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142 EMT RELATED MICRORNA-200 FAMILY FUNCTION AS TUMOR SUPPRESSORS IN RENAL CELL CARCINOMA
Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012
141 PI3K REGULATES P27 THROUGH MTORC2 IN RENAL CELL CARCINOMA Karthigayan Shanmugasundaram, Karen Block, Carolina Livi, Manjeri Venkatachalam, Sunil Sudarshan*, San Antonio, TX INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most lethal of all urologic malignancies. Moreover, its incidence is rising and it is among the ten most common malignancies in both men and women. Dysregulation of the cell cycle regulator p27, particularly reduced nuclear expression has been shown to inversely correlate with RCC tumor stage and grade. The PI3K/AKT signaling axis has been implicated in the molecular pathogenesis of RCC and has been previously shown to regulate p27 through a variety of mechanisms. For example, PI3K signaling may regulate AKT-mediated phosphorylation of p27 which can impact p27 localization and steadystate levels. In addition PI3K/ AKT signaling may regulate levels of SKP2, a component of the SCFSKP2 ubiquitin ligase complex that mediates p27 proteolysis. Recent evidence suggests that the mTOR signaling complex 2 (mTORC2), in conjunction with PI3K signaling, regulates AKT activation. Based on these data, we investigated the role of mTORC2 in p27 regulation in the context of RCC. METHODS: Both pharmacologic and genetic techniques were utilized to determine the effects of mTORC2 signaling on p27. Effects on p27 steady state levels were analyzed through the combined use of real time RT-PCR as well as immunoblotting. Effects on localization were determined through cell fractionation studies in addition to immunofluorescence. The impact of mTORC2 on cell proliferation was determined through cell cycle analysis in addition to thymidine incorporation assays. RESULTS: Similar to inhibition of PI3K signaling, inhibition of mTORC2, either via genetic or pharmacologic means, led to an increase in both total as well as nuclear p27 protein levels. mTORC2 signaling, through AKT activation, mediates p27 levels in part through the regulation of SKP2. Finally, mTORC2 blockade led to inhibition of cell cycle progression with concomitant reduced cellular proliferation in RCC cells. CONCLUSIONS: Our data demonstrate a role for mTORC2 in the regulation of p27 levels as well as localization in RCC cells. Furthermore, our data demonstrate that these effects mediate the proliferation of RCC cells. Given the established role of p27 as a prognostic biomarker for RCC, our data suggest a role for targeting mTORC2 in the treatment of kidney cancer. Source of Funding: AUA/Astellas Rising Star Award NIH K08 CA138774 Voelcker Fund Young Investigator Award
142 EMT RELATED MICRORNA-200 FAMILY FUNCTION AS TUMOR SUPPRESSORS IN RENAL CELL CARCINOMA Hirofumi Yoshino*, Hideki Enokida, Takeshi Yamasaki, Hideo Hidaka, Takeshi Chiyomaru, Kagoshima, Japan; Nijiro Nohata, Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC represents the most common renal cancer histology. However surgical treatment is provided for localized disease, relapse or metastasis of the patient is caused in a considerable ratio. At present, metastatic RCC is difficult to treat and the process of metastasis is not well understood. Therefore, it is crucial to find molecular mechanisms based on recent genome analysis in RCC oncogenesis and metastasis. Based on the microRNA (miRNA) expression signature of RCC revealed that miR-200 family significantly reduced in RCC cells. The miR-200 family (miR-200b, miR-200a and miR-429 are encoded by single polycistronic transcript on chromosome 1p36.33 and miR-200c and miR-
THE JOURNAL OF UROLOGY姞
e59
141 are cluster on chromosome 12 p13.31) of miRNA plays a major role to epithelial to mesenchymal transition (EMT) by targeting transcription repressors, zinc-finger E-box binding homeobox (ZEB1) and ZEB2. In this study, we investigated the functional significance of miR-200 family and identified the novel cancer pathways in RCC. METHODS: Cell proliferation and invasion assay was performed by restoration of mature miR-200 family (miR-200a, miR-200b, miR-200c, miR-429 and miR-141) in RCC cell lines. Genome-wide gene expression analysis was performed to identify the molecular networks of miR-200 family by microarray analysis. RESULTS: The mRNA expression levels of ZEB1 and ZEB2 were significantly decreased by miR-200 family (miR-200a, miR-200b, miR-200c, miR-429 and miR-141) transfectantion in RCC cells (P ⬍ 0.0001). Restoration of each miRNAs significantly inhibited cell proliferation and invasion in RCC cells (P ⬍ 0.0001). Interestingly, the morphological changes were recognized by miR-200 family transfection in RCC cell lines. Gene expression analysis showed more than ten candidate genes were searched for miR-200 family targets and these genes were up-regulated in RCC clinical specimens. CONCLUSIONS: Our data suggest that miR-200 family function as tumor suppressors in RCC. EMT-related tumor suppressive miR-200 family mediates novel molecular targets provide new insights into the potential mechanisms of RCC oncogenesis and metastasis. Source of Funding: None
143 HIGH-FAT DIET INCREASES RENAL CANCER GROWTH ACCOMPANIED BY LEPTIN INCREASE, AND SURVIVIN INHIBITION IS EFFECTIVE FOR SIMVASTATIN RESISTANT RENAL CANCER. Hidekazu Koike*, Maebashi, Japan; Takashi Nitta, Yoshitaka Sekine, Yosuke Furuya, Yasuyuki Morikawa, Hiroshi Matsui, Yasuhiro Shibata, Kazuhiro Suzuki, Maeabshi, Japan INTRODUCTION AND OBJECTIVES: Recently, there is a report that obesity is associated with a higher risk of clear-cell renal cell carcinoma. Statins might reduce risk of renal cell carcinoma in humans. In this study, we examined the mechanism of tumor growth by high-fat diet. Next, we studied the antitumor mechanism of simvastatin. Furthermore, in statin resistant renal cancer cells, we examined the role of survivin (a member of the inhibitor of apoptosis protein) and the antitumor sensitization effect due to survivin inhibition in statin treatment. METHODS: Mice transplanted s.c. with caki1 cells were injected intraperitoneally with 20mg/kg simvastatin every day under high-fat or normal diet. We examined tumor growth and Leptin density of blood. Next, we examined the anticancer mechanism of simvastatin associated with IGF-1r on Caki1 cells. Furthermore, we examined the antitumor sensitization effect due to survivin inhibition in statin treatment for statin resistant caki1 and KMRC1 cells. RESULTS: Tumor volume tended to be higher in the high-fat diet group than in the normal diet group. Leptin were increased in the high-fat diet group. Adiponectin tended to decrease in the high-fat diet group. Leptin increased cell proliferation. However, simvastatin did not decrease Leptin. In Caki1 cells, simvastatin inhibited cell proliferation decreasing gene expression levels of IGF-1r. Although simvastatin inhibited tumor growth, survivin mRNA were increased compared with control group. In simvastatin resistant caki1 cells, survivin gene expression levels were up-regulated, and simvastatin inhibited the cell proliferation in the presence of survivin siRNA. In KMRC1 cells, Low-dose simvastatin did not inhibit cell proliferation. However, in the presence of survivin siRNA, low-dose simvastatin inhibited KMRC1 cell proliferation. CONCLUSIONS: High-fat diet increased renal cell tumor growth increasing Leptin and decreasing Adiponectin density of blood. Simvastatin has anticancer effect being associated with IGF-1r. Survivin is associated with simvastatin resistance of renal cancer cells. So, the down-regulation of survivin would be also a potential candidate for a novel approach to treat simvastatin resistant renal cancer.
141 PI3K REGULATES P27 THROUGH MTORC2 IN RENAL CELL CARCINOMA Karthigayan Shanmugasundaram, Karen Block, Carolina Livi, Manjeri Venkatachalam, Sunil Sudarshan*, San Antonio, TX INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most lethal of all urologic malignancies. Moreover, its incidence is rising and it is among the ten most common malignancies in both men and women. Dysregulation of the cell cycle regulator p27, particularly reduced nuclear expression has been shown to inversely correlate with RCC tumor stage and grade. The PI3K/AKT signaling axis has been implicated in the molecular pathogenesis of RCC and has been previously shown to regulate p27 through a variety of mechanisms. For example, PI3K signaling may regulate AKT-mediated phosphorylation of p27 which can impact p27 localization and steadystate levels. In addition PI3K/ AKT signaling may regulate levels of SKP2, a component of the SCFSKP2 ubiquitin ligase complex that mediates p27 proteolysis. Recent evidence suggests that the mTOR signaling complex 2 (mTORC2), in conjunction with PI3K signaling, regulates AKT activation. Based on these data, we investigated the role of mTORC2 in p27 regulation in the context of RCC. METHODS: Both pharmacologic and genetic techniques were utilized to determine the effects of mTORC2 signaling on p27. Effects on p27 steady state levels were analyzed through the combined use of real time RT-PCR as well as immunoblotting. Effects on localization were determined through cell fractionation studies in addition to immunofluorescence. The impact of mTORC2 on cell proliferation was determined through cell cycle analysis in addition to thymidine incorporation assays. RESULTS: Similar to inhibition of PI3K signaling, inhibition of mTORC2, either via genetic or pharmacologic means, led to an increase in both total as well as nuclear p27 protein levels. mTORC2 signaling, through AKT activation, mediates p27 levels in part through the regulation of SKP2. Finally, mTORC2 blockade led to inhibition of cell cycle progression with concomitant reduced cellular proliferation in RCC cells. CONCLUSIONS: Our data demonstrate a role for mTORC2 in the regulation of p27 levels as well as localization in RCC cells. Furthermore, our data demonstrate that these effects mediate the proliferation of RCC cells. Given the established role of p27 as a prognostic biomarker for RCC, our data suggest a role for targeting mTORC2 in the treatment of kidney cancer. Source of Funding: AUA/Astellas Rising Star Award NIH K08 CA138774 Voelcker Fund Young Investigator Award
142 EMT RELATED MICRORNA-200 FAMILY FUNCTION AS TUMOR SUPPRESSORS IN RENAL CELL CARCINOMA Hirofumi Yoshino*, Hideki Enokida, Takeshi Yamasaki, Hideo Hidaka, Takeshi Chiyomaru, Kagoshima, Japan; Nijiro Nohata, Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC represents the most common renal cancer histology. However surgical treatment is provided for localized disease, relapse or metastasis of the patient is caused in a considerable ratio. At present, metastatic RCC is difficult to treat and the process of metastasis is not well understood. Therefore, it is crucial to find molecular mechanisms based on recent genome analysis in RCC oncogenesis and metastasis. Based on the microRNA (miRNA) expression signature of RCC revealed that miR-200 family significantly reduced in RCC cells. The miR-200 family (miR-200b, miR-200a and miR-429 are encoded by single polycistronic transcript on chromosome 1p36.33 and miR-200c and miR-
THE JOURNAL OF UROLOGY姞
e59
141 are cluster on chromosome 12 p13.31) of miRNA plays a major role to epithelial to mesenchymal transition (EMT) by targeting transcription repressors, zinc-finger E-box binding homeobox (ZEB1) and ZEB2. In this study, we investigated the functional significance of miR-200 family and identified the novel cancer pathways in RCC. METHODS: Cell proliferation and invasion assay was performed by restoration of mature miR-200 family (miR-200a, miR-200b, miR-200c, miR-429 and miR-141) in RCC cell lines. Genome-wide gene expression analysis was performed to identify the molecular networks of miR-200 family by microarray analysis. RESULTS: The mRNA expression levels of ZEB1 and ZEB2 were significantly decreased by miR-200 family (miR-200a, miR-200b, miR-200c, miR-429 and miR-141) transfectantion in RCC cells (P ⬍ 0.0001). Restoration of each miRNAs significantly inhibited cell proliferation and invasion in RCC cells (P ⬍ 0.0001). Interestingly, the morphological changes were recognized by miR-200 family transfection in RCC cell lines. Gene expression analysis showed more than ten candidate genes were searched for miR-200 family targets and these genes were up-regulated in RCC clinical specimens. CONCLUSIONS: Our data suggest that miR-200 family function as tumor suppressors in RCC. EMT-related tumor suppressive miR-200 family mediates novel molecular targets provide new insights into the potential mechanisms of RCC oncogenesis and metastasis. Source of Funding: None
143 HIGH-FAT DIET INCREASES RENAL CANCER GROWTH ACCOMPANIED BY LEPTIN INCREASE, AND SURVIVIN INHIBITION IS EFFECTIVE FOR SIMVASTATIN RESISTANT RENAL CANCER. Hidekazu Koike*, Maebashi, Japan; Takashi Nitta, Yoshitaka Sekine, Yosuke Furuya, Yasuyuki Morikawa, Hiroshi Matsui, Yasuhiro Shibata, Kazuhiro Suzuki, Maeabshi, Japan INTRODUCTION AND OBJECTIVES: Recently, there is a report that obesity is associated with a higher risk of clear-cell renal cell carcinoma. Statins might reduce risk of renal cell carcinoma in humans. In this study, we examined the mechanism of tumor growth by high-fat diet. Next, we studied the antitumor mechanism of simvastatin. Furthermore, in statin resistant renal cancer cells, we examined the role of survivin (a member of the inhibitor of apoptosis protein) and the antitumor sensitization effect due to survivin inhibition in statin treatment. METHODS: Mice transplanted s.c. with caki1 cells were injected intraperitoneally with 20mg/kg simvastatin every day under high-fat or normal diet. We examined tumor growth and Leptin density of blood. Next, we examined the anticancer mechanism of simvastatin associated with IGF-1r on Caki1 cells. Furthermore, we examined the antitumor sensitization effect due to survivin inhibition in statin treatment for statin resistant caki1 and KMRC1 cells. RESULTS: Tumor volume tended to be higher in the high-fat diet group than in the normal diet group. Leptin were increased in the high-fat diet group. Adiponectin tended to decrease in the high-fat diet group. Leptin increased cell proliferation. However, simvastatin did not decrease Leptin. In Caki1 cells, simvastatin inhibited cell proliferation decreasing gene expression levels of IGF-1r. Although simvastatin inhibited tumor growth, survivin mRNA were increased compared with control group. In simvastatin resistant caki1 cells, survivin gene expression levels were up-regulated, and simvastatin inhibited the cell proliferation in the presence of survivin siRNA. In KMRC1 cells, Low-dose simvastatin did not inhibit cell proliferation. However, in the presence of survivin siRNA, low-dose simvastatin inhibited KMRC1 cell proliferation. CONCLUSIONS: High-fat diet increased renal cell tumor growth increasing Leptin and decreasing Adiponectin density of blood. Simvastatin has anticancer effect being associated with IGF-1r. Survivin is associated with simvastatin resistance of renal cancer cells. So, the down-regulation of survivin would be also a potential candidate for a novel approach to treat simvastatin resistant renal cancer.