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716 Chemical Carcinogenesis in Mice With a Defective Epidermal Barrier – Exploring the Connection Between Skin Barrier and Cancer Susceptibility
S170
european journal of cancer 48, suppl. 5 (2012) S25–S288
validated and correlated with clinical and epidemiological data available for the study. Conclusion: This multi-marker approach identifies a set of different markers that can be assessed in plasma, and that together (i) may be useful to narrow down the subjects at high risk of development of HCC among chronic liver disease patients in populations where chronic HBV carriage is endemic and (ii) represent very promising tools for improved, low-cost detection and diagnosis of HCC in high incidence, low resource regions of the world. 716 Chemical Carcinogenesis in Mice With a Defective Epidermal Barrier − Exploring the Connection Between Skin Barrier and Cancer Susceptibility S. Cipolat1 , K. Natsuga1 , L.M. Sevilla2 , R. Nachat3 , F.M. Watt4 . 1 Cancer Research UK Cambridge Research Institute, Cambridge, United Kingdom, 2 University of Valencia, Instituto de Biomedicina de Valencia, Valencia, Spain, 3 ´ ` ´ Universite d’Auvergne, Nutrition Cancerogen ese et Therapie, Toulouse, France, 4 Cancer Research UK, Cambridge Research Institute, Cambridge, United Kingdom Introduction: The cornified envelope (CE) is the outermost layer of the skin and it constitutes the epidermal barrier that protects from the entrance of pathogens and antigens from the outside and minimizes the water loss form the inside. Involucrin, envoplakin, and periplakin create the essential protein scaffold on which the envelope assembles. Mice deficient in all the three genes (tKO) show a defective epidermal barrier and hyperkeratosis, resulting from impaired desquamation as compensation. In addition, tKO mice display a reduction in gd+ dendritic epidermal T cells (DETC) in the epidermis and infiltration of CD3+ CD4+ T cells in the dermis. The defective epidermal barrier and the changes in T cells composition prompted us to investigate the susceptibility of the triple ko mice to skin cancer. Material and Method: tKO and wt mice were subjected to 2-stage chemical carcinogenesis (1 painting with DMBA followed by 3 paintings per week/15 wks with TPA): the number of benign (papillomas) and malignant tumors (squamous cell carcinoma) were scored weekly up to 57 weeks. Results and Discussion: tKO mice are resistant to benign tumor development, showing reduced incidence and lower papillomas burden, compared with the relative wt controls. On the other hand, tKO mice show an increased malignant conversion from benign papillomas into malignant squamous cell carcinomas. Painting with DMBA induces the same levels of apoptosis, DNA breaks and proliferation in tKO and wt mice, claiming that the decreased tumor formation is not caused by defects in initiation. Promotion with TPA induces an exacerbated keratinocytes response (parakeratosis, hyperkeratosis, spongiosis and defective differentiation) and increased inflammation mainly made of CD3+ CD4+ T lymphocytes with a Th2/Th17 profile, neutrophils and eosinophils in tKO mice, associated with increased levels of TSLP and IgE in the serum. This phenotype resembles what is observed in atopic dermatitis patients. Conclusion: Mice ko for envoplakin, periplakin and involucrin represent a useful model of skin barrier defect with features similar to individuals with atopic dermatitis. tKO mice are resistant to benign tumour formation, but display the same number of malignant tumors in a two stage skin carcinogenesis experiment compared with the relative wt mice. The exacerbated keratinocytes and inflammatory responses observed after promoting the skin with TPA are responsible for the decreased tumour formation. The mechanisms governing the higher malignant conversion in the triple ko mice are now under investigation. 717 Combined Exposure to Multiple Carcinogens Enhances Development of Rat Mammary Cancers With Characteristic Gene Expression T. Imaoka1 , M. Nishimura1 , K. Ishikawa2 , S. Yamashita3 , Y. Ohmachi4 , H. Suzuki5 , K. Daino1 , T. Ushijima3 , T. Imai2 , Y. Shimada1 . 1 National Institute of Radiological Sciences, Radiobiology for Children’s Health Program, Chiba, Japan, 2 National Institute of Radiological Sciences, Advanced Radiation Biology Research Program, Chiba, Japan, 3 National Cancer Center, Research Institute, Tokyo, Japan, 4 National Institute of Radiological Sciences, Department of Radiation Emergency Medicine, Chiba, Japan, 5 Tokyo College of Medico-Pharmaco Technology, Department of Life Technology Engineering, Tokyo, Japan Introduction: Although mixed exposure to multiple carcinogens is common in human environment, the mechanism of cooperative carcinogenesis by two or more agents is poorly understood. In the present study, we undertook experiments using the rat mammary carcinogenesis model to assess the synergistic effect of combined exposure to g rays and chemical carcinogens and potential underlying mechanisms. Materials and Methods: Female Sprague-Dawley rats at 7 weeks of age were irradiated with g rays (0, 0.5, 1 or 2 Gy); three days after irradiation, rats were subjected to single i.p. injection with 1-methyl-1-nitrosourea (MNU; 20 or 40 mg/kg) or 10 oral administrations with 2-amino-1-methyl-6-
Sunday 8 − Tuesday 10 July 2012
phenylimidazo[4,5-b]pyridine (PhIP; 40 mg/kg over two weeks), or left without chemical treatment. Tumors were examined pathologically after autopsy at 50 weeks of age. H-ras gene mutation was assessed by a restriction fragment length assay. Microarrays were used to search for genes characteristically expressed in carcinomas induced by combined exposures, followed by quantitative RT-PCR analysis incorporating additional tumors. Results: Incidence of mammary carcinoma increased linearly as a function of radiation dose in the absence of chemicals. Administration of chemical carcinogens resulted in additive increase in cancer incidence. Significantly high (78%) prevalence of H-ras mutation was observed among carcinomas of rats exposed to g rays (1 Gy) plus MNU (40 mg/kg) as compared to rats exposed to either g rays (0%) or MNU (54%) only. Microarray and quantitative RT-PCR analyses identified characteristic gene expressions in carcinoma induced by combined exposures as compared to those induced by single carcinogens. For example, high expression of serum/glucocorticoid-regulated kinase (Sgk) was associated with carcinomas induced by g rays (1 Gy) plus MNU (40 mg/kg) harboring H-ras mutation, whereas matrix metalloproteinase 17 was highly expressed in those without the mutation. Sgk may be induced synergistically by radiation effects (e.g., oxidative stress, nitric oxide and TGF-b) and H-ras mutation and enhance proliferation of cancer cells. Carcinomas induced by g rays (1 Gy) plus PhIP contained abundant transcript of the transcription factor Lef1, which may activate Wnt-responsive genes and stimulate cancer progression. Conclusion: Interaction of oncogenic mechanisms at the molecular level may underlie the combined effect of multiple carcinogens on rat mammary carcinogenesis. 718 Tumor Suppressors and Tissue Specificity in Cancer − the Role of Rad51c in Sebaceous and Mammary Carcinomas M. Tumiati1 , S. Koopal1 , A. Hemmes1 , S. Kuznetsov1 . 1 Institute for Molecular Medicine Finland − FIMM, Helsinki, Finland Background: Mutations in tumor suppressors are known to predispose to cancer. Less known is why these cancers show a distinct preference for a particular set of target tissues. Rad51c, a major protein of Homologous Recombination (HR), has recently been identified as a novel hereditary breast and ovarian cancer predisposition gene in humans and Kuznetsov et al. previously described Rad51c as a tumor suppressor with a tissue-specific cancer phenotype in mice. Here, the loss of Rad51c in a p53-deficient background resulted in a strong prevalence of sebaceous carcinomas of preputial glands in males and mammary gland carcinomas in females, while the typical p53-related sarcomas were suppressed. The high incidence of preputial and mammary gland carcinomas seems therefore to be directly triggered by loss of Rad51c, making it a perfect animal model to investigate the mechanisms of tissue-specific tumorigenesis, with a special attention to the role of HR in cancer. Materials and Methods: Organs and tumors from skin-specific Rad51c, p53 and Brca2 knock-out mouse cohorts have been analyzed immunohistologically and gene expression profiling is being used to identify the events associated with the tissue-specific loss of Rad51c. Results: The loss of Rad51c is severely affecting preputial glands, causing depletion of mature sebaceous follicles, ectopic keratinization, increased apoptosis and impaired proliferation. Interestingly, loss of Brca2 does not lead to a similar phenotype, suggesting that Rad51c could have HR independent functions. Keeping in mind the findings from preputial glands, a similar approach was used to investigate the role of Rad51c in all developmental stages of the mammary gland. Preliminary data suggests that the loss of Rad51c alone in the mammary gland epithelium does not prevent the proliferation of the tissue, but further investigation is required. We have already collected several tumors and found a predominant sebaceous and squamous morphology. Currently, we are comparing data from the different cohorts and organs to pinpoint the events leading to tumorigenesis after Rad51c loss. Conclusions: Rad51c is, together with two other tumor suppressors playing a critical role in Homologous Recombination (HR), namely BRCA1 and BRCA2, a breast cancer predisposition gene. Thus, understanding the mechanisms of its role in tumorigenesis at the tissue level will shed light on a broader role of HR in cancer and provide the translational link to human patients harboring germline mutations in Rad51c. 720 Influence of Dietary Lipids on the Hepatic Xenobiotic Metabolism and its Importance on the Experimental Breast Cancer Initiation M. Manzanares1 , L. Acerete1 , M. Pelicano-Esqueta1 , M.C. Ruiz de Villa2 , ` de Barcelona, Cell Biology E. Escrich1 , M. Solanas1 . 1 Universitat Autonoma Physiology and Immunology, Barcelona, Spain, 2 Universitat de Barcelona, Statistics, Barcelona, Spain Background: Breast cancer is the most frequent cancer and one of the most important death causes in women worldwide. Diet, especially lipids, has an important role in its aetiology. In this study we have investigated the influence of a high corn oil (CO) and a high virgin olive oil (VOO) diets on
european journal of cancer 48, suppl. 5 (2012) S25–S288
validated and correlated with clinical and epidemiological data available for the study. Conclusion: This multi-marker approach identifies a set of different markers that can be assessed in plasma, and that together (i) may be useful to narrow down the subjects at high risk of development of HCC among chronic liver disease patients in populations where chronic HBV carriage is endemic and (ii) represent very promising tools for improved, low-cost detection and diagnosis of HCC in high incidence, low resource regions of the world. 716 Chemical Carcinogenesis in Mice With a Defective Epidermal Barrier − Exploring the Connection Between Skin Barrier and Cancer Susceptibility S. Cipolat1 , K. Natsuga1 , L.M. Sevilla2 , R. Nachat3 , F.M. Watt4 . 1 Cancer Research UK Cambridge Research Institute, Cambridge, United Kingdom, 2 University of Valencia, Instituto de Biomedicina de Valencia, Valencia, Spain, 3 ´ ` ´ Universite d’Auvergne, Nutrition Cancerogen ese et Therapie, Toulouse, France, 4 Cancer Research UK, Cambridge Research Institute, Cambridge, United Kingdom Introduction: The cornified envelope (CE) is the outermost layer of the skin and it constitutes the epidermal barrier that protects from the entrance of pathogens and antigens from the outside and minimizes the water loss form the inside. Involucrin, envoplakin, and periplakin create the essential protein scaffold on which the envelope assembles. Mice deficient in all the three genes (tKO) show a defective epidermal barrier and hyperkeratosis, resulting from impaired desquamation as compensation. In addition, tKO mice display a reduction in gd+ dendritic epidermal T cells (DETC) in the epidermis and infiltration of CD3+ CD4+ T cells in the dermis. The defective epidermal barrier and the changes in T cells composition prompted us to investigate the susceptibility of the triple ko mice to skin cancer. Material and Method: tKO and wt mice were subjected to 2-stage chemical carcinogenesis (1 painting with DMBA followed by 3 paintings per week/15 wks with TPA): the number of benign (papillomas) and malignant tumors (squamous cell carcinoma) were scored weekly up to 57 weeks. Results and Discussion: tKO mice are resistant to benign tumor development, showing reduced incidence and lower papillomas burden, compared with the relative wt controls. On the other hand, tKO mice show an increased malignant conversion from benign papillomas into malignant squamous cell carcinomas. Painting with DMBA induces the same levels of apoptosis, DNA breaks and proliferation in tKO and wt mice, claiming that the decreased tumor formation is not caused by defects in initiation. Promotion with TPA induces an exacerbated keratinocytes response (parakeratosis, hyperkeratosis, spongiosis and defective differentiation) and increased inflammation mainly made of CD3+ CD4+ T lymphocytes with a Th2/Th17 profile, neutrophils and eosinophils in tKO mice, associated with increased levels of TSLP and IgE in the serum. This phenotype resembles what is observed in atopic dermatitis patients. Conclusion: Mice ko for envoplakin, periplakin and involucrin represent a useful model of skin barrier defect with features similar to individuals with atopic dermatitis. tKO mice are resistant to benign tumour formation, but display the same number of malignant tumors in a two stage skin carcinogenesis experiment compared with the relative wt mice. The exacerbated keratinocytes and inflammatory responses observed after promoting the skin with TPA are responsible for the decreased tumour formation. The mechanisms governing the higher malignant conversion in the triple ko mice are now under investigation. 717 Combined Exposure to Multiple Carcinogens Enhances Development of Rat Mammary Cancers With Characteristic Gene Expression T. Imaoka1 , M. Nishimura1 , K. Ishikawa2 , S. Yamashita3 , Y. Ohmachi4 , H. Suzuki5 , K. Daino1 , T. Ushijima3 , T. Imai2 , Y. Shimada1 . 1 National Institute of Radiological Sciences, Radiobiology for Children’s Health Program, Chiba, Japan, 2 National Institute of Radiological Sciences, Advanced Radiation Biology Research Program, Chiba, Japan, 3 National Cancer Center, Research Institute, Tokyo, Japan, 4 National Institute of Radiological Sciences, Department of Radiation Emergency Medicine, Chiba, Japan, 5 Tokyo College of Medico-Pharmaco Technology, Department of Life Technology Engineering, Tokyo, Japan Introduction: Although mixed exposure to multiple carcinogens is common in human environment, the mechanism of cooperative carcinogenesis by two or more agents is poorly understood. In the present study, we undertook experiments using the rat mammary carcinogenesis model to assess the synergistic effect of combined exposure to g rays and chemical carcinogens and potential underlying mechanisms. Materials and Methods: Female Sprague-Dawley rats at 7 weeks of age were irradiated with g rays (0, 0.5, 1 or 2 Gy); three days after irradiation, rats were subjected to single i.p. injection with 1-methyl-1-nitrosourea (MNU; 20 or 40 mg/kg) or 10 oral administrations with 2-amino-1-methyl-6-
Sunday 8 − Tuesday 10 July 2012
phenylimidazo[4,5-b]pyridine (PhIP; 40 mg/kg over two weeks), or left without chemical treatment. Tumors were examined pathologically after autopsy at 50 weeks of age. H-ras gene mutation was assessed by a restriction fragment length assay. Microarrays were used to search for genes characteristically expressed in carcinomas induced by combined exposures, followed by quantitative RT-PCR analysis incorporating additional tumors. Results: Incidence of mammary carcinoma increased linearly as a function of radiation dose in the absence of chemicals. Administration of chemical carcinogens resulted in additive increase in cancer incidence. Significantly high (78%) prevalence of H-ras mutation was observed among carcinomas of rats exposed to g rays (1 Gy) plus MNU (40 mg/kg) as compared to rats exposed to either g rays (0%) or MNU (54%) only. Microarray and quantitative RT-PCR analyses identified characteristic gene expressions in carcinoma induced by combined exposures as compared to those induced by single carcinogens. For example, high expression of serum/glucocorticoid-regulated kinase (Sgk) was associated with carcinomas induced by g rays (1 Gy) plus MNU (40 mg/kg) harboring H-ras mutation, whereas matrix metalloproteinase 17 was highly expressed in those without the mutation. Sgk may be induced synergistically by radiation effects (e.g., oxidative stress, nitric oxide and TGF-b) and H-ras mutation and enhance proliferation of cancer cells. Carcinomas induced by g rays (1 Gy) plus PhIP contained abundant transcript of the transcription factor Lef1, which may activate Wnt-responsive genes and stimulate cancer progression. Conclusion: Interaction of oncogenic mechanisms at the molecular level may underlie the combined effect of multiple carcinogens on rat mammary carcinogenesis. 718 Tumor Suppressors and Tissue Specificity in Cancer − the Role of Rad51c in Sebaceous and Mammary Carcinomas M. Tumiati1 , S. Koopal1 , A. Hemmes1 , S. Kuznetsov1 . 1 Institute for Molecular Medicine Finland − FIMM, Helsinki, Finland Background: Mutations in tumor suppressors are known to predispose to cancer. Less known is why these cancers show a distinct preference for a particular set of target tissues. Rad51c, a major protein of Homologous Recombination (HR), has recently been identified as a novel hereditary breast and ovarian cancer predisposition gene in humans and Kuznetsov et al. previously described Rad51c as a tumor suppressor with a tissue-specific cancer phenotype in mice. Here, the loss of Rad51c in a p53-deficient background resulted in a strong prevalence of sebaceous carcinomas of preputial glands in males and mammary gland carcinomas in females, while the typical p53-related sarcomas were suppressed. The high incidence of preputial and mammary gland carcinomas seems therefore to be directly triggered by loss of Rad51c, making it a perfect animal model to investigate the mechanisms of tissue-specific tumorigenesis, with a special attention to the role of HR in cancer. Materials and Methods: Organs and tumors from skin-specific Rad51c, p53 and Brca2 knock-out mouse cohorts have been analyzed immunohistologically and gene expression profiling is being used to identify the events associated with the tissue-specific loss of Rad51c. Results: The loss of Rad51c is severely affecting preputial glands, causing depletion of mature sebaceous follicles, ectopic keratinization, increased apoptosis and impaired proliferation. Interestingly, loss of Brca2 does not lead to a similar phenotype, suggesting that Rad51c could have HR independent functions. Keeping in mind the findings from preputial glands, a similar approach was used to investigate the role of Rad51c in all developmental stages of the mammary gland. Preliminary data suggests that the loss of Rad51c alone in the mammary gland epithelium does not prevent the proliferation of the tissue, but further investigation is required. We have already collected several tumors and found a predominant sebaceous and squamous morphology. Currently, we are comparing data from the different cohorts and organs to pinpoint the events leading to tumorigenesis after Rad51c loss. Conclusions: Rad51c is, together with two other tumor suppressors playing a critical role in Homologous Recombination (HR), namely BRCA1 and BRCA2, a breast cancer predisposition gene. Thus, understanding the mechanisms of its role in tumorigenesis at the tissue level will shed light on a broader role of HR in cancer and provide the translational link to human patients harboring germline mutations in Rad51c. 720 Influence of Dietary Lipids on the Hepatic Xenobiotic Metabolism and its Importance on the Experimental Breast Cancer Initiation M. Manzanares1 , L. Acerete1 , M. Pelicano-Esqueta1 , M.C. Ruiz de Villa2 , ` de Barcelona, Cell Biology E. Escrich1 , M. Solanas1 . 1 Universitat Autonoma Physiology and Immunology, Barcelona, Spain, 2 Universitat de Barcelona, Statistics, Barcelona, Spain Background: Breast cancer is the most frequent cancer and one of the most important death causes in women worldwide. Diet, especially lipids, has an important role in its aetiology. In this study we have investigated the influence of a high corn oil (CO) and a high virgin olive oil (VOO) diets on