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242: Characterisation of retinoic acid effect on breast cancer cell plasticity
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
We found that ET-1was also involved in the VEGF-C-mediated proliferation of AML cells, and that recombinant ET-1 induced COX-2 mRNA and protein expressions in AML cells. Treatment with the endothelin receptor A (ETRA) antagonist, BQ 123, or ET-1 shRNAs inhibited VEGF-C-induced COX-2 expression. Flowcytometry and immunoblotting revealed that VEGF-C induces S phase accumulation through the inhibition of p27 and the upregulation of cyclin E and cyclin-dependent kinase-2 expressions. The cell-cycle-related effects of VEGF-C were reversed by the depletion of COX-2 or ET-1. The depletion of COX-2 or ET-1 also suppressed VEGF-C-induced increases in the bcl-2/bax ratio and chemoresistance against etoposide and cytosine arabinoside in AML cells. We also demonstrated VEGF-C/ET-1/COX-2 axismediated chemoresistance in an AML xenograft mouse model. Conclusions: Our findings suggest that VEGF-C induces COX-2-mediated resistance to chemotherapy through the induction of ET-1 expression. Acting as a key regulator in the VEGF-C/COX-2 axis, ET-1 represents a potential target for ameliorating resistance to chemotherapy in AML patients. No conflict of interest. 240 SOX14 downregulates SOX1 expression in HeLa cells I. Petrovic1 , J. Popovic1 , D. Stanisavljevic1 , M. Schwirtlich1 , A. Klajn1 , J. Marjanovic1 , N. Kovacevic Grujicic1 , V. Topalovic1 , M. Mojsin1 , M. Stevanovic1 . 1 Institute of Molecular Genetics and Genetic Engineering University of Belgrade, Laboratory for Human Molecular Genetics, Belgrade, Serbia Background: It has been reported that deregulation and/or amplification of many SOX genes are associated with a large number of tumor types. SOX14 is a largely unexplored member of the SOXB2 subgroup of transcription factors implicated mainly in neural development, while its closest relative, SOX21, apart from the role in neural development was also proposed as tumor suppressor. Having in mind redundant properties of SOX proteins, the similar role of SOX14 in cancer was not reported, especially in context of functional cross-talk between SOX14 and SOXB1 members. The aim of this study was to analyze its activator/repressor properties and to test whether SOX14 may affect SOXB1 members’ expression in HeLa cells. Material and Methods: In order to analyze functional role of SOX14 in cancer model system we generated SOX14 expression construct. The activation/repression property of human SOX14 protein was performed by co-transfection experiments with SOX14 expression construct and SOXresponsive luciferase reporter gene in HeLa cells. In order to analyze the potential cross-talk between SOXB1 and SOX14, HeLa cells were transiently transfected with SOX14 expression construct and the effect of its ectopic expression on SOXB members was analyzed by Western blot. Results: Our data demonstrated that SOX14 overexpression reduced SOX1 protein level, while no significant effects on SOX2, SOX3 and SOX21 expression was observed. It was shown that SOX1 is highly methylated in high grade squamous cell cervical carcinomas. On the other hand, a genome-wide RNA interference (RNAi) screen in K-ras transformed NIH 3T3 cells identified that SOX14 is one of 28 genes required for Ras-mediated epigenetic silencing of the pro-apoptotic Fas gene. Based on those findings, SOX14 could be involved in the positive or negative regulation of expression of genes implicated in epigenetic silencing, and its elevated expression could increase promoter hypermethylation of target genes, such as SOX1. Conclusion: This is the first report showing that SOX14 could affect the expression of SOX1. Since SOX1 is considered as tumor suppressor, the molecular mechanisms involved in the regulation of its expression, that relies on SOX14 gain additional significance. It would be interesting to explore how elevated expression of SOX14 influences HeLa cells’ proliferation and invasiveness, and what impact decreased expression of SOX1 has on the aforementioned processes. No conflict of interest. 241 Bcl-xL protein overexpression enhances tumor progression of human melanoma cells in zebrafish xenograft model: involvement of interleukin 8 1 C. Gabellini1 , E. Gomez-Abenza ´ , S. De Oliveira2 , D. Del Bufalo3 , V. Mulero1 . 1 University of Murcia, Department of Cell Biology and Histology Faculty of Biology, Murcia, Spain, 2 University of Lisbon, Microvascular Biology and Inflammation Unit Molecular Medicine Institute Biochemistry Institute Faculty of Medicine, Lisbon, Portugal, 3 Regina Elena National Cancer Institute, Experimental Chemotherapy Laboratory, Rome, Italy Background: The anti-apoptotic protein bcl-xL enhances metastatic potential in different tumor hystotypes and promotes tumor angiogenesis through enhancing pro-inflammatory chemokine interleukin 8 (CXCL8) expression. Using zebrafish as experimental model, we evaluated the impact of bclxL/CXCL8 axis in promoting melanoma angiogenesis and aggressiveness in vivo. Materials and Methods: To evaluate invasive and metastatic capability of human melanoma M14 cell line stably overexpressing bcl-xL protein, cells
were implanted into the yolk sac of 2 days post-fertilization (dpf) zebrafish embryos. To analyze the pro-angiogenic activity of recombinant human and zebrafish CXCL8 proteins, they were injected in the yolk sac of 2dpf transgenic fli1:EGFP embryos and tested for the capability to induce subintestinal vein (SIV) sprouting. To test the pro-inflammatory activity of human and zebrafish CXCL8 recombinant proteins, they were injected in the yolk sac of 2dpf transgenic lyz:DsRed embryos and tested for neutrophil recruitment ability. Results: Implantation of M14 trasfectants overexpressing bcl-xL protein into zebrafish embryos resulted in higher significant dissemination from primary site of injection and metastatization to distal parts of the larvae when compared to control cell line. Since the enhanced invasiveness showed by bcl-xL overexpressing cells might be due to their enhanced CXCL8 protein secretion, we tested the capability of human recombinant CXCL8 protein to induce angiogenesis in zebrafish, demonstrating that increasing doses of human CXCL8 protein induced a significant increased percentage of larvae positive for SIV sprouting. Next we demonstrated that also zebrafish Cxcl8-L2 elicits proangiogenic activity demonstrating that it is able to induce SIV sprouting, but at a lesser extent than human CXCL8. However, when comparing the capability of human and zebrafish CXCL8 to induce neutrophil recruitment, we found that human CXCL8 is not able to induce neutrophil recruitment to zebrafish yolk sac, in contrast to its zebrafish homologue. Next, we will investigate the involvement of CXCL8 receptors Cxcr1 and Cxcr2, highly conserved between humans and zebrafish, in the capability of bcl-xL protein to enhance melanoma cell invasion in zebrafish larvae and with the aim of elucidating the signaling pathways involved in the pro-angiogenic activity of human CXCL8 chemokine in zebrafish. Conclusions: These data elucidate the involvement of CXCL8 signalling in bcl-xL-induced melanoma progression, supporting future studies for developing new therapeutic approaches for tumors characterized by high bclxL expression. Moreover the conservation of pro-angiogenic activity of CXCL8 molecule in zebrafish establishes the possible use of this experimental model to study the efficacy of novel compounds inhibiting CXCL8 axis to counteract tumor progression. No conflict of interest. 242 Characterisation of retinoic acid effect on breast cancer cell plasticity G. Paroni1 , A. Zanetti1 , R. Affatato2 , E. Garattini1 . 1 Istituto di Ricerche Farmacologiche Mario Negri, Biochemistry and Molecular Biology, Milano, Italy, 2 Istituto di Ricerche Farmacologiche Mario Negri, Cardiovascular Research, Milano, Italy Introduction: In breast cancer, retinoids suppress tumor cell growth and prevent mammary cancer in rodent models. Their efficacy has been explained by growth inhibition and induction of apoptosis. Retinoids are required to maintain the differentiated state of adult epithelia. In cultures of breast cancer cell lines, we and others have shown that retinoids act as inducers of an epithelial-like phenotype whose implication in the therapeutic setting has been poorly investigated. Increasing evidence supports an aberrant regulation of the epithelial to mesenchymal transition (EMT) developmental process in breast tumour progression. Indeed, EMT is associated with augmented motility and invasion. Understanding the nature of retinoid induced modulation of breast cancer plasticity represents a crucial issue for the use of retinoids as anti-tumor agents. Material and Methods: The effect of ATRA (all-trans retinoic acid) on a panel of breast cancer cell lines was evaluated in 2D and 3D cultures. Western Blot and Immunofluorescence analysis of EMT associated genes were performed. As EMT cell model, the ATRA sensitive SKBR3 cell line was used. In this cell line EGF and Heregulin induce a mesenchymal phenotype increasing the migratory ability of the cells. The effect of ATRA in this experimental setting was investigated. Migration was evaluated by Boyden Chamber assay. qRTPCR of EMT associated genes were performed to identify genes modulated by ATRA treatment. ATRA mediated regulation of the identified genes was further validated by Immunofluorescence and Western Blot analysis. Results: ATRA causes a switch to an epithelial-like phenotype by inducing a rearrangement in both tight and adherens junction complexes and by modulating the expression of their constituent proteins. In cell lines amplified for the retinoic acid receptor a, ATRA induces the formation of polarized/organized structures reminiscent of mammary gland epithelium. Upon EMT induction by growth factors the mesenchimal phenotype and the increased migratory ability of the cells are counteracted by retinoids. A screening of EMT determinants demonstrated that NOTCH1, Snail and miR200c are regulated by ATRA. In particular, ATRA decreases EGF and Heregulin induced NOTCH1 expression, inhibits NOTCH1 transcriptional activity and phenocopies the migration impairment triggered by the g-secretase (NOTCH) inhibitor DAPT. Conclusion: Overall our data support a role for ATRA in breast cancer plasticity whose further investigation is essential for a rational use of retinoids in the clinics. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
We found that ET-1was also involved in the VEGF-C-mediated proliferation of AML cells, and that recombinant ET-1 induced COX-2 mRNA and protein expressions in AML cells. Treatment with the endothelin receptor A (ETRA) antagonist, BQ 123, or ET-1 shRNAs inhibited VEGF-C-induced COX-2 expression. Flowcytometry and immunoblotting revealed that VEGF-C induces S phase accumulation through the inhibition of p27 and the upregulation of cyclin E and cyclin-dependent kinase-2 expressions. The cell-cycle-related effects of VEGF-C were reversed by the depletion of COX-2 or ET-1. The depletion of COX-2 or ET-1 also suppressed VEGF-C-induced increases in the bcl-2/bax ratio and chemoresistance against etoposide and cytosine arabinoside in AML cells. We also demonstrated VEGF-C/ET-1/COX-2 axismediated chemoresistance in an AML xenograft mouse model. Conclusions: Our findings suggest that VEGF-C induces COX-2-mediated resistance to chemotherapy through the induction of ET-1 expression. Acting as a key regulator in the VEGF-C/COX-2 axis, ET-1 represents a potential target for ameliorating resistance to chemotherapy in AML patients. No conflict of interest. 240 SOX14 downregulates SOX1 expression in HeLa cells I. Petrovic1 , J. Popovic1 , D. Stanisavljevic1 , M. Schwirtlich1 , A. Klajn1 , J. Marjanovic1 , N. Kovacevic Grujicic1 , V. Topalovic1 , M. Mojsin1 , M. Stevanovic1 . 1 Institute of Molecular Genetics and Genetic Engineering University of Belgrade, Laboratory for Human Molecular Genetics, Belgrade, Serbia Background: It has been reported that deregulation and/or amplification of many SOX genes are associated with a large number of tumor types. SOX14 is a largely unexplored member of the SOXB2 subgroup of transcription factors implicated mainly in neural development, while its closest relative, SOX21, apart from the role in neural development was also proposed as tumor suppressor. Having in mind redundant properties of SOX proteins, the similar role of SOX14 in cancer was not reported, especially in context of functional cross-talk between SOX14 and SOXB1 members. The aim of this study was to analyze its activator/repressor properties and to test whether SOX14 may affect SOXB1 members’ expression in HeLa cells. Material and Methods: In order to analyze functional role of SOX14 in cancer model system we generated SOX14 expression construct. The activation/repression property of human SOX14 protein was performed by co-transfection experiments with SOX14 expression construct and SOXresponsive luciferase reporter gene in HeLa cells. In order to analyze the potential cross-talk between SOXB1 and SOX14, HeLa cells were transiently transfected with SOX14 expression construct and the effect of its ectopic expression on SOXB members was analyzed by Western blot. Results: Our data demonstrated that SOX14 overexpression reduced SOX1 protein level, while no significant effects on SOX2, SOX3 and SOX21 expression was observed. It was shown that SOX1 is highly methylated in high grade squamous cell cervical carcinomas. On the other hand, a genome-wide RNA interference (RNAi) screen in K-ras transformed NIH 3T3 cells identified that SOX14 is one of 28 genes required for Ras-mediated epigenetic silencing of the pro-apoptotic Fas gene. Based on those findings, SOX14 could be involved in the positive or negative regulation of expression of genes implicated in epigenetic silencing, and its elevated expression could increase promoter hypermethylation of target genes, such as SOX1. Conclusion: This is the first report showing that SOX14 could affect the expression of SOX1. Since SOX1 is considered as tumor suppressor, the molecular mechanisms involved in the regulation of its expression, that relies on SOX14 gain additional significance. It would be interesting to explore how elevated expression of SOX14 influences HeLa cells’ proliferation and invasiveness, and what impact decreased expression of SOX1 has on the aforementioned processes. No conflict of interest. 241 Bcl-xL protein overexpression enhances tumor progression of human melanoma cells in zebrafish xenograft model: involvement of interleukin 8 1 C. Gabellini1 , E. Gomez-Abenza ´ , S. De Oliveira2 , D. Del Bufalo3 , V. Mulero1 . 1 University of Murcia, Department of Cell Biology and Histology Faculty of Biology, Murcia, Spain, 2 University of Lisbon, Microvascular Biology and Inflammation Unit Molecular Medicine Institute Biochemistry Institute Faculty of Medicine, Lisbon, Portugal, 3 Regina Elena National Cancer Institute, Experimental Chemotherapy Laboratory, Rome, Italy Background: The anti-apoptotic protein bcl-xL enhances metastatic potential in different tumor hystotypes and promotes tumor angiogenesis through enhancing pro-inflammatory chemokine interleukin 8 (CXCL8) expression. Using zebrafish as experimental model, we evaluated the impact of bclxL/CXCL8 axis in promoting melanoma angiogenesis and aggressiveness in vivo. Materials and Methods: To evaluate invasive and metastatic capability of human melanoma M14 cell line stably overexpressing bcl-xL protein, cells
were implanted into the yolk sac of 2 days post-fertilization (dpf) zebrafish embryos. To analyze the pro-angiogenic activity of recombinant human and zebrafish CXCL8 proteins, they were injected in the yolk sac of 2dpf transgenic fli1:EGFP embryos and tested for the capability to induce subintestinal vein (SIV) sprouting. To test the pro-inflammatory activity of human and zebrafish CXCL8 recombinant proteins, they were injected in the yolk sac of 2dpf transgenic lyz:DsRed embryos and tested for neutrophil recruitment ability. Results: Implantation of M14 trasfectants overexpressing bcl-xL protein into zebrafish embryos resulted in higher significant dissemination from primary site of injection and metastatization to distal parts of the larvae when compared to control cell line. Since the enhanced invasiveness showed by bcl-xL overexpressing cells might be due to their enhanced CXCL8 protein secretion, we tested the capability of human recombinant CXCL8 protein to induce angiogenesis in zebrafish, demonstrating that increasing doses of human CXCL8 protein induced a significant increased percentage of larvae positive for SIV sprouting. Next we demonstrated that also zebrafish Cxcl8-L2 elicits proangiogenic activity demonstrating that it is able to induce SIV sprouting, but at a lesser extent than human CXCL8. However, when comparing the capability of human and zebrafish CXCL8 to induce neutrophil recruitment, we found that human CXCL8 is not able to induce neutrophil recruitment to zebrafish yolk sac, in contrast to its zebrafish homologue. Next, we will investigate the involvement of CXCL8 receptors Cxcr1 and Cxcr2, highly conserved between humans and zebrafish, in the capability of bcl-xL protein to enhance melanoma cell invasion in zebrafish larvae and with the aim of elucidating the signaling pathways involved in the pro-angiogenic activity of human CXCL8 chemokine in zebrafish. Conclusions: These data elucidate the involvement of CXCL8 signalling in bcl-xL-induced melanoma progression, supporting future studies for developing new therapeutic approaches for tumors characterized by high bclxL expression. Moreover the conservation of pro-angiogenic activity of CXCL8 molecule in zebrafish establishes the possible use of this experimental model to study the efficacy of novel compounds inhibiting CXCL8 axis to counteract tumor progression. No conflict of interest. 242 Characterisation of retinoic acid effect on breast cancer cell plasticity G. Paroni1 , A. Zanetti1 , R. Affatato2 , E. Garattini1 . 1 Istituto di Ricerche Farmacologiche Mario Negri, Biochemistry and Molecular Biology, Milano, Italy, 2 Istituto di Ricerche Farmacologiche Mario Negri, Cardiovascular Research, Milano, Italy Introduction: In breast cancer, retinoids suppress tumor cell growth and prevent mammary cancer in rodent models. Their efficacy has been explained by growth inhibition and induction of apoptosis. Retinoids are required to maintain the differentiated state of adult epithelia. In cultures of breast cancer cell lines, we and others have shown that retinoids act as inducers of an epithelial-like phenotype whose implication in the therapeutic setting has been poorly investigated. Increasing evidence supports an aberrant regulation of the epithelial to mesenchymal transition (EMT) developmental process in breast tumour progression. Indeed, EMT is associated with augmented motility and invasion. Understanding the nature of retinoid induced modulation of breast cancer plasticity represents a crucial issue for the use of retinoids as anti-tumor agents. Material and Methods: The effect of ATRA (all-trans retinoic acid) on a panel of breast cancer cell lines was evaluated in 2D and 3D cultures. Western Blot and Immunofluorescence analysis of EMT associated genes were performed. As EMT cell model, the ATRA sensitive SKBR3 cell line was used. In this cell line EGF and Heregulin induce a mesenchymal phenotype increasing the migratory ability of the cells. The effect of ATRA in this experimental setting was investigated. Migration was evaluated by Boyden Chamber assay. qRTPCR of EMT associated genes were performed to identify genes modulated by ATRA treatment. ATRA mediated regulation of the identified genes was further validated by Immunofluorescence and Western Blot analysis. Results: ATRA causes a switch to an epithelial-like phenotype by inducing a rearrangement in both tight and adherens junction complexes and by modulating the expression of their constituent proteins. In cell lines amplified for the retinoic acid receptor a, ATRA induces the formation of polarized/organized structures reminiscent of mammary gland epithelium. Upon EMT induction by growth factors the mesenchimal phenotype and the increased migratory ability of the cells are counteracted by retinoids. A screening of EMT determinants demonstrated that NOTCH1, Snail and miR200c are regulated by ATRA. In particular, ATRA decreases EGF and Heregulin induced NOTCH1 expression, inhibits NOTCH1 transcriptional activity and phenocopies the migration impairment triggered by the g-secretase (NOTCH) inhibitor DAPT. Conclusion: Overall our data support a role for ATRA in breast cancer plasticity whose further investigation is essential for a rational use of retinoids in the clinics. No conflict of interest.