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mTOR inhibition overcomes docetaxel chemoresistance in prostate cancer cell and mouse models through downregulation of sphingosine kinase 1
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 539 Oligonucleotide aptamers as innovative therapeutic tools for triple-negative breast cancers S. Camorani1 , E. Crescenzi1 , M. Passariello2 , R. Fontanella3 , M. Fedele1 , A. Zannetti3 , L. Cerchia1 . 1 Istituto di Endocrinologia e Oncologia Sperimentale “G. Salvatore”, CNR, Naples, Italy, 2 Dipartimento Di Medicina Molecolare e Biotecnologie Mediche, Universita` degli Studi di Napoli “Federico II”, Naples, Italy, 3 Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy Background: Triple-negative breast cancers (TNBCs), representing ~15% of all breast cancers, are characterized by the absence of estrogen and progesterone receptors and ErbB2, excluding the possibility of using efficacious targeted therapies developed against these proteins. Conversely, these tumors frequently overexpress the EGF receptor (EGFR), supporting the possibility of developing novel targeted therapies against this receptor. Selective molecules emerging for anti-cancer therapy are oligonucleotide aptamers, that, similarly to antibodies, interact at high affinity with their targets. They are characterized by small size, high stability, lack of immunogenicity and ready availability. We have previously selected a RNA-aptamer able to recognize the EGFR. Herein, we have characterized this aptamer as inhibitor of TNBC cells malignant phenotype. Further, we have recently applied a SELEX approach to live TNBC cells to select newly TNBC-specific aptamers. Material and Methods: The cell-SELEX screening has been performed starting from a library of 2 Fluoro-Pyrimidines RNAs by using human TNBC MDA-MB-231 cells for the positive selection and non-TNBC cells for counterselection steps. The biological effects of the anti-EGFR aptamer on TNBC cells have been determined by in vitro tumorigenicity-related functional assays including MTT, growth curves experiments and cell cycle analyses, Annexin V staining and immunoblotting for proteins involved in apoptosis, cell growth on Matrigel monolayer (vasculogenic mimicry,VM). Results: Therapies targeting EGFR, while showing efficacy in other tumors, have not delivered long term benefits to TNBC patients. In this study we prove that the anti-EGFR aptamer is able to drastically interfere with EGF-dependent EGFR activation in TNBC cells. Importantly, when applied to MDA-MB-231 and BT-549 cells, it significantly reduces cell proliferation that, conversely, is not affected by erlotinib and gefitinib treatment. It has been recently shown that aggressive TNBCs form de novo vascular networks that provide adequate blood supply for tumour growth. Consequently, the elimination of VM plays a crucial role in a successful treatment of TNBCs. We found that the antiEGFR aptamer, but not erlotinib, strongly hampers VM of TNBC cells. We are currently evaluating the aptamer therapeutic potential in animal models of TNBC. Further, we have generated a panel of aptamers that specifically bind to MDA-MB-231 target cells. The characterization of aptamers targeting properties and biological effects is on-going. Conclusions: Our results indicate TNBC selective aptamers as a potential tool to be used in alternative or in combination to conventional chemotherapy to manage TNBCs in the near future. Further, the cell-SELEX strategy that we developed holds a great promise in developing specific molecular probes for TNBC biomarker discovery. No conflict of interest.
540 Carnosol inhibits tumor growth and metastasis of breast cancer through inhibition of the histone acetytransferase activity of p300 and proteasome-dependent degradation of STAT3 R. Iratni1 , E.H. Hussain1 , A.D. Yusra1 , V. Ranjit1 . 1 UAE University, Biology, Al Ain, U.A.E. Introduction: Carnosol is a naturally occurring polyphenol (dietary diterpene) found in culinary herbs such as rosemary, sage, and oregano. Studies revealed that carnosol possesses many pharmacological activities, including antiinflammatory, anti-oxidant and anti-microbial properties. Here we investigated the in vivo anticancer effect of carnosol on triple negative breast cancer MDAMB-231 cells. Material and Method: Human breast cancer triple negative breast cancer (TNBC) MDA-MB-231 cell line was used. Protein expression and protein acetylation and phosphorylation was examined using Western blotting. Gelatin zymography was used to measure the activity of MMP-9. The in ovo chick chorioallantoic membrane (CAM) assay was used to study tumor growth and metastasis. Results: We found that carnosol significantly inhibited tumor growth and metastasis of breast cancer. Concentrations of 50 and 100 mM of carnosol led to significant reduction of tumor growth by 65 and 75% respectively. Moreover, carnosol inhibited invasion by downregulating MMP-9 expression. Mechanistically, we found that carnosol inhibited the STAT3 pathway by promoting the proteasome-dependent degradation of STAT3 protein. Interestingly, we found STAT3 rescued from proteasome degradation by proteasome inhibitors was neither phosphorylated nor acetylated. Additionally, we found that carnosol also inhibited the histone acetyltransferase activity of p300 and consequently target it to proteasome-dependent degradation. Moreover, molecular docking analysis indicate that carnosol appear to inhibit
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p300 HAT activity by blocking acetyl coenzyme A’s entry into its deep pocket. Carnosol was found to bind at the entrance to this active site cavity. Conclusion: Our findings identify the natural compound carnosol as a new inhibitor of p300 HAT with a potent anti-tumor growth of TNBC. No conflict of interest. 541 Plasma microRNA profiles; down-regulation of plasma miR-107 level contributes to poor outcomes in pancreatic cancer T. Imamura1 , S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , R. Morimura1 , H. Ikoma1 , H. Konishi1 , A. Shiozaki1 , H. Taniguchi2 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan, 2 Kyoto Second Red Cross Hospital, Department of Surgery, Kyoto, Japan Background: This study aimed to explore decreased tumor suppressive microRNAs (miRNAs) in plasma of pancreatic cancer (PCa) patients and detect their possible roles as a treatment target as well as a biomarker for PCa. Methods and Results: Six down-regulated miRNAs (miR-107, miR-126, miR-451, miR-145, miR-491-5p, and miR-146b-5p) were selected using a miRNAs microarray-based approach. Test- and large scale analyses revealed that plasma miR-107 level in PCa patients was most down-regulated among candidates compared to healthy volunteers (p<0.001). A low level of plasma miR-107 was significantly associated with advanced T- and N-stage, and significantly associated with a worse cause-specific survival, survival after surgery, and disease free survival rate after surgery. Multivariate analysis identified a low level of miR-107 as an independent poor prognostic factor in PCa patients (p = 0.012, Hazard ratio 4.56 (95% CI: 1.38–17.4)). Enforced expression of miR-107 in PCa cell lines induced the production of p21, which results in G1/S arrest, and inhibited cell proliferation, migration, and invasion. Furthermore, in vivo study demonstrated that the restoration of plasma miR-107 could suppress tumor growth. Conclusion: Down regulation of plasma miRNA-107, which serves as tumor suppressive functions in PCa contributes to disease progression and poor outcomes in PCa and preserving blood level of miR-107 might be a novel treatment candidate for PCa. No conflict of interest. 543 mTOR inhibition overcomes docetaxel chemoresistance in prostate cancer cell and mouse models through downregulation of sphingosine kinase 1 H. Alshaker1,2 , Q. Wang2 , C. Cooper2 , M. Winkler3 , D. Pchejetski2 . 1 University of Petra, Department of Pharmacology and Biomedical Sciences, Amman, Jordan, 2 University of East Anglia, School of Medicine, Norwich, United Kingdom, 3 Department of Surgery and Cancer, Imperial College London, London, United Kingdom Background: Sphingosine kinase 1 (SK1) and PI3K/Akt/mTOR pathways are widely implicated in cancer progression and docetaxel chemoresistance. Here we have investigated the chemosensitizing mechanism of mTOR inhibitor RAD001 (everolimus) in prostate cancer. Material and Methods: Cell viability and apoptosis assays were used to investigate the combined effects of RAD001 and docetaxel in prostate cancer cell cultures. SK1 activity was measured by radiolabelling and alterations in cell signalling and gene expression were measured by Western blotting and qRT-PCR. Mouse prostate cancer xenografts were used to confirm in vitro findings. Results: Treatment of hormone resistant prostate cancer cells with RAD001 induced a strong downregulation of HIF1a protein and SK1 mRNA expression. SK1 enzymatic activity was also inhibited, but in situ enzymatic assays demonstrated that RAD001 is not a direct SK1 inhibitor. Treatment with RAD001 alone did not significantly reduce PC-3 cell proliferation, but has sensitized them to docetaxel allowing a 4-fold reduction in the effective dose. The sensitization was SK1 dependent, as overexpression of SK1 has reduced caspase activation and rendered cells insensitive to RAD001 and docetaxel treatment. Increasing HIF1a expression with CoCl2 has only partially restored SK1 expression and prostate cancer cells chemoresistance. Combined treatment with low doses of docetaxel (5 mg/kg/biweekly) and RAD001 (5 mg/kg/3 times a week) has significantly reduced the tumour size of PC-3 xenografts established in nude mice (superior to individual treatments), which correlated with a significant reduction of SK1 expression and activity. Conclusion: Our results demonstrate a new molecular mechanism of interaction between mTOR and SK1 pathways that underlies the chemosensitizing properties of RAD001 in prostate cancer cell and mouse models. No conflict of interest.
540 Carnosol inhibits tumor growth and metastasis of breast cancer through inhibition of the histone acetytransferase activity of p300 and proteasome-dependent degradation of STAT3 R. Iratni1 , E.H. Hussain1 , A.D. Yusra1 , V. Ranjit1 . 1 UAE University, Biology, Al Ain, U.A.E. Introduction: Carnosol is a naturally occurring polyphenol (dietary diterpene) found in culinary herbs such as rosemary, sage, and oregano. Studies revealed that carnosol possesses many pharmacological activities, including antiinflammatory, anti-oxidant and anti-microbial properties. Here we investigated the in vivo anticancer effect of carnosol on triple negative breast cancer MDAMB-231 cells. Material and Method: Human breast cancer triple negative breast cancer (TNBC) MDA-MB-231 cell line was used. Protein expression and protein acetylation and phosphorylation was examined using Western blotting. Gelatin zymography was used to measure the activity of MMP-9. The in ovo chick chorioallantoic membrane (CAM) assay was used to study tumor growth and metastasis. Results: We found that carnosol significantly inhibited tumor growth and metastasis of breast cancer. Concentrations of 50 and 100 mM of carnosol led to significant reduction of tumor growth by 65 and 75% respectively. Moreover, carnosol inhibited invasion by downregulating MMP-9 expression. Mechanistically, we found that carnosol inhibited the STAT3 pathway by promoting the proteasome-dependent degradation of STAT3 protein. Interestingly, we found STAT3 rescued from proteasome degradation by proteasome inhibitors was neither phosphorylated nor acetylated. Additionally, we found that carnosol also inhibited the histone acetyltransferase activity of p300 and consequently target it to proteasome-dependent degradation. Moreover, molecular docking analysis indicate that carnosol appear to inhibit
S117
p300 HAT activity by blocking acetyl coenzyme A’s entry into its deep pocket. Carnosol was found to bind at the entrance to this active site cavity. Conclusion: Our findings identify the natural compound carnosol as a new inhibitor of p300 HAT with a potent anti-tumor growth of TNBC. No conflict of interest. 541 Plasma microRNA profiles; down-regulation of plasma miR-107 level contributes to poor outcomes in pancreatic cancer T. Imamura1 , S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , R. Morimura1 , H. Ikoma1 , H. Konishi1 , A. Shiozaki1 , H. Taniguchi2 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan, 2 Kyoto Second Red Cross Hospital, Department of Surgery, Kyoto, Japan Background: This study aimed to explore decreased tumor suppressive microRNAs (miRNAs) in plasma of pancreatic cancer (PCa) patients and detect their possible roles as a treatment target as well as a biomarker for PCa. Methods and Results: Six down-regulated miRNAs (miR-107, miR-126, miR-451, miR-145, miR-491-5p, and miR-146b-5p) were selected using a miRNAs microarray-based approach. Test- and large scale analyses revealed that plasma miR-107 level in PCa patients was most down-regulated among candidates compared to healthy volunteers (p<0.001). A low level of plasma miR-107 was significantly associated with advanced T- and N-stage, and significantly associated with a worse cause-specific survival, survival after surgery, and disease free survival rate after surgery. Multivariate analysis identified a low level of miR-107 as an independent poor prognostic factor in PCa patients (p = 0.012, Hazard ratio 4.56 (95% CI: 1.38–17.4)). Enforced expression of miR-107 in PCa cell lines induced the production of p21, which results in G1/S arrest, and inhibited cell proliferation, migration, and invasion. Furthermore, in vivo study demonstrated that the restoration of plasma miR-107 could suppress tumor growth. Conclusion: Down regulation of plasma miRNA-107, which serves as tumor suppressive functions in PCa contributes to disease progression and poor outcomes in PCa and preserving blood level of miR-107 might be a novel treatment candidate for PCa. No conflict of interest. 543 mTOR inhibition overcomes docetaxel chemoresistance in prostate cancer cell and mouse models through downregulation of sphingosine kinase 1 H. Alshaker1,2 , Q. Wang2 , C. Cooper2 , M. Winkler3 , D. Pchejetski2 . 1 University of Petra, Department of Pharmacology and Biomedical Sciences, Amman, Jordan, 2 University of East Anglia, School of Medicine, Norwich, United Kingdom, 3 Department of Surgery and Cancer, Imperial College London, London, United Kingdom Background: Sphingosine kinase 1 (SK1) and PI3K/Akt/mTOR pathways are widely implicated in cancer progression and docetaxel chemoresistance. Here we have investigated the chemosensitizing mechanism of mTOR inhibitor RAD001 (everolimus) in prostate cancer. Material and Methods: Cell viability and apoptosis assays were used to investigate the combined effects of RAD001 and docetaxel in prostate cancer cell cultures. SK1 activity was measured by radiolabelling and alterations in cell signalling and gene expression were measured by Western blotting and qRT-PCR. Mouse prostate cancer xenografts were used to confirm in vitro findings. Results: Treatment of hormone resistant prostate cancer cells with RAD001 induced a strong downregulation of HIF1a protein and SK1 mRNA expression. SK1 enzymatic activity was also inhibited, but in situ enzymatic assays demonstrated that RAD001 is not a direct SK1 inhibitor. Treatment with RAD001 alone did not significantly reduce PC-3 cell proliferation, but has sensitized them to docetaxel allowing a 4-fold reduction in the effective dose. The sensitization was SK1 dependent, as overexpression of SK1 has reduced caspase activation and rendered cells insensitive to RAD001 and docetaxel treatment. Increasing HIF1a expression with CoCl2 has only partially restored SK1 expression and prostate cancer cells chemoresistance. Combined treatment with low doses of docetaxel (5 mg/kg/biweekly) and RAD001 (5 mg/kg/3 times a week) has significantly reduced the tumour size of PC-3 xenografts established in nude mice (superior to individual treatments), which correlated with a significant reduction of SK1 expression and activity. Conclusion: Our results demonstrate a new molecular mechanism of interaction between mTOR and SK1 pathways that underlies the chemosensitizing properties of RAD001 in prostate cancer cell and mouse models. No conflict of interest.