- Email: [email protected]
1068 Potent, Cellular Inhibitors of Glucose-6-phosphate Dehydrogenase – Potential for Novel Therapeutic Intervention in Cancer
S258
european journal of cancer 48, suppl. 5 (2012) S25–S288
tumor. Together with the clinical trial data showing that low dose of mTOR inhibitor (3 mg/kg) had no evidence of efficacy in patients with recurrent GBM, it is concluded that systemic administration of rapamycin analogues may not be a treatment option for patients with malignant glioma due to their intolerable high dose, which may result in severe side effects. 1068 Potent, Cellular Inhibitors of Glucose-6-phosphate Dehydrogenase − Potential for Novel Therapeutic Intervention in Cancer D. James1 , G. Hopkins1 , N.M. Hamilton1 , N. Hamilton1 , J.R. Hitchin1 , A. Lyons1 , G. Thomson1 , I.D. Waddell1 , A.M. Jordan1 , D.J. Ogilvie1 . 1 Paterson Institute for Cancer Research, Drug Discovery, Manchester, United Kingdom Introduction: Glucose-6-phosphate dehydrogenase (G6PD) is the ratelimiting enzyme in the pentose phosphate pathway (PPP) and catalyses the oxidation of glucose-6-phosphate to glucono-d-lactone-6-phosphate, with the concomitant production of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Alongside an important role in the production of biosynthetic precursors such as ribonucleotides, this pathway is a major cellular source of NADPH. As such, the pathway plays a critical role in both fatty acid & cholesterol biosynthesis (thus supporting cell division) and maintaining glutathione in its reduced state (GSH), ameliorating cellular stress arising from reactive oxygen species (ROS). Given these key roles, modulation of the pathway has potential therapeutic application in cancer, and inhibition of G6PD is an attractive target, particularly in combination with standard of care radiotherapy or cytotoxic drugs which can increase ROS. Materials and Methods: The steroid nucleus of DHEA was used as a starting point for exploring SAR of novel steroid inhibitors of G6PD. Human G6PD was cloned into a His-tagged vector and expressed in E. coli. An enzyme assay was developed using purified G6PD which measured the amount of NADP reduced to NADPH in the presence of novel inhibitors. Inhibition of G6PD in cells was carried out with HEK293T cells. Novel inhibitors were added to cells together with 6-aminonicotinamide and incubated for 4−6 h. Inhibition of 6-phosphogluconate accumulation was measured, following cell lysis, by mass spectrometry. Results: Several compounds with approximately 10-fold improved potency over DHEA were identified and this improved activity translated to efficacy in a cellular assay. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. Conclusion: We have developed a series of novel steroid G6PD inhibitors which, in our hands, modulate flux through the PPP. These compounds may be useful for examining the role of G6PD inhibition in cells, and will assist the future design of more potent steroid inhibitors with potential therapeutic utility. 1069 Mouse p53-deficient Cancer Models as Platforms to Obtain Genomic Predictors for Human Cancer Clinical Outcome M. Duenas ˜ 1 , M. Santos1 , J.F. Aranda1 , A.B. Martinez-Cruz1 , C. Lorz1 , M. Taron2 , M. Martin3 , R. Rosell2 , J.M. Paramio1 , R. Garcia-Escudero1 . 1 Ciemat Madrid Spain, Molecular Oncology Unit, Madrid, Spain, 2 Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain, 3 ´ Oncology Department, Madrid, Spain Hospital General Gregorio Maran˜ on, Background: Our study aims to validate mouse models with p53-deficiency as tools to develop genomic-based predictors of human cancer clinical outcome. Material and Methods: Genome-wide gene expression microarray experiments were performed in skin carcinomas arising in p53-deficient transgenic mouse, and compared with microarray experiments from carcinomas performed in various mouse models of breast and lung cancer. Based on these models, and using various interspecies comparison and biostatistical tools, we have obtained genomic predictors of breast cancer and lung adenocarcinoma using training datasets containing microarray analysis and censored survival data of cancer patients. Validation was performed on different published testing datasets and in our cohort of FFPE blocks of tumor samples using qRT-PCR. Results: Previously, we have demonstrated that the transcriptome of p53-deficient skin carcinoma mouse models is similar to human cancers characterized by TP53-mutation and/or poor clinical outcome. Here we demonstrate that this skin transcriptome (682-gene signature) is significantly shared by carcinomas of mouse models of breast and lung cancer with p53inhibition. Furthermore, based on gene expression we have obtained and validated predictor tests for human breast and lung adenocarcinoma clinical outcome. Conclusions: Our results indicate that p53-pathway deficiency assessed using gene expression from mouse models could accurately stratify human cancer patients and provide new antitumor targets for p53-defective tumors.
Sunday 8 − Tuesday 10 July 2012
1070 Analysis of Lipid Composition in Human Cancer Cell Lines Before and After Treatment With Protolichesterinic Acid Isolated From Cetraria Islandica F. Eiriksson1 , B. Sigurdsson2 , S. Omarsdottir3 , H. Ogmundsdottir4 , M. Thorsteinsdottir3 . 1 University of Iceland, Medical and Pharmaceutical Sciences, Reykjavik, Iceland, 2 ArcticMass, Reykjavik, Iceland, 3 University of Iceland, Pharmaceutical Sciences, Reykjavik, Iceland, 4 University of Iceland, Medical, Reykjavik, Iceland Background: Cancer cells show differences from healthy normal cells in their metabolism, which contribute to their survival and growth. Lipids have numerous functions in biological processes, structural as well as regulatory. Hydroxyeicosatetraenoic acids (HETEs), the products of lipoxygenase (LOX) pathways have been implicated in cancer development and protolichesterinic acid (PA), a secondary lichen metabolite is a potent inhibitor of 5- and 12LOX. The purpose of this work was to develop a HPLC-MS/MS method for evaluation of lipid composition in human cancer cell lines, before and after treatment with PA. Material and Methods: PA was purified from petroleum ether extract of Cetraria islandica with preparative high-pressure liquid chromatography. A HPLC-MS/MS method was developed and optimized for quantification of HETEs and LTB4, utilizing chemometric approach. Different sample preparation methods were tested and parameters optimized with factorial experimental design. The amount of HETEs in both cultured cancer cells and cultural medium supernatants were analyzed with the optimized HPLC-MS/MS method. Cells were stimulated with calcium ionophore (A23187) and treated with PA. Results: Preparative HPLC was successfully used for crude-purification to obtain one of the major secondary metabolites in the lichen Cetraria islandica (L.) Ach., PA and its tautomer lichesterinic acid. NMR and analytical HPLC verified purity of 99.60% for PA. A HPLC-MS/MS method was developed and validated for quantification of the LOX pathway products; LTB4, 5and 12-HETE. An intraday validation assessment shows that quantitative determination of the analytes is linear in the range of 120–40000 pg/mL, and accuracy and precision met the acceptance criteria. Conclusion: Quantification of LTB4 and HETEs in Capan-2 cell line, before stimulation with A23187 showed a weak signal for the analytes, indicating the presence of these analytes in the cell samples and the feasibility of the method. Further optimization of the extraction method for intact cellular lipids is needed, including improved methods for membrane rupture and modifications of the final sample preparation method to increase the yield prior to HPLCMS/MS quantification. These optimizations are in progress and will then be applied to cells stimulated with A23187 with or without treatment with PA.
Sunday 8 − Tuesday 10 July 2012 Poster Session
Tumour Immunology 1072 Chitinase-3-like-1 Protein Overexpression Enhances Breast Cancer Metastasis to the Lung S. Libreros1 , R. Garcia-Areas1 , L. Onwuha-Ekpete1 , V. IragaravapuCharyulu1 . 1 Florida Atlantic University, College of Medicine, Boca Raton, USA Introduction: Breast cancer is the most common cancer in women. The prevalence of pulmonary metastasis is 3-fold greater in patients with chronic pulmonary inflammatory conditions. Chronic inflammation is one of the hallmarks of asthma, a disease of the airways. Currently one out of ten women suffers from asthma and will have a greater propensity to develop aggressive metastatic breast cancer. Published results indicated that one of the molecules that aggravate the inflammation in asthma is a glycoprotein known as Chitinase 3-like protein 1 (CHI3L1), a molecule that is associated with poor prognosis in metastatic breast cancer patients. We hypothesize that CHI3L1-induced pulmonary inflammation generates the proper environment for recruiting circulating breast cancer cells, thus increasing the rate of metastasis to the lung. Material and Method: To determine if inflammation associated with CHI3L1 in the lung alters the pulmonary environment to accelerate metastatic growth, pulmonary inflammation is induced in BALB/c mice by ragweed allergen sensitization and injected with overexpressing CHI3L1 breast cancer cells. Results and Discussion: We show that there are elevated levels of CHI3L1 in circulation with increased tumor growth, higher levels of metastasis to the lung with shorter survival rates in mice with asthma. However, the direct role of CHI3L1 in promoting metastasis is not clearly delineated. Conclusion: The studies proposed here will provide a greater understanding of the role of CHI3L1 in enhancing metastasis and the tissue-specific molecular signals that act to exacerbate metastasis under conditions of chronic inflammation.
european journal of cancer 48, suppl. 5 (2012) S25–S288
tumor. Together with the clinical trial data showing that low dose of mTOR inhibitor (3 mg/kg) had no evidence of efficacy in patients with recurrent GBM, it is concluded that systemic administration of rapamycin analogues may not be a treatment option for patients with malignant glioma due to their intolerable high dose, which may result in severe side effects. 1068 Potent, Cellular Inhibitors of Glucose-6-phosphate Dehydrogenase − Potential for Novel Therapeutic Intervention in Cancer D. James1 , G. Hopkins1 , N.M. Hamilton1 , N. Hamilton1 , J.R. Hitchin1 , A. Lyons1 , G. Thomson1 , I.D. Waddell1 , A.M. Jordan1 , D.J. Ogilvie1 . 1 Paterson Institute for Cancer Research, Drug Discovery, Manchester, United Kingdom Introduction: Glucose-6-phosphate dehydrogenase (G6PD) is the ratelimiting enzyme in the pentose phosphate pathway (PPP) and catalyses the oxidation of glucose-6-phosphate to glucono-d-lactone-6-phosphate, with the concomitant production of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Alongside an important role in the production of biosynthetic precursors such as ribonucleotides, this pathway is a major cellular source of NADPH. As such, the pathway plays a critical role in both fatty acid & cholesterol biosynthesis (thus supporting cell division) and maintaining glutathione in its reduced state (GSH), ameliorating cellular stress arising from reactive oxygen species (ROS). Given these key roles, modulation of the pathway has potential therapeutic application in cancer, and inhibition of G6PD is an attractive target, particularly in combination with standard of care radiotherapy or cytotoxic drugs which can increase ROS. Materials and Methods: The steroid nucleus of DHEA was used as a starting point for exploring SAR of novel steroid inhibitors of G6PD. Human G6PD was cloned into a His-tagged vector and expressed in E. coli. An enzyme assay was developed using purified G6PD which measured the amount of NADP reduced to NADPH in the presence of novel inhibitors. Inhibition of G6PD in cells was carried out with HEK293T cells. Novel inhibitors were added to cells together with 6-aminonicotinamide and incubated for 4−6 h. Inhibition of 6-phosphogluconate accumulation was measured, following cell lysis, by mass spectrometry. Results: Several compounds with approximately 10-fold improved potency over DHEA were identified and this improved activity translated to efficacy in a cellular assay. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. Conclusion: We have developed a series of novel steroid G6PD inhibitors which, in our hands, modulate flux through the PPP. These compounds may be useful for examining the role of G6PD inhibition in cells, and will assist the future design of more potent steroid inhibitors with potential therapeutic utility. 1069 Mouse p53-deficient Cancer Models as Platforms to Obtain Genomic Predictors for Human Cancer Clinical Outcome M. Duenas ˜ 1 , M. Santos1 , J.F. Aranda1 , A.B. Martinez-Cruz1 , C. Lorz1 , M. Taron2 , M. Martin3 , R. Rosell2 , J.M. Paramio1 , R. Garcia-Escudero1 . 1 Ciemat Madrid Spain, Molecular Oncology Unit, Madrid, Spain, 2 Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain, 3 ´ Oncology Department, Madrid, Spain Hospital General Gregorio Maran˜ on, Background: Our study aims to validate mouse models with p53-deficiency as tools to develop genomic-based predictors of human cancer clinical outcome. Material and Methods: Genome-wide gene expression microarray experiments were performed in skin carcinomas arising in p53-deficient transgenic mouse, and compared with microarray experiments from carcinomas performed in various mouse models of breast and lung cancer. Based on these models, and using various interspecies comparison and biostatistical tools, we have obtained genomic predictors of breast cancer and lung adenocarcinoma using training datasets containing microarray analysis and censored survival data of cancer patients. Validation was performed on different published testing datasets and in our cohort of FFPE blocks of tumor samples using qRT-PCR. Results: Previously, we have demonstrated that the transcriptome of p53-deficient skin carcinoma mouse models is similar to human cancers characterized by TP53-mutation and/or poor clinical outcome. Here we demonstrate that this skin transcriptome (682-gene signature) is significantly shared by carcinomas of mouse models of breast and lung cancer with p53inhibition. Furthermore, based on gene expression we have obtained and validated predictor tests for human breast and lung adenocarcinoma clinical outcome. Conclusions: Our results indicate that p53-pathway deficiency assessed using gene expression from mouse models could accurately stratify human cancer patients and provide new antitumor targets for p53-defective tumors.
Sunday 8 − Tuesday 10 July 2012
1070 Analysis of Lipid Composition in Human Cancer Cell Lines Before and After Treatment With Protolichesterinic Acid Isolated From Cetraria Islandica F. Eiriksson1 , B. Sigurdsson2 , S. Omarsdottir3 , H. Ogmundsdottir4 , M. Thorsteinsdottir3 . 1 University of Iceland, Medical and Pharmaceutical Sciences, Reykjavik, Iceland, 2 ArcticMass, Reykjavik, Iceland, 3 University of Iceland, Pharmaceutical Sciences, Reykjavik, Iceland, 4 University of Iceland, Medical, Reykjavik, Iceland Background: Cancer cells show differences from healthy normal cells in their metabolism, which contribute to their survival and growth. Lipids have numerous functions in biological processes, structural as well as regulatory. Hydroxyeicosatetraenoic acids (HETEs), the products of lipoxygenase (LOX) pathways have been implicated in cancer development and protolichesterinic acid (PA), a secondary lichen metabolite is a potent inhibitor of 5- and 12LOX. The purpose of this work was to develop a HPLC-MS/MS method for evaluation of lipid composition in human cancer cell lines, before and after treatment with PA. Material and Methods: PA was purified from petroleum ether extract of Cetraria islandica with preparative high-pressure liquid chromatography. A HPLC-MS/MS method was developed and optimized for quantification of HETEs and LTB4, utilizing chemometric approach. Different sample preparation methods were tested and parameters optimized with factorial experimental design. The amount of HETEs in both cultured cancer cells and cultural medium supernatants were analyzed with the optimized HPLC-MS/MS method. Cells were stimulated with calcium ionophore (A23187) and treated with PA. Results: Preparative HPLC was successfully used for crude-purification to obtain one of the major secondary metabolites in the lichen Cetraria islandica (L.) Ach., PA and its tautomer lichesterinic acid. NMR and analytical HPLC verified purity of 99.60% for PA. A HPLC-MS/MS method was developed and validated for quantification of the LOX pathway products; LTB4, 5and 12-HETE. An intraday validation assessment shows that quantitative determination of the analytes is linear in the range of 120–40000 pg/mL, and accuracy and precision met the acceptance criteria. Conclusion: Quantification of LTB4 and HETEs in Capan-2 cell line, before stimulation with A23187 showed a weak signal for the analytes, indicating the presence of these analytes in the cell samples and the feasibility of the method. Further optimization of the extraction method for intact cellular lipids is needed, including improved methods for membrane rupture and modifications of the final sample preparation method to increase the yield prior to HPLCMS/MS quantification. These optimizations are in progress and will then be applied to cells stimulated with A23187 with or without treatment with PA.
Sunday 8 − Tuesday 10 July 2012 Poster Session
Tumour Immunology 1072 Chitinase-3-like-1 Protein Overexpression Enhances Breast Cancer Metastasis to the Lung S. Libreros1 , R. Garcia-Areas1 , L. Onwuha-Ekpete1 , V. IragaravapuCharyulu1 . 1 Florida Atlantic University, College of Medicine, Boca Raton, USA Introduction: Breast cancer is the most common cancer in women. The prevalence of pulmonary metastasis is 3-fold greater in patients with chronic pulmonary inflammatory conditions. Chronic inflammation is one of the hallmarks of asthma, a disease of the airways. Currently one out of ten women suffers from asthma and will have a greater propensity to develop aggressive metastatic breast cancer. Published results indicated that one of the molecules that aggravate the inflammation in asthma is a glycoprotein known as Chitinase 3-like protein 1 (CHI3L1), a molecule that is associated with poor prognosis in metastatic breast cancer patients. We hypothesize that CHI3L1-induced pulmonary inflammation generates the proper environment for recruiting circulating breast cancer cells, thus increasing the rate of metastasis to the lung. Material and Method: To determine if inflammation associated with CHI3L1 in the lung alters the pulmonary environment to accelerate metastatic growth, pulmonary inflammation is induced in BALB/c mice by ragweed allergen sensitization and injected with overexpressing CHI3L1 breast cancer cells. Results and Discussion: We show that there are elevated levels of CHI3L1 in circulation with increased tumor growth, higher levels of metastasis to the lung with shorter survival rates in mice with asthma. However, the direct role of CHI3L1 in promoting metastasis is not clearly delineated. Conclusion: The studies proposed here will provide a greater understanding of the role of CHI3L1 in enhancing metastasis and the tissue-specific molecular signals that act to exacerbate metastasis under conditions of chronic inflammation.