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253 Role of NANOS Family Members in Tumor Progression
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european journal of cancer 48, suppl. 5 (2012) S25–S288
252 Homeobox Proteins Recruit Polycomb Repressive Complexes to Repress INK4a N. Popov1 , N. Martin1 , F. Aguilo2 , S. Raguz1 , A.P. Snijders1 , G. Dharmalingam1 , S. Li2 , T. Carroll1 , M.J. Walsh2 , J. Gil1 . 1 MRC Clinical Sciences Centre, Imperial College, London, United Kingdom, 2 Mount Sinai School of Medicine, New York, USA Background: Cellular senescence represents a crucial barrier against malignant transformation. p16 is one of the key tumor suppressors controlling cell proliferation and senescence. p16 is encoded by INK4a, which is frequently altered in human cancers. Polycomb repressive complexes (PRC) play an important role in INK4a epigenetic silencing but how they are recruited to the INK4a promoter is not well understood. We identified HLX1 (H2.0-like homeobox 1) in a screen for senescence regulators. Material and Methods: We combined cell proliferation assays with transcriptional and ChIP analysis in primary human fibroblasts to investigate the function of HLX1 in cellular senescence. Results: We observed that HLX1 extends replicative lifespan and impedes oncogenic RAS-induced senescence. HLX1 inhibits INK4a expression by recruiting Polycomb repressive complexes to the INK4a promoter and also regulates other PRC target genes. PRC-dependent repression of INK4a expression is a conserved property among Homeobox proteins as exemplified by HOXA9 (Homeobox A9). Conclusion: Altogether these data provide evidence for a collaboration between Homeobox proteins and Polycomb repressive complexes in transcriptional regulation. This mechanism could have general relevance in development, senescence and cancer. N. Popov and N. Martin contributed equally to this work. 253 Role of NANOS Family Members in Tumor Progression V. Andries1 , K. Staes1 , F. Van Roy1 . 1 VIB/UGent, Department for Molecular Biomedical Research, Zwijnaarde, Belgium Introduction: In carcinomas a recurrent molecular event of high prognostic value is inactivation of the cell-cell adhesion molecule E-cadherin. Differential gene expression analysis showed that the novel NANOS1 gene was downregulated in E-cadherin-transfected revertants of a breast cancer cell line (Strumane et al., 2006). In flies, Nanos is a maternal RNA-binding protein that acts together with Pumilio as a translational repressor. Genomic and transcriptomic analyses revealed the existence of three human Nanos homologs, hNanos-1 to -3. In human cancer, several lines of evidence indicate that hNanos members have the potential to confer malignancy. In vitro induction of hNanos-1 results in abrogation of cell aggregation, increased directional migration, and induction of invasion into type-I collagen gels (Strumane et al., 2006; Bonnomet et al., 2008). Material and Method: To study the malignancy of the Nanos homologs in vivo, we generated transgenic mouse lines conditionally expressing human NANOS-x (x = 1 to 3) genes by applying an improved transgenesis system (in collaboration with Dr. J. Haigh; Nyabi et al., 2009). To investigate the role of Nanos proteins in normal and cancerous cells, we identified new hNanos1 interacting proteins by a pull-down experiment, followed by mass spectrometric identification (Kris Gevaert, Ghent). Results and Discussion: We crossed our transgenic hNANOS-x mice with appropriate Cre lines in order to activate the transgenes in a tissue-specific manner. These mice are presently used in mouse tumor models, including a chemical hepatocarcinogenesis model and a transgenic prostate cancer model to investigate whether more malignant tumors are formed upon induction of hNanos-x expression in, for instance, the liver or the prostate. In addition, pull-down experiments of the overexpressed Myc-tagged hNanos1 identified several new interacting proteins for hNanos1. Interestingly, we identified among them the heat-shock protein 90 (Hsp90) as a putative interaction partner of hNanos1. Hsp90, also called the cancer chaperone, is a molecular chaperone required for the stability and function of numerous oncogenic signaling proteins. It has been shown that Hsp90 plays a role in metastasisrelated events and small-molecule Hsp90 inhibitors are currently being tested in clinical trials (reviewed by Tsutsumi, 2009). In further experiments, we want to scrutinize the functional implications of this Hsp90 binding as well as other interactions with hNanos proteins. Conclusion: The ultimate goal of this project is to verify conclusively whether hNanos proteins are novel potent promoters of tumor formation or progression. The use of transgenic mouse models and the identification and characterization of interacting proteins of hNanos-x will provide us more insight in this.
Sunday 8 − Tuesday 10 July 2012
254 HSP27 is Involved in Ovarian Cancer Cell Motility and Response to Paclitaxel S. Pavan1 , D. Musiani1 , E. Torchiaro1 , G. Migliardi1 , M. Olivero1 , M.F. Di Renzo1 . 1 Institute for Cancer Research and Treatment (IRCC), Department of Oncological Sciences, Candiolo, Italy Introduction: The Hepatocyte growth factor (HGF) triggers cell scattering, motility and invasiveness by binding its receptor encoded by the MET oncogene. In ovarian cancer cells lines, activation of the c-Met receptor by HGF results in activation of the p38 MAPK pathway and phosphorylation of the small Heat shock protein of 27 KDa (HSP27). HSP27 takes part in different biological functions such as cytoskeleton rearrangements and cell survival. HSP27 binds both actin and tubulin filaments and regulates their polymerization. HSP27 is often found over-expressed in epithelial tumors and might cause cancer cell resistance to chemotherapeutics. As ovarian cancer cells have elevated HSP27 levels, we investigated its role in mediating HGFinduced cell motility and invasion and its involvement in the cellular response to paclitaxel (a microtubule-stabilizing agent), a drug used in the treatment of ovarian carcinoma. Material and Method: HSP27 was stably silenced with short hairpin-RNAs in the ovarian carcinoma cells SKOV-3. Scatter, motility and invasion assays were performed in vitro to assess its involvement in HGF-induced motility. Immunofluorescence analysis by confocal microscopy was used to study HSP27 silencing effects on HGF-triggered actin polymerization and on tubulin filaments upon paclitaxel treatment. HSP27 involvement in SKOV-3 response to paclitaxel was evaluated both in vitro (apoptosis assay) and in vivo (regression trial). Results and Discussion: By the in vitro assays, it was evident that HSP27 silencing impaired SKOV-3 capability to scatter, move and invade in response to HGF. HSP27 knock-down prevented HGF-induced actin polymerization as shown by confocal microscopy images analysis. Besides, the apoptosis assay stressed that HSP27 silencing was able to sensitize SKOV-3 to paclitaxel by promoting cell death. This result was also confirmed by the in vivo experiments where HSP27-silenced tumors regressed with paclitaxel treatment at low doses while control cells were poorly affected. As observed by immunofluorescence, the effect of HSP27 knock-down in the response to paclitaxel was likely due to a massive reorganization of tubulin filaments, which were seen prevalently localized at the cell periphery. Conclusion: The results showed that HSP27 was involved in the regulation of HGF-induced actin polymerization required for SKOV-3 motility and invasiveness. Furthermore, HSP27 over-expression was responsible for SKOV-3 resistance to treatment with paclitaxel as its silencing promoted cell sensitization to the chemotherapeutic. This effect was likely related to the more pronounced tubulin filament reorganization, highlighting a possible cooperation between HSP27 knock-down and paclitaxel in the dysregulation of cytoskeleton assembly. Eventually, HSP27 can be considered as a valuable molecular target for ovarian cancer treatment. 255 The RNA-binding Protein RNPC1 Regulates Mdm2 Expression Via mRNA Stability X. Chen1 , E. Xu1 , J. Zhang1 . 1 UC Davis, Surgical and Radiological Sciences, Davis CA, USA Background: Mdm2, a target of p53 and an E3 ligase, is a primary negative regulator of p53. RNPC1, a RNA-binding protein, is a target of the p53 family, which is known to regulate p53 expression via translation and p63 expression via mRNA stability. Materials and Methods: Both molecular and Cellular techniques, including transcriptional studies, gene expression, and protein stability and degradation, were used to define how Mdm2 is regulated by RNPC1 in vitro and in vivo. Results: We found that over-expression of RNPC1 decreases, whereas knockdown of RNPC1 increases, Mdm2 expression independent of p53. We also showed that RNPC1 directly binds to a AU-rich element in Mdm2 3 UTR and promotes mRNA degradation. Conclusion: We uncover a novel mechanism by which Mdm2 is regulated by RNPC1 via mRNA stability. We postulate that by modulating Mdm2 expression, RNPC1 might be explored as a marker and/or a target for cancer therapy. 256 Evaluation of Rare Event Detection by Flow Cytometry for Cancer Prognosis A.E.L. Colo´ 1 , A. Del Giglio2 , L.C. Marti1 . 1 Hospital Israelita Albert Einstein, ˜ Paulo, Brazil, Instituto Israelita de Ensino e Pesquisa Albert Einstein, Sao 2 Faculdade de Medicina do ABC, Disciplina de Hematologia e Oncologia, ´ Brazil Santo Andre, Background: Metastatic disease is the major cause of cancer mortality. The formation of metastasis requires the migration of tumor cells from the primary sites to other organs. Therefore, the detection of circulating tumor cells (CTC) is target of several investigations once it could be useful to assess metastasis risk. In addition to CTC, studies on metastasis mechanisms in animal models suggest migration of hematopoietic bone marrow progenitor
european journal of cancer 48, suppl. 5 (2012) S25–S288
252 Homeobox Proteins Recruit Polycomb Repressive Complexes to Repress INK4a N. Popov1 , N. Martin1 , F. Aguilo2 , S. Raguz1 , A.P. Snijders1 , G. Dharmalingam1 , S. Li2 , T. Carroll1 , M.J. Walsh2 , J. Gil1 . 1 MRC Clinical Sciences Centre, Imperial College, London, United Kingdom, 2 Mount Sinai School of Medicine, New York, USA Background: Cellular senescence represents a crucial barrier against malignant transformation. p16 is one of the key tumor suppressors controlling cell proliferation and senescence. p16 is encoded by INK4a, which is frequently altered in human cancers. Polycomb repressive complexes (PRC) play an important role in INK4a epigenetic silencing but how they are recruited to the INK4a promoter is not well understood. We identified HLX1 (H2.0-like homeobox 1) in a screen for senescence regulators. Material and Methods: We combined cell proliferation assays with transcriptional and ChIP analysis in primary human fibroblasts to investigate the function of HLX1 in cellular senescence. Results: We observed that HLX1 extends replicative lifespan and impedes oncogenic RAS-induced senescence. HLX1 inhibits INK4a expression by recruiting Polycomb repressive complexes to the INK4a promoter and also regulates other PRC target genes. PRC-dependent repression of INK4a expression is a conserved property among Homeobox proteins as exemplified by HOXA9 (Homeobox A9). Conclusion: Altogether these data provide evidence for a collaboration between Homeobox proteins and Polycomb repressive complexes in transcriptional regulation. This mechanism could have general relevance in development, senescence and cancer. N. Popov and N. Martin contributed equally to this work. 253 Role of NANOS Family Members in Tumor Progression V. Andries1 , K. Staes1 , F. Van Roy1 . 1 VIB/UGent, Department for Molecular Biomedical Research, Zwijnaarde, Belgium Introduction: In carcinomas a recurrent molecular event of high prognostic value is inactivation of the cell-cell adhesion molecule E-cadherin. Differential gene expression analysis showed that the novel NANOS1 gene was downregulated in E-cadherin-transfected revertants of a breast cancer cell line (Strumane et al., 2006). In flies, Nanos is a maternal RNA-binding protein that acts together with Pumilio as a translational repressor. Genomic and transcriptomic analyses revealed the existence of three human Nanos homologs, hNanos-1 to -3. In human cancer, several lines of evidence indicate that hNanos members have the potential to confer malignancy. In vitro induction of hNanos-1 results in abrogation of cell aggregation, increased directional migration, and induction of invasion into type-I collagen gels (Strumane et al., 2006; Bonnomet et al., 2008). Material and Method: To study the malignancy of the Nanos homologs in vivo, we generated transgenic mouse lines conditionally expressing human NANOS-x (x = 1 to 3) genes by applying an improved transgenesis system (in collaboration with Dr. J. Haigh; Nyabi et al., 2009). To investigate the role of Nanos proteins in normal and cancerous cells, we identified new hNanos1 interacting proteins by a pull-down experiment, followed by mass spectrometric identification (Kris Gevaert, Ghent). Results and Discussion: We crossed our transgenic hNANOS-x mice with appropriate Cre lines in order to activate the transgenes in a tissue-specific manner. These mice are presently used in mouse tumor models, including a chemical hepatocarcinogenesis model and a transgenic prostate cancer model to investigate whether more malignant tumors are formed upon induction of hNanos-x expression in, for instance, the liver or the prostate. In addition, pull-down experiments of the overexpressed Myc-tagged hNanos1 identified several new interacting proteins for hNanos1. Interestingly, we identified among them the heat-shock protein 90 (Hsp90) as a putative interaction partner of hNanos1. Hsp90, also called the cancer chaperone, is a molecular chaperone required for the stability and function of numerous oncogenic signaling proteins. It has been shown that Hsp90 plays a role in metastasisrelated events and small-molecule Hsp90 inhibitors are currently being tested in clinical trials (reviewed by Tsutsumi, 2009). In further experiments, we want to scrutinize the functional implications of this Hsp90 binding as well as other interactions with hNanos proteins. Conclusion: The ultimate goal of this project is to verify conclusively whether hNanos proteins are novel potent promoters of tumor formation or progression. The use of transgenic mouse models and the identification and characterization of interacting proteins of hNanos-x will provide us more insight in this.
Sunday 8 − Tuesday 10 July 2012
254 HSP27 is Involved in Ovarian Cancer Cell Motility and Response to Paclitaxel S. Pavan1 , D. Musiani1 , E. Torchiaro1 , G. Migliardi1 , M. Olivero1 , M.F. Di Renzo1 . 1 Institute for Cancer Research and Treatment (IRCC), Department of Oncological Sciences, Candiolo, Italy Introduction: The Hepatocyte growth factor (HGF) triggers cell scattering, motility and invasiveness by binding its receptor encoded by the MET oncogene. In ovarian cancer cells lines, activation of the c-Met receptor by HGF results in activation of the p38 MAPK pathway and phosphorylation of the small Heat shock protein of 27 KDa (HSP27). HSP27 takes part in different biological functions such as cytoskeleton rearrangements and cell survival. HSP27 binds both actin and tubulin filaments and regulates their polymerization. HSP27 is often found over-expressed in epithelial tumors and might cause cancer cell resistance to chemotherapeutics. As ovarian cancer cells have elevated HSP27 levels, we investigated its role in mediating HGFinduced cell motility and invasion and its involvement in the cellular response to paclitaxel (a microtubule-stabilizing agent), a drug used in the treatment of ovarian carcinoma. Material and Method: HSP27 was stably silenced with short hairpin-RNAs in the ovarian carcinoma cells SKOV-3. Scatter, motility and invasion assays were performed in vitro to assess its involvement in HGF-induced motility. Immunofluorescence analysis by confocal microscopy was used to study HSP27 silencing effects on HGF-triggered actin polymerization and on tubulin filaments upon paclitaxel treatment. HSP27 involvement in SKOV-3 response to paclitaxel was evaluated both in vitro (apoptosis assay) and in vivo (regression trial). Results and Discussion: By the in vitro assays, it was evident that HSP27 silencing impaired SKOV-3 capability to scatter, move and invade in response to HGF. HSP27 knock-down prevented HGF-induced actin polymerization as shown by confocal microscopy images analysis. Besides, the apoptosis assay stressed that HSP27 silencing was able to sensitize SKOV-3 to paclitaxel by promoting cell death. This result was also confirmed by the in vivo experiments where HSP27-silenced tumors regressed with paclitaxel treatment at low doses while control cells were poorly affected. As observed by immunofluorescence, the effect of HSP27 knock-down in the response to paclitaxel was likely due to a massive reorganization of tubulin filaments, which were seen prevalently localized at the cell periphery. Conclusion: The results showed that HSP27 was involved in the regulation of HGF-induced actin polymerization required for SKOV-3 motility and invasiveness. Furthermore, HSP27 over-expression was responsible for SKOV-3 resistance to treatment with paclitaxel as its silencing promoted cell sensitization to the chemotherapeutic. This effect was likely related to the more pronounced tubulin filament reorganization, highlighting a possible cooperation between HSP27 knock-down and paclitaxel in the dysregulation of cytoskeleton assembly. Eventually, HSP27 can be considered as a valuable molecular target for ovarian cancer treatment. 255 The RNA-binding Protein RNPC1 Regulates Mdm2 Expression Via mRNA Stability X. Chen1 , E. Xu1 , J. Zhang1 . 1 UC Davis, Surgical and Radiological Sciences, Davis CA, USA Background: Mdm2, a target of p53 and an E3 ligase, is a primary negative regulator of p53. RNPC1, a RNA-binding protein, is a target of the p53 family, which is known to regulate p53 expression via translation and p63 expression via mRNA stability. Materials and Methods: Both molecular and Cellular techniques, including transcriptional studies, gene expression, and protein stability and degradation, were used to define how Mdm2 is regulated by RNPC1 in vitro and in vivo. Results: We found that over-expression of RNPC1 decreases, whereas knockdown of RNPC1 increases, Mdm2 expression independent of p53. We also showed that RNPC1 directly binds to a AU-rich element in Mdm2 3 UTR and promotes mRNA degradation. Conclusion: We uncover a novel mechanism by which Mdm2 is regulated by RNPC1 via mRNA stability. We postulate that by modulating Mdm2 expression, RNPC1 might be explored as a marker and/or a target for cancer therapy. 256 Evaluation of Rare Event Detection by Flow Cytometry for Cancer Prognosis A.E.L. Colo´ 1 , A. Del Giglio2 , L.C. Marti1 . 1 Hospital Israelita Albert Einstein, ˜ Paulo, Brazil, Instituto Israelita de Ensino e Pesquisa Albert Einstein, Sao 2 Faculdade de Medicina do ABC, Disciplina de Hematologia e Oncologia, ´ Brazil Santo Andre, Background: Metastatic disease is the major cause of cancer mortality. The formation of metastasis requires the migration of tumor cells from the primary sites to other organs. Therefore, the detection of circulating tumor cells (CTC) is target of several investigations once it could be useful to assess metastasis risk. In addition to CTC, studies on metastasis mechanisms in animal models suggest migration of hematopoietic bone marrow progenitor