- Email: [email protected]
Identification of chemotherapeutic resistant mutations in castration-resistant prostate cancer
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Results: In the current study, we observed increased expression levels of miR31-5p, miR-155-5p and miR-210-3p, respectively 60.55%, 50.00%, 39.47% in tumor tissue in comparison with paired normal tissue. In order to evaluate the miRNAs as biomarker for differentiation between tumour and metastatic tissue we performed the receiver operating characteristic (ROC) curve analysis. miR31-5p and miR-155-5p showed stronger discriminative accuracy, respectively with AUC = 0.808, p = 0.01 (95% CI: 0.650–0.967) and AUC = 0.744, p = 0.042 (95% CI: 0.575–0.913). miR-210-3p did not show statistically significant values for discriminative tumour tissue versus metastatic tissue potential. Spearman’s rho was performed in order to evaluate the expression levels of the three hypoxic miRNas, and HIF1a and VEGFA. The correlation analysis showed statistically significant correlation coefficient between miR-210-3p with HIF1a (0.452, p = 0.02) and VEGFA (0.349, p = 0.019). Moreover expression of miR-210-3p correlate with miR-31-5p (0.421, p = 0.04). However, miR-31-5p showed significant correlation with HIF1a, rho = 0.394, p = 0.007, but not with VEGFA. None of the studied microRNAs (miRNAs) showed correlation with age and lymph node metastasis. Conclusion: The current findings suggest miR-31-5p, miR-155-5p and miR210-3p have major role in hypoxic mechanisms. These results may contribute to future studies on elucidating the mechanisms of contribution of hypoxic miRNAs to LSCC. No conflict of interest. 115 Antitumor effect of pharmacological and genetic ablation of selected histone modifying enzymes in human glioma cells 1 1 . Nencki Institute of ´ S.K. Krol ´ 1 , M. Maleszewska1 , B. Wojta´s1 , B. Kaminska Experimental Biology, Laboratory of Molecular Neurobiology- Neurobiology Center, Warsaw, Poland
Background: Glioblastoma (GBM, WHO grade IV) is the most frequent, diffusive tumor, highly resistant to radio- and chemotherapy. Despite significant understanding the molecular mechanisms of GBM pathobiology, clinical prognoses remain poor and median survival of patients is 14 months. Recent findings from exome and RNA sequencing revealed many aberrations in sequences and/or expression of genes coding for epigenetic enzymes in GBM suggesting the important role of epigenetic dysfunction in GBM pathogenesis. Histone modifications and global aberrations at the histone level may result in dysregulated patterns of gene expression, and malfunction of proteins that regulate chromatin modification and remodeling. Histone modifications are reversible and the possibility of “resetting” of their abnormal patterns in cancer by genetic or pharmacological compounds is a promising, therapeutic strategy. Material and Methods: We determined the expression of epigenetic enzymes and chromatin modifiers in primary and established glioma cell lines using RT2 Epigenetic Profiler. The expression of selected enzymes and global levels of modified histones were analyzed by Western Blotting. We studied the effect of selected inhibitors (VPA − valproic acid, TSA − trichostatin A, 3DZNep − 3-Deazaneplanocin A) and siRNA-mediated knockdown of epigenetic enzymes: HDAC1, HDAC2, EZH2 on histone modifications by immunofluorescence staining and Western Blotting. Cell viability and proliferation of treated human glioma cells (LN18, U87) were studied using MTT metabolism and BrdU incorporation tests, respectively. Results: We demonstrate global downregulation of epigenetic enzymes in glioma cells lines when compared with non-transformed astrocytes. Only the levels of HDAC1, HDAC2 and EZH2 were significantly increased. We found that siRNA-mediated knockdown of HDAC1, HDAC2 and EZH2 results in inhibition of glioma cells proliferation. Similarly, inhibition of HDAC1, HDAC2 and EZH2 activities with VPA, TSA and 3DNZep has antitumor effects and reduced viability and proliferation of cultured glioma cells in dose- and time-dependent manner. Conclusions: Epigenetic mechanisms are significantly deregulated in glioma cells in a global scale. Inhibition of expression/activity of HDAC1, HDAC2 and EZH2, which are overexpressed in glioma cells, affects growth of tumor cells. These results provide a rationale for applying inhibitors of selected epigenetic enzymes in glioma therapy. No conflict of interest. 116 Identification of chemotherapeutic resistant mutations in castration-resistant prostate cancer F. Ozgun ¨ 1 , H. Sarac1 , N. Lack1 . 1 Koc University, Molecular Biology and Genetics, Istanbul, Turkey Introduction: Prostate cancer is an extremely common disease in Turkish and European men. The standard care for late-stage cancer is designed to inhibit the activation of Androgen Receptor (AR). One class of therapeutics is antiandrogens that directly inhibit AR activation. While this therapeutic approach is initially efficient, the cancer almost always develops resistance. When therapy fails, the median survival of patients who suffer from castrationresistant prostate cancer (CRPC) is less than 24 months. Enzalutamide has been shown to dramatically increase the survival of CRPC patients who have failed hormone therapy. Despite this success, recent clinical studies have
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demonstrated that cancer also develops resistance to enzalutamide. Point mutations on androgen receptor are one of the main causes of resistance. In an effort to identify those patients that will benefit from enzalutamide treatment we are developing a chemical screen to identify nearly all point mutations that cause drug resistance. Material and Method: We modified a well-validated screening technique that has been used to identify mutations that cause resistance to kinase inhibitors to study antiandrogens. We combine random mutagenesis of AR with a cellular selection in AR mutagenesis screen. AR mutants are tested in a cell line which survives only if there is functional AR in the presence of puromycin. In the presence of antiandrogen, AR function would be inhibited and the cells get the puromycin sensitivity back. We test more than 2 million individual mutants in the presence of antiandrogen and puromycin by using reporter cell line. AR gene from the resistant cells will be PCR amplified and sequenced to identify mutants. Results and Discussion: Initially, puromycin resistance of reporter cell line was confirmed by cell viability assay. It has been shown that the cells undergo death with increasing concentrations of enzalutamide. AR mutations were done by using XL-1 Red mutator strain. We performed blue/white screening taking advantage of LacZa gene to identify colonies that include AR mutation and calculate mutation rate. Conclusion: Chemical genetic technique offers a powerful technique to identify drug resistant mutations before it is given to patients. Once further optimized, this approach could be used to investigate the resistance mechanism for many clinical drugs. No conflict of interest. 117 Somatic mutation of the ATRX and gene in the ERBB4-Akt/mTOR pathway are frequent in cervical small cell neuroendocrine tumors M. Choi1 , S. Cho2 , H.J. Ban3 , C.H. Lee4 , H.K. Kim1 , C. Kim5 , J.Y. Lee1 . Korea University, Pathology, Seoul, Korea, 2 Seoul National University, Laboratory of Developmental Biology and Genomics, Seoul, Korea, 3 Hanyang University, 3Division of Molecular and Life Sciences, Ansan, Korea, 4 Thermo Fisher Scientific Corporation, Life Science Solutions Group, Seoul, Korea, 5 Department of Biotechnology, Konkuk University, Seoul, Korea 1
Background: Cervical small cell neuroendocrine tumors (CSCNETs) are extremely rare and aggressive form of neuroendocrine tumors (NETs). Because there is no sufficient standardized diagnostic or prognostic marker and consensus can be used so far, it is still difficult to diagnose and predict disease progress with limited treatment strategy. Material and Methods: This study included 16 formalin fixed paraffin embedded (FFPE) tissue blocks of CSCNET, which were diagnosed and collected between 1997 and 2012 in South Korea. Of 16 samples, five tumornormal paired CSCNETs were used to perform whole exome sequencing (WES) and subsequent immunohistochemistry staining to verify the finding of WES. Results: We first provide mutation profile of five tumor-adjacent normal paired CSCNETs using by whole exome sequencing. The most frequently mutated gene was the ATRX and genes in the Akt/mTOR pathway, which were indicated common mutation signature across NETs. Other frequently mutated gene was the ERBB4 encoding a receptor tyrosine kinase that is a member of the epidermal growth factor receptor (EGFR) and has diverse functions in the development of the nervous and neuroendocrine system by affecting downstream signaling pathway including Akt/mTOR pathway. Increased expression of ERBB4 was examined in the tumor tissue comparing normal counterpart by immunohistochemistry staining. Conclusion: This result suggested CSCNETs shares genetic characteristics of NETs and provided new insight for sufficient clinical management for patient, especially targeting ERBB4 and Akt/mTOR pathway. No conflict of interest. 118 Evolutionary trajectory of Asian EGFR mutation positive lung adenocarcinomas leads to “high intratumor heterogeneity” R. Nahar1 , W. Zhai2 , T. Zhang2 , A. Takano3 , A.J. Khng1 , Y.Y. Lee1 , X. Liu1 , C.H. Lim4 , T.K.H. Lim3 , T.P.T. Koh4 , Z.W. Aung5 , A.S.M. Teo1 , C.X. Chan1 , C.K. Toh6 , W.T. Lim6 , B. Lim7 , W.L. Tam7 , E.H. Tan6 , D.S.W. Tan6 , A.M. Hillmer1 . 1 Genome Institute of Singapore, Cancer Therapeutics and Stratified Oncology, Singapore, Singapore, 2 Genome Institute of Singapore, Human Genetics, Singapore, Singapore, 3 Singapore General Hospital, Department of Pathology, Singapore, Singapore, 4 National Heart Centre SIngapore, Department of Cardiothoracic Surgery, Singapore, Singapore, 5 National Cancer Centre Singapore, Department of Clinical Trials and Epidemiological Sciences, Singapore, Singapore, 6 National Cancer Centre Singapore, Division of Medical Oncology, Singapore, Singapore, 7 Genome Institute of Singapore, Cancer Stem Cell Biology, Singapore, Singapore Background: In contrast to western populations, activating mutations in EGFR are more frequent in Asian lung adenocarcinomas (LUADs) which are enriched for female gender and never-smokers. Although EGFR tyrosine
Background: Glioblastoma (GBM, WHO grade IV) is the most frequent, diffusive tumor, highly resistant to radio- and chemotherapy. Despite significant understanding the molecular mechanisms of GBM pathobiology, clinical prognoses remain poor and median survival of patients is 14 months. Recent findings from exome and RNA sequencing revealed many aberrations in sequences and/or expression of genes coding for epigenetic enzymes in GBM suggesting the important role of epigenetic dysfunction in GBM pathogenesis. Histone modifications and global aberrations at the histone level may result in dysregulated patterns of gene expression, and malfunction of proteins that regulate chromatin modification and remodeling. Histone modifications are reversible and the possibility of “resetting” of their abnormal patterns in cancer by genetic or pharmacological compounds is a promising, therapeutic strategy. Material and Methods: We determined the expression of epigenetic enzymes and chromatin modifiers in primary and established glioma cell lines using RT2 Epigenetic Profiler. The expression of selected enzymes and global levels of modified histones were analyzed by Western Blotting. We studied the effect of selected inhibitors (VPA − valproic acid, TSA − trichostatin A, 3DZNep − 3-Deazaneplanocin A) and siRNA-mediated knockdown of epigenetic enzymes: HDAC1, HDAC2, EZH2 on histone modifications by immunofluorescence staining and Western Blotting. Cell viability and proliferation of treated human glioma cells (LN18, U87) were studied using MTT metabolism and BrdU incorporation tests, respectively. Results: We demonstrate global downregulation of epigenetic enzymes in glioma cells lines when compared with non-transformed astrocytes. Only the levels of HDAC1, HDAC2 and EZH2 were significantly increased. We found that siRNA-mediated knockdown of HDAC1, HDAC2 and EZH2 results in inhibition of glioma cells proliferation. Similarly, inhibition of HDAC1, HDAC2 and EZH2 activities with VPA, TSA and 3DNZep has antitumor effects and reduced viability and proliferation of cultured glioma cells in dose- and time-dependent manner. Conclusions: Epigenetic mechanisms are significantly deregulated in glioma cells in a global scale. Inhibition of expression/activity of HDAC1, HDAC2 and EZH2, which are overexpressed in glioma cells, affects growth of tumor cells. These results provide a rationale for applying inhibitors of selected epigenetic enzymes in glioma therapy. No conflict of interest. 116 Identification of chemotherapeutic resistant mutations in castration-resistant prostate cancer F. Ozgun ¨ 1 , H. Sarac1 , N. Lack1 . 1 Koc University, Molecular Biology and Genetics, Istanbul, Turkey Introduction: Prostate cancer is an extremely common disease in Turkish and European men. The standard care for late-stage cancer is designed to inhibit the activation of Androgen Receptor (AR). One class of therapeutics is antiandrogens that directly inhibit AR activation. While this therapeutic approach is initially efficient, the cancer almost always develops resistance. When therapy fails, the median survival of patients who suffer from castrationresistant prostate cancer (CRPC) is less than 24 months. Enzalutamide has been shown to dramatically increase the survival of CRPC patients who have failed hormone therapy. Despite this success, recent clinical studies have
S13
demonstrated that cancer also develops resistance to enzalutamide. Point mutations on androgen receptor are one of the main causes of resistance. In an effort to identify those patients that will benefit from enzalutamide treatment we are developing a chemical screen to identify nearly all point mutations that cause drug resistance. Material and Method: We modified a well-validated screening technique that has been used to identify mutations that cause resistance to kinase inhibitors to study antiandrogens. We combine random mutagenesis of AR with a cellular selection in AR mutagenesis screen. AR mutants are tested in a cell line which survives only if there is functional AR in the presence of puromycin. In the presence of antiandrogen, AR function would be inhibited and the cells get the puromycin sensitivity back. We test more than 2 million individual mutants in the presence of antiandrogen and puromycin by using reporter cell line. AR gene from the resistant cells will be PCR amplified and sequenced to identify mutants. Results and Discussion: Initially, puromycin resistance of reporter cell line was confirmed by cell viability assay. It has been shown that the cells undergo death with increasing concentrations of enzalutamide. AR mutations were done by using XL-1 Red mutator strain. We performed blue/white screening taking advantage of LacZa gene to identify colonies that include AR mutation and calculate mutation rate. Conclusion: Chemical genetic technique offers a powerful technique to identify drug resistant mutations before it is given to patients. Once further optimized, this approach could be used to investigate the resistance mechanism for many clinical drugs. No conflict of interest. 117 Somatic mutation of the ATRX and gene in the ERBB4-Akt/mTOR pathway are frequent in cervical small cell neuroendocrine tumors M. Choi1 , S. Cho2 , H.J. Ban3 , C.H. Lee4 , H.K. Kim1 , C. Kim5 , J.Y. Lee1 . Korea University, Pathology, Seoul, Korea, 2 Seoul National University, Laboratory of Developmental Biology and Genomics, Seoul, Korea, 3 Hanyang University, 3Division of Molecular and Life Sciences, Ansan, Korea, 4 Thermo Fisher Scientific Corporation, Life Science Solutions Group, Seoul, Korea, 5 Department of Biotechnology, Konkuk University, Seoul, Korea 1
Background: Cervical small cell neuroendocrine tumors (CSCNETs) are extremely rare and aggressive form of neuroendocrine tumors (NETs). Because there is no sufficient standardized diagnostic or prognostic marker and consensus can be used so far, it is still difficult to diagnose and predict disease progress with limited treatment strategy. Material and Methods: This study included 16 formalin fixed paraffin embedded (FFPE) tissue blocks of CSCNET, which were diagnosed and collected between 1997 and 2012 in South Korea. Of 16 samples, five tumornormal paired CSCNETs were used to perform whole exome sequencing (WES) and subsequent immunohistochemistry staining to verify the finding of WES. Results: We first provide mutation profile of five tumor-adjacent normal paired CSCNETs using by whole exome sequencing. The most frequently mutated gene was the ATRX and genes in the Akt/mTOR pathway, which were indicated common mutation signature across NETs. Other frequently mutated gene was the ERBB4 encoding a receptor tyrosine kinase that is a member of the epidermal growth factor receptor (EGFR) and has diverse functions in the development of the nervous and neuroendocrine system by affecting downstream signaling pathway including Akt/mTOR pathway. Increased expression of ERBB4 was examined in the tumor tissue comparing normal counterpart by immunohistochemistry staining. Conclusion: This result suggested CSCNETs shares genetic characteristics of NETs and provided new insight for sufficient clinical management for patient, especially targeting ERBB4 and Akt/mTOR pathway. No conflict of interest. 118 Evolutionary trajectory of Asian EGFR mutation positive lung adenocarcinomas leads to “high intratumor heterogeneity” R. Nahar1 , W. Zhai2 , T. Zhang2 , A. Takano3 , A.J. Khng1 , Y.Y. Lee1 , X. Liu1 , C.H. Lim4 , T.K.H. Lim3 , T.P.T. Koh4 , Z.W. Aung5 , A.S.M. Teo1 , C.X. Chan1 , C.K. Toh6 , W.T. Lim6 , B. Lim7 , W.L. Tam7 , E.H. Tan6 , D.S.W. Tan6 , A.M. Hillmer1 . 1 Genome Institute of Singapore, Cancer Therapeutics and Stratified Oncology, Singapore, Singapore, 2 Genome Institute of Singapore, Human Genetics, Singapore, Singapore, 3 Singapore General Hospital, Department of Pathology, Singapore, Singapore, 4 National Heart Centre SIngapore, Department of Cardiothoracic Surgery, Singapore, Singapore, 5 National Cancer Centre Singapore, Department of Clinical Trials and Epidemiological Sciences, Singapore, Singapore, 6 National Cancer Centre Singapore, Division of Medical Oncology, Singapore, Singapore, 7 Genome Institute of Singapore, Cancer Stem Cell Biology, Singapore, Singapore Background: In contrast to western populations, activating mutations in EGFR are more frequent in Asian lung adenocarcinomas (LUADs) which are enriched for female gender and never-smokers. Although EGFR tyrosine