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435 Invasion and Lymphatic Dissemination of Human Squamous Cell Carcinoma Xenografts in Three-dimensional Murine Microenvironment
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
including reactivation of embryonic transcription factors and severe changes in miRNA expression. Cross-regulation between EMT-inducers and miRNAs was previously described like the Zeb1-miR-200 feed-back loop. Our goal is to determine whether specific expression networks between ETFs, miRNAs and mitogenic stresses exist in tumors, and represents a driving force towards transformation in breast tumorigenesis. Material and Methods: We performed oncogenic cooperation assays in immortalized human mammary epithelial cells (HMEC-hTert) by using an EMTinducer expression library in combination with various mitogenic proteins. As recently demonstrated in the laboratory, these combinations were efficient in transforming human cells, as assessed by soft-agar colony assay. EMTinducer expression was next analyzed in a large number of colonies to evaluate a potential specificity of EMT-inducers according to the mitogenic insult. In parallel, we developed an in silico approach utilizing predictive algorithms to identify novel miRNAs targeting embryonic transcription factors. Results: We found that Twist1 and Twist2 expression was specifically enriched in colonies generated in presence of a constitutively active from of b-catenin, whereas Snai2 expression was selected following PTEN depletion. The prediction of specificity of the identified combinations is currently being validated by bioinformatics tools. We additionally have identified novel miRNAs able to regulate EMT inducers and validated their down-regulation in a panel of basal B breast cancer cell lines. The interplay between miRNAs, mitogenic proteins and EMT inducers will be further examined by assessing their expression in a large cohort of Basal B/ Claudin-low human breast tumours, a recently identified subtype displaying a mammary stem cell signature and EMT features. Conclusions: Our results will widen our knowledge on the potential regulatory loops impacting on the epithelial/mesenchymal phenotype in order to evaluate their therapeutic value on drug resistance and stemness properties. 435 Invasion and Lymphatic Dissemination of Human Squamous Cell Carcinoma Xenografts in Three-dimensional Murine Microenvironment Y. Soeno1 , Y. Shirako1 , K. Fujita1 , Y. Taya1 , Y. Shimazu1 , K. Nakau1 , K. Sato1 , T. Aoba1 . 1 The Nippon Dental University, School of Life Dentistry at Tokyo, Department of Pathology Introduction: Squamous cell carcinoma (SCC) in tongue exhibits aggressive progression and early-stage lymphnode metastasis. The present study aimed at elucidating a putative linkage between carcinoma cell phenotypes, remodeling of microenvironment and malignant behavior in xenograft mouse model. Materials and Methods: Three tongue SCC cell-lines (HSC2, OSC19, and OSC20) and two other SCC cell-lines (gingival-derived KOSC2 and oral floor-derived HO-1u1) were used. Each cell-line grown in culture was transplanted into BALB/c nude mouse tongue. Tongue and regional lymphnodes were dissected from the animals at periodical intervals after inoculation and then processed for preparation of paraffin-embedded serial sections. Growth of primary tumor and nodal metastasis were evaluated histopathologically and loco-regional alterations of carcinoma cell phenotypes were validated in combination with immunohistochemistry and microdissectioncoupled gene expression analysis. We also conducted histology-based 3D reconstruction using 50–100 serial sections to visualize simultaneously invasion modes of carcinoma foci and angiogenesis/ lymphangiogenesis in the microenvironment. Results: All five cell-lines gave rise to visible tumor masses (>2 mm) in the tongue within 2−3 weeks, but individual cell-lines showed a diversity in proliferation activity assessed by Ki67(+), intra- and peri-tumor densities of blood/lymphatic vessels, and EMT-like phenotypic alterations. Notably, all the tongue SCC cell-lines yielded metastatic loci at high frequency as early as 6 days after inoculation, whereas the non-tongue SCC cell-lines showed modest proliferation in the inoculation site and lower metastatic potential. 3D visualization of tumor architecture disclosed discrete invasion modes between SCC cell-lines, such as from massive growth with pushing border (OSC19, OSC20) to branching (HSC2, KOSC2) and fingering infiltration (HO-1u1). Although all SCC cell-lines showed loss of E-cadherin at the invasion margin, in couple with upregulation of E-cadherin repressor ZEB1/2, vimentin expression varied markedly among tongue SCC cell-lines, namely null in OSC19, low in HSC2, and high in OSC20. These SCC phenotypes were carried over in metastatic nodal loci. Conclusion: The present xenograft model is suitable to understanding of early lymphatic dissemination of tongue SCC. The results also support the theory that SCC nodal metastasis is multi-modal phenomena taking place in the threedimensional microenvironment.
S105
436 Evaluating of BRCA1 and BRCA2 Germ-line Mutations and BRCA Risk Assessment of Mutation Carriers in Turkish Breast Cancer Patients G. Cecener1 , U. Egeli1 , B. Tunca1 , E. Erturk1 , S. Gokgoz2 , I. Tasdelen2 , S. Ak1 , E. Demirdogen1 , G. Tezcan1 , N. Bayram3 . 1 Uludag University Medical Faculty, Department of Medical Biology, Bursa, Turkey, 2 Uludag University Medical Faculty, Department of General Surgery, Bursa, Turkey, 3 Uludag University Faculty of Economics and Administrative Sciences, Department of Econometrics, Bursa, Turkey Background: A large number of germ-line mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known about the role of these inherited susceptibility genes in breast cancer risk among Turkish women. Furthermore, the founder mutations of our society are still unknown. Therefore the data of breast cancer patients may be beneficial to improve our knowledge about specific BRCA1 and BRCA2 mutations observed in Turkish population and their frequency. Material and Methods: Ninety seven women who had early age and/or a family history of breast cancer were included in the study. All coding regions and exon-intron boundaries of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis followed by direct sequencing of detected variants. These variations were evaluated in GVGD (http://agvgd.iarc.fr/), HSF (http://www.umd.be/HSF/) and ESEfinder (http://rulai.cshl.edu/cgi-bin/tools/ESE3/) web based programs to confirm whether these mutations affected Brca1and Brca2 proteins structure and splicing ability. In addition, BRCAPRO software was used for risk analysis. Results: Eleven different mutations were detected: two previously described truncating mutation (1643delC and 5382insC) and one previously described missense mutation (32 T to C), 2 novel truncating mutations (3545delA and 5248insT) and two novel missense mutations (4837 A to G and 5219 T to G) in BRCA1, and four previously reported missense mutations (1114 A to C, 5744 C to T, 7397 T to C and 8187 G to T) in BRCA2. These 11 different mutations were represented in 31.96 (31/97) percent of this study. Furthermore, the missense mutation (7397 T to C) in BRCA2 gene was observed in high frequency (10.3%) in this group. The effect on Brca1 and Brca2 proteins structure and splicing ability of determined novel alterations were analyzed by using GVGD, HSF and ESEfinder programs. The results of BRCAPRO software showed that the level of risk for both breast and ovarian cancer increased with age in women who carried the mutation. Statistical analysis of our findings and clinicopathological features of patients were performed using Mann-Whitney and Fisher’s Exact Tests by SPSS-16 web based program. There were significant differences in the tumor size and Ki67 expression between cases with or without the BRCA mutations (p = 0.035 and p = 0.014, respectively). Conclusions: These findings contribute significantly to what currently is known about the types and impact of germ-line BRCA1 and BRCA2 mutations in Turkish women. It is shown that determined novel alterations may have a role on breast carcinogenesis. The missense mutation (7397 T to C) in BRCA2 gene may have the founder effect on Turkish breast cancer patients. These informations may be helpful in guiding management in BRCA1 or BRCA2 patients considering breast-conserving therapy. 437 FADD Deficiency Causes Changes in Apoptosis and Necroptosis of Mouse Embryonic Fibroblasts J. Stambuk1 , M. Antunovic1 , K. Caput Mihalic1 , B. Nagy1 , I. Marijanovic1 . Faculty of Natural Sciences and Mathematics, Department of Molecular Biology, Zagreb, Croatia 1
Introduction: Necrosis, like apoptosis, can be strictly regulated and that form of necrosis is called necroptosis. FADD (Fas-associated death domain) protein is a key molecule of extrinsic apoptotic pathway that transduces signal from the membrane death receptors to caspase 8, but it also plays pivotal role in activation of necroptosis. UV irradiation triggers intrinsic apoptotic pathway via DNA damage and caspase 9 and extrinsic pathway via death receptor trimerization and caspase 8. Material and Methods: The effect of FADD deficiency on cell survival and activation of cell death was investigated using knock-out mouse embryonic fibroblasts (FADD−/− ) irradiated with different doses of UVB radiation. Cell viability was estimated using MTT test, caspase activity using commercial assays and detection of necroptosis using MTT test with necrostatin-1. Results and Discussion: Results showed that FADD−/− fibroblasts have lower proliferation rate than wild-type cells as their viability was reduced in comparison to wild-type cells, following the exposure to UVB radiation at intensity range 100–600 J/m2 . Increased activation of caspases 3/7 and caspase 9 was detected in the irradiated FADD−/− fibroblasts and these cells did not have an increased viability in the presence of necrostatin-1 in comparison to the wild type. Caspase 8 activation was not detected in either cell type after the exposure to 300 J/m2 of UVB. Conclusion: From results we can conclude that UVB radiation in FADD−/− fibroblasts causes stronger induction of apoptosis due to intrinsic pathway
european journal of cancer 48, suppl. 5 (2012) S25–S288
including reactivation of embryonic transcription factors and severe changes in miRNA expression. Cross-regulation between EMT-inducers and miRNAs was previously described like the Zeb1-miR-200 feed-back loop. Our goal is to determine whether specific expression networks between ETFs, miRNAs and mitogenic stresses exist in tumors, and represents a driving force towards transformation in breast tumorigenesis. Material and Methods: We performed oncogenic cooperation assays in immortalized human mammary epithelial cells (HMEC-hTert) by using an EMTinducer expression library in combination with various mitogenic proteins. As recently demonstrated in the laboratory, these combinations were efficient in transforming human cells, as assessed by soft-agar colony assay. EMTinducer expression was next analyzed in a large number of colonies to evaluate a potential specificity of EMT-inducers according to the mitogenic insult. In parallel, we developed an in silico approach utilizing predictive algorithms to identify novel miRNAs targeting embryonic transcription factors. Results: We found that Twist1 and Twist2 expression was specifically enriched in colonies generated in presence of a constitutively active from of b-catenin, whereas Snai2 expression was selected following PTEN depletion. The prediction of specificity of the identified combinations is currently being validated by bioinformatics tools. We additionally have identified novel miRNAs able to regulate EMT inducers and validated their down-regulation in a panel of basal B breast cancer cell lines. The interplay between miRNAs, mitogenic proteins and EMT inducers will be further examined by assessing their expression in a large cohort of Basal B/ Claudin-low human breast tumours, a recently identified subtype displaying a mammary stem cell signature and EMT features. Conclusions: Our results will widen our knowledge on the potential regulatory loops impacting on the epithelial/mesenchymal phenotype in order to evaluate their therapeutic value on drug resistance and stemness properties. 435 Invasion and Lymphatic Dissemination of Human Squamous Cell Carcinoma Xenografts in Three-dimensional Murine Microenvironment Y. Soeno1 , Y. Shirako1 , K. Fujita1 , Y. Taya1 , Y. Shimazu1 , K. Nakau1 , K. Sato1 , T. Aoba1 . 1 The Nippon Dental University, School of Life Dentistry at Tokyo, Department of Pathology Introduction: Squamous cell carcinoma (SCC) in tongue exhibits aggressive progression and early-stage lymphnode metastasis. The present study aimed at elucidating a putative linkage between carcinoma cell phenotypes, remodeling of microenvironment and malignant behavior in xenograft mouse model. Materials and Methods: Three tongue SCC cell-lines (HSC2, OSC19, and OSC20) and two other SCC cell-lines (gingival-derived KOSC2 and oral floor-derived HO-1u1) were used. Each cell-line grown in culture was transplanted into BALB/c nude mouse tongue. Tongue and regional lymphnodes were dissected from the animals at periodical intervals after inoculation and then processed for preparation of paraffin-embedded serial sections. Growth of primary tumor and nodal metastasis were evaluated histopathologically and loco-regional alterations of carcinoma cell phenotypes were validated in combination with immunohistochemistry and microdissectioncoupled gene expression analysis. We also conducted histology-based 3D reconstruction using 50–100 serial sections to visualize simultaneously invasion modes of carcinoma foci and angiogenesis/ lymphangiogenesis in the microenvironment. Results: All five cell-lines gave rise to visible tumor masses (>2 mm) in the tongue within 2−3 weeks, but individual cell-lines showed a diversity in proliferation activity assessed by Ki67(+), intra- and peri-tumor densities of blood/lymphatic vessels, and EMT-like phenotypic alterations. Notably, all the tongue SCC cell-lines yielded metastatic loci at high frequency as early as 6 days after inoculation, whereas the non-tongue SCC cell-lines showed modest proliferation in the inoculation site and lower metastatic potential. 3D visualization of tumor architecture disclosed discrete invasion modes between SCC cell-lines, such as from massive growth with pushing border (OSC19, OSC20) to branching (HSC2, KOSC2) and fingering infiltration (HO-1u1). Although all SCC cell-lines showed loss of E-cadherin at the invasion margin, in couple with upregulation of E-cadherin repressor ZEB1/2, vimentin expression varied markedly among tongue SCC cell-lines, namely null in OSC19, low in HSC2, and high in OSC20. These SCC phenotypes were carried over in metastatic nodal loci. Conclusion: The present xenograft model is suitable to understanding of early lymphatic dissemination of tongue SCC. The results also support the theory that SCC nodal metastasis is multi-modal phenomena taking place in the threedimensional microenvironment.
S105
436 Evaluating of BRCA1 and BRCA2 Germ-line Mutations and BRCA Risk Assessment of Mutation Carriers in Turkish Breast Cancer Patients G. Cecener1 , U. Egeli1 , B. Tunca1 , E. Erturk1 , S. Gokgoz2 , I. Tasdelen2 , S. Ak1 , E. Demirdogen1 , G. Tezcan1 , N. Bayram3 . 1 Uludag University Medical Faculty, Department of Medical Biology, Bursa, Turkey, 2 Uludag University Medical Faculty, Department of General Surgery, Bursa, Turkey, 3 Uludag University Faculty of Economics and Administrative Sciences, Department of Econometrics, Bursa, Turkey Background: A large number of germ-line mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known about the role of these inherited susceptibility genes in breast cancer risk among Turkish women. Furthermore, the founder mutations of our society are still unknown. Therefore the data of breast cancer patients may be beneficial to improve our knowledge about specific BRCA1 and BRCA2 mutations observed in Turkish population and their frequency. Material and Methods: Ninety seven women who had early age and/or a family history of breast cancer were included in the study. All coding regions and exon-intron boundaries of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis followed by direct sequencing of detected variants. These variations were evaluated in GVGD (http://agvgd.iarc.fr/), HSF (http://www.umd.be/HSF/) and ESEfinder (http://rulai.cshl.edu/cgi-bin/tools/ESE3/) web based programs to confirm whether these mutations affected Brca1and Brca2 proteins structure and splicing ability. In addition, BRCAPRO software was used for risk analysis. Results: Eleven different mutations were detected: two previously described truncating mutation (1643delC and 5382insC) and one previously described missense mutation (32 T to C), 2 novel truncating mutations (3545delA and 5248insT) and two novel missense mutations (4837 A to G and 5219 T to G) in BRCA1, and four previously reported missense mutations (1114 A to C, 5744 C to T, 7397 T to C and 8187 G to T) in BRCA2. These 11 different mutations were represented in 31.96 (31/97) percent of this study. Furthermore, the missense mutation (7397 T to C) in BRCA2 gene was observed in high frequency (10.3%) in this group. The effect on Brca1 and Brca2 proteins structure and splicing ability of determined novel alterations were analyzed by using GVGD, HSF and ESEfinder programs. The results of BRCAPRO software showed that the level of risk for both breast and ovarian cancer increased with age in women who carried the mutation. Statistical analysis of our findings and clinicopathological features of patients were performed using Mann-Whitney and Fisher’s Exact Tests by SPSS-16 web based program. There were significant differences in the tumor size and Ki67 expression between cases with or without the BRCA mutations (p = 0.035 and p = 0.014, respectively). Conclusions: These findings contribute significantly to what currently is known about the types and impact of germ-line BRCA1 and BRCA2 mutations in Turkish women. It is shown that determined novel alterations may have a role on breast carcinogenesis. The missense mutation (7397 T to C) in BRCA2 gene may have the founder effect on Turkish breast cancer patients. These informations may be helpful in guiding management in BRCA1 or BRCA2 patients considering breast-conserving therapy. 437 FADD Deficiency Causes Changes in Apoptosis and Necroptosis of Mouse Embryonic Fibroblasts J. Stambuk1 , M. Antunovic1 , K. Caput Mihalic1 , B. Nagy1 , I. Marijanovic1 . Faculty of Natural Sciences and Mathematics, Department of Molecular Biology, Zagreb, Croatia 1
Introduction: Necrosis, like apoptosis, can be strictly regulated and that form of necrosis is called necroptosis. FADD (Fas-associated death domain) protein is a key molecule of extrinsic apoptotic pathway that transduces signal from the membrane death receptors to caspase 8, but it also plays pivotal role in activation of necroptosis. UV irradiation triggers intrinsic apoptotic pathway via DNA damage and caspase 9 and extrinsic pathway via death receptor trimerization and caspase 8. Material and Methods: The effect of FADD deficiency on cell survival and activation of cell death was investigated using knock-out mouse embryonic fibroblasts (FADD−/− ) irradiated with different doses of UVB radiation. Cell viability was estimated using MTT test, caspase activity using commercial assays and detection of necroptosis using MTT test with necrostatin-1. Results and Discussion: Results showed that FADD−/− fibroblasts have lower proliferation rate than wild-type cells as their viability was reduced in comparison to wild-type cells, following the exposure to UVB radiation at intensity range 100–600 J/m2 . Increased activation of caspases 3/7 and caspase 9 was detected in the irradiated FADD−/− fibroblasts and these cells did not have an increased viability in the presence of necrostatin-1 in comparison to the wild type. Caspase 8 activation was not detected in either cell type after the exposure to 300 J/m2 of UVB. Conclusion: From results we can conclude that UVB radiation in FADD−/− fibroblasts causes stronger induction of apoptosis due to intrinsic pathway