- Email: [email protected]
525: Reactive oxygen species and nitric oxide in irradiated and irradiation-induced bystander cells
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 525 Reactive oxygen species and nitric oxide in irradiated and irradiation-induced bystander cells M. Skonieczna1 , K. Gajda1 , K. Biernacki1 , D. Hudy1 , A. Krzywon1 , S. Student1 , I. Slezak-Prochazka1 , M. Widel1 , J. Rzeszowska-Wolny1 . 1 Silesian University of Technology, Atomatic Control, Gliwice, Poland Background: We and others have reported that cells irradiated in vitro influence unirradiated neighbors by so-called bystander effects that can imitate the interactions between a tumor and surrounding tissues during cancer therapy with ionizing radiation. These effects are mediated by soluble factors released by irradiated cells and intra-cellular signaling pathways whose nature is not yet well elucidated. Here, to explore the possible role of reactive oxygen species (ROS) and nitric oxide (NO) in irradiated and bystander cells we measured their levels after different times of co-cultivation of irradiated and unirradiated cells. Materials and Methods: Human NHDF fibroblasts, Me45 melanoma cells, and HCT116 colon carcinoma cells were co-cultivated with irradiated (4 Gy of X-rays) or untreated cells of the same or a different type but separated by an 0.4 mm-pore membrane. Cellular ROS, superoxide, and NO levels and cell cycle effects were measured by specific fluorescent dyes and flow cytometry or microscopy. Results and Discussion: Cellular ROS levels change with time in cultured cells and depend strongly on the cell cycle, with an increase in G2 phase. Cocultivation of unirradiated fibroblasts with melanoma cells did not significantly influence cellular ROS and NO levels, but the levels of NO released during co-cultivation were higher in presence of melanoma cells. Production of mRNA for inducible NO synthase was markedly suppressed in fibroblasts cocultivated with either irradiated or un-irradiated melanoma cells, and increased in bystander HCT116 cells with a knockout of the P53 gene. Conclusions: Our observations suggest that NO released by cells may participate in bystander signaling though regulation of inducible NO synthase and that this regulation depends on the status of the TP53 gene. This work was financially supported by the National Science Center (NCN, Poland) under Decision No DEC-2012/05/NZ2/01618; DEC-2012/05/ B/ST6/03472 and by grant No. BKM/514/RAU-1/2013 t.26 from Silesian University of Technology. No conflict of interest. 526 TRAP1 is responsible for the co-translational regulation of BRAF and the downstream attenuation of ERK phosphorylation and cell cycle progression: A novel molecular target for human BRAF-mutated colorectal carcinomas L. Sisinni1 , V. Condelli1 , A. Piscazzi2 , D.S. Matassa3 , F. Maddalena1 , G. Lettini1 , G. Palladino2 , M.R. Amoroso3 , F. Esposito3 , M. Landriscina2 . 1 CROB − IRCCS, Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture (PZ), Italy, 2 University of Foggia, Clinical Oncology Unit Department of Medical and Surgical Sciences, Foggia, Italy, 3 University of Naples Federico II, Department of Molecular Medicine and Medical Biotechnology, Naples, Italy Background: Human BRAF-driven tumors are aggressive malignancies with poor clinical outcome and lack of sensitivity to standard therapies. TRAP1 is a HSP90 molecular chaperone deregulated in human tumors and responsible for several features of cancer cells, i.e. protection from apoptosis and drug resistance, cell metabolic regulation, protein quality control and ubiquitination of specific client proteins. Materials and Methods: Starting from the evidence that BRAF stability is under the control of HSP90 chaperones, we tested the hypothesis that TRAP1 plays a regulatory function on BRAF pathway in human breast and colorectal carcinoma cell lines. Results: TRAP1 is responsible for the co-translational regulation of TRAP1 synthesis/ubiquitination without affecting its long term stability. Indeed, BRAF synthesis is facilitated in a rich-TRAP1 background, whereas its protein levels are downreguled upon TRAP1 silencing and this correlates with increased co-translational ubiquitination. This regulatory activity on BRAF levels modulates its downstream pathway, since TRAP1 silencing induces the attenuation of ERK phosphorylation and the progression through the cell cycle with accumulation of cells in G0-G1 and G2-M transitions and a wide reprogramming of gene expression. Interestingly, a genome-wide profiling of TRAP1-silenced cells identified cell cycle regulation as the most significant function controlled by TRAP1. Noteworthy, TRAP1 regulation on BRAF is conserved in human CRC since a significant co-expression between the two proteins was observed. Finally, the dual HSP90/TRAP1 inhibitor HSP990 showed specific anti-TRAP1 activity and demonstrated to be highly active in human BRAF-mutated CRC cell lines. Conclusion: This novel function of TRAP1 on BRAF protein synthesis/ubiquitination open an attractive therapeutic window to target the dependency of BRAF-driven tumor cells on the TRAP1-dependent translational/quality control machinery. No conflict of interest.
S127
527 A hormone-dependent feedback-loop controls androgen receptor levels by limiting Midline1, a novel translation enhancer and promoter of oncogenic signaling U. Demir1 , A. Koehler2 , E. Kickstein3 , B. Aranda-Orgilles ´ 3 , H. Bu4 , M.R. Schweiger5 , G. Schaefer6 , S. Schweiger7 , H. Klocker1 , R. Schneider2 . 1 Univ.-Klinik Innsbruck, Dept. of Urology, Innsbruck, Austria, 2 Innsbruck University, Institute of Biochemistry, Innsbruck, Austria, 3 Max-Planck Institute for Molecular Genetics, Berlin, Germany, 4 Innsbruck University, Institute for Biomedical Aging, Innsbruck, Austria, 5 Max-Planck Institute for Molecular Genetics, 6Institute of Vertebrate Genetics, Berlin, Germany, 6 Univ.-Klinik Innsbruck, Dept. of Pathology, Innsbruck, Austria, 7 Medical University Mainz, Institute for Human Genetics, Mainz, Germany Introduction: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFkB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer (CRPCa) during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFkB and Hh signaling. Results and Discussion: Here we show that MID1 also strongly enhances the translation of AR. Modulations of MID1, experimentally in PCa cells or by natural mutations occurring in Opitz-BBB/G patients, directly correlate with AR protein levels. AR on the other hand, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. Concurrent promotion of AR-, Akt/mTOR-, NFkB- and Hh-activities by sustained MID1-upregulation during antiandrogen therapy therefore would provide a powerful proliferative scenario for PCa progression toward CRPCa. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. Conclusion: Thus, MID1 represents a novel, multi-faceted player in PCa and a promising target to treat CRPCa. No conflict of interest. 528 Cooperation between Rho-ROCK and STAT 3 signaling modulates colon cancer cell proliferation R. Peres-Moreira1 , F. Leve2 , R. Binato3 , E. Abdelhay3 , J.A. Morgado-D´ıaz1 . 1 ˆ Instituto Nacional de Cancer, Biologia Celular, Rio de Janeiro, Brazil, 2 ´ Instituto Nacional de Metrologia Qualidade e Tecnologia, Laboratorio de ˆ Biotecnologia, Rio de Janeiro, Brazil, 3 Instituto Nacional de Cancer, Centro ´ de Transplante de Medula Ossea, Rio de Janeiro, Brazil Background: Lysophosphatidic acid (LPA) activates specific receptors triggering pathways involved in colorectal cancer progression, but the role that it plays downstream its receptors in this cancer type is poorly understood. The aim of this study was to investigate the role of LPA in cell proliferation of colon cancer cell lines with different invasive potentials as well as the cell signaling pathways underlying this event. Material and Method: Cells derived from colorretal cancer, Caco-2, HT-29 and HCT-116 were treated with LPA (10 mM). Cell proliferation was analyzed by crystal violet technique and cell cycle progression by flow cytometry. The pathways involved in proliferation were investigated using the pharmacological inhibitors Y27632 (ROCK) and STA 21 (STAT3). The proteins expression and localization were monitored by immunoblotting and immunofluorescence, respectively. The TCF/LEF luciferase assay was used to ensure transcriptional activity of b-catenin. The global gene expression analysis was performed by ChipArray. Results: First, we observed that the three cell lines express LPA receptors (LPA 1−3), but in a differential manner, and that the treatment with LPA induced proliferation only in HCT-116 cells, in a Rho-ROCK dependent way. Next, we investigated the downstream signaling pathways that could modulate this event. We found that neither the transcriptional activity of b-catenin nor its localization was affected by LPA treatment. However, LPA promoted activation of STAT 3 in HCT-116 cells, as observed by increased levels of its phosphorylated form and its localization into the nucleus. This activation was Rho-ROCK-independent, since Y27632 did not prevent this effect. Interestingly, the ROCK and STAT 3 inhibitors, Y27632 and STA 21 respectively, when used separately, prevented the proliferation induced by LPA, and the associated inhibition of both proteins prevented the cell cycle progression in a stronger way than isolated inhibition with these inhibitors. Finally, the global gene expression analysis after LPA treatment showed increased expression of the cyclins E1, A2 and B1, which was confirmed by immunoblotting. Besides, the associated inhibition of ROCK and STAT 3 also prevented this effect. Conclusion: LPA increases the proliferative potential of HCT-116 cells, a cell line with high invasive potential, through a mechanism involving cooperation between the Rho-ROCK and STAT 3 pathways in the cell cycle control. No conflict of interest.
S127
527 A hormone-dependent feedback-loop controls androgen receptor levels by limiting Midline1, a novel translation enhancer and promoter of oncogenic signaling U. Demir1 , A. Koehler2 , E. Kickstein3 , B. Aranda-Orgilles ´ 3 , H. Bu4 , M.R. Schweiger5 , G. Schaefer6 , S. Schweiger7 , H. Klocker1 , R. Schneider2 . 1 Univ.-Klinik Innsbruck, Dept. of Urology, Innsbruck, Austria, 2 Innsbruck University, Institute of Biochemistry, Innsbruck, Austria, 3 Max-Planck Institute for Molecular Genetics, Berlin, Germany, 4 Innsbruck University, Institute for Biomedical Aging, Innsbruck, Austria, 5 Max-Planck Institute for Molecular Genetics, 6Institute of Vertebrate Genetics, Berlin, Germany, 6 Univ.-Klinik Innsbruck, Dept. of Pathology, Innsbruck, Austria, 7 Medical University Mainz, Institute for Human Genetics, Mainz, Germany Introduction: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFkB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer (CRPCa) during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFkB and Hh signaling. Results and Discussion: Here we show that MID1 also strongly enhances the translation of AR. Modulations of MID1, experimentally in PCa cells or by natural mutations occurring in Opitz-BBB/G patients, directly correlate with AR protein levels. AR on the other hand, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. Concurrent promotion of AR-, Akt/mTOR-, NFkB- and Hh-activities by sustained MID1-upregulation during antiandrogen therapy therefore would provide a powerful proliferative scenario for PCa progression toward CRPCa. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. Conclusion: Thus, MID1 represents a novel, multi-faceted player in PCa and a promising target to treat CRPCa. No conflict of interest. 528 Cooperation between Rho-ROCK and STAT 3 signaling modulates colon cancer cell proliferation R. Peres-Moreira1 , F. Leve2 , R. Binato3 , E. Abdelhay3 , J.A. Morgado-D´ıaz1 . 1 ˆ Instituto Nacional de Cancer, Biologia Celular, Rio de Janeiro, Brazil, 2 ´ Instituto Nacional de Metrologia Qualidade e Tecnologia, Laboratorio de ˆ Biotecnologia, Rio de Janeiro, Brazil, 3 Instituto Nacional de Cancer, Centro ´ de Transplante de Medula Ossea, Rio de Janeiro, Brazil Background: Lysophosphatidic acid (LPA) activates specific receptors triggering pathways involved in colorectal cancer progression, but the role that it plays downstream its receptors in this cancer type is poorly understood. The aim of this study was to investigate the role of LPA in cell proliferation of colon cancer cell lines with different invasive potentials as well as the cell signaling pathways underlying this event. Material and Method: Cells derived from colorretal cancer, Caco-2, HT-29 and HCT-116 were treated with LPA (10 mM). Cell proliferation was analyzed by crystal violet technique and cell cycle progression by flow cytometry. The pathways involved in proliferation were investigated using the pharmacological inhibitors Y27632 (ROCK) and STA 21 (STAT3). The proteins expression and localization were monitored by immunoblotting and immunofluorescence, respectively. The TCF/LEF luciferase assay was used to ensure transcriptional activity of b-catenin. The global gene expression analysis was performed by ChipArray. Results: First, we observed that the three cell lines express LPA receptors (LPA 1−3), but in a differential manner, and that the treatment with LPA induced proliferation only in HCT-116 cells, in a Rho-ROCK dependent way. Next, we investigated the downstream signaling pathways that could modulate this event. We found that neither the transcriptional activity of b-catenin nor its localization was affected by LPA treatment. However, LPA promoted activation of STAT 3 in HCT-116 cells, as observed by increased levels of its phosphorylated form and its localization into the nucleus. This activation was Rho-ROCK-independent, since Y27632 did not prevent this effect. Interestingly, the ROCK and STAT 3 inhibitors, Y27632 and STA 21 respectively, when used separately, prevented the proliferation induced by LPA, and the associated inhibition of both proteins prevented the cell cycle progression in a stronger way than isolated inhibition with these inhibitors. Finally, the global gene expression analysis after LPA treatment showed increased expression of the cyclins E1, A2 and B1, which was confirmed by immunoblotting. Besides, the associated inhibition of ROCK and STAT 3 also prevented this effect. Conclusion: LPA increases the proliferative potential of HCT-116 cells, a cell line with high invasive potential, through a mechanism involving cooperation between the Rho-ROCK and STAT 3 pathways in the cell cycle control. No conflict of interest.