- Email: [email protected]
154 Overcoming Acquired Resistance to Afatinib in HCC827, a Non-small Cell Lung Cancer Cell Line
Poster Session – Drug Resistance and Modifiers induced apoptosis. It was also observed that Andrographolide could act as an inhibitor of glutathione S-transferase enzyme as evidenced from kinetic studies. Enzyme kinetic studies also revealed that the type of inhibition was competitive. Molecular simulation studies using ArgusLab 4.0.1 has predicted the binding of Andrographolide to the H-site (Hydrophobic site) of Glutathione S-transferase. The data obtained from the present study indicates that Andrographolide is a competitive inhibitor of glutathione s-transferase. Conclusions: The study shows that Andrographolide is acting as a competitive inhibitor of glutathione S-transferase enzyme. Hence, Andrographolide could be used as adjuvant in chemotherapy against drug resistant cancers. 152 POSTER Molecular Mechanisms and Cell Death Pathways in Targeted Therapy-Resistant Breast Cancer R. Montaser1 , H.M. Coley1 , T.M. Crook2 , G.A. Ajabnoor3 . 1 University of Surrey, Faculty of health and medical scienses, Guildford Surrey, United Kingdom; 2 University of Dundee, Dundee Cancer Centre, Dundee, United Kingdom; 3 King Abdulaziz University, Faculty of Medicine, Jeddah, Saudi Arabia Targeted therapies, which include anti-endocrine agents, monoclonal antibodies and small molecule inhibitors directed against growth factor receptors, have significantly changed the treatment of breast cancer. However, as for conventional cancer chemotherapeutic agents, resistance to targeted therapies is a major problem in the management of breast cancer. In order to address these issues we have developed a number of drug-resistant cell line models. We first used the hormone-responsive breast cancer cell lines MCF7 and T47D with acquired resistance (approximately 3-fold) against tamoxifen (MCF-7TR) and fulvestrant (T47DFR) respectively, to elucidate the mechanisms of endocrine resistance. For both resistant cell line variants we have been able to demonstrate marked down-regulation of the oestrogen receptor ERa and abolition of p27kip2 trans-activation following treatment with either anti-endocrine agent. In addition, we have shown that HER receptor status may change as a consequence of anti-endocrine therapy resistance. For both resistant cell lines we have seen clear up-regulation of HER2 expression, relative to drug sensitive counterparts. Treatment with the HER1/2 inhibitor lapatinib indicated collateral sensitivity (2-fold) to the drug in both MCF-7TR and T47DFR, relative to their respective parental counterparts. Our current studies are focusing on clarifying further the underlying mechanisms associated with these important findings, including analysis of the cell death pathways used in drug resistance. Previously, we have shown that chemotherapy resistance in breast cancer may be associated with a switch from apoptotic to autophagic cell death (Ajabnoor et al. 2012) where caspase activity is largely abolished. Using scanning laser confocal microscopy we have demonstrated increased levels of LC3II in tamoxifen-treated MCF-7TR cells compared with MCF-7 cells in the same experiments. In agreement with our previous finding in taxaneresistant MCF-7 cells, we see a marked down-regulation of BCl2 in antiendocrine resistant cell lines MCF-7TR and T47DFR. These novel findings are suggestive of an adaptive response by drug resistant cells to bring about survival strategies which culminate in a markedly reduced apoptotic response whilst amplifying autophagic pathways. In order to look further into the problem of targeted therapy resistance, we have recently developed breast cancer cell lines with approximate 3-fold acquired resistance to lapatinib (defined as -LapR): SKBR3LapR and MDA-MB-231LapR and both cell lines show decreased expression of HER2. Our current work is aimed at looking further into mechanisms of lapatinib resistance using a microarray directed approach. References Ajabnoor GM, Crook T, Coley HM (2012), Cell Death and Disease 3: doi 1038/cddis.2011.139. 153 POSTER Heterogeneity of Genetic Changes Associated with Acquired Crizotinib Resistance in Anaplastic Lymphoma Kinase-Rearranged Lung Cancer S. Kim1 , T.m. Kim1 , S.h. Lee1 , B. Keam1 , D.w. Kim1 , D.s. Heo1 . 1 Cancer Research Institute, Department of Internal Medicine Seoul National University College of Medicine, Seoul, Korea Background: Anaplastic lymphoma kinase (ALK)-rearranged lung cancers are markedly responsive to treatment with the ALK tyrosine kinase inhibitor crizotinib. Unfortunately, patients with this type of cancer can acquire resistance to crizotinib through several mechanisms, including ALK
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amplification and mutations at L1196M and G1269A. In this paper, we evaluated whether a specific gene mutation in ALK triggers resistance to crizotinib in ALK -rearranged lung cancer patients. Material and Methods: Tumor samples were derived from seven ALKpositive NSCLC patients who showed acquired resistance to crizotinib and these were analyzed for ALK, EGFR, and K-ras mutations. In addition, available tumor tissues before crizotinib were retrieved for these molecular tests. In vitro cytotoxicity and ALK downstream signaling pathways were compared between EML4-ALK-positive (NCI-H3122) and crizotinibresistant (SNU-2535) cell lines. Results: The specimens of seven patients had evaluable material for secondary mutations that were reported previously. Three patients had developed either a G1269A or an L1196M mutation in the ALK tyrosine kinase domain, but no identifiable genetic mutation was found in the other four patients. One patient had both the L1196M and G1269A mutations. Interestingly, one patient who did not have any resistance mutation exhibited an EGFR L858R mutation in a post-crizotinib biopsy sample in addition to an ALK gene rearrangement, as determined by FISH analysis. Furthemore, this patient was examined for the suspicious amplification of ALK in the post-treatment specimen compared with the pre-treatment specimen We showed that the L1196M and G1269A mutations inhibited crizotinib-mediated downregulation of ALK signaling and blocked cell growth inhibition in EML4-ALK L1196M- and G1269Amutated cells. Furthermore, we developed crizotinib-resistant SNU-2535 cells from the pleural effusion of a patient who harbored a G1269A mutation. In addition, we found that crizotinib inhibited cell proliferation, induced apoptosis and inhibited signaling pathways, including Akt and ERK, in a wild-type EML4-ALK -positive cell line but not a crizotinib-resistant cell line. Conclusions: Clinically, crizotinib is effective against ALK -rearranged lung cancer, but drug resistance is problematic. These current data demonstrate that the L1196M and G1269A mutations of the ALK tyrosine kinase domain or both the presence of an EGFR mutation (L858R) and ALK amplification are associated with resistance to crizotinib in some of ALK -rearranged lung cancer. Our findings offer information to improve therapeutic outcomes of patients with crizotinib-resistant ALK -rearranged lung cancer. 154 POSTER Overcoming Acquired Resistance to Afatinib in HCC827, a Non-small Cell Lung Cancer Cell Line J. Hsu1 , W. Lin1 , T. Lien1 , W. Chang2 , M. Sun2 , S. Jiang2 , K. Yen1 , Y. Chao1 . 1 National Health Research Institutes, Biotechnology and Pharmaceutical Research, Miaoli County, Taiwan; 2 National Health Research Institutes, National Cancer Institutes, Miaoli County, Taiwan Background: The first-generation EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib or erlotinib, have demonstrated tremendous clinical benefits in non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. However, such impressive responses to EGFR TKIs are limited to the emergence of resistance due to a variety of mechanisms including a secondary mutation of T790M in EGFR that abrogates the binding of the drugs. It has been postulated that second-generation EGFR TKIs, such as afatinib (BIBW2992), with activity against the T790M mutant EGFR kinase might overcome the drug resistance problem due to T790M mutation after treatment with current EGFR TKIs or, when used as the first-line treatment, could delay or suppress the emergence of resistance in EGFR. Therefore, it is important to understand how cancer cells may respond to treatment with the second-generation EGFR TKIs. Material and Methods: Inasmuch as cultured cancer cells may be able to recapitulate the clinically relevant mechanisms of drug resistance, we performed this study to generate BIBW2992-resistant cells, HCC827-BR1 and HCC827-BR2, from the parental HCC827 cells that were continuously exposed to BIBW2992 with increasing concentration. The IC50 s of BIBW2992-resistant cells are ~1,700-fold higher than that of the parental HCC827 cells. In HCC827-BR cells, EGFR, MET, and Erb2 were down-regulated and no secondary mutation was found to be present in the coding region of EGFR. Microarray gene expression analysis was also applied to gain insight into the underlying resistance mechanism. We noted an obvious induction of genes marked for epithelial to mesenchymal transition (EMT) in the drug resistant cells. The consequential effects of EMT on cell motility and invasiveness were analyzed and showed that the HCC827-BR cells exhibited more migration and invasion capability. Most importantly, we strived to seek if alternative medicine might be applied alone or in combination to treat the resistant cells or to diminish the emergence of resistant cells. We discovered that, compared to the parental cells, the HCC827-BR cells were more sensitive to another FDAapproved tyrosine kinase inhibitor (TKI). Furthermore, as revealed in the clonogenicity assay, the emergence of colonies resistance to BIBW2992 could be thwarted when low concentration of this TKI was combined with BIBW2992 for the treatment of parental HCC827 cells.
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Conclusion: This study has shown that emergence of EMT in HCC827 is accompanied by resistance to afatinib. Results from this study may also suggest alternative medicines with a potential to diminish the emergence of resistant cells. 155 POSTER Sorafenib Synergizes with the Antidiabetic Drug Metformin in Non-Small Cell Lung Cancer (NSCLC) F.H. Groenendijk1 , W.W. Mellema2 , M.M. Van Den Heuvel3 , E.F. Smit2 , R.L. Beijersbergen1 , R. Bernards1 . 1 NKI-AvL, Molecular Carcinogenesis, Amsterdam, The Netherlands; 2 VUMC, Department of Pulmonary Diseases, Amsterdam, The Netherlands; 3 NKI-AvL, Department of Thoracic Oncology, Amsterdam, The Netherlands Background: Sorafenib (Nexavar® ) is a small molecule multitargeted kinase inhibitor targeting several tyrosine protein kinases (e.g. VEGFR, PDGFR) and Raf kinases. The drug is FDA-approved for the treatment of advanced hepatocellular carcinoma and advanced clear cell renal cell carcinoma. Sorafenib is currently tested in multiple phase I-III studies for a broad range of solid tumors, including Non-Small Cell Lung Cancer (NSCLC). In a phase II clinical trial in KRASmt advanced NSCLC with single agent sorafenib overall survival was different in 5/57 patients using the oral antidiabetic drug metformin (medians 9.0 vs 4.8 months, p = 0.13). Furthermore, it was observed that the two patients with the longest progression free survival were patients using metformin. Metformin has been described as 5 AMP-activated protein kinase (AMPK)-activator. Results: In NSCLC cell lines, sorafenib in combination with metformin was found to act synergistically in inhibiting cellular proliferation. The synergistic effect closely paralleled AMPKalpha phosphorylation on the activation site threonine-172, that can be performed by the tumor suppressor LKB1 and the Ca2+ -activated protein kinase CaMKKII. Consistent with our findings, the drug combination of sorafenib with the synthetic allosteric AMPK activator A-769662 led to an identical synergistic growth inhibition of NSCLC cells. AMPK acts as a cellular energy sensor regulating several intracellular metabolic systems and activation leads to suppression of mTOR signalling. The combination treatment showed a synergistic effect on inhibiting the phosphorylation of the downstream mTOR targets 4E-BP1 and the ribosomal protein S6. Conclusion: Our results suggest that the combination of sorafenib with metformin could have beneficial effects on tumor regression by synergistically activating AMPK. We are currently testing this concept in a NSCLC-xenograft study of sorafenib in combination with metformin.
Poster Session – Drug Resistance and Modifiers 157 POSTER Blockage of Nrf2/AKR1C Axis Enhanced Therapeutic Efficacy of Oxaliplatin in Oxaliplatin Resistant Gastric Cancer Cells C. Kuo1 , C. Chu1 , C. Huang2 , J. Chang1 , W. Pan1 , L. Chen1 . 1 National Health Research Institutes, National Institute of Cancer Research, Tainan City, Taiwan; 2 National Yang-Ming University, Institute of Biopharmaceutical Sciences, Taipei City, Taiwan Background: Oxaliplatin is one of the major cytotoxic chemotherapeutic agents for the treatment of advanced gastrointestinal tract cancer, including pancreatic cancer, but the underlying mechanisms of acquired oxaliplatin resistance remain obscured. We previously established an oxaliplatin resistant subline, named TSGH-S3 (S3), from human gastric adenocarcinoma TSGH cells by stepwise exposure to increasing concentration of oxaliplatin. We found that enhanced copper efflux transporter ATP7A and global DNA repair capacity, at least in part, contributed to the development of such phenotype in S3 cells (British Journal of Cancer 97:334–344, 2007). In the present study, we applied global analysis using gene array technology to further investigate the difference between S3 and TSGH cells. Materials and Methods: S3, an oxaliplatin resistant subline, was made resistant to oxaliplatin by continuous selection against increasing drug concentrations from the human gastric adenocarcinoma TSGH cells. Growth inhibitory assay, Western blot analysis, quantitative RT-PCR (qRTPCR), flow cytometry analysis, and RNA interference technology were used to reveal molecular events in this study. Results: According to the result from microarray analysis, the transcripts of aldo-keto reductase 1C subfamily members, including AKR1C1 and AKR1C3, were noted to be highly enriched in S3 cells as compared to TSGH. The enhanced expression of these molecules in S3 cells were further confirmed by qRT-PCR and western blotting. Therefore, we aimed to elucidate the role of AKR1Cs in the development of oxaliplatin resistance. Notably, manipulation of AKR1C activity with specific AKR1C inhibitors or AKR1C-targeted siRNA significantly reversed the oxaliplatin resistance in S3 cells. Since AKR1Cs are classical ARE genes and can be trans-activated by redox-sensitive transcription factor Nrf2, we therefore examined the role of Nrf2 in S3 cells. As the result, knockdown of Nrf2 expression not only decreased the expression level of AKR1C1, AKR1C2, AKR1C3, and other ARE genes, but also significantly reversed oxaliplatin resistance in S3 cells. Conclusions: Taken together, we proposed that activation of Nrf2/AKR1C axis serves as an important regulator of oxaliplatin resistance in this human gastric carcinomas cell model. Thus, manipulation of Nrf2/AKR1C activity may have potential in management of oxaliplatin-refractory gastrointestinal cancers.
156 POSTER Aldehyde Dehydrogenase 1A1 Mediates Resistance to Temozolomide in Glioblastoma
158 POSTER Overcoming Docetaxel Resistance in Prostate Cancer by Targeting FAK Phosphorylation, and Its Mode of Action
A. Schaefer1 , J. Teufel1 , F. Ringel2 , J. Schlegel1 . 1 Klinikum Rechts der ¨ Munchen, Isar − Technische Universitat ¨ Division of Neuropathology, ¨ Munich, Germany; 2 Klinikum Rechts der Isar − Technische Universitat Munchen, ¨ Department of Neurosurgery, Munich, Germany
B.Y. Lee1 , F. Hochgrafe ¨ 1 , H.M. Lin1 , L. Castillo1 , M. Raftery2 , L.G. Horvath3 , R.J. Daly1 . 1 Garvan Institute of Medical Research, Cancer Program, Sydney, Australia; 2 University of New South Wales, Bioanalytical Mass Spectrometry Facility, Sydney, Australia; 3 Royal Prince Alfred Hospital, Sydney Cancer Centre, Sydney, Australia
Background: Implementation of the chemotherapy drug temozolomide increased the overall survival of glioblastoma patients. Nevertheless, the prognosis remains poor and glioblastomas recur regularly. This clinical behavior seems to be due to a therapy-resistant sub-population of cells being able to escape therapy and to induce tumor re-growth. We analyzed the role of the stem-like marker aldehyde dehydrogenase 1A1 (ALDH1A1) in mediating temozolomide resistance and its value as a predictor of clinical outcome in glioblastoma patients. Materials and Methods: Nine primary and established glioblastoma cell lines were treated with mere temozolomide or in combination with 4-diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH1A1, or with ALDH1A1 shRNA. Further, ALDH1A1 expression of seventy primary GBM patients was correlated with median survival. Results: ALDH1A1 overexpression predicted temozolomide resistance in vitro. Sensitivity of ALDH1A1 positive cells could be restored by inhibition of ALDH1A1 by DEAB or by knockdown with shRNA, as indicated by increased cytotoxicity, reduced clonogenicity and accumulation in G2/M cell cycle phase after treatment with temozolomide. Further, the prognosis of patients with high-level ALDH1A1 expression was poor compared to those with low levels (p < 0.0001). Conclusions: ALDH1A1 is a new mediator for resistance of glioblastomas to temozolomide, a predictor of clinical outcome, and may serve as a potential target to improve therapy in human glioma.
Background: Docetaxel (DTX) is the standard-of-care for patients diagnosed with metastatic castration resistant prostate cancer (CRPC). Inevitably, all patients develop resistance against DTX-based therapy. This study aims to characterize the perturbed tyrosine phosphorylation events, which contribute to the chemoresistant phenotype, and thereby identify therapeutic targets, and design a strategy to overcome the chemoresistance in CRPC. Material and Methods: We used a dose-escalation protocol to establish DTX-resistant (Rx) prostate cancer cell lines, PC3-Rx and DU145-Rx, derived from their parental PC3 and DU145 cells, respectively. We performed phosphotyrosine profiling with a quantitative mass spectrometry (MS)based approach using LC-MS/MS (LTQ Velos Orbitrap) in combination with stable isotope labeling by amino acids in cell culture (SILAC). Results: Quantitative phosphotyrosine profiling of the parental and DTXRx cell lines identified 10 significantly upregulated phosphopeptides in both Rx models, including that of focal adhesion kinase (FAK). Pathway enrichment analysis revealed that ‘Focal Adhesion’ and ‘Regulation of Actin Cytoskeleton’ pathways are enriched in both Rx cell lines. In addition, we discovered significantly enhanced intrinsic FAK (Y397 & Y576) and AKT (S473 & T308) phosphorylation with or without DTX in both Rx cell lines. Importantly, a novel therapeutic strategy of co-treating Rx cells with DTX and PF562271, a small molecule FAK inhibitor, further inhibited the phosphorylation of FAK (Y397 & Y576) and AKT (S473 & T308) than DTX alone, and reversed the chemoresistant phenotype (35 & 28-fold IC50 decrease p < 0.045; less clonogenic p < 0.016). Furthermore, the cotreatment also recapitulated more efficacious antitumour effect in vivo (50%
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amplification and mutations at L1196M and G1269A. In this paper, we evaluated whether a specific gene mutation in ALK triggers resistance to crizotinib in ALK -rearranged lung cancer patients. Material and Methods: Tumor samples were derived from seven ALKpositive NSCLC patients who showed acquired resistance to crizotinib and these were analyzed for ALK, EGFR, and K-ras mutations. In addition, available tumor tissues before crizotinib were retrieved for these molecular tests. In vitro cytotoxicity and ALK downstream signaling pathways were compared between EML4-ALK-positive (NCI-H3122) and crizotinibresistant (SNU-2535) cell lines. Results: The specimens of seven patients had evaluable material for secondary mutations that were reported previously. Three patients had developed either a G1269A or an L1196M mutation in the ALK tyrosine kinase domain, but no identifiable genetic mutation was found in the other four patients. One patient had both the L1196M and G1269A mutations. Interestingly, one patient who did not have any resistance mutation exhibited an EGFR L858R mutation in a post-crizotinib biopsy sample in addition to an ALK gene rearrangement, as determined by FISH analysis. Furthemore, this patient was examined for the suspicious amplification of ALK in the post-treatment specimen compared with the pre-treatment specimen We showed that the L1196M and G1269A mutations inhibited crizotinib-mediated downregulation of ALK signaling and blocked cell growth inhibition in EML4-ALK L1196M- and G1269Amutated cells. Furthermore, we developed crizotinib-resistant SNU-2535 cells from the pleural effusion of a patient who harbored a G1269A mutation. In addition, we found that crizotinib inhibited cell proliferation, induced apoptosis and inhibited signaling pathways, including Akt and ERK, in a wild-type EML4-ALK -positive cell line but not a crizotinib-resistant cell line. Conclusions: Clinically, crizotinib is effective against ALK -rearranged lung cancer, but drug resistance is problematic. These current data demonstrate that the L1196M and G1269A mutations of the ALK tyrosine kinase domain or both the presence of an EGFR mutation (L858R) and ALK amplification are associated with resistance to crizotinib in some of ALK -rearranged lung cancer. Our findings offer information to improve therapeutic outcomes of patients with crizotinib-resistant ALK -rearranged lung cancer. 154 POSTER Overcoming Acquired Resistance to Afatinib in HCC827, a Non-small Cell Lung Cancer Cell Line J. Hsu1 , W. Lin1 , T. Lien1 , W. Chang2 , M. Sun2 , S. Jiang2 , K. Yen1 , Y. Chao1 . 1 National Health Research Institutes, Biotechnology and Pharmaceutical Research, Miaoli County, Taiwan; 2 National Health Research Institutes, National Cancer Institutes, Miaoli County, Taiwan Background: The first-generation EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib or erlotinib, have demonstrated tremendous clinical benefits in non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. However, such impressive responses to EGFR TKIs are limited to the emergence of resistance due to a variety of mechanisms including a secondary mutation of T790M in EGFR that abrogates the binding of the drugs. It has been postulated that second-generation EGFR TKIs, such as afatinib (BIBW2992), with activity against the T790M mutant EGFR kinase might overcome the drug resistance problem due to T790M mutation after treatment with current EGFR TKIs or, when used as the first-line treatment, could delay or suppress the emergence of resistance in EGFR. Therefore, it is important to understand how cancer cells may respond to treatment with the second-generation EGFR TKIs. Material and Methods: Inasmuch as cultured cancer cells may be able to recapitulate the clinically relevant mechanisms of drug resistance, we performed this study to generate BIBW2992-resistant cells, HCC827-BR1 and HCC827-BR2, from the parental HCC827 cells that were continuously exposed to BIBW2992 with increasing concentration. The IC50 s of BIBW2992-resistant cells are ~1,700-fold higher than that of the parental HCC827 cells. In HCC827-BR cells, EGFR, MET, and Erb2 were down-regulated and no secondary mutation was found to be present in the coding region of EGFR. Microarray gene expression analysis was also applied to gain insight into the underlying resistance mechanism. We noted an obvious induction of genes marked for epithelial to mesenchymal transition (EMT) in the drug resistant cells. The consequential effects of EMT on cell motility and invasiveness were analyzed and showed that the HCC827-BR cells exhibited more migration and invasion capability. Most importantly, we strived to seek if alternative medicine might be applied alone or in combination to treat the resistant cells or to diminish the emergence of resistant cells. We discovered that, compared to the parental cells, the HCC827-BR cells were more sensitive to another FDAapproved tyrosine kinase inhibitor (TKI). Furthermore, as revealed in the clonogenicity assay, the emergence of colonies resistance to BIBW2992 could be thwarted when low concentration of this TKI was combined with BIBW2992 for the treatment of parental HCC827 cells.
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Conclusion: This study has shown that emergence of EMT in HCC827 is accompanied by resistance to afatinib. Results from this study may also suggest alternative medicines with a potential to diminish the emergence of resistant cells. 155 POSTER Sorafenib Synergizes with the Antidiabetic Drug Metformin in Non-Small Cell Lung Cancer (NSCLC) F.H. Groenendijk1 , W.W. Mellema2 , M.M. Van Den Heuvel3 , E.F. Smit2 , R.L. Beijersbergen1 , R. Bernards1 . 1 NKI-AvL, Molecular Carcinogenesis, Amsterdam, The Netherlands; 2 VUMC, Department of Pulmonary Diseases, Amsterdam, The Netherlands; 3 NKI-AvL, Department of Thoracic Oncology, Amsterdam, The Netherlands Background: Sorafenib (Nexavar® ) is a small molecule multitargeted kinase inhibitor targeting several tyrosine protein kinases (e.g. VEGFR, PDGFR) and Raf kinases. The drug is FDA-approved for the treatment of advanced hepatocellular carcinoma and advanced clear cell renal cell carcinoma. Sorafenib is currently tested in multiple phase I-III studies for a broad range of solid tumors, including Non-Small Cell Lung Cancer (NSCLC). In a phase II clinical trial in KRASmt advanced NSCLC with single agent sorafenib overall survival was different in 5/57 patients using the oral antidiabetic drug metformin (medians 9.0 vs 4.8 months, p = 0.13). Furthermore, it was observed that the two patients with the longest progression free survival were patients using metformin. Metformin has been described as 5 AMP-activated protein kinase (AMPK)-activator. Results: In NSCLC cell lines, sorafenib in combination with metformin was found to act synergistically in inhibiting cellular proliferation. The synergistic effect closely paralleled AMPKalpha phosphorylation on the activation site threonine-172, that can be performed by the tumor suppressor LKB1 and the Ca2+ -activated protein kinase CaMKKII. Consistent with our findings, the drug combination of sorafenib with the synthetic allosteric AMPK activator A-769662 led to an identical synergistic growth inhibition of NSCLC cells. AMPK acts as a cellular energy sensor regulating several intracellular metabolic systems and activation leads to suppression of mTOR signalling. The combination treatment showed a synergistic effect on inhibiting the phosphorylation of the downstream mTOR targets 4E-BP1 and the ribosomal protein S6. Conclusion: Our results suggest that the combination of sorafenib with metformin could have beneficial effects on tumor regression by synergistically activating AMPK. We are currently testing this concept in a NSCLC-xenograft study of sorafenib in combination with metformin.
Poster Session – Drug Resistance and Modifiers 157 POSTER Blockage of Nrf2/AKR1C Axis Enhanced Therapeutic Efficacy of Oxaliplatin in Oxaliplatin Resistant Gastric Cancer Cells C. Kuo1 , C. Chu1 , C. Huang2 , J. Chang1 , W. Pan1 , L. Chen1 . 1 National Health Research Institutes, National Institute of Cancer Research, Tainan City, Taiwan; 2 National Yang-Ming University, Institute of Biopharmaceutical Sciences, Taipei City, Taiwan Background: Oxaliplatin is one of the major cytotoxic chemotherapeutic agents for the treatment of advanced gastrointestinal tract cancer, including pancreatic cancer, but the underlying mechanisms of acquired oxaliplatin resistance remain obscured. We previously established an oxaliplatin resistant subline, named TSGH-S3 (S3), from human gastric adenocarcinoma TSGH cells by stepwise exposure to increasing concentration of oxaliplatin. We found that enhanced copper efflux transporter ATP7A and global DNA repair capacity, at least in part, contributed to the development of such phenotype in S3 cells (British Journal of Cancer 97:334–344, 2007). In the present study, we applied global analysis using gene array technology to further investigate the difference between S3 and TSGH cells. Materials and Methods: S3, an oxaliplatin resistant subline, was made resistant to oxaliplatin by continuous selection against increasing drug concentrations from the human gastric adenocarcinoma TSGH cells. Growth inhibitory assay, Western blot analysis, quantitative RT-PCR (qRTPCR), flow cytometry analysis, and RNA interference technology were used to reveal molecular events in this study. Results: According to the result from microarray analysis, the transcripts of aldo-keto reductase 1C subfamily members, including AKR1C1 and AKR1C3, were noted to be highly enriched in S3 cells as compared to TSGH. The enhanced expression of these molecules in S3 cells were further confirmed by qRT-PCR and western blotting. Therefore, we aimed to elucidate the role of AKR1Cs in the development of oxaliplatin resistance. Notably, manipulation of AKR1C activity with specific AKR1C inhibitors or AKR1C-targeted siRNA significantly reversed the oxaliplatin resistance in S3 cells. Since AKR1Cs are classical ARE genes and can be trans-activated by redox-sensitive transcription factor Nrf2, we therefore examined the role of Nrf2 in S3 cells. As the result, knockdown of Nrf2 expression not only decreased the expression level of AKR1C1, AKR1C2, AKR1C3, and other ARE genes, but also significantly reversed oxaliplatin resistance in S3 cells. Conclusions: Taken together, we proposed that activation of Nrf2/AKR1C axis serves as an important regulator of oxaliplatin resistance in this human gastric carcinomas cell model. Thus, manipulation of Nrf2/AKR1C activity may have potential in management of oxaliplatin-refractory gastrointestinal cancers.
156 POSTER Aldehyde Dehydrogenase 1A1 Mediates Resistance to Temozolomide in Glioblastoma
158 POSTER Overcoming Docetaxel Resistance in Prostate Cancer by Targeting FAK Phosphorylation, and Its Mode of Action
A. Schaefer1 , J. Teufel1 , F. Ringel2 , J. Schlegel1 . 1 Klinikum Rechts der ¨ Munchen, Isar − Technische Universitat ¨ Division of Neuropathology, ¨ Munich, Germany; 2 Klinikum Rechts der Isar − Technische Universitat Munchen, ¨ Department of Neurosurgery, Munich, Germany
B.Y. Lee1 , F. Hochgrafe ¨ 1 , H.M. Lin1 , L. Castillo1 , M. Raftery2 , L.G. Horvath3 , R.J. Daly1 . 1 Garvan Institute of Medical Research, Cancer Program, Sydney, Australia; 2 University of New South Wales, Bioanalytical Mass Spectrometry Facility, Sydney, Australia; 3 Royal Prince Alfred Hospital, Sydney Cancer Centre, Sydney, Australia
Background: Implementation of the chemotherapy drug temozolomide increased the overall survival of glioblastoma patients. Nevertheless, the prognosis remains poor and glioblastomas recur regularly. This clinical behavior seems to be due to a therapy-resistant sub-population of cells being able to escape therapy and to induce tumor re-growth. We analyzed the role of the stem-like marker aldehyde dehydrogenase 1A1 (ALDH1A1) in mediating temozolomide resistance and its value as a predictor of clinical outcome in glioblastoma patients. Materials and Methods: Nine primary and established glioblastoma cell lines were treated with mere temozolomide or in combination with 4-diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH1A1, or with ALDH1A1 shRNA. Further, ALDH1A1 expression of seventy primary GBM patients was correlated with median survival. Results: ALDH1A1 overexpression predicted temozolomide resistance in vitro. Sensitivity of ALDH1A1 positive cells could be restored by inhibition of ALDH1A1 by DEAB or by knockdown with shRNA, as indicated by increased cytotoxicity, reduced clonogenicity and accumulation in G2/M cell cycle phase after treatment with temozolomide. Further, the prognosis of patients with high-level ALDH1A1 expression was poor compared to those with low levels (p < 0.0001). Conclusions: ALDH1A1 is a new mediator for resistance of glioblastomas to temozolomide, a predictor of clinical outcome, and may serve as a potential target to improve therapy in human glioma.
Background: Docetaxel (DTX) is the standard-of-care for patients diagnosed with metastatic castration resistant prostate cancer (CRPC). Inevitably, all patients develop resistance against DTX-based therapy. This study aims to characterize the perturbed tyrosine phosphorylation events, which contribute to the chemoresistant phenotype, and thereby identify therapeutic targets, and design a strategy to overcome the chemoresistance in CRPC. Material and Methods: We used a dose-escalation protocol to establish DTX-resistant (Rx) prostate cancer cell lines, PC3-Rx and DU145-Rx, derived from their parental PC3 and DU145 cells, respectively. We performed phosphotyrosine profiling with a quantitative mass spectrometry (MS)based approach using LC-MS/MS (LTQ Velos Orbitrap) in combination with stable isotope labeling by amino acids in cell culture (SILAC). Results: Quantitative phosphotyrosine profiling of the parental and DTXRx cell lines identified 10 significantly upregulated phosphopeptides in both Rx models, including that of focal adhesion kinase (FAK). Pathway enrichment analysis revealed that ‘Focal Adhesion’ and ‘Regulation of Actin Cytoskeleton’ pathways are enriched in both Rx cell lines. In addition, we discovered significantly enhanced intrinsic FAK (Y397 & Y576) and AKT (S473 & T308) phosphorylation with or without DTX in both Rx cell lines. Importantly, a novel therapeutic strategy of co-treating Rx cells with DTX and PF562271, a small molecule FAK inhibitor, further inhibited the phosphorylation of FAK (Y397 & Y576) and AKT (S473 & T308) than DTX alone, and reversed the chemoresistant phenotype (35 & 28-fold IC50 decrease p < 0.045; less clonogenic p < 0.016). Furthermore, the cotreatment also recapitulated more efficacious antitumour effect in vivo (50%