sun, Yunguang; Wang, Zixuan; Palmer, Joshua; Fortina, Paolo M; Solomides, Charalambos; Werner-Wasik, Maria; Dicker, Adam P; Axelrod, Rita; Campling, Barbara; Evans III, Nathaniel; Cowan, Scott; Lu, Bo

    2013-01-01

    RTOG 0839 is a Phase II study of pre-operative chemoradiotherapy with or without panitumumab in potentially operable locally advanced non-small cell lung cancer (NSCLC). The investigational agent, panitumumab, is an anti-epithelial growth factor receptor (EGFR) antibody that improves progression-free survival in chemorefractory metastatic colorectal cancer (mCRC). Recently, both KRAS mutational status (i.e., mutated or not) and subtype (i.e., activating or inactivating) have been shown to be predictive of response to anti-EGFR therapy in mCRC. However, in NSCLC, it is unknown if KRAS mutational status or subtype predict benefit to anti-EGFR therapies because of unique genetic and epigenetic factors unique to each cancer. We present a patient with stage III NSCLC containing a KRAS G12D activating mutation who had a partial pathologic response, with disappearance of a minor KRAS mutant clone. This case suggests possible eradication of the G12D KRAS lung cancer clones by concurrent chemoradiation with panitumumab. PMID:23917487

  20. Outcomes of patients with advanced cancer and KRAS mutations in phase I clinical trials

    PubMed Central

    Said, Rabih; Ye, Yang; Falchook, Gerald Steven; Janku, Filip; Naing, Aung; Zinner, Ralph; Blumenschein, George R.; Fu, Siqing; Hong, David S.; Piha-Paul, Sarina Anne; Wheler, Jennifer J.; Kurzrock, Razelle; Palmer, Gary A.; Aldape, Kenneth; Hess, Kenneth R.; Tsimberidou, Apostolia Maria

    2014-01-01

    Background KRAS mutation is common in human cancer. We assessed the clinical factors, including type of KRAS mutation and treatment, of patients with advanced cancer and tumor KRAS mutations and their association with treatment outcomes. Methods Patients referred to the Phase I Clinic for treatment who underwent testing for KRAS mutations were analyzed. Results Of 1,781 patients, 365 (21%) had a KRAS mutation. The G12D mutation was the most common mutation (29%). PIK3CA mutations were found in 24% and 10% of patients with and without KRAS mutations (p<0.0001). Of 223 patients with a KRAS mutation who were evaluable for response, 56 were treated with a MEK inhibitor-containing therapy and 167 with other therapies. The clinical benefit (partial response and stable disease lasting ≥ 6 months) rates were 23% and 9%, respectively, for the MEK inhibitor versus other therapies (p=0.005). The median progression-free survival (PFS) was 3.3 and 2.2 months, respectively (p=0.09). The respective median overall survival was 8.4 and 7.0 months (p=0.38). Of 66 patients with a KRAS mutation and additional alterations, higher rates of clinical benefit (p=0.04), PFS (p=0.045), and overall survival (p=0.02) were noted in patients treated with MEK inhibitor-containing therapy (n=9) compared to those treated with targeted therapy matched to the additional alterations (n=24) or other therapy (n=33). Conclusions MEK inhibitors in patients with KRAS-mutated advanced cancer were associated with higher clinical benefit rates compared to other therapies. Therapeutic strategies that include MEK inhibitors or novel agents combined with other targeted therapies or chemotherapy need further investigation. PMID:25313136

  1. Nitrative and oxidative DNA damage caused by K-ras mutation in mice

    SciTech Connect

    Ohnishi, Shiho; Saito, Hiromitsu; Suzuki, Noboru; Ma, Ning; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke

    2011-09-23

    Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

  2. Modulation of EZH2 Expression by MEK-ERK or PI3K-AKT Signaling in Lung Cancer Is Dictated by Different KRAS Oncogene Mutations.

    PubMed

    Riquelme, Erick; Behrens, Carmen; Lin, Heather Y; Simon, George; Papadimitrakopoulou, Vassiliki; Izzo, Julie; Moran, Cesar; Kalhor, Neda; Lee, J Jack; Minna, John D; Wistuba, Ignacio I

    2016-02-01

    EZH2 overexpression promotes cancer by increasing histone methylation to silence tumor suppressor genes, but how EZH2 levels become elevated in cancer is not understood. In this study, we investigated the mechanisms by which EZH2 expression is regulated in non-small cell lung carcinoma cells by oncogenic KRAS. In cells harboring KRAS(G12C) and KRAS(G12D) mutations, EZH2 expression was modulated by MEK-ERK and PI3K/AKT signaling, respectively. Accordingly, MEK-ERK depletion decreased EZH2 expression in cells harboring the KRAS(G12C) mutation, whereas PI3K/AKT depletion decreased EZH2 expression, EZH2 phosphorylation, and STAT3 activity in KRAS(G12D)-mutant cell lines. Combined inhibition of EZH2 and MEK-ERK or PI3K/AKT increased the sensitivity of cells with specific KRAS mutations to MEK-ERK and PI3K/AKT-targeted therapies. Our work defines EZH2 as a downstream effector of KRAS signaling and offers a rationale for combining EZH2 inhibitory strategies with MEK-ERK- or PI3K/AKT-targeted therapies to treat lung cancer patients, as stratified into distinct treatment groups based on specific KRAS mutations. PMID:26676756

  3. Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

    PubMed Central

    Kerner, Gerald S. M. A.; Schuuring, Ed; Sietsma, Johanna; Hiltermann, Thijo J. N.; Pieterman, Remge M.; de Leede, Gerard P. J.; van Putten, John W. G.; Liesker, Jeroen; Renkema, Tineke E. J.; van Hengel, Peter; Platteel, Inge; Timens, Wim; Groen, Harry J. M.

    2013-01-01

    Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival. PMID:23922984

  4. Dietary acrylamide intake and the risk of colorectal cancer with specific mutations in KRAS and APC.

    PubMed

    Hogervorst, Janneke G F; de Bruijn-Geraets, Daisy; Schouten, Leo J; van Engeland, Manon; de Kok, Theo M C M; Goldbohm, R Alexandra; van den Brandt, Piet A; Weijenberg, Matty P

    2014-05-01

    Acrylamide, a probable human carcinogen, is present in heat-treated carbohydrate-rich foods. Epidemiological studies have not shown a clear association between acrylamide intake and colorectal cancer (CRC) risk. This may be due to the molecular heterogeneity in colorectal tumors, which was not taken into consideration before. Since the acrylamide metabolite glycidamide induces specific DNA mutations in rodents, we investigated whether acrylamide is associated with CRC risk characterized by mutations in Kirsten-ras (KRAS) and adenomatous polyposis coli (APC); key genes in colorectal carcinogenesis. This case-cohort analysis, within the Netherlands Cohort Study on diet and cancer, was based on 7.3 years of follow-up. Acrylamide intake was assessed with a food frequency questionnaire. Mutation analysis of codons 1286-1520 in exon 15 in APC and codons 12 and 13 in exon 1 in KRAS was performed on tumor tissue of 733 cases. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Among men, acrylamide intake was statistically significantly associated with an increased risk of particularly tumors with an activating KRAS mutation {HR fourth versus first quartile: 2.12 [95% confidence interval (CI): 1.16-3.87], P trend: 0.01}. Among women, acrylamide intake was statistically significantly associated with a decreased risk of particularly tumors with a truncating APC mutation (fourth versus first quartile: 0.47 (95% CI: 0.23-0.94), P trend: 0.02), but only in the highest quartile of intake. This is the first study to show that acrylamide might be associated with CRC with specific somatic mutations, differentially in men and women. More research is needed to corroborate or refute these findings. PMID:24398672

  5. Activated Kras, but Not Hras or Nras, May Initiate Tumors of Endodermal Origin via Stem Cell Expansion▿

    PubMed Central

    Quinlan, Margaret P.; Quatela, Steven E.; Philips, Mark R.; Settleman, Jeffrey

    2008-01-01

    The three closely related human Ras genes, Hras, Nras, and Kras, are all widely expressed, engage a common set of downstream effectors, and can each exhibit oncogenic activity. However, the vast majority of activating Ras mutations in human tumors involve Kras. Moreover, Kras mutations are most frequently seen in tumors of endodermally derived tissues (lung, pancreas, and colon), suggesting that activated Kras may affect an endodermal progenitor to initiate oncogenesis. Using a culture model of retinoic acid (RA)-induced stem cell differentiation to endoderm, we determined that while activated HrasV12 promotes differentiation and growth arrest in these endodermal progenitors, KrasV12 promotes their proliferation. Furthermore, KrasV12-expressing endodermal progenitors fail to differentiate upon RA treatment and continue to proliferate and maintain stem cell characteristics. NrasV12 neither promotes nor prevents differentiation. A structure-function analysis demonstrated that these distinct effects of the Ras isoforms involve their variable C-terminal domains, implicating compartmentalized signaling, and revealed a requirement for several established Ras effectors. These findings indicate that activated Ras isoforms exert profoundly different effects on endodermal progenitors and that mutant Kras may initiate tumorigenesis by expanding a susceptible stem/progenitor cell population. These results potentially explain the high frequency of Kras mutations in tumors of endodermal origin. PMID:18268007

  6. Prognostic role of KRAS mutations in Sardinian patients with colorectal carcinoma

    PubMed Central

    Palomba, Grazia; Cossu, Antonio; Paliogiannis, Panagiotis; Pazzola, Antonio; Baldino, Giovanni; Scartozzi, Mario; Ionta, Maria Teresa; Ortu, Salvatore; Capelli, Francesca; Lanzillo, Annamaria; Sedda, Tito; Sanna, Giovanni; Barca, Michela; Virdis, Luciano; Budroni, Mario; Palmieri, Giuseppe

    2016-01-01

    The presence of mutations in the KRAS gene is a predictor of a poor clinical response to EGFR-targeted agents in patients affected by colorectal cancer (CRC), but its significance as a global prognostic factor remains unclear. The aim of the present study was to evaluate the impact of the KRAS mutational status on time to first metastasis (TTM) and overall survival (OS) in a cohort of Sardinian CRC patients. A total of 551 patients with metastatic CRC at the time of enrolment were included. Clinical and pathological features of the disease, including follow-up information, were obtained from medical records and cancer registry data. For mutational analysis formalin-fixed paraffin-embedded tissue samples were processed using a standard protocol. The coding sequence and splice junctions of exons 2 and 3 of the KRAS gene were screened for mutations by direct automated sequencing. Overall, 186 KRAS mutations were detected in 183/551 (33%) patients: 125 (67%) were located in codon 12, 36 (19%) in codon 13, and 18 (10%) in codon 61. The remaining mutations (7; 4%) were detected in uncommonly-affected codons. No significant correlation between KRAS mutations and gender, age, anatomical location and stage of the disease at the time of diagnosis was identified. Furthermore, no prognostic value of KRAS mutations was found considering either TTM or OS. When patients were stratified by KRAS mutational status and gender, males were significantly associated with a longer TTM. The results of the present study indicate that KRAS mutation correlated with a slower metastatic progression in males with CRC from Sardinia, irrespective of the age at diagnosis and the codon of the mutation. PMID:27446446

  7. Lin28-let7 Modulates Radiosensitivity of Human Cancer Cells With Activation of K-Ras

    SciTech Connect

    Oh, Jee-Sun.; Kim, Jae-Jin; Byun, Ju-Yeon; Kim, In-Ah

    2010-01-15

    Purpose: To evaluate the potential of targeting Lin28-let7 microRNA regulatory network for overcoming the radioresistance of cancer cells having activated K-Ras signaling. Methods and Materials: A549 lung carcinoma cells and ASPC1 pancreatic cancer cells possessing K-RAS mutation were transfected with pre-let7a microRNA or Lin28 siRNA, respectively. Clonogenic assay, quantitative reverse transcription polymerase chain reaction, and Western analysis were performed. The effects of Lin28 on SQ20B cells having wild-type K-RAS, and a normal fibroblast were also assessed. Results: The overexpression of let-7a decreased expression of K-Ras and radiosensitized A549 cells. Inhibition of Lin28, a repressor of let-7, attenuated K-Ras expression and radiosensitized A549 and ASPC1 cells. Neither SQ20B cells expressing wild-type K-RAS nor HDF, the normal human fibroblasts, were radiosensitized by this approach. Conclusions: The Lin28-let7 regulatory network may be a potentially useful therapeutic target for overcoming the radioresistance of human cancers having activated K-Ras signaling.

  8. IKK is a therapeutic target in KRAS-Induced lung cancer with disrupted p53 activity

    PubMed Central

    Bassères, Daniela S.; Ebbs, Aaron; Cogswell, Patricia C.; Baldwin, Albert S.

    2014-01-01

    Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-KrasG12D) combined with loss of p53 (LSL-KrasG12D/p53fl/fl). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity. PMID:24955217

  9. Mathematical modeling of drug resistance due to KRAS mutation in colorectal cancer.

    PubMed

    Sameen, Sheema; Barbuti, Roberto; Milazzo, Paolo; Cerone, Antonio; Del Re, Marzia; Danesi, Romano

    2016-01-21

    The most challenging task in colorectal cancer research nowadays is to understand the development of acquired resistance to anti-EGFR drugs. The key reason for this problem is the KRAS mutations appearance after the treatment with monoclonal antibodies (moAb). Here we present a mathematical model for the analysis of KRAS mutations behavior in colorectal cancer with respect to moAb treatments. To evaluate the drug performance we have developed equations for two types of tumors cells, KRAS mutated and KRAS wild-type. Both tumor cell populations were treated with a combination of moAb and chemotherapy drugs. It was observed that even the minimal initial concentration of KRAS mutation before the treatment has the ability to make the tumor refractory to the treatment. Minor population of KRAS mutations has strong influence on large number of wild-type cells as well rendering them resistant to chemotherapy. Patient׳s immune responses are specifically taken into considerations and it is found that, in case of KRAS mutations, the immune strength does not affect medication efficacy. Finally, cetuximab (moAb) and irinotecan (chemotherapy) drugs are analyzed as first-line treatment of colorectal cancer with few KRAS mutated cells. Results show that this combined treatment could be only effective for patients with high immune strengths and it should not be recommended as first-line therapy for patients with moderate immune strengths or weak immune systems because of a potential risk of relapse, with KRAS mutant cells acquired resistance involved with them. PMID:26551156

  10. Clinical Relevance of KRAS in Human Cancers

    PubMed Central

    Jančík, Sylwia; Drábek, Jiří; Radzioch, Danuta; Hajdúch, Marián

    2010-01-01

    The KRAS gene (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is an oncogene that encodes a small GTPase transductor protein called KRAS. KRAS is involved in the regulation of cell division as a result of its ability to relay external signals to the cell nucleus. Activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states, leading to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors. This review highlights some of the features of the KRAS gene and the KRAS protein and summarizes current knowledge of the mechanism of KRAS gene regulation. It also underlines the importance of activating mutations in the KRAS gene in relation to carcinogenesis and their importance as diagnostic biomarkers, providing clues regarding human cancer patients' prognosis and indicating potential therapeutic approaches. PMID:20617134

  11. The potential utility of re-mining results of somatic mutation testing: KRAS status in lung adenocarcinoma.

    PubMed

    Biernacka, Anna; Tsongalis, Peter D; Peterson, Jason D; de Abreu, Francine B; Black, Candice C; Gutmann, Edward J; Liu, Xiaoying; Tafe, Laura J; Amos, Christopher I; Tsongalis, Gregory J

    2016-05-01

    KRAS mutant non-small cell lung cancers (NSCLCs) vary in clinical outcome depending on which specific KRAS mutation is present. Shorter progression free survival has been associated with KRAS variants G12C and G12V. Cell lines with these variants depend to a greater extent on the RAS/RAF/MEK/ERK signaling pathway and become more susceptible to MEK inhibition. Because different KRAS mutations may lead to altered drug sensitivity, we aimed to determine specific KRAS mutation status in a NSCLC patient cohort at our institution. A total of 502 NSCLC samples were screened for somatic mutations using the 50 gene AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2). However only samples positive for variants in the KRAS gene were included in this study. Variants identified in the KRAS genes were curated using publicly available databases. The overall mutation rate in the KRAS gene was 32.7% (164/502). The most common KRAS mutations were G12C (41%), G12V (19%), and G12D (14%) along with less frequent variants. After re-mining our sequencing data, we found that more than a half of our KRAS mutant NSCLC patients could potentially benefit from the addition of a MEK inhibitor such as selumetinib to standard chemotherapeutic agents. Due to mutated KRAS, these patients will likely fail traditional anti-EGFR therapies but be eligible for newer combination therapies. PMID:27068338

  12. KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment

    PubMed Central

    Normanno, Nicola; Pinto, Carmine; Castiglione, Francesca; Bardelli, Alberto; Gambacorta, Marcello; Botti, Gerardo; Nappi, Oscar; Siena, Salvatore; Ciardiello, Fortunato; Taddei, GianLuigi; Marchetti, Antonio

    2011-01-01

    Background Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients. Methodology/Principal Findings In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass. Conclusions The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers

  13. The value of KRAS mutation testing with CEA for the diagnosis of pancreatic mucinous cysts

    PubMed Central

    Kadayifci, Abdurrahman; Al-Haddad, Mohammad; Atar, Mustafa; Dewitt, John M.; Forcione, David G.; Sherman, Stuart; Casey, Brenna W.; Fernandez-del Castillo, Carlos; Schmidt, C. Max; Pitman, Martha B.; Brugge, William R.

    2016-01-01

    Background and aims: Pancreatic cyst fluid (PCF) CEA has been shown to be the most accurate preoperative test for detection of cystic mucinous neoplasms (CMNs). This study aimed to assess the added value of PCF KRAS mutational analysis to CEA for diagnosis of CMNs. Patients and methods: This is a retrospective study of prospectively collected endoscopic ultrasonography (EUS) fine-needle aspiration (FNA) data. KRAS mutation was determined by direct sequencing or equivalent methods. Cysts were classified histologically (surgical cohort) or by clinical (EUS or FNA) findings (clinical cohort). Performance characteristics of KRAS, CEA and their combination for detection of a cystic mucinous neoplasm (CMN) and malignancy were calculated. Results: The study cohort consisted of 943 patients: 147 in the surgical cohort and 796 in the clinical cohort. Overall, KRAS and CEA each had high specificity (100 % and 93.2 %), but low sensitivity (48.3 % and 56.3 %) for the diagnosis of a CMN. The positivity of KRAS or CEA increased the diagnostic accuracy (80.8 %) and AUC (0.84) significantly compared to KRAS (65.3 % and 0.74) or CEA (65.8 % and 0.74) alone, but only in the clinical cohort (P < 0.0001 for both). KRAS mutation was significantly more frequent in malignant CMNs compared to histologically confirmed non-malignant CMNs (73 % vs. 37 %, P = 0.001). The negative predictive value of KRAS mutation was 77.6 % in differentiating non-malignant cysts. Conclusions: The detection of a KRAS mutation in PCF is a highly specific test for mucinous cysts. It outperforms CEA for sensitivity in mucinous cyst diagnosis, but the data does not support its routine use. PMID:27092317

  14. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients

    PubMed Central

    Hsu, Hung-Chih; Thiam, Tan Kien; Lu, Yen-Jung; Yeh, Chien Yuh; Tsai, Wen-Sy; You, Jeng Fu; Hung, Hsin Yuan; Tsai, Chi-Neu; Hsu, An; Chen, Hua-Chien; Chen, Shu-Jen; Yang, Tsai-Sheng

    2016-01-01

    Approximately 45% of metastatic colorectal cancer (mCRC) patients with wild-type KRAS exon 2 are resistant to cetuximab treatment. We set out to identify additional genetic markers that might predict the response to cetuximab treatment. Fifty-three wild-type KRAS exon 2 mCRC patients were treated with cetuximab/irinotecan-based chemotherapy as a first- or third-line therapy. The mutational statuses of 10 EGFR pathway genes were analyzed in primary tumors using next-generation sequencing. BRAF, PIK3CA, KRAS (exons 3 and 4), NRAS, PTEN, and AKT1 mutations were detected in 6, 6, 5, 4, 1, and 1 patient, respectively. Four of the BRAF mutations were non-V600 variants. Four tumors harbored multiple co-existing (complex) mutations. All patients with BRAF mutations or complex mutation patterns were cetuximab non-responders. All patients but one harboring KRAS, NRAS, or BRAF mutations were non-responders. Mutations in any one of these three genes were associated with a poor response rate (7.1%) and reduced survival (PFS = 8.0 months) compared to wild-type patients (74.4% and 11.6 months). Our data suggest that KRAS, NRAS, and BRAF mutations predict response to cetuximab treatment in mCRC patients. PMID:26989027

  15. KRAS mutations: variable incidences in a Brazilian cohort of 8,234 metastatic colorectal cancer patients

    PubMed Central

    2014-01-01

    Background KRAS mutations are frequently found in colorectal cancer (CRC) indicating the importance of its genotyping in the study of the molecular mechanisms behind this disease. Although major advances have occurred over the past decade, there are still important gaps in our understanding of CRC carcinogenesis, particularly whether sex-linked factors play any role. Methods The profile of KRAS mutations in the Brazilian population was analyzed by conducting direct sequencing of KRAS codons 12 and 13 belonging to 8,234 metastatic CRC patient samples. DNA was extracted from paraffin-embedded tissue, exon 1 was amplified by PCR and submitted to direct sequencing. The data obtained was analysed comparing different geographical regions, gender and age. Results The median age was 59 years and the overall percentage of wild-type and mutated KRAS was 62.8% and 31.9%, respectively. Interestingly, different percentages of mutated KRAS patients were observed between male and female patients (32.5% versus 34.8%, respectively; p = 0.03). KRAS Gly12Asp mutation was the most prevalent for both genders and for most regions, with the exception of the North where Gly12Val was the most frequent mutation found. Conclusions To the best of our knowledge this is one of the largest cohorts of KRAS genotyping in CRC patients and the largest to indicate a higher incidence of KRAS mutation in females compared to males in Brazil. Nevertheless, further research is required to better address the impact of gender differences in colorectal cancer. PMID:24720724

  16. G12V Kras mutations in cervical cancer under virtual microscope of molecular dynamics simulations.

    PubMed

    Chen, X P; Xu, W H; Xu, D F; Fu, S M; Ma, Z C

    2016-01-01

    Kras mutations and cancers are common and their role in the progression of cancer is well known and elucidated. The present work is searching for the most deleterious mutation of the four found at codon 12 and 13 of Kras in cervical cancers using prediction servers; different servers were used to look into different factors that govern the protein function. The in silico results predicted G12V to be the most devastating; this particular mutation was then subjected to molecular dynamics simulation (MDS) for further analysis. The authors' approach of MDSs helped them to place the native and mutant structure under virtual microscope and observe their dynamics over time. The results generated are enlightening the effect of G12V variation on the dynamics of Kras. The structural variation between the native and mutant Kras over 50 nanoseconds (ns) run varied at every parameter checked and the results are in excellent agreement with the available experimental data. PMID:27048113

  17. A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens

    PubMed Central

    Gonzalez de Castro, D; Angulo, B; Gomez, B; Mair, D; Martinez, R; Suarez-Gauthier, A; Shieh, F; Velez, M; Brophy, V H; Lawrence, H J; Lopez-Rios, F

    2012-01-01

    Background: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. Methods: We conducted a two-site comparison of two commercial KRAS mutation kits – the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit – and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. Results: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. Conclusion: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation. PMID:22713664

  18. Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer

    PubMed Central

    Saber, Ali; van der Wekken, Anthonie J.; Kerner, Gerald S. M. A.; van den Berge, Maarten; Timens, Wim; Schuuring, Ed; ter Elst, Arja; van den Berg, Anke; Hiltermann, T. Jeroen N.; Groen, Harry J. M.

    2016-01-01

    Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56–4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14–14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04–0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients. PMID:27008036

  19. The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B.

    PubMed

    Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

    2016-01-01

    Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4B(WT)-GTP/GDP) catalytic domain, the K-Ras4B(WT)-GTP-GAP complex, and the mutants (K-Ras4B(G12C/G12D/G12V)-GTP/GDP, K-Ras4B(G13D)-GTP/GDP, K-Ras4B(Q61H)-GTP/GDP) and their complexes with GAP. In addition, we simulated 'exchanged' nucleotide states. These comprehensive simulations reveal that in solution K-Ras4B(WT)-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4B(G12C/G12D)-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer. PMID:26902995

  20. Antitumoral Efficacy of the Protease Inhibitor Gabexate Mesilate in Colon Cancer Cells Harbouring KRAS, BRAF and PIK3CA Mutations

    PubMed Central

    Brandi, Giovanni; Tavolari, Simona; De Rosa, Francesco; Di Girolamo, Stefania; Agostini, Valentina; Barbera, Maria Aurelia; Frega, Giorgio; Biasco, Guido

    2012-01-01

    The employment of anti-epidermal growth factor receptor (EGFR) antibodies represents a backbone of the therapeutic options for the treatment of metastatic colorectal cancer (mCRC). However, this therapy is poorly effective or ineffective in unselected patients. Mutations in KRAS, BRAF and PIK3CA genes have recently emerged as the best predictive factors of low/absent response to EGFR-targeted therapy. Due to the need for efficacious treatment options for mCRC patients bearing these mutations, in this short report we examined the antitumoral activity of the protease inhibitor gabexate mesilate, alone and in combination with the anti-EGFR monoclonal antibody cetuximab, in a panel of human CRC cell lines harbouring a different expression pattern of wild-type/mutated KRAS, BRAF and PIK3CA genes. Results obtained showed that gabexate mesilate significantly inhibited the growth, invasive potential and tumour-induced angiogenesis in all the CRC cells employed in this study (including those ones harbouring dual KRAS/PIK3CA or BRAF/PIK3CA mutation), while cetuximab affected these parameters only in CRC cells with KRAS, BRAF and PIK3CA wild-type. Notably, the antitumoral efficacy of gabexate mesilate and cetuximab in combination was found to be not superior than that observed with gabexate mesilate as single agent. Overall, these preliminary findings suggest that gabexate mesilate could represent a promising therapeutic option for mCRC patients, particularly for those harbouring KRAS, BRAF and PIK3CA mutations, either as mono-therapy or in addition to standard chemotherapy regimens. Further studies to better elucidate gabexate mesilate mechanism of action in CRC cells are therefore warranted. PMID:22911782

  1. Sex differences in the prognostic significance of KRAS codons 12 and 13, and BRAF mutations in colorectal cancer: a cohort study

    PubMed Central

    2013-01-01

    Background Activating KRAS and BRAF mutations predict unresponsiveness to EGFR-targeting therapies in colorectal cancer (CRC), but their prognostic value needs further validation. In this study, we investigated the impact of KRAS codons 12 and 13, and BRAF mutations on survival from CRC, overall and stratified by sex, in a large prospective cohort study. Methods KRAS codons 12 and 13, and BRAF mutations were analysed by pyrosequencing of tumours from 525 and 524 incident CRC cases in The Malmö Diet and Cancer Study. Associations with cancer-specific survival (CSS) were explored by Cox proportional hazards regression, unadjusted and adjusted for age, TNM stage, differentiation grade, vascular invasion and microsatellite instability (MSI) status. Results KRAS and BRAF mutations were mutually exclusive. KRAS mutations were found in 191/ 525 (36.4%) cases, 82.2% of these mutations were in codon 12, 17.3% were in codon 13, and 0.5% cases had mutations in both codons. BRAF mutations were found in 78/524 (14.9%) cases. Overall, mutation in KRAS codon 13, but not codon 12, was associated with a significantly reduced CSS in unadjusted, but not in adjusted analysis, and BRAF mutation did not significantly affect survival. However, in microsatellite stable (MSS), but not in MSI tumours, an adverse prognostic impact of BRAF mutation was observed in unadjusted, but not in adjusted analysis. While KRAS mutation status was not significantly associated with sex, BRAF mutations were more common in women. BRAF mutation was not prognostic in women; but in men, BRAF mutation was associated with a significantly reduced CSS in overall adjusted analysis (HR = 3.50; 95% CI = 1.41–8.70), but not in unadjusted analysis. In men with MSS tumours, BRAF mutation was an independent factor of poor prognosis (HR = 4.91; 95% CI = 1.99–12.12). KRAS codon 13 mutation was associated with a significantly reduced CSS in women, but not in men in unadjusted, but not in adjusted analysis. Conclusions

  2. [KRAS mutation assay on EUS-FNA specimens from pacients with pancreatic mass].

    PubMed

    Bunganič, Bohuš; Hálková, Tereza; Benešová, Lucie; Belšánová, Barbora; Laclav, Martin; Hrůzová, Martina; Traboulsi, Eva; Frič, Přemysl; Suchánek, Štěpán; Minárik, Marek; Zavoral, Miroslav

    2016-01-01

    Differential diagnosis of solid pancreatic masses using EUS FNA is in 1015 % of cases still challenging. Promising method, which helps to distinguish between chronic pancreatitis and cancer, is point mutations of the proto-oncogene KRAS test. This method is not established in routine clinical practice yet.Objectives were the determination of the sensitivity of the KRAS assay using various kinds of samples of patients with pancreatic mass and testing the effect of the presence of KRAS mutations on the prognosis of survival. 147 patients underwent EUS-FNA examination of pancreatic mass, accompanied by blood sampling with subsequent separation of plasma for the detection of circulating tumor DNA. Part of biopsy sample was left native in a stabilizing solution and part as cytological smear. Samples (native aspirates, cytological smears, plasma) were examined for the presence of KRAS mutation by heteroduplex analysis, denaturing capillary electrophoresis.Among 147 patients with pancreatic masses, 118 were diagnosed as a cancer, 26 chronic pancreatitis, 3 neuroendocrine tumor. In total 147 native aspirates, 118 cytological smears and 94 plasma samples were examined. The highest sensitivity of KRAS mutation was reached in the group of pancreatic cancer patients using cytology, in which 90 % of KRAS mutation was detected (106/118 of the samples). When using the native cellular aspirates, mutation was detected in 78 % (92/118 samples), and examination of plasma was positive in 27 % (24/90 samples). In four patients with chronic pancreatitis KRAS mutations was detected, although none has been cytologically confirmed as a cancer. Two of these four patients were confirmed in the course of the disease as a cancer, one patient died because of alcoholic delirium and the last one was indicated for surgery recently.Examination of KRAS mutations can be performed in all patients undergoing EUS-FNA, with the cytology being the most reliable type of sample for genetic tests. KRAS

  3. KRAS (but not BRAF) mutations in ovarian serous borderline tumor are associated with recurrent low-grade serous carcinoma

    PubMed Central

    Tsang, Yvonne T.; Deavers, Michael T.; Sun, Charlotte C.; Kwan, Suet-Yan; Kuo, Eric; Malpica, Anais; Mok, Samuel C.; Gershenson, David M.; Wong, Kwong-Kwok

    2014-01-01

    BRAF and KRAS mutations in ovarian serous borderline tumors (OSBTs) and ovarian low-grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumor samples from 23 recurrent LGSC patients with known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for 5 patients, and either OSBT or LGSC were available for another 18 patients. Tumor cells from paraffin-embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumors that appeared to have wild-type KRAS by conventional PCR–Sanger sequencing were further analyzed by full COLD (coamplification at lower denaturation temperature)-PCR and deep sequencing. Full COLD-PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in 10 patients. Full COLD-PCR deep sequencing detected low-abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in 7 OSBT samples and 6 LGSC samples. To our surprise, patients with the KRAS G12V mutation appeared to have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumor cells with or without detectable KRAS mutations. PMID:24549645

  4. ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition

    PubMed Central

    Toulany, Mahmoud; Minjgee, Minjmaa; Saki, Mohammad; Holler, Marina; Meier, Friedegund; Eicheler, Wolfgang; Rodemann, H Peter

    2014-01-01

    K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors. PMID:24351425

  5. K-Ras Mutations in Non-Small-Cell Lung Cancer: Prognostic and Predictive Value

    PubMed Central

    D'Arcangelo, Manolo; Cappuzzo, Federico

    2012-01-01

    Non-small-cell lung cancer (NSCLC) is a heterogeneous disease due to the presence of different clinically relevant molecular subtypes. Until today, several biological events have been identified in lung adenocarcinoma, including epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations, offering new hopes to patients with metastatic disease. Unfortunately, in approximately 50% of adenocarcinoma and for those harbouring K-RAS mutations, the most frequent mutation in Caucasian lung adenocarcinoma, so far no specific drug demonstrated efficacy. The rat sarcoma (RAS) genes, including H-RAS, K-RAS, and N-RAS, encode a family of proteins regulating cell growth, differentiation, and apoptosis. K-RAS mutations are present in 20–30% of NSCLC and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. Although in colorectal cancer patients K-RAS mutations represent a validated negative predictive biomarker for treatment with anti-EGFR monoclonal antibodies, their role in selecting specific treatment for NSCLC patients remains undefined. Aim of the present paper is to critically analyze the prognostic and predictive value of K-RAS mutations in NSCLC.

  6. Impact of genetic profiles on the efficacy of anti-EGFR antibodies in metastatic colorectal cancer with KRAS mutation.

    PubMed

    Kishiki, Tomokazu; Ohnishi, Hiroaki; Masaki, Tadahiko; Ohtsuka, Kouki; Ohkura, Yasuo; Furuse, Jyunji; Sugiyama, Masanori; Watanabe, Takashi

    2014-07-01

    Reports indicate that, even in KRAS-mutated colon cancer, there are subsets of patients who benefit from anti-EGFR monoclonal antibody (MoAb) treatment. The aim of the present study was to identify genetic profiles that contribute to the responsiveness of metastatic colorectal cancer (mCRC) to anti-EGFR MoAb. We retrospectively evaluated the efficacy of anti-EGFR MoAb in mCRC patients with KRAS mutations according to KRAS mutational subtypes, BRAF and PIK3CA mutational status and PTEN and MET expression. Among 21 patients with KRAS-mutant tumors, 8 (38%) harbored p.G13D, 7 (33%) harbored p.G12V, 5 (24%) harbored p.G12D, and 1 (5%) harbored p.G12C mutation. Patients with the p.G13D mutation exhibited a significantly higher disease control rate than patients with other KRAS mutations (P=0.042), and tended to show a longer progression-free survival (PFS) than patients with other KRAS mutations with marginal significance (P=0.074). Patients with loss of PTEN had significantly shorter PFS than those with normal PTEN expression in patients with KRAS mutations (P=0.044). MET overexpression was significantly associated with shorter PFS compared to normal MET expression in patients with KRAS mutations (P=0.016). Our data demonstrated the potential utility of alterations in PTEN and MET expression as predictive markers for response to anti-EGFR MoAbs in mCRC patients with KRAS mutations. In addition, we confirmed the predictive value of the KRAS p.G13D mutation for better response to anti-EGFR therapies in comparison with other KRAS mutations. PMID:24839940

  7. Prevalence of K-Ras mutations in hepatocellular carcinoma: A Turkish Oncology Group pilot study

    PubMed Central

    TURHAL, NAZIM SERDAR; SAVAŞ, BERNA; ÇOŞKUN, ÖZNUR; BAŞ, EMINE; KARABULUT, BÜLENT; NART, DENIZ; KORKMAZ, TANER; YAVUZER, DILEK; DEMIR, GÖKHAN; DOĞUSOY, GÜLEN; ARTAÇ, MEHMET

    2015-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common male-predominant type of cancer worldwide. There is no effective treatment regimen available for advanced-stage disease and chemotherapy is generally ineffective in these patients. The number of studies on the prevalence of K-Ras mutations in HCC patients is currently limited. A total of 58 patients from 6 comprehensive cancer centers in 4 metropolitan cities of Turkey were enrolled in this study. Each center committed to enroll approximately 10 random patients whose formalin-fixed paraffin-embedded tumor tissues were available for K-Ras, exon 2 genotyping. Two methods were applied based on the availability of adequate amounts of tumor DNA. In the first method, the samples were processed using TheraScreen. The genomic DNA was further used to detect the 7 most frequent somatic mutations (35G>A; 35G>C; 35G>T; 34G>A; 34G>C; 34G>T and 38G>A) in codons 12 and 13 in exon 2 of the K-Ras oncogene by quantitative polymerase chain reaction (PCR). In the second method, the genomic DNA was amplified by PCR using primers specific for K-Ras exon 2 with the GML SeqFinder Sequencing System's KRAS kit. The identified DNA sequence alterations were confirmed by sequencing both DNA strands in two independent experiments with forward and reverse primers. A total of 40 samples had adequate tumor tissue for the mutation analysis. A total of 33 (82.5%) of the investigated samples harbored no mutations in exon 2. All the mutations were identified via a direct sequencing technique, whereas none were identified by TheraScreen. In conclusion, in our patients, HCC exhibited a remarkably low (<20%) K-Ras mutation rate. Patients harboring K-Ras wild-type tumors may be good candidates for treatment with epidermal growth factor inhibitors, such as cetuximab. PMID:26807232

  8. K-ras mutations in beryllium-induced mouse lung tumors

    SciTech Connect

    Belinsky, S.A.; Mitchell, C.E.

    1994-11-01

    Previous studies at ITRI have shown that single, nose-only exposure of F344/N rats to beryllium metal (Be) produced a 64% incidence of lung tumors over the lifetime of the rat. Long tumors induced by Be metal were subsequently analyzed for alterations in the K-ras and p53 genes. Mutation of the K-ras gene was both a rare (2 of 24 tumors) and late event in Be-induced carcinogenesis. In addition, no mutations were detected in exons 5 - 8 of the p53 gene. These results indicated that the mechanisms underlying the development of Be-induced lung cancer in rats did not involve gene dysfunction commonly associated with human non-small-cell lung cancer. The purpose of this study was to determine and compare the prevalence and specificity for mutation of the K-ras gene in lung tumors induced in the A/J mouse by Be to mutations in spontaneous tumors.

  9. A comparison of four methods for detecting KRAS mutations in formalin-fixed specimens from metastatic colorectal cancer patients

    PubMed Central

    MATSUNAGA, MOTOTSUGU; KANETA, TOSHIKADO; MIWA, KEISUKE; ICHIKAWA, WATARU; FUJITA, KEN-ICHI; NAGASHIMA, FUMIO; FURUSE, JUNJI; KAGE, MASAYOSHI; AKAGI, YOSHITO; SASAKI, YASUTSUNA

    2016-01-01

    There is currently no standard method for the detection of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status in colorectal tumors. In the present study, we compared the KRAS mutation detection ability of four methods: direct sequencing, Scorpion-ARMS assaying, pyrosequencing and multi-analyte profiling (Luminex xMAP). We evaluated 73 cases of metastatic colorectal cancer (mCRC) resistant to irinotecan, oxaliplatin and fluoropyrimidine that were enrolled in an all-case study of cetuximab. The KRAS mutation detection capacity of the four analytical methods was compared using DNA samples extracted from tumor tissue, and the detection success rate and concordance of the detection results were evaluated. KRAS mutations were detected by direct sequencing, Scorpion-ARMS assays, pyrosequencing and Luminex xMAP at success rates of 93.2%, 97.3%, 95.9% and 94.5%, respectively. The concordance rates of the detection results by Scorpion-ARMS, pyrosequencing and Luminex xMAP with those of direct sequencing were 0.897, 0.923 and 0.900 (κ statistics), respectively. The direct sequencing method could not determine KRAS mutation status in five DNA samples. Of these, Scorpion-ARMS, pyrosequencing and Luminex xMAP successfully detected three, two and one KRAS mutation statuses, respectively. Three cases demonstrated inconsistent results, whereby Luminex xMAP detected mutated KRAS in two samples while wild-type KRAS was detected by the other methods. In the remaining case, direct sequencing detected wild-type KRAS, which was identified as mutated KRAS by the other methods. In conclusion, we confirmed that Scorpion-ARMS, pyrosequencing and Luminex xMAP were equally reliable in detecting KRAS mutation status in mCRC. However, in rare cases, the KRAS status was differentially diagnosed using these methods. PMID:27347117

  10. Sensitive detection of KRAS mutations using enhanced-ice-COLD-PCR mutation enrichment and direct sequence identification.

    PubMed

    How Kit, Alexandre; Mazaleyrat, Nicolas; Daunay, Antoine; Nielsen, Helene Myrtue; Terris, Benoît; Tost, Jörg

    2013-11-01

    A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing-based read-out desirable in clinical practice. Here, we present a modified version of the ice-COLD-PCR assay called Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild-type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E-ice-COLD-PCR is performed to identify the mutated base. This developed two-step method is rapid and cost-effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies. PMID:24038839

  11. p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma.

    PubMed

    Moskaluk, C A; Hruban, R H; Kern, S E

    1997-06-01

    Pancreatic adenocarcinoma is thought to arise from a noninvasive neoplastic precursor, the pancreatic intraductal lesion (PIL). Mutations of the K-ras gene are known to occur in PILs, but their high prevalence among PILs within the general population probably limit the use of K-ras as a marker of eventual clinical risk. In search of genetic constellations that might indicate the progression of some PILs toward an invasive phenotype, mutations at both the K-ras and p16 genes were sought within PILs of 10 pancreata resected for adenocarcinoma. K-ras mutations were present in most PILs and in nearly all PILs having nuclear atypia. In half of the patients, two or more unique K-ras mutations were identified among distinct PILs, which is evidence for the separate clonal evolution of multiple pancreatic neoplasms within individual patients. p16 alterations (one homozygous deletion and three point mutations) were found in 4 of the 10 carcinomas; these four pancreata harbored p16 alterations in three of nine PILs, of which one was a "histologically early" lesion. Two patients had p16 alterations in PILs matching those of the associated carcinomas. p16 mutations were not found in PILs of pancreata having wild-type p16 in the carcinoma, nor were they found in ducts having normal histology. It is suggested that alterations of the p16 gene affect a subset of PILs that contain mutations of the K-ras gene and that these mutations might identify high-risk precursors of the invasive malignancy. PMID:9187111

  12. Oncogenic KRAS sensitizes premalignant, but not malignant cells, to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway

    PubMed Central

    Conti, Annalisa; Majorini, Maria Teresa; Elliott, Richard; Ashworth, Alan; Lord, Christopher J.; Cancelliere, Carlotta; Bardelli, Alberto; Seneci, Pierfausto; Walczak, Henning; Delia, Domenico; Lecis, Daniele

    2015-01-01

    KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation. PMID:26028667

  13. Hotspot-Mutation Analysis of the EGFR/KRAS/BRAF Pathway Using Mutation Surveyor® Software

    PubMed Central

    Hulce, D.; LeVan, K.; Shouyong, N.; Liu, J.

    2011-01-01

    Hotspot-mutation analysis of the EGFR/KRAS/BRAF pathway (or other clinically relevant pathway) can quickly genotype patients as candidates who may respond favorably to specific drug treatments and therapies or into other groups where treatment options are limited and less favorable. Sanger sequencing analysis using Mutation Surveyor software provides high-throughput, high-sensitivity variation detection. Increased efficiency can be achieved using flexible and customizable reporting-sequencing results can be organized by patient identifiers, variation type (reported or unreported, pathogenic or benign or drug sensitive), by gene/exon/amplicon, or quality metrics, and other options. GenBank sequence files from NCBI for EGFR exons 18, 19, 20, and 21; KRAS exons 2 and 3; and BRAF exon 15 were edited to contain reported variations. These reported variations included polymorphisms from dbSNP (downloaded with the GenBank file), pathogenic and drug-sensitivity variations for EGFR (obtained from http://www.egfr.org/), activating mutations for KRAS, and constitutive mutations for BRAF. Bidirectional sequencing data for twelve, simulated (mutations obtained from sequencing reports in the scientific literature), patients were developed and compared to the customized GenBank sequences. Sequencing analysis results were grouped by patient-specific identifiers. Any unmatched or low quality data files are identified in the report, indicating which samples require resequencing. Mutations that match reported variations added to the GenBank sequences are highlighted-SNP identifiers or color coding of SNP type quickly indicate which variations are pathogenic or drug-sensitive or reported in dbSNP. Unreported variations are not highlighted and may be benign or variations of unknown significance. The gene column displays the gene and accession number for that gene used for the analysis. The exon column displays the exon number of the gene, and accession numbers of the mRNA and protein

  14. Elderly male smokers with right lung tumors are viable candidates for KRAS mutation screening.

    PubMed

    Yang, Yang; Shi, Chun; Sun, Hui; Yin, Wei; Zhou, Xiao; Zhang, Lei; Jiang, Gening

    2016-01-01

    Genetic aberrations in tumor driver genes provide specific molecular targets for therapeutic intervention, which can greatly improve therapeutic outcomes. Here, we analyzed the mutational frequency of EGFR and KRAS gene, as well as EML4-ALK rearrangement, and summarized the clinicopathological characters of Chinese lung cancer patients. We detected the mutation spectrum of 1033 primary lung cancer patients. The analyzed clinicopathological parameters included gender, age at diagnosis, smoking status, pathological TNM stage, tumor morphology and location, visceral pleural invasion, and histological type. A total of 618 patients had mutations in EGFR or KRAS gene as well as rearrangement of EML4-ALK. Exon 19 deletions and L858R in the EGFR gene were the most frequent mutations. Left-side lung cancer was more common in female patients carrying the KRAS mutation. Rearrangement of EML4-ALK was more common in non-tobacco-using male patients, who also exhibited a higher likelihood of visceral pleura invasion. Elderly females who never smoked and possessed 1-20 mm stage I adenocarcinomas in the right side exhibited a higher frequency of EGFR mutations. Elderly male smokers with right lung tumors were viable candidates for KRAS mutation screening. PMID:26739511

  15. Genomic and Transcriptional Alterations in Lung Adenocarcinoma in Relation to EGFR and KRAS Mutation Status

    PubMed Central

    Planck, Maria; Edlund, Karolina; Botling, Johan; Micke, Patrick; Isaksson, Sofi; Staaf, Johan

    2013-01-01

    Introduction In lung adenocarcinoma, the mutational spectrum is dominated by EGFR and KRAS mutations. Improved knowledge about genomic and transcriptional alterations in and between mutation-defined subgroups may identify genes involved in disease development or progression. Methods Genomic profiles from 457 adenocarcinomas, including 113 EGFR-mutated, 134 KRAS-mutated and 210 EGFR and KRAS-wild type tumors (EGFRwt/KRASwt), and gene expression profiles from 914 adenocarcinomas, including 309 EGFR-mutated, 192 KRAS-mutated, and 413 EGFRwt/KRASwt tumors, were assembled from different repositories. Genomic and transcriptional differences between the three mutational groups were analyzed by both supervised and unsupervised methods. Results EGFR-mutated adenocarcinomas displayed a larger number of copy number alterations and recurrent amplifications, a higher fraction of total loss-of-heterozygosity, higher genomic complexity, and a more distinct expression pattern than EGFR-wild type adenocarcinomas. Several of these differences were also consistent when the three mutational groups were stratified by stage, gender and smoking status. Specific copy number alterations were associated with mutation status, predominantly including regions of gain with the highest frequency in EGFR-mutated tumors. Differential regions included both large and small regions of gain on 1p, 5q34-q35.3, 7p, 7q11.21, 12p12.1, 16p, and 21q, and losses on 6q16.3-q21, 8p, and 9p, with 20-40% frequency differences between the mutational groups. Supervised gene expression analyses identified 96 consistently differentially expressed genes between the mutational groups, and together with unsupervised analyses these analyses highlighted the difficulty in broadly resolving the three mutational groups into distinct transcriptional entities. Conclusions We provide a comprehensive overview of the genomic and transcriptional landscape in lung adenocarcinoma stratified by EGFR and KRAS mutations. Our analyses

  16. KRAS G12D Mutation Subtype Is A Prognostic Factor for Advanced Pancreatic Adenocarcinoma

    PubMed Central

    Bournet, Barbara; Muscari, Fabrice; Buscail, Camille; Assenat, Eric; Barthet, Marc; Hammel, Pascal; Selves, Janick; Guimbaud, Rosine; Cordelier, Pierre; Buscail, Louis

    2016-01-01

    Objectives: There is no molecular biomarker available in the clinical practice to assess the prognosis of advanced pancreatic carcinoma. This multicenter prospective study aimed to investigate the role of KRAS mutation subtypes within the primary tumor to determine the prognosis of advanced pancreatic cancer. Methods: The exon-2 KRAS mutation status was tested on endoscopic ultrasound-guided fine-needle aspiration biopsy material (primary tumor; restriction fragment-length polymorphism plus sequencing and TaqMan allelic discrimination) of patients with proven locally advanced and/or metastatic pancreatic ductal carcinoma. We used the Kaplan–Meier method, log-rank test, and Cox's model to evaluate the impact of KRAS status on the overall survival (OS), adjusting for age, stage of disease, clinical performance status, CA 19-9 levels, and treatment. Results: A total of 219 patients (men: 116; mean age: 67±9.4 years) were included: 147 harbored a codon-12 KRAS mutation (G12D: 73; G12V: 53; G12R: 21) and 72 had a wild-type KRAS. There was no difference in the OS between patients with a mutant KRAS (8 months; 95% confidence interval (95% CI): 8.7–12.3) and the wild-type (9 months; 95% CI: 8.7–12.8; hazard ratio (HR): 1.03; P=0.82). However, the patients with a G12D mutation had a significantly shorter OS (6 months; 95% CI: 6.4–9.7) compared with the other patients (OS: 9 months; 95% CI: 10–13; HR: 1.47; P=0.003; i.e., wild type: 9 months, G12V: 9 months, G12R: 14 months). Similar results were observed in the subgroup of 162 patients who received chemotherapy (HR: 1.66; P=0.0013; G12D (n=49): 8 months, wild type (n=56): 10 months, G12V (n=38): 10 months, G12R (n=19): 14 months). Multivariate analyses identified KRAS G12D as an independent predictor for worse prognosis within the entire series (HR: 1.44; P=0.01) and in the subgroup of patients that received chemotherapy (HR: 1.84; P=0.02). Conclusions: The KRAS G12D mutation subtype is an independent prognostic

  17. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

    PubMed Central

    Chattopadhyay, Nibedita; Berger, Allison J.; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  18. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

    PubMed

    Chattopadhyay, Nibedita; Berger, Allison J; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  19. Hybridization-Induced Aggregation Technology for Practical Clinical Testing: KRAS Mutation Detection in Lung and Colorectal Tumors.

    PubMed

    Sloane, Hillary S; Landers, James P; Kelly, Kimberly A

    2016-07-01

    KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways. PMID:27289420

  20. Experiences from treatment-predictive KRAS testing; high mutation frequency in rectal cancers from females and concurrent mutations in the same tumor

    PubMed Central

    Jönsson, Mats; Ekstrand, Anna; Edekling, Thomas; Eberhard, Jakob; Grabau, Dorthe; Borg, David; Nilbert, Mef

    2009-01-01

    Background KRAS mutations represent key alterations in colorectal cancer development and lead to constitutive EGFR signaling. Since EGFR inhibition represents a therapeutic strategy in advanced colorectal cancer, KRAS mutation analysis has quickly been introduced as a treatment-predictive test. Methods We used a real-time PCR based method to determine KRAS mutations in 136 colorectal cancers with mutations identified in 53 (39%) tumors. Results KRAS mutations were significantly more often found in rectal cancer (21/38, 55%) than in colon cancer (32/98, 33%) (P = 0.02). This finding was explained by marked differences mutation rates in female patients who showed mutations in 33% of the colon cancers and in 67% of the rectal cancers (P = 0.01). Concurrent KRAS mutations were identified in three tumors; two colorectal cancers harbored Gly12Asp/Gly13Asp and Gly12Cys/Gly13Asp and a third tumor carried Gly12Cys/Gly12Asp in an adenomatous component and additionally acquired Gly12Val in the invasive component. Conclusion The demonstration of a particularly high KRAS mutation frequency among female rectal cancer patients suggests that this subset is the least likely to respond to anti-EGFR therapies, whereas the observation of concurrent KRAS mutations imply that repeated KRAS targeting may occur during tumor progression in a subset of colorectal cancers. PMID:19832985

  1. A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations

    PubMed Central

    Angulo, Bárbara; García-García, Elena; Martínez, Rebeca; Suárez-Gauthier, Ana; Conde, Esther; Hidalgo, Manuel; López-Ríos, Fernando

    2010-01-01

    KRAS mutation testing has become a standard procedure in the management of patients with carcinomas. The most frequently used method for KRAS testing is direct sequencing of PCR products. The development of commercial real-time quantitative PCR kits offers a useful alternative since they are in theory much more sensitive than direct sequencing and they avoid post- PCR handling. We present our experience as a reference center for the study of KRAS mutations, comparing direct sequencing and the use of a commercial real-time quantitative PCR kit, as well as determining the sensitivity of both procedures in clinical practice. The TheraScreen K-RAS Mutation Kit identified mutations in 75 (44%) of the 170 tumors. Three cases were tested positive using TheraScreen K-RAS Mutation Kit and negative by direct sequencing. We then compared the sensitivity of the kit and that of direct sequencing using 74 mutant tumors. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in 13.5% of the tumors and, in 84%, KRAS mutation was identified at a dilution of 5%. Sequencing was able to detect KRAS mutations when the mutant DNA represented 10% of the total DNA in 20/74 (27%) of the tumors. When the mutant DNA represented 30% of the total DNA, sequencing could detect mutations in 56/74 (76%). PMID:20203003

  2. Clinicopathologic distribution of KRAS and BRAF mutations in a Chinese population with colorectal cancer precursor lesions

    PubMed Central

    Yi, Chenghao; Huang, Yanqing; Yu, Xing; Li, Xiaofen; Zheng, Shu; Ding, Kefeng; Xu, Jinghong

    2016-01-01

    Investigating the clinical features and corresponding histomorphologic and molecular profiles of precursor lesions of colorectal cancer in a natural population provides new insights into the nature of colorectal cancer, uncovers new screening markers and establishes new prevention strategies for colorectal cancer. In this study, 4302 patients with at least one colorectal polyp from a large colorectal cancer screening program were evaluated and genetic mutations in either KRAS or BRAF were detected in 495 patients. The population-based mutation rates of KRAS and BRAF genes in colorectal polyps within this Chinese patient population were 21.8% and 12.1% respectively. Interestingly, considerable variability in the KRAS and BRAF mutations rates were found among different types of polyps. In a multivariate analysis, presence of villous histology and high-grade dysplasia was associated with KRAS mutations (OR, 3.0; 95% CI, 1.7-5.4 and OR, 3.5; 95% CI 1.9-6.5, respectively), while serrated adenomas and hyperplastic polyps were associated with BRAF V600E mutations (OR, 20.6; 95% CI, 8.2–51.8 and OR, 11.9; 95% CI 4.9–29.0, respectively). KRAS mutations may, in part, drive the histologic progression of adenomas toward a villous histology and higher grades of dysplasia. Mutant BRAF may, in part, drive the histologic progression of adenomas toward serrated histology. Dysplasia may arise from hyperplastic polyps, resulting in the formation of serrated adenomas and potentially the development of colorectal carcinoma. PMID:26910894

  3. K-ras mutation at codon 12 in stage I pancreatic adenocarcinoma: analysis by laser capture microdissection and direct sequencing.

    PubMed

    Chang, M C; Chang, Y T; Wu, M S; Shun, C T; Tien, Y W; Lin, J T

    2001-05-01

    Pancreatic ductal adenocarcinoma has been reported to carry a rate mutation high in codon 12 of the K-ras oncogene. To avoid the pitfalls of conventional methods of tissue dissection that might affect the sensitivity and specificity of detecting K-ras mutation, laser capture microdissection (LCM) technique was used. Pancreatic adenocarcinoma tissues were obtained from 15 patients who underwent Whipple's procedure. Selected tissues procured by LCM were analyzed by direct sequencing after polymerase chain reaction amplification of K-ras sequences at codon 12. K-ras mutation was noted in nine patients. All mutations showed G to A substitution at codon 12. The mutational pattern (GGT to GAT) is similar in both western and eastern reports. LCM is a feasible method to effectively obtain pure tumor cells from a surgical specimen. It remains to be determined whether this low mutation rate is a result of relatively early stage of disease or different carcinogenesis in different geographic regions. PMID:11432318

  4. Locally advanced rectal cancers with simultaneous occurrence of KRAS mutation and high VEGF expression show invasive characteristics.

    PubMed

    Krajnović, Milena; Marković, Bojana; Knežević-Ušaj, Slavica; Nikolić, Ivan; Stanojević, Maja; Nikolić, Valentina; Šiljić, Marina; Jovanović Ćupić, Snežana; Dimitrijević, Bogomir

    2016-07-01

    In this study, we investigated the mutation status of KRAS gene in pretherapeutic and preoperative biopsies in 63 specimens of locally advanced rectal cancers in order to evaluate its potential predictive and/or prognostic role. Regions of interest of KRAS exon 2 were amplified and visualized on 2% agarose gel. Obtained PCR products were subjected to direct sequencing. KRAS mutations were detected in 35% of patients, 91% of which were located in codon 12 and 9% in codon 13. In general, KRAS mutation status did not affect the response to neoadjuvant chemoradiotherapy (CRT). However, patients harboring mutated KRAS gene, simultaneously with high vascular endothelial growth factor (VEGF) expression, exhibited a worse response to CRT (p=0.030), a more frequent appearance of local recurrences and distant metastasis (p=0.003), and shorter overall survival (p=0.001) compared to all others. On the contrary, patients with GGT>GCT KRAS mutation exhibited a significantly better response to CRT than those with any other type of KRAS mutation (p=0.017). Moreover, the presence of GGT>GCT mutation was associated with low VEGF and Ki67 expression (p=0.012 in both cases), parameters related to less aggressiveness of the disease. Our results suggest that KRAS mutation status could have some predictive and prognostic importance in rectal cancer when analyzed together with other parameters, such as VEGF and Ki67 expression. In addition, it seems that not only the presence but the type of KRAS mutation is important for examining its impact on CRT response. PMID:27184911

  5. A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion.

    PubMed

    Ikenoue, Tsuneo; Terakado, Yumi; Nakagawa, Hayato; Hikiba, Yohko; Fujii, Tomoaki; Matsubara, Daisuke; Noguchi, Rei; Zhu, Chi; Yamamoto, Keisuke; Kudo, Yotaro; Asaoka, Yoshinari; Yamaguchi, Kiyoshi; Ijichi, Hideaki; Tateishi, Keisuke; Fukushima, Noriyoshi; Maeda, Shin; Koike, Kazuhiko; Furukawa, Yoichi

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC. PMID:27032374

  6. A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion

    PubMed Central

    Ikenoue, Tsuneo; Terakado, Yumi; Nakagawa, Hayato; Hikiba, Yohko; Fujii, Tomoaki; Matsubara, Daisuke; Noguchi, Rei; Zhu, Chi; Yamamoto, Keisuke; Kudo, Yotaro; Asaoka, Yoshinari; Yamaguchi, Kiyoshi; Ijichi, Hideaki; Tateishi, Keisuke; Fukushima, Noriyoshi; Maeda, Shin; Koike, Kazuhiko; Furukawa, Yoichi

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC. PMID:27032374

  7. Mutation of NRAS but not KRAS significantly reduces myeloma sensitivity to single-agent bortezomib therapy

    PubMed Central

    Lichter, David I.; Di Bacco, Alessandra; Blakemore, Stephen J.; Berger, Allison; Koenig, Erik; Bernard, Hugues; Trepicchio, William; Li, Bin; Neuwirth, Rachel; Chattopadhyay, Nibedita; Bolen, Joseph B.; Dorner, Andrew J.; van de Velde, Helgi; Ricci, Deborah; Jagannath, Sundar; Berenson, James R.; Richardson, Paul G.; Stadtmauer, Edward A.; Orlowski, Robert Z.; Lonial, Sagar; Anderson, Kenneth C.; Sonneveld, Pieter; San Miguel, Jesús F.; Esseltine, Dixie-Lee; Schu, Matthew

    2014-01-01

    Various translocations and mutations have been identified in myeloma, and certain aberrations, such as t(4;14) and del17, are linked with disease prognosis. To investigate mutational prevalence in myeloma and associations between mutations and patient outcomes, we tested a panel of 41 known oncogenes and tumor suppressor genes in tumor samples from 133 relapsed myeloma patients participating in phase 2 or 3 clinical trials of bortezomib. DNA mutations were identified in 14 genes. BRAF as well as RAS genes were mutated in a large proportion of cases (45.9%) and these mutations were mutually exclusive. New recurrent mutations were also identified, including in the PDGFRA and JAK3 genes. NRAS mutations were associated with a significantly lower response rate to single-agent bortezomib (7% vs 53% in patients with mutant vs wild-type NRAS, P = .00116, Bonferroni-corrected P = .016), as well as shorter time to progression in bortezomib-treated patients (P = .0058, Bonferroni-corrected P = .012). However, NRAS mutation did not impact outcome in patients treated with high-dose dexamethasone. KRAS mutation did not reduce sensitivity to bortezomib or dexamethasone. These findings identify a significant clinical impact of NRAS mutation in myeloma and demonstrate a clear example of functional differences between the KRAS and NRAS oncogenes. PMID:24335104

  8. Three-dimensionally specific inhibition of DNA repair-related genes by activated KRAS in colon crypt model.

    PubMed

    Tsunoda, Toshiyuki; Takashima, Yasuo; Fujimoto, Takahiro; Koyanagi, Midori; Yoshida, Yasuhiro; Doi, Keiko; Tanaka, Yoko; Kuroki, Masahide; Sasazuki, Takehiko; Shirasawa, Senji

    2010-05-01

    Growth and differentiation of colonic epithelium are regulated in the three-dimensional (3D) physiological architecture, colonic crypt, and deregulation of 3D interactions is involved in tumorigenesis. Cell-based 3D culture systems provide a suitable approach bridging the gap between two-dimensional (2D) culture and animal models. KRAS mutations are found at high frequencies in human colorectal cancer (CRC); however, KRAS-targeted cancer therapy has not been developed. Here, we have established a 3D cell culture model resembling the colonic crypt by use of HKe3 cells, human CRC HCT116 cells disrupted at activated KRAS. In this 3D colonic crypt model, HKe3 cells showed the features of time course-dependent transit-amplifying and terminal-differentiated stages, which are characteristic of normal colonic crypt. On the basis of the features of HCT116 cells, activated KRAS inhibited normal cell polarity and apoptosis in 3D culture. The expression of DNA repair-related tumor suppressor genes including TP53, BRCA1, BRCA2, and EXO-1 was markedly suppressed by activated KRAS in 3D culture but not in 2D culture. These results together suggest that activated KRAS plays critical roles in the accumulation of genetic alterations through inhibition of DNA repair genes and apoptosis and that this 3D culture model will provide a useful tool for investigating the molecular mechanisms of CRC development. PMID:20454511

  9. Three-dimensionally Specific Inhibition of DNA Repair-Related Genes by Activated KRAS in Colon Crypt Model1 2

    PubMed Central

    Tsunoda, Toshiyuki; Takashima, Yasuo; Fujimoto, Takahiro; Koyanagi, Midori; Yoshida, Yasuhiro; Doi, Keiko; Tanaka, Yoko; Kuroki, Masahide; Sasazuki, Takehiko; Shirasawa, Senji

    2010-01-01

    Growth and differentiation of colonic epithelium are regulated in the three-dimensional (3D) physiological architecture, colonic crypt, and deregulation of 3D interactions is involved in tumorigenesis. Cell-based 3D culture systems provide a suitable approach bridging the gap between two-dimensional (2D) culture and animal models. KRAS mutations are found at high frequencies in human colorectal cancer (CRC); however, KRAS-targeted cancer therapy has not been developed. Here, we have established a 3D cell culture model resembling the colonic crypt by use of HKe3 cells, human CRC HCT116 cells disrupted at activated KRAS. In this 3D colonic crypt model, HKe3 cells showed the features of time course-dependent transit-amplifying and terminal-differentiated stages, which are characteristic of normal colonic crypt. On the basis of the features of HCT116 cells, activated KRAS inhibited normal cell polarity and apoptosis in 3D culture. The expression of DNA repair-related tumor suppressor genes including TP53, BRCA1, BRCA2, and EXO-1 was markedly suppressed by activated KRAS in 3D culture but not in 2D culture. These results together suggest that activated KRAS plays critical roles in the accumulation of genetic alterations through inhibition of DNA repair genes and apoptosis and that this 3D culture model will provide a useful tool for investigating the molecular mechanisms of CRC development. PMID:20454511

  10. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

    PubMed

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. PMID:27043547

  11. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    PubMed Central

    Olmedillas López, Susana; García-Olmo, Dolores C.; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. PMID:27043547

  12. KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

    SciTech Connect

    Erben, Philipp; Stroebel, Philipp; Horisberger, Karoline; Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin; Kaehler, Georg; Kienle, Peter; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas

    2011-11-15

    Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan-Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

  13. INVOLVEMENT OF KRAS G12A MUTATION IN THE IL-2-INDEPENDENT GROWTH OF A HUMAN T-LGL LEUKEMIA CELL LINE, PLT-2

    PubMed Central

    MIZUTANI, NAOKI; ITO, HIROMI; HAGIWARA, KAZUMI; KOBAYASHI, MISA; HOSHIKAWA, ASUKA; NISHIDA, YAYOI; TAKAGI, AKIRA; KOJIMA, TETSUHITO; SUZUKI, MOTOSHI; OSAWA, YOSUKE; OHNISHI, KAZUNORI; DAIBATA, MASANORI; MURATE, TAKASHI

    2012-01-01

    ABSTRACT Cytokine-dependent cell lines have been used to analyze the cytokine-induced cellular signaling and the mechanism of oncogenesis. In the current study, we analyzed MOTN-1 and PLT-2 cell lines established from different stages of a T-cell large granular lymphocyte leukemia patient (Daibata et al. 2004). MOTN-1 is IL-2-dependent derived from the chronic phase, whereas IL-2-independent PLT-2 is from the aggressive and terminal stage. They shared considerable chromosome abnormalities and the pattern of T-cell receptor rearrangement, presuming that the cytokine independence of PLT-2 was due to the additive genetic abnormality. Besides IL-2, IL-15 supported MOTN-1 cell growth, because these receptors share β- and γ-subunits. IL-2 activated ERK, AKT and STAT pathway of MOTN-1. STAT3 pathway of PLT-2 was also activated by IL-2, suggesting intact IL-2 induces signal transduction of PLT-2. However, ERK1/2 but not AKT, was continuously activated in PLT-2, consistent with the increased Ras-activity of PLT-2. Sequence analysis revealed KRAS G12A mutation but not NRAS and HRAS mutation of PLT-2 but not MOTN-1. Another signaling molecule affecting Ras-signaling pathway, SHP2, which has been frequently mutated in juvenile myelomonocytic leukemia (JMML), did not show mutation. Moreover, MEK inhibitor, PD98059, as well as farnesylation inhibitor inhibited PLT-2 cell growth. Using NIH3T3 and MOTN-1, ERK activation, increased cell proliferation and survival by KRAS G12A were shown, suggesting the important role of KRAS G12A in IL-2-independent growth of PLT-2. Taken together, KRAS G12A is important for IL-2-independent growth of PLT-2 cells and suggests the possibility of involvement of KRAS mutation with disease progression. PMID:23092099

  14. EGFR and KRAS mutation status in non-small-cell lung cancer occurring in HIV-infected patients.

    PubMed

    Créquit, Perrine; Ruppert, Anne-Marie; Rozensztajn, Nathalie; Gounant, Valérie; Vieira, T; Poulot, Virginie; Antoine, Martine; Chouaid, Christos; Wislez, Marie; Cadranel, Jacques; Lavole, Armelle

    2016-06-01

    Non-small-cell lung cancer (NSCLC) is the most common non-acquired immune deficiency syndrome-related malignancy responsible for death. Mutational status is crucial for choosing treatment of advanced NSCLC, yet no data is available on the frequency of epidermal growth factor receptor (EGFR) and Kirsten ras (KRAS) mutations and their impact on NSCLC in human immunodeficiency virus (HIV)-infected patients (HIV-NSCLC). All consecutive HIV-NSCLC patients diagnosed between June 1996 and August 2013 at two Paris university hospitals were reviewed, with tumor samples analyzed for EGFR and KRAS mutational status. Overall, 63 tumor samples were analyzed out of 73 HIV-NSCLC cases, with 63% of advanced NSCLC. There were 60 non-squamous and nine squamous cell carcinomas, with EGFR and KRAS mutations identified in two (3.3%) and seven (11.5%) tumors, respectively. The proportion of KRAS mutations was 29% if solely the more sensitive molecular techniques were considered. The two patients with advanced adenocarcinoma harboring EGFR mutations exhibited lasting partial response to EGFR-tyrosine kinase inhibitors. Overall survival for patients with advanced NSCLC were >30 months for those with EGFR mutations, <3 months for KRAS mutations (n=2), and the median was 9 months [4.1-14.3] for wild-type (n=34). In multivariate analysis, KRAS mutation and CD4<200 cells/μL were associated with poor prognosis (hazard ratio (HR): 24 [4.1-140.2], p=0.0004; HR: 3.1 [1.3-7.5], p=0.01, respectively). EGFR mutation must be investigated in HIV-NSCLC cases due to its predictive and prognostic impact, whereas KRAS mutation is of poor prognostic value. Clinicians should search for drugs dedicated to this target population. PMID:27133754

  15. Combined use of PI3K and MEK inhibitors synergistically inhibits lung cancer with EGFR and KRAS mutations.

    PubMed

    Jiang, Ze-Bo; Huang, Jun; Xie, Chun; Li, Xia; Liu, Liping; He, Jiaxi; Pan, Hui; Huang, Liyan; Fan, Xing-Xing; Yao, Xiao-Jun; Xie, Ying; Li, Na; Liu, Liang; He, Jian-Xing; Leung, Elaine Lai-Han

    2016-07-01

    EGFR and KRAS mutations are the two most common driver mutations in non-small cell lung cancer (NSCLC). Molecular target-based therapy using small molecules such as gefitinib has been used for inhibiting EGFR with good initial responses; however, drug resistance is common when using a mono-targeting strategy. At present, KRAS remains an undruggable target. As such, the development of new drugs targeting the downstream of KRAS and EGFR and their crosstalk pathways is critically needed to effectively treat NSCLC. The present study aimed to elucidate the anticancer effects of PI3K (BKM120) and MEK (PD1056309) inhibitors on NSCLC cell lines with KRAS or EGFR mutations. Inhibition of the EGFR and KRAS downstream P13K pathway using BKM120 significantly inhibited the growth of NSCLC cell lines with either EGFR or KRAS mutations. In addition, significant cell cycle arrest and induction of apoptosis were observed following BKM120 treatment. Notably, although the A549 and H358 NSCLC cell lines harbor the same KRAS mutation, A549 cells were less sensitive than H358 cells in the response to BKM120 treatment. Similarly, PC-9 and H1650 cells harbor the same EGFR mutation, however, H1650 was less sensitive to BKM120. Different sensitivity between NSCLC cell lines with the same oncogenic mutation suggests that multiple crosstalk pathways exit. Combined usage of BKM120 and PD1056309 synergistically enhanced apoptosis in the A549 cells and mildly enhanced apoptosis in the H1650 and H358 cells, suggesting the crosstalk of the MEK pathway with the P13K/Akt pathways in these cell lines. Overall, our findings suggest that inhibition of EGFR and KRAS downstream with a P13K/Akt inhibitor could be useful for treating NSCLC. However, for NSCLC exhibiting crosstalk with other survival pathways, such as the MEK pathway, combination treatment is required. PMID:27121230

  16. TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

    PubMed Central

    Luu, Carrie; Heinrich, Eileen L.; Duldulao, Marjun; Arrington, Amanda K.; Fakih, Marwan; Garcia-Aguilar, Julio; Kim, Joseph

    2013-01-01

    Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRASmut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53−/−, TP53+/−, TP53mut/+, and TP53mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRASmut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (−/− or −/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer. PMID:23936455

  17. Cetuximab treatment for metastatic colorectal cancer with KRAS p.G13D mutations improves progression-free survival

    PubMed Central

    OSUMI, HIROKI; SHINOZAKI, EIJI; OSAKO, MASAHIKO; KAWAZOE, YOSHIMASA; OBA, MASARU; MISAKA, TAKAHARU; GOTO, TAKASHI; KAMO, HITOMI; SUENAGA, MITSUKUNI; KUMEKAWA, YOSUKE; OGURA, MARIKO; OZAKA, MASATO; MATSUSAKA, SATOSHI; CHIN, KEISHO; HATAKE, KIYOHIKO; MIZUNUMA, NOBUYUKI

    2015-01-01

    A number of previous studies have reported that 30–50% of patients with colorectal cancer (CRC) harbor Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, which is a major predictive biomarker of resistance to epidermal growth factor (EGFR)-targeted therapy. Treatment with an anti-EGFR inhibitor is recommended for patients with KRAS wild-type metastatic colorectal cancer (mCRC). A recent retrospective study of cetuximab reported that patients with KRAS p.G13D mutations had better outcomes compared with those with other mutations. The aim of this retrospective study was to assess the prevalence of KRAS p.G13D mutations and evaluate the effectiveness of cetuximab in mCRC patients with KRAS p.G13D or other KRAS mutations. We reviewed the clinical records of 98 mCRC patients with KRAS mutations who were treated between August, 2004 and January, 2011 in four hospitals located in Tokyo and Kyushu Island. We also investigated KRAS mutation subtypes and patient characteristics. In the patients who received cetuximab, univariate and multivariate analyses were performed to assess the effect of KRAS p.G13D mutations on progression-free survival (PFS) and overall survival (OS). Of the 98 patients, 23 (23.5%) had KRAS p.G13D-mutated tumors, whereas 75 (76.5%) had tumors harboring other mutations. Of the 31 patients who received cetuximab, 9 (29.0%) had KRAS p.G13D mutations and 22 (71.0%) had other mutations. There were no significant differences in age, gender, primary site, pathological type, history of chemotherapy, or the combined use of irinotecan between either of the patient subgroups. The univariate analysis revealed no significant difference in PFS or OS between the patients with KRAS p.G13D mutations and those with other mutations (median PFS, 4.5 vs. 2.8 months, respectively; P=0.65; and median OS, 15.3 vs. 8.9 months, respectively; P=0.51). However, the multivariate analysis revealed a trend toward better PFS among patients harboring p.G13D mutations (PFS

  18. Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection

    PubMed Central

    Ostrow, Sheli L.; Simon, Einav; Prinz, Elad; Bick, Tova; Shentzer, Talia; Nagawkar, Sima S.; Sabo, Edmond; Ben-Izhak, Ofer; Hershberg, Ruth; Hershkovitz, Dov

    2016-01-01

    Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor. PMID:26902163

  19. KRAS mutation positive mucinous adenocarcinoma originating in mature ovarian teratoma: case report and review of literature.

    PubMed

    Hershkovitz, Dov; Vlodavsky, Euvgeni; Simon, Einav; Ben-Izhak, Ofer

    2013-12-01

    Mature ovarian teratomas rarely undergo transformation into malignancy. Carcinomas, mostly squamous cell carcinoma, are the most common malignancy arising in mature cystic teratoma. In the present report we describe a 13-year-old girl who developed a large mass in her ovary. Fine needle biopsy identified intestinal type mucinous adenocarcinoma, which was also identified in the full surgical specimen. Extensive sampling of the surgical specimen also identified areas of mature cystic teratoma. Interestingly, molecular analysis of DNA extracted from various components of the lesion identified KRAS mutation in the carcinoma, borderline mucinous tumor and benign intestinal-type epithelium but not in the epidermal component of the teratoma. To the best of our knowledge this is the first report of KRAS mutation in mucinous carcinoma originating in mature cystic teratoma. We discuss the importance of extensive tissue sampling to differentiate between carcinoma originating in teratoma and metastatic colorectal carcinoma to the ovary. Additionally, the identification of KRAS mutation in the morphologically benign intestinal-type epithelium indicated that it is an early event in the carcinogenic sequence and that the molecular pathway of carcinogenesis in teratoma is similar to that in the carcinogenic process of somatic tissue. PMID:24422958

  20. Coordinate direct input of both KRAS and IGF1 receptor to activation of PI 3-kinase in KRAS mutant lung cancer

    PubMed Central

    Molina-Arcas, Miriam; Hancock, David C.; Sheridan, Clare; Kumar, Madhu S.; Downward, Julian

    2013-01-01

    SUMMARY Using a panel of non-small cell lung cancer (NSCLC) lines, we show here that MEK and RAF inhibitors are selectively toxic for the KRAS mutant genotype, while PI 3-kinase (PI3K), AKT and mTOR inhibitors are not. IGF1 receptor (IGF1R) tyrosine kinase inhibitors also show selectivity for KRAS mutant lung cancer lines. Combinations of IGF1R and MEK inhibitors resulted in strengthened inhibition of KRAS mutant lines and also showed improved effectiveness in autochthonous mouse models of Kras induced NSCLC. PI3K pathway activity is dependent on basal IGF1R activity in KRAS mutant, but not wild-type, lung cancer cell lines. KRAS is needed for both MEK and PI3K pathway activity in KRAS mutant, but not wild-type, lung cancer cells, while acute activation of KRAS causes stimulation of PI3K dependent upon IGF1R kinase activity. Coordinate direct input of both KRAS and IGF1R is thus required to activate PI3K in KRAS mutant lung cancer cells. PMID:23454899

  1. Pooled Analysis of the Prognostic and Predictive Effects of KRAS Mutation Status and KRAS Mutation Subtype in Early-Stage Resected Non–Small-Cell Lung Cancer in Four Trials of Adjuvant Chemotherapy

    PubMed Central

    Shepherd, Frances A.; Domerg, Caroline; Hainaut, Pierre; Jänne, Pasi A.; Pignon, Jean-Pierre; Graziano, Stephen; Douillard, Jean-Yves; Brambilla, Elizabeth; Le Chevalier, Thierry; Seymour, Lesley; Bourredjem, Abderrahmane; Teuff, Gwénaël Le; Pirker, Robert; Filipits, Martin; Rosell, Rafael; Kratzke, Robert; Bandarchi, Bizhan; Ma, Xiaoli; Capelletti, Marzia; Soria, Jean-Charles; Tsao, Ming-Sound

    2013-01-01

    Purpose We undertook this analysis of KRAS mutation in four trials of adjuvant chemotherapy (ACT) versus observation (OBS) to clarify the prognostic/predictive roles of KRAS in non–small-cell lung cancer (NSCLC). Methods KRAS mutation was determined in blinded fashion. Exploratory analyses were performed to characterize relationships between mutation status and subtype and survival outcomes using a multivariable Cox model. Results Among 1,543 patients (763 OBS, 780 ACT), 300 had KRAS mutations (codon 12, n = 275; codon 13, n = 24; codon 14, n = 1). In OBS patients, there was no prognostic difference for overall survival for codon-12 (mutation v wild type [WT] hazard ratio [HR] = 1.04; 95% CI, 0.77 to 1.40) or codon-13 (HR = 1.01; 95% CI, 0.47 to 2.17) mutations. No significant benefit from ACT was observed for WT-KRAS (ACT v OBS HR = 0.89; 95% CI, 0.76 to 1.04; P = .15) or codon-12 mutations (HR = 0.95; 95% CI, 0.67 to 1.35; P = .77); with codon-13 mutations, ACT was deleterious (HR = 5.78; 95% CI, 2.06 to 16.2; P < .001; interaction P = .002). There was no prognostic effect for specific codon-12 amino acid substitution. The effect of ACT was variable among patients with codon-12 mutations: G12A or G12R (HR = 0.66; P = .48), G12C or G12V (HR = 0.94; P = .77) and G12D or G12S (HR = 1.39; P = .48; comparison of four HRs, including WT, interaction P = .76). OBS patients with KRAS-mutated tumors were more likely to develop second primary cancers (HR = 2.76, 95% CI, 1.34 to 5.70; P = .005) but not ACT patients (HR = 0.66; 95% CI, 0.25 to 1.75; P = .40; interaction, P = .02). Conclusion KRAS mutation status is not significantly prognostic. The potential interaction in patients with codon-13 mutations requires validation. At this time, KRAS status cannot be recommended to select patients with NSCLC for ACT. PMID:23630215

  2. Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma.

    PubMed

    Schabath, M B; Welsh, E A; Fulp, W J; Chen, L; Teer, J K; Thompson, Z J; Engel, B E; Xie, M; Berglund, A E; Creelan, B C; Antonia, S J; Gray, J E; Eschrich, S A; Chen, D-T; Cress, W D; Haura, E B; Beg, A A

    2016-06-16

    While mutations in the KRAS oncogene are among the most prevalent in human cancer, there are few successful treatments to target these tumors. It is also likely that heterogeneity in KRAS-mutant tumor biology significantly contributes to the response to therapy. We hypothesized that the presence of commonly co-occurring mutations in STK11 and TP53 tumor suppressors may represent a significant source of heterogeneity in KRAS-mutant tumors. To address this, we utilized a large cohort of resected tumors from 442 lung adenocarcinoma patients with data including annotation of prevalent driver mutations (KRAS and EGFR) and tumor suppressor mutations (STK11 and TP53), microarray-based gene expression and clinical covariates, including overall survival (OS). Specifically, we determined impact of STK11 and TP53 mutations on a new KRAS mutation-associated gene expression signature as well as previously defined signatures of tumor cell proliferation and immune surveillance responses. Interestingly, STK11, but not TP53 mutations, were associated with highly elevated expression of KRAS mutation-associated genes. Mutations in TP53 and STK11 also impacted tumor biology regardless of KRAS status, with TP53 strongly associated with enhanced proliferation and STK11 with suppression of immune surveillance. These findings illustrate the remarkably distinct ways through which tumor suppressor mutations may contribute to heterogeneity in KRAS-mutant tumor biology. In addition, these studies point to novel associations between gene mutations and immune surveillance that could impact the response to immunotherapy. PMID:26477306

  3. Activated KRAS Cooperates with MLL-AF4 to Promote Extramedullary Engraftment and Migration of Cord Blood CD34+ HSPC But Is Insufficient to Initiate Leukemia.

    PubMed

    Prieto, Cristina; Stam, Ronald W; Agraz-Doblas, Antonio; Ballerini, Paola; Camos, Mireia; Castaño, Julio; Marschalek, Rolf; Bursen, Aldeheid; Varela, Ignacio; Bueno, Clara; Menendez, Pablo

    2016-04-15

    The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematologic malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood-derived CD34(+) hematopoietic stem/progenitor cells (HSPC). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34(+) HSPCs. Intrabone marrow transplantation into immunodeficient mice further showed that MA4 and KRAS(G12V) alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34(+) HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)(+) B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS affects the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4(+) B-ALL is important for tumor maintenance rather than initiation. Cancer Res; 76(8); 2478-89. ©2016 AACR. PMID:26837759

  4. Detection of KRAS mutations using double-stranded toehold-exchange probes.

    PubMed

    Wu, Zhenhua; Ma, Tianle; Coll, Jean-Luc; Liu, Fangming; Zhang, Honglian; Ma, Yunfei; Wang, Zhishuo; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2016-06-15

    Detection of KRAS mutations in cancer tissues is immensely valuable for the identification of personalized genotype-based therapy. Here, we employed a double-stranded toehold-exchange probe, which is labeled with fluorescent molecules (FAM) and quenchers (Dabcyl), to detect KRAS mutations in cancer tissues. This probe was able to differentiate the intended mutation in a sample containing as little as 5% mutant alleles in a background of wild-type DNA. This probe also performed robustly at a wide range of conditions, for examples, from 4 °C to 37 °C, from 200 mM Na(+) to 1M Na(+), and from 200 mM K(+) to 500 mM K(+). Furthermore, we validated the practicality of this probe in a clinical setting using 8 pairs of cancer tissue samples and their NT (corresponding adjacent nontumorous tissue) samples. All the results generated from the probe detection agreed with those from direct sequencing. Combining features of extreme high specificity and robustness, this probe is a valuable tool for reliable diagnosis of cancer-related mutations. PMID:26829579

  5. The Prognostic Value of Microsatellite Instability, KRAS, BRAF and PIK3CA Mutations in Stage II Colon Cancer Patients

    PubMed Central

    Vogelaar, F Jeroen; N van Erning, Felice; Reimers, Marlies S; van der Linden, Hans; Pruijt, Hans; C van den Brule, Adriaan J; Bosscha, Koop

    2015-01-01

    In the era of personalized cancer medicine, identifying mutations within patient tumors plays an important role in defining high-risk stage II colon cancer patients. The prognostic role of BRAF V600E mutation, microsatellite instability (MSI) status, KRAS mutation and PIK3CA mutation in stage II colon cancer patients is not settled. We retrospectively analyzed 186 patients with stage II colon cancer who underwent an oncological resection but were not treated with adjuvant chemotherapy. KRAS mutations, PIK3CA mutation, V600E BRAF mutation and MSI status were determined. Survival analyses were performed. Mutations were found in the patients with each mutation in the following percentages: 23% (MSI), 35% (KRAS), 19% (BRAF) and 11% (PIK3CA). A trend toward worse overall survival (OS) was seen in patients with an MSI (5-year OS 74% versus 82%, adjusted hazard ratio [HR] 1.8, 95% confidence interval [CI] 0.6–4.9) and a KRAS-mutated tumor (5-year OS 77% versus 82%, adjusted HR 1.7, 95% CI 0.8–3.5). MSI and BRAF-mutated tumors tended to correlate with poorer disease-free survival (DFS) (5-year DFS 60% versus 78%, adjusted HR 1.6, 95% CI 0.5–2.1 and 5-year DFS 57% versus 77%, adjusted HR 1.1, 95% CI 0.4–2.6 respectively). In stage II colon cancer patients not treated with adjuvant chemotherapy, BRAF mutation and MSI status both tended to have a negative prognostic effect on disease-free survival. KRAS and MSI status also tended to be correlated with worse overall survival. PMID:26716438

  6. NVP-BKM120, a novel PI3K inhibitor, shows synergism with a STAT3 inhibitor in human gastric cancer cells harboring KRAS mutations

    PubMed Central

    PARK, EUNJU; PARK, JINAH; HAN, SAE-WON; IM, SEOCK-AH; KIM, TAE-YOU; OH, DO-YOUN; BANG, YUNG-JUE

    2012-01-01

    Aberrations of Phosphoinositide 3-kinase (PI3K)/AKT signaling are frequently observed in many types of cancer, promoting its emergence as a promising target for cancer treatment. PI3K can become activated by various pathways, one of which includes RAS. RAS can not only directly activate the PI3K/AKT pathway via binding to p110 of PI3K, but also regulates mTOR via ERK or RSK independently of the PI3K/AKT pathway. Thus, actively mutated RAS can constitutively activate PI3K signaling. Additionally, in RAS tumorigenic transformation, signal transducer and activator of transcription 3 (STAT3) has been known also to be required. In this study, we examined the efficacy of NVP-BKM120, a pan-class I PI3K inhibitor in human gastric cancer cells and hypothesized that the combined inhibition of PI3K and STAT3 would be synergistic in KRAS mutant gastric cancer cells. NVP-BKM120 demonstrated anti-proliferative activity in 11 human gastric cancer cell lines by decreasing mTOR downstream signaling. But NVP-BKM120 treatment increased p-AKT by subsequent abrogation of feedback inhibition by stabilizing insulin receptor substrate-1. In KRAS mutant gastric cancer cells, either p-ERK or p-STAT3 was also increased upon treatment of NVP-BKM120. The synergistic efficacy study demonstrated that dual PI3K and STAT3 blockade showed a synergism in cells harboring mutated KRAS by inducing apoptosis. The synergistic effect was not seen in KRAS wild-type cells. Together, these findings suggest for the first time that the dual inhibition of PI3K and STAT3 signaling may be an effective therapeutic strategy for KRAS mutant gastric cancer patients. PMID:22159814

  7. A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

    PubMed

    Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

    2015-07-01

    It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency. PMID:25936694

  8. KRAS mutational subtype and copy number predict in vitro response of human pancreatic cancer cell lines to MEK inhibition

    PubMed Central

    Hamidi, H; Lu, M; Chau, K; Anderson, L; Fejzo, M; Ginther, C; Linnartz, R; Zubel, A; Slamon, D J; Finn, R S

    2014-01-01

    Background: To study the molecular mechanism regulating sensitivity to MEK inhibition in pancreatic cancer cell lines. Methods: A growth inhibition assay determined sensitivity to MEK162 in a panel of 29 pancreatic cancer cell lines. For the same panel, KRAS mutational status and copy-number variation (CNV) was determine using PCR, array CGH and FISH. Two sensitive and two resistant cell lines were further interrogated for difference in baseline and MEK162-induced gene expression, as well as signal transduction using microarray and western blotting. Cell cycle and apoptosis analysis was measured by flow cytometry. Results: We report a strong correlation between both specific KRAS mutational subtype and CNV, and sensitivity to MEK inhibition. Cell lines with a KRAS (V12) mutation and KRAS gains or loss (n=7) are ∼10 times more resistant than those having neither a KRAS (V12) mutation nor KRAS CNV (n=14). Significant differences in baseline and MEK162-induced gene expression exist between the sensitive and resistant lines, especially in genes involved in RAS, EGF receptor and PI3K pathways. This was further supported by difference in signal transduction. MEK 162 blocked ERK1/2, as well as inhibited PI3K and S6 and increased p27KIP1 levels in the sensitive lines. Conclusions: Given the potency of MEK162, it may be a promising new therapy for patients with pancreatic cancer and KRAS mutational subtypes, and CNV may serve as important biomarkers for selecting patients that benefit from MEK-targeting based on these preclinical data. PMID:25167228

  9. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  10. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

    PubMed Central

    Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  11. LUNG TUMOR KRAS AND TP53 MUTATIONS IN NON-SMOKERS REFLECT EXPOSURE TO PAH-RICH COAL COMBUSTION EMISSIONS

    EPA Science Inventory


    Abstract

    We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G -+ T transver...

  12. Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma

    PubMed Central

    Unni, Arun M; Lockwood, William W; Zejnullahu, Kreshnik; Lee-Lin, Shih-Queen; Varmus, Harold

    2015-01-01

    Human lung adenocarcinomas (LUAD) contain mutations in EGFR in ∼15% of cases and in KRAS in ∼30%, yet no individual adenocarcinoma appears to carry activating mutations in both genes, a finding we have confirmed by re-analysis of data from over 600 LUAD. Here we provide evidence that co-occurrence of mutations in these two genes is deleterious. In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene. We also show that forced expression of a second oncogene in human cancer cell lines with an endogenous mutated oncogene is deleterious. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype. Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities. DOI: http://dx.doi.org/10.7554/eLife.06907.001 PMID:26047463

  13. Concordance of KRAS mutation status between luminal and peripheral regions of primary colorectal cancer. A laser-capture microdissection-based study.

    PubMed

    Lewandowska, M; Hybiak, J; Domagala, W

    2016-03-01

    The presence of KRAS mutation in colorectal cancer (CRC) is a marker of resistance to anti-EGFR therapy. However, there are conflicting reports concerning intratumoral heterogeneity of KRAS mutations. The aim of this study was to determine whether within primary CRCs with KRAS mutations intratumoral KRAS mutation heterogeneity can be detected between two strictly defined areas, i.e. the luminal (mucosa/submucosa) and peripheral invasive front of the tumor. Using laser-capture microdissection, from every tumor about 400-500 nests of cancer cells were excised from each of the examined areas (luminal and peripheral) and PNAClamp, a high-sensitivity real-time PCR-based diagnostic assay for KRAS mutation testing, was used for molecular analysis. KRAS mutations were detected in codon 12 in both luminal and peripheral regions in all tumors examined. We conclude that from the point of view of practical KRAS mutation testing for predictive purposes in patients with CRC (i.e. testing mutations in codons 12 and 13) sampling errors are unlikely to occur if in CRCs with KRAS mutations only the luminal (as in biopsy tissue) or peripheral region is examined, provided a sensitive system of detection is applied and an appropriate number of tumor cells with minimal contamination by benign cells is analyzed. PMID:27179269

  14. Non-small Cell Lung Cancer with Concomitant EGFR, KRAS, and ALK Mutation: Clinicopathologic Features of 12 Cases

    PubMed Central

    Lee, Taebum; Lee, Boram; Choi, Yoon-La; Han, Joungho; Ahn, Myung-Ju; Um, Sang-Won

    2016-01-01

    Background: Although epidermal growth factor receptor (EGFR), v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and anaplastic lymphoma kinase (ALK) mutations in non-small cell lung cancer (NSCLC) were thought to be mutually exclusive, some tumors harbor concomitant mutations. Discovering a driver mutation on the basis of morphologic features and therapeutic responses with mutation analysis can be used to understand pathogenesis and predict resistance in targeted therapy. Methods: In 6,637 patients with NSCLC, 12 patients who had concomitant mutations were selected and clinicopathologic features were reviewed. Clinical characteristics included sex, age, smoking history, previous treatment, and targeted therapy with response and disease-free survival. Histologic features included dominant patterns, nuclear and cytoplasmic features. Results: All patients were diagnosed with adenocarcinoma and had an EGFR mutation. Six patients had concomitant KRAS mutations and the other six had KRAS mutations. Five of six EGFR-KRAS mutation patients showed papillary and acinar histologic patterns with hobnail cells. Three of six received EGFR tyrosine kinase inhibitor (TKI) and showed partial response for 7–29 months. All six EGFR-ALK mutation patients showed solid or cribriform patterns and three had signet ring cells. Five of six EGFR-ALK mutation patients received EGFR TKI and/or ALK inhibitor and four showed partial response or stable disease, except for one patient who had acquired an EGFR mutation. Conclusions: EGFR and ALK mutations play an important role as driver mutations in double mutated NSCLC, and morphologic analysis can be used to predict treatment response. PMID:27086595

  15. Comparison of KRAS mutation analysis of colorectal cancer samples by standard testing and next-generation sequencing

    PubMed Central

    Kothari, Nishi; Schell, Michael J; Teer, Jamie K; Yeatman, Timothy; Shibata, David; Kim, Richard

    2016-01-01

    Aims Based on KRAS testing, the subset of patients with metastatic colorectal cancer (CRC) that could benefit from anti-EGFR therapy can be better delineated. Though KRAS testing has become significantly more prevalent over the last few years, methods for testing remain heterogeneous and discordance has been reported between methods. Methods In this study, we examined a CRC patient population and compared KRAS testing done in Clinical Laboratory Improvement Amendments (CLIA) approved laboratories as part of standard clinical care and by next-generation sequencing (NGS) using the Illumina platform. Discordances were further evaluated with manual review of the NGS testing. Results Out of 468 CRC patient samples, 77 had KRAS testing done by both CLIA assay and NGS. There were concordant results between testing methodologies in 74 out of 77 patients, or 96% (95% CI 89% to 99%). There were three patient samples that showed discordant results between the two methods of testing. Upon further investigation of the NGS results for the three discordant cases, one sample showed a low level of the mutation seen in the standard testing, one sample showed low tumour fraction and a third did not show any evidence of the mutation that was found with the standard assay. Five patients had KRAS mutations not typically tested with standard testing. Conclusions Overall there was a high concordance rate between NGS and standard testing for KRAS. However, NGS revealed mutations that are not tested for with standard KRAS assays that might have clinical impact with regards to the role for anti-EGFR therapy. PMID:25004944

  16. Expression of c-myc and mutation of the KRAS gene in patients with ovarian mucinous tumors.

    PubMed

    Li, X S; Sun, J; He, X L

    2015-01-01

    We examined the expression of c-myc and mutations in the KRAS gene in ovarian mucinous tumors to explore the pathogenesis of these tumors and the feasibility of targeted gene therapy. Expression of c-myc protein and mutations in the KRAS gene in 24 cases of ovarian mucinous cystadenoma, 46 cases of ovarian borderline mucinous cystadenoma, and 46 cases of ovarian mucinous cystadenocarcinoma were detected using the immunohistochemistry PV-9000 2-step method and polymerase chain reaction-restriction fragment length polymorphism. The positive expression rates of c-myc in ovarian mucinous cystadenoma, borderline mucinous cystadenoma, and cystadenocarcinoma were 0, 39.1, and 65.2%, respectively (P < 0.01), while the mutation rates in KRAS were 0, 39.1 and 13.0%, respectively. The mutation rate of the borderline group was significantly higher, while rates in the other 2 groups were similar (P > 0.05). c-myc was not correlated with clinical stage, pathological grade, or age of patients with ovarian mucinous cystadenocarcinoma or borderline mucinous cystadenoma (P > 0.05), but was correlated with tumor size (P < 0.05). Mutations in KRAS were not correlated with clinical stage or tumor size in patients with borderline mucinous cystadenoma (P > 0.05), whereas it was correlated with age (P < 0.05). In borderline mucinous cystadenoma, c-myc expression and KRAS mutations were not correlated (P > 0.05). c-myc is involved in the formation of ovarian borderline mucinous cystadenoma and mucinous cystadenocarcinoma, and the KRAS gene may contribute to the formation of borderline mucinous cystadenoma. PMID:26400304

  17. Prevalence of KRAS, BRAF, PI3K and EGFR mutations among Asian patients with metastatic colorectal cancer

    PubMed Central

    PHUA, LEE CHENG; NG, HUI WEN; YEO, ANGIE HUI LING; CHEN, ELYA; LO, MICHELLE SHU MEI; CHEAH, PEH YEAN; CHAN, ERIC CHUN YONG; KOH, POH KOON; HO, HAN KIAT

    2015-01-01

    Mutations in oncogenes along the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the resistance to cetuximab in patients with metastatic colorectal cancer (mCRC). However, the relative significance of these mutations based on their frequencies of occurrence in the Singaporean population remains unclear. In the present study, the prevalence of Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B (BRAF), phosphoinositide 3-kinase (PI3K) and EGFR somatic mutations were determined among Singaporean patients with mCRC. DNA extracted from 45 pairs of surgically resected tumor and normal mucosa samples was subjected to direct sequencing or restriction fragment length polymorphism. Associations of the genetic mutations with various clinicopathological parameters were further explored. Mutations in either codon 12 or 13 of KRAS were confirmed as prominent phenomena among the included Singaporean mCRC patients, at a prevalence comparable with that of Caucasian and patients of other Asian ethnicities [33.3% (90% confidence interval, 21.8–44.9%)]. KRAS mutation was not associated with clinicopathological features, including age, gender and ethnicity of patients, or the tumor site, differentiation and mucinous status. Conversely, the prevalence of BRAF (0%), PI3K (2.2%) and EGFR (0%) mutations were low. The results of the present study indicate that KRAS mutations are prevalent among the studied population, and confirm the low prevalence of BRAF, PI3K and EGFR mutations. KRAS should be prioritized as an investigational gene for future studies of predictive biomarkers of cetuximab response among Singaporean patients with mCRC. PMID:26622882

  18. Immunophenotype and K-RAS mutation in mucinous ovarian adenocarcinoma with mural nodule of high-grade sarcoma: case report.

    PubMed

    Desouki, Mohamed M; Fadare, Oluwole; Kanbour, Anisa; Kanbour-Shakir, Amal

    2014-03-01

    Ovarian mucinous tumors with mural nodules are rare. The mural nodules are microscopically divergent neoplasms of varying sizes that may be benign (eg, sarcoma-like and carcinosarcoma-like), or malignant (eg, anaplastic carcinoma and sarcoma). The K-RAS gene mutation in ovarian mucinous neoplasms with mural nodules has not been previously reported. This is a case report of a 25-year-old female diagnosed with ovarian invasive mucinous adenocarcinoma with mural nodule of high-grade sarcoma. The mucinous tumor component demonstrated a K-RAS codon 12/13 mutation (p.G12V, c.35 G>T), whereas the sarcomatous component demonstrated a K-RAS codon 12/13 mutation (p.G12D, c.35 G>A). Although both tumor components revealed a mutation in codon 12 of K-RAS, they were of different nucleotide substitutions, indicating that these 2 tumor components were of different clonal origins. However, the fact that the 2 mutations identified in the tumor components are the most common mutations reported in mucinous tumors of the ovary, raises the possibility that sarcomatous mural nodules simply represent a form of dedifferentiation in mucinous tumors. PMID:24487474

  19. Patient and Tumor Characteristics and BRAF and KRAS Mutations in Colon Cancer, NCCTG/Alliance N0147

    PubMed Central

    Gonsalves, Wilson I.; Mahoney, Michelle R.; Sargent, Daniel J.; Nelson, Garth D.; Alberts, Steven R.; Sinicrope, Frank A.; Goldberg, Richard M.; Limburg, Paul J.; Thibodeau, Stephen N.; Grothey, Axel; Hubbard, Joleen M.; Chan, Emily; Nair, Suresh; Berenberg, Jeffrey L.

    2014-01-01

    Background KRAS and BRAF V600E mutations are important predictive and prognostic markers, respectively, in colon cancer, but little is known about patient and clinical factors associated with them. Methods Two thousand three hundred twenty-six of 3397 patients in the N0147 phase III adjuvant trial for stage III colon cancer completed a patient questionnaire. Primary tumors were assessed for KRAS and BRAF V600E mutations and defective mismatch repair (dMMR) status. Logistic regression models and categorical data analysis were used to identify associations of patient and tumor characteristics with mutation status. All statistical tests were two-sided. Results KRAS (35%) and BRAF V600E (14%) mutations were nearly mutually exclusive. KRAS mutations were more likely to be present in patients without a family history of colon cancer and never smokers. Tumors with KRAS mutations were less likely to have dMMR (odds ratio [OR] = 0.21; 95% confidence interval [CI] = 0.15 to 0.31; P < .001) and high-grade histology (OR = 0.73; 95% CI = 0.59 to 0.92; P < .001) but were more often right-sided. Among KRAS-mutated tumors, those with a Gly13Asp mutation tended to have dMMR and high-grade histology. Tumors with BRAF V600E mutations were more likely to be seen in patients who were aged 70 years or older (OR = 3.33; 95% CI = 2.50 to 4.42; P < .001) and current or former smokers (OR = 1.64; 95% CI = 1.26 to 2.14; P < .001) but less likely in non-whites and men. Tumors with BRAF V600E mutations were more likely to be right-sided and to have four or more positive lymph nodes, high-grade histology, and dMMR. Conclusions Specific patient and tumor characteristics are associated with KRAS and BRAF V600E mutations. PMID:24925349

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

    PubMed Central

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

    2014-01-01

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

  1. MUTATION SPECTRA OF SMOKY COAL COMBUSTION EMMISSIONS IN SALMONELLA REFLECTS THE TP53 AND KRAS MUTATIONS IN LUNG TUMORS FROM SMOKY COAL EXPOSED INDIVIDUALS

    EPA Science Inventory


    Mutation Spectra of Smoky Coal Combustion Emissions in Salmonella Reflect the TP53
    and KRAS Mutations in Lung Tumors from Smoky Coal-Exposed Individuals

    Abstract
    Nonsmoking women in Xuan Wei County, Yunnan Province, China who use smoky coal for cooking and h...

  2. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    EPA Science Inventory

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

    Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
    <...

  3. Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis.

    PubMed

    Rau, Kun-Ming; Chen, Han-Ku; Shiu, Li-Yen; Chao, Tsai-Ling; Lo, Yi-Ping; Wang, Chin-Chou; Lin, Meng-Chih; Huang, Chao-Cheng

    2016-01-01

    Mutations on epidermal growth factor receptor (EGFR) of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5%) were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6%) were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy. PMID:27070580

  4. Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis

    PubMed Central

    Rau, Kun-Ming; Chen, Han-Ku; Shiu, Li-Yen; Chao, Tsai-Ling; Lo, Yi-Ping; Wang, Chin-Chou; Lin, Meng-Chih; Huang, Chao-Cheng

    2016-01-01

    Mutations on epidermal growth factor receptor (EGFR) of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5%) were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6%) were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy. PMID:27070580

  5. Detection of K-ras Mutations in Predicting Efficacy of Epidermal Growth Factor Receptor Tyrosine Kinase (EGFR-TK) Inhibitor in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Li, Ze; Liu, Xue-Wei; Chi, Zhao-Cheng; Sun, Bao-Sheng; Cheng, Ying; Cheng, Long-Wei

    2015-01-01

    Epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors are useful in treating different advanced human cancers; however, their clinical efficacy varies. This study detected K-ras mutations to predict the efficacy of EGFR-TK inhibitor cetuximab treatment on Chinese patients with metastatic colorectal cancer (mCRC). A total of 87 patients with metastatic colorectal cancer were treated with cetuximab for 2-16 months, in combination with chemotherapy between August 2008 and July 2012, and tissue samples were used to detect K-ras mutations. The data showed that K-ras mutation occurred in 27/87 (31%). The objective response rates and disease control rate in K-ras wild type and mutant patients were 42% (25/60) versus 11% (3/27) (p<0.05) and 60% (36/60) versus 26% (7/27) (p<0.05), respectively. Patients with the wild-type K-ras had significantly higher median survival times and progression-free survival, than patients with mutated K-ras (21 months versus 17 months, p=0.017; 10 months versus 6 months, p=0.6). These findings suggest that a high frequency of K-ras mutations occurs in Chinese mCRC patients and that K-ras mutation is required to select patients for eligibility for cetuximab therapy. Further prospective studies using a large sample size are needed to confirm these preliminary findings. PMID:25950441

  6. ALTERNATE PATHWAY TO LUNG CANCER INDICATED BY KRAS AND P53 MUTATIONS IN NONSMOKERS EXPOSED TO INDOOR SMOKY COAL EMISSIONS

    EPA Science Inventory

    Alternate Pathway to Lung Cancer Indicated by KRAS and P53 Mutations in Nonsmokers Exposed to Indoor Smoky Coal Emissions

    Use of smoky coal in unvented homes in Xuan Wei County, Yunnan Province, China, is
    associated with lung cancer among nonsmoking females. Such wome...

  7. LUNG TUMOR KRAS AND TP53 MUTATIONS IN NONSMOKERS REFLECT EXPOSURE TO PAH-RICH COAL COMBUSTION EMISSIONS

    EPA Science Inventory

    Lung Tumor KRAS and TP53 Mutations in Nonsmokers Reflect Exposure to PAH-Rich
    Coal Combustion Emissions

    Use of smoky coal in unvented homes in Xuan Wei County, Yunnan Province, China, is associated with lung cancer among nonsmoking females. Such women have the highest...

  8. Survival outcome according to KRAS mutation status in newly diagnosed patients with stage IV non-small cell lung cancer treated with platinum doublet chemotherapy

    PubMed Central

    Brady, Anna K.; McNeill, Jonathan D.; Judy, Brendan; Bauml, Joshua; Evans, Tracey L.; Cohen, Roger B.; Langer, Corey; Vachani, Anil; Aggarwal, Charu

    2015-01-01

    Introduction Mutations (MT) of the KRAS gene are the most common mutation in non-small cell lung cancer (NSCLC), seen in about 20–25% of all adenocarcinomas. Effect of KRAS MT on response to cytotoxic chemotherapy is unclear. Methods We undertook a single-institution retrospective analysis of 93 consecutive patients with stage IV NSCLC adenocarcinoma with known KRAS and EGFR MT status to determine the association of KRAS MT with survival. All patients were treated between January 1, 2008 and December 31, 2011 with standard platinum based chemotherapy at the University of Pennsylvania. Overall and progression free survival were analyzed using Kaplan-Meier and Cox proportional hazard methods. Results All patients in this series received platinum doublet chemotherapy, and 42 (45%) received bevacizumab. Overall survival and progression free survival for patients with KRAS MT was no worse than for patients with wild type KRAS. Median overall survival for patients with KRAS MT was 19 months (mo) vs. 15.6 mo for KRAS WT, p = 0.34, and progression-free survival was 6.2 mo in patients with KRAS MT vs. 7mo in patients with KRAS WT, p = 0.51. In multivariable analysis including age, race, gender, and ECOG PS, KRAS MT was not associated with overall survival (HR 1.12, 95% CI 0.58–2.16, p = 0.74) or progression free survival (HR 0.80, 95% CI 0.48–1.34, p = 41). Of note, receipt of bevacizumab was associated with improved overall survival only in KRAS WT patients (HR 0.34, p = 0.01). Conclusions KRAS MT are not associated with inferior progression-free and overall survival in advanced NSCLC patients treated with standard first-line platinum-based chemotherapy. PMID:26471290

  9. KRAS mutation in adenocarcinoma of the gastrointestinal type arising from a mature cystic teratoma of the ovary.

    PubMed

    Li, Yan; Zhang, Ruiguang; Pan, Danzhen; Huang, Bangxing; Weng, Mixia; Nie, Xiu

    2014-01-01

    Mature cystic teratomas (MCT) in the ovary rarely undergo malignant transformation. Moreover, adenocarcinoma of the gastrointestinal type is much rarer. We present two cases of perimenopausal female pateints with mature cystic teratoma of single ovary, while local adenocarcinoma arising in the MCT. The malignancies showed immunohistochemical features of intestinal differentiation, such as strong positivity for CDX-2, villin and CK-20, and negativity for CK-7. Furthermore, the mutation analysis of molecular alteration revealed a KRAS gene mutation in the intestinal adenocarcinoma part, extending into benign intestinal-type epithelium linings. Yet the mutation was not present in the epidermal component of the teratoma. We present these as two unique cases of mucinous adenocarcinoma of the intestinal type arising from mature cystic teratoma. Moreover, we also submit that this KRAS mutation might contribute to identify malignant transformation of a MCT and suggest possible effect on targeted treatment decisions for anti-epidermal growth factor receptor (EGFR) therapy in metastasized patients. PMID:25297496

  10. Associations of Anthropometric Factors with KRAS and BRAF Mutation Status of Primary Colorectal Cancer in Men and Women: A Cohort Study

    PubMed Central

    Brändstedt, Jenny; Wangefjord, Sakarias; Nodin, Björn; Eberhard, Jakob; Sundström, Magnus; Manjer, Jonas; Jirström, Karin

    2014-01-01

    Obesity is a well-established risk factor for colorectal cancer (CRC), and accumulating evidence suggests a differential influence of sex and anthropometric factors on the molecular carcinogenesis of the disease. The aim of the present study was to investigate the relationship between height, weight, bodyfat percentage, waist- and hip circumference, waist-hip ratio (WHR), body mass index (BMI) and CRC risk according to KRAS and BRAF mutation status of the tumours, with particular reference to potential sex differences. KRAS and BRAF mutations were analysed by pyrosequencing in tumours from 494 incident CRC cases in the Malmö Diet and Cancer Study. Hazard ratios of CRC risk according to anthropometric factors and mutation status were calculated using multivariate Cox regression models. While all anthropometric measures except height were associated with an increased risk of KRAS-mutated tumours, only BMI was associated with an increased risk of KRAS wild type tumours overall. High weight, hip, waist, WHR and BMI were associated with an increased risk of BRAF wild type tumours, but none of the anthropometric factors were associated with risk of BRAF-mutated CRC, neither in the overall nor in the sex-stratified analysis. In men, several anthropometric measures were associated with both KRAS-mutated and KRAS wild type tumours. In women, only a high WHR was significantly associated with an increased risk of KRAS-mutated CRC. A significant interaction was found between sex and BMI with respect to risk of KRAS-mutated tumours. In men, all anthropometric factors except height were associated with an increased risk of BRAF wild type tumours, whereas in women, only bodyfat percentage was associated with an increased risk of BRAF wild type tumours. The results from this prospective cohort study further support an influence of sex and lifestyle factors on different pathways of colorectal carcinogenesis, defined by KRAS and BRAF mutation status of the tumours. PMID:24918610

  11. Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer

    PubMed Central

    Guo, Kang; Wang, Wu-Ping; Jiang, Tao; Wang, Ju-Zheng; Chen, Zhao; Li, Yong; Zhou, Yong-An; Li, Xiao-Fei

    2016-01-01

    Background The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. Methods EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients’ survival. Results A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0

  12. [Long-Term Survival of a Patient with KRAS Mutated Colon Cancer Successfully Treated with Regorafenib].

    PubMed

    Ohta, Ryo; Sekikawa, Koji; Yamazaki, Masato; Goto, Manabu; Narita, Kazuhiro; Ikeda, Hironari; Oneyama, Masataka; Nakayama, Mikihiro; Shimoda, Yota; Sato, Shun; Inoue, Takahiro

    2015-10-01

    A 65-year-old woman underwent iliocecal resection for cecal cancer. During post-operative follow-up, she was diagnosed with metastasis to the abdominal wall and a curative resection was performed. After 12 courses of adjuvant chemotherapy with a modified combination of folinic acid, 5-fluorouracil, and oxaliplatin (mFOLFOX6), recurrence was noted in the lung. A curative resection was successfully performed and she was subsequently treated with bevacizumab and a combination of folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI). One year after surgical resection, recurrence in the remnant lung was diagnosed. Because of the KRAS mutation, she could not be treated with anti-epidermal growth factor antibodies. The metastatic lung tumor continued to enlarge. Therefore, we selected regorafenib as third-line chemotherapy. After treatment with regorafenib, the size of the target lesion decreased significantly. PMID:26489551

  13. KRAS promoter oligonucleotide with decoy activity dimerizes into a unique topology consisting of two G-quadruplex units

    PubMed Central

    Podbevšek, Peter; Plavec, Janez

    2016-01-01

    Mutations of the KRAS proto-oncogene are associated with several tumor types, which is why it is being considered as a target for anti-cancer drug development. The human KRAS promoter contains a nuclease hypersensitive element (NHE), which can bind to nuclear proteins and is believed to form G-quadruplex structures. Previous studies showed that a 32-nt oligonucleotide (32R-3n) mimicking the KRAS NHE can reduce gene transcription by sequestering MAZ, a crucial transcription factor. Here we show that 32R-3n has to dimerize in order to fold into a G-quadruplex structure. Individual 5′- and 3′-end G-quadruplex units are formed and both feature a symmetric head-to-head topology with edge-type loops. The MAZ binding sequence is located within the 3′-end unit. Nuclear magnetic resonance data complemented by CD and UV spectra show that nucleotides of the MAZ binding G-rich motif are dynamic and could be available for sequence or structure based recognition. Both stable G-quadruplex structures could protect 5′- and 3′-ends of 32R-3n and enhance its anti-cancer activity. Single stranded genomic KRAS NHE including nucleotides flanking the 32R-3n sequence could favor a different monomeric fold, which remains unknown. PMID:26656490

  14. KRAS promoter oligonucleotide with decoy activity dimerizes into a unique topology consisting of two G-quadruplex units.

    PubMed

    Podbevšek, Peter; Plavec, Janez

    2016-01-29

    Mutations of the KRAS proto-oncogene are associated with several tumor types, which is why it is being considered as a target for anti-cancer drug development. The human KRAS promoter contains a nuclease hypersensitive element (NHE), which can bind to nuclear proteins and is believed to form G-quadruplex structures. Previous studies showed that a 32-nt oligonucleotide (32R-3n) mimicking the KRAS NHE can reduce gene transcription by sequestering MAZ, a crucial transcription factor. Here we show that 32R-3n has to dimerize in order to fold into a G-quadruplex structure. Individual 5'- and 3'-end G-quadruplex units are formed and both feature a symmetric head-to-head topology with edge-type loops. The MAZ binding sequence is located within the 3'-end unit. Nuclear magnetic resonance data complemented by CD and UV spectra show that nucleotides of the MAZ binding G-rich motif are dynamic and could be available for sequence or structure based recognition. Both stable G-quadruplex structures could protect 5'- and 3'-ends of 32R-3n and enhance its anti-cancer activity. Single stranded genomic KRAS NHE including nucleotides flanking the 32R-3n sequence could favor a different monomeric fold, which remains unknown. PMID:26656490

  15. Targeting Mutant KRAS for Anticancer Therapeutics: A Review of Novel Small Molecule Modulators

    PubMed Central

    Wang, Yuanxiang; Kaiser, Christine E.; Frett, Brendan; Li, Hong-yu

    2015-01-01

    The RAS proteins play a role in cell differentiation, proliferation, and survival. Aberrant RAS signaling has been found to play a role in 30% of all cancers. KRAS, a key member of the RAS protein family, is an attractive cancer target, as frequent point mutations in the KRAS gene render the protein constitutively active. A number of attempts have been made to target aberrant KRAS signaling by identifying small molecule compounds that (1) are synthetic lethal to mutant KRAS, (2) block KRAS/GEF interactions, (3) inhibit downstream KRAS effectors, or (4) inhibit the post-translational processing of RAS proteins. In addition, inhibition of novel targets outside the main KRAS signaling pathway, specifically the cell cycle related kinase PLK1, has been shown have an effect in cells that harbor mutant KRAS. Herein we review the use of various high-throughput screening assays utilized to identify new small-molecule compounds capable of targeting mutant KRAS-driven cancers. PMID:23566315

  16. PIK3CA Amplification Is Common in Left Side-Tubular Adenomas but Uncommon Sessile Serrated Adenomas Exclusively with KRAS Mutation

    PubMed Central

    Lee, Hyunsu; Lee, Jae-Ho; Kim, Dae-Kwang; Choi, In-Jang; Hwang, Ilseon; Kang, Yu-Na; Kim, Shin

    2015-01-01

    Colorectal cancer is a heterogeneous disorder than arises via multiple distinct pathways, from tubular adenomas (TAs) and sessile serrated adenomas (SSAs), which are clinically, morphologically, and molecularly different. We examined PIK3CA amplification in colorectal precancerous legions, including TAs and SSAs. DNA was isolated from paired normal and tumoral tissues in 64 TAs and 32 SSAs. PIK3CA amplification, KRAS mutation, and BRAF mutation were analyzed by real-time PCR and pyrosequencing. PIK3CA amplification was found in 25% of TAs and 9.4% of SSAs, respectively. KRAS and BRAF mutations were mutually exclusive in both TAs and SSAs. In TAs, PIK3CA amplification was associated with left side and it was mutually exclusive with KRAS mutation. These results suggest that PIK3CA amplification may be early and important event in colorectal carcinogenesis and may drive the development of left-side TAs independently with KRAS mutation. PMID:26019684

  17. BRAF, PIK3CA, and HER2 Oncogenic Alterations According to KRAS Mutation Status in Advanced Colorectal Cancers with Distant Metastasis

    PubMed Central

    Koh, Jiwon; Kwak, Yoonjin; Seo, An Na; Park, Kyoung Un; Kim, Duck-Woo; Kang, Sung-Bum; Kim, Woo Ho; Lee, Hye Seung

    2016-01-01

    Background Anti-EGFR antibody–based treatment is an important therapeutic strategy for advanced colorectal cancer (CRC); despite this, several mutations—including KRAS, BRAF, and PIK3CA mutations, and HER2 amplification—are associated with the mechanisms underlying the development of resistance to anti-EGFR therapy. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC. Methods KRAS, BRAF, and PIK3CA mutations were determined by Cobas real-time polymerase chain reaction (PCR) in 191 advanced CRC patients with distant metastasis. Microsatellite instability (MSI) status was determined by a fragmentation assay and HER2 amplification was assessed by silver in situ hybridization. In addition, KRAS mutations were investigated by the Sanger sequencing method in 97 of 191 CRC cases. Results Mutations in KRAS, BRAF, and PIK3CA were found in 104 (54.5%), 6 (3.1%), and 25 (13.1%) cases of advanced CRC, respectively. MSI-high status and HER2 amplification were observed in 3 (1.6%) and 16 (8.4%) cases, respectively. PIK3CA mutations were more frequently found in KRAS mutant type (18.3%) than KRAS wild type (6.9%) (P = 0.020). In contrast, HER2 amplifications and BRAF mutations were associated with KRAS wild type with borderline significance (P = 0.052 and 0.094, respectively). In combined analyses with KRAS, BRAF and HER2 status, BRAF mutations or HER2 amplifications were associated with the worst prognosis in the wild type KRAS group (P = 0.004). When comparing the efficacy of detection methods, the results of real time PCR analysis revealed 56 of 97 (57.7%) CRC cases with KRAS mutations, whereas Sanger sequencing revealed 49 cases (50.5%). Conclusions KRAS mutations were found in 54.5% of advanced CRC patients. Our results support that subgrouping using PIK3CA and BRAF mutation or HER2 amplification status, in addition to KRAS mutation status, is helpful for managing advanced CRC patients. PMID

  18. Clinical and metabolic parameters in non-small cell lung carcinoma and colorectal cancer patients with and without KRAS mutations.

    PubMed

    Yilmaz, Ahmet; Mohamed, Nehad; Patterson, Kara A; Tang, Yan; Shilo, Konstantin; Villalona-Calero, Miguel A; Davis, Michael E; Zhou, Xiao-Ping; Frankel, Wendy; Otterson, Gregory A; Zhao, Weiqiang

    2014-09-01

    Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those

  19. Clinical Validation of KRAS, BRAF, and EGFR Mutation Detection Using Next-Generation Sequencing

    PubMed Central

    Lin, Ming-Tseh; Mosier, Stacy L.; Thiess, Michele; Beierl, Katie F.; Debeljak, Marija; Tseng, Li-Hui; Chen, Guoli; Yegnasubramanian, Srinivasan; Ho, Hao; Cope, Leslie; Wheelan, Sarah J.; Gocke, Christopher D.; Eshleman, James R.

    2015-01-01

    Objectives To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth. Methods We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA). Results We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations. Conclusions Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts. PMID:24838331

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

    PubMed Central

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

    2013-01-01

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

  1. Impact of KRAS mutation on response and outcome of patients with stage III non-squamous non-small cell lung cancer

    PubMed Central

    Yagishita, Shigehiro; Horinouchi, Hidehito; Sunami, Kuniko S; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sumi, Minako; Shiraishi, Kouya; Kohno, Takashi; Furuta, Koh; Tsuta, Koji; Tamura, Tomohide; Ohe, Yuichiro

    2015-01-01

    The frequency and clinical profile of patients with stage III non-small cell lung cancer harboring KRAS mutations have not yet been well documented. Here, we analyzed hotspot KRAS mutations using high-resolution melting analyses in tumor specimens from patients who received chemoradiotherapy between January 2001 and December 2010 at the National Cancer Center Hospital. The associations between the presence of KRAS mutations and the response rate, relapse-free survival, first relapse sites, survival post-progression and overall survival were investigated. A total of 274 non-squamous non-small cell lung cancer patients received chemoradiotherapy at our hospital. After excluding 121 patients for whom tumor specimens were not available and 34 patients with EGFR mutations, the remaining 119 patients were included in the analysis. KRAS mutations were found at a frequency of 13%. Patients with KRAS mutations had a shorter median relapse-free survival (6.1 vs 10.9 months) and a lower response rate (63% vs 81%). As for the first relapse site, patients with KRAS mutations had fewer local relapses (8% vs 23%) and more brain metastases (46% vs 12%). After disease progression, patients with KRAS mutations had a significantly shorter median survival post-progression (2.5 vs 7.3 months, P = 0.028) and median overall survival (15.1 vs 29.1 months, P = 0.022). Our results suggested that KRAS mutation could be associated with a reduced efficacy of chemoradiotherapy and a shortened survival time. PMID:26177347

  2. Impact of KRAS mutation on response and outcome of patients with stage III non-squamous non-small cell lung cancer.

    PubMed

    Yagishita, Shigehiro; Horinouchi, Hidehito; Sunami, Kuniko S; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sumi, Minako; Shiraishi, Kouya; Kohno, Takashi; Furuta, Koh; Tsuta, Koji; Tamura, Tomohide; Ohe, Yuichiro

    2015-10-01

    The frequency and clinical profile of patients with stage III non-small cell lung cancer harboring KRAS mutations have not yet been well documented. Here, we analyzed hotspot KRAS mutations using high-resolution melting analyses in tumor specimens from patients who received chemoradiotherapy between January 2001 and December 2010 at the National Cancer Center Hospital. The associations between the presence of KRAS mutations and the response rate, relapse-free survival, first relapse sites, survival post-progression and overall survival were investigated. A total of 274 non-squamous non-small cell lung cancer patients received chemoradiotherapy at our hospital. After excluding 121 patients for whom tumor specimens were not available and 34 patients with EGFR mutations, the remaining 119 patients were included in the analysis. KRAS mutations were found at a frequency of 13%. Patients with KRAS mutations had a shorter median relapse-free survival (6.1 vs 10.9 months) and a lower response rate (63% vs 81%). As for the first relapse site, patients with KRAS mutations had fewer local relapses (8% vs 23%) and more brain metastases (46% vs 12%). After disease progression, patients with KRAS mutations had a significantly shorter median survival post-progression (2.5 vs 7.3 months, P = 0.028) and median overall survival (15.1 vs 29.1 months, P = 0.022). Our results suggested that KRAS mutation could be associated with a reduced efficacy of chemoradiotherapy and a shortened survival time. PMID:26177347

  3. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-01

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

  4. Dovitinib (TKI258), a multi-target angiokinase inhibitor, is effective regardless of KRAS or BRAF mutation status in colorectal cancer

    PubMed Central

    Lee, Choong-Kun; Lee, Myung Eun; Lee, Won Suk; Kim, Jeong Min; Park, Kyu Hyun; Kim, Tae Soo; Lee, Kang Young; Ahn, Joong Bae; Chung, Hyun Cheol; Rha, Sun Young

    2015-01-01

    Introduction: We aimed to determine whether KRAS and BRAF mutant colorectal cancer (CRC) cells exhibit distinct sensitivities to the multi-target angiokinase inhibitor, TKI258 (dovitinib). Materials and methods: We screened 10 CRC cell lines by using receptor tyrosine kinase (RTK) array to identify activated RTKs. MTT assays, anchorage-independent colony-formation assays, and immunoblotting assays were performed to evaluate the in vitro anti-tumor effects of TKI258. In vivo efficacy study followed by pharmacodynamic evaluation was done. Results: Fibroblast Growth Factor Receptor 1 (FGFR1) and FGFR3 were among the most highly activated RTKs in CRC cell lines. In in vitro assays, the BRAF mutant HT-29 cells were more resistant to the TKI258 than the KRAS mutant LoVo cells. However, in xenograft assays, TKI258 equally delayed the growth of tumors induced by both cell lines. TUNEL assays showed that the apoptotic index was unchanged following TKI258 treatment, but staining for Ki-67 and CD31 was substantially reduced in both xenografts, implying an anti-angiogenic effect of the drug. TKI258 treatment was effective in delaying CRC tumor growth in vivo regardless of the KRAS and BRAF mutation status. Conclusions: Our results identify FGFRs as potential targets in CRC treatment and suggest that combined targeting of multiple RTKs with TKI258 might serve as a novel approach to improve outcome of patients with CRC. PMID:25628921

  5. Impact of KRAS, BRAF, PIK3CA, TP53 status and intraindividual mutation heterogeneity on outcome after liver resection for colorectal cancer metastases.

    PubMed

    Løes, Inger Marie; Immervoll, Heike; Sorbye, Halfdan; Angelsen, Jon-Helge; Horn, Arild; Knappskog, Stian; Lønning, Per Eystein

    2016-08-01

    We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p < 0.001, 0.002 and 0.023, respectively). KRAS and BRAF mutations also predicted a reduced median disease-specific survival (DSS) (29 vs. 51 and 16 vs. 49 months; p <0.001 and 0.008, respectively). No effect of TP53 (60.4%) mutation status was observed. Postoperative, but not preoperative chemotherapy improved both TTR and DSS (p < 0.001 for both) with no interaction with gene mutation status. Among 94 patients harboring two or more metastatic deposits, 13 revealed mutation heterogeneity across metastatic deposits for at least one gene. Mutation heterogeneity predicted reduced median DSS compared to homogeneous mutations (18 vs. 37 months; p = 0.011 for all genes; 16 vs. 26 months; p < 0.001 analyzing BRAF or KRAS mutations separately). In multivariate analyses, KRAS or BRAF mutations consistently predicted poor TRR and DSS. Mutation heterogeneity robustly predicted DSS but not TTR, while postoperative chemotherapy improved both TTR and DSS. Our findings indicate that BRAF and KRAS mutations as well as mutation heterogeneity predict poor outcome in CRC patients subsequent to liver resections and might help guide treatment decisions. PMID:26991344

  6. Mutant Kras Promotes Hyperplasia and Alters Differentiation in the Colon Epithelium But Does Not Expand the Presumptive Stem Cell Pool

    PubMed Central

    Feng, Ying; Bommer, Guido T.; Zhao, Jenny; Green, Maranne; Sands, Evan; Zhai, Yali; Brown, Kelly; Burberry, Aaron; Cho, Kathleen R.; Fearon, Eric R.

    2011-01-01

    BACKGROUND & AIMS Adenomatous polyps are precursors to colorectal cancer (CRC), whereas hyperplastic polyps (HPPs) have a small risk of progression to CRC. Mutations in KRAS are found in ~40% of CRCs and large adenomas and a subset of HPPs. We investigated the reasons that HPPs with KRAS mutations lack malignant potential; we compared the effects of Kras/KRAS activation to those of Adenomatous polyposis coli (Apc)/APC inactivation, which promotes adenoma formation. METHODS We activated a KrasG12D mutant allele or inactivated Apc alleles in mouse colon epithelium and analyzed phenotypes and expression of selected genes and proteins. The mouse data were validated using samples of human HPPs and adenomas. Signaling pathways and factors that contribute to Kras/KRAS-induced phenotypes were studied in intestinal epithelial cells. RESULTS Activation of Kras led to hyperplasia and serrated crypt architecture akin to that observed in human HPPs. We also observed loss of Paneth cells and increases in goblet cell numbers. Abnormalities in Kras-mediated differentiation and proliferation required mitogen-activated protein kinase (MAPK) signaling and were linked to activation of the Hes1 transcription factor. Human HPPs also had activation of HES1. In contrast to Apc/APC inactivation, Kras/KRAS activation did not increase expression of crypt stem cell markers in colon epithelium or colony formation in vitro. Kras/KRAS activation was not associated with substantial induction of p16INK4a protein expression in mouse colon epithelium or human HPPs. CONCLUSIONS Although Kras/KRAS mutation promotes serrated and hyperplastic morphological features in colon epithelium, it is not able to initiate adenoma development, perhaps in part because activated Kras/KRAS signaling does not increase the number of presumptive stem cells in affected crypts. PMID:21699772

  7. KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia

    PubMed Central

    Malinowska-Ozdowy, K; Frech, C; Schönegger, A; Eckert, C; Cazzaniga, G; Stanulla, M; zur Stadt, U; Mecklenbräuker, A; Schuster, M; Kneidinger, D; von Stackelberg, A; Locatelli, F; Schrappe, M; Horstmann, M A; Attarbaschi, A; Bock, C; Mann, G; Haas, O A; Panzer-Grümayer, R

    2015-01-01

    High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18–30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone. PMID:25917266

  8. KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia.

    PubMed

    Malinowska-Ozdowy, K; Frech, C; Schönegger, A; Eckert, C; Cazzaniga, G; Stanulla, M; zur Stadt, U; Mecklenbräuker, A; Schuster, M; Kneidinger, D; von Stackelberg, A; Locatelli, F; Schrappe, M; Horstmann, M A; Attarbaschi, A; Bock, C; Mann, G; Haas, O A; Panzer-Grümayer, R

    2015-08-01

    High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone. PMID:25917266

  9. Combined point mutation in KRAS or EGFR genes and EML4-ALK translocation in lung cancer patients.

    PubMed

    Jürgens, Jessica; Engel-Riedel, Walburga; Prickartz, Alexander; Ludwig, Corinna; Schildgen, Oliver; Tillmann, Ramona-Liza; Stoelben, Erich; Brockmann, Michael; Schildgen, Verena

    2014-03-01

    A total of three cases with novel constellations regarding mutation patterns in non-small-cell lung cancer (NSCLC) are reported. The mutation patterns that are observed are novel and unexpected. First, a combined simultaneous KRAS mutation and EML4-ALK translocation, both in the main tumor and a bone metastasis, were observed, these mutations are assumed to mutually exclude each other. A further two cases include a father and a daughter, both of whom are suffering from NSCLC with different EGFR mutation patterns. A common cause was assumed; however, could not be deduced to mutations in the KRAS, BRAF and EGFR genes. The aforementioned cases are important, as it must be taken into account that mutations previously assumed to be exclusive can occur in combination, may influence the clinical outcome and may require different therapy compared with single mutated tumors. It has to be discussed whether diagnostic algorithms need to be adapted. The cases of father and daughter show that further unknown factors can influence development of NSCLC. PMID:24754584

  10. MGMT promoter hypermethylation and K-RAS, PTEN and TP53 mutations in tamoxifen-exposed and non-exposed endometrial cancer cases

    PubMed Central

    Nagy, E; Gajjar, K B; Patel, I I; Taylor, S; Martin-Hirsch, P L; Stringfellow, H F; Martin, F L; Phillips, D H

    2014-01-01

    Background: Tamoxifen has anti-oestrogenic and anti-tumour activity in the breast, but is oestrogenic and carcinogenic in the endometrium. It can induce experimental tumours by both hormonal and DNA-damaging mechanisms, but its carcinogenic mode of action in human endometrium remains unclear. Methods: We investigated whether an epigenetic mechanism, involving promoter hypermethylation of the gene for the DNA repair enzyme MGMT (O6-methylguanine DNA methyltransferase), was associated with K-RAS, TP53 and PTEN mutations in endometrial tumours from women treated with tamoxifen (TAM, n=30) or unexposed to the drug (EC, n=38). Results: There were significant (P<0.05) differences in tumour grade between the TAM and EC groups, with more favourable morphology in the latter. K-RAS mutations, predominantly G>A, occurred in small numbers in both groups. TP53 mutations were of mainly A>G, C>T and indel modifications in both groups, but more frequent in TAM cases. PTEN mutations dominated in EC tumours and were of the type that has large impact on protein function, such as indel or nonsense mutations. These observations alongside the mutational spectrum in PTEN suggest that the malignancies arise from different backgrounds, hence pointing to an effect of tamoxifen. Both groups displayed MGMT promoter hypermethylation. This coincided with mutations more frequently in the TAM (78%) than in the EC (50%) group, even though there were significantly (P<0.05) fewer mutations and methylations in TAM cases. Conclusions: Although the difference in coincidence did not reach significance with the current sample size, the findings suggest that epigenetic processes may play a role in the way tamoxifen induces endometrial cancer. PMID:24853176

  11. K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

    PubMed

    Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin

    2016-07-01

    K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p < 0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p < 0.01) and 3.16 (95 % CI 2.1-4.71; p < 0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p < 0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples. PMID:27225938

  12. Effects of RAL signal transduction in KRAS- and BRAF-mutated cells and prognostic potential of the RAL signature in colorectal cancer.

    PubMed

    Győrffy, Balázs; Stelniec-Klotz, Iwona; Sigler, Christian; Kasack, Katharina; Redmer, Torben; Qian, Yu; Schäfer, Reinhold

    2015-05-30

    Our understanding of oncogenic signaling pathways has strongly fostered current concepts for targeted therapies in metastatic colorectal cancer. The RALA pathway is novel candidate due to its independent role in controlling expression of genes downstream of RAS.We compared RALA GTPase activities in three colorectal cancer cell lines by GTPase pull-down assay and analyzed the transcriptional and phenotypic effects of transient RALA silencing. Knocking-down RALA expression strongly diminished the active GTP-bound form of the protein. Proliferation of KRAS mutated cell lines was significantly reduced, while BRAF mutated cells were mostly unaffected. By microarray analysis we identified common genes showing altered expression upon RALA silencing in all cell lines. None of these genes were affected when the RAF/MAPK or PI3K pathways were blocked.To investigate the potential clinical relevance of the RALA pathway and its associated transcriptome, we performed a meta-analysis interrogating progression-free survival of colorectal cancer patients of five independent data sets using Cox regression. In each dataset, the RALA-responsive signature correlated with worse outcome.In summary, we uncovered the impact of the RAL signal transduction on genetic program and growth control in KRAS- and BRAF-mutated colorectal cells and demonstrated prognostic potential of the pathway-responsive gene signature in cancer patients. PMID:26033452

  13. Synergistic effects of sorafenib in combination with gemcitabine or pemetrexed in lung cancer cell lines with K-ras mutations

    PubMed Central

    Wang, Shu; Su, Zeng-feng; Yuan, Yuan

    2016-01-01

    K-ras is currently accepted as the most frequently mutated oncogene in non-small cell lung cancer (NSCLC, including squamous carcinoma, adenocarcinoma, and large cell carcinoma). NSCLC patients with the K-ras mutation appear to be refractory to the majority of systemic therapies. In the present study, the in vitro antitumor effects and correlated molecular mechanisms of sorafenib combined with gemcitabine or pemetrexed were explored in the K-ras mutation-positive NSCLC A549 cell line. Sorafenib was seen to exhibit dose-dependent growth inhibition in the A549 cells, while sorafenib combined with pemetrexed demonstrated a greater synergism compared with sorafenib combined with gemcitabine. Sorafenib arrested the cell cycle at the G1 phase, while gemcitabine and pemetrexed caused arrest at the S phase. The molecular mechanism of this synergism was due to the downstream signalling pathways, which were efficiently suppressed by sorafenib, therefore increasing the incidence of the entry of the chemotherapeutic drugs into the apoptotic pathways. Moreover, sorafenib and pemetrexed demonstrated stronger synergism, demonstrating that inhibiting the Ras/Raf/Mek/Erk and Ras/PI3K/Akt pathways concurrently may achieve improved antitumor effects. PMID:27095937

  14. Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy.

    PubMed

    Herling, Carmen Diana; Klaumünzer, Marion; Rocha, Cristiano Krings; Altmüller, Janine; Thiele, Holger; Bahlo, Jasmin; Kluth, Sandra; Crispatzu, Giuliano; Herling, Marco; Schiller, Joanna; Engelke, Anja; Tausch, Eugen; Döhner, Hartmut; Fischer, Kirsten; Goede, Valentin; Nürnberg, Peter; Reinhardt, Hans Christian; Stilgenbauer, Stephan; Hallek, Michael; Kreuzer, Karl-Anton

    2016-07-21

    Genetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment. PMID:27226433

  15. Somatic c.34G>T KRAS mutation: a new prescreening test for MUTYH-associated polyposis?

    PubMed

    Aimé, Adeline; Coulet, Florence; Lefevre, Jeremie H; Colas, Chrystelle; Cervera, Pascale; Flejou, Jean-François; Lascols, Olivier; Soubrier, Florent; Parc, Yann

    2015-01-01

    We investigated the somatic c.34G>T KRAS transversion as a marker suggestive of MUTYH-associated polyposis (MAP). We compared 86 adenomas and 19 colorectal cancers (CRCs) of 30 MAP patients to 135 adenomas and five CRCs of 47 familial adenomatous polyposis (FAP) patients. The c.34G>T mutation was investigated by DNA sequencing. Secondly, the germline MUTYH gene sequence was analyzed in patients carrying c.34G>T in CRCs diagnosed between 2008 and 2012. The c.34G>T was present in 39.7% of MAP adenomas versus 1.6% of FAP adenomas (P < 0.01). Sensitivity and specificity for detecting MAP were 39.7% and 98%, respectively. Sensitivity increased with the number of adenomas tested (P = 0.039). KRAS exon 2 analysis was performed on 2239 CRC and 2.2% harbored the c.34G>T transversion. Among 28 carriers of the c.34G>T mutation, biallelic MUTYH mutations were detected in seven patients (25%). One patient did not have any polyp or family history and did not fulfill criteria for MUTYH testing. With high specificity, the c.34G>T mutation seems to be a useful and promising test for MAP. For polyposis, it may guide genetic testing toward APC or MUTYH. If routinely performed in CRC patients, it could help to diagnose MUTYH-mutation carriers, even when they don't fulfill genetic testing criteria. PMID:26056087

  16. Cutaneous metastasis as an initial presentation of lung adenocarcinoma with KRAS mutation: a case report and literature review

    PubMed Central

    Wu, Shenhong; Karbowitz, Stephen R.; Morgenstern, Nora; Rose, David R.

    2014-01-01

    Cutaneous metastasis as an initial presentation occurs in 0.8% of patients with internal malignancies, and is poorly understood in its molecular pathogenesis. We reported a case in which a 61-year-old male patient initially presented with rapidly growing skin nodule on his left chest wall, then developed dyspnea and loss of weight. Echocardiogram showed a large pericardial effusion with right ventricular collapse. PET/CT revealed moderate pleural effusion and multiple lymphadenopathies with hypermetabolic concentration of radiotracer in the lymph nodes as well as in the chest wall skin mass. Biopsy of the skin mass and pericardial/pleural fluids revealed metastatic adenocarcinoma consistent with lung primary with KRAS mutation. Palliative chemotherapy was administered without resulting in any improvement. This is the first case report to show that KRAS-mutant lung adenocarcinoma can be associated with cutaneous metastasis.

  17. Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity

    PubMed Central

    Cuddington, Breanne P.

    2014-01-01

    identified bovine herpesvirus 1 as a novel oncolytic virus with many unique and clinically relevant features. Here, we show that BHV-1 can target a wide range of human cancer types, most potently lung cancer. In addition, we show that enhanced KRAS activity, a hallmark of many cancers, is one of the factors that increases BHV-1 oncolytic capacity. These findings hold potential for future treatments, particularly in the context of lung cancer, where KRAS mutations are a negative predictor of treatment efficacy. PMID:24696490

  18. Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing.

    PubMed

    Stranska, Jana; Jancik, Sylwia; Slavkovsky, Rastislav; Holinkova, Veronika; Rabcanova, Miroslava; Vojta, Petr; Hajduch, Marian; Drabek, Jiri

    2015-03-01

    Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification. PMID:25655305

  19. ALK-rearranged adenocarcinoma with extensive mucin production can mimic mucinous adenocarcinoma: clinicopathological analysis and comprehensive histological comparison with KRAS-mutated mucinous adenocarcinoma.

    PubMed

    Cha, Yoon Jin; Han, Joungho; Hwang, Soo Hyun; Lee, Tae Bum; Kim, Hojoong; Zo, Jea Ill

    2016-06-01

    We aimed to investigate clinicopathological features and histology of ALK-rearranged adenocarcinomas with extensive mucin production (AEM) that mimic mucinous adenocarcinoma (MA). Retrospectively, 12 cases of AEM and 25 cases of MA harbouring KRAS mutation were retrieved. The clinicopathological profile and detailed histological features were analysed and compared based on the ALK and KRAS status. AEMs occurred in younger patients (p = 0.044) and were characterised by floating tubulopapillary pattern (p < 0.001), prominent nucleolus (p < 0.001), and apical cytoplasmic snouts (p < 0.001). In contrast, KRAS-mutated MAs lacked ALK-specific histological patterns (p < 0.05). Instead, tumour-infiltrating leukocytes (p = 0.018) and smooth cytoplasmic borders (p < 0.001) with vesicular nuclei (p = 0.004) were prominent in KRAS-mutated MAs. AEMs demonstrated characteristic tubulopapillary pattern and apical snouts, which were distinguishing features from MAs with KRAS mutation. Apical snouts can be a useful histological surrogate for ALK rearrangement in the pulmonary adenocarcinomas showing extensive mucin that mimic MA. PMID:27114375

  20. Higher metastatic efficiency of KRas G12V than KRas G13D in a colorectal cancer model.

    PubMed

    Alamo, Patricia; Gallardo, Alberto; Di Nicolantonio, Federica; Pavón, Miguel Angel; Casanova, Isolda; Trias, Manuel; Mangues, María Antonia; Lopez-Pousa, Antonio; Villaverde, Antonio; Vázquez, Esther; Bardelli, Alberto; Céspedes, María Virtudes; Mangues, Ramón

    2015-02-01

    Although all KRas (protein that in humans is encoded by the KRas gene) point mutants are considered to have a similar prognostic capacity, their transformation and tumorigenic capacities vary widely. We compared the metastatic efficiency of KRas G12V (Kirsten rat sarcoma viral oncogene homolog with valine mutation at codon 12) and KRas G13D (Kirsten rat sarcoma viral oncogene homolog with aspartic mutation at codon 13) oncogenes in an orthotopic colorectal cancer (CRC) model. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild-type (Kras WT)] expressing the KRas G12V or KRas G13D allele were microinjected in the mouse cecum. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Microscopic, macroscopic, and visible lymphatic foci were 1.5- to 3.0-fold larger in KRas G12V than in KRas G13D mice (P < 0.05). In the lung, only microfoci were developed in both groups. KRas G12V primary tumors had lower apoptosis (7.0 ± 1.2 vs. 7.4 ± 1.0 per field, P = 0.02), higher tumor budding at the invasion front (1.2 ± 0.2 vs. 0.6 ± 0.1, P = 0.04), and a higher percentage of C-X-C chemokine receptor type 4 (CXCR4)-overexpressing intravasated tumor emboli (49.8 ± 9.4% vs. 12.8 ± 4.4%, P < 0.001) than KRas G13D tumors. KRas G12V primary tumors showed Akt activation, and β5 integrin, vascular endothelial growth factor A (VEGFA), and Serpine-1 overexpression, whereas KRas G13D tumors showed integrin β1 and angiopoietin 2 (Angpt2) overexpression. The increased cell survival, invasion, intravasation, and specific molecular regulation observed in KRas G12V tumors is consistent with the higher aggressiveness observed in patients with CRC expressing this oncogene. PMID:25359494

  1. Immunoglobulin G fragment C receptor polymorphisms and KRAS mutations: are they useful biomarkers of clinical outcome in advanced colorectal cancer treated with anti-EGFR-based therapy?

    PubMed

    Paez, David; Paré, Laia; Espinosa, Iñigo; Salazar, Juliana; del Rio, Elisabeth; Barnadas, Agustí; Marcuello, Eugenio; Baiget, Montserrat

    2010-09-01

    KRAS mutations have been identified as a strong predictor of resistance to anti-epidermal growth factor receptor (EGFR) therapies. Besides inhibiting the EGFR pathway, anti-EGFR monoclonal antibodies may exert antitumor effects through antibody-dependent cell-mediated cytotoxicity (ADCC). Through this mechanism, the antibody fragment C portion (Fcγ) interacts with Fc receptors (FcγRs) expressed by immune effectors cells. We investigated the association of FcγR polymorphisms and KRAS mutation with the clinical outcome of 104 refractory metastatic colorectal cancer (mCRC) patients treated with anti-EGFR antibodies. FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms were analyzed in genomic DNA using a 48.48 dynamic array on the BioMark system (Fluidigm, South Sanfrancisco, CA, USA). Tumor tissues from 96 cases were screened for KRAS mutations. KRAS mutation was associated with a lower response rate (RR) (P = 0.035) and a shorter progression-free survival (PFS) (3 vs 7 months; P = 0.36). FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms did not show statistically significant associations with response, PFS, or KRAS status. In the logistic regression analysis, KRAS status (P = 0.04) and skin toxicity (P = 0.03) were associated with RR. By multivariate analysis, the clinical risk classification (P = 0.006) and skin toxicity (P < 0.0001) were found to be independent risk factors for PFS. In conclusion, the FcγRIIa and FcγRIIIa polymorphisms are not useful as molecular markers for clinical outcome in mCRC patients. To date, the EORTC (European Organization for Research and Treatment of Cancer Classification), skin toxicity, and KRAS status are the only reliable biomarkers to identify patients that would benefit from anti-EGFR therapy. PMID:20550522

  2. KRAS mutant NSCLC, a new opportunity for the synthetic lethality therapeutic approach

    PubMed Central

    Belda-Iniesta, Cristóbal

    2013-01-01

    K-RAS accounts for 90% of RAS mutations in lung adenocarcinomas, the most commonly mutated oncogene in NSCLC, with mutations detected in about 25% of all tumors. Direct inhibition of KRAS has proven clinically challenging. So far, no successful targeted therapy has been developed and remains an elusive target for cancer therapy. Despite significant efforts, currently there are no drugs directly targeting mutated KRAS. Thus, new strategies have emerged for targeting RAS including the use of synthetic lethality. A specific knowledge of individual tumor molecular abnormalities that result in oncogene-specific “synthetic lethal” interactions will allow the rationale to combine promising targeted therapies for KRAS-mutated NSCLC. In this article, we review the new approach based on testing drugs or combinations of agents that work downstream of activated K-RAS. PMID:25806225

  3. KRAS mutant NSCLC, a new opportunity for the synthetic lethality therapeutic approach.

    PubMed

    de Castro Carpeño, Javier; Belda-Iniesta, Cristóbal

    2013-04-01

    K-RAS accounts for 90% of RAS mutations in lung adenocarcinomas, the most commonly mutated oncogene in NSCLC, with mutations detected in about 25% of all tumors. Direct inhibition of KRAS has proven clinically challenging. So far, no successful targeted therapy has been developed and remains an elusive target for cancer therapy. Despite significant efforts, currently there are no drugs directly targeting mutated KRAS. Thus, new strategies have emerged for targeting RAS including the use of synthetic lethality. A specific knowledge of individual tumor molecular abnormalities that result in oncogene-specific "synthetic lethal" interactions will allow the rationale to combine promising targeted therapies for KRAS-mutated NSCLC. In this article, we review the new approach based on testing drugs or combinations of agents that work downstream of activated K-RAS. PMID:25806225

  4. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis; the role of p53 and p21

    PubMed Central

    Escandell, José M.; Kaler, Pawan; Recio, M. Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija

    2008-01-01

    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke3 cells. However, the presence of oncogenic k-Ras significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransfromed intestinal epithelial cells with inducible expression of k-RasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  5. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21.

    PubMed

    Escandell, José M; Kaler, Pawan; Recio, M Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija

    2008-07-15

    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  6. Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma

    PubMed Central

    Park, Jangchul; Kobayashi, Yoshihisa; Urayama, Kevin Y.; Yamaura, Hidekazu; Yatabe, Yasushi; Hida, Toyoaki

    2016-01-01

    This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB–IV) and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups’ characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159), KRAS mutations (n = 55), or ALK rearrangements (n = 51). Among the three groups, we evaluated only patients with stage IIIB–IV lung adenocarcinoma who had EGFR mutations (n = 126), KRAS mutations (n = 35), or ALK rearrangements (n = 47). We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009). Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003). Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively). Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049). Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively). Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively). In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

  7. Comparison of a PNA clamp PCR and an ARMS/Scorpion PCR assay for the detection of K-ras mutations.

    PubMed

    Nordgård, Oddmund; Oltedal, Satu; Janssen, Emiel A M; Gilje, Bjørnar; Kørner, Hartwig; Tjensvoll, Kjersti; Smaaland, Rune

    2012-03-01

    Point mutations in the K-ras gene have been shown to confer resistance against epidermal growth factor receptor-directed therapy of metastatic colorectal cancer. Accordingly, K-ras mutation testing has become mandatory in hospitals offering such treatment. We compared the performance and reagent costs of 2 sensitive methods for detection of K-ras mutations: a peptide nucleic acid (PNA) clamp polymerase chain reaction (PCR) assay and a commercially available amplification refractory mutation system/Scorpion (ARMS/S) PCR assay. Both methods were applied in parallel to 101 formalin-fixed, paraffin-embedded tumor and metastasis samples from patients with colon cancer. The PNA clamp PCR assay detected K-ras mutations in 35% (35 of 101) of the samples, whereas the ARMS/S PCR assay detected mutations in 27% (27 of 101) of them. There was 92% (93 of 101) concordance between the 2 methods and the κ coefficient for the comparison was 0.82. The 8 discordant cases were exclusively positive by PNA clamp PCR. Finally, the reagent costs of the PNA clamp PCR assay were estimated to be at least 20 times lower than the ARMS/S assay. We concluded that the high performance and low costs associated with the PNA clamp PCR assay encourage its use in the administration of personalized epidermal growth factor receptor-directed therapy. PMID:22306670

  8. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

    PubMed Central

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID

  9. Higher quality of molecular testing, an unfulfilled priority: Results from external quality assessment for KRAS mutation testing in colorectal cancer.

    PubMed

    Tembuyser, Lien; Ligtenberg, Marjolijn J L; Normanno, Nicola; Delen, Sofie; van Krieken, J Han; Dequeker, Elisabeth M C

    2014-05-01

    Precision medicine is now a key element in clinical oncology. RAS mutational status is a crucial predictor of responsiveness to anti-epidermal growth factor receptor agents in metastatic colorectal cancer. In an effort to guarantee high-quality testing services in molecular pathology, the European Society of Pathology has been organizing an annual KRAS external quality assessment program since 2009. In 2012, 10 formalin-fixed, paraffin-embedded samples, of which 8 from invasive metastatic colorectal cancer tissue and 2 artificial samples of cell line material, were sent to more than 100 laboratories from 26 countries with a request for routine KRAS testing. Both genotyping and clinical reports were assessed independently. Twenty-seven percent of the participants genotyped at least 1 of 10 samples incorrectly. In total, less than 5% of the distributed specimens were genotyped incorrectly. Genotyping errors consisted of false negatives, false positives, and incorrectly genotyped mutations. Twenty percent of the laboratories reported a technical error for one or more samples. A review of the written reports showed that several essential elements were missing, most notably a clinical interpretation of the test result, the method sensitivity, and the use of a reference sequence. External quality assessment serves as a valuable educational tool in assessing and improving molecular testing quality and is an important asset for monitoring quality assurance upon incorporation of new biomarkers in diagnostic services. PMID:24631467

  10. Capturing the metabolomic diversity of KRAS mutants in non-small-cell lung cancer cells

    PubMed Central

    Marabese, Mirko; Broggini, Massimo; Pastorelli, Roberta

    2014-01-01

    In non-small-cell lung cancer (NSCLC), one-fifth of patients have KRAS mutations, which are considered a negative predictive factor to first-line therapy. Evidence is emerging that not all KRAS mutations have the same biological activities and possible remodeling of cell metabolism by KRAS activation might complicate the scenario. An open question is whether different KRAS mutations at codon-12 affect cellular metabolism differently with possible implications for different responses to cancer treatments. We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 (G12C, G12D, G12V) and the wild-type. The glutamine deprivation assay and real-time PCR were used to confirm the involvement of some of the metabolic pathways highlighted. Cell clones indicated distinct metabolomic profiles in KRAS wild-type and mutants. Clones harboring different KRAS mutations at codon-12 also had different metabolic remodeling, such as a different redox buffering system and different glutamine-dependency not driven by the transcriptional state of enzymes involved in glutaminolysis. These findings indicate that KRAS mutations at codon-12 are associated with different metabolomic profiles that might affect the responses to cancer treatments. PMID:24952473

  11. Oncogenic KRAS activates an embryonic stem cell-like program in human colon cancer initiation

    PubMed Central

    Le Rolle, Anne-France; Chiu, Thang K.; Zeng, Zhaoshi; Shia, Jinru; Weiser, Martin R.; Paty, Philip B.; Chiu, Vi K.

    2016-01-01

    Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer. PMID:26744320

  12. Analysis of p53, K-ras gene mutation & Helicobacter pylori infection in patients with gastric cancer & peptic ulcer disease at a tertiary care hospital in north India

    PubMed Central

    Saxena, Ashish; Shukla, Sanket Kumar; Prasad, Kashi Nath; Ghoshal, Uday Chand

    2012-01-01

    Background & objectives: Mutations in the oncogene and tumour suppressor genes play an important role in carcinogenesis. We investigated the association of p53 and K-ras gene mutation and Helicobacter pylori infection in patients with gastric cancer (GC) and peptic ulcer disease (PUD) attending a tertiary care hospital in north India. Methods: In total, 348 adult patients [62 GC, 45 PUD and 241 non-ulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR. Mutation in the exon 5-8 of p53 gene was analyzed by PCR-single stranded conformational polymorphism (SSCP) and confirmed by sequence analysis. K-ras gene codon 12 mutation was analyzed by PCR-based restriction fragment length polymorphism. Results: Overall p53 gene mutation was found in 4.6 per cent of the study population, and its distribution in GC, PUD and NUD was 21, 4.4 and 0.4 per cent, respectively. p53 gene mutation was significantly higher in patients with GC than PUD (P<0.05) and NUD (P<0.001). No difference in p53 gene mutation was observed between H. pylori infected and non-infected individuals. K-ras gene mutation was absent in all the patients. Interpretation & conclusions: Our results show that p53 gene mutation may be associated with gastric carcinogenesis independent to H. pylori infection and absence of K-ras gene mutation questions its role in the pathogenesis of GC and PUD in Indian patients. PMID:23168708

  13. IRS2 copy number gain, KRAS and BRAF mutation status as predictive biomarkers for response to the IGF-1R/IR inhibitor BMS-754807 in colorectal cancer cell lines.

    PubMed

    Huang, Fei; Chang, Han; Greer, Ann; Hillerman, Stephen; Reeves, Karen A; Hurlburt, Warren; Cogswell, John; Patel, Dharmesh; Qi, Zhenhao; Fairchild, Craig; Ryseck, Rolf-Peter; Wong, Tai W; Finckenstein, Friedrich G; Jackson, Jeffrey; Carboni, Joan M

    2015-02-01

    Insulin-like growth factor receptor 1 (IGF-1R)-targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAF(V600E) or KRAS(G13D) mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit. PMID:25527633

  14. KRAS Mouse Models

    PubMed Central

    O’Hagan, Rónán C.; Heyer, Joerg

    2011-01-01

    KRAS is a potent oncogene and is mutated in about 30% of all human cancers. However, the biological context of KRAS-dependent oncogenesis is poorly understood. Genetically engineered mouse models of cancer provide invaluable tools to study the oncogenic process, and insights from KRAS-driven models have significantly increased our understanding of the genetic, cellular, and tissue contexts in which KRAS is competent for oncogenesis. Moreover, variation among tumors arising in mouse models can provide insight into the mechanisms underlying response or resistance to therapy in KRAS-dependent cancers. Hence, it is essential that models of KRAS-driven cancers accurately reflect the genetics of human tumors and recapitulate the complex tumor-stromal intercommunication that is manifest in human cancers. Here, we highlight the progress made in modeling KRAS-dependent cancers and the impact that these models have had on our understanding of cancer biology. In particular, the development of models that recapitulate the complex biology of human cancers enables translational insights into mechanisms of therapeutic intervention in KRAS-dependent cancers. PMID:21779503

  15. Outcomes by Tumor Histology and KRAS Mutation Status After Lung Stereotactic Body Radiation Therapy for Early-Stage Non–Small-Cell Lung Cancer

    PubMed Central

    Mak, Raymond H.; Hermann, Gretchen; Lewis, John H.; Aerts, Hugo J.W.L.; Baldini, Elizabeth H.; Chen, Aileen B.; Colson, Yolonda L.; Hacker, Fred H.; Kozono, David; Wee, Jon O.; Chen, Yu-Hui; Catalano, Paul J.; Wong, Kwok-Kin; Sher, David J.

    2015-01-01

    We analyzed outcomes after lung stereotactic body radiotherapy (SBRT) for early-stage non–small-cell lung carcinoma in patients by histology and KRAS mutation status. Histology was not associated with outcomes, but KRAS mutation was associated with lower freedom from recurrence on univariable analysis and decreased cancer-specific survival on multivariable analysis. Given the small sample sizes, these results are hypothesis generating, and further study of SBRT outcomes by tumor genotype in larger data sets is needed. Background We analyzed outcomes after lung stereotactic body radiotherapy (SBRT) for early-stage non–small cell lung-carcinoma (NSCLC) by histology and KRAS genotype. Patients and Methods We included 75 patients with 79 peripheral tumors treated with SBRT (18 Gy × 3 or 10 to 12 Gy × 5) at our institution from 2009 to 2012. Genotyping for KRAS mutations was performed in 10 patients. Outcomes were analyzed by the Kaplan-Meier method/Cox regression, or cumulative incidence method/Fine-Gray analysis. Results The median patient age was 74 (range, 46 to 93) years, and Eastern Cooperative Oncology Group performance status was 0 to 1 in 63%. Tumor histology included adenocarcinoma (44%), squamous cell carcinoma (25%), and NSCLC (18%). Most tumors were T1a (54%). Seven patients had KRAS-mutant tumors (9%). With a median follow-up of 18.8 months among survivors, the 1-year estimate of overall survival was 88%, cancer-specific survival (CSS) 92%, primary tumor control 94%, and freedom from recurrence (FFR) 67%. In patients with KRAS-mutant tumors, there was a significantly lower tumor control (67% vs. 96%; P = .04), FFR (48% vs. 69%; P = .03), and CSS (75% vs. 93%; P = .05). On multivariable analysis, histology was not associated with outcomes, but KRAS mutation (hazard ratio, 10.3; 95% confidence interval, 2.3–45.6; P = .0022) was associated with decreased CSS after adjusting for age. Conclusion In this SBRT series, histology was not associated with

  16. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

    PubMed

    Cai, Xin; Sheng, Jianhui; Tang, Chuanning; Nandakumar, Vijayalakshmi; Ye, Hua; Ji, Hong; Tang, Haiying; Qin, Yu; Guan, Hongwei; Lou, Feng; Zhang, Dandan; Sun, Hong; Dong, Haichao; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Yan, He; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Wu, Taihua; Lin, Hongli

    2014-01-01

    Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations. PMID:24760004

  17. Frequent Mutations in EGFR, KRAS and TP53 Genes in Human Lung Cancer Tumors Detected by Ion Torrent DNA Sequencing

    PubMed Central

    Cai, Xin; Sheng, Jianhui; Tang, Chuanning; Nandakumar, Vijayalakshmi; Ye, Hua; Ji, Hong; Tang, Haiying; Qin, Yu; Guan, Hongwei; Lou, Feng; Zhang, Dandan; Sun, Hong; Dong, Haichao; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Yan, He; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Jones, Lindsey; Huang, Xue F.; Chen, Si-Yi; Wu, Taihua; Lin, Hongli

    2014-01-01

    Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations. PMID:24760004

  18. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. PMID:26461287

  19. Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to pro-apoptotic agents.

    PubMed

    Klingbeil, Olaf; Lesche, Ralf; Gelato, Kathy A; Haendler, Bernard; Lejeune, Pascale

    2016-01-01

    Non-small cell lung cancer (NSCLC) has the highest incidence of cancer-related death worldwide and a high medical need for more effective therapies. Small-molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1, I-BET762 and OTX-015 are active in a wide range of different cancer types, including lung cancer. Although their activity on oncogene expression such as c-Myc has been addressed in many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. Here we evaluated the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptosis response. Using a panel of 12 KRAS-mutated NSCLC models, we found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with downregulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. In the H1373 xenograft model, treatment with JQ1 significantly reduced tumor growth and downregulated the expression of c-Myc. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and we found the expression of the two key apoptosis regulators FLIP and XIAP to be highly BET dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Further we showed that combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether, these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment. They also demonstrate that BET inhibition primes NSCLC cells for induction of apoptosis and that a combination with pro

  20. k-RAS mutations in non-small cell lung cancer patients treated with TKIs among smokers and non-smokers: a meta-analysis

    PubMed Central

    Lu, Hui-Yu

    2016-01-01

    Aim of the study Recent studies have suggested that k-RAS mutations are related to the response to epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitions (TKIs) in advanced non-small cell lung cancer (NSCLC) treatment. The aim of this meta-analysis was to assess the relationship between smoking history and k-RAS mutations in NSCLC treated with TKIs. Material and methods We searched MEDLINE and Web of Science up to 15 March 2014. The pooled relative risk (RR) was estimated by using fixed effect model or random effect model, according to heterogeneity between studies. We also carried out power analyses. Results We identified 12 studies with 1193 patients, including 196 patients (16.4%) with k-RAS mutations. The pooled k-RAS mutations incidence was 22.8% (174/764) in patients with smoke expose vs. 5.4% (23/429) in those with no smoke exposure. The pooled RR was 2.991 (95% CI: 1.884–4.746; Z = 4.65, p = 0.000). No publication bias was found (Begg's test: z = 1.09, p = 0.274 and Egger's test: t = 1.38, p = 0.201). In subgroup analyses, the pooled RR was 3.336 (95% CI: 1.925–5.779; Z = 4.30, p = 0.000) in the Caucasian subgroup, while in the Asian subgroup the pooled RR was 2.093 (95% CI: 0.909–4.822; Z = 1.73, p = 0.083), but the sample size was underpowered (0.465). Conclusions The current meta-analysis found that smoking was related to increased incidence of k-RAS mutations in non-small cell lung cancer treated with TKIs. This may be further evidence that smoking will lead to a worse prognosis in NSCLC patients treated with TKIs. PMID:27358590

  1. Correlation of immunohistochemical staining p63 and TTF-1 with EGFR and K-ras mutational spectrum and diagnostic reproducibility in non small cell lung carcinoma.

    PubMed

    Thunnissen, Erik; Boers, Evan; Heideman, Daniëlle A M; Grünberg, Katrien; Kuik, Dirk J; Noorduin, Arnold; van Oosterhout, Matthijs; Pronk, Divera; Seldenrijk, Cees; Sietsma, Hannie; Smit, Egbert F; van Suylen, Robertjan; von der Thusen, Jan; Vrugt, Bart; Wiersma, Anne; Witte, Birgit I; den Bakker, Michael

    2012-12-01

    For treatment purposes, distinction between squamous cell carcinoma and adenocarcinoma is important. The aim of this study is to examine the diagnostic accuracy on lung cancer small biopsies for the distinction between adenocarcinoma and squamous cell carcinoma and relate these to immunohistochemical and KRAS and EGFR mutation analysis. An interobserver study was performed on 110 prospectively collected biopsies obtained by bronchoscopy or transthoracic needle biopsy of patients with non-small cell lung cancer. The diagnosis was correlated with immunohistochemical (IHC) analysis for markers of adeno- (TTF1 and/or mucin positivity) and squamous cell differentiation (P63 and CK5/6) as well as KRAS and EGFR mutation analysis. Eleven observers independently read H&E-stained slides of 110 cases, resulting in a kappa value of 0.55 ± 0.10. The diagnosis non-small cell lung cancer not otherwise specified was given on average on 29.5 % of the biopsies. A high concordance was observed between hematoxylin-eosin-based consensus diagnosis (≥8/11 readings concordant) and IHC markers. In all cases with EGFR (n = 1) and KRAS (n = 20) mutations, adenodifferentiation as determined by IHC was present and p63 staining was absent. In 2 of 25 cases with a consensus diagnosis of squamous cell carcinoma, additional stainings favored adenodifferentation, and a KRAS mutation was present. P63 is most useful for distinction between EGFR/KRAS mutation positive and negative patients. In the diagnostic work-up of non-small cell lung carcinoma the limited reproducibility on small biopsies is optimized with immunohistochemical analysis, resulting in reliable delineation for predictive analysis. PMID:23064619

  2. Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1.

    PubMed

    Ohashi, Kadoaki; Sequist, Lecia V; Arcila, Maria E; Moran, Teresa; Chmielecki, Juliann; Lin, Ya-Lun; Pan, Yumei; Wang, Lu; de Stanchina, Elisa; Shien, Kazuhiko; Aoe, Keisuke; Toyooka, Shinichi; Kiura, Katsuyuki; Fernandez-Cuesta, Lynnette; Fidias, Panos; Yang, James Chih-Hsin; Miller, Vincent A; Riely, Gregory J; Kris, Mark G; Engelman, Jeffrey A; Vnencak-Jones, Cindy L; Dias-Santagata, Dora; Ladanyi, Marc; Pao, William

    2012-07-31

    Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment. PMID:22773810

  3. Application of PCR methods to evaluate EGFR, KRAS and BRAF mutations in a small number of tumor cells in cytological material from lung cancer patients

    PubMed Central

    LEWANDOWSKA, MARZENA ANNA; JÓŹWICKI, WOJCIECH; JOCHYMSKI, CEZARY; KOWALEWSKI, JANUSZ

    2013-01-01

    The epidermal growth factor receptor (EGFR) mutation status in the tyrosine kinase domain is known to be a predictor of the response to gefitinib or erlotinib in lung cancer; thus, a non-surgical procedure of tumor specimen collection is critical for mutation analysis. The aim of the present study was to analyze the EGFR, KRAS and BRAF status in limited cytological material. To the best of our knowledge, this is the first time that the quantitative scale of tumor cells and the percentage of tumor cells in cytological material were evaluated at the early stages of pathomorphological material qualification for EGFR, KRAS and BRAF mutation analysis. Our results revealed that even 100–1,000 tumor cells from fine needle aspiration (FNA) samples provided reliable results of mutation analysis when sensitive real-time polymerase chain reaction (PCR) methods were used. EGFR mutations were detected in 10% (7/71) and KRAS mutations were detected in 35% (19/54) of the lung adenocarcinoma cases. In addition, we reported the most common inhibiting mutation (p.T790M) found in coexistence with p.L858R in an FNA sample from a patient, for whom short-term improvement after erlotinib treatment was observed before further progression of the disease. Subsequently, mutual exclusion of EGFR and KRAS mutations was observed. Cytological samples with a small number of tumor cells obtained via FNA, endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) or brushing are suggested to be used for diagnostic purposes after careful selection by cytopathologists and analysis using a validated, sensitive real-time PCR method. PMID:23817662

  4. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    PubMed Central

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  5. Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group

    PubMed Central

    2012-01-01

    Background To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. Methods Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). Results 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. Conclusions Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. Trial registration number 2002-04-017 PMID:22876876

  6. K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin.

    PubMed

    Birchenall-Roberts, Maria C; Fu, Tao; Kim, Soo-Gyung; Huang, Ying K; Dambach, Michael; Resau, James H; Ruscetti, Francis W

    2006-09-22

    Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin. PMID:16889753

  7. K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin

    SciTech Connect

    Birchenall-Roberts, Maria C. . E-mail: birchena@mail.ncifcrf.gov; Fu, Tao; Kim, Soo-Gyung; Huang, Ying K.; Dambach, Michael; Resau, James H.; Ruscetti, Francis W.

    2006-09-22

    Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.

  8. KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: A meta-analysis of 41 studies

    PubMed Central

    Pan, Wei; Yang, Yan; Zhu, Hongcheng; Zhang, Youcheng; Zhou, Rongping; Sun, Xinchen

    2016-01-01

    Mutation of oncogene KRAS is common in non-small cell lung cancer (NSCLC), however, its clinical significance is still controversial. Independent studies evaluating its prognostic and predictive value usually drew inconsistent conclusions. Hence, We performed a meta-analysis with 41 relative publications, retrieved from multi-databases, to reconcile these controversial results and to give an overall impression of KRAS mutation in NSCLC. According to our findings, KRAS mutation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in early stage resected NSCLC (hazard ratio or HR=1.56 and 1.57, 95% CI 1.39-1.76 and 1.17-2.09 respectively), and with inferior outcomes of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) treatment and chemotherapy (relative risk or RR=0.21 and 0.66 for objective response rate or ORR, 95% CI 0.12-0.39 and 0.54-0.81 respectively; HR=1.46 and 1.30 for progression-free survival or PFS, 95%CI 1.23-1.74 and 1.14-1.50 respectively) in advanced NSCLC. When EGFR mutant patients were excluded, KRAS mutation was still significantly associated with worse OS and PFS of EGFR-TKIs (HR=1.40 and 1.35, 95 % CI 1.21-1.61 and 1.11-1.64). Although KRAS mutant patients presented worse DFS and PFS of chemotherapy (HR=1.33 and 1.11, 95% CI 0.97-1.84 and 0.95-1.30), and lower response rate to EGFR-TKIs or chemotherapy (RR=0.55 and 0.88, 95 % CI 0.27-1.11 and 0.76-1.02), statistical differences were not met. In conclusion, KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC. There's a need for developing target therapies for KRAS mutant lung cancer and other tumors. PMID:26840022

  9. Activated K-Ras, But Not H-Ras or N-Ras, Regulates Brain Neural Stem Cell Proliferation in a Raf/Rb-Dependent Manner

    PubMed Central

    Bender, R. Hugh F.; Haigis, Kevin M.; Gutmann, David H.

    2016-01-01

    Neural stem cells (NSCs) give rise to all the major cell types in the brain, including neurons, oligodendrocytes, and astrocytes. However, the intracellular signaling pathways that govern brain NSC proliferation and differentiation have been incompletely characterized to date. Since some neurodevelopmental brain disorders (Costello syndrome and Noonan syndrome) are caused by germline activating mutations in the RAS genes, Ras small GTPases are likely critical regulators of brain NSC function. In the mammalian brain, Ras exists as three distinct molecules (H-Ras, K-Ras, and N-Ras), each with different subcellular localizations, downstream signaling effectors, and biological effects. Leveraging a novel series of conditional-activated Ras molecule-expressing genetically engineered mouse strains, we demonstrate that activated K-Ras, but not H-Ras or N-Ras, expression increases brain NSC growth in a Raf-dependent, but Mek-independent, manner. Moreover, we show that activated K-Ras regulation of brain NSC proliferation requires Raf binding and suppression of retinoblastoma (Rb) function. Collectively, these observations establish tissue-specific differences in activated Ras molecule regulation of brain cell growth that operate through a noncanonical mechanism. PMID:25788415

  10. Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK

    PubMed Central

    Wang, Jieqiong; Hu, Kewen; Guo, Jiawei; Cheng, Feixiong; Lv, Jing; Jiang, Wenhao; Lu, Weiqiang; Liu, Jinsong; Pang, Xiufeng; Liu, Mingyao

    2016-01-01

    No effective targeted therapies exist for cancers with somatic KRAS mutations. Here we develop a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results show that dual inhibition of polo-like kinase 1 and RhoA/Rho kinase (ROCK) leads to the synergistic effects in KRAS-mutant cancers. Microarray analysis reveals that this combinatory inhibition significantly increases transcription and activity of cyclin-dependent kinase inhibitor p21WAF1/CIP1, leading to specific G2/M phase blockade in KRAS-mutant cells. Overexpression of p21WAF1/CIP1, either by cDNA transfection or clinical drugs, preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and p21WAF1/CIP1. Co-administration of BI-2536 and fasudil either in the LSL-KRASG12D mouse model or in a patient tumour explant mouse model of KRAS-mutant lung cancer suppresses tumour growth and significantly prolongs mouse survival, suggesting a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers. PMID:27193833

  11. Oncogenic KRAS confers chemoresistance by upregulating NRF2

    PubMed Central

    Tao, Shasha; Wang, Shue; Moghaddam, Seyed Javad; Ooi, Aikseng; Chapman, Eli; Wong, Pak K.; Zhang, Donna D.

    2014-01-01

    Oncogenic KRAS mutations found in 20–30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in the NRF2 promoter. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Co-treatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors. PMID:25339352

  12. Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations

    PubMed Central

    Duan, Guang-Jie; Shi, Yan; Deng, Guo-Hong; Xia, Han; Xu, Han-Qing; Zhao, Na; Fu, Wei-Ling; Huang, Qing

    2015-01-01

    The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene. PMID:26701781

  13. Whole genome sequencing reveals potential targets for therapy in patients with refractory KRAS mutated metastatic colorectal cancer

    PubMed Central

    2014-01-01

    Background The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy is poor, with median survival of less than one year. The purpose of this study was to identify candidate therapeutically targetable somatic events in mCRC patient samples by whole genome sequencing (WGS), so as to obtain targeted treatment strategies for individual patients. Methods Four patients were recruited, all of whom had received > 2 prior therapy regimens. Percutaneous needle biopsies of metastases were performed with whole blood collection for the extraction of constitutional DNA. One tumor was not included in this study as the quality of tumor tissue was not sufficient for further analysis. WGS was performed using Illumina paired end chemistry on HiSeq2000 sequencing systems, which yielded coverage of greater than 30X for all samples. NGS data were processed and analyzed to detect somatic genomic alterations including point mutations, indels, copy number alterations, translocations and rearrangements. Results All 3 tumor samples had KRAS mutations, while 2 tumors contained mutations in the APC gene and the PIK3CA gene. Although we did not identify a TCF7L2-VTI1A translocation, we did detect a TCF7L2 mutation in one tumor. Among the other interesting mutated genes was INPPL1, an important gene involved in PI3 kinase signaling. Functional studies demonstrated that inhibition of INPPL1 reduced growth of CRC cells, suggesting that INPPL1 may promote growth in CRC. Conclusions Our study further supports potential molecularly defined therapeutic contexts that might provide insights into treatment strategies for refractory mCRC. New insights into the role of INPPL1 in colon tumor cell growth have also been identified. Continued development of appropriate targeted agents towards specific events may be warranted to help improve outcomes in CRC. PMID:24943349

  14. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan

    PubMed Central

    Maitra, Radhashree; Seetharam, Raviraja; Tesfa, Lydia; Augustine, Titto A.; Klampfer, Lidija; Coffey, Matthew C.; Mariadason, John M.; Goel, Sanjay

    2014-01-01

    Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC. PMID:24798549

  15. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan.

    PubMed

    Maitra, Radhashree; Seetharam, Raviraja; Tesfa, Lydia; Augustine, Titto A; Klampfer, Lidija; Coffey, Matthew C; Mariadason, John M; Goel, Sanjay

    2014-05-15

    Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC. PMID:24798549

  16. K-ras oncogene DNA sequences in pink salmon in streams impacted by the Exxon Valdez oil spill: no evidence of oil-induced heritable mutations.

    PubMed

    Cronin, Matthew A; Wickliffe, Jeffrey K; Dunina, Yelena; Baker, Robert J

    2002-08-01

    It was hypothesized in previous studies that the Exxon Valdez oil spill in Prince William Sound, Alaska, induced heritable mutations and resulted in mortality of pink salmon (Oncorhynchus gorbuscha) embryos. In one of these studies, laboratory exposure of pink salmon embryos to crude oil resulted in apparent mutation-induction in exon 1 and exon 2 of the K-ras oncogene, but no fish from the area impacted by the oil spill were analyzed. We assessed K-ras exon 1 and exon 2 DNA sequences in pink salmon from five streams that were oiled and five streams that were not oiled by the Exxon Valdez oil spill in Prince William Sound, and two streams with natural oil seeps and one stream without seeps on the Alaska Peninsula. Of the 79 fish analyzed for exon 1 and the 89 fish analyzed for exon 2, none had the nucleotide substitutions representing the mutations induced in the laboratory study. Other variable nucleotides occurred in similar proportions in oiled and non-oiled streams and probably represent natural allelic variation. These data do not support the hypothesis that heritable mutations in the K-ras gene were induced by the Exxon Valdez oil spill or oil seeps. PMID:12211696

  17. 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

  18. Amplification-free In Situ KRAS Point Mutation Detection at 60 copies/mL in Urine in a Background of 1000-fold Wild Type

    PubMed Central

    KirimLi, Ceyhun E.; Shih, Wei-Heng; Shih, Wan Y.

    2016-01-01

    We have examined in situ detection of single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance in situ mutant (MT) DNA detection specificity against the wild type (WT), the detection was carried out in a flow with a flow rate of 4 mL/min and at 63°C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies/mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, the detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations. PMID:26783561

  19. Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2016-02-21

    We have examined the in situ detection of a single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance the in situ mutant (MT) DNA detection specificity against the wild type (WT), detection was carried out in a flow with a flow rate of 4 mL min(-1) and at 63 °C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies per mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, this detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations. PMID:26783561

  20. Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

    PubMed

    Zdanov, Stephanie; Mandapathil, Magis; Abu Eid, Rasha; Adamson-Fadeyi, Saudat; Wilson, Willie; Qian, Jiahua; Carnie, Andrea; Tarasova, Nadya; Mkrtichyan, Mikayel; Berzofsky, Jay A; Whiteside, Theresa L; Khleif, Samir N

    2016-04-01

    Constitutive activation of theKRASoncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes,KRASacts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutantKRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutantKRASoncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.Cancer Immunol Res; 4(4); 354-65. ©2016 AACR. PMID:26880715

  1. Lauric acid can improve the sensitization of Cetuximab in KRAS/BRAF mutated colorectal cancer cells by retrievable microRNA-378 expression.

    PubMed

    Weng, Wen-Hui; Leung, Wai-Hung; Pang, Yeu-Jye; Hsu, Hsi-Hsien

    2016-01-01

    EGFR-inhibitor (Cetuximab) is one of the main targeted drugs used for metastatic colorectal carcinoma (CRC). The benefit from Cetuximab appears to be limited to a subtype of patients, not for the patients with tumors harboring mutated BRAF or KRAS genes; unfortunately, it accounts for ~40-50% of CRC cases. Previous studies have connected higher expression levels of miR-378 to be commonly presented in patients without BRAF or KRAS mutants than in mutated CRCs. The microRNA-378 (miR-378) is coexpressed with PGC-1β and can be easily induced by fatty acid, for example lauric acid. Therefore, we hypothesized that elevation of miR-378 expression in mutated CRCs may stimulate the cell response to Cetuximab. Herein, seven CRC cell lines with confirmed mutation status were involved in two parallel experiments; directly in vitro transfected miR-378 mimics, and using lauric acid to indirectly induce the level of miR-378 in cells. After the increase of miR-378 in cells by either direct or indirect approaches, sensitivity to Cetuximab was restored in all BRAF mutants (p-value <0.0001-0.0003), and half of KRAS mutants CRC (p-value 0.039-0.007). Further evidence was gained by decreasing expression of MEK and ERK2 proteins after transfection with miR-378; it was similar to the indirect induction by lauric acid approach. In conclusion, the present study demonstrated that lauric acid may efficiently induce miR-378 expression in CRC mutants, and both BRAF and a subtype of KRAS mutants presented significantly improved sensitivity to Cetuximab. Notably, BRAF mutants could even be inhibited in cell proliferation after elevated concentration of miR-378 in cells without combining with targeted therapy. This new approach may shed new light on BRAF or KRAS mutation in CRC patients for clinical trial, since lauric acid may easily be obtain from natural food, and it is supposed to be harmless to the cardiovascular system. PMID:26496897

  2. Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation

    PubMed Central

    McKinnon, Timothy; Venier, Rosemarie; Dickson, Brendan C.; Kabaroff, Leah; Alkema, Manon; Chen, Li; Shern, Jack F.; Yohe, Marielle E.; Khan, Javed; Gladdy, Rebecca A.

    2015-01-01

    While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53−/− myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53−/− myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum. PMID:25992772

  3. MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Alam, Maroof; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal. PMID:25245423

  4. KRAS Mutation Status and Clinical Outcome of Preoperative Chemoradiation With Cetuximab in Locally Advanced Rectal Cancer: A Pooled Analysis of 2 Phase II Trials

    SciTech Connect

    Kim, Sun Young; Shim, Eun Kyung; Yeo, Hyun Yang; Baek, Ji Yeon; Hong, Yong Sang; Kim, Dae Yong; Kim, Tae Won; Kim, Jee Hyun; Im, Seock-Ah; Jung, Kyung Hae; Chang, Hee Jin

    2013-01-01

    Purpose: Cetuximab-containing chemotherapy is known to be effective for KRAS wild-type metastatic colorectal cancer; however, it is not clear whether cetuximab-based preoperative chemoradiation confers an additional benefit compared with chemoradiation without cetuximab in patients with locally advanced rectal cancer. Methods and Materials: We analyzed EGFR, KRAS, BRAF, and PIK3CA mutation status with direct sequencing and epidermal growth factor receptor (EGFR) and Phosphatase and tensin homolog (PTEN) expression status with immunohistochemistry in tumor samples of 82 patients with locally advanced rectal cancer who were enrolled in the IRIX trial (preoperative chemoradiation with irinotecan and capecitabine; n=44) or the ERBIRIX trial (preoperative chemoradiation with irinotecan and capecitabine plus cetuximab; n=38). Both trials were similarly designed except for the administration of cetuximab; radiation therapy was administered at a dose of 50.4 Gy/28 fractions and irinotecan and capecitabine were given at doses of 40 mg/m{sup 2} weekly and 1650 mg/m{sup 2}/day, respectively, for 5 days per week. In the ERBIRIX trial, cetuximab was additionally given with a loading dose of 400 mg/m{sup 2} on 1 week before radiation, and 250 mg/m{sup 2} weekly thereafter. Results: Baseline characteristics before chemoradiation were similar between the 2 trial cohorts. A KRAS mutation in codon 12, 13, and 61 was noted in 15 (34%) patients in the IRIX cohort and 5 (13%) in the ERBIRIX cohort (P=.028). Among 62 KRAS wild-type cancer patients, major pathologic response rate, disease-free survival and pathologic stage did not differ significantly between the 2 cohorts. No mutations were detected in BRAF exon 11 and 15, PIK3CA exon 9 and 20, or EGFR exon 18-24 in any of the 82 patients, and PTEN and EGFR expression were not predictive of clinical outcome. Conclusions: In patients with KRAS wild-type locally advanced rectal cancer, the addition of cetuximab to the chemoradiation with

  5. Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Zhang, Weiying; Nandakumar, Nivedita; Shi, Yuhao; Manzano, Mark; Smith, Alias; Graham, Garrett; Gupta, Swati; Vietsch, Eveline E.; Laughlin, Sean Z.; Wadhwa, Mandheer; Chetram, Mahandranauth; Joshi, Mrinmayi; Wang, Fen; Kallakury, Bhaskar; Toretsky, Jeffrey; Wellstein, Anton; Yi, Chunling

    2014-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D: Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras: Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers. PMID:24803537

  6. Adenocarcinoma arising from intracranial recurrent mature teratoma and featuring mutated KRAS and wild-type BRAF genes.

    PubMed

    Kim, Eun Soo; Kwon, Mi Jung; Song, Joon Ho; Kim, Dong Hoon; Park, Hye-Rim

    2015-02-01

    Malignant transformation or recurrence of intracranial mature teratoma is an extremely rare occurrence, compared to the usual ovarian counterpart. Previously, yolk sac tumor elements have been considered to be selective progenitors of enteric-type adenocarcinoma arising from intracranial germ cell tumors. However, the present case demonstrates the occurrence of enteric-type adenocarcinoma in recurrent intracranial mature cystic teratoma 12 years after gross total removal, a case of which has not previously been documented in the literature. The 11.5-cm long, dura mater-based tumor on the right fronto-temporal lobe displaced the brain; however, the patient had no neurologic symptoms or discomfort other than pus-like discharge on the scalp. Microscopic examinations revealed a small focus of adenocarcinoma and dysplastic colonic mucosa in the mature cystic teratoma. No immature elements were seen. The cystic wall was almost denuded and showed an exuberant xanthogranulomatous reaction with foreign-body type giant cells engulfing keratin materials and cholesterol clefts, suggesting that chronic inflammation due to repeated cyst wall rupture and the previous resection may contribute to malignant transformation. The adenocarcinoma showed strong immunohistochemical expression of CK20 and p53, but CK7 in patches. The molecular profile of the adenocarcinoma showed a mutation in KRAS and wild-type BRAF, which might be associated with malignant transformation of intracranial mature teratomas. In conclusion, the intracranial mature teratomas should require long-term follow-up, and clinicians, radiologists and pathologists should be aware of the potential for malignant progression of recurrent intracranial mature cystic teratoma despite gross total resection and no neurologic symptoms. PMID:25039399

  7. A vertically-stacked, polymer, microfluidic point mutation analyzer: Rapid, high accuracy detection of low-abundance K-ras mutations

    PubMed Central

    Han, Kyudong; Lee, Tae Yoon; Nikitopoulos, Dimitris E.; Soper, Steven A.; Murphy, Michael C.

    2011-01-01

    Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well-trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user-base for these types of molecular assays, a vertically-stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employed a primary PCR coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers and ExoSAP-IT® purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in ~1 h, an 80% reduction in time compared to conventional bench-top instrumentation. Purifying the post-PCR products with the ExoSAP-IT® enzyme led to optimized LDR performance minimizing false positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25 μL sample, equivalent to DNA from 42 mutant cells. PMID:21771577

  8. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

    PubMed Central

    Seekhuntod, Sirirat; Thavarungkul, Paninee; Chaichanawongsaroj, Nuntaree

    2016-01-01

    Background Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. PMID:26812617

  9. Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas.

    PubMed

    Myers, Meagan B; Banda, Malathi; McKim, Karen L; Wang, Yiying; Powell, Michael J; Parsons, Barbara L

    2016-04-01

    Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measureable levels of at least three of the five mutations. HRAS G12D was significantly increased in DCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10(-3) to 4.6 × 10(-2)) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047R mutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells. PMID:27108388

  10. Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas12

    PubMed Central

    Myers, Meagan B.; Banda, Malathi; McKim, Karen L.; Wang, Yiying; Powell, Michael J.; Parsons, Barbara L.

    2016-01-01

    Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measureable levels of at least three of the five mutations. HRAS G12D was significantly increased in DCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10− 3 to 4.6 × 10− 2) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047R mutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells. PMID:27108388

  11. Targeting the KRAS variant for treatment of non-small cell lung cancer: potential therapeutic applications.

    PubMed

    Ricciuti, Biagio; Leonardi, Giulia Costanza; Metro, Giulio; Grignani, Francesco; Paglialunga, Luca; Bellezza, Guido; Baglivo, Sara; Mencaroni, Clelia; Baldi, Alice; Zicari, Daniela; Crinò, Lucio

    2016-01-01

    Lung cancer is the leading cause of cancer deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for 80% of all lung cancers. Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the deadliest cancer-related proteins and plays a pivotal role in the most aggressive and lethal human cancers, including lung adenocarcinoma where it represents one of the most frequently mutated oncogene. Although therapeutic progresses have made an impact over the last decade, median survival for patients with advanced lung cancer remains disappointing, with a 5-year worldwide survival rate of <15%. For more than 20 years it has been recognized that constitutively active signaling downstream of KRAS is a fundamental driver of lung tumorigenesis. However, years of pursuit have failed to yield a drug that can safely curb KRAS activity; up to now no approved therapies exist for KRAS-mutant NSCLC. The aim of this review is to discuss the current knowledge of KRAS-mutated NSCLC, touching upon KRAS clinical relevance as a prognostic and predictive biomarker, with an emphasis on novel therapeutic approaches for the treatment of KRAS-variant NSCLC. PMID:26714748

  12. Oncogenic KRAS triggers MAPK-dependent errors in mitosis and MYC-dependent sensitivity to anti-mitotic agents.

    PubMed

    Perera, David; Venkitaraman, Ashok R

    2016-01-01

    Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs. PMID:27412232

  13. Oncogenic KRAS triggers MAPK-dependent errors in mitosis and MYC-dependent sensitivity to anti-mitotic agents

    PubMed Central

    Perera, David; Venkitaraman, Ashok R.

    2016-01-01

    Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs. PMID:27412232

  14. Transition between morule-like and solid components may occur in solid-predominant adenocarcinoma of the lung: report of 2 cases with EGFR and KRAS mutations

    PubMed Central

    Tajima, Shogo; Koda, Kenji

    2015-01-01

    A limited number of pulmonary adenocarcinoma cases with morule-like components have been described to date, and the most frequent histological subtype is papillary-predominant adenocarcinoma. Occasionally, this type of adenocarcinoma is associated with solid-predominant adenocarcinoma. EGFR mutations are predominant in adenocarcinoma with morule-like components, followed by ALK rearrangements. Herein, we present 2 cases of solid-predominant adenocarcinoma with morule-like components harboring either an EGFR or KRAS mutation. This KRAS-mutant case is the first to be associated with morule-like components, to the best of our knowledge. Both cases showed transition between micropapillary and morule-like components. Transition between morule-like and solid components was also observed in both cases. Although a few cases of solid-predominant adenocarcinoma have been shown to harbor morule-like components, this type of transition has not been previously well described. We surmised that the solid components of some EGFR-mutant adenocarcinomas might be derived from morule-like components. PMID:26261656

  15. Statins augment efficacy of EGFR-TKIs in patients with advanced-stage non-small cell lung cancer harbouring KRAS mutation.

    PubMed

    Fiala, Ondrej; Pesek, Milos; Finek, Jindrich; Minarik, Marek; Benesova, Lucie; Bortlicek, Zbynek; Topolcan, Ondrej

    2015-08-01

    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) represent novel effective agents approved for the treatment of patients with advanced-stage NSCLC. KRAS mutations have been reported as a negative prognostic and predictive factor in patients with NSCLC treated with EGFR-TKIs. Several studies have recently shown that statins can block tumour cell growth, invasion and metastatic potential. We analysed clinical data of 67 patients with locally advanced (IIIB) or metastatic stage (IV) NSCLC harbouring Kirsten rat sarcoma viral oncogene (KRAS) mutation treated with erlotinib or gefitinib. Twelve patients were treated with combination of EGFR-TKI and statin and 55 patients were treated with EGFR-TKI alone. Comparison of patients' survival (progression-free survival (PFS) and overall survival (OS)) according to the treatment used was performed using the Gehan-Wilcoxon test. The median of PFS and OS for patients treated with EGFR-TKI alone was 1.0 and 5.4 months compared to 2.0 and 14.0 months for patients treated with combination of EGFR-TKI and statin (p = 0.025, p = 0.130). In conclusion, the study results suggest significant improvement of PFS for patients treated with combination of statin and EGFR-TKI, and the difference in OS was not significant. PMID:25702091

  16. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

    PubMed Central

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  17. Loss of integrin alpha1beta1 ameliorates Kras-induced lung cancer.

    PubMed

    Macias-Perez, Ines; Borza, Corina; Chen, Xiwu; Yan, Xuexian; Ibanez, Raquel; Mernaugh, Glenda; Matrisian, Lynn M; Zent, Roy; Pozzi, Ambra

    2008-08-01

    The collagen IV binding receptor integrin alpha1beta1 has been shown to regulate lung cancer due to its proangiogenic properties; however, it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin alpha1-null mice were crossed with KrasLA2 mice that carry an oncogenic mutation of the Kras gene (G12D) and develop spontaneous primary tumors with features of non-small cell lung cancer. We provide evidence that KrasLA2/alpha1-null mice have a decreased incidence of primary lung tumors and longer survival compared with KrasLA2/alpha1 wild-type controls. Tumors from KrasLA2/alpha1-null mice were also smaller, less vascularized, and exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end staining, respectively. Moreover, tumors from the KrasLA2/alpha1-null mice showed diminished extracellular signal-regulated kinase (ERK) but enhanced p38 mitogen-activated protein kinase activation. Primary lung tumor epithelial cells isolated from KrasLA2/alpha1-null mice showed a significant decrease in anchorage-independent colony formation, collagen-mediated cell proliferation, ERK activation, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/alpha1 wild-type tumor cells. These results indicate that loss of the integrin alpha1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin alpha1beta1 cooperate to drive the growth of non-small cell lung cancer in vivo. PMID:18676835

  18. Mutational Hotspot of TET2, IDH1, IDH2, SRSF2, SF3B1, KRAS, and NRAS from Human Systemic Mastocytosis Are Not Conserved in Canine Mast Cell Tumors

    PubMed Central

    Zorzan, Eleonora; Hanssens, Katia; Giantin, Mery; Dacasto, Mauro; Dubreuil, Patrice

    2015-01-01

    Introduction Both canine cutaneous mast cell tumor (MCT) and human systemic mastocytosis (SM) are characterized by abnormal proliferation and accumulation of mast cells in tissues and, frequently, by the presence of activating mutations in the receptor tyrosine kinase V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), albeit at different incidence (>80% in SM and 10–30% in MCT). In the last few years, it has been discovered that additional mutations in other genes belonging to the methylation system, the splicing machinery and cell signaling, contribute, with c-KIT, to SM pathogenesis and/or phenotype. In the present study, the mutational profile of the Tet methylcytosine dioxygenase 2 (TET2), the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), the serine/arginine-rich splicing factor 2 (SRSF2), the splicing factor 3b subunit 1 (SF3B1), the Kirsten rat sarcoma viral oncogene homolog (KRAS) and the neuroblastoma RAS viral oncogene homolog (NRAS), commonly mutated in human myeloid malignancies and mastocytosis, was investigated in canine MCTs. Methods Using the Sanger sequencing method, a cohort of 75 DNA samples extracted from MCT biopsies already investigated for c-KIT mutations were screened for the “human-like” hot spot mutations of listed genes. Results No mutations were ever identified except for TET2 even if with low frequency (2.7%). In contrast to what is observed in human TET2 no frame-shift mutations were found in MCT samples. Conclusion Results obtained in this preliminary study are suggestive of a substantial difference between human SM and canine MCT if we consider some target genes known to be involved in the pathogenesis of human SM. PMID:26562302

  19. Inhibition of KRAS-driven tumorigenicity by interruption of an autocrine cytokine circuit.

    PubMed

    Zhu, Zehua; Aref, Amir R; Cohoon, Travis J; Barbie, Thanh U; Imamura, Yu; Yang, Shenghong; Moody, Susan E; Shen, Rhine R; Schinzel, Anna C; Thai, Tran C; Reibel, Jacob B; Tamayo, Pablo; Godfrey, Jason T; Qian, Zhi Rong; Page, Asher N; Maciag, Karolina; Chan, Edmond M; Silkworth, Whitney; Labowsky, Mary T; Rozhansky, Lior; Mesirov, Jill P; Gillanders, William E; Ogino, Shuji; Hacohen, Nir; Gaudet, Suzanne; Eck, Michael J; Engelman, Jeffrey A; Corcoran, Ryan B; Wong, Kwok-Kin; Hahn, William C; Barbie, David A

    2014-04-01

    Although the roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)-related kinases Tank-binding kinase-1 (TBK1) and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS. PMID:24444711

  20. HER2 activating mutations are targets for colorectal cancer treatment

    PubMed Central

    Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

    2015-01-01

    The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

  1. Inhibition of KRAS-driven tumorigenicity by interruption of an autocrine cytokine circuit

    PubMed Central

    Zhu, Zehua; Aref, Amir R.; Cohoon, Travis J.; Barbie, Thanh U.; Imamura, Yu; Yang, Shenghong; Moody, Susan E.; Shen, Rhine R.; Schinzel, Anna C.; Thai, Tran C.; Reibel, Jacob B.; Tamayo, Pablo; Godfrey, Jason T.; Qian, Zhi Rong; Page, Asher N.; Maciag, Karolina; Chan, Edmond M.; Silkworth, Whitney; Labowsky, Mary T.; Rozhansky, Lior; Mesirov, Jill P.; Gillanders, William E.; Ogino, Shuji; Hacohen, Nir; Gaudet, Suzanne; Eck, Michael J.; Engelman, Jeffrey A.; Corcoran, Ryan B.; Wong, Kwok-Kin; Hahn, William C.; Barbie, David A.

    2014-01-01

    Although the roles of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here we show that the IKK-related kinases TBK1 and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and IL-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 and MEK inhibitor therapy induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, JAK, and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS. PMID:24444711

  2. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices

    PubMed Central

    Leshchiner, Elizaveta S.; Parkhitko, Andrey; Bird, Gregory H.; Luccarelli, James; Bellairs, Joseph A.; Escudero, Silvia; Opoku-Nsiah, Kwadwo; Godes, Marina; Perrimon, Norbert; Walensky, Loren D.

    2015-01-01

    Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85DV12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer. PMID:25624485

  3. Targeting KRAS for diagnosis, prognosis, and treatment of pancreatic cancer: Hopes and realities.

    PubMed

    Bournet, Barbara; Buscail, Camille; Muscari, Fabrice; Cordelier, Pierre; Buscail, Louis

    2016-02-01

    Mutation of the KRAS oncogene in pancreatic cancer is responsible for permanent activation of the P21 RAS protein and the cascade of signalling pathways. Consequently, multiple cellular processes, such as transformation, proliferation, invasion, and survival are activated. The aim of this review was to present all potential clinical applications of targeting KRAS in terms of diagnosis and management of pancreatic adenocarcinoma. Quantitative polymerase chain reaction technology provides reliable assessment of KRAS mutations, both in tissues and from fine-needle aspiration biopsies. Numerous studies report that the combination of endoscopic ultrasound-guided cytopathology and a KRAS mutation assay can improve the positive and differential diagnosis of pancreatic cancer, differentiating between benign versus malignant solid pancreatic cancer, and reducing false-negative results compared to cytopathology alone. In addition, the presence of a KRAS mutation is frequently associated with a worse prognosis, both in cases of advanced and resected tumours. However, the KRAS mutation assay is not as efficient at predicting a response to both anti-epidermal growth factor receptor treatments and/or chemotherapy. Targeting of KRAS to treat pancreatic adenocarcinoma has been applied at different stages of RAS molecular intracellular processes: at the transcription level with antisense or interference RNA, at the posttranslational level with inhibitors of farnesyl transferase or anti-RAS vaccination peptides, and to target multiple signalling pathways using inhibitors of mitogen-activated protein kinase, phosphoinositide 3-kinase, AKT, mammalian target of rapamycin, RAF. Despite some encouraging results at pre-clinical and phase I stages, no significant clinical benefits have been observed. Combinatory approaches with standard chemotherapy will be welcome. PMID:26735353

  4. A CDK4/6 inhibitor enhances cytotoxicity of paclitaxel in lung adenocarcinoma cells harboring mutant KRAS as well as wild-type KRAS.

    PubMed

    Zhang, Xiang-Hua; Cheng, Ying; Shin, Jung-Young; Kim, Jeong-Oh; Oh, Ji-Eun; Kang, Jin-Hyoung

    2013-07-01

    The KRAS gain-of-function mutation confers intrinsic resistance to targeted anti-cancer drugs and cytotoxic chemotherapeutic agents, ultimately leading to treatment failure. KRAS mutation frequency in lung adenocarcinoma is ~15-30%. Novel therapeutic strategies should be developed to improve clinical outcomes in these cases. Deregulation of the p16/cyclin-dependent kinase (CDK) 4/retinoblastoma (Rb) pathway is frequently observed in various cancers and it represents an attractive therapeutic target. We compared the anti-tumor efficacy of genetically knocked-down CDK4 and a pharmacological inhibitor of CDK4/6, CINK4, in KRAS mutation-positive lung adenocarcinoma cells. We also investigated changes in anti-proliferative activity and downstream molecules with these treatments in combination with paclitaxel. CDK4 short interfering RNA (siRNA) significantly increased paclitaxel sensitivity in KRAS mutation-positive H23 cells. CINK4 demonstrated concentration- and time-dependent anti-proliferative activity in 5 adenocarcinoma lines. CINK4 induced G 1 arrest by downregulating the p16/cyclin D1/Rb pathway, resulting in apoptotic induction via increased expression of cleaved caspase3, cleaved PARP and Bax. Combined CINK4 and paclitaxel produced synergistic anti-proliferative activity and increased apoptosis through reduced cyclin D1 and Bcl-2 in KRAS mutation-positive cancer cells. These data suggest CDK4 is a promising target for development of anti-cancer drugs and CINK4 combined with paclitaxel may be an effective therapeutic strategy for enhancing anti-tumor efficacy in KRAS mutation-positive lung adenocarcinoma. PMID:23792647

  5. Oncogenic K-ras confers SAHA resistance by up-regulating HDAC6 and c-myc expression

    PubMed Central

    Zhang, Yi; Tan, Zhiping; Su, Bing; Li, Yu

    2016-01-01

    Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance. PMID:26848526

  6. Oncogenic K-ras confers SAHA resistance by up-regulating HDAC6 and c-myc expression.

    PubMed

    Wang, Qun; Tan, Rong; Zhu, Xin; Zhang, Yi; Tan, Zhiping; Su, Bing; Li, Yu

    2016-03-01

    Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance. PMID:26848526

  7. Prognostic relevance of KRAS genotype in metastatic colorectal cancer patients unfit for FIr-B/FOx intensive regimen

    PubMed Central

    BRUERA, GEMMA; CANNITA, KATIA; GIORDANO, ALDO VICTOR; VICENTINI, ROBERTO; FICORELLA, CORRADO; RICEVUTO, ENRICO

    2014-01-01

    First-line triplet chemotherapy plus bevacizumab (FIr-B/FOx) can improve efficacy of metastatic colorectal cancer (MCRC), KRAS wild-type and mutant. Prognostic relevance of KRAS genotype was evaluated in patients unfit for FIr-B/FOx, treated with conventional medical treatments. Consecutive MCRC patients not eligible for FIr-B/FOx regimen due to age (≥75 years) and/or comorbidities were treated with tailored conventional first-line treatments. KRAS codon 12/13 mutations were screened by direct sequencing. Activity and efficacy were evaluated and compared according to medical treatments, age (non-elderly and elderly ≥65 years), comorbidity stage (Cumulative Illness Rating Scale), metastatic extension (liver-limited and other/multiple metastatic), and KRAS genotype, using log-rank. Selected first line treatments were medical in 37 patients (92.5%), and surgical in 3 patients (7.5%). Medical treatment regimens: triplet, 18 (45%); doublet, 15 (37.5%); mono-therapy, 4 (10%). At median follow-up of 8 months, objective response rate (ORR) was 37%, median progression-free survival (PFS) 7 months, liver metastasectomies 8% (liver-limited disease 37.5%), median overall survival (OS) 13 months. Triplet regimens failed to significantly affect clinical outcome, compared to doublet. According to KRAS genotype, ORR, PFS and OS were, respectively: wild-type 50%, 8 months, 13 months; mutant 25%, 6 months, 9 months. KRAS genotype wild-type compared to mutant significantly affected PFS, while not OS. KRAS c.35 G>A mutation (G12D) significantly affected worse PFS and OS compared to wild-type and/or other mutations. KRAS genotype, specifically the c.35 G>A KRAS mutation, may indicate poor prognosis in MCRC patients unfit for intensive medical treatments. PMID:24715238

  8. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.

    PubMed

    Barbie, David A; Tamayo, Pablo; Boehm, Jesse S; Kim, So Young; Moody, Susan E; Dunn, Ian F; Schinzel, Anna C; Sandy, Peter; Meylan, Etienne; Scholl, Claudia; Fröhling, Stefan; Chan, Edmond M; Sos, Martin L; Michel, Kathrin; Mermel, Craig; Silver, Serena J; Weir, Barbara A; Reiling, Jan H; Sheng, Qing; Gupta, Piyush B; Wadlow, Raymond C; Le, Hanh; Hoersch, Sebastian; Wittner, Ben S; Ramaswamy, Sridhar; Livingston, David M; Sabatini, David M; Meyerson, Matthew; Thomas, Roman K; Lander, Eric S; Mesirov, Jill P; Root, David E; Gilliland, D Gary; Jacks, Tyler; Hahn, William C

    2009-11-01

    The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer. PMID:19847166

  9. Preclinical Activity of the Rational Combination of Selumetinib (AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models

    PubMed Central

    Morelli, M. Pia; Tentler, John J.; Kulikowski, Gillian N.; Tan, Aik-Choon; Bradshaw-Pierce, Erica L.; Pitts, Todd M.; Brown, Amy M.; Nallapareddy, Sujatha; Arcaroli, John J.; Serkova, Natalie J.; Hidalgo, Manuel; Ciardiello, Fortunato; Eckhardt, S. Gail

    2013-01-01

    Purpose Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%,supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. Experimental Design Transcriptional profiling and gene set enrichment analysis (baseline and post-treatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, 18F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. Results In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G1, and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups. Conclusion These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative

  10. Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

    PubMed Central

    Schrumpf, Melanie; Talebian Yazdi, Mehrdad; Ruano, Dina; Forte, Giusi I.; Nederlof, Petra M.; Veselic, Maud; Rabe, Klaus F.; Annema, Jouke T.; Smit, Vincent; Morreau, Hans; van Wezel, Tom

    2011-01-01

    Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients. PMID:21408138

  11. Genetic Evidence for XPC-KRAS Interactions During Lung Cancer Development.

    PubMed

    Zhang, Xiaoli; He, Nonggao; Gu, Dongsheng; Wickliffe, Jeff; Salazar, James; Boldogh, Istavan; Xie, Jingwu

    2015-10-20

    Lung cancer causes more deaths than breast, colorectal and prostate cancers combined. Despite major advances in targeted therapy in a subset of lung adenocarcinomas, the overall 5-year survival rate for lung cancer worldwide has not significantly changed for the last few decades. DNA repair deficiency is known to contribute to lung cancer development. In fact, human polymorphisms in DNA repair genes such as xeroderma pigmentosum group C (XPC) are highly associated with lung cancer incidence. However, the direct genetic evidence for the role of XPC for lung cancer development is still lacking. Mutations of the Kirsten rat sarcoma viral oncogene homolog (Kras) or its downstream effector genes occur in almost all lung cancer cells, and there are a number of mouse models for lung cancer with these mutations. Using activated Kras, Kras(LA1), as a driver for lung cancer development in mice, we showed for the first time that mice with Kras(LA1) and Xpc knockout had worst outcomes in lung cancer development, and this phenotype was associated with accumulated DNA damage. Using cultured cells, we demonstrated that induced expression of oncogenic KRAS(G12V) led to increased levels of reactive oxygen species (ROS) as well as DNA damage, and both can be suppressed by anti-oxidants. Our results suggest that XPC may help repair DNA damage caused by KRAS-mediated production of ROS. PMID:26554912

  12. KRAS Testing for Anti-EGFR Therapy in Advanced Colorectal Cancer

    PubMed Central

    2010-01-01

    tumour node metastasis (TNM) system or stage D in the Duke’s classification system. Patients with advanced colorectal cancer (mCRC) either present with metastatic disease or develop it through disease progression. KRAS (Kristen-RAS, a member of the rat sarcoma virus (ras) gene family of oncogenes) is frequently mutated in epithelial cancers such as colorectal cancer, with mutations occurring in mutational hotspots (codons 12 and 13) of the KRAS protein. Involved in EGFR-mediated signalling of cellular processes such as cell proliferation, resistance to apoptosis, enhanced cell motility and neoangiogenesis, a mutation in the KRAS gene is believed to be involved in cancer pathogenesis. Such a mutation is also hypothesized to be involved in resistance to targeted anti-EGFR (epidermal growth factor receptor with tyrosine kinase activity) treatments such as cetuximab and panitumumab, hence, the important in evaluating the evidence on the predictive value of KRAS testing in this context. KRAS Mutation Testing in Advanced Colorectal Cancer Both cetuximab and panitumumab are indicated by Health Canada in the treatment of patients with metastatic colorectal cancer whose tumours are WT for the KRAS gene. Cetuximab may be offered as monotherapy in patients intolerant to irinotecan-based chemotherapy or in patients who have failed both irinotecan and oxaliplatin-based regimens and who received a fluoropyrimidine. It can also be administered in combination with irinotecan in patients refractory to other irinotecan-based chemotherapy regimens. Panitumumab is only indicated as a single agent after failure of fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens. In Ontario, patients with advanced colorectal cancer who are refractory to chemotherapy may be offered the targeted anti-EGFR treatments cetuximab or panitumumab. Eligibility for these treatments is based on the KRAS status of their tumour, derived from tissue collected from surgical or biopsy

  13. Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells.

    PubMed

    Scholl, Claudia; Fröhling, Stefan; Dunn, Ian F; Schinzel, Anna C; Barbie, David A; Kim, So Young; Silver, Serena J; Tamayo, Pablo; Wadlow, Raymond C; Ramaswamy, Sridhar; Döhner, Konstanze; Bullinger, Lars; Sandy, Peter; Boehm, Jesse S; Root, David E; Jacks, Tyler; Hahn, William C; Gilliland, D Gary

    2009-05-29

    An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations. PMID:19490892

  14. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

    PubMed Central

    Azoitei, Ninel; Hoffmann, Christopher M.; Ellegast, Jana M.; Ball, Claudia R.; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A.; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno

    2012-01-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential. PMID:22451720

  15. Kras is Required for Adult Hematopoiesis.

    PubMed

    Damnernsawad, Alisa; Kong, Guangyao; Wen, Zhi; Liu, Yangang; Rajagopalan, Adhithi; You, Xiaona; Wang, Jinyong; Zhou, Yun; Ranheim, Erik A; Luo, Hongbo R; Chang, Qiang; Zhang, Jing

    2016-07-01

    Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its role in adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow (BM) is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced thrombopoietin (TPO) signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while stem cell factor-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias toward myeloid differentiation in MPPs. Although granulocyte macrophage colony-stimulating factor (GM-CSF)-evoked ERK1/2 activation is only moderately decreased in Kras(-/-) myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9-12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras(-/-) BM cells is greatly compromised, which is attributable to defects in the self-renewal of Kras(-/-) HSCs and defects in differentiated hematopoietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. Stem Cells 2016;34:1859-1871. PMID:26972179

  16. Roles of the Pas1 and Par2 genes in determination of the unique, intermediate susceptibility of BALB/cByJ mice to urethane-induction of lung carcinogenesis: differential effects on tumor multiplicity, size and Kras2 mutations.

    PubMed

    Karasaki, H; Obata, M; Ogawa, K; Lee, G H

    1997-10-01

    The C3H/HeJ (C3H), A/J and BALB/cByJ (BALB) mouse strains are respectively resistant, sensitive and intermediate regarding the induction of lung tumors by urethane. The phenotypic difference between C3H and A/J is largely determined by the Pas1 (Pulmonary adenoma susceptibility 1) gene on chromosome 6, the A/J allele of which dominantly increases the tumor burden. We recently found that BALB mice possess a unique lung tumor resistance gene on chromosome 18, designated Par2 (Pulmonary adenoma resistance 2), which partially, but dominantly suppresses the sensitive phenotype of A/J mice (Oncogene 13: 1599-1604, 1996). It has, however, remained unclear why BALB mice carrying the Par2 gene are significantly more sensitive to urethane-induced lung carcinogenesis than C3H mice that have no dominant lung tumor resistance genes. In the present study, using (C3H x BALB)F1 x C3H backcross mice treated with urethane, we demonstrated that BALB mice possess the disease allele of the Pas1 gene despite their 15-fold more resistance relative to A/J mice (LOD = 22.6). The BALB Par2 allele only significantly reduced the mean lung tumor multiplicity (LOD = 4.4) in the backcross population carrying the BALB allele of Pas1, indicating that the intermediate BALB phenotype may at least in part be the result of interactions between these two dominant genes. While the BALB Pas1 allele increased both the mean multiplicity and size of lung tumors, the BALB Par2 allele affected only the mean tumor multiplicity, implying that they are involved in different stages of multi-step lung carcinogenesis. In addition, we found that 68% of lung tumors from the BALB Pas1-positive backcross mice contained activating point mutations of the Kras2 oncogene, tightly linked to the Pas1 locus, whereas these genetic alterations were absent in tumors from BALB Pas1-negative mice. The Par2 genotype exhibited no effect on this parameter. Since the activating point mutations were observed exclusively in the BALB

  17. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions

    PubMed Central

    Gillette, William K.; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H.; Grose, Carissa; Jones, Jane E.; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V.; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G.

    2015-01-01

    Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5–10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ. PMID:26522388

  18. Glycoproteomic Approach Identifies KRAS as a Positive Regulator of CREG1 in Non-small Cell Lung Cancer Cells

    PubMed Central

    Clark, David J.; Mei, Yuping; Sun, Shisheng; Zhang, Hui; Yang, Austin J.; Mao, Li

    2016-01-01

    Protein glycosylation plays a fundamental role in a multitude of biological processes, and the associated aberrant expression of glycoproteins in cancer has made them attractive biomarkers and therapeutic targets. In this study, we examined differentially expressed glycoproteins in cell lines derived from three different states of lung tumorigenesis: an immortalized bronchial epithelial cell (HBE) line, a non-small cell lung cancer (NSCLC) cell line harboring a Kirsten rat sarcoma viral oncogene homolog (KRAS) activation mutation and a NSCLC cell line harboring an epidermal growth factor receptor (EGFR) activation deletion. Using a Triple SILAC proteomic quantification strategy paired with hydrazide chemistry N-linked glycopeptide enrichment, we quantified 118 glycopeptides in the three cell lines derived from 82 glycoproteins. Proteomic profiling revealed 27 glycopeptides overexpressed in both NSCLC cell lines, 6 glycopeptides overexpressed only in the EGFR mutant cells and 19 glycopeptides overexpressed only in the KRAS mutant cells. Further investigation of a panel of NSCLC cell lines found that Cellular repressor of E1A-stimulated genes (CREG1) overexpression was closely correlated with KRAS mutation status in NSCLC cells and could be down-regulated by inhibition of KRAS expression. Our results indicate that CREG1 is a down-stream effector of KRAS in a sub-type of NSCLC cells and a novel candidate biomarker or therapeutic target for KRAS mutant NSCLC. PMID:26722374

  19. Alterations in the K-ras and p53 genes in rat lung tumors

    SciTech Connect

    Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E.

    1997-06-01

    Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

  20. Evaluation of KRAS Gene Expression and LCS6 Variant in Genomic and Cell-Free DNA of Iranian Women With Endometriosis

    PubMed Central

    Farahani, Maryam Shahrabi; Moghaddam, Soheila Amini; Mahdian, Reza

    2015-01-01

    Since the activation of KRAS results in de novo endometriosis in mice, KRAS is regarded as a crucial gene in ectopic endometrial implantation. Recently, it has been reported that 31% of women with endometriosis have KRAS let-7 complementary binding site 6 single-nucleotide polymorphism (LCS6 SNP). This study addresses the correlation between KRAS LCS6 SNP and endometriosis in a case–control study. To detect probable somatic mutation in ectopic endometrial tissue, we evaluated LCS6 SNP in cell-free DNA samples. Quantitative real-time reverse transcription-polymerase chain reaction was performed to determine the expression of KRAS transcripts in eutopic endometrial tissue. Our results suggest that the variant is not associated with the development of endometriosis in Iranian women. We observed higher levels of KRAS messenger RNA (mRNA) expression in eutopic endometrium of patients with endometriosis compared to controls. Although, the KRAS LCS6 is neither constitutional nor somatic biomarker for endometriosis, increased expression ratio of KRAS mRNA indicates its role in the implantation of endometrial tissue outside the uterine cavity. PMID:25361550

  1. Evaluation of KRAS Gene Expression and LCS6 Variant in Genomic and Cell-Free DNA of Iranian Women With Endometriosis.

    PubMed

    Farahani, Maryam Shahrabi; Shahbazi, Shirin; Moghaddam, Soheila Amini; Mahdian, Reza

    2015-06-01

    Since the activation of KRAS results in de novo endometriosis in mice, KRAS is regarded as a crucial gene in ectopic endometrial implantation. Recently, it has been reported that 31% of women with endometriosis have KRAS let-7 complementary binding site 6 single-nucleotide polymorphism (LCS6 SNP). This study addresses the correlation between KRAS LCS6 SNP and endometriosis in a case-control study. To detect probable somatic mutation in ectopic endometrial tissue, we evaluated LCS6 SNP in cell-free DNA samples. Quantitative real-time reverse transcription-polymerase chain reaction was performed to determine the expression of KRAS transcripts in eutopic endometrial tissue. Our results suggest that the variant is not associated with the development of endometriosis in Iranian women. We observed higher levels of KRAS messenger RNA (mRNA) expression in eutopic endometrium of patients with endometriosis compared to controls. Although, the KRAS LCS6 is neither constitutional nor somatic biomarker for endometriosis, increased expression ratio of KRAS mRNA indicates its role in the implantation of endometrial tissue outside the uterine cavity. PMID:25361550

  2. Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

    PubMed

    Foster, Scott A; Whalen, Daniel M; Özen, Ayşegül; Wongchenko, Matthew J; Yin, JianPing; Yen, Ivana; Schaefer, Gabriele; Mayfield, John D; Chmielecki, Juliann; Stephens, Philip J; Albacker, Lee A; Yan, Yibing; Song, Kyung; Hatzivassiliou, Georgia; Eigenbrot, Charles; Yu, Christine; Shaw, Andrey S; Manning, Gerard; Skelton, Nicholas J; Hymowitz, Sarah G; Malek, Shiva

    2016-04-11

    Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy. PMID:26996308

  3. Similar but different: distinct roles for KRAS and BRAF oncogenes in colorectal cancer development and therapy resistance

    PubMed Central

    Morkel, Markus; Riemer, Pamela; Bläker, Hendrik; Sers, Christine

    2015-01-01

    Colorectal cancer (CRC) is characterized by recurrent mutations deregulating key cell signaling cascades and providing the cancer cells with novel functional traits. Among the most frequent mutations in CRC are gain-of-function missense mutations in KRAS and BRAF. Oncogenic activation of KRAS and BRAF is mutually exclusive and occurs in approximately 40% and 10% of all CRCs, respectively. Here we summarize genetic alterations currently described in the literature and databases, indicating overlapping but also specific co-occurrences with either mutated BRAF or KRAS. We describe common and potentially specific biological functions of KRAS and BRAF oncoproteins in the intestinal epithelial cells and during initiation and progression of CRC. We discuss signal transduction networks, highlighting individual functions of oncogenic KRAS and BRAF in terms of feedback loops and their impact on treatment outcome. Finally, we give an update on current strategies of targeted therapeutic intervention in oncogenic RAS-RAF signaling networks for the treatment of metastatic CRC and outline future directions. PMID:26299805

  4. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

    PubMed Central

    Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

    2015-01-01

    Background Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies. PMID:26540293

  5. Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias

    PubMed Central

    2010-01-01

    Background Gene mutation is an important mechanism of myeloid leukemogenesis. However, the number and combination of gene mutated in myeloid malignancies is still a matter of investigation. Methods We searched for mutations in the ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in 65 myelodysplastic syndromes (MDSs) and 64 acute myeloid leukemias (AMLs) without balanced translocation or complex karyotype. Results Mutations in ASXL1 and CBL were frequent in refractory anemia with excess of blasts. Mutations in TET2 occurred with similar frequency in MDSs and AMLs and associated equally with either ASXL1 or NPM1 mutations. Mutations of RUNX1 were mutually exclusive with TET2 and combined with ASXL1 but not with NPM1. Mutations in FLT3 (mutation and internal tandem duplication), IDH1, IDH2, NPM1 and WT1 occurred primarily in AMLs. Conclusion Only 14% MDSs but half AMLs had at least two mutations in the genes studied. Based on the observed combinations and exclusions we classified the 12 genes into four classes and propose a highly speculative model that at least a mutation in one of each class is necessary for developing AML with simple or normal karyotype. PMID:20678218

  6. Genetic events that limit the efficacy of MEK and RTK inhibitor therapies in a mouse model of KRAS-driven pancreatic cancer

    PubMed Central

    Pettazzoni, Piergiorgio; Viale, Andrea; Shah, Parantu; Carugo, Alessandro; Ying, Haoqiang; Wang, Huamin; Genovese, Giannicola; Seth, Sahil; Minelli, Rosalba; Green, Tessa; Huang-Hobbs, Emmet; Corti, Denise; Sanchez, Nora; Nezi, Luigi; Marchesini, Matteo; Kapoor, Avnish; Yao, Wantong; Di Francesco, Maria Emilia; Petrocchi, Alessia; Deem, Angela K.; Scott, Kenneth; Colla, Simona; Mills, Gordon B.; Fleming, Jason B.; Heffernan, Timothy P.; Jones, Philip; Toniatti, Carlo; DePinho, Ronald A.; Draetta, Giulio F.

    2014-01-01

    Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction to pharmacological inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis while MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTKs) expressed in a large proportion of human PDAC samples analyzed. While single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three co-activated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. Additionally, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. PMID:25736685

  7. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    PubMed Central

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  8. The role of antiangiogenic agents in the treatment of patients with advanced colorectal cancer according to K-RAS status.

    PubMed

    García-Alfonso, Pilar; Grande, Enrique; Polo, Eduardo; Afonso, Ruth; Reina, Juan José; Jorge, Mónica; Campos, Juan Manuel; Martínez, Virginia; Angeles, Cristina; Montagut, Clara

    2014-10-01

    Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer worldwide. Recently, it has been found that about 40 % of patients with CRC have mutations in the K-RAS gene. Several clinical trials have showed that patients with metastatic colorectal cancer (mCRC) who present tumour-promoting mutations in signalling pathways involving the epidermal growth factor receptor (EGFR), which includes activating K-RAS mutations, do not respond to anti-EGFR drugs such as panitumumab and cetuximab. Hence, K-RAS status is now considered an important negative predictive factor for response to anti-EGFR drugs. Moreover, K-RAS status seems to have also a prognostic role in CRC, but this fact is somewhat controversial. Activity of antiangiogenic agents seems not to be influenced by K-RAS gene status. Tumour angiogenesis has attracted interest in attempts to improve the management of mCRC. The vascular endothelial growth factor (VEGF) pathway is fundamental to the regulation of angiogenesis, and research has focused on developing agents that selectively target it. In this way, the anti-VEGF antibody bevacizumab in combination with chemotherapy has provided important clinical benefits in terms of response rate, progression-free survival and overall survival to patients with mCRC. Efficacy data of bevacizumab in K-RAS wild-type patients seem to be comparable with the efficacy data observed with anti-EGFR therapies in a cross-trial comparison. Although there is a lack of prospective and randomized data in this setting, the combination of chemotherapy plus antiangiogenic agents could be considered as an effective alternative for the treatment of mCRC with independence of K-RAS gene status. Here, we review the available data we have in the literature of the use of antiangiogenic strategies in the treatment of mCRC nowadays. PMID:24793846

  9. A combination therapy for KRAS-driven lung adenocarcinomas using lipophilic bisphosphonates and rapamycin

    PubMed Central

    Xia, Yifeng; Liu, Yi-Liang; Xie, Yonghua; Zhu, Wei; Guerra, Francisco; Shen, Shen; Yeddula, Narayana; Fischer, Wolfgang; Low, William; Zhou, Xiaoying; Zhang, Yonghui; Oldfield, Eric; Verma, Inder M.

    2015-01-01

    Lung cancer is the most common human malignancy and leads to about one-third of all cancer-related deaths. Lung adenocarcinomas harboring KRAS mutations, in contrast to those with EGFR and EML4-ALK mutations, have not yet been successfully targeted. Here, we describe a combination therapy for treating these malignancies using two agents: a lipophilic bisphosphonate and rapamycin. This drug combination is much more effective than either agent acting alone in the KRAS G12D induced mouse lung model. Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of the KRAS and other small G-proteins critical for tumor growth and cell survival. Bisphosphonate treatment of cells initiated autophagy but was ultimately unsuccessful and led to p62 accumulation and concomitant NF-κB activation, resulting in dampened efficacy in vivo. However, we found that rapamycin, in addition to inhibiting the mTOR pathway, facilitated autophagy and prevented p62 accumulation-induced NF-κB activation and tumor cell proliferation. Overall, these results suggest that using lipophilic bisphosphonates in combination with rapamycin may provide an effective strategy for targeting lung adenocarcinomas harboring KRAS mutations. PMID:25411474

  10. LYTAK1, a novel TAK1 inhibitor, suppresses KRAS mutant colorectal cancer cell growth in vitro and in vivo.

    PubMed

    Zhou, Jundong; Zheng, Bing; Ji, Jiansong; Shen, Fei; Min, Han; Liu, Biao; Wu, Jinchang; Zhang, Shuyu

    2015-05-01

    KRAS mutation in colorectal cancer (CRC) activates transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) to promote tumor progression. In the current study, we explored the potential effect of LYTAK1, a novel TAK1 inhibitor, against KRAS mutant CRC cells in vitro and in vivo. We found that LYTAK1 dose-dependently inhibited KRAS mutant CRC cell (HT-29 and SW-620 lines) growth, and induced cell cycle G1-S arrest. Further, LYTAK1 activated apoptosis in HT-29 cells and SW-620 cells, and apoptosis inhibitors almost reversed LYTAK1-mediated growth inhibition. While in KRAS wild-type (WT) CRC cell lines (DLD-1 and HCT-116), LYTAK1 had almost no effect on cell growth, cell cycle progression, or cell apoptosis. In KRAS mutant HT-29 cells and SW-260 cells, LYTAK1 blocked TAK1 activation or phosphorylation at Thr-184/187. Activation of nuclear factor κB (NF-κB) in these cells, detected by phosphorylations of p65 and IκB kinase α (IKKα) as well as expression of NF-κB-regulated gene cyclin D1, was significantly inhibited by LYTAK1. Further, LYTAK1 treatment resulted in downregulation of β-catenin and Wnt response gene Axin 2, indicating Wnt inactivation. In vivo, oral LYTAK1 significantly inhibited HT-29 xenograft growth in nude mice. Together, these results show that LYTAK1 inhibits KRAS mutant CRC cell growth both in vitro and in vivo. LYTAK1 might be investigated as a novel agent against CRC with KRAS mutation. PMID:25524577

  11. Performance and Cost Efficiency of KRAS Mutation Testing for Metastatic Colorectal Cancer in Routine Diagnosis: The MOKAECM Study, a Nationwide Experience

    PubMed Central

    Chatellier, Gilles; Côté, Jean-François; Pages, Jean-Christophe; de Fraipont, Florence; Boyer, Jean-Christophe; Merlio, Jean Philippe; Morel, Alain; Gorisse, Marie-Claude; de Cremoux, Patricia; Leroy, Karen; Milano, Gérard; Ouafik, L’Houcine; Merlin, Jean-Louis; Le Corre, Delphine; Aucouturier, Pascaline; Sabourin, Jean-Christophe; Nowak, Frédérique; Frebourg, Thierry; Emile, Jean-François; Durand-Zaleski, Isabelle; Laurent-Puig, Pierre

    2013-01-01

    Purpose Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs. Methods 96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists. Results This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0. Conclusion The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment. PMID:23935912

  12. KRAS Engages AGO2 to Enhance Cellular Transformation.

    PubMed

    Shankar, Sunita; Pitchiaya, Sethuramasundaram; Malik, Rohit; Kothari, Vishal; Hosono, Yasuyuki; Yocum, Anastasia K; Gundlapalli, Harika; White, Yasmine; Firestone, Ari; Cao, Xuhong; Dhanasekaran, Saravana M; Stuckey, Jeanne A; Bollag, Gideon; Shannon, Kevin; Walter, Nils G; Kumar-Sinha, Chandan; Chinnaiyan, Arul M

    2016-02-16

    Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling. PMID:26854235

  13. GNA14 Somatic Mutation Causes Congenital and Sporadic Vascular Tumors by MAPK Activation.

    PubMed

    Lim, Young H; Bacchiocchi, Antonella; Qiu, Jingyao; Straub, Robert; Bruckner, Anna; Bercovitch, Lionel; Narayan, Deepak; McNiff, Jennifer; Ko, Christine; Robinson-Bostom, Leslie; Antaya, Richard; Halaban, Ruth; Choate, Keith A

    2016-08-01

    Vascular tumors are among the most common neoplasms in infants and children; 5%-10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS, KRAS, NRAS, GNAQ, and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention. PMID:27476652

  14. Effectors and potential targets selectively upregulated in human KRAS-mutant lung adenocarcinomas

    PubMed Central

    Li, Jinyu; Sordella, Raffaella; Powers, Scott

    2016-01-01

    Genetic and proteomic analysis of human tumor samples can provide an important compliment to information obtained from model systems. Here we examined protein and gene expression from the Cancer Genome and Proteome Atlases (TCGA and TCPA) to characterize proteins and protein-coding genes that are selectively upregulated in KRAS-mutant lung adenocarcinomas. Phosphoprotein activation of several MAPK signaling components was considerably stronger in KRAS-mutants than any other group of tumors, even those with activating mutations in receptor tyrosine kinases (RTKs) and BRAF. Co-occurring mutations in KRAS-mutants were associated with differential activation of PDK1 and PKC-alpha. Genes showing strong activation in RNA-seq data included negative regulators of RTK/RAF/MAPK signaling along with potential oncogenic effectors including activators of Rac and Rho proteins and the receptor protein-tyrosine phosphatase genes PTPRM and PTPRE. These results corroborate RAF/MAPK signaling as an important therapeutic target in KRAS-mutant lung adenocarcinomas and pinpoint new potential targets. PMID:27301828

  15. A randomized Phase II clinical study of combining panitumumab and bevacizumab, plus irinotecan, 5-fluorouracil, and leucovorin (FOLFIRI) compared with FOLFIRI alone as second-line treatment for patients with metastatic colorectal cancer and KRAS mutation

    PubMed Central

    Liu, Yuguo; Luan, Lijuan; Wang, Xingli

    2015-01-01

    Background This study investigated the efficacy and safety of a new treatment strategy of combining panitumumab and bevacizumab, plus irinotecan, 5-fluorouracil, and leucovorin (FOLFIRI) versus FOLFIRI alone as second-line chemotherapy for metastatic colorectal cancer (mCRC) patients with known V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) mutation status. Methods Patients with mCRC who had known KRAS tumor status and unsuccessful previous oxaliplatin-based chemotherapy were included in the study. They were randomly assigned to two groups to receive panitumumab and bevacizumab plus FOLFIRI, or FOLFIRI alone. In panitumumab and bevacizumab plus FOLFIRI group, patients were given 4 mg/kg panitumumab and bevacizumab plus FOLFIRI every 2 weeks. Results In all, 65 patients were assigned to panitumumab and bevacizumab plus FOLFIRI group, and 77 to FOLFIRI alone group. For WT KRAS patients, the median progression-free survival (PFS) was 5.7 months (95% confidence interval [CI], 2.4–7.5 months) for panitumumab and bevacizumab plus FOLFIRI and 3.8 months (95% CI, 3.0–6.7 months) for FOLFIRI alone; median overall survival (OS) was 15.2 months (95% CI, 8.9–19.7 months) for panitumumab and bevacizumab plus FOLFIRI and 11.0 months (95% CI, 8.2–15.4 months) for FOLFIRI alone. For MU KRAS patients, median PFS was 5.1 months (95% CI, 2.7–10.2 months) for panitumumab and bevacizumab plus FOLFIRI and 4.1 months (95% CI, 2.5–8.4 months) for FOLFIRI alone; median OS was 12.8 months (95% CI, 7.8–15.8 months) for panitumumab and bevacizumab plus FOLFIRI and 10.5 months (95% CI, 6.1–15.3 months) for FOLFIRI alone. Grade 3 and 4 adverse events were associated with panitumumab and bevacizumab plus FOLFIRI but tolerable among patients. Conclusion Patients with mCRC can be safely and efficiently treated with second-line chemotherapy of combining panitumumab and bevacizumab plus FOLFIRI, despite their KRAS mutation status. PMID:25999741

  16. Wild-type KRAS inhibits oncogenic KRAS-induced T-ALL in mice.

    PubMed

    Staffas, A; Karlsson, C; Persson, M; Palmqvist, L; Bergo, M O

    2015-05-01

    The role of hyperactive RAS signaling is well established in myeloid malignancies but less clear in T-cell malignancies. The Kras2(LSL)Mx1-Cre (KM) mouse model expresses endogenous KRAS(G12D) in hematopoietic cells and is widely used to study mechanisms and treatment of myeloproliferative neoplasms (MPN). The model displays an intriguing shift from MPN to acute T-cell leukemia (T-ALL) after transplantation to wild-type mice, but the mechanisms underlying this lineage shift is unknown. Here, we show that KRAS(G12D) increases proliferation of both myeloid and T-cell progenitors, but whereas myeloid cells differentiate, T-cell differentiation is inhibited at early stages. Secondary mutations in the expanded pool of T-cell progenitors accompany T-ALL development, and our results indicate that the shift from myeloid to T-lymphoid malignancy after transplantation is explained by the increased likelihood for secondary mutations when the tumor lifespan is increased. We demonstrate that tumor lifespan increases after transplantation because primary KM mice die rapidly, not from MPN, but from KRAS(G12D) expression in nonhematopoietic cells, which causes intestinal bleeding and severe anemia. We also identify loss of the wild-type KRAS allele as a secondary mutation in all T-ALL cells and provide evidence that wild-type KRAS acts as a tumor suppressor in the T-cell lineage in mice. PMID:25371176

  17. Selective Targeting of the KRAS G12C Mutant: Kicking KRAS When It's Down.

    PubMed

    Hobbs, G Aaron; Wittinghofer, Alfred; Der, Channing J

    2016-03-14

    Two recent studies evaluated a small molecule that specifically binds to and inactivates the KRAS G12C mutant. The new findings argue that the perception that mutant KRAS is persistently frozen in its active GTP-bound form may not be accurate. PMID:26977877

  18. The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

    PubMed

    Zaanan, Aziz; Okamoto, Koichi; Kawakami, Hisato; Khazaie, Khashayarsha; Huang, Shengbing; Sinicrope, Frank A

    2015-09-25

    In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response. PMID:26245900

  19. Comparative analysis of radiosensitizers for K-RAS mutant rectal cancers.

    PubMed

    Kleiman, Laura B; Krebs, Angela M; Kim, Stephen Y; Hong, Theodore S; Haigis, Kevin M

    2013-01-01

    Approximately 40% of rectal cancers harbor activating K-RAS mutations, and these mutations are associated with poor clinical response to chemoradiotherapy. We aimed to identify small molecule inhibitors (SMIs) that synergize with ionizing radiation (IR) ("radiosensitizers") that could be incorporated into current treatment strategies for locally advanced rectal cancers (LARCs) expressing mutant K-RAS. We first optimized a high-throughput assay for measuring individual and combined effects of SMIs and IR that produces similar results to the gold standard colony formation assay. Using this screening platform and K-RAS mutant rectal cancer cell lines, we tested SMIs targeting diverse signaling pathways for radiosensitizing activity and then evaluated our top hits in follow-up experiments. The two most potent radiosensitizers were the Chk1/2 inhibitor AZD7762 and the PI3K/mTOR inhibitor BEZ235. The chemotherapeutic agent 5-fluorouracil (5-FU), which is used to treat LARC, synergized with AZD7762 and enhanced radiosensitization by AZD7762. This study is the first to compare different SMIs in combination with IR for the treatment of K-RAS mutant rectal cancer, and our findings suggest that Chk1/2 inhibitors should be evaluated in new clinical trials for LARC. PMID:24349411

  20. GNAS mutation as an alternative mechanism of activation of the Wnt/β-catenin signaling pathway in gastric adenocarcinoma of the fundic gland type.

    PubMed

    Nomura, Ryosuke; Saito, Tsuyoshi; Mitomi, Hiroyuki; Hidaka, Yasuhiro; Lee, Se-yong; Watanabe, Sumio; Yao, Takashi

    2014-12-01

    Gastric adenocarcinoma of the fundic gland type (GAFG) is a rare variant of gastric tumor. We have recently reported the frequent accumulation of β-catenin in GAFGs and showed that approximately half of the cases studied harbored at least 1 mutation in CTNNB1/AXINs/APC, leading to the constitutive activation of the Wnt/β-catenin pathway. However, the mechanisms of Wnt signaling activation in the remaining cases are unknown. Accumulating evidence showed that the activating mutation in GNAS promotes tumorigenesis via the activation of the Wnt/β-catenin pathway or the ERK1/2 MAPK pathway. Therefore, we analyzed the mutations in GNAS (exons 8 and 9) and in KRAS (exon 2) in 26 GAFGs. Immunohistochemistry revealed nuclear β-catenin expression in 22 of 26 GAFGs, and 10 (38.5%) of 26 cases harbored at least 1 mutation in CTNNB1/AXINs/APC. Activating mutations in GNAS were found in 5 (19.2%) of 26 GAFGs, all of which harbored R201C mutations. Activating mutations in KRAS were found in 2 (7.7%) of 26 GAFGs, and both of these also contained GNAS activating mutations. Four of 5 cases with GNAS mutation showed nuclear β-catenin expression, and presence of GNAS mutation was associated with β-catenin nuclear expression (P = .01). Furthermore, 3 of these 4 cases did not harbor mutations in CTNNB1, APC, or AXINs, suggesting that mutations in the Wnt component genes and those in GNAS occur almost exclusively. These results suggest that GNAS mutation might occur in a small subset of GAFG as an alternative mechanism of activating the Wnt/β-catenin signaling pathway. PMID:25288233

  1. Colorectal cancer-related mutant KRAS alleles function as positive regulators of autophagy

    PubMed Central

    Alves, Sara; Castro, Lisandra; Fernandes, Maria Sofia; Francisco, Rita; Castro, Paula; Priault, Muriel; Chaves, Susana Rodrigues; Moyer, Mary Pat; Oliveira, Carla; Seruca, Raquel; Côrte-Real, Manuela

    2015-01-01

    The recent interest to modulate autophagy in cancer therapy has been hampered by the dual roles of this conserved catabolic process in cancer, highlighting the need for tailored approaches. Since RAS isoforms have been implicated in autophagy regulation and mutation of the KRAS oncogene is highly frequent in colorectal cancer (CRC), we questioned whether/how mutant KRAS alleles regulate autophagy in CRC and its implications. We established two original models, KRAS-humanized yeast and KRAS-non-cancer colon cells and showed that expression of mutated KRAS up-regulates starvation-induced autophagy in both. Accordingly, KRAS down-regulation inhibited autophagy in CRC-derived cells harboring KRAS mutations. We further show that KRAS-induced autophagy proceeds via up-regulation of the MEK/ERK pathway in both colon models and that KRAS and autophagy contribute to CRC cell survival during starvation. Since KRAS inhibitors have proven difficult to develop, our results suggest using autophagy inhibitors as a combined/alternative therapeutic approach in CRCs with mutant KRAS. PMID:26418750

  2. MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice

    PubMed Central

    Cogoi, Susanna; Zorzet, Sonia; Rapozzi, Valentina; Géci, Imrich; Pedersen, Erik B.; Xodo, Luigi E.

    2013-01-01

    KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3′-end, whereas to enhance the folding and stability, two polycyclic aromatic hydrocarbon units (TINA or AMANY) were inserted internally, to cap the quadruplex. The most active G4-decoy (2998), which had two para-TINAs, strongly suppressed KRAS expression in Panc-1 cells. It also repressed their metabolic activity (IC50 = 520 nM), and it inhibited cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased the Kaplan–Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy. PMID:23471001

  3. Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer.

    PubMed

    Hutton, Josiah E; Wang, Xiaojing; Zimmerman, Lisa J; Slebos, Robbert J C; Trenary, Irina A; Young, Jamey D; Li, Ming; Liebler, Daniel C

    2016-09-01

    Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu. PMID:27340238

  4. Targeting cytokine networks in KRAS-driven tumorigenesis.

    PubMed

    Golay, Hadrien G; Barbie, David A

    2014-08-01

    KRAS is one of the most commonly mutated oncogenes in human tumors, and is typically associated with aggressive disease. Despite intensive study and years of effort, KRAS has remained refractory to targeted inhibition. Given the challenge of inhibiting KRAS directly, current approaches to KRAS targeted therapy have involved the disruption of downstream signaling pathways. However, combinations of drugs that target RAF/MEK and PI3K/AKT signaling have failed to live up to expectations in the clinic. Here we summarize the evidence that the cytokine signaling circuitry of KRAS-driven tumors represents an equally tractable drug target. Indeed, the incorporation of novel therapeutics that disrupts these cytokine signaling networks may hold the key to overcoming this seemingly impenetrable treatment barrier. PMID:24928447

  5. ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

    PubMed Central

    Wang, Lidong; Yang, Huibin; Abel, Ethan V.; Ney, Gina M.; Palmbos, Phillip L.; Bednar, Filip; Zhang, Yaqing; Leflein, Jacob; Waghray, Meghna; Owens, Scott; Wilkinson, John E.; Prasad, Jayendra; Ljungman, Mats; Rhim, Andrew D.; Pasca di Magliano, Marina

    2015-01-01

    The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT. PMID:25593307

  6. KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes.

    PubMed

    McKenzie, Andrew J; Hoshino, Daisuke; Hong, Nan Hyung; Cha, Diana J; Franklin, Jeffrey L; Coffey, Robert J; Patton, James G; Weaver, Alissa M

    2016-05-01

    Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes. PMID:27117408

  7. Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance | Poster

    Cancer.gov

    Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

  8. Overview on how oncogenic Kras promotes pancreatic carcinogenesis by inducing low intracellular ROS levels.

    PubMed

    Kong, Bo; Qia, Chengjia; Erkan, Mert; Kleeff, Jörg; Michalski, Christoph W

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease without clearly known disease causes. Recent epidemiological and animal studies suggest that the supplementation of dietary antioxidants (e.g., vitamins C and E) decreases cancer risk, implying that increased reactive oxygen species (ROS) may play a role in pancreatic carcinogenesis. However, oncogenic Kras mutations (e.g., Kras(G12D)), which are present in more than 90% of PDAC, have been proven to foster low intracellular ROS levels. Here, oncogenic Kras activates expression of a series of anti-oxidant genes via Nrf2 (nuclear factor, erythroid derived 2, like 2) and also mediates an unusual metabolic pathway of glutamine to generate NADPH. This can then be used as the reducing power for ROS detoxification, leading collectively to low ROS levels in pancreatic pre-neoplastic cells and in cancer cells. In adult stem cells and cancer stem cells, low ROS levels have been associated with the formation of a proliferation-permissive intracellular environment and with perseverance of self-renewal capacities. Therefore, it is conceivable that low intracellular ROS levels may contribute significantly to oncogenic Kras-mediated PDAC formation. PMID:24062691

  9. Pancreatitis promotes oncogenic KrasG12D-induced pancreatic transformation through activation of Nupr1

    PubMed Central

    Grasso, Daniel; Garcia, Maria Noé; Hamidi, Tewfik; Cano, Carla; Calvo, Ezequiel; Lomberk, Gwen; Urrutia, Raul; Iovanna, Juan L

    2014-01-01

    During the initiation stage of pancreatic adenocarcinoma induced by oncogenic Kras, pancreatic cells are exposed to both a protumoral effect and an opposing tumor suppressive process known as oncogene-induced senescence. Pancreatitis disrupts this balance in favor of the transforming effect of oncogenes by lowering the tumor suppressive threshold of oncogene-induced senescence through expression of the stress protein Nupr1. PMID:27308320

  10. Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma.

    PubMed

    Yeddula, Narayana; Xia, Yifeng; Ke, Eugene; Beumer, Joep; Verma, Inder M

    2015-11-24

    Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS-RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers. PMID:26542681

  11. Cten Is Targeted by Kras Signalling to Regulate Cell Motility in the Colon and Pancreas

    PubMed Central

    Al-Ghamdi, Saleh; Albasri, Abdulkader; Cachat, Julien; Ibrahem, Salih; Muhammad, Belal A.; Jackson, Darryl; Nateri, Abdolrahman S.; Kindle, Karin B.; Ilyas, Mohammad

    2011-01-01

    CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down (using small interfering (si)RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown (transwell migration and wounding assay, p<0.001 for both). Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 (transwell migration assay, p<0.001). We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility. PMID:21698197

  12. Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

    PubMed

    Tape, Christopher J; Ling, Stephanie; Dimitriadi, Maria; McMahon, Kelly M; Worboys, Jonathan D; Leong, Hui Sun; Norrie, Ida C; Miller, Crispin J; Poulogiannis, George; Lauffenburger, Douglas A; Jørgensen, Claus

    2016-05-01

    Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT. PMID:27087446

  13. KRAS-Mutant Lung Cancers in the Era of Targeted Therapy.

    PubMed

    Naidoo, Jarushka; Drilon, Alexander

    2016-01-01

    KRAS-mutant lung cancers account for approximately 25% of non-small cell lung carcinomas, thus representing an enormous burden of cancer worldwide. KRAS mutations are clear drivers of tumor growth and are characterized by a complex biology involving the interaction between mutant KRAS, various growth factor pathways, and tumor suppressor genes. While KRAS mutations are classically associated with a significant smoking history, they are also identified in a substantial proportion of never-smokers. These mutations are found largely in lung adenocarcinomas with solid growth patterns and tumor-infiltrating lymphocytes. A variety of tools are available for diagnosis including Sanger sequencing, multiplex mutational hotspot profiling, and next-generation sequencing. The prognostic and predictive roles of KRAS status remain controversial. It has become increasingly clear, however, that KRAS mutations drive primary resistance to EGFR tyrosine kinase inhibition. Until recently, mutant KRAS was not thought of as a clinically-targetable driver in lung cancers. With the expansion of our knowledge regarding the biology of KRAS-mutant lung cancers and the role of MEK and PI3K/mTOR inhibition, the face of targeted therapeutics for this genomic subset of patients is slowly beginning to change. PMID:26667343

  14. KRAS Genotypic Changes of Circulating Tumor Cells during Treatment of Patients with Metastatic Colorectal Cancer

    PubMed Central

    Kalikaki, Aristea; Politaki, Helen; Souglakos, John; Apostolaki, Stella; Papadimitraki, Elisavet; Georgoulia, Nefeli; Tzardi, Maria; Mavroudis, Dimitris; Georgoulias, Vassilis; Voutsina, Alexandra

    2014-01-01

    Introduction Circulating tumor cells (CTCs) could represent a non-invasive source of cancer cells used for longitudinal monitoring of the tumoral mutation status throughout the course of the disease. The aims of the present study were to investigate the detection of KRAS mutations in CTCs from patients with metastatic colorectal cancer (mCRC) and to compare their mutation status during treatment or disease progression with that of the corresponding primary tumors. Materials and Methods Identification of the seven most common KRAS mutations on codons 12 and 13 was performed by Peptide Nucleic Acid (PNA)-based qPCR method. The sensitivity of the assay was determined after isolation of KRAS mutant cancer cells spiked into healthy donors' blood, using the CellSearch Epithelial Cell kit. Consistent detection of KRAS mutations was achieved in samples containing at least 10 tumor cells/7.5 ml of blood. Results The clinical utility of the assay was assessed in 48 blood samples drawn from 31 patients with mCRC. All patients had PIK3CA and BRAF wild type primary tumors and 14 KRAS mutant tumors. CTCs were detected in 65% of specimens obtained from 74% of patients. KRAS mutation analysis in CTC-enriched specimens showed that 45% and 16.7% of patients with mutant and wild type primary tumors, respectively, had detectable mutations in their CTCs. Assessing KRAS mutations in serial blood samples revealed that individual patient's CTCs exhibited different mutational status of KRAS during treatment. Conclusions The current findings support the rationale for using the CTCs as a dynamic source of tumor cells which, by re-evaluating their KRAS mutation status, could predict, perhaps more accurately, the response of mCRC patients to targeted therapy. PMID:25137394

  15. Estrogen withdrawal, increased breast cancer risk and the KRAS-variant

    PubMed Central

    McVeigh, Terri P; Jung, Song-Yi; Kerin, Michael J; Salzman, David W; Nallur, Sunitha; Nemec, Antonio A; Dookwah, Michelle; Sadofsky, Jackie; Paranjape, Trupti; Kelly, Olivia; Chan, Elcie; Miller, Nicola; Sweeney, Karl J; Zelterman, Daniel; Sweasy, Joann; Pilarski, Robert; Telesca, Donatello; Slack, Frank J; Weidhaas, Joanne B

    2015-01-01

    The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42–37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer. PMID:25961464

  16. Estrogen withdrawal, increased breast cancer risk and the KRAS-variant.

    PubMed

    McVeigh, Terri P; Jung, Song-Yi; Kerin, Michael J; Salzman, David W; Nallur, Sunitha; Nemec, Antonio A; Dookwah, Michelle; Sadofsky, Jackie; Paranjape, Trupti; Kelly, Olivia; Chan, Elcie; Miller, Nicola; Sweeney, Karl J; Zelterman, Daniel; Sweasy, Joann; Pilarski, Robert; Telesca, Donatello; Slack, Frank J; Weidhaas, Joanne B

    2015-01-01

    The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42-37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer. PMID:25961464

  17. KRAS as a Therapeutic Target

    PubMed Central

    McCormick, Frank

    2015-01-01

    KRAS proteins play a major role in human cancer, but have not yielded to therapeutic attack. New technologies in drug discovery and insights into signaling pathways that KRAS controls have promoted renewed efforts to develop therapies, either through direct targeting of KRAS itself, new ways of blocking KRAS processing, or by identifying targets that KRAS cancers depend on for survival. While drugs that block the well-established downstream pathways, RAF-MAPK and PI 3 kinase, are being tested in the clinic, new efforts are underway to exploit previously unrecognized vulnerabilities, such as altered metabolic networks, or novel pathways identified through synthetic lethal screens. Furthermore, new ways of suppressing KRAS gene expression and of harnessing the immune system offer further hope that new ways of treating KRAS are finally coming into view. These issues are discussed in this edition of CCR Focus. PMID:25878360

  18. Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells.

    PubMed

    Chu, P-C; Yang, M-C; Kulp, S K; Salunke, S B; Himmel, L E; Fang, C-S; Jadhav, A M; Shan, Y-S; Lee, C-T; Lai, M-D; Shirley, L A; Bekaii-Saab, T; Chen, C-S

    2016-07-28

    Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer. PMID:26616862

  19. CXCR2 signaling regulates KRAS(G12D)-induced autocrine growth of pancreatic cancer

    PubMed Central

    Purohit, Abhilasha; Varney, Michelle; Rachagani, Satyanarayana; Ouellette, Michel M.; Batra, Surinder K.; Singh, Rakesh K.

    2016-01-01

    Pharmacological inhibition of RAS, the master regulator of pancreatic ductal adenocarcinoma (PDAC), continues to be a challenge. Mutations in various isoforms of RAS gene, including KRAS are known to upregulate CXC chemokines; however, their precise role in KRAS-driven pancreatic cancer remains unclear. In this report, we reveal a previously unidentified tumor cell-autonomous role of KRAS(G12D)-induced CXCR2 signaling in mediating growth of neoplastic PDAC cells. Progressively increasing expression of mCXCR2 and its ligands was detected in the malignant ductal cells of Pdx1-cre;LSL-Kras(G12D) mice. Knocking-down CXCR2 in KRAS(G12D)-bearing human pancreatic duct-derived cells demonstrated a significant decrease in the in vitro and in vivo tumor cell proliferation. Furthermore, CXCR2 antagonists showed selective growth inhibition of KRAS(G12D)-bearing cells in vitro. Intriguingly, both genetic and pharmacological inhibition of CXCR2 signaling in KRAS(G12D)-bearing pancreatic ductal cells reduced the levels of KRAS protein, strongly implying the presence of a KRAS-CXCR2 feed-forward loop. Together, these data demonstrate the role of CXCR2 signaling in KRAS(G12D)-induced growth transformation and progression in PDAC. PMID:26771140

  20. Pulmonary adenocarcinoma in situ: analyses of a large series with reference to smoking, driver mutations, and receptor tyrosine kinase pathway activation.

    PubMed

    Sato, Seijiro; Motoi, Noriko; Hiramatsu, Miyako; Miyauchi, Eisaku; Ono, Hiroshi; Saito, Yuichi; Nagano, Hiroko; Ninomiya, Hironori; Inamura, Kentaro; Uehara, Hirofumi; Mun, Mingyon; Sakao, Yukinori; Okumura, Sakae; Tsuchida, Masanori; Ishikawa, Yuichi

    2015-07-01

    Lung adenocarcinomas in situ (AISs) often occur in individuals who have never smoked, although smoking is one of the main causes of lung cancer. To characterize AIS and, in particular, determine how AIS might be related to smoking, we collected a large number of AIS cases and examined clinicopathologic features, EGFR and KRAS mutation status, and activation status of receptor tyrosine kinase downstream signal pathways, including pAkt, pERK, and pStat3, using immunohistochemistry. We identified 110 AISs (36 smokers and 74 nonsmokers) among 1549 adenocarcinomas resected surgically during 1995 to 2010. Between the AIS of smokers and nonsmokers, only the sex ratio was significantly different; all the other clinicopathologic factors including TTF-1 and driver mutations were not significantly different: EGFR and KRAS mutation rates (smokers:nonsmokers) were 61:58 (%) (P=0.7) and 6.1:1.4 (%) (P=0.2), respectively, whereas, in invasive adenocarcinomas, the rates were 41:69 (%) (P<0.001) and 9.4:2.3 (%) (P<0.04), respectively. For pAkt and pERK, around 40% to 50% of AISs were positive, and for pStat3, >80% were positive, with no significant differences between smokers and nonsmokers with AIS. Mucinous AIS (n=8) rarely harbored KRAS mutations and expressed significantly less pStat3 (P<0.001) than nonmucinous AIS. Taken together, AIS occurs predominantly in female individuals and nonsmokers. However, characteristics of AIS arising in smokers and nonsmokers were similar in terms of cell lineage, driver mutations, and receptor tyrosine kinase pathway activation. Our results suggest that smoking is not a major cause of AIS. Rather, smoking may play a role in progression of AIS to invasive adenocarcinoma with AIS features. PMID:25970685

  1. Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

    PubMed

    Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y

    2016-06-01

    The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. PMID:26811072

  2. Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.

    PubMed

    Cho, Kwang-Jin; van der Hoeven, Dharini; Zhou, Yong; Maekawa, Masashi; Ma, Xiaoping; Chen, Wei; Fairn, Gregory D; Hancock, John F

    2015-01-01

    K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization. PMID:26572827

  3. Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

    PubMed Central

    Cho, Kwang-jin; van der Hoeven, Dharini; Zhou, Yong; Maekawa, Masashi; Ma, Xiaoping; Chen, Wei

    2015-01-01

    K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization. PMID:26572827

  4. Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

    PubMed Central

    Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J.; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O’Brien, Mary; Turner, Nicholas C.; Popat, Sanjay; Downward, Julian

    2015-01-01

    Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

  5. Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

    PubMed

    Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O'Brien, Mary; Turner, Nicholas C; Popat, Sanjay; Downward, Julian

    2015-01-01

    Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

  6. A combinatorial strategy for treating KRAS-mutant lung cancer.

    PubMed

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T; Gainor, Justin F; Corcoran, Ryan B; Engelman, Jeffrey A; Rudin, Charles M; Rosen, Neal; Lowe, Scott W

    2016-06-30

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

  7. Multidimensional Screening Platform for Simultaneously Targeting Oncogenic KRAS and Hypoxia-Inducible Factors Pathways in Colorectal Cancer.

    PubMed

    Bousquet, Michelle S; Ma, Jia Jia; Ratnayake, Ranjala; Havre, Pamela A; Yao, Jin; Dang, Nam H; Paul, Valerie J; Carney, Thomas J; Dang, Long H; Luesch, Hendrik

    2016-05-20

    Colorectal cancer (CRC) is a genetic disease, due to progressive accumulation of mutations in oncogenes and tumor suppressor genes. Large scale genomic sequencing projects revealed >100 mutations in any individual CRC. Many of these mutations are likely passenger mutations, and fewer are driver mutations. Of these, activating mutations in RAS proteins are essential for cancer initiation, progression, and/or resistance to therapy. There has been significant interest in developing drugs targeting mutated cancer gene products or downstream signaling pathways. Due to the number of mutations involved and inherent redundancy in intracellular signaling, drugs targeting one mutation or pathway have been either ineffective or led to rapid resistance. We have devised a strategy whereby multiple cancer pathways may be simultaneously targeted for drug discovery. For proof-of-concept, we targeted the oncogenic KRAS and HIF pathways, since oncogenic KRAS has been shown to be required for cancer initiation and progression, and HIF-1α and HIF-2α are induced by the majority of mutated oncogenes and tumor suppressor genes in CRC. We have generated isogenic cell lines defective in either oncogenic KRAS or both HIF-1α and HIF-2α and subjected them to multiplex genomic, siRNA, and high-throughput small molecule screening. We have identified potential drug targets and compounds for preclinical and clinical development. Screening of our marine natural product library led to the rediscovery of the microtubule agent dolastatin 10 and the class I histone deacetylase (HDAC) inhibitor largazole to inhibit oncogenic KRAS and HIF pathways. Largazole was further validated as an antiangiogenic agent in a HIF-dependent manner in human cells and in vivo in zebrafish using a genetic model with activated HIF. Our general strategy, coupling functional genomics with drug susceptibility or chemical-genetic interaction screens, enables the identification of potential drug targets and candidates with

  8. GNAS(R201H) and Kras(G12D) cooperate to promote murine pancreatic tumorigenesis recapitulating human intraductal papillary mucinous neoplasm.

    PubMed

    Taki, K; Ohmuraya, M; Tanji, E; Komatsu, H; Hashimoto, D; Semba, K; Araki, K; Kawaguchi, Y; Baba, H; Furukawa, T

    2016-05-01

    Intraductal papillary mucinous neoplasm (IPMN), the most common pancreatic cystic neoplasm, is known to progress to invasive ductal adenocarcinoma. IPMNs commonly harbor activating somatic mutations in GNAS and KRAS, primarily GNAS(R201H) and KRAS(G12D). GNAS encodes the stimulatory G-protein α subunit (Gsα) that mediates a stimulatory signal to adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP), subsequently activating cAMP-dependent protein kinase A. The GNAS(R201H) mutation results in constitutive activation of Gsα. To study the potential role of GNAS in pancreatic tumorigenesis in vivo, we generated lines of transgenic mice in which the transgene consisted of Lox-STOP-Lox (LSL)-GNAS(R201H) under the control of the CAG promoter (Tg(CAG-LSL-GNAS)). These mice were crossed with pancreatic transcription factor 1a (Ptf1a)-Cre mice (Ptf1a(Cre/+)), generating Tg(CAG-LSL-GNAS);Ptf1a(Cre/+) mice. This mouse line showed elevated cAMP levels, small dilated tubular complex formation, loss of acinar cells and fibrosis in the pancreas; however, no macroscopic tumorigenesis was apparent by 2 months of age. We then crossed Tg(CAG-LSL-GNAS);Ptf1a(Cre/+) mice with LSL-Kras(G12D) mice, generating Tg(CAG-LSL-GNAS);LSL-Kras(G12D);Ptf1a(Cre/+) mice. We used these mice to investigate a possible cooperative effect of GNAS(R201H) and Kras(G12D) in pancreatic tumorigenesis. Within 5 weeks, Tg(CAG-LSL-GNAS);LSL-Kras(G12D);Ptf1a(Cre/+) mice developed a cystic tumor consisting of marked dilated ducts lined with papillary dysplastic epithelia in the pancreas, which closely mimicked the human IPMN. Our data strongly suggest that activating mutations in GNAS and Kras cooperatively promote murine pancreatic tumorigenesis, which recapitulates IPMN. Our mouse model may serve as a unique in vivo platform to find biomarkers and effective drugs for diseases associated with GNAS mutations. PMID:26257060

  9. Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma

    PubMed Central

    Yeddula, Narayana; Xia, Yifeng; Ke, Eugene; Beumer, Joep; Verma, Inder M.

    2015-01-01

    Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS–RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers. PMID:26542681

  10. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.

    PubMed

    Lito, Piro; Solomon, Martha; Li, Lian-Sheng; Hansen, Rasmus; Rosen, Neal

    2016-02-01

    It is thought that KRAS oncoproteins are constitutively active because their guanosine triphosphatase (GTPase) activity is disabled. Consequently, drugs targeting the inactive or guanosine 5'-diphosphate-bound conformation are not expected to be effective. We describe a mechanism that enables such drugs to inhibit KRAS(G12C) signaling and cancer cell growth. Inhibition requires intact GTPase activity and occurs because drug-bound KRAS(G12C) is insusceptible to nucleotide exchange factors and thus trapped in its inactive state. Indeed, mutants completely lacking GTPase activity and those promoting exchange reduced the potency of the drug. Suppressing nucleotide exchange activity downstream of various tyrosine kinases enhanced KRAS(G12C) inhibition, whereas its potentiation had the opposite effect. These findings reveal that KRAS(G12C) undergoes nucleotide cycling in cancer cells and provide a basis for developing effective therapies to treat KRAS(G12C)-driven cancers. PMID:26841430

  11. Cis-acting elements in its 3' UTR mediate post-transcriptional regulation of KRAS.

    PubMed

    Kim, Minlee; Kogan, Nicole; Slack, Frank J

    2016-03-15

    Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3' untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3' UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3' UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3' UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3' UTR that is required for KRAS 3' UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3' UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3' UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression. PMID:26930719

  12. ACB-PCR MEASUREMENT OF K-RAS CODON 12 MUTATION IN A/J MOUSE LUNG EXPOSED TO BENZO[A]PYRENE: A DOSE-RESPONSE ASSESSMENT

    EPA Science Inventory

    Benzo[a]pyrene (B[a]P) is a known human carcinogen and environmental contaminant. The direct measurement of K-Ras mutant fraction (MF) was developed as a metric with which to examine the default assumption of low dose linearity in the mutational response to B...

  13. Killing of Kras mutant colon cancer cells via Rac-independent actin remodeling by the βGBP cytokine a physiological PI3K inhibitor therapeutically effective in vivo

    PubMed Central

    Mallucci, Livio; Shi, Dong-yun; Davies, Derek; Jordan, Peter; Nicol, Alastair; Lotti, Lavinia; Mariani-Costantini, Renato; Verginelli, Fabio; Wells, Valerie; Zicha, Daniel

    2012-01-01

    Activating mutations in Kras are the most frequent mutations in human cancer. They define a subset of patients who do not respond to current therapies and for whom prognosis is poor. Oncogenic Kras has been shown to deregulate numerous signalling pathways of which the most intensively studied are the Ras-ERK cascade and the PI3K-Akt cascade. However, to date there are no effective targeted therapies in the clinic against Kras-mutant cancers. Here we report that the βGBP cytokine, a physiological inhibitor of class I PI3Ks is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when co-harboring mutant-activated PIK3CA. Our study unveils an elective route to intrinsic and extrinsic apoptosis which involves the cytoskeleton. Early events are inhibition of PI3K activity and Rac-independent actin rearrangement assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis and Chk2 activation underscore events critical to the activation of an intrinsic apoptotic program. Clustering of CD95/Fas death receptors underscore events critical to the activation of extrinsic apoptosis. In nude mice we present the first evidence that xenograft tumor development is strongly inhibited by Hu-r-βGBP. Taken together our results open a new therapeutic opportunity against a subset of patients refractive to current treatments. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu-r-βGBP may also be therapeutically effective against other cancers harbouring activating Ras mutations as well as PIK3CA mutations. PMID:22752425

  14. Killing of Kras-mutant colon cancer cells via Rac-independent actin remodeling by the βGBP cytokine, a physiological PI3K inhibitor therapeutically effective in vivo.

    PubMed

    Mallucci, Livio; Shi, Dong-yun; Davies, Derek; Jordan, Peter; Nicol, Alastair; Lotti, Lavinia; Mariani-Costantini, Renato; Verginelli, Fabio; Wells, Valerie; Zicha, Daniel

    2012-09-01

    Activating mutations in Kras are the most frequent mutations in human cancer. They define a subset of patients who do not respond to current therapies and for whom prognosis is poor. Oncogenic Kras has been shown to deregulate numerous signaling pathways of which the most intensively studied are the Ras/extracellular signal-regulated kinase cascade and the phosphoinositide 3-kinase (PI3K)/Akt cascade. However, to date, there are no effective targeted therapies in the clinic against Kras-mutant cancers. Here, we report that the β-galactoside-binding protein (βGBP) cytokine, a physiologic inhibitor of class I PI3Ks, is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when coharboring mutant-activated PIK3CA. Our study unveils an elective route to intrinsic and extrinsic apoptosis, which involves the cytoskeleton. Early events are inhibition of PI3K activity and Rac-independent actin rearrangement assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis, and checkpoint kinase 2 activation underscore events critical to the activation of an intrinsic apoptotic program. Clustering of CD95/Fas death receptors underscore events critical to the activation of extrinsic apoptosis. In nude mice, we present the first evidence that xenograft tumor development is strongly inhibited by Hu-r-βGBP. Taken together, our results open a new therapeutic opportunity to a subset of patients refractive to current treatments. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu-r-βGBP may also be therapeutically effective against other cancers harboring activating Ras mutations as well as PIK3CA mutations. PMID:22752425

  15. KRAS/BRAF Analysis in Ovarian Low-Grade Serous Carcinoma Having Synchronous All Pathological Precursor Regions

    PubMed Central

    Nakamura, Kohei; Nakayama, Kentaro; Ishibashi, Tomoka; Ishikawa, Noriyoshi; Ishikawa, Masako; Katagiri, Hiroshi; Minamoto, Toshiko; Sato, Emi; Sanuki, Kaori; Yamashita, Hitomi; Iida, Kouji; Sultana, Razia; Kyo, Satoru

    2016-01-01

    Ovarian low-grade serous carcinoma is thought to begin as a serous cystadenoma or adenofibroma that progresses in a slow stepwise fashion. Among the low-grade serous carcinomas, there is a high frequency of activating mutations in the KRAS or BRAF genes; however, it remains unclear as to how these mutations contribute to tumor progression. This is the first report to track the histopathological progression of serous adenofibroma to low-grade serous carcinoma. Each stage was individually analyzed by pathological and molecular genetic methods to determine what differences occur between the distinct stages of progression. PMID:27128903

  16. EGFR, KRAS, BRAF, and HER-2 molecular status in brain metastases from 77 NSCLC patients

    PubMed Central

    Villalva, Claire; Duranton-Tanneur, Valérie; Guilloteau, Karline; Burel-Vandenbos, Fanny; Wager, Michel; Doyen, Jérôme; Levillain, Pierre Marie; Fontaine, Denys; Blons, Hélène; Pedeutour, Florence; Karayan-Tapon, Lucie

    2013-01-01

    The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER-2 mutations in brain metastases from non-small cell lung carcinomas (BM-NSCLC). A total of 77 samples of BM-NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER-2 mutations in BM-NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM-NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM-NSCLC whereas KRAS mutations are as frequent in BM-NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern. PMID:23930206

  17. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers

    PubMed Central

    Haley, Lisa; Tseng, Li-Hui; Zheng, Gang; Dudley, Jonathan; Anderson, Derek A; Azad, Nilofer S; Gocke, Christopher D; Eshleman, James R; Lin, Ming-Tseh

    2015-01-01

    Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2–20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2–4, 48% when testing for KRAS and NRAS exons 2–4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a

  18. Activating STAT6 mutations in follicular lymphoma

    PubMed Central

    Yildiz, Mehmet; Li, Hongxiu; Bernard, Denzil; Amin, Nisar A.; Ouillette, Peter; Jones, Siân; Saiya-Cork, Kamlai; Parkin, Brian; Jacobi, Kathryn; Shedden, Kerby; Wang, Shaomeng; Chang, Alfred E.; Kaminski, Mark S.

    2015-01-01

    Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell–based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) –induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4–induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis. PMID:25428220

  19. Proteome analysis for downstream targets of oncogenic KRAS--the potential participation of CLIC4 in carcinogenesis in the lung.

    PubMed

    Okudela, Koji; Katayama, Akira; Woo, Tetsukan; Mitsui, Hideaki; Suzuki, Takehisa; Tateishi, Yoko; Umeda, Shigeaki; Tajiri, Michihiko; Masuda, Munetaka; Nagahara, Noriyuki; Kitamura, Hitoshi; Ohashi, Kenichi

    2014-01-01

    This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated. PMID:24503901

  20. Proteome Analysis for Downstream Targets of Oncogenic KRAS - the Potential Participation of CLIC4 in Carcinogenesis in the Lung

    PubMed Central

    Okudela, Koji; Katayama, Akira; Woo, Tetsukan; Mitsui, Hideaki; Suzuki, Takehisa; Tateishi, Yoko; Umeda, Shigeaki; Tajiri, Michihiko; Masuda, Munetaka; Nagahara, Noriyuki; Kitamura, Hitoshi; Ohashi, Kenichi

    2014-01-01

    This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated. PMID:24503901

  1. RAS Mutations as Predictive Biomarkers in Clinical Management of Metastatic Colorectal Cancer.

    PubMed

    Waring, Paul; Tie, Jeanne; Maru, Dipen; Karapetis, Christos S

    2016-06-01

    The use of anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies in patients with metastatic colorectal cancer is guided by the presence of activating point mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS and NRAS genes in the primary tumor. Although these mutations have been incorporated into the prescribing information for both cetuximab and panitumumab, highlighted in the National Comprehensive Cancer Network Guidelines, and routinely tested, a number of controversial issues and unanswered questions related to these mutations and their clinical significance remain. In the present review, we explored the contradictory data related to the prognostic value of KRAS mutations, the reported frequent discordance of KRAS mutations, and the reported nonequivalence of some of these mutations. We also considered the issues related to incorporating additional mutations into the already accredited and approved assays and the challenges created by changing an assay's analytical and clinical limits of detection. We also discuss the lack of biologic data supporting the pathogenicity of newly described clinically actionable mutations and explore the uncertainty regarding the clinical significance of low-frequency mutations, highlighting the importance of correcting allele frequencies for tumor purity. We also considered the importance of distinguishing the significance of low-frequency RAS mutations in tumors previously not treated or treated with anti-EGFR therapies and explore new technologies capable of detecting emerging polyclonal RAS mutations that appear to confer drug resistance. PMID:26952655

  2. Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms

    PubMed Central

    Nishikawa, G; Sekine, S; Ogawa, R; Matsubara, A; Mori, T; Taniguchi, H; Kushima, R; Hiraoka, N; Tsuta, K; Tsuda, H; Kanai, Y

    2013-01-01

    Background: The molecular basis for the development of appendiceal mucinous tumours, which can be a cause of pseudomyxoma peritonei, remains largely unknown. Methods: Thirty-five appendiceal mucinous neoplasms were analysed for GNAS and KRAS mutations. A functional analysis of mutant GNAS was performed using a colorectal cancer cell line. Results: A mutational analysis identified activating GNAS mutations in 16 of 32 low-grade appendiceal mucinous neoplasms (LAMNs) but in none of three mucinous adenocarcinomas (MACs). KRAS mutations were found in 30 LAMNs and in all MACs. We additionally analysed a total of 186 extra-appendiceal mucinous tumours and found that GNAS mutations were highly prevalent in intraductal papillary mucinous tumours of the pancreas (88%) but were rare or absent in mucinous tumours of the colorectum, ovary, lung and breast (0–9%). The prevalence of KRAS mutations was quite variable among the tumours. The introduction of the mutant GNAS into a colorectal cancer cell line markedly induced MUC2 and MUC5AC expression, but did not promote cell growth either in vitro or in vivo. Conclusion: Activating GNAS mutations are a frequent and characteristic genetic abnormality of LAMN. Mutant GNAS might play a direct role in the prominent mucin production that is a hallmark of LAMN. PMID:23403822

  3. KRAS and YAP1 converge to regulate EMT and tumor survival

    PubMed Central

    Shao, Diane D.; Xue, Wen; Krall, Elsa B.; Bhutkar, Arjun; Piccioni, Federica; Wang, Xiaoxing; Schinzel, Anna C.; Sood, Sabina; Rosenbluh, Joseph; Kim, Jong W.; Zwang, Yaara; Roberts, Thomas M.; Root, David E.; Jacks, Tyler; Hahn, William C.

    2014-01-01

    Summary Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional co-activator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling. PMID:24954536

  4. Retrospective analysis of KRAS status in metastatic colorectal cancer patients: a single-center feasibility study

    PubMed Central

    Montomoli, Jonathan; Hamilton-Dutoit, Stephen Jacques; Frøslev, Trine; Taylor, Aliki; Erichsen, Rune

    2012-01-01

    Background: The occurrence of KRAS mutations and their association with prognosis in metastatic colorectal cancer patients is not well documented in population-based studies. Objectives: To examine the feasibility of identifying archived colorectal cancer specimens, and through linkage with nationwide Danish population-based databases to investigate the prevalence of KRAS mutations and their association with colorectal cancer survival. Methods: We used the Danish Pathology Database to identify the physical location of primary (or in some cases secondary) tumor specimens from selected metastatic colorectal cancer patients referred to our hospital for palliative chemotherapy between November 1, 2008 and September 30, 2009. Routinely stored paraffin tissue blocks were obtained from the pathology archives of the originating hospital. KRAS mutation tumor status was assessed for each patient using the commercialized TheraScreen KRAS Mutation Kit. Using the unique identifier number, we linked the patients to the Danish National Registry of Patients and the Danish Civil Registration System to obtain data on date of first colorectal cancer diagnosis and follow-up status. We estimated prevalence of KRAS mutations and the 1-, 2-, and 5-year survival after colorectal cancer diagnosis using the Kaplan–Meier technique. Results: We identified 106 metastatic colorectal cancer patients (64% males). All were successfully linked to the registries, and archived tumor-tissue samples were obtained and analyzed in each case. The overall prevalence of KRAS mutations was 55%, and 1-, 2-, and 5-year overall survival after colorectal cancer diagnosis was 91%, 68%, and 25%, respectively. Conclusion: It is feasible to use Danish population-based registries to obtain archived tissue samples from metastatic colorectal cancer patients, and to estimate prevalence of KRAS mutation and subsequently evaluate the association with colorectal cancer survival. PMID:23028236

  5. Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors

    PubMed Central

    Baldelli, Elisa; Bellezza, Guido; Haura, Eric B.; Crinó, Lucio; Cress, W. Douglas; Deng, Jianghong; Ludovini, Vienna; Sidoni, Angelo; Schabath, Matthew B.; Puma, Francesco; Vannucci, Jacopo; Siggillino, Annamaria; Liotta, Lance A.; Petricoin, Emanuel F.; Pierobon, Mariaelena

    2015-01-01

    Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets. Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to laser capture microdissection and reverse phase protein microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC). Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02). This finding was verified in an independent population by IHC (p=0.03). KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications. PMID:26468985

  6. Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors.

    PubMed

    Baldelli, Elisa; Bellezza, Guido; Haura, Eric B; Crinó, Lucio; Cress, W Douglas; Deng, Jianghong; Ludovini, Vienna; Sidoni, Angelo; Schabath, Matthew B; Puma, Francesco; Vannucci, Jacopo; Siggillino, Annamaria; Liotta, Lance A; Petricoin, Emanuel F; Pierobon, Mariaelena

    2015-10-20

    Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets.Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to Laser Capture Microdissection and Reverse Phase Protein Microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC).Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02).This finding was verified in an independent population by IHC (p=0.03).KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications. PMID:26468985

  7. Impaired JNK signaling cooperates with KrasG12D expression to accelerate pancreatic ductal adenocarcinoma

    PubMed Central

    Davies, Clare C.; Harvey, Emma; McMahon, Raymond F.T.; Finegan, Katherine G.; Connor, Frances; Davis, Roger J.; Tuveson, David A.; Tournier, Cathy

    2014-01-01

    The c-Jun N-terminal protein kinase (JNK) and its two direct activators, namely the mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) and MKK7, constitute a signaling node frequently mutated in human pancreatic ductal adenocarcinoma (PDAC). Here we demonstrate the cooperative interaction of endogenous expression of KrasG12D with loss-of-function mutations in mkk4 or both, mkk4 and mkk7 genes in the pancreas. More specifically, impaired JNK signaling in a subpopulation of Pdx1-expressing cells dramatically accelerated the appearance of KrasG12D-induced acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasias, which rapidly progressed to invasive PDAC within 10 weeks of age. Furthermore, inactivation of mkk4/mkk7 compromised acinar regeneration following acute inflammatory stress by locking damaged exocrine cells in a permanently de-differentiated state. Therefore, we propose that JNK signaling exerts its tumor suppressive function in the pancreas by antagonising the metaplastic conversion of acinar cells towards a ductal fate capable of responding to oncogenic stimulation. PMID:24713432

  8. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

    PubMed Central

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-01-01

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

  9. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    DOE PAGESBeta

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

  10. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    SciTech Connect

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

  11. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

    PubMed

    Yun, Jihye; Mullarky, Edouard; Lu, Changyuan; Bosch, Kaitlyn N; Kavalier, Adam; Rivera, Keith; Roper, Jatin; Chio, Iok In Christine; Giannopoulou, Eugenia G; Rago, Carlo; Muley, Ashlesha; Asara, John M; Paik, Jihye; Elemento, Olivier; Chen, Zhengming; Pappin, Darryl J; Dow, Lukas E; Papadopoulos, Nickolas; Gross, Steven S; Cantley, Lewis C

    2015-12-11

    More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations. PMID:26541605

  12. Sorafenib enhances the therapeutic efficacy of rapamycin in colorectal cancers harboring oncogenic KRAS and PIK3CA

    PubMed Central

    Evers, B. Mark

    2012-01-01

    Activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling is associated with tumorigenesis and metastasis of colorectal cancer (CRC). The mammalian target of rapamycin (mTOR) kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis and metastasis of CRCs, indicating that mTOR inhibition may have therapeutic potential. Notwithstanding, many cancers, including CRC, demonstrate resistance to the antitumorigenic effects of rapamycin. In this study, we show that inhibition of mTORC1 with rapamycin leads to feedback activation of PI3K/Akt and Ras-MAPK signaling, resulting in cell survival and possible contribution to rapamycin resistance. Combination with the multikinase inhibitor, sorafenib, abrogates rapamycin-induced activation of PI3K/Akt and Ras-MAPK signaling pathways. Combination of rapamycin with sorafenib synergistically inhibits proliferation of CRC cells. CRCs harboring coexistent KRAS and PIK3CA mutations are partially sensitive to either rapamycin or sorafenib monotherapy, but highly sensitive to combination treatment with rapamycin and sorafenib. Combination with sorafenib enhances therapeutic efficacy of rapamycin on induction of apoptosis and inhibition of cell-cycle progression, migration and invasion of CRCs. We demonstrate efficacy and safety of concomitant treatment with rapamycin and sorafenib at inhibiting growth of xenografts from CRC cells with coexistent mutations in KRAS and PIK3CA. The efficacy and tolerability of combined treatment with rapamycin and sorafenib provides rationale for use in treating CRC patients, particularly those with tumors harboring coexistent KRAS and PIK3CA mutations. Abbreviations:CIcombination indexCRCcolorectal cancerIHCimmunohistochemistryMAPKmitogen activated protein kinasemTORmammalian target of rapamycinPI3Kphosphatidylinositol 3-kinase. PMID:22696593

  13. Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation.

    PubMed

    Banda, Malathi; McKim, Karen L; Haber, Lynne T; MacGregor, Judith A; Gollapudi, B Bhaskar; Parsons, Barbara L

    2015-08-01

    This study investigated whether Kras mutation is an early event in the development of lung tumors induced by inhalation of particulate vanadium pentoxide (VP) aerosols. A National Toxicology Program tumor bioassay of inhaled particulate VP aerosols established that VP-induced alveolar/bronchiolar carcinomas of the B6C3F1 mouse lung carried Kras mutations at a higher frequency than observed in spontaneous mouse lung tumors. Therefore, this study sought to: (1) characterize any Kras mutational response with respect to VP exposure concentration, and (2) investigate the possibility that amplification of preexisting Kras mutation is an early event in VP-induced mouse lung tumorigenesis. Male Big Blue B6C3F1 mice (6 mice/group) were exposed to aerosolized particulate VP by inhalation, 6h/day, 5 days/week for 4 or 8 weeks, using VP exposure concentrations of 0, 0.1, and 1 mg/m(3). The levels of two different Kras codon 12 mutations [GGT → GAT (G12D) and GGT → GTT (G12V)] were measured in lung DNAs by Allele-specific Competitive Blocker PCR (ACB-PCR). For both exposure concentrations (0.1 and 1.0mg/m(3)) and both time points (4 and 8 weeks), the mutant fractions observed in VP-exposed mice were not significantly different from the concurrent controls. Given that 8 weeks of inhalation of a tumorigenic concentration of particulate aerosols of VP did not result in a significant change in levels of lung Kras mutation, the data do not support either a direct genotoxic effect of VP on Kras or early amplification of preexisting mutation as being involved in the genesis of VP-induced mouse lung tumors under the exposure conditions used. Rather, the data suggest that accumulation of Kras mutation occurs later with chronic VP exposure and is likely not an early event in VP-induced mouse lung carcinogenesis. PMID:26232258

  14. A mouse model of melanoma driven by oncogenic KRAS

    PubMed Central

    Milagre, Carla; Dhomen, Nathalie; Geyer, Felipe C; Hayward, Robert; Lambros, Maryou; Reis-Filho, Jorge S; Marais, Richard

    2010-01-01

    The small G-protein NRAS is mutated in 22% of human melanomas, whereas the related proteins, KRAS and HRAS are mutated in only 2% and 1% of melanomas respectively. We have developed a mouse models of melanoma in which Cre recombinase/loxP technology is used to drive inducible expression of G12VKRAS in the melanocytic lineage. The mice develop skin hyper-pigmentation, nevi and tumors that bear many of the cardinal histopathology features and molecular characteristics of human melanoma. These tumors invade and destroy the underlying muscles and cells derived from them can grow as subcutaneous tumors and colonise the lungs of nude mice. These data establish that oncogenic KRAS can be a founder event in melanomagenesis. PMID:20516123

  15. Identification and characterization of a new G-quadruplex forming region within the kRAS promoter as a transcriptional regulator.

    PubMed

    Morgan, Rhianna K; Batra, Harshul; Gaerig, Vanessa C; Hockings, Jennifer; Brooks, Tracy A

    2016-02-01

    kRAS is one of the most prevalent oncogenic aberrations. It is either upregulated or mutationally activated in a multitude of cancers, including pancreatic, lung, and colon cancers. While a significant effort has been made to develop drugs that target kRAS, their clinical activity has been disappointing due to a variety of mechanistic hurdles. The presented works describe a novel mechanism and molecular target to downregulate kRAS expression--a previously undescribed G-quadruplex (G4) secondary structure within the proximal promoter acting as a transcriptional silencer. There are three distinct guanine-rich regions within the core kRAS promoter, including a previously examined region (G4near). Of these regions, the most distal region does not form an inducible and stable structure, whereas the two more proximal regions (termed near and mid) do form strong G4s. G4near is predominantly a tri-stacked structure with a discontinuous guanine run incorporated; G4mid consists of seven distinct runs of continuous guanines and forms numerous competing isoforms, including a stable three-tetrad stacked mixed parallel and antiparallel loop structures with longer loops of up to 10 nucleotides. Comprehensive analysis of the regulation of transcription by higher order structures has revealed that the guanine-rich region in the middle of the core promoter, termed G4mid, is a stronger repressor of promoter activity than G4near. Using the extensive guanine-rich region of the kRAS core promoter, and particularly the G4mid structure, as the primary target, future drug discovery programs will have potential to develop a potent, specifically targeted small molecule to be used in the treatment of pancreatic, ovarian, lung, and colon cancers. PMID:26597160

  16. Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

    PubMed Central

    Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

    2014-01-01

    The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

  17. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

    PubMed

    Malhotra, Pooja; Anwar, Mumtaz; Nanda, Neha; Kochhar, Rakesh; Wig, Jai Dev; Vaiphei, Kim; Mahmood, Safrun

    2013-06-01

    The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort. PMID:23526092

  18. Porocarcinomas harbor recurrent HRAS-activating mutations and tumor suppressor inactivating mutations.

    PubMed

    Harms, Paul W; Hovelson, Daniel H; Cani, Andi K; Omata, Kei; Haller, Michaela J; Wang, Michael L; Arps, David; Patel, Rajiv M; Fullen, Douglas R; Wang, Min; Siddiqui, Javed; Andea, Aleodor; Tomlins, Scott A

    2016-05-01

    Porocarcinomas are a rare eccrine carcinoma with significant metastatic potential. Oncogenic drivers of porocarcinomas have been underexplored, with PIK3CA-activating mutation reported in 1 case. We analyzed 5 porocarcinomas by next-generation sequencing using the DNA component of the Oncomine Comprehensive Assay, which provides data on copy number changes and mutational events in 126 cancer-relevant genes through multiplex polymerase chain reaction. We detected an average of 3.3 high-confidence nonsynonymous mutations per tumor (range, 1-6), including a spectrum of oncogenic activation and tumor suppressor inactivation events. Tumor suppressor mutations included TP53 (4/5, 80%), RB1 (3/5, 60%), ATM (2/5, 40%), ARID1A (1/5, 20%), and CDKN2A (1/5, 20%). In 4 (80%) of 5 tumors, at least 1 potential oncogenic driver was identified. Activating HRAS mutations were detected in 2 (40%) of 5, including G13D and Q61L hotspot mutations. Mutations of EGFR were identified in 2 (40%) of 5; these mutations have been previously reported in cancer but did not affect classic activation hotspot sites. EGFR and HRAS mutations were mutually exclusive. HRAS mutations were detected by targeted sequencing in a minority of benign eccrine poromas (2/17; 11.7%), suggesting that HRAS activation may rarely be an early event in sweat gland neoplasia. Together, our data suggest roles for HRAS and EGFR as drivers in a subset of poroma and porocarcinoma. TP53 and RB1 inactivation events are also likely to contribute to tumorigenesis. These findings suggest that porocarcinomas display diversity with respect to oncogenic drivers, which may have implications for targeted therapy in metastatic or unresectable cases. PMID:27067779

  19. Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies

    PubMed Central

    de Mello, Ramon Andrade; Marques, Dânia Sofia; Medeiros, Rui; Araújo, António MF

    2011-01-01

    Lung cancer is currently the leading cause of cancer death in Western nations. Non-small cell lung cancer (NSCLC) represents 80% of all lung cancers, and adenocarcinoma is the predominant histological type. Despite the intensive research carried out on this field and therapeutic advances, the overall prognosis of these patients remains unsatisfactory, with a 5-year overall survival rate of less than 15%. Nowadays, pharmacogenetics and pharmacogenomics represent the key to successful treatment. Recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinoma: one associated with smoking and activation of the K-Ras oncogene and the other not associated with smoking and activation of the epidermal growth factor receptor (EGFR). The K-ras mutation is mainly responsible for primary resistance to new molecules which inhibit tyrosine kinase EGFR (erlotinib and gefitinib) and most of the EGFR mutations are responsible for increased tumor sensitivity to these drugs. This article aims to conduct a systematic review of the literature regarding the molecular pathways involving the EGFR, K-Ras and EGFR targeted therapies in NSCLC tumor behavior. PMID:22087435

  20. Frequent PIK3CA-activating mutations in hidradenoma papilliferums.

    PubMed

    Liau, Jau-Yu; Lan, Jui; Hong, Jin-Bon; Tsai, Jia-Huei; Kuo, Kuan-Tin; Chu, Chia-Yu; Sheen, Yi-Shuan; Huang, Wen-Chang

    2016-09-01

    Hidradenoma papilliferum (HP) is a benign epithelial tumor most commonly seen in the vulva. It is proposed to be derived from the anogenital mammary-like glands and is histologically very similar to the mammary intraductal papilloma (IP). Approximately 60% of mammary IPs have activating mutations in either PIK3CA or AKT1, with each gene accounting for 30% of cases. In this study, we screened the mutation statuses of PIK3CA, AKT1, RAS, and BRAF in 30 HPs. The results showed that activating mutations in either PIK3CA or AKT1 were identified in 20 tumors (67%); 19 tumors had PIK3CA mutations (63%; 13 in exon 20 and 6 in exon 9), and 1 had an AKT1 E17K mutation (3%). BRAF V600E mutation was found in an HP that also had a PIK3CA H1047R mutation. No RAS mutation was found. The mutation status was not correlated with the degree of epithelial cell hyperplasia. We conclude that although there might be site-related variations in the mutation frequencies of PIK3CA and AKT1 genes, HP is histologically and also genetically very similar to the mammary IP, suggesting that HP can be viewed as the extramammary counterpart of mammary IP. PMID:27184479

  1. KRAS-driven ROS promote malignant transformation

    PubMed Central

    Suh, Yongjoon; Lee, Su-Jae

    2015-01-01

    The mechanism underlying KRAS (Kirsten rat sarcoma viral oncogene homolog)-driven cellular transformation remains unclear because of the complexity of its downstream effectors. Park et al. recently reported that levels of reactive oxygen species (ROS) are increased by KRAS and are responsible for KRAS-driven malignant transformation, and further identified the signaling cascade involved as KRAS/p38/PDPK1/PKCδ/p47phox/NOX1. These findings provide new insight into the molecular mechanisms governing KRAS-driven malignant transformation. PMID:27308397

  2. Cis-acting elements in its 3′ UTR mediate post-transcriptional regulation of KRAS

    PubMed Central

    Kim, Minlee; Kogan, Nicole; Slack, Frank J.

    2016-01-01

    Multiple RNA-binding proteins and non-coding RNAs, such as microRNAs (miRNAs), are involved in post-transcriptional gene regulation through recognition motifs in the 3′ untranslated region (UTR) of their target genes. The KRAS gene encodes a key signaling protein, and its messenger RNA (mRNA) contains an exceptionally long 3′ UTR; this suggests that it may be subject to a highly complex set of regulatory processes. However, 3′ UTR-dependent regulation of KRAS expression has not been explored in detail. Using extensive deletion and mutational analyses combined with luciferase reporter assays, we have identified inhibitory and stabilizing cis-acting regions within the KRAS 3′ UTR that may interact with miRNAs and RNA-binding proteins, such as HuR. Particularly, we have identified an AU-rich 49-nt fragment in the KRAS 3′ UTR that is required for KRAS 3′ UTR reporter repression. This element contains a miR-185 complementary element, and we show that overexpression of miR-185 represses endogenous KRAS mRNA and protein in vitro. In addition, we have identified another 49-nt fragment that is required to promote KRAS 3′ UTR reporter expression. These findings indicate that multiple cis-regulatory motifs in the 3′ UTR of KRAS finely modulate its expression, and sequence alterations within a binding motif may disrupt the precise functions of trans-regulatory factors, potentially leading to aberrant KRAS expression. PMID:26930719

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

    PubMed Central

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

    2012-01-01

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

  4. Prognostic impact of KRAS mutant type and MET amplification in metastatic and recurrent gastric cancer patients treated with first-line S-1 plus cisplatin chemotherapy

    PubMed Central

    Matsusaka, Satoshi; Kobunai, Takashi; Yamamoto, Noriko; Chin, Keisho; Ogura, Mariko; Tanaka, Gotaro; Matsuoka, Kazuaki; Ishikawa, Yuichi; Mizunuma, Nobuyuki; Yamaguchi, Toshiharu

    2016-01-01

    Receptor tyrosine kinase (RTK)-related genes, including HER2, EGFR, MET, FGFR2 and KRAS, are target molecules that are clinically beneficial in gastric cancer (GC). We investigated the correlation between RTK-related genes and the curative effect of first-line S-1 plus cisplatin (SP) combination chemotherapy in metastatic and recurrent GC. We enrolled 150 patients with histopathologically confirmed metastatic and recurrent GC treated with SP. KRAS mutation was detected using direct sequencing. DNA copy number was measured by real-time PCR. Formalin-fixed paraffin-embedded specimens were examined immunohistochemically for HER2, EGFR, FGFR2 and MET. Among 144 patients, KRAS mutation was detected in five (3.5%) at codon 12 and one (0.7%) at codon 13. FGFR2, EGFR, HER2, MET and KRAS gene amplification was suggested in 4.4%, 5.9%, 9%, 3.7% and 10.3% of patients, respectively. KRAS mutation, but not KRAS amplification, was associated with significantly shorter overall and progression-free survival. MET membranous overexpression was associated with a significantly higher tumor response. MET amplification was associated with significantly shorter overall survival. We show for the first time that KRAS mutation and MET amplification are promising predictive markers in metastatic and recurrent GC patients treated with SP. KRAS status may be a useful prognostic marker in patients treated with SP. PMID:27014419

  5. Glucocerebrosidase enzyme activity in GBA mutation Parkinson's disease.

    PubMed

    Ortega, Roberto A; Torres, Paola A; Swan, Matthew; Nichols, William; Boschung, Sarah; Raymond, Deborah; Barrett, Matthew J; Johannes, Brooke A; Severt, Lawrence; Shanker, Vicki; Hunt, Ann L; Bressman, Susan; Pastores, Gregory M; Saunders-Pullman, Rachel

    2016-06-01

    Mutations in the glucocerebrosidase (GBA1) gene, the most common genetic contributor to Parkinson's disease (PD), are associated with an increased risk of PD in heterozygous and homozygous carriers. While glucocerebrosidase enzyme (GCase) activity is consistently low in Gaucher disease, there is a range of leukocyte GCase activity in healthy heterozygous GBA1 mutation carriers. To determine whether GCase activity may be a marker for PD with heterozygous GBA1 mutations (GBA1 mutation PD, GBA PD), GBA PD patients (n=15) were compared to PD patients without heterozygous GBA1 mutations (idiopathic PD; n=8), heterozygous GBA1 carriers without PD (asymptomatic carriers; n=4), and biallelic mutation carriers with PD (Gaucher disease with PD, GD1 PD; n=3) in a pilot study. GCase activity (nmol/mg protein/hour) in GD1 PD (median [interquartile range]; minimum-maximum: 6.4 [5.7]; 5.3-11) was lower than that of GBA PD (16.0 [7.0]; 11-40) (p=0.01), while GCase activity in GBA PD was lower than idiopathic PD (28.5 [15.0]; 16-56) (p=0.01) and asymptomatic carriers (25.5 [2.5]; 23-27) (p=0.04). Therefore, GCase activity appears to be a possible marker of heterozygous GBA1 mutation PD, and larger studies are warranted. Prospective studies are also necessary to determine whether lower GCase activity precedes development of PD. PMID:26857292

  6. Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to KRAS mutant NSCLC dissociates GAB1-SHP2 post oncogene knockdown.

    PubMed

    Srikar, R; Suresh, Dhananjay; Zambre, Ajit; Taylor, Kristen; Chapman, Sarah; Leevy, Matthew; Upendran, Anandhi; Kannan, Raghuraman

    2016-01-01

    A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation. PMID:27530552

  7. Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to KRAS mutant NSCLC dissociates GAB1-SHP2 post oncogene knockdown

    PubMed Central

    Srikar, R.; Suresh, Dhananjay; Zambre, Ajit; Taylor, Kristen; Chapman, Sarah; Leevy, Matthew; Upendran, Anandhi; Kannan, Raghuraman

    2016-01-01

    A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation. PMID:27530552

  8. KRAS(G12D)-mediated oncogenic transformation of thyroid follicular cells requires long-term TSH stimulation and is regulated by SPRY1.

    PubMed

    Zou, Minjing; Baitei, Essa Y; Al-Rijjal, Roua A; Parhar, Ranjit S; Al-Mohanna, Futwan A; Kimura, Shioko; Pritchard, Catrin; BinEssa, Huda; Alanazi, Azizah A; Alzahrani, Ali S; Akhtar, Mohammed; Assiri, Abdullah M; Meyer, Brian F; Shi, Yufei

    2015-11-01

    KRAS(G12D) can cause lung cancer rapidly, but is not sufficient to induce thyroid cancer. It is not clear whether long-term serum thyroid stimulating hormone (TSH) stimulation can promote KRAS(G12D)-mediated thyroid follicular cell transformation. In the present study, we investigated the effect of long-term TSH stimulation in KRAS(G12D) knock-in mice and the role of Sprouty1 (SPRY1) in KRAS(G12D)-mediated signaling. We used TPO-KRAS(G12D) mice for thyroid-specific expression of KRAS(G12D) under the endogenous KRAS promoter. Twenty TPO-KRAS(G12D) mice were given anti-thyroid drug propylthiouracil (PTU, 0.1% w/v) in drinking water to induce serum TSH and 20 mice were without PTU treatment. Equal number of wild-type littermates (TPO-KRAS(WT)) was given the same treatment. The expression of SPRY1, a negative regulator of receptor tyrosine kinase (RTK) signaling, was analyzed in both KRAS(G12D)-and BRAF(V600E)-induced thyroid cancers. Without PTU treatment, only mild thyroid enlargement and hyperplasia were observed in TPO-KRAS(G12D) mice. With PTU treatment, significant thyroid enlargement and hyperplasia occurred in both TPO-KRAS(G12D) and TPO-KRAS(WT) littermates. Thyroids from TPO-KRAS(G12D) mice were six times larger than TPO-KRAS(WT) littermates. Distinct thyroid histology was found between TPO-KRAS(G12D) and TPO-KRAS(WT) mice: thyroid from TPO-KRAS(G12D) mice showed hyperplasia with well-maintained follicular architecture whereas in TPO-KRAS(WT) mice this structure was replaced by papillary hyperplasia. Among 10 TPO-KRAS(G12D) mice monitored for 14 months, two developed follicular thyroid cancer (FTC), one with pulmonary metastasis. Differential SPRY1 expression was demonstrated: increased in FTC and reduced in papillary thyroid cancer (PTC). The increased SPRY1 expression in FTC promoted TSH-RAS signaling through PI3K/AKT pathway whereas downregulation of SPRY1 by BRAF(V600E) in PTC resulted in both MAPK and PI3K/AKT activation. We conclude that chronic TSH

  9. 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

  10. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  11. Predictive value of K-ras and PIK3CA in non-small cell lung cancer patients treated with EGFR-TKIs: a systemic review and meta-analysis

    PubMed Central

    Chen, Jie-Ying; Cheng, Ya-Nan; Han, Lei; Wei, Feng; Yu, Wen-Wen; Zhang, Xin-Wei; Cao, Shui; Yu, Jin-Pu

    2015-01-01

    Objective A meta-analysis was performed to augment the insufficient data on the impact of mutative EGFR downstream phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on the clinical efficiency of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment of non-small cell lung cancer (NSCLC) patients. Methods Network databases were explored in April, 2015. Papers that investigated the clinical outcomes of NSCLC patients treated with EGFR-TKIs according to the status of K-ras and/or PIK3CA gene mutation were included. A quantitative meta-analysis was conducted using standard statistical methods. Odds ratios (ORs) for objective response rate (ORR) and hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) were calculated. Results Mutation in K-ras significantly predicted poor ORR [OR =0.22; 95% confidence interval (CI), 0.13-0.35], shorter PFS (HR =1.56; 95% CI, 1.27-1.92), and shorter OS (HR =1.59; 95% CI, 1.33-1.91) in NSCLC patients treated with EGFR-TKIs. Mutant PIK3CA significantly predicted shorter OS (HR =1.83; 95% CI, 1.05-3.20), showed poor ORR (OR =0.70; 95% CI, 0.22-2.18), and shorter PFS (HR =1.79; 95% CI, 0.91-3.53) in NSCLC patients treated with EGFR-TKIs. Conclusion K-ras mutation adversely affected the clinical response and survival of NSCLC patients treated with EGFR-TKIs. PIK3CA mutation showed similar trends. In addition to EGFR, adding K-ras and PIK3CA as routine gene biomarkers in clinical genetic analysis is valuable to optimize the effectiveness of EGFR-TKI regimens and identify optimal patients who will benefit from EGFR-TKI treatment. PMID:26175928

  12. Driver Gene Mutations in Stools of Colorectal Carcinoma Patients Detected by Targeted Next-Generation Sequencing.

    PubMed

    Armengol, Gemma; Sarhadi, Virinder K; Ghanbari, Reza; Doghaei-Moghaddam, Masoud; Ansari, Reza; Sotoudeh, Masoud; Puolakkainen, Pauli; Kokkola, Arto; Malekzadeh, Reza; Knuutila, Sakari

    2016-07-01

    Detection of driver gene mutations in stool DNA represents a promising noninvasive approach for screening colorectal cancer (CRC). Amplicon-based next-generation sequencing (NGS) is a good option to study mutations in many cancer genes simultaneously and from a low amount of DNA. Our aim was to assess the feasibility of identifying mutations in 22 cancer driver genes with Ion Torrent technology in stool DNA from a series of 65 CRC patients. The assay was successful in 80% of stool DNA samples. NGS results showed 83 mutations in cancer driver genes, 29 hotspot and 54 novel mutations. One to five genes were mutated in 75% of cases. TP53, KRAS, FBXW7, and SMAD4 were the top mutated genes, consistent with previous studies. Of samples with mutations, 54% presented concomitant mutations in different genes. Phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes were mutated in 70% of samples, with 58% having alterations in KRAS, NRAS, or BRAF. Because mutations in these genes can compromise the efficacy of epidermal growth factor receptor blockade in CRC patients, identifying mutations that confer resistance to some targeted treatments may be useful to guide therapeutic decisions. In conclusion, the data presented herein show that NGS procedures on stool DNA represent a promising tool to detect genetic mutations that could be used in the future for diagnosis, monitoring, or treating CRC. PMID:27155048

  13. Carnosine inhibits KRAS-mediated HCT116 proliferation by affecting ATP and ROS production.

    PubMed

    Iovine, Barbara; Iannella, Maria Luigia; Nocella, Francesca; Pricolo, Maria Rosaria; Bevilacqua, Maria Assunta

    2012-02-28

    Carnosine is a natural dipeptide that has generated particular interest for its antioxidant, anti-aging and especially for its antiproliferative properties. In this study, we demonstrate that carnosine inhibits the proliferation of human HCT116 colon cancer cells. In this cell line, the activating KRAS mutation induces mitochondrial ROS, the signaling molecules for cell proliferation. We observed that 50-100 mM carnosine decreases ATP and ROS concentration and induces cell cycle arrest in G1 phase. In HCT116 cells these effects are related to decreased ERK1/2 phosphorylation and increased p21waf1 protein. Our findings support the concept that carnosine could inhibit HCT116 cell growth via its antioxidant activity and its ability to affect glycolysis. PMID:22137144

  14. Bacteriophage lambda cro mutations: effects on activity and intracellular degradation.

    PubMed Central

    Pakula, A A; Young, V B; Sauer, R T

    1986-01-01

    Following random mutagenesis of the bacteriophage lambda cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type. Images PMID:2947238

  15. Gain-of-function SOS1 mutations cause a distinctive form of noonansyndrome

    SciTech Connect

    Tartaglia, Marco; Pennacchio, Len A.; Zhao, Chen; Yadav, KamleshK.; Fodale, Valentina; Sarkozy, Anna; Pandit, Bhaswati; Oishi, Kimihiko; Martinelli, Simone; Schackwitz, Wendy; Ustaszewska, Anna; Martin, Joes; Bristow, James; Carta, Claudio; Lepri, Francesca; Neri, Cinzia; Vasta,Isabella; Gibson, Kate; Curry, Cynthia J.; Lopez Siguero, Juan Pedro; Digilio, Maria Cristina; Zampino, Giuseppe; Dallapiccola, Bruno; Bar-Sagi, Dafna; Gelb, Brude D.

    2006-09-01

    Noonan syndrome (NS) is a developmental disordercharacterized by short stature, facial dysmorphia, congenital heartdefects and skeletal anomalies1. Increased RAS-mitogenactivated proteinkinase (MAPK) signaling due to PTPN11 and KRAS mutations cause 50 percentof NS2-6. Here, we report that 22 of 129 NS patients without PTPN11 orKRAS mutation (17 percent) have missense mutations in SOS1, which encodesa RAS-specific guanine nucleotide exchange factor (GEF). SOS1 mutationscluster at residues implicated in the maintenance of SOS1 in itsautoinhibited form and ectopic expression of two NS-associated mutantsinduced enhanced RAS activation. The phenotype associated with SOS1defects is distinctive, although within NS spectrum, with a highprevalence of ectodermal abnormalities but generally normal developmentand linear growth. Our findings implicate for the first timegain-of-function mutations in a RAS GEF in inherited disease and define anew mechanism by which upregulation of the RAS pathway can profoundlychange human development.

  16. The end of KRAS, and other, cancers? A new way forward.

    PubMed

    Mallucci, Livio; Wells, Valerie

    2014-04-01

    Mutant KRAS, as well as other mutant driver genes and epidriver genes, is a dominant determinant of resistance to cancer therapeutics. The recent introduction of targeting therapies based on drugs that inhibit the kinase catalytic function of nodal points along the Ras/extracellular-signal-regulated kinase (ERK) and the phosphatidylinositol-3-kinase (PI3K)/Akt cascades is meeting with limited success. Against this background, recent evidence shows that the β-galactoside-binding protein (βGBP) molecule, a physiological PI3K inhibitor, is a potent inducer of apoptosis in KRAS-mutant cancer cells (along with other aggressive cancer cells of different genetic makeup) and that it is therapeutically effective in vivo. Absence of p53 or phosphatase and tensin homolog (PTEN) tumor suppressor function or added activating PI3K mutations does not affect βGBP function. In contrast to the concept of one drug against one target, βGBP operates through alternative physiological routes. PMID:24291216

  17. EGFR-Mediated Chromatin Condensation Protects KRAS-Mutant Cancer Cells Against Ionizing Radiation

    PubMed Central

    Wang, Meng; Kern, Ashley M.; Hülskötter, Marieke; Greninger, Patricia; Singh, Anurag; Pan, Yunfeng; Chowdhury, Dipanjan; Krause, Mechthild; Baumann, Michael; Benes, Cyril H.; Efstathiou, Jason A.; Settleman, Jeff; Willers, Henning

    2014-01-01

    Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in-vitro and in-vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like co-localization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is co-dependent upon EGFR and PKCα. PKCα, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. PMID:24648348

  18. Activating mutations in CTNNB1 in aldosterone producing adenomas.

    PubMed

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K; Dralle, Henning; Walz, Martin K; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5-10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  19. Activating mutations in CTNNB1 in aldosterone producing adenomas

    PubMed Central

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K.; Dralle, Henning; Walz, Martin K.; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  20. Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models.

    PubMed

    Davis, S Lindsey; Robertson, Kelli M; Pitts, Todd M; Tentler, John J; Bradshaw-Pierce, Erica L; Klauck, Peter J; Bagby, Stacey M; Hyatt, Stephanie L; Selby, Heather M; Spreafico, Anna; Ecsedy, Jeffrey A; Arcaroli, John J; Messersmith, Wells A; Tan, Aik Choon; Eckhardt, S Gail

    2015-01-01

    Aurora A kinase and MEK inhibitors induce different, and potentially complementary, effects on the cell cycle of malignant cells, suggesting a rational basis for utilizing these agents in combination. In this work, the combination of an Aurora A kinase and MEK inhibitor was evaluated in pre-clinical colorectal cancer models, with a focus on identifying a subpopulation in which it might be most effective. Increased synergistic activity of the drug combination was identified in colorectal cancer cell lines with concomitant KRAS and PIK3CA mutations. Anti-proliferative effects were observed upon treatment of these double-mutant cell lines with the drug combination, and tumor growth inhibition was observed in double-mutant human tumor xenografts, though effects were variable within this subset. Additional evaluation suggests that degree of G2/M delay and p53 mutation status affect apoptotic activity induced by combination therapy with an Aurora A kinase and MEK inhibitor in KRAS and PIK3CA mutant colorectal cancer. Overall, in vitro and in vivo testing was unable to identify a subset of colorectal cancer that was consistently responsive to the combination of a MEK and Aurora A kinase inhibitor. PMID:26136684

  1. Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models

    PubMed Central

    Davis, S. Lindsey; Robertson, Kelli M.; Pitts, Todd M.; Tentler, John J.; Bradshaw-Pierce, Erica L.; Klauck, Peter J.; Bagby, Stacey M.; Hyatt, Stephanie L.; Selby, Heather M.; Spreafico, Anna; Ecsedy, Jeffrey A.; Arcaroli, John J.; Messersmith, Wells A.; Tan, Aik Choon; Eckhardt, S. Gail

    2015-01-01

    Aurora A kinase and MEK inhibitors induce different, and potentially complementary, effects on the cell cycle of malignant cells, suggesting a rational basis for utilizing these agents in combination. In this work, the combination of an Aurora A kinase and MEK inhibitor was evaluated in pre-clinical colorectal cancer models, with a focus on identifying a subpopulation in which it might be most effective. Increased synergistic activity of the drug combination was identified in colorectal cancer cell lines with concomitant KRAS and PIK3CA mutations. Anti-proliferative effects were observed upon treatment of these double-mutant cell lines with the drug combination, and tumor growth inhibition was observed in double-mutant human tumor xenografts, though effects were variable within this subset. Additional evaluation suggests that degree of G2/M delay and p53 mutation status affect apoptotic activity induced by combination therapy with an Aurora A kinase and MEK inhibitor in KRAS and PIK3CA mutant colorectal cancer. Overall, in vitro and in vivo testing was unable to identify a subset of colorectal cancer that was consistently responsive to the combination of a MEK and Aurora A kinase inhibitor. PMID:26136684

  2. Somatic Activating PIK3CA Mutations Cause Venous Malformation.

    PubMed

    Limaye, Nisha; Kangas, Jaakko; Mendola, Antonella; Godfraind, Catherine; Schlögel, Matthieu J; Helaers, Raphael; Eklund, Lauri; Boon, Laurence M; Vikkula, Miikka

    2015-12-01

    Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects. PMID:26637981

  3. A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk.

    PubMed

    Ratner, Elena; Lu, Lingeng; Boeke, Marta; Barnett, Rachel; Nallur, Sunitha; Chin, Lena J; Pelletier, Cory; Blitzblau, Rachel; Tassi, Renata; Paranjape, Trupti; Hui, Pei; Godwin, Andrew K; Yu, Herbert; Risch, Harvey; Rutherford, Thomas; Schwartz, Peter; Santin, Alessandro; Matloff, Ellen; Zelterman, Daniel; Slack, Frank J; Weidhaas, Joanne B

    2010-08-15

    Ovarian cancer (OC) is the single most deadly form of women's cancer, typically presenting as an advanced disease at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The KRAS oncogene and altered levels of the microRNA (miRNA) let-7 are associated with an increased risk of developing solid tumors. In this study, we investigated a hypothesized association between an increased risk of OC and a variant allele of KRAS at rs61764370, referred to as the KRAS-variant, which disrupts a let-7 miRNA binding site in this oncogene. Specimens obtained were tested for the presence of the KRAS-variant from nonselected OC patients in three independent cohorts, two independent ovarian case-control studies, and OC patients with hereditary breast and ovarian cancer syndrome (HBOC) as well as their family members. Our results indicate that the KRAS-variant is associated with more than 25% of nonselected OC cases. Further, we found that it is a marker for a significant increased risk of developing OC, as confirmed by two independent case-control analyses. Lastly, we determined that the KRAS-variant was present in 61% of HBOC patients without BRCA1 or BRCA2 mutations, previously considered uninformative, as well as in their family members with cancer. Our findings strongly support the hypothesis that the KRAS-variant is a genetic marker for increased risk of developing OC, and they suggest that the KRAS-variant may be a new genetic marker of cancer risk for HBOC families without other known genetic abnormalities. PMID:20647319

  4. Bi-Directional SIFT Predicts a Subset of Activating Mutations

    PubMed Central

    Lee, William; Lazarus, Robert A.; Zhang, Zemin

    2009-01-01

    Advancements in sequencing technologies have empowered recent efforts to identify polymorphisms and mutations on a global scale. The large number of variations and mutations found in these projects requires high-throughput tools to identify those that are most likely to have an impact on function. Numerous computational tools exist for predicting which mutations are likely to be functional, but none that specifically attempt to identify mutations that result in hyperactivation or gain-of-function. Here we present a modified version of the SIFT (Sorting Intolerant from Tolerant) algorithm that utilizes protein sequence alignments with homologous sequences to identify functional mutations based on evolutionary fitness. We show that this bi-directional SIFT (B-SIFT) is capable of identifying experimentally verified activating mutants from multiple datasets. B-SIFT analysis of large-scale cancer genotyping data identified potential activating mutations, some of which we have provided detailed structural evidence to support. B-SIFT could prove to be a valuable tool for efforts in protein engineering as well as in identification of functional mutations in cancer. PMID:20011534

  5. TERT promoter mutations and monoallelic activation of TERT in cancer

    PubMed Central

    Huang, F W; Bielski, C M; Rinne, M L; Hahn, W C; Sellers, W R; Stegmeier, F; Garraway, L A; Kryukov, G V

    2015-01-01

    Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer. PMID:26657580

  6. Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers.

    PubMed

    Mayers, Jared R; Torrence, Margaret E; Danai, Laura V; Papagiannakopoulos, Thales; Davidson, Shawn M; Bauer, Matthew R; Lau, Allison N; Ji, Brian W; Dixit, Purushottam D; Hosios, Aaron M; Muir, Alexander; Chin, Christopher R; Freinkman, Elizaveta; Jacks, Tyler; Wolpin, Brian M; Vitkup, Dennis; Vander Heiden, Matthew G

    2016-09-01

    Tumor genetics guides patient selection for many new therapies, and cell culture studies have demonstrated that specific mutations can promote metabolic phenotypes. However, whether tissue context defines cancer dependence on specific metabolic pathways is unknown. Kras activation and Trp53 deletion in the pancreas or the lung result in pancreatic ductal adenocarinoma (PDAC) or non-small cell lung carcinoma (NSCLC), respectively, but despite the same initiating events, these tumors use branched-chain amino acids (BCAAs) differently. NSCLC tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source, whereas PDAC tumors have decreased BCAA uptake. These differences are reflected in expression levels of BCAA catabolic enzymes in both mice and humans. Loss of Bcat1 and Bcat2, the enzymes responsible for BCAA use, impairs NSCLC tumor formation, but these enzymes are not required for PDAC tumor formation, arguing that tissue of origin is an important determinant of how cancers satisfy their metabolic requirements. PMID:27609895

  7. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort.

    PubMed

    Ines, Chaar; Donia, Ounissi; Rahma, Boughriba; Ben Ammar, Azza; Sameh, Amara; Khalfallah, Taher; Abdelmajid, Ben Hmida; Sabeh, Mzabi; Saadia, Bouraoui

    2014-07-01

    According to the multistep route of genetic alterations in the colorectal adenoma-carcinoma sequence, the complex K-ras/p53 mutation is one of the first alterations to occur and represent an important genetic event in colorectal cancer (CRC). An evaluation of the mutation spectra in K-ras and p53 gene was effected in 167 Tunisian patients with sporadic CRC to determine whether our populations have similar pattern of genetic alteration as in Maghrebin's population. Mutation patterns of codon 12-13 of K-ras and exon 5-8 of p53 were analyzed by immunohistochemistry and PCR-SSCP and confirmed by sequencing. Mutations in the K-ras gene were detected in 31.13 % and affect the women more than the men (p = 0.008). Immunostaining showed that expression of p21 ras was correlated with the advanced age (p = 0.004), whereas loss of signal was associated with mucinous histotype (p = 0.003). Kaplan-Meier survival curve found that patients with the K-ras mutation had a shorter survival compared with patients without mutation (p = 0.005). Alteration in p53 was seen in 17.4 % of patients and affects three hot spot codons such as 175, 245, and 248. Overexpression of p53 was seen in 34.1 % and correlated with tumor node metastasis (TNM) advanced stage (p = 0.037) and mucinous histotype (p = 0.001). A high concordance between p53 expression and alteration (p<0.005) was shown. Concomitant mutations in K-ras and p53 gene were detected in only 4 % of tumors. K-ras and p53 undergo separate pathways in colorectal tumorogenesis. Interestingly, mutations in the K-ras gene might be considered a valuable prognostic factor correlated to poor outcome. p53 gene alterations were rather low in our set, and methylation pattern of p53 is required to elucidate the molecular basis of this protein in CRC. PMID:24763823

  8. Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

    PubMed Central

    Fuentes-Calvo, Isabel; Blázquez-Medela, Ana M.; Santos, Eugenio; López-Novoa, José M.; Martínez-Salgado, Carlos

    2010-01-01

    Background Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells. Methodology/Principal Findings Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras−/−,N-ras−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras−/−) embrionary fibroblasts by mating of K-ras+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras−/− fibroblasts. Conclusions/Significance We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations. PMID:20090846

  9. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    PubMed Central

    Pathan, Akbar Ali Khan; Panthi, Bhavana; Khan, Zahid; Koppula, Purushotham Reddy; Alanazi, Mohammed Saud; Sachchidanand; Parine, Narasimha Reddy; Chourasia, Mukesh

    2016-01-01

    Objective Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein. PMID:27217775

  10. KRAS, BRAF and PIK3CA Status in Squamous Cell Anal Carcinoma (SCAC)

    PubMed Central

    Giannini, Massimo; Freier, Eva; Tamberi, Stefano; Scarpi, Emanuela; Passardi, Alassandro; Zoli, Wainer; Ragazzini, Angela; Amadori, Dino; Frassineti, Giovanni Luca

    2014-01-01

    Anti-EGFR therapy appears to be a potential treatment option for squamous cell anal carcinoma (SCAC). KRAS mutation is a rare event in SCAC, indicating the absence of the principal mechanism of resistance to this type of therapy. However, no information is available from the literature regarding the status of BRAF or PIK3CA in this cancer type. We analysed KRAS, BRAF and PIK3CA status in SCAC patients in relation to the clinical-pathological characteristics of patients and to the presence of the human papilloma virus (HPV). One hundred and three patients were treated with the Nigro scheme for anal cancer from March 2001 to August 2012. Fifty patients were considered for the study as there was insufficient paraffin-embedded tumour tissue to perform molecular analysis the remaining 53. DNA was extracted from paraffin-embedded sections. KRAS, BRAF and PIK3CA gene status and HPV genotype were evaluated by pyrosequencing. KRAS and BRAF genes were wild-type in all cases. Conversely, PIK3CA gene was found to be mutated in 11 (22%) cases. In particular, 8 mutations occurred in exon 9 and 3 in exon 20 of the PIK3CA gene. These findings suggest that SCAC could potentially respond to an anti-EGFR drug. PIK3CA mutation may be involved in the process of carcinogenesis in some cases of SCAC. PMID:24642661

  11. GNAS gene mutation may be present only transiently during colorectal tumorigenesis

    PubMed Central

    Zauber, Peter; Marotta, Stephen P; Sabbath-Solitare, Marlene

    2016-01-01

    Mutations of the gene GNAS have been shown to activate the adenylate cyclase gene and lead to constitutive cAMP signaling. Several preliminary reports have suggested a role for GNAS gene mutations during colorectal carcinogenesis, particularly mucinous carcinomas. The aim of this study was to clarify the incidence of GNAS mutations in adenomas (tubular, tubulovillous, and villous), carcinomas with residual adenoma, and carcinomas, and to relate these findings to mutations of the KRAS gene and to the mucinous status of the tumors. We used standard PCR techniques and direct gene sequencing to evaluate tumors for gene mutations. No GNAS mutations were identified in 25 tubular adenomas, but were present in 6.4% of tubulovillous adenomas and 11.2% of villous adenomas. A GNAS mutation was found in 9.7% of the benign portion of carcinoma with residual adenoma, but in none of 86 carcinomas. A similar trend was seen for KRAS mutation across the five groups of tumors. GNAS mutations may function as an important driver mutation during certain phases of colorectal carcinogenesis, but may then be lost once the biological advantage gained by the mutated gene is no longer necessary to sustain or advance tumor development. PMID:27186325

  12. Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis.

    PubMed

    Padanad, Mahesh S; Konstantinidou, Georgia; Venkateswaran, Niranjan; Melegari, Margherita; Rindhe, Smita; Mitsche, Matthew; Yang, Chendong; Batten, Kimberly; Huffman, Kenneth E; Liu, Jingwen; Tang, Ximing; Rodriguez-Canales, Jaime; Kalhor, Neda; Shay, Jerry W; Minna, John D; McDonald, Jeffrey; Wistuba, Ignacio I; DeBerardinis, Ralph J; Scaglioni, Pier Paolo

    2016-08-01

    KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain. PMID:27477280

  13. Retraction: "Activated K-Ras and INK4a/Arf Deficiency Promote Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With Cancer Stem Cell Phenotype" by Wang et al.

    PubMed

    2016-10-01

    The above article, published online on November 23, 2012 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 4B and C to be inappropriately manipulated and re-labeled. Literature Cited Wang Z, Ali S, Banerjee S, Bao B, Li Y, Azmi AS, Korc M, Sarkar FH. 2013. Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype. J Cell Physiol 228:556-562; doi: 10.1002/jcp.24162. PMID:27315162

  14. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  15. Multigene mutational profiling of cholangiocarcinomas identifies actionable molecular subgroups

    PubMed Central

    Mafficini, Andrea; Wood, Laura D.; Corbo, Vincenzo; Melisi, Davide; Malleo, Giuseppe; Vicentini, Caterina; Malpeli, Giorgio; Antonello, Davide; Sperandio, Nicola; Capelli, Paola; Tomezzoli, Anna; Iacono, Calogero; Lawlor, Rita T.; Bassi, Claudio; Hruban, Ralph H.; Guglielmi, Alfredo; Tortora, Giampaolo; de Braud, Filippo; Scarpa, Aldo

    2014-01-01

    One-hundred-fifty-three biliary cancers, including 70 intrahepatic cholangiocarcinomas (ICC), 57 extrahepatic cholangiocarcinomas (ECC) and 26 gallbladder carcinomas (GBC) were assessed for mutations in 56 genes using multigene next-generation sequencing. Expression of EGFR and mTOR pathway genes was investigated by immunohistochemistry. At least one mutated gene was observed in 118/153 (77%) cancers. The genes most frequently involved were KRAS (28%), TP53 (18%), ARID1A (12%), IDH1/2 (9%), PBRM1 (9%), BAP1 (7%), and PIK3CA (7%). IDH1/2 (p=0.0005) and BAP1 (p=0.0097) mutations were characteristic of ICC, while KRAS (p=0.0019) and TP53 (p=0.0019) were more frequent in ECC and GBC. Multivariate analysis identified tumour stage and TP53 mutations as independent predictors of survival. Alterations in chromatin remodeling genes (ARID1A, BAP1, PBRM1, SMARCB1) were seen in 31% of cases. Potentially actionable mutations were seen in 104/153 (68%) cancers: i) KRAS/NRAS/BRAF mutations were found in 34% of cancers; ii) mTOR pathway activation was documented by immunohistochemistry in 51% of cases and by mutations in mTOR pathway genes in 19% of cancers; iii) TGF-ß/Smad signaling was altered in 10.5% cancers; iv) mutations in tyrosine kinase receptors were found in 9% cases. Our study identified molecular subgroups of cholangiocarcinomas that can be explored for specific drug targeting in clinical trials. PMID:24867389

  16. Multigene mutational profiling of cholangiocarcinomas identifies actionable molecular subgroups.

    PubMed

    Simbolo, Michele; Fassan, Matteo; Ruzzenente, Andrea; Mafficini, Andrea; Wood, Laura D; Corbo, Vincenzo; Melisi, Davide; Malleo, Giuseppe; Vicentini, Caterina; Malpeli, Giorgio; Antonello, Davide; Sperandio, Nicola; Capelli, Paola; Tomezzoli, Anna; Iacono, Calogero; Lawlor, Rita T; Bassi, Claudio; Hruban, Ralph H; Guglielmi, Alfredo; Tortora, Giampaolo; de Braud, Filippo; Scarpa, Aldo

    2014-05-15

    One-hundred-fifty-three biliary cancers, including 70 intrahepatic cholangiocarcinomas (ICC), 57 extrahepatic cholangiocarcinomas (ECC) and 26 gallbladder carcinomas (GBC) were assessed for mutations in 56 genes using multigene next-generation sequencing. Expression of EGFR and mTOR pathway genes was investigated by immunohistochemistry. At least one mutated gene was observed in 118/153 (77%) cancers. The genes most frequently involved were KRAS (28%), TP53 (18%), ARID1A (12%), IDH1/2 (9%), PBRM1 (9%), BAP1 (7%), and PIK3CA (7%). IDH1/2 (p=0.0005) and BAP1 (p=0.0097) mutations were characteristic of ICC, while KRAS (p=0.0019) and TP53 (p=0.0019) were more frequent in ECC and GBC. Multivariate analysis identified tumour stage and TP53 mutations as independent predictors of survival. Alterations in chromatin remodeling genes (ARID1A, BAP1, PBRM1, SMARCB1) were seen in 31% of cases. Potentially actionable mutations were seen in 104/153 (68%) cancers: i) KRAS/NRAS/BRAF mutations were found in 34% of cancers; ii) mTOR pathway activation was documented by immunohistochemistry in 51% of cases and by mutations in mTOR pathway genes in 19% of cancers; iii) TGF-ß/Smad signaling was altered in 10.5% cancers; iv) mutations in tyrosine kinase receptors were found in 9% cases. Our study identified molecular subgroups of cholangiocarcinomas that can be explored for specific drug targeting in clinical trials. PMID:24867389

  17. The hypervariable region of K-Ras4B is responsible for its specific interactions with Calmodulin

    PubMed Central

    Abraham, Sherwin J.; Nolet, Ryan P.; Calvert, Richard J.; Anderson, Lucy M.; Gaponenko, Vadim

    2009-01-01

    K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival and motility. The p21 Ras proteins such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry we demonstrate that the hypervariable region of K-Ras contributes in a major way to the interaction with calmodulin while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca2+-loaded calmodulin with micromolar affinity, while the GTP-γ-S loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin. PMID:19583261

  18. Receptor tyrosine kinases exert dominant control over PI3K signaling in human KRAS mutant colorectal cancers

    PubMed Central

    Ebi, Hiromichi; Corcoran, Ryan B.; Singh, Anurag; Chen, Zhao; Song, Youngchul; Lifshits, Eugene; Ryan, David P.; Meyerhardt, Jeffrey A.; Benes, Cyril; Settleman, Jeffrey; Wong, Kwok-Kin; Cantley, Lewis C.; Engelman, Jeffrey A.

    2011-01-01

    Therapies inhibiting receptor tyrosine kinases (RTKs) are effective against some human cancers when they lead to simultaneous downregulation of PI3K/AKT and MEK/ERK signaling. However, mutant KRAS has the capacity to directly activate ERK and PI3K signaling, and this is thought to underlie the resistance of KRAS mutant cancers to RTK inhibitors. Here, we have elucidated the molecular regulation of both the PI3K/AKT and MEK/ERK signaling pathways in KRAS mutant colorectal cancer cells and identified combination therapies that lead to robust cancer cell apoptosis. KRAS knockdown using shRNA suppressed ERK signaling in all of the human KRAS mutant colorectal cancer cell lines examined. However, no decrease, and actually a modest increase, in AKT phosphorylation was often seen. By performing PI3K immunoprecipitations, we determined that RTKs, often IGF-IR, regulated PI3K signaling in the KRAS mutant cell lines. This conclusion was also supported by the observation that specific RTK inhibition led to marked suppression of PI3K signaling and biochemical assessment of patient specimens. Interestingly, combination of RTK and MEK inhibitors led to concomitant inhibition of PI3K and MEK signaling, marked growth suppression, and robust apoptosis of human KRAS mutant colorectal cancer cell lines in vitro and upon xenografting in mice. These findings provide a framework for utilizing RTK inhibitors in the treatment of KRAS mutant colorectal cancers. PMID:21985784

  19. RHOA-FAK is a required signaling axis for the maintenance of KRAS-driven adenocarcinomas

    PubMed Central

    Konstantinidou, Georgia; Ramadori, Giorgio; Torti, Francesca; Kangasniemi, Kim; Ramirez, Rachel E.; Cai, Yiran; Behrens, Carmen; Dellinger, Michael T.; Brekken, Rolf A.; Wistuba, Ignacio I.; Heguy, Adriana; Teruya-Feldstein, Julie; Scaglioni, Pier Paolo

    2013-01-01

    Non-small cell lung cancer (NSCLC) often expresses mutant KRAS together with tumor-associated mutations of the CDKN2A locus, which are associated with aggressive, therapy-resistant tumors. Here, we unravel specific requirements for the maintenance of NSCLC that carry this genotype. We establish that the ERK/RHOA/focal adhesion kinase (FAK) network is deregulated in high-grade lung tumors. Suppression of RHOA or FAK induces cell death selectively in mutant KRAS;INK4a/ARF deficient lung cancer cells. Furthermore, pharmacological inhibition of FAK caused tumor regression specifically in the high-grade lung cancer that developed in mutant Kras;Cdkn2a-null mice. Our findings provide the rationale for the rapid implementation of genotype-specific targeted therapies utilizing FAK inhibitors in cancer patients. PMID:23358651

  20. Comparison of KRAS and PIK3CA gene status between primary tumors and paired metastases in colorectal cancer

    PubMed Central

    He, Qiong; Xu, Qi; Wu, Wei; Chen, Lei; Sun, Weijing; Ying, Jieer

    2016-01-01

    Purpose In metastatic or recurrent colorectal cancer (MRCRC), the concordance of Kirsten rat sarcoma viral oncogene homolog (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation status between the primary tumors and metastases is still controversial. The purpose of this study was to evaluate the association between KRAS and PIK3CA mutational status and various clinicopathologic features, and compare their genotype in primary tumors with that of the paired metastatic tumors. Method We compared the mutation status of KRAS and PIK3CA between the primary tumors and the paired metastases of 59 MRCRC patients with available tissues (resection or biopsy). The presence of KRAS and PIK3CA mutations were determined by direct sequencing analysis. Results Seventeen patients (28.8%) had the KRAS mutation and 46 patients (80.0%) had the PIK3CA mutation when considering both the primary and metastatic sites. KRAS mutation was observed in ten primary tumors and eleven related metastases (16.9% vs 18.6%), while PIK3CA mutation was found in 26 primary tumors and 32 related metastases (44.1% vs 54.2%). KRAS status was concordant between primary and metastatic sites in 45 patients (76.3%, kappa =0.157), while the concordance of PIK3CA status was only found in 25 patients (42.4%, kappa =−0.141). The PIK3CA status discordance rate was significantly higher in 40 patients undergoing metachronous resection of primary tumor or metastasis, compared with that in 19 patients with synchronous resection of primary tumor or metastasis (67.5% [27/40] vs 36.8% [7/19]; P=0.026). Conclusion Our results demonstrate that low concordance of KRAS and high discordance of PIK3CA mutational status exist between the primary tumors and paired metastasis, and these findings remind us to have second thoughts about the need to evaluate metastatic tumors separately rather than only based on the primary tumor data when targeted therapy is considered. PMID:27143928

  1. Colorectal cancer prognosis: is it all mutation, mutation, mutation?

    PubMed Central

    Hassan, A B; Paraskeva, C

    2005-01-01

    For the 500 000 new cases of colorectal cancer in the world each year, identification of patients with a worse prognosis and those who are more likely to respond to treatment is a challenge. There is an increasing body of evidence correlating genetic mutations with outcome in tumours derived from human colorectal cancer cohorts. K-ras, but not p53 or APC, mutations appear to be associated with poorer overall survival in colorectal cancer patients. PMID:16099785

  2. Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis

    PubMed Central

    Grabner, Beatrice; Schramek, Daniel; Mueller, Kristina M.; Moll, Herwig P.; Svinka, Jasmin; Hoffmann, Thomas; Bauer, Eva; Blaas, Leander; Hruschka, Natascha; Zboray, Katalin; Stiedl, Patricia; Nivarthi, Harini; Bogner, Edith; Gruber, Wolfgang; Mohr, Thomas; Zwick, Ralf Harun; Kenner, Lukas; Poli, Valeria; Aberger, Fritz; Stoiber, Dagmar; Egger, Gerda; Esterbauer, Harald; Zuber, Johannes; Moriggl, Richard; Eferl, Robert; Győrffy, Balázs; Penninger, Josef M.; Popper, Helmut; Casanova, Emilio

    2015-01-01

    STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased KrasG12D-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3–NF-κB–IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance. PMID:25734337

  3. Pivotal Role of the Chromatin Protein Nupr1 in Kras-Induced Senescence and Transformation.

    PubMed

    Grasso, Daniel; Bintz, Jennifer; Lomberk, Gwen; Molejon, Maria Ines; Loncle, Celine; Garcia, Maria Noé; Lopez, Maria Belen; Urrutia, Raul; Iovanna, Juan L

    2015-01-01

    Nupr1 is a chromatin protein, which cooperates with Kras(G12D) to induce PanIN formation and pancreatic cancer development in mice, though the molecular mechanisms underlying this effect remain to be fully characterized. In the current study, we report that Nupr1 acts as a gene modifier of the effect of Kras(G12D)-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation. Congruently, 5-aza-2'-deoxycytydine, a general inhibitor of DNA methylation, reverses the Kras(G12D)-induced PanIN development by promoting senescence. This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1(-/-) mice the expression of Kras(G12D) induces senescence instead of transformation. Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as Kras(G12D), during carcinogenesis. The biomedical relevance of these findings lies in that they support the rational for developing similar therapeutic interventions in human aimed at controlling either the initiation or progression of cancer. PMID:26617245

  4. Pivotal Role of the Chromatin Protein Nupr1 in Kras-Induced Senescence and Transformation

    PubMed Central

    Grasso, Daniel; Bintz, Jennifer; Lomberk, Gwen; Molejon, Maria Ines; Loncle, Celine; Garcia, Maria Noé; Lopez, Maria Belen; Urrutia, Raul; Iovanna, Juan L.

    2015-01-01

    Nupr1 is a chromatin protein, which cooperates with KrasG12D to induce PanIN formation and pancreatic cancer development in mice, though the molecular mechanisms underlying this effect remain to be fully characterized. In the current study, we report that Nupr1 acts as a gene modifier of the effect of KrasG12D-induced senescence by regulating Dnmt1 expression and consequently genome-wide levels of DNA methylation. Congruently, 5-aza-2′-deoxycytydine, a general inhibitor of DNA methylation, reverses the KrasG12D-induced PanIN development by promoting senescence. This requirement of Nupr1 expression, however, is not restricted to the pancreas since in lung of Nupr1–/– mice the expression of KrasG12D induces senescence instead of transformation. Therefore, mechanistically this data reveals that epigenetic events, at least at the level of DNA methylation, modulate the functional outcome of common genetic mutations, such as KrasG12D, during carcinogenesis. The biomedical relevance of these findings lies in that they support the rational for developing similar therapeutic interventions in human aimed at controlling either the initiation or progression of cancer. PMID:26617245

  5. Targeting pathways downstream of KRAS in lung adenocarcinoma.

    PubMed

    Zhu, Zehua; Golay, Hadrien G; Barbie, David A

    2014-08-01

    Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure. PMID:25303301

  6. Targeting pathways downstream of KRAS in lung adenocarcinoma

    PubMed Central

    Zhu, Zehua; Golay, Hadrien G; Barbie, David A

    2014-01-01

    Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure. PMID:25303301

  7. EGFR, HER-2 and KRAS in canine gastric epithelial tumors: a potential human model?

    PubMed

    Terragni, Rossella; Casadei Gardini, Andrea; Sabattini, Silvia; Bettini, Giuliano; Amadori, Dino; Talamonti, Chiara; Vignoli, Massimo; Capelli, Laura; Saunders, Jimmy H; Ricci, Marianna; Ricci, Marianna; Ulivi, Paola; Ulivi, Paola

    2014-01-01

    Epidermal growth factor receptor (EGFR or HER-1) and its analog c-erbB-2 (HER-2) are protein tyrosine kinases correlated with prognosis and response to therapy in a variety of human cancers. KRAS mediates the transduction of signals between EGFR and the nucleus, and its mutation has been identified as a predictor of resistance to anti-EGFR drugs. In human oncology, the importance of the EGFR/HER-2/KRAS signalling pathway in gastric cancer is well established, and HER-2 testing is required before initiating therapy. Conversely, this pathway has never been investigated in canine gastric tumours. A total of 19 canine gastric epithelial neoplasms (5 adenomas and 14 carcinomas) were retrospectively evaluated for EGFR/HER-2 immunohistochemical expression and KRAS mutational status. Five (35.7%) carcinomas were classified as intestinal-type and 9 (64.3%) as diffuse-type. EGFR was overexpressed (≥ 1+) in 8 (42.1%) cases and HER-2 (3+) in 11 (57.9%) cases, regardless of tumour location or biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type (80%) than in the diffuse-type (11.1%, p = 0.023). KRAS gene was wild type in 18 cases, whereas one mucinous carcinoma harboured a point mutation at codon 12 (G12R). EGFR and HER-2 may be promising prognostic and therapeutic targets in canine gastric epithelial neoplasms. The potential presence of KRAS mutation should be taken into account as a possible mechanism of drug resistance. Further studies are necessary to evaluate the role of dog as a model for human gastric cancer. PMID:24454858

  8. EGFR, HER-2 and KRAS in Canine Gastric Epithelial Tumors: A Potential Human Model?

    PubMed Central

    Bettini, Giuliano; Amadori, Dino; Talamonti, Chiara; Vignoli, Massimo; Capelli, Laura; Saunders, Jimmy H.; Ricci, Marianna; Ulivi, Paola

    2014-01-01

    Epidermal growth factor receptor (EGFR or HER-1) and its analog c-erbB-2 (HER-2) are protein tyrosine kinases correlated with prognosis and response to therapy in a variety of human cancers. KRAS mediates the transduction of signals between EGFR and the nucleus, and its mutation has been identified as a predictor of resistance to anti-EGFR drugs. In human oncology, the importance of the EGFR/HER-2/KRAS signalling pathway in gastric cancer is well established, and HER-2 testing is required before initiating therapy. Conversely, this pathway has never been investigated in canine gastric tumours. A total of 19 canine gastric epithelial neoplasms (5 adenomas and 14 carcinomas) were retrospectively evaluated for EGFR/HER-2 immunohistochemical expression and KRAS mutational status. Five (35.7%) carcinomas were classified as intestinal-type and 9 (64.3%) as diffuse-type. EGFR was overexpressed (≥1+) in 8 (42.1%) cases and HER-2 (3+) in 11 (57.9%) cases, regardless of tumour location or biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type (80%) than in the diffuse-type (11.1%, p = 0.023). KRAS gene was wild type in 18 cases, whereas one mucinous carcinoma harboured a point mutation at codon 12 (G12R). EGFR and HER-2 may be promising prognostic and therapeutic targets in canine gastric epithelial neoplasms. The potential presence of KRAS mutation should be taken into account as a possible mechanism of drug resistance. Further studies are necessary to evaluate the role of dog as a model for human gastric cancer. PMID:24454858

  9. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

    PubMed

    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-10-01

    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc. PMID:26873620

  10. G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription

    PubMed Central

    Cogoi, Susanna; Xodo, Luigi E.

    2006-01-01

    In human and mouse, the promoter of the KRAS gene contains a nuclease hypersensitive polypurine–polypyrimidine element (NHPPE) that is essential for transcription. An interesting feature of the polypurine G-rich strand of NHPPE is its ability to assume an unusual DNA structure that, according to circular dichroism (CD) and DMS footprinting experiments, is attributed to an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops. The human and mouse KRAS NHPPE G-rich strands display melting temperature of 64 and 73°C, respectively, as well as a K+-dependent capacity to arrest DNA polymerase. Photocleavage and CD experiments showed that the cationic porphyrin TMPyP4 stacks to the external G-tetrads of the KRAS quadruplexes, increasing the Tm by ∼20°C. These findings raise the intriguing question that the G-quadruplex formed within the NHPPE of KRAS may be involved in the regulation of transcription. Indeed, transfection experiments showed that the activity of the mouse KRAS promoter is reduced to 20% of control, in the presence of the quadruplex-stabilizing TMPyP4. In addition, we found that G-rich oligonucleotides mimicking the KRAS quadruplex, but not the corresponding 4-base mutant sequences or oligonucleotides forming quadruplexes with different structures, competed with the NHPPE duplex for binding to nuclear proteins. When vector pKRS-413, containing CAT driven by the mouse KRAS promoter, and KRAS quadruplex oligonucleotides were co-transfected in 293 cells, the expression of CAT was found to be downregulated to 40% of the control. On the basis of these data, we propose that the NHPPE of KRAS exists in equilibrium between a double-stranded form favouring transcription and a folded quadruplex form, which instead inhibits transcription. Such a mechanism, which is probably adopted by other growth-related genes, provides useful hints for the rational design of anticancer drugs against the KRAS oncogene. PMID:16687659

  11. Profiling of transcripts and proteins modulated by K-ras oncogene in the lung tissues of K-ras transgenic mice by omics approaches.

    PubMed

    Lee, Sojung; Kang, Jungwoo; Cho, Minchul; Seo, Eunhee; Choi, Heesook; Kim, Eunjin; Kim, Junghee; Kim, Heejong; Kang, Gum Yong; Kim, Kwang Pyo; Park, Young-Ho; Yu, Dae-Yeul; Yum, Young Na; Park, Sue-Nie; Yoon, Do-Young

    2009-01-01

    The mutated K-ras gene is involved in approximately 30% of human cancers. In order to search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we performed microarray and proteomics (LC/ESI-MS/MS) analysis. Genes (RAB27b RAS family, IL-1RA, IL-33, chemokine ligand 6, epiregulin, EGF-like domain and cathepsin) related to cancer development (Wnt signaling pathway) and inflammation (chemokine/cytokine signaling pathway, Toll receptor signaling) were up-regulated while genes (troponin, tropomodulin 2, endothelial lipase, FGFR4, integrin alpha8 and adenylate cyclase 8) related to the tumor suppression such as p53 pathway, TGF-beta signaling pathway and cadherin signaling pathway were down-regulated by K-ras oncogene. Proteomics approach revealed that up-regulated proteins in lung adenomas of K-ras mice were classified as follows: proteins related to the metabolism/catabolism (increased from 7 to 22% by K-ras gene), proteins related to translation/transcription and nucleotide (from 4 to 6%), proteins related to signal transduction (from 3 to 5%), proteins related to phosphorylation (from 1 to 2%). ATP synthase, Ras oncogene family, cytochrome c oxidase, flavoprotein, TEF 1, adipoprotein A-1 BP, glutathione oxidase, fatty acid BP 4, diaphorase 1, MAPK4 and transgelin were up-regulated by K-ras oncogene. However, integrin alpha1, Ras-interacting protein (Rain), endothelin-converting enzyme-1d and splicing factor 3b were down-regulated. These studies suggest that genes related to cancer development and inflammation were up-regulated while genes related to the tumor suppression were down-regulated by K-ras, resulting in the tumor growth. Putative biomarkers such as cell cycle related genes (Cdc37), cancer cell adhesion (Glycam 1, integrin alpha8, integrin alphaX and Clec4n), signal transduction (Tlr2, IL-33, and Ccbp2), migration (Ccr1, Ccl6, and diaphorase 1 (Cyb5r3) and cancer development (epiregulin) can be useful for diagnosis and as

  12. Role of Kras Status in Patients with Metastatic Colorectal Cancer Receiving First-Line Chemotherapy plus Bevacizumab: A TTD Group Cooperative Study

    PubMed Central

    Díaz-Rubio, Eduardo; Gómez-España, Auxiliadora; Massutí, Bartomeu; Sastre, Javier; Reboredo, Margarita; Manzano, José Luis; Rivera, Fernando; Safont, MªJosé; Montagut, Clara; González, Encarnación; Benavides, Manuel; Marcuello, Eugenio; Cervantes, Andrés; Martínez de Prado, Purificación; Fernández-Martos, Carlos; Arrivi, Antonio; Bando, Inmaculada; Aranda, E.; Gómez, A.; Massutí, B.; Yuste, A.; Rubio, E. Díaz; Sastre, J.; Valladares, M.; Abad, A.; Rivera, F.; Safont, MªJosé; Gallén, M.; González, E.; Benavides, M.; Marcuello, E.; Tobeña, M.; Cervantes, A.; Martínez de Prado, P.; Fernández-Martos, C.; Arrivi, A.; López-Ladrón, A.; Lacasta, A.; Llanos, M.; Remón, J.; Anton, A.; Vicent, J. Mª.; Gala´n, A.; Dueñas, R.; Tabernero, J. Mª.; Manzano, H.; Gómez, Mª. J.; Alfaro, J.; Losa, F.; Escudero, P.; García, T.; García López, J. L.; de Paredes, Mª L. García; Velasco, A.; Almenar, D.; Vera, R.; García Puche, J. L.; Carrato, A.; Lescure, A. Rodriguez; Jiménez, E.; Alberola, V.; García-Foncillas, J.; Constenla, M.; Ruiz, A.; Bueso, P.; Cabrera, E.; del Río,, L.; Ponce, J.; Oltra, A.; Checa, T.; Etxeberría, A.; Alonso, C.

    2012-01-01

    Background In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates. Methodology/Principal Findings KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p = 0.0038; HR: 1.40; 95% CI:1.12–1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p = 0.0002; HR: 1.55; 95% CI: 1.23–1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p = 0.0054; OR: 1.77; 95% CI: 1.18–2.64). Conclusions/Significance This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses. PMID:23174912

  13. Oncogenically active MYD88 mutations in human lymphoma

    PubMed Central

    Ngo, Vu N.; Young, Ryan M.; Schmitz, Roland; Jhavar, Sameer; Xiao, Wenming; Lim, Kian-Huat; Kohlhammer, Holger; Xu, Weihong; Yang, Yandan; Zhao, Hong; Shaffer, Arthur L.; Romesser, Paul; Wright, George; Powell, John; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Ott, German; Gascoyne, Randy D.; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Fisher, Richard I.; Braziel, Rita M.; Tubbs, Raymond R.; Cook, J. R.; Weisenburger, Denny D.; Chan, Wing C.; Staudt, Louis M.

    2016-01-01

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy1. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling2,3, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt’s lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, theMYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations

  14. Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy.

    PubMed

    Eng, Christina H; Wang, Zuncai; Tkach, Diane; Toral-Barza, Lourdes; Ugwonali, Savuth; Liu, Shanming; Fitzgerald, Stephanie L; George, Elizabeth; Frias, Elizabeth; Cochran, Nadire; De Jesus, Rowena; McAllister, Gregory; Hoffman, Gregory R; Bray, Kevin; Lemon, LuAnna; Lucas, Judy; Fantin, Valeria R; Abraham, Robert T; Murphy, Leon O; Nyfeler, Beat

    2016-01-01

    Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy. PMID:26677873

  15. Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

    PubMed Central

    Eng, Christina H.; Wang, Zuncai; Tkach, Diane; Toral-Barza, Lourdes; Ugwonali, Savuth; Liu, Shanming; Fitzgerald, Stephanie L.; George, Elizabeth; Frias, Elizabeth; Cochran, Nadire; De Jesus, Rowena; McAllister, Gregory; Hoffman, Gregory R.; Bray, Kevin; Lemon, LuAnna; Lucas, Judy; Fantin, Valeria R.; Abraham, Robert T.; Murphy, Leon O.; Nyfeler, Beat

    2016-01-01

    Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy. PMID:26677873

  16. Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer

    PubMed Central

    Chen, Zhao; Zhang, Jishuai; Wang, Yanxiao; Chen, Jicheng; Li, Xiubin; Ye, Hui; Tang, Chuanhao; Cheng, Xuan; Hou, Ning; Yang, Xiao; Wong, Kwok-Kin

    2013-01-01

    Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6-/- mice with KrasG12D mutant mice, which develop lung tumors after activation of mutant KrasG12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. KrasG12D; IL-6-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than KrasG12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated KrasG12D; p53flox/flox; IL-6-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than KrasG12D; p53flox/flox mice. Tumors from KrasG12D; IL-6-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than KrasG12D mice; however, these changes were not present between tumors from KrasG12D; p53flox/flox; IL-6-/- and KrasG12D; p53flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in KrasG12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion. PMID:24260500

  17. The epithelial polarity regulator LGALS9/galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux

    PubMed Central

    Wiersma, Valerie R; de Bruyn, Marco; Wei, Yunwei; van Ginkel, Robert J; Hirashima, Mitsuomi; Niki, Toshiro; Nishi, Nozomu; Zhou, Jin; Pouwels, Simon D; Samplonius, Douwe F; Nijman, Hans W; Eggleton, Paul; Helfrich, Wijnand; Bremer, Edwin

    2015-01-01

    Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRASmut) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRASmut CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAFmut). In KRASmut CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRASmut cancer cells. Thus, rLGALS9 has potent antitumor activity toward refractory KRASmut CRC cells that may be exploitable for therapeutic use. PMID:26086204

  18. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis.

    PubMed

    Bennett, James T; Tan, Tiong Yang; Alcantara, Diana; Tétrault, Martine; Timms, Andrew E; Jensen, Dana; Collins, Sarah; Nowaczyk, Malgorzata J M; Lindhurst, Marjorie J; Christensen, Katherine M; Braddock, Stephen R; Brandling-Bennett, Heather; Hennekam, Raoul C M; Chung, Brian; Lehman, Anna; Su, John; Ng, SuYuen; Amor, David J; Majewski, Jacek; Biesecker, Les G; Boycott, Kym M; Dobyns, William B; O'Driscoll, Mark; Moog, Ute; McDonell, Laura M

    2016-03-01

    Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis. PMID:26942290

  19. Copy number variants and rasopathies: germline KRAS duplication in a patient with syndrome including pigmentation abnormalities.

    PubMed

    Gilbert-Dussardier, Brigitte; Briand-Suleau, Audrey; Laurendeau, Ingrid; Bilan, Frédéric; Cavé, Hélène; Verloes, Alain; Vidaud, Michel; Vidaud, Dominique; Pasmant, Eric

    2016-01-01

    RAS/MAPK pathway germline mutations were described in Rasopathies, a class of rare genetic syndromes combining facial abnormalities, heart defects, short stature, skin and genital abnormalities, and mental retardation. The majority of the mutations identified in the Rasopathies are point mutations which increase RAS/MAPK pathway signaling. Duplications encompassing RAS/MAPK pathway genes (PTPN11, RAF1, MEK2, or SHOC2) were more rarely described. Here we report, a syndromic familial case of a 12p duplication encompassing the dosage sensitive gene KRAS, whose phenotype overlapped with rasopathies. The patient was referred because of a history of mild learning disabilities, small size, facial dysmorphy, and pigmentation abnormalities (café-au-lait and achromic spots, and axillar lentigines). This phenotype was reminiscent of rasopathies. No mutation was identified in the most common genes associated with Noonan, cardio-facio-cutaneous, Legius, and Costello syndromes, as well as neurofibromatosis type 1. The patient constitutional DNA exhibited a ~10.5 Mb duplication at 12p, including the KRAS gene. The index case's mother carried the same chromosome abnormality and also showed development delay with short stature, and numerous café-au-lait spots. Duplication of the KRAS gene may participate in the propositus phenotype, in particular of the specific pigmentation abnormalities. Array-CGH or some other assessment of gene/exon CNVs of RAS/MAPK pathway genes should be considered in the evaluation of individuals with rasopathies. PMID:27450488

  20. Base substitution mutations induced by metabolically activated aflatoxin B1.

    PubMed

    Foster, P L; Eisenstadt, E; Miller, J H

    1983-05-01

    We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli. By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions. One possible pathway leading to this base change involves depurination at guanine residues. We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo[a]pyrene diol epoxide and N-acetoxyacetylaminofluorene also specifically induce GxC leads to TxA transversions. PMID:6405385

  1. Insulin receptor substrate-1 deficiency drives a proinflammatory phenotype in KRAS mutant lung adenocarcinoma.

    PubMed

    Metz, Heather E; Kargl, Julia; Busch, Stephanie E; Kim, Kyoung-Hee; Kurland, Brenda F; Abberbock, Shira R; Randolph-Habecker, Julie; Knoblaugh, Sue E; Kolls, Jay K; White, Morris F; Houghton, A McGarry

    2016-08-01

    Insulin receptor substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many pathways activated in lung cancer. It has been assumed that IRS-1 promotes tumor growth through its ability to activate PI3K signaling downstream of the insulin-like growth factor receptor. Surprisingly, tumors with reduced IRS-1 staining in a human lung adenocarcinoma tissue microarray displayed a significant survival disadvantage, especially within the Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant subgroup. Accordingly, adenoviral Cre recombinase (AdCre)-treated LSL-Kras/Irs-1(fl/fl) (Kras/Irs-1(-/-)) mice displayed increased tumor burden and mortality compared with controls. Mechanistically, IRS-1 deficiency promotes Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling via the IL-22 receptor, resulting in enhanced tumor-promoting inflammation. Treatment of Kras/Irs-1(+/+) and Kras/Irs-1(-/-) mice with JAK inhibitors significantly reduced tumor burden, most notably in the IRS-1-deficient group. PMID:27439864

  2. Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

    SciTech Connect

    Yu, S.-H.; Wang, T.-H.; Au, L.-C.

    2009-01-09

    Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU {yields} GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.

  3. Activation of proto-oncogenes in human and mouse lung tumors

    SciTech Connect

    Reynolds, S.H.; Anderson, M.W. )

    1991-06-01

    Lung cancer is a leading cause of cancer-related deaths in several nations. Epidemiological studies have indicated that 85% of all lung cancer deaths and 30% of all cancer deaths in the US are associated with tobacco smoking. Various chemicals in tobacco smoke are thought to react with DNA and to ultimately yield heritable mutations. In an effort to understand the molecular mechanisms involved in lung tumorigenesis, the authors have analyzed proto-oncogene activation in a series of human lung tumors from smokers and spontaneously occurring and chemically induced lung tumors in mice. Approximately 86% of the human lung tumors and > 90% of the mouse lung tumors were found to contain activated oncogenes. ras Oncogenes activated by point mutations were detected in many of the human lung adenocarcinomas and virtually all of the mouse lung adenomas and adenocarcinomas. The mutation profiles of the activated K-ras genes detected in the chemically induced mouse lung tumors suggest that the observed mutations result from genotoxic effects of the chemicals. Comparison of the K-ras mutations observed in the human lung adenocarcinomas with mutation profiles observed in the mouse lung tumors suggest that bulky hydrophobic DNA adducts may be responsible for the majority of the mutations observed in the activated human K-ras genes. Other data indicate that approximately 20% of human lung tumors contain potentially novel transforming genes that may also be targets for mutagens in cigarette smoke.

  4. Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

    PubMed Central

    Ulivi, Paola; Delmonte, Angelo; Chiadini, Elisa; Calistri, Daniele; Papi, Maximilian; Mariotti, Marita; Verlicchi, Alberto; Ragazzini, Angela; Capelli, Laura; Gamboni, Alessandro; Puccetti, Maurizio; Dubini, Alessandra; Burgio, Marco Angelo; Casanova, Claudia; Crinò, Lucio; Amadori, Dino; Dazzi, Claudio

    2014-01-01

    Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity. PMID:25561229

  5. Frederick National Lab and the Pancreatic Cancer Action Network Award Fellowships for KRAS Research | Poster

    Cancer.gov

    By Nancy Parrish, Staff Writer The Frederick National Laboratory for Cancer Research (FNLCR) recently formed a partnership with the Pancreatic Cancer Action Network (PanCAN) to award a one-year fellowship to two scientists whose research will help lead to new therapies for pancreatic cancer. The scientists will focus on KRAS, a gene in the RAS family that is mutated in 95 percent of pancreatic cancers, according to the National Cancer Institute (NCI).

  6. Slug inhibits pancreatic cancer initiation by blocking Kras-induced acinar-ductal metaplasia

    PubMed Central

    Ebine, Kazumi; Chow, Christina R.; DeCant, Brian T.; Hattaway, Holly Z.; Grippo, Paul J.; Kumar, Krishan; Munshi, Hidayatullah G.

    2016-01-01

    Cells in the pancreas that have undergone acinar-ductal metaplasia (ADM) can transform into premalignant cells that can eventually become cancerous. Although the epithelial-mesenchymal transition regulator Snail (Snai1) can cooperate with Kras in acinar cells to enhance ADM development, the contribution of Snail-related protein Slug (Snai2) to ADM development is not known. Thus, transgenic mice expressing Slug and Kras in acinar cells were generated. Surprisingly, Slug attenuated Kras-induced ADM development, ERK1/2 phosphorylation and proliferation. Co-expression of Slug with Kras also attenuated chronic pancreatitis-induced changes in ADM development and fibrosis. In addition, Slug attenuated TGF-α-induced acinar cell metaplasia to ductal structures and TGF-α-induced expression of ductal markers in ex vivo acinar explant cultures. Significantly, blocking the Rho-associated protein kinase ROCK1/2 in the ex vivo cultures induced expression of ductal markers and reversed the effects of Slug by inducing ductal structures. In addition, blocking ROCK1/2 activity in Slug-expressing Kras mice reversed the inhibitory effects of Slug on ADM, ERK1/2 phosphorylation, proliferation and fibrosis. Overall, these results increase our understanding of the role of Slug in ADM, an early event that can eventually lead to pancreatic cancer development. PMID:27364947

  7. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    PubMed

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. PMID:26867649

  8. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    PubMed

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  9. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

    PubMed Central

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease. PMID:27570485

  10. Management and future directions in non-small cell lung cancer with known activating mutations.

    PubMed

    Gerber, David E; Gandhi, Leena; Costa, Daniel B

    2014-01-01

    Lung cancer accounts for a quarter of all cancer deaths. Non-small cell lung cancer (NSCLC) is currently segregated by the presence of actionable driver oncogenes. This review will provide an overview of molecular subsets of lung cancer, including descriptions of the defining oncogenes (EGFR, ALK, KRAS, ROS1, RET, BRAF, ERBB2, NTRK1, FGFR, among others) and how these predict for response to small molecule tyrosine kinase inhibitors (TKIs) that are either clinically available or in clinical trial development for advanced NSCLC. Particular focus will be placed on subsets with EGFR mutated and ALK rearranged NSCLC. Somatic TKI-sensitizing EGFR mutations (such as exon 19 deletions and L858R substitutions) are the most robust predictive biomarker for symptom improvement, radiographic response, and increment in progression-free survival (PFS) when EGFR TKIs (gefitinib, erlotinib, and afatinib) are used for patients with advanced NSCLC. However, the palliative benefits that EGFR TKIs afford are limited by multiple biologic mechanisms of tumor adaptation/resistance (such as the EGFR-T790M mutation and oncogene bypass tracks), and future efforts toward delaying, preventing, and treating resistance are underway. Similar to EGFR mutations, ALK rearrangements exemplify an oncogene-driven NSCLC that can be effectively palliated with a precision TKI therapy (the multitargeted ALK/MET/ROS1 TKI crizotinib). When resistance to first-line crizotinib therapy occurs, multiple second generation ALK TKIs have demonstrated impressive rates of disease control in clinical trials, and these may modify long-term outcomes for patients with ALK-positive NSCLC. The development of TKIs for other oncogene-driven NSCLCs may expand the portfolio of precision therapies for this recalcitrant cancer. PMID:24857124

  11. JAK-2 V617F mutation increases heparanase procoagulant activity.

    PubMed

    Kogan, Inna; Chap, Dafna; Hoffman, Ron; Axelman, Elena; Brenner, Benjamin; Nadir, Yona

    2016-01-01

    Patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are at increased risk of arterial and venous thrombosis. In patients with ET a positive correlation was observed between JAK-2 V617F mutation, that facilitates erythropoietin receptor signalling, and thrombotic events, although the mechanism involved is not clear. We previously demonstrated that heparanase protein forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF) which leads to increased factor Xa production and subsequent activation of the coagulation system. The present study was aimed to evaluate heparanase procoagulant activity in myeloproliferative neoplasms. Forty bone marrow biopsies of patients with ET, PV, PMF and chronic myelogenous leukaemia (CML) were immunostained to heparanase, TF and TF pathway inhibitor (TFPI). Erythropoietin receptor positive cell lines U87 human glioma and MCF-7 human breast carcinoma were studied. Heparanase and TFPI staining were more prominent in ET, PV and PMF compared to CML. The strongest staining was in JAK-2 positive ET biopsies. Heparanase level and procoagulant activity were higher in U87 cells transfected to over express JAK-2 V617F mutation compared to control and the effect was reversed using JAK-2 inhibitors (Ruxolitinib, VZ3) and hydroxyurea, although the latter drug did not inhibit JAK-2 phosphorylation. Erythropoietin increased while JAK-2 inhibitors decreased the heparanase level and procoagulant activity in U87 and MCF-7 parental cells. In conclusion, JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor. The present findings may potentially point to a new mechanism of thrombosis in JAK-2 positive ET patients. PMID:26489695

  12. KrasG12D induces EGFR-MYC cross signaling in murine primary pancreatic ductal epithelial cells

    PubMed Central

    Diersch, Sandra; Wirth, Matthias; Schneeweis, Christian; Jörs, Simone; Geisler, Fabian; Siveke, Jens T.; Rad, Roland; Schmid, Roland M.; Saur, Dieter; Rustgi, Anil K.; Reichert, Maximilian; Schneider, Günter

    2015-01-01

    Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic KrasG12D from the endogenous promoter. We show that primary PDECs are susceptible to KrasG12D-driven transformation and form pancreatic ductal adenocarcinomas (PDAC) in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of KrasG12D induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease KrasG12D-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby, explain the crucial role of EGFR for KrasG12D-driven carcinogenesis in the pancreas. PMID:26592448

  13. Liquid Biopsy for Detection of Actionable Oncogenic Mutations in Human Cancers and Electric Field Induced Release and Measurement Liquid Biopsy (eLB)

    PubMed Central

    Tu, Michael; Chia, David; Wei, Fang; Wong, David

    2015-01-01

    Oncogenic activations by mutations in key cancer genes such as EGFR and KRAS are frequently associated with human cancers. Molecular targeting of specific oncogenic mutations in human cancer is a major therapeutic inroad for anti-cancer drug therapy. In addition, progressive developments of oncogene mutations lead to drug resistance. Therefore, the ability to detect and continuously monitor key actionable oncogenic mutations is important to guide the use of targeted molecular therapies to improve long-term clinical outcomes in cancer patients. Current oncogenic mutation detection is based on direct sampling of cancer tissue by surgical resection or biopsy. Oncogenic mutations were recently shown to be detectable in circulating bodily fluids of cancer patients. This field of investigation, termed liquid biopsy, permits a less invasive means of assessing the oncogenic mutation profile of a patient. This paper will review the analytical strategies used to assess oncogenic mutations from biofluid samples. Clinical applications will also be discussed. PMID:26645892

  14. Ataxia telangiectasia mutated influences cytochrome c oxidase activity.

    PubMed

    Patel, Akshar Y; McDonald, Todd M; Spears, Larry D; Ching, James Kain; Fisher, Jonathan S

    2011-02-25

    Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1h with the ATM inhibitor KU55933 caused a ∼50% reduction (P<0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P<0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity. PMID:21266166

  15. Oncogenic Kras-induced GM-CSF production promotes the development of pancreatic neoplasia

    PubMed Central

    Pylayeva-Gupta, Yuliya; Lee, Kyoung Eun; Hajdu, Cristina H.; Miller, George; Bar-Sagi, Dafna

    2013-01-01

    Summary Stromal responses elicited by early stage neoplastic lesions can promote tumor growth. However, the molecular mechanisms that underlie the early recruitment of stromal cells to sites of neoplasia remain poorly understood. Here we demonstrate an oncogenic KrasG12D-dependent upregulation of GM-CSF in mouse pancreatic ductal epithelial cells (PDEC). An enhanced GM-CSF production is also observed in human PanIN lesions. KrasG12D-dependent production of GM-CSF in vivo is required for the recruitment of Gr1+CD11b+ myeloid cells. The suppression of GM-CSF production inhibits the in vivo growth of KrasG12D-PDECs and, consistent with the role of GM-CSF in Gr1+CD11b+ mobilization, this effect is mediated by CD8+ T cells. These results identify a pathway that links oncogenic activation to the evasion of anti-tumor immunity. PMID:22698407

  16. The Significance of Ras Activity in Pancreatic Cancer Initiation

    PubMed Central

    Logsdon, Craig D.; Lu, Weiqin

    2016-01-01

    The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of K-RAS. However, oncogenic K-Rasmt alone is not sufficient to lead to pancreatic ductal adenocarcinoma (PDAC) in either human or in genetically modified adult mouse models. Many stimulants, such as high fat diet, CCK, LPS, PGE2 and others, have physiological effects at low concentrations that are mediated in part through modest increases in K-Ras activity. However, at high concentrations, they induce inflammation that, in the presence of oncogenic K-Ras expression, substantially accelerates PDAC formation. The mechanism involves increased activity of oncogenic K-Rasmt. Unlike what has been proposed in the standard paradigm for the role of Ras in oncogenesis, oncogenic K-Rasmt is now known to not be constitutively active. Rather, it can be activated by standard mechanisms similar to wild-type K-Ras, but its activity is sustained for a prolonged period. Furthermore, if the level of K-Ras activity exceeds a threshold at which it begins to generate its own activators, then a feed-forward loop is formed between K-Ras activity and inflammation and pathological processes including oncogenesis are initiated. Oncogenic K-Rasmt activation, a key event in PDAC initiation and development, is subject to complex regulatory mechanisms. Reagents which inhibit inflammation, such as the Cox2 inhibitor celecoxib, block the feed-forward loop and prevent induction of PDAC in models with endogenous oncogenic K-Rasmt. Increased understanding of the role of activating and inhibitory mechanisms on oncogenic K-Rasmt activity is of paramount importance for the development of preventive and therapeutic strategies to fight against this lethal disease. PMID:26929740

  17. Frequent PTPRK-RSPO3 fusions and RNF43 mutations in colorectal traditional serrated adenoma.

    PubMed

    Sekine, Shigeki; Yamashita, Satoshi; Tanabe, Taro; Hashimoto, Taiki; Yoshida, Hiroshi; Taniguchi, Hirokazu; Kojima, Motohiro; Shinmura, Kazuya; Saito, Yutaka; Hiraoka, Nobuyoshi; Ushijima, Toshikazu; Ochiai, Atsushi

    2016-06-01

    The molecular mechanisms underlying the serrated pathway of colorectal tumourigenesis, particularly those related to traditional serrated adenomas (TSAs), are still poorly understood. In this study, we analysed genetic alterations in 188 colorectal polyps, including hyperplastic polyps, sessile serrated adenomas/polyps (SSA/Ps), TSAs, tubular adenomas, and tubulovillous adenomas by using targeted next-generation sequencing and reverse transcription-PCR. Our analyses showed that most TSAs (71%) contained genetic alterations in WNT pathway components. In particular, PTPRK-RSPO3 fusions (31%) and RNF43 mutations (24%) were frequently and almost exclusively observed in TSAs. Consistent with the WNT pathway activation, immunohistochemical analysis showed diffuse and focal nuclear accumulation of β-catenin in 53% and 30% of TSAs, respectively. APC mutations were observed in tubular and tubulovillous adenomas and in a subset of TSAs. BRAF mutations were exclusively and frequently encountered in serrated lesions. KRAS mutations were observed in all types of polyps, but were most commonly encountered in tubulovillous adenomas and TSAs. This study has demonstrated that TSAs frequently harbour genetic alterations that lead to WNT pathway activation, in addition to BRAF and KRAS mutations. In particular, PTPRK-RSPO3 fusions and RNF43 mutations were found to be characteristic genetic features of TSAs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26924569

  18. Mutational analysis of primary and metastatic colorectal cancer samples underlying the resistance to cetuximab-based therapy

    PubMed Central

    Nemecek, Radim; Berkovcova, Jitka; Radova, Lenka; Kazda, Tomas; Mlcochova, Jitka; Vychytilova-Faltejskova, Petra; Slaby, Ondrej; Svoboda, Marek

    2016-01-01

    Purpose Although several molecular markers predicting resistance to cetuximab- or panitumumab-based therapy of metastatic colorectal cancer were described, mutations in RAS proto-oncogenes remain the only predictors being used in daily clinical practice. However, 35%–45% of wild-type RAS patients still do not respond to this anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody-based therapy, and therefore the definition of other predictors forms an important clinical need. The aim of the present retrospective single-institutional study was to evaluate potential genes responsible for resistance to anti-EGFR therapy in relation to mutational analysis of primary versus metastatic lesions. Patients and methods Twenty-four paired primary and corresponding metastatic tissue samples from eight nonresponding and four responding metastatic colorectal cancer patients treated with cetuximab-based therapy were sequenced using a next-generation sequencing panel of 26 genes involved in EGFR signaling pathway and colorectal carcinogenesis. Results Mutational status of primary tumors and metastatic lesions was highly concordant in TP53, APC, CTNNB1, KRAS, PIK3CA, PTEN, and FBXW7 genes. Metastatic samples harbor significantly more mutations than primary tumors. Potentially negative predictive value of FBXW7 mutations in relationship to anti-EGFR treatment outcomes was confirmed. Finally, new occurrences of activating KRAS mutations were identified in a group of patients initially determined as wild-type RAS by routinely used qPCR-based RAS mutational tests. All newly detected activating KRAS mutations most likely led to cetuximab treatment failure. Conclusion The results of the present study suggest a need of careful consideration of previously published results of anti-EGFR-targeted therapy with regard to potentially inaccurate diagnostic tools used in the past. Based on our findings, we recommend more extensive use of next-generation sequencing testing in daily

  19. Three faces of recombination activating gene 1 (RAG1) mutations.

    PubMed

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation. PMID:26689875

  20. Determination of synthetic lethal interactions in KRAS oncogene-dependent cancer cells reveals novel therapeutic targeting strategies

    PubMed Central

    Steckel, Michael; Molina-Arcas, Miriam; Weigelt, Britta; Marani, Michaela; Warne, Patricia H; Kuznetsov, Hanna; Kelly, Gavin; Saunders, Becky; Howell, Michael; Downward, Julian; Hancock, David C

    2012-01-01

    Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Cancer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy. PMID:22613949

  1. An extra copy of p53 suppresses development of spontaneous Kras-driven but not radiation-induced cancer

    PubMed Central

    Moding, Everett J.; Min, Hooney D.; Castle, Katherine D.; Ali, Moiez; Woodlief, Loretta; Williams, Nerissa; Ma, Yan; Kim, Yongbaek; Lee, Chang-Lung; Kirsch, David G.

    2016-01-01

    The tumor suppressor p53 blocks tumor progression in multiple tumor types. Radiation-induced cancer following exposure to radiation therapy or space travel may also be regulated by p53 because p53 has been proposed to respond to DNA damage to suppress tumorigenesis. Here, we investigate the role of p53 in lung carcinogenesis and lymphomagenesis in LA-1 KrasG12D mice with wild-type p53 or an extra copy of p53 (super p53) exposed to fractionated total body irradiation with low linear energy transfer (low-LET) X-rays or high-LET iron ions and compared tumor formation in these mice with unirradiated controls. We found that an additional copy of p53 suppressed both Kras-driven lung tumor and lymphoma development in the absence of radiation. However, an additional copy of p53 did not affect lymphoma development following low- or high-LET radiation exposure and was unable to suppress radiation-induced expansion of thymocytes with mutated Kras. Moreover, radiation exposure increased lung tumor size in super p53 but not wild-type p53 mice. These results demonstrate that although p53 suppresses the development of spontaneous tumors expressing KrasG12D, in the context of exposure to ionizing radiation, an extra copy of p53 does not protect against radiation-induced lymphoma and may promote KrasG12D mutant lung cancer. PMID:27453951

  2. ¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

    PubMed

    Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

    2013-10-01

    K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein. PMID:22886485

  3. KRAS genomic status predicts the sensitivity of ovarian cancer cells to decitabine

    PubMed Central

    Stewart, ML; Tamayo, P; Wilson, AJ; Wang, S; Chang, YM; Kim, JW; Khabele, D; Shamji, AF; Schreiber, SL

    2015-01-01

    Decitabine, a cancer therapeutic that inhibits DNA methylation, produces variable antitumor response rates in patients with solid tumors that might be leveraged clinically with identification of a predictive biomarker. In this study, we profiled the response of human ovarian, melanoma and breast cancer cells treated with decitabine, finding that RAS/MEK/ERK pathway activation and DNMT1 expression correlated with cytotoxic activity. Further, we showed that KRAS genomic status predicted decitabine sensitivity in low and high-grade serous ovarian cancer cells. Pre-treatment with decitabine decreased the cytotoxic activity of MEK inhibitors in KRAS-mutant ovarian cancer cells, with reciprocal downregulation of DNMT1 and MEK/ERK phosphorylation. In parallel with these responses, decitabine also upregulated the pro-apoptotic BCL-2 family member BNIP3, which is known to be regulated by MEK and ERK, and heightened the activity of pro-apoptotic small molecule navitoclax, a BCL-2 family inhibitor. In a xenograft model of KRAS-mutant ovarian cancer, combining decitabine and navitoclax heighted antitumor activity beyond administration of either compound alone. Our results define the RAS/MEK/DNMT1 pathway as a determinant of sensitivity to DNA methyltransferase inhibition, specifically implicating KRAS status as a biomarker of drug response in ovarian cancer. PMID:25968887

  4. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  5. Comprehensive mutational profiling of core binding factor acute myeloid leukemia.

    PubMed

    Duployez, Nicolas; Marceau-Renaut, Alice; Boissel, Nicolas; Petit, Arnaud; Bucci, Maxime; Geffroy, Sandrine; Lapillonne, Hélène; Renneville, Aline; Ragu, Christine; Figeac, Martin; Celli-Lebras, Karine; Lacombe, Catherine; Micol, Jean-Baptiste; Abdel-Wahab, Omar; Cornillet, Pascale; Ifrah, Norbert; Dombret, Hervé; Leverger, Guy; Jourdan, Eric; Preudhomme, Claude

    2016-05-19

    Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML. PMID:26980726

  6. KRAS Mutant Pancreatic Cancer: No Lone Path to an Effective Treatment

    PubMed Central

    Zeitouni, Daniel; Pylayeva-Gupta, Yuliya; Der, Channing J.; Bryant, Kirsten L.

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers with a dismal 7% 5-year survival rate and is projected to become the second leading cause of cancer-related deaths by 2020. KRAS is mutated in 95% of PDACs and is a well-validated driver of PDAC growth and maintenance. However, despite comprehensive efforts, an effective anti-RAS drug has yet to reach the clinic. Different paths to inhibiting RAS signaling are currently under investigation in the hope of finding a successful treatment. Recently, direct RAS binding molecules have been discovered, challenging the perception that RAS is an “undruggable” protein. Other strategies currently being pursued take an indirect approach, targeting proteins that facilitate RAS membrane association or downstream effector signaling. Unbiased genetic screens have identified synthetic lethal interactors of mutant RAS. Most recently, metabolic targets in pathways related to glycolytic signaling, glutamine utilization, autophagy, and macropinocytosis are also being explored. Harnessing the patient’s immune system to fight their cancer is an additional exciting route that is being considered. The “best” path to inhibiting KRAS has yet to be determined, with each having promise as well as potential pitfalls. We will summarize the state-of-the-art for each direction, focusing on efforts directed toward the development of therapeutics for pancreatic cancer patients with mutated KRAS. PMID:27096871

  7. KRAS Mutant Pancreatic Cancer: No Lone Path to an Effective Treatment.

    PubMed

    Zeitouni, Daniel; Pylayeva-Gupta, Yuliya; Der, Channing J; Bryant, Kirsten L

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers with a dismal 7% 5-year survival rate and is projected to become the second leading cause of cancer-related deaths by 2020. KRAS is mutated in 95% of PDACs and is a well-validated driver of PDAC growth and maintenance. However, despite comprehensive efforts, an effective anti-RAS drug has yet to reach the clinic. Different paths to inhibiting RAS signaling are currently under investigation in the hope of finding a successful treatment. Recently, direct RAS binding molecules have been discovered, challenging the perception that RAS is an "undruggable" protein. Other strategies currently being pursued take an indirect approach, targeting proteins that facilitate RAS membrane association or downstream effector signaling. Unbiased genetic screens have identified synthetic lethal interactors of mutant RAS. Most recently, metabolic targets in pathways related to glycolytic signaling, glutamine utilization, autophagy, and macropinocytosis are also being explored. Harnessing the patient's immune system to fight their cancer is an additional exciting route that is being considered. The "best" path to inhibiting KRAS has yet to be determined, with each having promise as well as potential pitfalls. We will summarize the state-of-the-art for each direction, focusing on efforts directed toward the development of therapeutics for pancreatic cancer patients with mutated KRAS. PMID:27096871

  8. Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase).

    PubMed

    Ilinskaya, Olga N; Singh, Indrabahadur; Dudkina, Elena; Ulyanova, Vera; Kayumov, Airat; Barreto, Guillermo

    2016-07-01

    RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease. PMID:27066977