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701 Escherichia Coli and Colorectal Carcinogenesis
S166
european journal of cancer 48, suppl. 5 (2012) S25–S288
701 Escherichia Coli and Colorectal Carcinogenesis M. Bonnet1 , E. Buc2 , P. Sauvanet2 , C. Darcha2 , B. Pereira2 , D. Pezet2 , A. Darfeuille-Michaud1 . 1 UMR1071 Inserm UdA, M2iSH, Clermont Ferrand, France Introduction: Tumor microenvironment of colorectal carcinoma (CRC) is a complex association of non neoplastic and cancer cells and a large amount of microorganisms that could be linked to cancer by various mechanisms including chronic inflammation and production of carcinogenic metabolites. E. coli is a consistent commensal of the human gut microbiota but some pathogenic strains have acquired the ability to produce toxins that can interfere with eukaryotic cell cycle or directly induce DNA damages. In this study, we analyzed the E. coli population associated with the colonic mucosa of human colorectal cancers and investigated the ability of CRC-associated E. coli to induce colonic tumorigenesis in multiple intestinal neoplasia (Min) mice with mutation in the APC gene, which is altered in the majority of CRC. Material and Methods: Tumors and normal mucosa from CRC patients (n = 52) and healthy mucosa for diverticulosis controls (n = 30) were collected. Mucosa-associated and internalized E. coli were quantified and characterized. Min mice were inoculated with a CRC-associated E. coli strain (strain CRC20), non pathogenic E. coli (strain K-12 MG1655) or PBS alone. Fecal bacterial colonization was quantified. Colonic samples were analyzed (macroscopy, histology). Results: Significantly higher numbers of mucosa-adherent and mucosainternalized E. coli were observed in tumors compared to normal tissue from control and CRC patients (at distance of the tumor). A statistically significant relationship was found between the presence of mucosa-associated E. coli and poor prognostic factors for CRC as tumor staging, lymph node status. In vitro, CRC-associated E. coli strains were able to invade and to persist in intestinal epithelial cells. Electron microscopy analysis of infected cultured cells indicated that bacteria induce the elongation of eukaryotic cell membranes and are internalized within endocytic vacuoles. Infections of Min mice with CRC20 E. coli indicated that this strain was able to persist at a high level in the gut up to 10 weeks after infection in comparison to non pathogenic E. coli that was no longer detected 2 weeks after infection. CRC20-colonized mice showed a marked increased number of visible colonic polyps at 10 weeks compared to controls. Histological analyzes confirmed that all these polyps were adenocarcinoma. Conclusion: These data support that mucosa-adherent pathogenic E. coli may be a cofactor in the pathogenesis of colorectal cancer. 702 Role of the Transcription Factor Forkhead Box P3 in Breast Cancer and Metastasis A. Cataldo1 , V. Uva2 , T. Triulzi1 , P. Casalini1 , L. Sfondrini2 , E. Tagliabue1 , A. Balsari2 . 1 Fondazione IRCCS “Istituto Nazionale dei Tumori”, Molecular Targeting Unit, Milan, Italy, 2 University of Milan, Department of Human of Morphology and Biomedical Science “Citta` Studi”, Milan, Italy Background: The transcription factor forkhead box P3 (FOXP3) is implicated in the regulation of immune system development and function and it has been recently reported to be expressed in tumor cells. We recently reported that FOXP3 expression in breast cancer was associated with worse overall survival probability and the risk increased with increasing FOXP3 immunostaining intensity. FOXP3 was also a strong prognostic factor for distant metastasesfree survival, but not for local recurrence risk. GSEA analysis of a published dataset relative to MCF7 breast cancer cells expressing or not FOXP3, indicated that this transcription factor induces expression of several genes implicated in migration and metastasis. We investigated the involvement of FOXP3 in the metastatic process using in vivo and in vitro models. Material and Methods: MDA-MB-231 breast cancer cell line, expressing low levels of FOXP3, was used to establish a tet-off inducible expression system, in which full-length (WT) FOXP3 or splice variant form (D2) were induced upon doxycycline removal. The consequences of FOXP3 up-regulation were examined by proliferation, migration and invasion assays. D2 FOXP3-tet-off stable clone was injected subcutaneously in SCID mice with or without the administration of doxycyline. Tumor weight and the number of spontaneous lung metastases were compared between the two experimental groups. Results: Real Time PCR and Western Blot analysis confirmed that FOXP3 mRNA and protein levels were significantly increased in both WT and D2 FOXP3-tet-off MDA-MB-231 cell lines upon doxycycline removal. Up-regulation of WT and D2 FOXP3 inhibited proliferation, increased migration and invasion in vitro. In our in vivo model, tumor weight and volume in Doxi-taking mice were significantly larger than corresponding measurements in Doxi-free mice. At the end of the observation period, a high percentage of Doxi-free mice developed a local secondary tumor whereas no secondary tumor development was noted in the corresponding Doxi-taking group. Furthermore, an increased number of lung metastases was observed in Doxifree mice compared to Doxi-taking mice.
Sunday 8 − Tuesday 10 July 2012
Conclusions: Metastasis is a process requiring the activation of several pathways, and a transcriptional factor such as FOXP3, able to regulate numerous genes, might be a relevant player. Our data suggest that FOXP3 expression in breast tumor cells inhibits tumor growth and promotes metastatic capability. 703 K-Ras and CDKN2a Mutations in Pancreatic Cancer S. Rachakonda1 , A. Bauer2 , F. Canzian1 , A. Scarpa3 , J. Neoptolemos4 , J. Werner5 , N. Giese5 , A. Heller5 , J. Hoheisel2 , R. Kumar1 . 1 German Cancer Research Center, Division of Molecular Genetic Epidemiology, Heidelberg, Germany, 2 German Cancer Research Center, Division of Functional Genome Analysis, Heidelberg, Germany, 3 University of Verona, Department of Pathology and Diagnostics, Verona, Italy, 4 National Institute for Health Research, Pancreas Biomedical Research Unit and the Liverpool Experimental Cancer Medicine Centre, Liverpool, United Kingdom, 5 University Hospital Heidelberg, Department of General Surgery, Heidelberg, Germany Introduction: Pancreatic cancer is the most fatal of all gastro-intestinal malignancies with a poor median survival of less than 5%. The tumor heterogeneity, lack of prognostic biomarkers coupled to therapeutic resistance is responsible for the poor outcome. Manifestation of the disease as a result of series of accumulated genetic alterations could potentially be used for prognostic purposes. Materials and Methods: Mutations were screened in four important cancer associated genes K-RAS, CDKN2A, B-RAF and GNAS in 171 resected exocrine tumors with at least 10% tumor content. Point mutations were investigated using single stranded conformation polymorphism (SSCP) in the codon 11, 12, 13 and 61 of K-RAS, exon 1 and 2 of CDKN2A, codon 201 of GNAS, and codon 600 of B-RAF genes. The mutations were confirmed by DNA sequencing. The deletions in CDKN2A locus were characterized using multiplex ligation-dependent probe amplification (MLPA) and analysed using Coffalyser software. All the experiments were repeated for reproducibility. Statistical analyses were done to determine proportional hazard ratios using SAS 9.2. Results: Mutations in K-RAS were found in 134 tumors (78%) with 131 of those in codon 12 and only 3 in codon 61. The G12D mutation accounted to 61%, followed by G12R (18%) and G12V (17%). Alterations in CDKN2A were detected in 43 tumors; GNAS mutations were present in two tumors and none in B-RAF. Presence of mutations in K-RAS gene were associated with a reduced patient overall survival by half (17 vs 30 months, log-rank P = 0.07) with a multivariate hazard ratio (HR) of 1.87 (95% CI 0.99–3.51). The patients with G12D mutation in tumors showed a median overall survival of 16 months (log-rank P = 0.02; HR 1.99 (95% CI 1.02–3.90)). Though, the association of survival in patients with CDKN2A aberrations in tumors was not statistically significant, the sub-set of patients with concomitant K-RAS mutations and CDKN2A alterations in tumors showed poorest median overall survival (13 vs 30 months, log-rank P = 0.03) and corresponding HR 2.77 (95% CI 1.23– 6.23). Conclusions: Mutations in K-RAS are frequent but not universal in pancreatic tumors. The presence of specific G12D K-RAS mutation was associated with poor survival and could potentially be used as predictive marker. Concomitant occurrence of K-RAS mutations and aberrations in CDKN2A resulted in a subgroup of patients with lowest survival. 704 Animal Model of the Papillary Thyroid Carcinoma Induced by BRAFV600E Mutation D. Rusinek1 , E. Chmielik2 , M. Kowal1 , M. Swierniak1 , M. Kowalska1 , M. Oczko-Wojciechowska1 , C. Przeorek1 , W. Widlak3 , B. Jarzab1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Department of Nuclear Medicine and Endocrine Oncology, Gliwice, Poland, 2 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Department of Tumor Pathology, Gliwice, Poland, 3 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Center for Translational Research and Molecular Biology of Cancer, Gliwice, Poland Introduction: A significant increase in the frequency of BRAFV600E mutation in papillary thyroid carcinomas (PTC) and a decrease in the incidence of the second major genetic alteration in PTC, RET rearrangements were recently observed. Association of BRAF mutation with aggressive phenotype of PTC and poor prognosis strengthen the need of getting know better the molecular background of PTC induced by the BRAFV600E mutation. The aim of our study was to obtain the mouse model of the BRAFT1799A induced papillary thyroid carcinoma for the analysis of BRAFT1799A influence on the gene expression profile of PTC. Material and Methods: Transgenic mice were obtained by injection of the mutated human BRAF gene (pMEV-2HA) plasmid; Biomyx) under the bovine thyroglobulin promoter (obtained by courtesy of Prof. J.E. Dumont) into the pronucleus of one-cell mouse embryos (FVB/N). After 6−12 months the
european journal of cancer 48, suppl. 5 (2012) S25–S288
701 Escherichia Coli and Colorectal Carcinogenesis M. Bonnet1 , E. Buc2 , P. Sauvanet2 , C. Darcha2 , B. Pereira2 , D. Pezet2 , A. Darfeuille-Michaud1 . 1 UMR1071 Inserm UdA, M2iSH, Clermont Ferrand, France Introduction: Tumor microenvironment of colorectal carcinoma (CRC) is a complex association of non neoplastic and cancer cells and a large amount of microorganisms that could be linked to cancer by various mechanisms including chronic inflammation and production of carcinogenic metabolites. E. coli is a consistent commensal of the human gut microbiota but some pathogenic strains have acquired the ability to produce toxins that can interfere with eukaryotic cell cycle or directly induce DNA damages. In this study, we analyzed the E. coli population associated with the colonic mucosa of human colorectal cancers and investigated the ability of CRC-associated E. coli to induce colonic tumorigenesis in multiple intestinal neoplasia (Min) mice with mutation in the APC gene, which is altered in the majority of CRC. Material and Methods: Tumors and normal mucosa from CRC patients (n = 52) and healthy mucosa for diverticulosis controls (n = 30) were collected. Mucosa-associated and internalized E. coli were quantified and characterized. Min mice were inoculated with a CRC-associated E. coli strain (strain CRC20), non pathogenic E. coli (strain K-12 MG1655) or PBS alone. Fecal bacterial colonization was quantified. Colonic samples were analyzed (macroscopy, histology). Results: Significantly higher numbers of mucosa-adherent and mucosainternalized E. coli were observed in tumors compared to normal tissue from control and CRC patients (at distance of the tumor). A statistically significant relationship was found between the presence of mucosa-associated E. coli and poor prognostic factors for CRC as tumor staging, lymph node status. In vitro, CRC-associated E. coli strains were able to invade and to persist in intestinal epithelial cells. Electron microscopy analysis of infected cultured cells indicated that bacteria induce the elongation of eukaryotic cell membranes and are internalized within endocytic vacuoles. Infections of Min mice with CRC20 E. coli indicated that this strain was able to persist at a high level in the gut up to 10 weeks after infection in comparison to non pathogenic E. coli that was no longer detected 2 weeks after infection. CRC20-colonized mice showed a marked increased number of visible colonic polyps at 10 weeks compared to controls. Histological analyzes confirmed that all these polyps were adenocarcinoma. Conclusion: These data support that mucosa-adherent pathogenic E. coli may be a cofactor in the pathogenesis of colorectal cancer. 702 Role of the Transcription Factor Forkhead Box P3 in Breast Cancer and Metastasis A. Cataldo1 , V. Uva2 , T. Triulzi1 , P. Casalini1 , L. Sfondrini2 , E. Tagliabue1 , A. Balsari2 . 1 Fondazione IRCCS “Istituto Nazionale dei Tumori”, Molecular Targeting Unit, Milan, Italy, 2 University of Milan, Department of Human of Morphology and Biomedical Science “Citta` Studi”, Milan, Italy Background: The transcription factor forkhead box P3 (FOXP3) is implicated in the regulation of immune system development and function and it has been recently reported to be expressed in tumor cells. We recently reported that FOXP3 expression in breast cancer was associated with worse overall survival probability and the risk increased with increasing FOXP3 immunostaining intensity. FOXP3 was also a strong prognostic factor for distant metastasesfree survival, but not for local recurrence risk. GSEA analysis of a published dataset relative to MCF7 breast cancer cells expressing or not FOXP3, indicated that this transcription factor induces expression of several genes implicated in migration and metastasis. We investigated the involvement of FOXP3 in the metastatic process using in vivo and in vitro models. Material and Methods: MDA-MB-231 breast cancer cell line, expressing low levels of FOXP3, was used to establish a tet-off inducible expression system, in which full-length (WT) FOXP3 or splice variant form (D2) were induced upon doxycycline removal. The consequences of FOXP3 up-regulation were examined by proliferation, migration and invasion assays. D2 FOXP3-tet-off stable clone was injected subcutaneously in SCID mice with or without the administration of doxycyline. Tumor weight and the number of spontaneous lung metastases were compared between the two experimental groups. Results: Real Time PCR and Western Blot analysis confirmed that FOXP3 mRNA and protein levels were significantly increased in both WT and D2 FOXP3-tet-off MDA-MB-231 cell lines upon doxycycline removal. Up-regulation of WT and D2 FOXP3 inhibited proliferation, increased migration and invasion in vitro. In our in vivo model, tumor weight and volume in Doxi-taking mice were significantly larger than corresponding measurements in Doxi-free mice. At the end of the observation period, a high percentage of Doxi-free mice developed a local secondary tumor whereas no secondary tumor development was noted in the corresponding Doxi-taking group. Furthermore, an increased number of lung metastases was observed in Doxifree mice compared to Doxi-taking mice.
Sunday 8 − Tuesday 10 July 2012
Conclusions: Metastasis is a process requiring the activation of several pathways, and a transcriptional factor such as FOXP3, able to regulate numerous genes, might be a relevant player. Our data suggest that FOXP3 expression in breast tumor cells inhibits tumor growth and promotes metastatic capability. 703 K-Ras and CDKN2a Mutations in Pancreatic Cancer S. Rachakonda1 , A. Bauer2 , F. Canzian1 , A. Scarpa3 , J. Neoptolemos4 , J. Werner5 , N. Giese5 , A. Heller5 , J. Hoheisel2 , R. Kumar1 . 1 German Cancer Research Center, Division of Molecular Genetic Epidemiology, Heidelberg, Germany, 2 German Cancer Research Center, Division of Functional Genome Analysis, Heidelberg, Germany, 3 University of Verona, Department of Pathology and Diagnostics, Verona, Italy, 4 National Institute for Health Research, Pancreas Biomedical Research Unit and the Liverpool Experimental Cancer Medicine Centre, Liverpool, United Kingdom, 5 University Hospital Heidelberg, Department of General Surgery, Heidelberg, Germany Introduction: Pancreatic cancer is the most fatal of all gastro-intestinal malignancies with a poor median survival of less than 5%. The tumor heterogeneity, lack of prognostic biomarkers coupled to therapeutic resistance is responsible for the poor outcome. Manifestation of the disease as a result of series of accumulated genetic alterations could potentially be used for prognostic purposes. Materials and Methods: Mutations were screened in four important cancer associated genes K-RAS, CDKN2A, B-RAF and GNAS in 171 resected exocrine tumors with at least 10% tumor content. Point mutations were investigated using single stranded conformation polymorphism (SSCP) in the codon 11, 12, 13 and 61 of K-RAS, exon 1 and 2 of CDKN2A, codon 201 of GNAS, and codon 600 of B-RAF genes. The mutations were confirmed by DNA sequencing. The deletions in CDKN2A locus were characterized using multiplex ligation-dependent probe amplification (MLPA) and analysed using Coffalyser software. All the experiments were repeated for reproducibility. Statistical analyses were done to determine proportional hazard ratios using SAS 9.2. Results: Mutations in K-RAS were found in 134 tumors (78%) with 131 of those in codon 12 and only 3 in codon 61. The G12D mutation accounted to 61%, followed by G12R (18%) and G12V (17%). Alterations in CDKN2A were detected in 43 tumors; GNAS mutations were present in two tumors and none in B-RAF. Presence of mutations in K-RAS gene were associated with a reduced patient overall survival by half (17 vs 30 months, log-rank P = 0.07) with a multivariate hazard ratio (HR) of 1.87 (95% CI 0.99–3.51). The patients with G12D mutation in tumors showed a median overall survival of 16 months (log-rank P = 0.02; HR 1.99 (95% CI 1.02–3.90)). Though, the association of survival in patients with CDKN2A aberrations in tumors was not statistically significant, the sub-set of patients with concomitant K-RAS mutations and CDKN2A alterations in tumors showed poorest median overall survival (13 vs 30 months, log-rank P = 0.03) and corresponding HR 2.77 (95% CI 1.23– 6.23). Conclusions: Mutations in K-RAS are frequent but not universal in pancreatic tumors. The presence of specific G12D K-RAS mutation was associated with poor survival and could potentially be used as predictive marker. Concomitant occurrence of K-RAS mutations and aberrations in CDKN2A resulted in a subgroup of patients with lowest survival. 704 Animal Model of the Papillary Thyroid Carcinoma Induced by BRAFV600E Mutation D. Rusinek1 , E. Chmielik2 , M. Kowal1 , M. Swierniak1 , M. Kowalska1 , M. Oczko-Wojciechowska1 , C. Przeorek1 , W. Widlak3 , B. Jarzab1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Department of Nuclear Medicine and Endocrine Oncology, Gliwice, Poland, 2 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Department of Tumor Pathology, Gliwice, Poland, 3 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Center for Translational Research and Molecular Biology of Cancer, Gliwice, Poland Introduction: A significant increase in the frequency of BRAFV600E mutation in papillary thyroid carcinomas (PTC) and a decrease in the incidence of the second major genetic alteration in PTC, RET rearrangements were recently observed. Association of BRAF mutation with aggressive phenotype of PTC and poor prognosis strengthen the need of getting know better the molecular background of PTC induced by the BRAFV600E mutation. The aim of our study was to obtain the mouse model of the BRAFT1799A induced papillary thyroid carcinoma for the analysis of BRAFT1799A influence on the gene expression profile of PTC. Material and Methods: Transgenic mice were obtained by injection of the mutated human BRAF gene (pMEV-2HA) plasmid; Biomyx) under the bovine thyroglobulin promoter (obtained by courtesy of Prof. J.E. Dumont) into the pronucleus of one-cell mouse embryos (FVB/N). After 6−12 months the