- Email: [email protected]
118: Interaction of C-FABP and PPARS in prostate cancer
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
its well-defined role in CNS ontogeny. Targeting beta1-mediated adhesion interactions may have potential as a novel anti-cancer therapy. No conflict of interest. 114 An aqueous extract of Limoniastrum guyonianum gall induces anti-tumor effects in melanoma-injected mice via modulation of the immune response M. Krifa1 , I. Skandrani1 , A. Pizzi2 , N. Nasr1 , K. Ghedira1 , L. Chekir3 . 1 Faculty of Pharmacy, Monastir, Tunisia, 2 ENSTIB/LERMAB Epinal, ENSTIB, Monastir, Tunisia, 3 Faculty of Dental Medicine, Monastir, Tunisia Background: Several studies have reported that plant-derived natural products have cancer chemopreventive and chemotherapeutic properties. The objectives of this study were to evaluate the in vitro and in vivo anti-tumor potential of the aqueous gall extract (G extract) from Limoniastrum guyonianum and to elucidate its immunological mechanisms, in part, by assessing its effects on the growth of transplanted tumors and the immune response in these tumorbearing mice. Effects of G extract on cellular anti-oxidant activity in the mice were also studied. Materials and Methods: Mice were inoculated with B16F10 mouse tumor cells and then treated intraperitoneally with G extract at 25 or 50 mg extract/kg BW for 7, 14, or 21 days. At each timepoint, effects of the extract on the growth of the tumor, splenocyte proliferation, natural killer (NK) cell activity, and CTL activity among splenocytes isolated from the mice were measured. Results: The G extract-induced tumor growth inhibition was associated with characteristic apoptotic changes in the tumor cells, like nuclear condensation. In addition, the extract inhibited melanin synthesis and tyrosinase activity among the melanoma cells in a concentration-related manner. In situ, G extract did not only significantly inhibit the growth of the transplantable tumor, but also remarkably increased splenocyte proliferation and both NK and CTL activities in tumor-bearing mice. The extract was also seen to have promoted lysosomal activity of host macrophages and gave rise to enhanced cellular anti-oxidant activity in several cell types (blood, splenocytes and macrophages) in the mice. Conclusions: Results indicated that use of the G extract could improve cellular and humoral immune responses in situ and that the extract could potentially be employed as an anti-tumor agent that acts, in part, by causing immunostimulatory and anti-oxidant-status enhancing effects. These effects could be ascribed to the presence of condensed tannins and polyphenols such as epicatechin and epigallocatechin gallate. Abbreviations: G extract: aqueous gall extract; NK: natural killer; CTL: cytotoxic T-lymphocyte. No conflict of interest. 115 The role of sumoylation in the regulation of p53 response D. Marouco1 , N.A. Barlev1 . 1 University of Leicester, Biochemistry, Leicester, United Kingdom Background: p53 is a major tumour suppressor protein implicated in many cellular processes, regulating cell cycle arrest, apoptosis and DNA repair. The regulation of p53 can be achieved by post-translational modifications (PTMs) which alter the function of the protein in the cell. Among such PTMs, p53 can be modified via sumoylation − the covalent binding of SUMO (Small Ubiquitin MOdifier) protein − on lysine 386. Materials and Methods: The aim of this project is to define the role of sumoylation in regulation of the activity of p53. For that, we combined realtime qPCR and luciferase reporter assays to analyse the function of p53 sumoylation in cellulo. In vitro binding and modification studies were used to investigate the interplay between p53 sumoylation and other PTMs. Results: Here we show that sumoylation of p53 with both SUMO-1 and SUMO-2 significantly decreases the transcriptional ability of p53 for its target genes p21 and PUMA, in both U2OS and HCT116 cell lines. Furthermore, the overexpression of a wild type p53 protein fused with the sumoylation conjugating enzyme ubc9 (p53-ubc9) in p53 null cells H1299 shows a reduced ability to activate p53-target genes, when compared to the sumoylation deficient mutant (p53K386R-ubc9). In addition, in vitro assays show that p53 acetylation by CBP/p300, as well as the methylation of lysine 372 by Set9, is affected by the presence of sumoylation of lysine 386. Conclusion: These results highlight the importance of Sumoylation as a modulator of p53 activity, and confirm the idea of a post-translational modifications network, where covalent modifications interplay with other modifications for the regulation of p53. No conflict of interest.
116 The mRNA-binding protein LARP1 is a pro-survival factor that promotes tumourigenicity and chemotherapy resistance in ovarian cancer T.G. Hopkins1 , J. Weir2 , M. Mura1 , N. Abd-Latip1 , K. Sweeney1 , S. Ghaem-Maghami1 , G. Gabra1 , S.P. Blagden1 . 1 Imperial College, Surgery and Cancer, LONDON, United Kingdom, 2 Imperial College Healthcare NHS Trust, Cellular Pathology, LONDON, United Kingdom Background: There is growing evidence that mRNA-binding proteins can be key post-transcriptional drivers of cancer progression. We have recently shown that LARP1 is highly expressed in cervical and lung malignancies. In cervical cancer cells, LARP1 is in complex with an mRNA interactome enriched for key cancer-related transcripts, including mTOR. We investigated the extent to which LARP1 regulates cancer progression in ovarian malignancies and used high-throughput strategies to identify novel LARP1-regulated genes. Material and Methods: Expression array data was obtained from the TCGA, Oncomine and kmplot repositories. For in vivo xenograft studies, SKOV3 cells with stable expression of a control or LARP1-targeting short hairpin sequence were injected subcutaneously into NOD-SCID mice. Apoptosis was assessed with Annexin V and Caspase 3/7 assays. Stem marker expression was assessed by flow cytometry. For transcriptome analysis, total RNA was extracted from OVCAR8 cells following treatment with control or LARP1targeting siRNA. Three independent repeats underwent deep sequencing using the Illumina HiSeq200 platform. Gene expression was assessed by RTPCR and western blotting. Results: In silico analysis of publicly available mRNA expression data demonstrated that LARP1 was highly expressed in ovarian cancers (n = 735) compared to the normal ovary, and high levels were predictive of poor outcome (n = 557). Xenograft experiments, in which ovarian cancer cell lines were implanted subcutaneously into immunocompromised mice, demonstrated that stable LARP1 knockdown dramatically reduced tumour growth. In vitro, we found that reduced LARP1 expression was associated with a reduction in clonogenicity and anchorage-independent growth. Knockdown of LARP1 led to reductions in populations positive for putative cancer stem cell markers, including CD133, and with high aldehydyde dehydrogenase activity. Transient knockdown of LARP1 was sufficient to restore platinum sensitivity in resistant lines. To identify LARP1-regulated targets we performed transcriptome deep sequencing. There was a significant enrichment (p < 0.001) for LARP1 interactome partners in the genes that displayed altered expression following knockdown. Functional annotation clustering revealed multiple genes linked to survival and evasion of apoptosis, notably the anti-apoptotic protein BCL2. We confirmed that LARP1 knockdown led to reduction in BCL2 mRNA and protein expression. BCL2 transcripts are in complex with LARP1 protein in ovarian cancer cells and LARP1 is required for transcript stability. Conclusions: LARP1 appears to be an important driver of malignant progression in ovarian cancer. LARP1 promotion of BCL2 transcript stability identifies it as a key pro-survival factor, and the protein may have potential as a therapeutic target. No conflict of interest. 118 Interaction of C-FABP and PPARS in prostate cancer F. Seyed Forootan1 , S. Seyed Forootan Shiva1 , Y. Ke1 . 1 University of Liverpool, Molecular and Clinical Cancer Medicine (Pathology), Liverpool, United Kingdom Background: How C-FABP promotes tumourigenicity of prostate cancer is not fully understood. We hypothesize that C-FABP may transport an excessive amount of intracellular fatty acids into cancer cells to activate their nuclear receptor PPARs to trigger a chain of molecular events which may lead to an facilitated malignant progression of the cancer cells. Method: Expression of C-FABP, PPARb/d and PPARg in cell lines were detected by Western blot at protein level and by RT-PCR at mRNA level. The expression of C-FABP, PPARb/d and PPARg in BPH and in prostate carcinoma tissues were detected by immunohistochemical staining. Then the relevant PPAR gene was suppressed transiently (siRNA technique) and stably (shRNA technique) to assess its effect on malignant progression in P Ca, in vitro (Proliferation assay, Invasion assay, Soft agar assay) and in vivo. Results: Although the expression of PPARb/d in carcinoma tissues was significantly higher than that in BPH, no significant difference in its expression between benign and malignant cell lines was observed. For PPARg and C-FABP, not only that their expression levels in malignant cell lines and tissues (cytoplasm and nucleus) were significantly higher than those expressed in benign cells and in BPH respectively, it appeared that their expression levels were increased as the increasing malignancies of the cell lines and tissues. While the increased expression of PPARb/d in carcinomas was not correlated with the prostate cancer patient survival time, the increased expression of both C-FABP and PPARg were significantly correlated to a reduced survival period of time. Multi variant analysis further showed that C-FABP and PPARg were not independent when used as markers to predict the patient survival. Suppression of PPARg showed to be correlated with significant reduction of
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 proliferation rate, invasiveness and the ability of colony formation of P Ca cells in vitro. Furthermore, suppression of PPARg expression in the highly malignant prostate cancer cells significantly inhibited the tumourigenicity in nude mice. Conclusion: This study showed that the prognostic value of PPARg is dependent to C-FABP in prostate carcinomas so they are two independent factors which can be used to predict the patient outcomes. Our previous work on C-FABP and this study suggested that suppression of both CFABP and PPARg genes can cause similar degree of reductions on the tumourigenicity of P Ca cells. These results strongly support our hypothesis that C-FABP promotes tumourigenicity by transporting an excessive amount of intracellular fatty acids into cancer cells to activate their nuclear receptor PPARg which may then activate the down-stream cancer-promoting genes. No conflict of interest. 120 Autocrine human growth hormone promotes the oncogenic behaviour of hepatocellular carcinoma cells Y.J. Chen1 , X.J. Kong2 , Z.S. Wu2 , Y.C. Lau1 , J.J. Wang1 , T. Zhu2 , P.E. Lobie1,3 . 1 National University of Singapore, Pharmacology, Singapore, Singapore, 2 University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China, 3 National University of Singapore, Cancer Science Institute, Singapore Introduction: The liver is an essential target tissue of growth hormone (GH), which is involved in somatic growth promotion and metabolic processes. Aberrant GH signalling has been implicated in the development of metabolic liver diseases, as well as liver cancer. In addition to its endocrine actions, GH is widely recognized as a local growth factor in a number of tissues. The oncogenic potential of autocrine human GH (hGH) has been intensively investigated in human mammary and endometrial cancer cells. However, the oncogenic potential of autocrine hGH in hepatocellular carcinoma (HCC) is still unclear. We have recently observed that tumour expression of hGH associated with poor clinical outcomes in HCC patients. Hence, we have proceeded to determine whether autocrine hGH promoted oncogenic behaviour in HCC cells, and deciphered the potential mechanisms. Material and Method: HCC cells were stably transfected with the pcDNA3hGH plasmid or vector control. Would healing and Transwell assays were performed to investigate the invasive potential of autocrine hGH in HCC cells. The cancer stem cell (CSC) like properties promoted by autocrine hGH were investigated by spheroid and Aldefluor assays. Western blot, luciferase reporter assay and chromatin immunoprecipitation (ChIP) assays were performed to determine the potential signalling mechanism utilized by autocrine hGH in HCC cells. Results and Discussion: Using wound healing and Transwell assays, we observed that autocrine hGH significantly increased the invasive potential of HCC cells. Furthermore, autocrine hGH increased spheroid formation in suspension culture, indicative of CSC-like activity. Aldefluor assay demonstrated that autocrine hGH increased the ALDH1 positive population of HCC cells. Western blot analysis showed that autocrine hGH activated STAT3 in HCC cells. Autocrine activation of STAT3 in HCC cells reduced the expression of Claudin-1, a tight junction protein. Luciferase reporter assays demonstrated that autocrine hGH activated STAT3 inhibited the transcriptional activity of the Claudin-1 promoter. ChIP assay revealed that STAT3 directly bound to the promoter region of Claudin-1 gene. And this binding was enhanced by autocrine hGH. We identified three putative STAT3 binding sites in the Claudin-1 promoter. Furthermore, AG490, an inhibitor of JAK2, restored Claudin-1 expression in HCC cells with forced expression of hGH. Forced expression of Claudin-1 abrogated autocrine hGH stimulation of invasive and CSC-like behaviour. Conclusion: These results indicate that autocrine hGH induces invasive and CSC-like properties in HCC cells. hGH-induced oncogenicity in HCC cells is mediated by STAT3 dependent inhibition of Claudin-1 expression. No conflict of interest. 122 Poly(I:C) and chemotherapeutics synergistically induce cell death in head and neck cancer cell lines T. Matijevic Glavan1 , B. Verillaud2 , P. Busson2 , J. Pavelic1 . 1 Rudjer Boskovic Institute, Department of Molecular Medicine, Zagreb, Croatia, 2 Gustave Roussy Institute, UMR 8126, Paris-Villejuif, France Introduction: Toll-like receptors (TLRs) recognize pathogen-derived molecules and also products of inflamed tissue, resulting in the activation of the immune response. TLR ligands are already being used in clinical studies because of their ability to induce apoptosis and trigger the immune system. In a previous study we showed that TLR3 activation by synthetic dsRNA [poly(I:C)] in cancer cell line Detroit 562 (pharynx carcinoma) induced caspasedependent apoptosis. Additionally, when combined with chemotherapeutics this treatment acts synergistically by enhancing cancer cell death. In this study we tried to clarify the mechanism of the observed synergy.
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Material and Method: Cell lines used were Detroit 562 and SQ20B. Cell survival was determined by using cytotoxicity assay and colony formation assay while protein expression was obtained by western blot. Results and Discussion: We have shown here that the combination of poly(I:C) and cisplatin cytotoxicity is TLR3-dependent while methotrexate and hydroxyurea act through other dsRNA receptors (RIG-I and MDA5). Additionally, poly(I:C) has a dual effect in Detroit 562 cells: it induces both pro-apoptotic and anti-apoptotic (an increase in the expression of c-IAP2) events. Thus, combined treatment by poly(I:C) and c-IAP inhibitor significantly decreased cell survival even at low concentrations. However, our results showed that cisplatin has the ability to down regulate the c-IAP2 expression induced by poly(I:C) in the Detroit 262 cells thus increasing the pro-apoptotic response. In this study we also used laryngeal cell line SQ20B stably transfected with inducible shRNA for TLR3 which is a good model for poly(I:C) combinational therapy research. We confirmed that synergistic effect of poly(A:U) and cisplatin on cell death is TLR3-dependent. Additionally, as SQ20B is radiation resistant cell line, we explored here whether it can be sensitized to radiotherapy by poly(I:C)/cisplatin and which molecular pathways are involved in this process. Conclusion: Synthetic dsRNAs, like poly(I:C), have the potential to help overcoming the resistance of some human malignant cells to classical modalities of radiotherapy and chemotherapy. No conflict of interest. 123 M30 assay may not be an accurate method for apoptosis in the cancer cells expressing low level of cytokeratin B. Cevatemre1 , F. Ari1 , M. Sarimahmut1 , A. Yilmaztepe Oral2 , E. Ulukaya2 . 1 Faculty of Arts and Sciences, Biology, Bursa, Turkey, 2 Faculty of Medicine, Medical Biochemistry, Bursa, Turkey Background: Caspase-cleaved fragment of cytokeratin 18 (M30), which is present only in epithelial cells, has been regarded as a biomarker of apoptotic cell death because it is released from the cells during apoptosis. It is therefore believed that it reflects cell death of epithelial tumors. Based on this, studies suggest that M30 may have important clinical biomarker utility, as increased levels of M30 may be prognostic and/or predict tumour response to chemotherapy. However, it does not increase in the sera of some patients although those patients respond to the treatment well. In the current study A549, H1299 and PC3 lung cancer cell lines were used to determine the correlation between apoptosis and M30 levels. Material and Methods: The MTT and ATP viability assays were used to determine the cytotoxic activity of some anti cancer agents that are also used in the clinics. We used fluorescence imaging of nuclei (Hoechst 33342 staining) as well as Annexin V-FITC staining to detect apoptosis. M30 levels were measured by ELISA. Results: M30 levels were clearly increased in A549 cells, but remained unchanged in H1299 and PC3 cells although these cells underwent apoptosis. These two cell lines were subsequently found to express low level of cytokeratin 18. Conclusions: Our results suggest that M30 may not be a universal marker for apoptosis in all cancer types. Therefore the data should be interpreted with caution. No conflict of interest. 124 Vincristine induces autophagy-mediated HMGB1 release via transcriptional regulation of Mcl-1 antagonizes apoptosis in human oral cancer cells S. Yang1 , C. Lin2 , M. Hsieh3 . 1 Chung Shan Medical University, Institute of Medicine, Taichung, Taiwan, 2 Chung Shan Medical University, Institute of Oral Sciences, Taichung, Taiwan, 3 Changhua Christian Hospital, Cancer Research Center, Changhua, Taiwan Background: The autophagy-associated release of HMGB1 (high-mobility group box 1) is known to protect cancer cells from numerous chemotherapeutics. However, the related molecular mechanism, involved in the protection of oral cancer cells remains not clear. Material and Methods: Cell viability was examined by MTT assay, whereas cell cycle analysis and quantification of acidic vesicular organelle (AVO) formation were measured by flow cytometry. Real-time PCR confirmed the effects of vincristine on HMGB1 mRNA level in SCC-9 cells. Western blotting was performed for detecting changes of autophagy-associated proteins. Results: In this study, we determined that HMGB1 released by oral cancer cells protected the cells against apoptosis caused by vincristine by upregulating the transcription of Mcl-1. Extracellular HMGB1 seem to be required for the autophagy-mediated inhibition of apoptosis because HMGB1 knockdown by siRNA abolished the effect of autophagy protective. Vincristine treatment increased the expression of Mcl-1 mRNA, but did not alter the protein expression levels of Mcl-1. Inhibiting HMGB1 expression blocked the increase in the Mcl-1 transcription and reduced Mcl-1 protein levels, demonstrate that HMGB1-mediated signaling is required for the upregulation
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
its well-defined role in CNS ontogeny. Targeting beta1-mediated adhesion interactions may have potential as a novel anti-cancer therapy. No conflict of interest. 114 An aqueous extract of Limoniastrum guyonianum gall induces anti-tumor effects in melanoma-injected mice via modulation of the immune response M. Krifa1 , I. Skandrani1 , A. Pizzi2 , N. Nasr1 , K. Ghedira1 , L. Chekir3 . 1 Faculty of Pharmacy, Monastir, Tunisia, 2 ENSTIB/LERMAB Epinal, ENSTIB, Monastir, Tunisia, 3 Faculty of Dental Medicine, Monastir, Tunisia Background: Several studies have reported that plant-derived natural products have cancer chemopreventive and chemotherapeutic properties. The objectives of this study were to evaluate the in vitro and in vivo anti-tumor potential of the aqueous gall extract (G extract) from Limoniastrum guyonianum and to elucidate its immunological mechanisms, in part, by assessing its effects on the growth of transplanted tumors and the immune response in these tumorbearing mice. Effects of G extract on cellular anti-oxidant activity in the mice were also studied. Materials and Methods: Mice were inoculated with B16F10 mouse tumor cells and then treated intraperitoneally with G extract at 25 or 50 mg extract/kg BW for 7, 14, or 21 days. At each timepoint, effects of the extract on the growth of the tumor, splenocyte proliferation, natural killer (NK) cell activity, and CTL activity among splenocytes isolated from the mice were measured. Results: The G extract-induced tumor growth inhibition was associated with characteristic apoptotic changes in the tumor cells, like nuclear condensation. In addition, the extract inhibited melanin synthesis and tyrosinase activity among the melanoma cells in a concentration-related manner. In situ, G extract did not only significantly inhibit the growth of the transplantable tumor, but also remarkably increased splenocyte proliferation and both NK and CTL activities in tumor-bearing mice. The extract was also seen to have promoted lysosomal activity of host macrophages and gave rise to enhanced cellular anti-oxidant activity in several cell types (blood, splenocytes and macrophages) in the mice. Conclusions: Results indicated that use of the G extract could improve cellular and humoral immune responses in situ and that the extract could potentially be employed as an anti-tumor agent that acts, in part, by causing immunostimulatory and anti-oxidant-status enhancing effects. These effects could be ascribed to the presence of condensed tannins and polyphenols such as epicatechin and epigallocatechin gallate. Abbreviations: G extract: aqueous gall extract; NK: natural killer; CTL: cytotoxic T-lymphocyte. No conflict of interest. 115 The role of sumoylation in the regulation of p53 response D. Marouco1 , N.A. Barlev1 . 1 University of Leicester, Biochemistry, Leicester, United Kingdom Background: p53 is a major tumour suppressor protein implicated in many cellular processes, regulating cell cycle arrest, apoptosis and DNA repair. The regulation of p53 can be achieved by post-translational modifications (PTMs) which alter the function of the protein in the cell. Among such PTMs, p53 can be modified via sumoylation − the covalent binding of SUMO (Small Ubiquitin MOdifier) protein − on lysine 386. Materials and Methods: The aim of this project is to define the role of sumoylation in regulation of the activity of p53. For that, we combined realtime qPCR and luciferase reporter assays to analyse the function of p53 sumoylation in cellulo. In vitro binding and modification studies were used to investigate the interplay between p53 sumoylation and other PTMs. Results: Here we show that sumoylation of p53 with both SUMO-1 and SUMO-2 significantly decreases the transcriptional ability of p53 for its target genes p21 and PUMA, in both U2OS and HCT116 cell lines. Furthermore, the overexpression of a wild type p53 protein fused with the sumoylation conjugating enzyme ubc9 (p53-ubc9) in p53 null cells H1299 shows a reduced ability to activate p53-target genes, when compared to the sumoylation deficient mutant (p53K386R-ubc9). In addition, in vitro assays show that p53 acetylation by CBP/p300, as well as the methylation of lysine 372 by Set9, is affected by the presence of sumoylation of lysine 386. Conclusion: These results highlight the importance of Sumoylation as a modulator of p53 activity, and confirm the idea of a post-translational modifications network, where covalent modifications interplay with other modifications for the regulation of p53. No conflict of interest.
116 The mRNA-binding protein LARP1 is a pro-survival factor that promotes tumourigenicity and chemotherapy resistance in ovarian cancer T.G. Hopkins1 , J. Weir2 , M. Mura1 , N. Abd-Latip1 , K. Sweeney1 , S. Ghaem-Maghami1 , G. Gabra1 , S.P. Blagden1 . 1 Imperial College, Surgery and Cancer, LONDON, United Kingdom, 2 Imperial College Healthcare NHS Trust, Cellular Pathology, LONDON, United Kingdom Background: There is growing evidence that mRNA-binding proteins can be key post-transcriptional drivers of cancer progression. We have recently shown that LARP1 is highly expressed in cervical and lung malignancies. In cervical cancer cells, LARP1 is in complex with an mRNA interactome enriched for key cancer-related transcripts, including mTOR. We investigated the extent to which LARP1 regulates cancer progression in ovarian malignancies and used high-throughput strategies to identify novel LARP1-regulated genes. Material and Methods: Expression array data was obtained from the TCGA, Oncomine and kmplot repositories. For in vivo xenograft studies, SKOV3 cells with stable expression of a control or LARP1-targeting short hairpin sequence were injected subcutaneously into NOD-SCID mice. Apoptosis was assessed with Annexin V and Caspase 3/7 assays. Stem marker expression was assessed by flow cytometry. For transcriptome analysis, total RNA was extracted from OVCAR8 cells following treatment with control or LARP1targeting siRNA. Three independent repeats underwent deep sequencing using the Illumina HiSeq200 platform. Gene expression was assessed by RTPCR and western blotting. Results: In silico analysis of publicly available mRNA expression data demonstrated that LARP1 was highly expressed in ovarian cancers (n = 735) compared to the normal ovary, and high levels were predictive of poor outcome (n = 557). Xenograft experiments, in which ovarian cancer cell lines were implanted subcutaneously into immunocompromised mice, demonstrated that stable LARP1 knockdown dramatically reduced tumour growth. In vitro, we found that reduced LARP1 expression was associated with a reduction in clonogenicity and anchorage-independent growth. Knockdown of LARP1 led to reductions in populations positive for putative cancer stem cell markers, including CD133, and with high aldehydyde dehydrogenase activity. Transient knockdown of LARP1 was sufficient to restore platinum sensitivity in resistant lines. To identify LARP1-regulated targets we performed transcriptome deep sequencing. There was a significant enrichment (p < 0.001) for LARP1 interactome partners in the genes that displayed altered expression following knockdown. Functional annotation clustering revealed multiple genes linked to survival and evasion of apoptosis, notably the anti-apoptotic protein BCL2. We confirmed that LARP1 knockdown led to reduction in BCL2 mRNA and protein expression. BCL2 transcripts are in complex with LARP1 protein in ovarian cancer cells and LARP1 is required for transcript stability. Conclusions: LARP1 appears to be an important driver of malignant progression in ovarian cancer. LARP1 promotion of BCL2 transcript stability identifies it as a key pro-survival factor, and the protein may have potential as a therapeutic target. No conflict of interest. 118 Interaction of C-FABP and PPARS in prostate cancer F. Seyed Forootan1 , S. Seyed Forootan Shiva1 , Y. Ke1 . 1 University of Liverpool, Molecular and Clinical Cancer Medicine (Pathology), Liverpool, United Kingdom Background: How C-FABP promotes tumourigenicity of prostate cancer is not fully understood. We hypothesize that C-FABP may transport an excessive amount of intracellular fatty acids into cancer cells to activate their nuclear receptor PPARs to trigger a chain of molecular events which may lead to an facilitated malignant progression of the cancer cells. Method: Expression of C-FABP, PPARb/d and PPARg in cell lines were detected by Western blot at protein level and by RT-PCR at mRNA level. The expression of C-FABP, PPARb/d and PPARg in BPH and in prostate carcinoma tissues were detected by immunohistochemical staining. Then the relevant PPAR gene was suppressed transiently (siRNA technique) and stably (shRNA technique) to assess its effect on malignant progression in P Ca, in vitro (Proliferation assay, Invasion assay, Soft agar assay) and in vivo. Results: Although the expression of PPARb/d in carcinoma tissues was significantly higher than that in BPH, no significant difference in its expression between benign and malignant cell lines was observed. For PPARg and C-FABP, not only that their expression levels in malignant cell lines and tissues (cytoplasm and nucleus) were significantly higher than those expressed in benign cells and in BPH respectively, it appeared that their expression levels were increased as the increasing malignancies of the cell lines and tissues. While the increased expression of PPARb/d in carcinomas was not correlated with the prostate cancer patient survival time, the increased expression of both C-FABP and PPARg were significantly correlated to a reduced survival period of time. Multi variant analysis further showed that C-FABP and PPARg were not independent when used as markers to predict the patient survival. Suppression of PPARg showed to be correlated with significant reduction of
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 proliferation rate, invasiveness and the ability of colony formation of P Ca cells in vitro. Furthermore, suppression of PPARg expression in the highly malignant prostate cancer cells significantly inhibited the tumourigenicity in nude mice. Conclusion: This study showed that the prognostic value of PPARg is dependent to C-FABP in prostate carcinomas so they are two independent factors which can be used to predict the patient outcomes. Our previous work on C-FABP and this study suggested that suppression of both CFABP and PPARg genes can cause similar degree of reductions on the tumourigenicity of P Ca cells. These results strongly support our hypothesis that C-FABP promotes tumourigenicity by transporting an excessive amount of intracellular fatty acids into cancer cells to activate their nuclear receptor PPARg which may then activate the down-stream cancer-promoting genes. No conflict of interest. 120 Autocrine human growth hormone promotes the oncogenic behaviour of hepatocellular carcinoma cells Y.J. Chen1 , X.J. Kong2 , Z.S. Wu2 , Y.C. Lau1 , J.J. Wang1 , T. Zhu2 , P.E. Lobie1,3 . 1 National University of Singapore, Pharmacology, Singapore, Singapore, 2 University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China, 3 National University of Singapore, Cancer Science Institute, Singapore Introduction: The liver is an essential target tissue of growth hormone (GH), which is involved in somatic growth promotion and metabolic processes. Aberrant GH signalling has been implicated in the development of metabolic liver diseases, as well as liver cancer. In addition to its endocrine actions, GH is widely recognized as a local growth factor in a number of tissues. The oncogenic potential of autocrine human GH (hGH) has been intensively investigated in human mammary and endometrial cancer cells. However, the oncogenic potential of autocrine hGH in hepatocellular carcinoma (HCC) is still unclear. We have recently observed that tumour expression of hGH associated with poor clinical outcomes in HCC patients. Hence, we have proceeded to determine whether autocrine hGH promoted oncogenic behaviour in HCC cells, and deciphered the potential mechanisms. Material and Method: HCC cells were stably transfected with the pcDNA3hGH plasmid or vector control. Would healing and Transwell assays were performed to investigate the invasive potential of autocrine hGH in HCC cells. The cancer stem cell (CSC) like properties promoted by autocrine hGH were investigated by spheroid and Aldefluor assays. Western blot, luciferase reporter assay and chromatin immunoprecipitation (ChIP) assays were performed to determine the potential signalling mechanism utilized by autocrine hGH in HCC cells. Results and Discussion: Using wound healing and Transwell assays, we observed that autocrine hGH significantly increased the invasive potential of HCC cells. Furthermore, autocrine hGH increased spheroid formation in suspension culture, indicative of CSC-like activity. Aldefluor assay demonstrated that autocrine hGH increased the ALDH1 positive population of HCC cells. Western blot analysis showed that autocrine hGH activated STAT3 in HCC cells. Autocrine activation of STAT3 in HCC cells reduced the expression of Claudin-1, a tight junction protein. Luciferase reporter assays demonstrated that autocrine hGH activated STAT3 inhibited the transcriptional activity of the Claudin-1 promoter. ChIP assay revealed that STAT3 directly bound to the promoter region of Claudin-1 gene. And this binding was enhanced by autocrine hGH. We identified three putative STAT3 binding sites in the Claudin-1 promoter. Furthermore, AG490, an inhibitor of JAK2, restored Claudin-1 expression in HCC cells with forced expression of hGH. Forced expression of Claudin-1 abrogated autocrine hGH stimulation of invasive and CSC-like behaviour. Conclusion: These results indicate that autocrine hGH induces invasive and CSC-like properties in HCC cells. hGH-induced oncogenicity in HCC cells is mediated by STAT3 dependent inhibition of Claudin-1 expression. No conflict of interest. 122 Poly(I:C) and chemotherapeutics synergistically induce cell death in head and neck cancer cell lines T. Matijevic Glavan1 , B. Verillaud2 , P. Busson2 , J. Pavelic1 . 1 Rudjer Boskovic Institute, Department of Molecular Medicine, Zagreb, Croatia, 2 Gustave Roussy Institute, UMR 8126, Paris-Villejuif, France Introduction: Toll-like receptors (TLRs) recognize pathogen-derived molecules and also products of inflamed tissue, resulting in the activation of the immune response. TLR ligands are already being used in clinical studies because of their ability to induce apoptosis and trigger the immune system. In a previous study we showed that TLR3 activation by synthetic dsRNA [poly(I:C)] in cancer cell line Detroit 562 (pharynx carcinoma) induced caspasedependent apoptosis. Additionally, when combined with chemotherapeutics this treatment acts synergistically by enhancing cancer cell death. In this study we tried to clarify the mechanism of the observed synergy.
S27
Material and Method: Cell lines used were Detroit 562 and SQ20B. Cell survival was determined by using cytotoxicity assay and colony formation assay while protein expression was obtained by western blot. Results and Discussion: We have shown here that the combination of poly(I:C) and cisplatin cytotoxicity is TLR3-dependent while methotrexate and hydroxyurea act through other dsRNA receptors (RIG-I and MDA5). Additionally, poly(I:C) has a dual effect in Detroit 562 cells: it induces both pro-apoptotic and anti-apoptotic (an increase in the expression of c-IAP2) events. Thus, combined treatment by poly(I:C) and c-IAP inhibitor significantly decreased cell survival even at low concentrations. However, our results showed that cisplatin has the ability to down regulate the c-IAP2 expression induced by poly(I:C) in the Detroit 262 cells thus increasing the pro-apoptotic response. In this study we also used laryngeal cell line SQ20B stably transfected with inducible shRNA for TLR3 which is a good model for poly(I:C) combinational therapy research. We confirmed that synergistic effect of poly(A:U) and cisplatin on cell death is TLR3-dependent. Additionally, as SQ20B is radiation resistant cell line, we explored here whether it can be sensitized to radiotherapy by poly(I:C)/cisplatin and which molecular pathways are involved in this process. Conclusion: Synthetic dsRNAs, like poly(I:C), have the potential to help overcoming the resistance of some human malignant cells to classical modalities of radiotherapy and chemotherapy. No conflict of interest. 123 M30 assay may not be an accurate method for apoptosis in the cancer cells expressing low level of cytokeratin B. Cevatemre1 , F. Ari1 , M. Sarimahmut1 , A. Yilmaztepe Oral2 , E. Ulukaya2 . 1 Faculty of Arts and Sciences, Biology, Bursa, Turkey, 2 Faculty of Medicine, Medical Biochemistry, Bursa, Turkey Background: Caspase-cleaved fragment of cytokeratin 18 (M30), which is present only in epithelial cells, has been regarded as a biomarker of apoptotic cell death because it is released from the cells during apoptosis. It is therefore believed that it reflects cell death of epithelial tumors. Based on this, studies suggest that M30 may have important clinical biomarker utility, as increased levels of M30 may be prognostic and/or predict tumour response to chemotherapy. However, it does not increase in the sera of some patients although those patients respond to the treatment well. In the current study A549, H1299 and PC3 lung cancer cell lines were used to determine the correlation between apoptosis and M30 levels. Material and Methods: The MTT and ATP viability assays were used to determine the cytotoxic activity of some anti cancer agents that are also used in the clinics. We used fluorescence imaging of nuclei (Hoechst 33342 staining) as well as Annexin V-FITC staining to detect apoptosis. M30 levels were measured by ELISA. Results: M30 levels were clearly increased in A549 cells, but remained unchanged in H1299 and PC3 cells although these cells underwent apoptosis. These two cell lines were subsequently found to express low level of cytokeratin 18. Conclusions: Our results suggest that M30 may not be a universal marker for apoptosis in all cancer types. Therefore the data should be interpreted with caution. No conflict of interest. 124 Vincristine induces autophagy-mediated HMGB1 release via transcriptional regulation of Mcl-1 antagonizes apoptosis in human oral cancer cells S. Yang1 , C. Lin2 , M. Hsieh3 . 1 Chung Shan Medical University, Institute of Medicine, Taichung, Taiwan, 2 Chung Shan Medical University, Institute of Oral Sciences, Taichung, Taiwan, 3 Changhua Christian Hospital, Cancer Research Center, Changhua, Taiwan Background: The autophagy-associated release of HMGB1 (high-mobility group box 1) is known to protect cancer cells from numerous chemotherapeutics. However, the related molecular mechanism, involved in the protection of oral cancer cells remains not clear. Material and Methods: Cell viability was examined by MTT assay, whereas cell cycle analysis and quantification of acidic vesicular organelle (AVO) formation were measured by flow cytometry. Real-time PCR confirmed the effects of vincristine on HMGB1 mRNA level in SCC-9 cells. Western blotting was performed for detecting changes of autophagy-associated proteins. Results: In this study, we determined that HMGB1 released by oral cancer cells protected the cells against apoptosis caused by vincristine by upregulating the transcription of Mcl-1. Extracellular HMGB1 seem to be required for the autophagy-mediated inhibition of apoptosis because HMGB1 knockdown by siRNA abolished the effect of autophagy protective. Vincristine treatment increased the expression of Mcl-1 mRNA, but did not alter the protein expression levels of Mcl-1. Inhibiting HMGB1 expression blocked the increase in the Mcl-1 transcription and reduced Mcl-1 protein levels, demonstrate that HMGB1-mediated signaling is required for the upregulation