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468 Clinicopathologic implication of c-MYC gene copy number gain and overexpression in colorectal cancer
Poster Session – Molecular Targeted Agents II The peptide was additionally linked with poly-arginine cell penetrating domain to increase efficiency of its internalization. To allow separation of TRAIL/Apo2L domain and the effector peptide after reaching the tumor, we linked these two domains with a sequence motif recognized by MMPs and uPa proteases. Targeted effector should induce cancer cell death by triggering mitochondrial membrane permeabilization and swelling, resulting in the induction of apoptosis. AD-O56.9 protein was produced using E. coli as expression system, and purified from insoluble inclusion bodies. The obtained molecule was characterized biophysically. MTT cell viability assay was used to assess AD-O56.9 mediated killing of carcinoma cells. Flow cytometric analysis were used to evaluate influence of the AD-O56.9 on mitochondrial membrane integrity, and other apoptosis biomarkers. The SPR was used to confirm interaction with cellular targets − death receptors. The tumoricidal activity of AD-O56.9 was evaluated in NOD/SCID mice bearing different types of human cancers xenografts. AD-O56.9 exhibits cytotoxic effect on various cancer cell lines (established and primary and TRAIL-sensitive and TRAIL-resistant), but showed no toxic effect on normal cells. The ‘KLA’ peptide that enables to overcome resistance to TRAIL is effective only being a part of AD-O56.9. Analyzing apoptosis markers as PARP cleavage and caspases activity, on sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549) we showed that AD-O56.9 induced apoptosis in these cells. AD-O56.9 also caused strong depolarization of mitochondrial membrane that is a cellular target for ‘KLA peptide’. Importantly, AD-O56.9 administration caused significant regression on mice xenograft models of TRAIL sensitive human pancreatic carcinoma MIA PaCa-2, human oesophageal adenocarcinoma OE19, TRAIL-resistant human hepatocellular carcinoma HepG2 and human uterine sarcoma MES\erhyphen;SA/Dx5. Our novel fusion protein AD-O56.9 is able to induce apoptosis in many cancer cell lines, that are TRAIL resistant and causes tumor regression in mice bearing human cancer cells. The obtained results show that selection and linking two anticancer molecules that displays some therapeutic deficiency but with complementary properties could be the promising strategy in development of new therapeutics. 468 POSTER Clinicopathologic implication of c-MYC gene copy number gain and overexpression in colorectal cancer K. Lee1 , Y. Kwak2 , G. Choe1 , W. Kim2 , D. Kim3 , S. Kang3 , H. Lee1 . 1 Seoul National University Bundang Hospital, Pathology, Seongnam-si, South Korea; 2 Seoul National University Hospital, Pathology, Seoul, South Korea; 3 Seoul National University Bundang Hospital, Surgery, Seongnam-si, South Korea Background: The aim of this study was to determine the incidence and clinical implications of c-MYC gene copy number (GCN) gain and overexpression in the patients with primary colorectal cancer (CRC). Materials and Methods: c-MYC status was determined by performing dual-color silver in-situ hybridisation (SISH), mRNA in-situ hybridisation (ISH), and immunohistochemistry (IHC) in a retrospective cohort of 367 consecutive CRC patients. In addition, b-catenin expression was investigated by IHC and microsatellite instability (MSI) was analysed with 5 microsatellite markers. Results: c-MYC amplification, defined as a c-MYC:CEP8 ratio 2.0, was observed in 31 patients (8.4%) and GCN gain, defined as a c-MYC copies/nucleus 4.0, was found in 63 patients (17.2%). c-MYC GCN gain was more frequently observed in CRCs located in the sigmoid colon and rectum than other location (P = 0.034). c-MYC GCN gain was associated with large tumour size (P = 0.041) and microsatellite stable or MSI-low (P = 0.029). Although c-MYC amplification did not predict patients’ prognosis (P = 0.142), c-MYC GCN gain was significantly associated with worse patients’ survival (P = 0.015). By multivariate Cox regression analysis, the hazard ratio of c-MYC GCN gain was 2.350 (95% CI, 1.453 to 3.802; P < 0.001). In a subgroup of stage II and III, c-MYC GCN gain was also significantly associated with worse prognosis by univariate (P = 0.034) and multivariate (P = 0.040) survival analysis. The incidence of c-MYC protein overexpression was approximately 201 of 367 patients (54.8%). c-MYC protein overexpression was more correlated with mRNA overtranscription [correlation coefficient (CC) = 0.479] and b-catenin nuclear expression (CC = 0.282) than c-MYC/CEP ratio (CC = 0.195) and c-MYC GCN gain (CC = 0.211). In contrast to c-MYC GCN gain, c-MYC protein overexpression, mRNA overtranscription, and b-catenin nuclear expression were significantly associated with better prognosis by univariate survival analysis (P = 0.010, P = 0.042, and P = 0.011, respectively), but not significant by multivariate analysis. Conclusions: c-MYC GCN gain was an independent prognostic factor in both consecutive primary CRC patients and in the subgroup of stage II and III CRC patients. However, there was low correlation between c-MYC protein overexpression and c-MYC GCN gain or amplification. Our findings
Friday 21 November 2014 153 may serve as a basis for future studies on predicting poor outcome in CRC patients and on selecting candidates for c-MYC targeted therapy. 469 POSTER OTX015, a novel BET-bromodomain (BET-BRD) inhibitor, is a promising anticancer agent for human glioblastoma L. Ouafik1 , C. Berenguer1 , M. Cayol1 , L. Astorgues-Xerri2 , M. Bekradda3 , E. Odore4 , K. Rezai5 , M.E. Riveiro3 , E. Cvitkovic6 . 1 UMR911-CRO2, Team 4, Marseille Cedex 05, France; 2 Oncology Therapeutic ˆ Development, Hopital Beaujon Department of Medical Oncology, Clichy Cedex, France; 3 Oncology Therapeutic Development, Department of Medical Oncology, Clichy Cedex, France; 4 Oncology Therapeutic Development, Institut Rene´ Huguenin Hospital, Saint Cloud, France; 5 Institute Curie, Rene´ Huguenin Hospital, Saint Cloud, France; 6 Oncology Therapeutic Development, Head of Consulting, Clichy Cedex, France Background: BRD2 and BRD4, members of the BET family of bromodomains, were recently described to be significantly increased in glioblastoma multiforme (GBM), the most common primary adult brain cancer. Small molecules antagonizing BET proteins binding to acetylated histones are thus candidate therapies for GBM. We report in vitro and in vivo antitumoral effects of OTX015 (OncoEthix SA, Switzerland), a potent BET-inhibitor, in U87MG cells, a p53 wild-type human glioblastoma-astrocytoma model with an epithelial-like phenotype, sensitive to temozolomide (TMZ). Material and Methods: OTX015 GI50 was determined with MTT assays in human U87MG cells after 72 h. GBM cells were also treated with OTX015 500nM for 4 h and 24 h and RNA levels of 9 genes were evaluated (C-MYC; N-MYC; MTHFD1L; HEXIM1; HIST2H2BE; HIST1H2BJ; SESN3; HIST1H2BK; HIST2H2BF, HIST2H4A). qRT-PCR were performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. 105 U87MG cells were injected in the frontal lobe of male athymic NMRI nude mice and 5 days later mice were randomized to 4 groups (6 mice/group); vehicle (PBS, once daily, ip, continuous), OTX015 (25 or 50 mg/kg/bidaily, gavage, continuous), or TMZ (100 mg/kg/daily, ip, days 6−10). Animals were sacrificed upon apparent symptoms. Median survival was determined by Kaplan–Meier method, with a log-rank Mantel-Cox test. Results: OTX015 and JQ1, the benchmark compound, displayed antiproliferative effects in U87MG after 72 h with GI50 values of 0.9 and 2.1 mM, respectively. C-MYC mRNA levels remained unchanged up to 24 hexposure to OTX015, while HIST2H2BE, HIST1H2BK, HIST2H2BJ and HIST2H4A were upregulated. OTX015 significantly increased survival in U87MG-bearing mice with median survival of 27 and 29 days for 25 and 50 mg/kg OTX015, respectively vs 21 days in control mice (p < 0.05). No major side effects were seen during OTX015 treatment, whereas TMZ led to pronounced weight loss over 5 days treatment. Conclusion: C-MYC appears to play a minor role in mediating OTX015 antitumor effects while p21 and HEXIM-related genes coding for cell cycle regulation and histones are upregulated, indicating chromatin remodeling. Oral OTX015 significantly slowed progression in an orthotopic human glioblastoma mouse model, highlighting the therapeutic potential of OTX015 in GBM. 470 POSTER Transcriptional regulation of cancer stem cells marker CD133 by p53 E.K. Park1 , S.Y. Bang1 , S.A. Yi1 , J.W. Han1 . 1 Research Center for Epigenome Regulation (RCER) School of pharmacy Sungkyunkwan University, Department of Biochemistry and Molecular Biology, Suwon Gyeunggi-do, Korea Background: Cancer stem cells (CSCs) are known as a rare population of tumor cells that are responsible for tumor initiation and self-renewal capacity. Based on these characteristics, CSCs have been considered as a potential target for cancer therapy. CD133 is currently one of the bestcharacterized CSCs markers in various tumor lines. However, the precise function and regulation mechanism of CD133 in CSC are still unclear. In this study, we investigated the role of p53 in transcriptional regulation of CD133. Methods: We treated doxorubicin to the various cancer cell lines and examined expression level of CD133 by Immunocytochemistry, qRT-PCR and Western blot analysis. Luciferase reporter gene assay and Chromatin Immunoprecipitation assay was performed for analyzing CD133 promoter activity and p53 promoter binding sites in the CD133 promoter. The functions of CD133 were measured using cell counting, colony formation assay and Brdu Incorporation assay. Results: We found opposite expression pattern of CD133 and tumor suppressor p53 in several cancer cell lines. CD133 expression is decreased by either induction or overexpression of p53, indicating that p53 acts as a
Friday 21 November 2014 153 may serve as a basis for future studies on predicting poor outcome in CRC patients and on selecting candidates for c-MYC targeted therapy. 469 POSTER OTX015, a novel BET-bromodomain (BET-BRD) inhibitor, is a promising anticancer agent for human glioblastoma L. Ouafik1 , C. Berenguer1 , M. Cayol1 , L. Astorgues-Xerri2 , M. Bekradda3 , E. Odore4 , K. Rezai5 , M.E. Riveiro3 , E. Cvitkovic6 . 1 UMR911-CRO2, Team 4, Marseille Cedex 05, France; 2 Oncology Therapeutic ˆ Development, Hopital Beaujon Department of Medical Oncology, Clichy Cedex, France; 3 Oncology Therapeutic Development, Department of Medical Oncology, Clichy Cedex, France; 4 Oncology Therapeutic Development, Institut Rene´ Huguenin Hospital, Saint Cloud, France; 5 Institute Curie, Rene´ Huguenin Hospital, Saint Cloud, France; 6 Oncology Therapeutic Development, Head of Consulting, Clichy Cedex, France Background: BRD2 and BRD4, members of the BET family of bromodomains, were recently described to be significantly increased in glioblastoma multiforme (GBM), the most common primary adult brain cancer. Small molecules antagonizing BET proteins binding to acetylated histones are thus candidate therapies for GBM. We report in vitro and in vivo antitumoral effects of OTX015 (OncoEthix SA, Switzerland), a potent BET-inhibitor, in U87MG cells, a p53 wild-type human glioblastoma-astrocytoma model with an epithelial-like phenotype, sensitive to temozolomide (TMZ). Material and Methods: OTX015 GI50 was determined with MTT assays in human U87MG cells after 72 h. GBM cells were also treated with OTX015 500nM for 4 h and 24 h and RNA levels of 9 genes were evaluated (C-MYC; N-MYC; MTHFD1L; HEXIM1; HIST2H2BE; HIST1H2BJ; SESN3; HIST1H2BK; HIST2H2BF, HIST2H4A). qRT-PCR were performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. 105 U87MG cells were injected in the frontal lobe of male athymic NMRI nude mice and 5 days later mice were randomized to 4 groups (6 mice/group); vehicle (PBS, once daily, ip, continuous), OTX015 (25 or 50 mg/kg/bidaily, gavage, continuous), or TMZ (100 mg/kg/daily, ip, days 6−10). Animals were sacrificed upon apparent symptoms. Median survival was determined by Kaplan–Meier method, with a log-rank Mantel-Cox test. Results: OTX015 and JQ1, the benchmark compound, displayed antiproliferative effects in U87MG after 72 h with GI50 values of 0.9 and 2.1 mM, respectively. C-MYC mRNA levels remained unchanged up to 24 hexposure to OTX015, while HIST2H2BE, HIST1H2BK, HIST2H2BJ and HIST2H4A were upregulated. OTX015 significantly increased survival in U87MG-bearing mice with median survival of 27 and 29 days for 25 and 50 mg/kg OTX015, respectively vs 21 days in control mice (p < 0.05). No major side effects were seen during OTX015 treatment, whereas TMZ led to pronounced weight loss over 5 days treatment. Conclusion: C-MYC appears to play a minor role in mediating OTX015 antitumor effects while p21 and HEXIM-related genes coding for cell cycle regulation and histones are upregulated, indicating chromatin remodeling. Oral OTX015 significantly slowed progression in an orthotopic human glioblastoma mouse model, highlighting the therapeutic potential of OTX015 in GBM. 470 POSTER Transcriptional regulation of cancer stem cells marker CD133 by p53 E.K. Park1 , S.Y. Bang1 , S.A. Yi1 , J.W. Han1 . 1 Research Center for Epigenome Regulation (RCER) School of pharmacy Sungkyunkwan University, Department of Biochemistry and Molecular Biology, Suwon Gyeunggi-do, Korea Background: Cancer stem cells (CSCs) are known as a rare population of tumor cells that are responsible for tumor initiation and self-renewal capacity. Based on these characteristics, CSCs have been considered as a potential target for cancer therapy. CD133 is currently one of the bestcharacterized CSCs markers in various tumor lines. However, the precise function and regulation mechanism of CD133 in CSC are still unclear. In this study, we investigated the role of p53 in transcriptional regulation of CD133. Methods: We treated doxorubicin to the various cancer cell lines and examined expression level of CD133 by Immunocytochemistry, qRT-PCR and Western blot analysis. Luciferase reporter gene assay and Chromatin Immunoprecipitation assay was performed for analyzing CD133 promoter activity and p53 promoter binding sites in the CD133 promoter. The functions of CD133 were measured using cell counting, colony formation assay and Brdu Incorporation assay. Results: We found opposite expression pattern of CD133 and tumor suppressor p53 in several cancer cell lines. CD133 expression is decreased by either induction or overexpression of p53, indicating that p53 acts as a