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375 Expression Profile of SETD Methyltransferase Family Genes in Breast Cancer Cell Lines
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
resulting in a less-pronounced differential effect. Pathway inhibition analysis showed that, whereas MEK and PI3K inhibitors respectively decreased ERK and AKT phosphorylation in all cell lines, IGF1R inhibitors decreased AKT phosphorylation only in KRAS mutant cell lines. Inhibition of AKT phosphorylation correlates with effectiveness of IGF-1R inhibitor in blocking cell proliferation. Conclusions: Our results indicate that mutant KRAS activation of PI3K requires coordinate input from IGF-1R. These findings suggest potential therapeutic strategies for NSCLC tumours harbouring KRAS mutations. 375 Expression Profile of SETD Methyltransferase Family Genes in Breast Cancer Cell Lines M.S. Estrela1 , C.A. Moura1 , A.B. Motoyama1 , F. Pittella Silva1 . 1 Faculdade ˆ ´ de Ciencias da Saude, ´ Laboratorio de Farmacologia Molecular, Brasilia, Brazil Background: Lysine-methyltransferases are key enzymes that posttranslationally modify lysine residues in histones and play a key role in gene expression, epigenetic regulation, and as determinants of survival in malignant cells. The involvement of epigenetic modifying enzymes in carcinogenesis has gained a great deal of attention in the past few years. However, little is known about the role of SETD methyltransferase family genes in this process. In this study we investigate the expression of all SETD family genes in a panel of 7 breast cancer cell lines and a non-tumor counterpart. Material and Methods: We selected seven breast cancer cell lines (MCF7, CAMA1, SKBR3, MDA-MB-231, MDA-MB-436, MDA-MB-468 and HCC1954) and a matching cell line from normal B lymphoblastoid (HCC1954-BL) for the expression analysis. Total RNA was isolated and cDNAs were synthesized. Quantification of 9 SETD family genes (SETD1A, SETD1B, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7 and SETD8) was performed by semiquantitative PCR and Real Time PCR (qPCR). All PCRs were normalized with b-actin. Results were analyzed by the comparative 2−DDCt method and each cell line was compared with the control group of normal cells HCC1954-BL. Results: In accordance to previous reports, we found SETD7 to be overexpressed in all 7 cell lines. SETD6 is highly expressed in all cell lines, except in MCF7 and MDA-MB-436, compared to the normal cells. SETD8 was also found to be highly expressed in HCC1954, MCF7, MDAMB-231 and MDA-MB-436. All together, this data points for a possible role of SETD6 and SETD8 in breast carcinogenesis. However, further studies are necessary. Interestingly, SETD4 was specifically highly expressed in the ER (−) breast cancer cell lines HCC1954, MDA-MB-231 and MDA-MB-436, but not significantly found in ER (+) cells such as MCF7. This suggests SETD4 expression as a possible specific molecular marker. We found SETD1B to be 4 fold more expressed in 3 cell lines, including HCC1954, in comparison to the matching normal cell line. SETD1A was slightly more expressed in HCC1954 and MDA-MB-436 cells. The role of these two genes in carcinogenesis has also not been fully elucidated. The genes SETD2, SETD3 and SETD5 were not significantly expressed in the majority of breast cancer cell lines. Their expression was only found to be significant in HCC1954 in comparison to its matching normal cell line. SETD2 was recently reported as a tumor suppressor, being significantly less expressed in breast cancer and in later tumor stages. The result obtained in this study may reflect the characteristic of the primary stage IIA cell line HCC1954. Conclusions: The comparative analysis of SETD family gene expression in cancer cell lines provides new insights on the relation between SETD genes and tumorigenesis. These findings can serve as platform for further analysis in clinical samples from patients, aiming to point new potential molecular markers or targets for therapeutic intervention in breast cancer. 376 Apoptotic Intrinsic Pathway as an Essential Key for the Human Salivary Gland Branching Morphogenesis T.H.N. Teshima1 , R.C.F. Ianez2 , C.M. Coutinho-Camillo2 , S.V. Louren¸co1 . 1 ˜ Paulo − Dentistry School, Oral Pathology, Sao ˜ Paulo / SP, University of Sao ˜ Paulo / SP, Brazil Brazil, 2 AC Camargo Hospital, Pathologic Anatomy, Sao Introduction: Since the disclosure of apoptosis and its relation to tissue developing, homeostasis and pathogenesis, the mechanisms supposedly involved in this process have been investigated. Then, it has been well established that apoptosis occurs by two major pathways, intrinsic and extrinsic, sometimes showing a cross-reaction. Bcl-2 family proteins can control the intrinsic pathway through the balance between their pro and anti-apoptotic factors. Considering apoptosis as an essential key for the development of many tissues, this study purposes to illustrate the presence of Bax, Bak, Bcl-2, Bad, Bid, Bcl-XL and Bcl-X during the stages of human salivary glands (HSG) branching morphogenesis. Material and Methods: Samples from 20 fetuses of natural miscarriages were submitted to immunohistochemical reactions for Bax, Bak, Bad, Bid, Bcl-2, BclXL and Bcl-X. Normal mature salivary glands and some other tissues were employed as controls.
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Results and Discussion: Bax, Bak and Bcl-XL were significantly expressed during almost all stages of salivary gland development and Bcl-X, Bad and Bid showed only a focal positivity, being present in ductal and epithelial (buds) cells, both in nucleus and cytoplasm. Mature HSG were employed as controls and were negative as well as Bcl-2 and acinar cells from fetuses. Bax and Bak predominance at earlier phases suggests that they guide the formation of the luminal space, whilst the nuclear expression of Bcl-XL was more concentrated at latter stages. Regarding that the intrinsic pathway depends on the balance between Bcl-2 family pro and anti-apoptotic factors, those results were consistent, once Bcl-XL seems to control the apoptotic rate after lumen formation. Besides, evidences indicate a mutual regulation between Bcl-2 and Bcl-XL, being a possible reason for the absence of Bcl-2. Bad in turn acts binding to anti-apoptotic factors, Bcl-XL is an isoform of Bcl-X and Bid transmits death signals from the extrinsic pathway to the intrinsic. Thus, their almost negative expression indicates a secondary role during the human salivary gland morphogenesis, reinforcing the importance of the intrinsic apoptotic pathway during this process. Conclusion: Apoptosis seems to have an important role in the formation of the ductal network of HSG mainly through the intrinsic pathway. Associated with other proteins, Bcl-2 family members can contribute for the gland development as well as for the acquirement of new data. 377 Extra-cellular Matrix Stiffness and Immune Cells Infiltrate Are Associated With Breast Tumor Phenotype I. Acerbi1 , S.Y. Zheng1 , B. Ruffell2 , A. Au3 , Q. Shi4 , J.T. Liphardt4 , L.M. Coussens2 , Y.Y. Chen5 , E.S. Hwang6 , V.M. Weaver1 . 1 University of California San Francisco, Surgery, San Francisco CA, USA, 2 University of California San Francisco, Pathology, San Francisco CA, USA, 3 University of California San Francisco, Cancer Center, San Francisco CA, USA, 4 University of California Berkeley, Physics, Berkeley CA, USA, 5 University of California San Francisco, Pathology and Laboratory Medicine, San Francisco CA, USA, 6 Duke University, Surgery, Durham NC, USA Introduction: Local tumor progression is influenced by the dynamic interplay between the genetically-modified epithelium and the associated microenvironment. Data indicate that the extra-cellular matrix (ECM) stiffens progressively as mammary tumors evolve and enhancing ECM stiffness promotes mammary tumorigenesis while inhibiting ECM stiffening reduces tumor progression (Butcher et al., 2009; Levental et al., 2009). In addition, tumor progression is associated with altered inflammatory infiltrate (Ruffell et al., 2011), which can influence ECM remodeling. This poses the interesting possibility that inflammation could regulate tumor progression by inducing ECM stiffness. Accordingly, we hypothesize that immune infiltrate and ECM stiffness may be integrated into a tumor histology phenotype associated to breast cancer progression and subtypes. Material and Methods: We obtained breast tissue with benign and invasive ER positive and negative cancer from treated and non-treated patients. Tissues were subjected to immune profiling, IHC histological and mechanosignaling analysis (H&E; 397FAK; YAP-TAZ; LOX; FN) and biomechanical assessment (Atomic Force Microscopy; Structured Illumination Polarized Imaging; Two Photon Imaging). Results and Discussion: Preliminary data show a significant increase in ECM stiffness in tumor tissue. The highest stiffness is located at the tumor edge (2−4 fold greater). Intriguingly, we observed that ER negative tumors are stiffer than ER positive tumors. ECM stiffness also correlates with orientated parallel collagen fibers and ECM birefringence. Moreover, ECM stiffness is lower in treated tumors (40% lower) with the most striking reduction in ECM stiffness in ER negative samples. Our preliminary data suggest that ECM stiffening may be correlated with the percentage of macrophages in the immune infiltrate. Conclusions: (1) tumor progression is associated with marked fibrosis, accompanied by collagen deposition and linearization and an increase in LOX and FN expression; (2) tumor progression is associated with high tissue stiffness; and (3) ECM stiffening and macrophage infiltration level may be correlated with ER/PR status. This data should lead to a deeper understanding of breast cancer microenvironment and its role in tumor response to therapy. Supported by: NCI SPORE P50 CA058207 to VMW, CP, & SH; U01 ES019458−02 to ZW; NCI R01 CA138818−01A1 to VMW; & U54 CA143836−01 to VMW & JL. 378 Transformation and Aeging of Human IPSC Teratoma-derived Cells T. Matsuo1 , M. Kamada1 , T. Kumazaki1 , Y. Kawahara1 , T. Takahashi1 , Y. Mitsui1 . 1 Tokushima Bunri University, Pharmaceutical Science at Kagawa Campus, Sanuki, Japan Background: Generation of human cancer cell lines from normal cells is hardly successful without oncovirus infection. Tumorigenicity of induced pluripotent stem cell (iPSC) is considered as a risk factor of their transplant. We aimed to generate transformed human cells from human iPSC teratoma.
european journal of cancer 48, suppl. 5 (2012) S25–S288
resulting in a less-pronounced differential effect. Pathway inhibition analysis showed that, whereas MEK and PI3K inhibitors respectively decreased ERK and AKT phosphorylation in all cell lines, IGF1R inhibitors decreased AKT phosphorylation only in KRAS mutant cell lines. Inhibition of AKT phosphorylation correlates with effectiveness of IGF-1R inhibitor in blocking cell proliferation. Conclusions: Our results indicate that mutant KRAS activation of PI3K requires coordinate input from IGF-1R. These findings suggest potential therapeutic strategies for NSCLC tumours harbouring KRAS mutations. 375 Expression Profile of SETD Methyltransferase Family Genes in Breast Cancer Cell Lines M.S. Estrela1 , C.A. Moura1 , A.B. Motoyama1 , F. Pittella Silva1 . 1 Faculdade ˆ ´ de Ciencias da Saude, ´ Laboratorio de Farmacologia Molecular, Brasilia, Brazil Background: Lysine-methyltransferases are key enzymes that posttranslationally modify lysine residues in histones and play a key role in gene expression, epigenetic regulation, and as determinants of survival in malignant cells. The involvement of epigenetic modifying enzymes in carcinogenesis has gained a great deal of attention in the past few years. However, little is known about the role of SETD methyltransferase family genes in this process. In this study we investigate the expression of all SETD family genes in a panel of 7 breast cancer cell lines and a non-tumor counterpart. Material and Methods: We selected seven breast cancer cell lines (MCF7, CAMA1, SKBR3, MDA-MB-231, MDA-MB-436, MDA-MB-468 and HCC1954) and a matching cell line from normal B lymphoblastoid (HCC1954-BL) for the expression analysis. Total RNA was isolated and cDNAs were synthesized. Quantification of 9 SETD family genes (SETD1A, SETD1B, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7 and SETD8) was performed by semiquantitative PCR and Real Time PCR (qPCR). All PCRs were normalized with b-actin. Results were analyzed by the comparative 2−DDCt method and each cell line was compared with the control group of normal cells HCC1954-BL. Results: In accordance to previous reports, we found SETD7 to be overexpressed in all 7 cell lines. SETD6 is highly expressed in all cell lines, except in MCF7 and MDA-MB-436, compared to the normal cells. SETD8 was also found to be highly expressed in HCC1954, MCF7, MDAMB-231 and MDA-MB-436. All together, this data points for a possible role of SETD6 and SETD8 in breast carcinogenesis. However, further studies are necessary. Interestingly, SETD4 was specifically highly expressed in the ER (−) breast cancer cell lines HCC1954, MDA-MB-231 and MDA-MB-436, but not significantly found in ER (+) cells such as MCF7. This suggests SETD4 expression as a possible specific molecular marker. We found SETD1B to be 4 fold more expressed in 3 cell lines, including HCC1954, in comparison to the matching normal cell line. SETD1A was slightly more expressed in HCC1954 and MDA-MB-436 cells. The role of these two genes in carcinogenesis has also not been fully elucidated. The genes SETD2, SETD3 and SETD5 were not significantly expressed in the majority of breast cancer cell lines. Their expression was only found to be significant in HCC1954 in comparison to its matching normal cell line. SETD2 was recently reported as a tumor suppressor, being significantly less expressed in breast cancer and in later tumor stages. The result obtained in this study may reflect the characteristic of the primary stage IIA cell line HCC1954. Conclusions: The comparative analysis of SETD family gene expression in cancer cell lines provides new insights on the relation between SETD genes and tumorigenesis. These findings can serve as platform for further analysis in clinical samples from patients, aiming to point new potential molecular markers or targets for therapeutic intervention in breast cancer. 376 Apoptotic Intrinsic Pathway as an Essential Key for the Human Salivary Gland Branching Morphogenesis T.H.N. Teshima1 , R.C.F. Ianez2 , C.M. Coutinho-Camillo2 , S.V. Louren¸co1 . 1 ˜ Paulo − Dentistry School, Oral Pathology, Sao ˜ Paulo / SP, University of Sao ˜ Paulo / SP, Brazil Brazil, 2 AC Camargo Hospital, Pathologic Anatomy, Sao Introduction: Since the disclosure of apoptosis and its relation to tissue developing, homeostasis and pathogenesis, the mechanisms supposedly involved in this process have been investigated. Then, it has been well established that apoptosis occurs by two major pathways, intrinsic and extrinsic, sometimes showing a cross-reaction. Bcl-2 family proteins can control the intrinsic pathway through the balance between their pro and anti-apoptotic factors. Considering apoptosis as an essential key for the development of many tissues, this study purposes to illustrate the presence of Bax, Bak, Bcl-2, Bad, Bid, Bcl-XL and Bcl-X during the stages of human salivary glands (HSG) branching morphogenesis. Material and Methods: Samples from 20 fetuses of natural miscarriages were submitted to immunohistochemical reactions for Bax, Bak, Bad, Bid, Bcl-2, BclXL and Bcl-X. Normal mature salivary glands and some other tissues were employed as controls.
S91
Results and Discussion: Bax, Bak and Bcl-XL were significantly expressed during almost all stages of salivary gland development and Bcl-X, Bad and Bid showed only a focal positivity, being present in ductal and epithelial (buds) cells, both in nucleus and cytoplasm. Mature HSG were employed as controls and were negative as well as Bcl-2 and acinar cells from fetuses. Bax and Bak predominance at earlier phases suggests that they guide the formation of the luminal space, whilst the nuclear expression of Bcl-XL was more concentrated at latter stages. Regarding that the intrinsic pathway depends on the balance between Bcl-2 family pro and anti-apoptotic factors, those results were consistent, once Bcl-XL seems to control the apoptotic rate after lumen formation. Besides, evidences indicate a mutual regulation between Bcl-2 and Bcl-XL, being a possible reason for the absence of Bcl-2. Bad in turn acts binding to anti-apoptotic factors, Bcl-XL is an isoform of Bcl-X and Bid transmits death signals from the extrinsic pathway to the intrinsic. Thus, their almost negative expression indicates a secondary role during the human salivary gland morphogenesis, reinforcing the importance of the intrinsic apoptotic pathway during this process. Conclusion: Apoptosis seems to have an important role in the formation of the ductal network of HSG mainly through the intrinsic pathway. Associated with other proteins, Bcl-2 family members can contribute for the gland development as well as for the acquirement of new data. 377 Extra-cellular Matrix Stiffness and Immune Cells Infiltrate Are Associated With Breast Tumor Phenotype I. Acerbi1 , S.Y. Zheng1 , B. Ruffell2 , A. Au3 , Q. Shi4 , J.T. Liphardt4 , L.M. Coussens2 , Y.Y. Chen5 , E.S. Hwang6 , V.M. Weaver1 . 1 University of California San Francisco, Surgery, San Francisco CA, USA, 2 University of California San Francisco, Pathology, San Francisco CA, USA, 3 University of California San Francisco, Cancer Center, San Francisco CA, USA, 4 University of California Berkeley, Physics, Berkeley CA, USA, 5 University of California San Francisco, Pathology and Laboratory Medicine, San Francisco CA, USA, 6 Duke University, Surgery, Durham NC, USA Introduction: Local tumor progression is influenced by the dynamic interplay between the genetically-modified epithelium and the associated microenvironment. Data indicate that the extra-cellular matrix (ECM) stiffens progressively as mammary tumors evolve and enhancing ECM stiffness promotes mammary tumorigenesis while inhibiting ECM stiffening reduces tumor progression (Butcher et al., 2009; Levental et al., 2009). In addition, tumor progression is associated with altered inflammatory infiltrate (Ruffell et al., 2011), which can influence ECM remodeling. This poses the interesting possibility that inflammation could regulate tumor progression by inducing ECM stiffness. Accordingly, we hypothesize that immune infiltrate and ECM stiffness may be integrated into a tumor histology phenotype associated to breast cancer progression and subtypes. Material and Methods: We obtained breast tissue with benign and invasive ER positive and negative cancer from treated and non-treated patients. Tissues were subjected to immune profiling, IHC histological and mechanosignaling analysis (H&E; 397FAK; YAP-TAZ; LOX; FN) and biomechanical assessment (Atomic Force Microscopy; Structured Illumination Polarized Imaging; Two Photon Imaging). Results and Discussion: Preliminary data show a significant increase in ECM stiffness in tumor tissue. The highest stiffness is located at the tumor edge (2−4 fold greater). Intriguingly, we observed that ER negative tumors are stiffer than ER positive tumors. ECM stiffness also correlates with orientated parallel collagen fibers and ECM birefringence. Moreover, ECM stiffness is lower in treated tumors (40% lower) with the most striking reduction in ECM stiffness in ER negative samples. Our preliminary data suggest that ECM stiffening may be correlated with the percentage of macrophages in the immune infiltrate. Conclusions: (1) tumor progression is associated with marked fibrosis, accompanied by collagen deposition and linearization and an increase in LOX and FN expression; (2) tumor progression is associated with high tissue stiffness; and (3) ECM stiffening and macrophage infiltration level may be correlated with ER/PR status. This data should lead to a deeper understanding of breast cancer microenvironment and its role in tumor response to therapy. Supported by: NCI SPORE P50 CA058207 to VMW, CP, & SH; U01 ES019458−02 to ZW; NCI R01 CA138818−01A1 to VMW; & U54 CA143836−01 to VMW & JL. 378 Transformation and Aeging of Human IPSC Teratoma-derived Cells T. Matsuo1 , M. Kamada1 , T. Kumazaki1 , Y. Kawahara1 , T. Takahashi1 , Y. Mitsui1 . 1 Tokushima Bunri University, Pharmaceutical Science at Kagawa Campus, Sanuki, Japan Background: Generation of human cancer cell lines from normal cells is hardly successful without oncovirus infection. Tumorigenicity of induced pluripotent stem cell (iPSC) is considered as a risk factor of their transplant. We aimed to generate transformed human cells from human iPSC teratoma.