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COX-2 gene variations and risk of developing breast cancer
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
S35
196 Reprogramming bladder cancer cells for studying cancer initiation and progression
199 The role of Fanconi Anaemia pathway in sporadic non-FA associated head and neck squamous cell carcinoma
B. Iskender Izgi1 , K. Izgi2 , H. Canatan1 . 1 Erciyes University Faculty of Medicine, Department of Medical Biology/ Betul-Ziya Eren Genome and Stem Cell Centre, Kayseri, Turkey, 2 Erciyes University Faculty of Medicine, Department of Medical Biochemistry/ Betul-Ziya Eren Genome and Stem Cell Centre, Kayseri, Turkey
F. Alsobahi1 , S. Collis2 , K. Hunter1 . 1 University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom, 2 University of Sheffield, Department of Oncology and Metabolism, Sheffield, United Kingdom
Background: The induced pluripotent stem cell (iPSC) technology is the forced expression of specific transcription factors in somatic cells resulting in transformation into self-renewing, pluripotent cells which possess the ability to differentiate into any type of cells in the human body. The iPSC technology not only offers an invaluable source for cell replacement therapies but also provides a system to study the disease mechanism and drug development/toxicity testing. Malignant cells could also be reprogrammed into iPSC-like cells with lower efficiency but due to the genetic and epigenetic barriers in cancer cells only a limited number of cancer cell types could be successfully reprogrammed until today. In the present study we aimed at reprogramming bladder cancer cells and this is the first study focusing on the reprogramming susceptibility of two different bladder cancer cell lines to reprogramming. Materials and Methods: By using non-integrating Sendai virus (SeV) system we transfected two bladder cancer cell lines (T24 and HTB-9) with reprogramming factors − OCT4, SOX2, c-MYC and KLF4. Pluripotencyassociated features were demonstrated using immunofluorescence staining, qRT-PCR and western blotting. In vitro differentiation ability of iPS-like cells was assessed by embryoid body assay. Results: We generated iPSC-like cells from T24 bladder cancer cell line which represented pluripotent features both at gene and protein level. Reprogrammed T24 cells were demonstrated to differentiate into cells representing three embryonic germ layers in vitro. Using the same technique, we have also transfected HTB-9 cells with reprogramming factors which were unable to give rise to iPS-like cells but only upregulated some pluripotencyassociated marker expression. Conclusion: To the best of our knowledge, this is the first study describing the generation of pluripotent stem cells using iPSC technology from bladder cancer cells. Our study provides a valuable tool for cancer studies with the potential to represent the early steps of bladder carcinogenesis and for understanding the origin of cancer stem cells. No conflict of interest. 198 COX-2 gene variations and risk of developing breast cancer G. Ozhan1 , Z. Kara2 , H. Kara3 . 1 Istanbul University, Faculty of Pharmacy, Istanbul, Turkey, 2 Istanbul University, Pharmacology, Istanbul, Turkey, 3 Acibadem University, Faculty of Medicine, Istanbul, Turkey Introduction: Breast cancer is the most frequent cancer and responsible for the deaths from 33% of all cancers and from 20% of all cancer related deaths in women. Beside the general risk factors such as gender, age, age of menarche and menopause, obesity, hormone replacement therapy, oral contraceptive use, high and low penetrance genes, and epigenetic and modifier genes have a important role in its carcinogenesis. Cyclooxygenases (COXs), central enzymes in the prostaglandin pathway, play important role in several biologic processes relevant to cancer development and progression such as inflammation, tumour cell invasion and metastasis. COX-2 over-expression in carcinomas has been associated with a more aggressive behavior and a worse prognosis. While expression of COX-2 is nearly undetectable in normal breast tissue, the gene is over-expressed in 40% of invasive breast tumors. There are some studies on the association between COX-2 genetic variants and breast cancer risks, however, the findings have been inconsistent. Therefore, our aim was to investigate the relationship between COX-2 genotypes and haplotypes in a Turkish population-based case–control study on breast cancer susceptibility. Material and Method: It was evaluated COX-2 (G765C, T8473C, G306C) genotypes in 104 patients and 100 healthy controls by using RT-PCR. Result and Discussion: It was observed that only COX-2 T8473C might be associated with breast cancer risk (OR = 1.86±0.79–4.38), however the association did not reach statistical significance (p = 0.151). Clinicopathological characteristics and haplotype analysis were not associated with breast cancer risk. Conclusion: We believe that the findings are the first results of COX-2 allele distributions in the Turkish population and may provide an understanding of aetiology in breast cancer. No conflict of interest.
Introduction: Fanconi Anaemia (FA) is a rare inherited disease caused by mutations in genes of the Fanconi Anaemia pathway. The FA pathway is involved in the DNA damage response and the mutilations result in chromosomal instability. Patients with FA who survive bone marrow transplantation are found to be at high risk of developing Head and Neck squamous cell carcinoma (HNSCC) with 500–700 times the risk of normal individuals. Given the relative specificity of this effect, we hypothesize that alterations in the expression and posttranslational activation of FANC genes may contribute to the pathogenesis of sporadic non-FA HNSCC in addition to FA-HNSCC. Aims: To determine whether FANC genes are altered in non-FA HNSCC, and to define the effect of such alterations on the DNA damage response in HNSCC. Materials and Methods: The expression of FANCA, FANCC, FANCD2, and KI67 proteins was assessed normal oral mucosa and HNSCC tissue using immunohistochemistry. Expression of FANCA, FANCC and FANCD2 was assessed by qPCR and Western blot in a panel of Cisplatin treated and untreated sporadic HNSCC and FA-HNSCC cells. Immunofluorescence was used to assess DNA damage (53BP1, gH2AX) and repair and FANC pathway activation (FANCD2 foci) while Comet assay was used to assess DNA strand breaks and repair. Results: Higher FANCD2, FANCA and FANCC expression was found in HNSCC tissues compared to normal tissue and this correlates with the proliferation marker Ki67. The expression of FANCA, FANCC and FANCD2 are higher in HNSCC and FA-HNSCC cells compared to normal cells and this increased further after treatment with Cisplatin. FANCD2, 53BP1 and gH2AX nuclear foci and DNA fragmentation were increased after cisplatin treatment in all cells and the DNA damage was largely repaired in non-FA HNSCC cells over 48 hours, but not in FA-HNSCC cells. Conclusions: These data demonstrate that functional activation of the FA pathway in response to DNA damage occurs in non-FA HNSCC, but not FAHNSCC after treatment with Cisplatin. No conflict of interest. 200 HER2 cooperates with YWHAZ to promote to invasive esophagogastric cancer by inducing epithelial–mesenchymal transition S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , T. Kosuga1 , H. Konishi1 , A. Shiozaki1 , H. Fujiwara1 , K. Okamoto1 , H. Tsuda2 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan, 2 National Defense Medical College, Department of Basic Pathology, Saitama, Japan Background: Co-overexpression of HER2 and YWHAZ was significantly correlated with poor outcomes in breast cancer patients (Lu et al., Cancer Cell 2009). Material and Methods: We used MKN74 cells, which present cooverexpression of HER2 and YWHA Z and 104 primary tumors of esophagogastric adenocaricinoma, to clarify the clinical significance and biological functions of co-overexpressions. Results: In MKN74 cells, knockdown of both HER2 and YWHAZ using each specific small interfering RNA more strongly inhibited migration and invasion with enhancing E-cadherin expression and reducing Vimentin and ZEB1 expressions than knockdown of either. Importantly, patients whose tumor was overexpressed both HER2 (H) and YWHAZ (Y) significantly had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one (5-year survival rate; H+Y− vs. H−Y− vs. H−Y+ vs. H+Y+: 78% vs. 80% vs. 56% vs. 22%). Conclusion: Co-overexpression of YWHAZ in HER2-overexpressing esophago-gastric cancer contributes to tumor progression and poor outcomes. YWHAZ may be a key molecule for selecting prospective patients with malignant outcomes in patients undergoing chemotherapy for HER2 overexpression. No conflict of interest. 201 Plasma microRNA profiles; down-regulation of tumor suppressive miR-X level in plasma relates to poor outcomes and is a novel treatment target in gastric cancer S. Komatsu1 , D. Ichikawa1 , T. Imamura1 , W. Okajima1 , T. Ohashi1 , J. Kiuchi1 , T. Arita1 , H. Konishi1 , A. Shiozaki1 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan Background: This study aimed to explore decreased tumor suppressive microRNAs (miRNAs) in plasma of gastric cancer (GC) patients and detect their possible roles as a treatment target as well as a biomarker for GC.
S35
196 Reprogramming bladder cancer cells for studying cancer initiation and progression
199 The role of Fanconi Anaemia pathway in sporadic non-FA associated head and neck squamous cell carcinoma
B. Iskender Izgi1 , K. Izgi2 , H. Canatan1 . 1 Erciyes University Faculty of Medicine, Department of Medical Biology/ Betul-Ziya Eren Genome and Stem Cell Centre, Kayseri, Turkey, 2 Erciyes University Faculty of Medicine, Department of Medical Biochemistry/ Betul-Ziya Eren Genome and Stem Cell Centre, Kayseri, Turkey
F. Alsobahi1 , S. Collis2 , K. Hunter1 . 1 University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom, 2 University of Sheffield, Department of Oncology and Metabolism, Sheffield, United Kingdom
Background: The induced pluripotent stem cell (iPSC) technology is the forced expression of specific transcription factors in somatic cells resulting in transformation into self-renewing, pluripotent cells which possess the ability to differentiate into any type of cells in the human body. The iPSC technology not only offers an invaluable source for cell replacement therapies but also provides a system to study the disease mechanism and drug development/toxicity testing. Malignant cells could also be reprogrammed into iPSC-like cells with lower efficiency but due to the genetic and epigenetic barriers in cancer cells only a limited number of cancer cell types could be successfully reprogrammed until today. In the present study we aimed at reprogramming bladder cancer cells and this is the first study focusing on the reprogramming susceptibility of two different bladder cancer cell lines to reprogramming. Materials and Methods: By using non-integrating Sendai virus (SeV) system we transfected two bladder cancer cell lines (T24 and HTB-9) with reprogramming factors − OCT4, SOX2, c-MYC and KLF4. Pluripotencyassociated features were demonstrated using immunofluorescence staining, qRT-PCR and western blotting. In vitro differentiation ability of iPS-like cells was assessed by embryoid body assay. Results: We generated iPSC-like cells from T24 bladder cancer cell line which represented pluripotent features both at gene and protein level. Reprogrammed T24 cells were demonstrated to differentiate into cells representing three embryonic germ layers in vitro. Using the same technique, we have also transfected HTB-9 cells with reprogramming factors which were unable to give rise to iPS-like cells but only upregulated some pluripotencyassociated marker expression. Conclusion: To the best of our knowledge, this is the first study describing the generation of pluripotent stem cells using iPSC technology from bladder cancer cells. Our study provides a valuable tool for cancer studies with the potential to represent the early steps of bladder carcinogenesis and for understanding the origin of cancer stem cells. No conflict of interest. 198 COX-2 gene variations and risk of developing breast cancer G. Ozhan1 , Z. Kara2 , H. Kara3 . 1 Istanbul University, Faculty of Pharmacy, Istanbul, Turkey, 2 Istanbul University, Pharmacology, Istanbul, Turkey, 3 Acibadem University, Faculty of Medicine, Istanbul, Turkey Introduction: Breast cancer is the most frequent cancer and responsible for the deaths from 33% of all cancers and from 20% of all cancer related deaths in women. Beside the general risk factors such as gender, age, age of menarche and menopause, obesity, hormone replacement therapy, oral contraceptive use, high and low penetrance genes, and epigenetic and modifier genes have a important role in its carcinogenesis. Cyclooxygenases (COXs), central enzymes in the prostaglandin pathway, play important role in several biologic processes relevant to cancer development and progression such as inflammation, tumour cell invasion and metastasis. COX-2 over-expression in carcinomas has been associated with a more aggressive behavior and a worse prognosis. While expression of COX-2 is nearly undetectable in normal breast tissue, the gene is over-expressed in 40% of invasive breast tumors. There are some studies on the association between COX-2 genetic variants and breast cancer risks, however, the findings have been inconsistent. Therefore, our aim was to investigate the relationship between COX-2 genotypes and haplotypes in a Turkish population-based case–control study on breast cancer susceptibility. Material and Method: It was evaluated COX-2 (G765C, T8473C, G306C) genotypes in 104 patients and 100 healthy controls by using RT-PCR. Result and Discussion: It was observed that only COX-2 T8473C might be associated with breast cancer risk (OR = 1.86±0.79–4.38), however the association did not reach statistical significance (p = 0.151). Clinicopathological characteristics and haplotype analysis were not associated with breast cancer risk. Conclusion: We believe that the findings are the first results of COX-2 allele distributions in the Turkish population and may provide an understanding of aetiology in breast cancer. No conflict of interest.
Introduction: Fanconi Anaemia (FA) is a rare inherited disease caused by mutations in genes of the Fanconi Anaemia pathway. The FA pathway is involved in the DNA damage response and the mutilations result in chromosomal instability. Patients with FA who survive bone marrow transplantation are found to be at high risk of developing Head and Neck squamous cell carcinoma (HNSCC) with 500–700 times the risk of normal individuals. Given the relative specificity of this effect, we hypothesize that alterations in the expression and posttranslational activation of FANC genes may contribute to the pathogenesis of sporadic non-FA HNSCC in addition to FA-HNSCC. Aims: To determine whether FANC genes are altered in non-FA HNSCC, and to define the effect of such alterations on the DNA damage response in HNSCC. Materials and Methods: The expression of FANCA, FANCC, FANCD2, and KI67 proteins was assessed normal oral mucosa and HNSCC tissue using immunohistochemistry. Expression of FANCA, FANCC and FANCD2 was assessed by qPCR and Western blot in a panel of Cisplatin treated and untreated sporadic HNSCC and FA-HNSCC cells. Immunofluorescence was used to assess DNA damage (53BP1, gH2AX) and repair and FANC pathway activation (FANCD2 foci) while Comet assay was used to assess DNA strand breaks and repair. Results: Higher FANCD2, FANCA and FANCC expression was found in HNSCC tissues compared to normal tissue and this correlates with the proliferation marker Ki67. The expression of FANCA, FANCC and FANCD2 are higher in HNSCC and FA-HNSCC cells compared to normal cells and this increased further after treatment with Cisplatin. FANCD2, 53BP1 and gH2AX nuclear foci and DNA fragmentation were increased after cisplatin treatment in all cells and the DNA damage was largely repaired in non-FA HNSCC cells over 48 hours, but not in FA-HNSCC cells. Conclusions: These data demonstrate that functional activation of the FA pathway in response to DNA damage occurs in non-FA HNSCC, but not FAHNSCC after treatment with Cisplatin. No conflict of interest. 200 HER2 cooperates with YWHAZ to promote to invasive esophagogastric cancer by inducing epithelial–mesenchymal transition S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , T. Kosuga1 , H. Konishi1 , A. Shiozaki1 , H. Fujiwara1 , K. Okamoto1 , H. Tsuda2 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan, 2 National Defense Medical College, Department of Basic Pathology, Saitama, Japan Background: Co-overexpression of HER2 and YWHAZ was significantly correlated with poor outcomes in breast cancer patients (Lu et al., Cancer Cell 2009). Material and Methods: We used MKN74 cells, which present cooverexpression of HER2 and YWHA Z and 104 primary tumors of esophagogastric adenocaricinoma, to clarify the clinical significance and biological functions of co-overexpressions. Results: In MKN74 cells, knockdown of both HER2 and YWHAZ using each specific small interfering RNA more strongly inhibited migration and invasion with enhancing E-cadherin expression and reducing Vimentin and ZEB1 expressions than knockdown of either. Importantly, patients whose tumor was overexpressed both HER2 (H) and YWHAZ (Y) significantly had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one (5-year survival rate; H+Y− vs. H−Y− vs. H−Y+ vs. H+Y+: 78% vs. 80% vs. 56% vs. 22%). Conclusion: Co-overexpression of YWHAZ in HER2-overexpressing esophago-gastric cancer contributes to tumor progression and poor outcomes. YWHAZ may be a key molecule for selecting prospective patients with malignant outcomes in patients undergoing chemotherapy for HER2 overexpression. No conflict of interest. 201 Plasma microRNA profiles; down-regulation of tumor suppressive miR-X level in plasma relates to poor outcomes and is a novel treatment target in gastric cancer S. Komatsu1 , D. Ichikawa1 , T. Imamura1 , W. Okajima1 , T. Ohashi1 , J. Kiuchi1 , T. Arita1 , H. Konishi1 , A. Shiozaki1 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan Background: This study aimed to explore decreased tumor suppressive microRNAs (miRNAs) in plasma of gastric cancer (GC) patients and detect their possible roles as a treatment target as well as a biomarker for GC.