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662: HER2 isoforms in mammary carcinogenesis and targeted therapy susceptibility
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
Conclusion: The mutational landscape of CRCs that metastasizes to the ovary is similar to consecutive series of CRC, although the low frequency of PIK3CA and PBXW7 should be further studied. Passenger mutations are more often discordant between the primary CRC and the ovarian metastasis than driver mutations. No conflict of interest. 660 In vitro model for DNA double-strand break (DSB) repair analysis in cells derived from breast cancer specimens identifies new prognostic markers M. Deniz1 , T. Gundelach1 , J. Kaufmann1 , M. Keimling1 , W. Janni1 , ¨ L. Wiesmuller ¨ 1 . 1 Universitatsfrauenklinik, Ulm, Germany Background: All currently known breast cancer susceptibility genes are linked to DSB repair. To find novel markers for breast cancer risk, we analyzed DSB repair in peripheral blood lymphocytes (PBLs) with known mutations in predisposing genes revealing a functional signature indicative of errorprone repair. Our first case–control study for prospective evaluation of this signature revealed highly significant increases of the error-prone mechanisms non-homologous end joining (NHEJ) and single-strand annealing (SSA) in women with familial risk. These findings underscore the potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility. To additionally capture non-hereditary factors for identification of prognostic markers, we established a new primary cell model resembling breast cancer specimens and scrutinized DSB repair. Material and Methods: We established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial mesenchymal transition cells (EMTs) and fibroblasts from fresh breast cancer tissue. Our study included 148 patients donating tumor and 91 additionally blood samples. We analyzed distinct DSB repair mechanisms in these cell types and PBLs and we investigated DNA lesion processing by quantitative immunofluorescence microscopy of nuclear signals (foci) after radiomimetic treatment. The data for each variable were correlated with clinically relevant parameters to determine association of repair changes with age, receptor status and recommendation of adjuvant chemotherapy integrating various prognostic characteristics. Results and Discussion: We show that our protocol for isolation of different primary cell types derived from breast cancer specimens enables both foci analyses as an estimate of repair capacity and fluorescence-based analysis of distinct DSB repair qualities. Our study reveals an age-dependent effect on overall DSB removal, namely a gradual decline of repair capacity in epithelial cells from patients with increasing age. Intriguingly, we previously demonstrated elevated error-prone DSB repair in PBLs from patients of young age (<50), i.e. predominantly predisposed patients. Most interestingly, we found elevated NHEJ in EMTs from patients with recommendation of adjuvant chemotherapy, i.e. with high risk tumors. Conclusion: We carved out differences between hereditary and acquired DSB repair characteristics in breast cancer patients, which could be useful as a method to further classify breast cancer to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors. Our discovery of errorprone DSB repair activities in EMTs from high risk tumors may serve as novel prognostic marker beyond the limitations of genotyping prompting a prospective study to correlate DSB repair quality with response to adjuvant chemotherapy. No conflict of interest. 661 Wnt/b-catenin signalling inhibition decreases cell proliferation in Wnt autocrine-soft tissue sarcoma cells A. Obrador-Hevia1 , S. Calabuig-Farinas ˜ 2 , E. Mart´ınez3 , A. Ochoa3 , I. Felipe-Abrio3 , J. Mart´ın3 , R. Alemany4 . 1 Hospital Universitari Son Espases, Oncohaematology, Palma, Spain, 2 Hospital General Universitario de Valencia, Molecular Oncology Laboratory, Valencia, Spain, 3 Hospital Universitari Son Espases, Oncohematology, Palma, Spain, 4 Universitat de les Illes Balears, Cell Biology, Palma, Spain Introduction: Soft Tissue Sarcomas (STS) are malignant tumors of mesenchymal origin that affect around 200,000 individuals per year in the entire world, more than 50 recognized subtypes. This group of tumours is characterized by molecular alterations in cellular processes such as cell cycle regulation and apoptosis in which b-catenin has been recently involved. b-catenin is the key molecule in the Wnt/APC/b-catenin signalling pathway and it regulates the transcriptional program activated by the secreted protein Wnt. The aims of this study were: (1) to evaluate the role of the Wnt/APC/b-catenin pathway in the pathogenesis of STS; (2) to study its role as a functional target of therapeutic agents. Materials and Methods: A panel of STS cell lines was used. Cells were treated with different inhibitors of the Wnt/APC/b-catenin pathway (XAV939, IWR-1, IWP-2; Sigma) for further analysis. Conventional drugs such as Doxorubicin were also used to evaluate different combination strategies. Cell viability (CellTiter 96® AQueous One Solution, Promega; xCELLigence System; ACEA Biosciences) and cell cycle analysis (Coulter Epics XL-MSL;
Beckman Coulter) were performed. Expression and cellular localization of different proteins were studied by means of Western Blot, quantitative PCR and immunocytochemistry. Results and Discussion: The role of the Wnt/APC/b-catenin pathway in a panel of STS cell lines was studied. Cells with APC mutations or other alterations of the pathway were resistant to inhibition of the Wnt/APC/b-catenin pathway. On the contrary, inhibition of b-catenin was capable of inducing cell death and apoptosis in Wnt autocrine cells with wild-type APC and b-catenin, suggesting that b-catenin overexpression has an important role in sarcomagenesis. Moreover, downregulation of CDC25A, a recently defined b-catenin target gene in sarcomas, was involved in the response to these inhibitors. Conclusions: The inhibition of b-catenin and its transcriptional program represents a potential therapy that could enhance the effects of conventional drugs in soft tissue sarcomas. No conflict of interest. 662 HER2 isoforms in mammary carcinogenesis and targeted therapy susceptibility A. Palladini1 , M. Dall’Ora1 , V. Grosso1 , M.L. Ianzano1 , D. Ranieri1 , T. Balboni1 , M. Iezzi2 , C. De Giovanni1 , P.L. Lollini1 , P. Nanni1 . 1 University of Bologna, Department of Experimental Diagnostic and Specialty Medicine, Bologna, Italy, 2 G. D’Annunzio University, Aging Research Centre, Chieti, Italy Background: About 50% of HER2+ mammary tumors coexpress wild-type HER2 (wtHER2) and its splice isoform Delta16 (D16), and coexpression correlates with local dissemination. Transgenic mice for either isoform, as well as transduced cell lines, have been used to study the role of HER2 isoforms, but a model of coexpression was still lacking. Here we describe for the first time a new animal model of breast cancer driven by the coexpression of wt and D16 HER2 isoforms, obtained by crossing wtHER2 and D16 transgenic mice (F1 mice). Cell lines established from F1 mammary tumors were used to study the role of coexpressed isoforms in targeted drug susceptibility. Material and Methods: F1 mice were obtained by crossing wtHER2 female mice with D16 male mice. Animal experiments were authorized by the local animal use and care committee. In vitro drug sensitivity of transgenic cell lines was evaluated in anchorage independent conditions. Expression levels of wtHER2, D16 and angiogenesis mediators were assessed by Real Time PCR. Results: Kinetics of spontaneous tumor onset of F1 mice coexpressing wtHER2 and D16 isoforms was similar to that of D16 mice (100% incidence, median latency 17 weeks) and faster than that of wtHER2 mice (85% incidence, median latency 46 weeks). Mean number of tumors per mouse at death were: wtHER2, 2.6±0.2; F1, 6.8±0.4; D16, 4.8±0.5. Metastatic spread was similar in all transgenic mice. Different tumors of F1 mice showed heterogeneous levels of wtHER2 and D16 transcripts. Most tumors (>80%) had a high level of D16 (D16high ) and a low level of wtHER2 (wtHER2low ). Some tumors presented a high level of both transcripts or D16low and wtHER2high . Tumors of wtHER2 mice frequently displayed angiogenesis with endotheliumfree lacunae, while D16 seemed to sustain a more regular angiogenesis. F1 tumors had aspects of both wtHER2 and D16 angiogenesis. We also observed a correlation between HER2high tumors and low expression of angiogenesis mediators such as VEGF-a, VEGFR2, CD146 and CD31. Expression of D16 isoform did not confer in vitro resistance to HER2-targeted drugs (trastuzumab and lapatinib) in comparison to wtHER2. Conclusions: The study of F1 mice coexpressing wtHER2 and D16 isoform suggested that D16 isoform plays a role in early tumor onset but did not increase resistance to targeted therapy. No conflict of interest. 663 Inter- and intra-tumoral heterogeneity in oncogenic activation of PI3K and MAPK pathways in aggressive thyroid carcinomas (PDC and ATC) - Cohabitation rate R. Munoz ˜ Mart´ınez1 , A.M. Costa2 , J. Cameselle Teijeiro3 , T. Alvarez Gago4 , G. Garcia-Rostan1 . 1 Institute of Molecular Biology and Genetics (IBGM), Molecular Genetics of Disease, Vallodolid, Spain, 2 Institute of Molecular Pathology and Immunology of Porto University (IPATIMUP), Molecular targets in cancer, Porto, Portugal, 3 School of Medicine − Santiago de Compostela University, Department of Pathology, Santiago de Compostela, Spain, 4 School of Medicine − Valladolid University, Department of Pathology, Valladolid, Spain The MAPK and the PI3K pathways are viewed as therapeutic targets of the future due to their pivotal role in cellular proliferation, growth regulation, survival, adhesion, motility and spreading. While genetic alterations of effectors along the MAPK pathway are early events in thyroid follicular cell carcinogenesis, being some of them (oncogenic BRAF and RAS activation) also involved in tumour dedifferentiation and progression, the
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
Conclusion: The mutational landscape of CRCs that metastasizes to the ovary is similar to consecutive series of CRC, although the low frequency of PIK3CA and PBXW7 should be further studied. Passenger mutations are more often discordant between the primary CRC and the ovarian metastasis than driver mutations. No conflict of interest. 660 In vitro model for DNA double-strand break (DSB) repair analysis in cells derived from breast cancer specimens identifies new prognostic markers M. Deniz1 , T. Gundelach1 , J. Kaufmann1 , M. Keimling1 , W. Janni1 , ¨ L. Wiesmuller ¨ 1 . 1 Universitatsfrauenklinik, Ulm, Germany Background: All currently known breast cancer susceptibility genes are linked to DSB repair. To find novel markers for breast cancer risk, we analyzed DSB repair in peripheral blood lymphocytes (PBLs) with known mutations in predisposing genes revealing a functional signature indicative of errorprone repair. Our first case–control study for prospective evaluation of this signature revealed highly significant increases of the error-prone mechanisms non-homologous end joining (NHEJ) and single-strand annealing (SSA) in women with familial risk. These findings underscore the potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility. To additionally capture non-hereditary factors for identification of prognostic markers, we established a new primary cell model resembling breast cancer specimens and scrutinized DSB repair. Material and Methods: We established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial mesenchymal transition cells (EMTs) and fibroblasts from fresh breast cancer tissue. Our study included 148 patients donating tumor and 91 additionally blood samples. We analyzed distinct DSB repair mechanisms in these cell types and PBLs and we investigated DNA lesion processing by quantitative immunofluorescence microscopy of nuclear signals (foci) after radiomimetic treatment. The data for each variable were correlated with clinically relevant parameters to determine association of repair changes with age, receptor status and recommendation of adjuvant chemotherapy integrating various prognostic characteristics. Results and Discussion: We show that our protocol for isolation of different primary cell types derived from breast cancer specimens enables both foci analyses as an estimate of repair capacity and fluorescence-based analysis of distinct DSB repair qualities. Our study reveals an age-dependent effect on overall DSB removal, namely a gradual decline of repair capacity in epithelial cells from patients with increasing age. Intriguingly, we previously demonstrated elevated error-prone DSB repair in PBLs from patients of young age (<50), i.e. predominantly predisposed patients. Most interestingly, we found elevated NHEJ in EMTs from patients with recommendation of adjuvant chemotherapy, i.e. with high risk tumors. Conclusion: We carved out differences between hereditary and acquired DSB repair characteristics in breast cancer patients, which could be useful as a method to further classify breast cancer to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors. Our discovery of errorprone DSB repair activities in EMTs from high risk tumors may serve as novel prognostic marker beyond the limitations of genotyping prompting a prospective study to correlate DSB repair quality with response to adjuvant chemotherapy. No conflict of interest. 661 Wnt/b-catenin signalling inhibition decreases cell proliferation in Wnt autocrine-soft tissue sarcoma cells A. Obrador-Hevia1 , S. Calabuig-Farinas ˜ 2 , E. Mart´ınez3 , A. Ochoa3 , I. Felipe-Abrio3 , J. Mart´ın3 , R. Alemany4 . 1 Hospital Universitari Son Espases, Oncohaematology, Palma, Spain, 2 Hospital General Universitario de Valencia, Molecular Oncology Laboratory, Valencia, Spain, 3 Hospital Universitari Son Espases, Oncohematology, Palma, Spain, 4 Universitat de les Illes Balears, Cell Biology, Palma, Spain Introduction: Soft Tissue Sarcomas (STS) are malignant tumors of mesenchymal origin that affect around 200,000 individuals per year in the entire world, more than 50 recognized subtypes. This group of tumours is characterized by molecular alterations in cellular processes such as cell cycle regulation and apoptosis in which b-catenin has been recently involved. b-catenin is the key molecule in the Wnt/APC/b-catenin signalling pathway and it regulates the transcriptional program activated by the secreted protein Wnt. The aims of this study were: (1) to evaluate the role of the Wnt/APC/b-catenin pathway in the pathogenesis of STS; (2) to study its role as a functional target of therapeutic agents. Materials and Methods: A panel of STS cell lines was used. Cells were treated with different inhibitors of the Wnt/APC/b-catenin pathway (XAV939, IWR-1, IWP-2; Sigma) for further analysis. Conventional drugs such as Doxorubicin were also used to evaluate different combination strategies. Cell viability (CellTiter 96® AQueous One Solution, Promega; xCELLigence System; ACEA Biosciences) and cell cycle analysis (Coulter Epics XL-MSL;
Beckman Coulter) were performed. Expression and cellular localization of different proteins were studied by means of Western Blot, quantitative PCR and immunocytochemistry. Results and Discussion: The role of the Wnt/APC/b-catenin pathway in a panel of STS cell lines was studied. Cells with APC mutations or other alterations of the pathway were resistant to inhibition of the Wnt/APC/b-catenin pathway. On the contrary, inhibition of b-catenin was capable of inducing cell death and apoptosis in Wnt autocrine cells with wild-type APC and b-catenin, suggesting that b-catenin overexpression has an important role in sarcomagenesis. Moreover, downregulation of CDC25A, a recently defined b-catenin target gene in sarcomas, was involved in the response to these inhibitors. Conclusions: The inhibition of b-catenin and its transcriptional program represents a potential therapy that could enhance the effects of conventional drugs in soft tissue sarcomas. No conflict of interest. 662 HER2 isoforms in mammary carcinogenesis and targeted therapy susceptibility A. Palladini1 , M. Dall’Ora1 , V. Grosso1 , M.L. Ianzano1 , D. Ranieri1 , T. Balboni1 , M. Iezzi2 , C. De Giovanni1 , P.L. Lollini1 , P. Nanni1 . 1 University of Bologna, Department of Experimental Diagnostic and Specialty Medicine, Bologna, Italy, 2 G. D’Annunzio University, Aging Research Centre, Chieti, Italy Background: About 50% of HER2+ mammary tumors coexpress wild-type HER2 (wtHER2) and its splice isoform Delta16 (D16), and coexpression correlates with local dissemination. Transgenic mice for either isoform, as well as transduced cell lines, have been used to study the role of HER2 isoforms, but a model of coexpression was still lacking. Here we describe for the first time a new animal model of breast cancer driven by the coexpression of wt and D16 HER2 isoforms, obtained by crossing wtHER2 and D16 transgenic mice (F1 mice). Cell lines established from F1 mammary tumors were used to study the role of coexpressed isoforms in targeted drug susceptibility. Material and Methods: F1 mice were obtained by crossing wtHER2 female mice with D16 male mice. Animal experiments were authorized by the local animal use and care committee. In vitro drug sensitivity of transgenic cell lines was evaluated in anchorage independent conditions. Expression levels of wtHER2, D16 and angiogenesis mediators were assessed by Real Time PCR. Results: Kinetics of spontaneous tumor onset of F1 mice coexpressing wtHER2 and D16 isoforms was similar to that of D16 mice (100% incidence, median latency 17 weeks) and faster than that of wtHER2 mice (85% incidence, median latency 46 weeks). Mean number of tumors per mouse at death were: wtHER2, 2.6±0.2; F1, 6.8±0.4; D16, 4.8±0.5. Metastatic spread was similar in all transgenic mice. Different tumors of F1 mice showed heterogeneous levels of wtHER2 and D16 transcripts. Most tumors (>80%) had a high level of D16 (D16high ) and a low level of wtHER2 (wtHER2low ). Some tumors presented a high level of both transcripts or D16low and wtHER2high . Tumors of wtHER2 mice frequently displayed angiogenesis with endotheliumfree lacunae, while D16 seemed to sustain a more regular angiogenesis. F1 tumors had aspects of both wtHER2 and D16 angiogenesis. We also observed a correlation between HER2high tumors and low expression of angiogenesis mediators such as VEGF-a, VEGFR2, CD146 and CD31. Expression of D16 isoform did not confer in vitro resistance to HER2-targeted drugs (trastuzumab and lapatinib) in comparison to wtHER2. Conclusions: The study of F1 mice coexpressing wtHER2 and D16 isoform suggested that D16 isoform plays a role in early tumor onset but did not increase resistance to targeted therapy. No conflict of interest. 663 Inter- and intra-tumoral heterogeneity in oncogenic activation of PI3K and MAPK pathways in aggressive thyroid carcinomas (PDC and ATC) - Cohabitation rate R. Munoz ˜ Mart´ınez1 , A.M. Costa2 , J. Cameselle Teijeiro3 , T. Alvarez Gago4 , G. Garcia-Rostan1 . 1 Institute of Molecular Biology and Genetics (IBGM), Molecular Genetics of Disease, Vallodolid, Spain, 2 Institute of Molecular Pathology and Immunology of Porto University (IPATIMUP), Molecular targets in cancer, Porto, Portugal, 3 School of Medicine − Santiago de Compostela University, Department of Pathology, Santiago de Compostela, Spain, 4 School of Medicine − Valladolid University, Department of Pathology, Valladolid, Spain The MAPK and the PI3K pathways are viewed as therapeutic targets of the future due to their pivotal role in cellular proliferation, growth regulation, survival, adhesion, motility and spreading. While genetic alterations of effectors along the MAPK pathway are early events in thyroid follicular cell carcinogenesis, being some of them (oncogenic BRAF and RAS activation) also involved in tumour dedifferentiation and progression, the