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PD33-05 TUMOR-SUPPRESSIVE MICRORNA-29S-MEDIATED NOVEL MOLECULAR PATHWAYS IN RENAL CELL CARCINOMA
THE JOURNAL OF UROLOGYâ
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
insulin signaling in the progression of a murine RCC model using metformin to treat hyperinsulinemia induced by a high-carbohydrate diet. METHODS: pT1-T4 tumor specimens from 99 patients with RCC were evaluated for IR expression by immunohistochemistry. Serum insulin and C-peptide levels were measured. The association between biomarker expression and serum concentration, prognostic factors, and clinical outcomes were assessed. C57BL/6 mice were randomized into four groups: low-carbohydrate diet, high-carbohydrate diet, low-carbohydrate diet þ metformin, and high-carbohydrate diet þ metformin groups. RENCA cells were injected subcutaneously. Body weight, glucose tolerance test, serum insulin level, and tumor volume were evaluated. Immunoblotting was performed to assess the insulin signaling pathway in the murine RCC model. RESULTS: The IR expression level was significantly lower in patients with tumor stage pT2-4 and with a preoperative serum Cpeptide level more than 5.14 ng/mL. High IR expression was an independent predictor of favorable progression-free survival after radical nephrectomy. In vivo progression of RENCA in the murine model was not significantly stimulated by hyperinsulinemia induced by a high-carbohydrate diet. However, in vivo progression of RENCA was significantly inhibited by metformin in both the low- and highcarbohydrate diet groups. IR expression was significantly higher in the tumors from the low-carbohydrate diet group and high-carbohydrate diet plus metformin group than that from the high-carbohydrate diet group. CONCLUSIONS: IR expression in RCC was inversely associated with cancer progression and serum insulin levels in both clinical and murine models. The anticancer effect of metformin in the murine RCC model might be derived from the direct effect of the agent on cancer cells rather than the effect of the lower insulin level. The lower expression of IR in the tumor from patients with a poor outcome might be the result of a higher serum insulin level, which is not strong cause of cancer progression. Source of Funding: none
PD33-05 TUMOR-SUPPRESSIVE MICRORNA-29S-MEDIATED NOVEL MOLECULAR PATHWAYS IN RENAL CELL CARCINOMA Shuichi Tatarano*, Kagoshima, Japan; Rika Nishikawa, Chiba, Japan; Takeshi Chiyomaru, Hideki Enokida, Satoru Inoguchi, Tomoaki Ishihara, Hirofumi Yoshino, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) have been shown to play major roles in carcinogenesis in a variety of cancers, including renal cell carcinoma (RCC). Our recent study of miRNA expression signature in RCC revealed that miR-29s (miR29a/b/c) expression was significantly reduced in RCC tissues, suggesting miR-29s function as tumor suppressive miRNAs in RCC cells. The aim of this study was to investigate the functional significance of miR-29s and to identify these miRNAs-mediated oncogenic pathways in RCC. METHODS: Expression levels of miR-29s and these candidate target genes were evaluated in RCC cell lines (786-O and A498) and RCC clinical specimens (24 ccRCC and 24 non-cancerous tissues) by PCR methods. Gain-of-function studies using mature miR-29s were performed to investigate cell proliferation, migration and invasion in RCC cell lines. To identify the molecular pathways potentially regulated by the miR-29s, we applied gene expression data and in silico analysis using TargetScan database [http://www.targetscan.org/] and publicly available gene expression data set in GEO. Loss-of-function assays were performed to investigate the functional significance of miR-29s target genes. RESULTS: The expression level of miR-29a/b/c was significantly lower in RCC tissues and cell lines compared to normal tissues (P < 0.0002, P < 0.0003 and P < 0.0001). Restoration of miR-29s in RCC cell lines revealed significant inhibition of cancer cell proliferation,
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migration and invasion. In silico analysis and oligomicroarray analysis showed that Lysyl oxidase homolog 2 (LOXL2) was candidate target of miR-29s regulation. Silencing of LOXL2 significantly inhibited cancer cell migration and invasion. LOXL2 was direct regulation of miR-29s by luciferase reporter assay. Up-regulation of LOXL2 was detected in RCC specimens (p ¼ 0.0012). CONCLUSIONS: Down-regulation of miR-29s cause activation of LOXL2 signaling in RCC cells and these events contributes to RCC oncogenesis and metastasis. LOXL2 signaling plays a variety of cellular biological processes such as ECM stabilization and promoting EMT. Therefore, recognition of tumor suppressive miRNAmediated RCC pathways provides new therapeutic strategies for the disease. Source of Funding: none
PD33-06 A RETROSPECTIVE COHORT STUDY OF RENAL CANCER MOLECULAR HETEROGENEITY AND DNA METHYLATION PROGNOSTIC MARKERS. Alexander Laird*, Duncan Sproul, Grant Stewart, Fiach O’Mahony, Edinburgh, United Kingdom; Antony Riddick, Cambridge, United Kingdom; Richard Meehan, Edinburgh, United Kingdom; David Harrison, St Andrew’s, United Kingdom INTRODUCTION AND OBJECTIVES: Predicting which renal cell cancer (RCC) patients recur after surgery remains difficult and there are currently no molecular prognostic markers in clinical use. Genetic Intra-Tumoural Heterogeneity (genetic ITH) has been described in RCC and may limit clinical translation of biomarkers. There has been no assessment of ITH at other molecular levels. We aimed to define and compare morphological, proteomic, transcriptomic and DNA methylation ITH in RCC and identify potential prognostic biomarkers. METHODS: Tissue sampling was performed from multiple areas of primary tumours from 23 RCC patients (cohort 1) for intratumoural proteomic, transcrptomic and DNA methylation heterogeneity analysis. ITH was assessed using the ANOVA test and by comparison of unsupervised hierarchical clustering. Identification and validation of prognostic differentially methylated regions (DMR) was performed on 3 cohorts, 2 database and 1 experimental (cohort 2 TCGA, n¼154; cohort 3 Edinburgh, n¼46; cohort 4 CGP, n¼194). RESULTS: RCC exhibits protein, gene expression and methylation ITH. Protein ITH is greater than transcriptomic and methylation ITH for targets implicated in RCC pathogenesis (mean variance (MV [IQR]) 0.08[0.08], 0.07 [0.04], 0.07 [0.05] respectively; p<0.0001). Methylation ITH (MV 0.08 [0.05]) is significantly less than inter-patient variance of methylation (20.9 [8.5], p<0.0001). There were 322 DMRs between recurrent and non-recurrent patients in cohort 2 but only 5 DMRs were validated in cohort 3. NEFM gene promoter methylation was the only DMR associated with cancer specific survival (mean CSS; unmethylated-NEFM¼107months, methylated-NEFM¼49months; p<0.001), which was independent of TNM stage and nuclear grade on multivariate analysis (HR1.8 [1.1-3.0]; p¼0.02). This was confirmed on cohort 4. CONCLUSIONS: RCC intra-tumoural protein heterogeneity is significantly greater than methylation and gene-expression ITH. While methylation ITH exists, its occurrence is significantly less than interpatient variance. This led us to investigate methylation associated prognostic markers in RCC and we found promoter methylation of NEFM is associated with poorer cancer specific survival independent of tumour stage, grade and node status. Source of Funding: Medical Research Council, The RCSEd Robertson’s Trust and The Melville Trust
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
insulin signaling in the progression of a murine RCC model using metformin to treat hyperinsulinemia induced by a high-carbohydrate diet. METHODS: pT1-T4 tumor specimens from 99 patients with RCC were evaluated for IR expression by immunohistochemistry. Serum insulin and C-peptide levels were measured. The association between biomarker expression and serum concentration, prognostic factors, and clinical outcomes were assessed. C57BL/6 mice were randomized into four groups: low-carbohydrate diet, high-carbohydrate diet, low-carbohydrate diet þ metformin, and high-carbohydrate diet þ metformin groups. RENCA cells were injected subcutaneously. Body weight, glucose tolerance test, serum insulin level, and tumor volume were evaluated. Immunoblotting was performed to assess the insulin signaling pathway in the murine RCC model. RESULTS: The IR expression level was significantly lower in patients with tumor stage pT2-4 and with a preoperative serum Cpeptide level more than 5.14 ng/mL. High IR expression was an independent predictor of favorable progression-free survival after radical nephrectomy. In vivo progression of RENCA in the murine model was not significantly stimulated by hyperinsulinemia induced by a high-carbohydrate diet. However, in vivo progression of RENCA was significantly inhibited by metformin in both the low- and highcarbohydrate diet groups. IR expression was significantly higher in the tumors from the low-carbohydrate diet group and high-carbohydrate diet plus metformin group than that from the high-carbohydrate diet group. CONCLUSIONS: IR expression in RCC was inversely associated with cancer progression and serum insulin levels in both clinical and murine models. The anticancer effect of metformin in the murine RCC model might be derived from the direct effect of the agent on cancer cells rather than the effect of the lower insulin level. The lower expression of IR in the tumor from patients with a poor outcome might be the result of a higher serum insulin level, which is not strong cause of cancer progression. Source of Funding: none
PD33-05 TUMOR-SUPPRESSIVE MICRORNA-29S-MEDIATED NOVEL MOLECULAR PATHWAYS IN RENAL CELL CARCINOMA Shuichi Tatarano*, Kagoshima, Japan; Rika Nishikawa, Chiba, Japan; Takeshi Chiyomaru, Hideki Enokida, Satoru Inoguchi, Tomoaki Ishihara, Hirofumi Yoshino, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) have been shown to play major roles in carcinogenesis in a variety of cancers, including renal cell carcinoma (RCC). Our recent study of miRNA expression signature in RCC revealed that miR-29s (miR29a/b/c) expression was significantly reduced in RCC tissues, suggesting miR-29s function as tumor suppressive miRNAs in RCC cells. The aim of this study was to investigate the functional significance of miR-29s and to identify these miRNAs-mediated oncogenic pathways in RCC. METHODS: Expression levels of miR-29s and these candidate target genes were evaluated in RCC cell lines (786-O and A498) and RCC clinical specimens (24 ccRCC and 24 non-cancerous tissues) by PCR methods. Gain-of-function studies using mature miR-29s were performed to investigate cell proliferation, migration and invasion in RCC cell lines. To identify the molecular pathways potentially regulated by the miR-29s, we applied gene expression data and in silico analysis using TargetScan database [http://www.targetscan.org/] and publicly available gene expression data set in GEO. Loss-of-function assays were performed to investigate the functional significance of miR-29s target genes. RESULTS: The expression level of miR-29a/b/c was significantly lower in RCC tissues and cell lines compared to normal tissues (P < 0.0002, P < 0.0003 and P < 0.0001). Restoration of miR-29s in RCC cell lines revealed significant inhibition of cancer cell proliferation,
e711
migration and invasion. In silico analysis and oligomicroarray analysis showed that Lysyl oxidase homolog 2 (LOXL2) was candidate target of miR-29s regulation. Silencing of LOXL2 significantly inhibited cancer cell migration and invasion. LOXL2 was direct regulation of miR-29s by luciferase reporter assay. Up-regulation of LOXL2 was detected in RCC specimens (p ¼ 0.0012). CONCLUSIONS: Down-regulation of miR-29s cause activation of LOXL2 signaling in RCC cells and these events contributes to RCC oncogenesis and metastasis. LOXL2 signaling plays a variety of cellular biological processes such as ECM stabilization and promoting EMT. Therefore, recognition of tumor suppressive miRNAmediated RCC pathways provides new therapeutic strategies for the disease. Source of Funding: none
PD33-06 A RETROSPECTIVE COHORT STUDY OF RENAL CANCER MOLECULAR HETEROGENEITY AND DNA METHYLATION PROGNOSTIC MARKERS. Alexander Laird*, Duncan Sproul, Grant Stewart, Fiach O’Mahony, Edinburgh, United Kingdom; Antony Riddick, Cambridge, United Kingdom; Richard Meehan, Edinburgh, United Kingdom; David Harrison, St Andrew’s, United Kingdom INTRODUCTION AND OBJECTIVES: Predicting which renal cell cancer (RCC) patients recur after surgery remains difficult and there are currently no molecular prognostic markers in clinical use. Genetic Intra-Tumoural Heterogeneity (genetic ITH) has been described in RCC and may limit clinical translation of biomarkers. There has been no assessment of ITH at other molecular levels. We aimed to define and compare morphological, proteomic, transcriptomic and DNA methylation ITH in RCC and identify potential prognostic biomarkers. METHODS: Tissue sampling was performed from multiple areas of primary tumours from 23 RCC patients (cohort 1) for intratumoural proteomic, transcrptomic and DNA methylation heterogeneity analysis. ITH was assessed using the ANOVA test and by comparison of unsupervised hierarchical clustering. Identification and validation of prognostic differentially methylated regions (DMR) was performed on 3 cohorts, 2 database and 1 experimental (cohort 2 TCGA, n¼154; cohort 3 Edinburgh, n¼46; cohort 4 CGP, n¼194). RESULTS: RCC exhibits protein, gene expression and methylation ITH. Protein ITH is greater than transcriptomic and methylation ITH for targets implicated in RCC pathogenesis (mean variance (MV [IQR]) 0.08[0.08], 0.07 [0.04], 0.07 [0.05] respectively; p<0.0001). Methylation ITH (MV 0.08 [0.05]) is significantly less than inter-patient variance of methylation (20.9 [8.5], p<0.0001). There were 322 DMRs between recurrent and non-recurrent patients in cohort 2 but only 5 DMRs were validated in cohort 3. NEFM gene promoter methylation was the only DMR associated with cancer specific survival (mean CSS; unmethylated-NEFM¼107months, methylated-NEFM¼49months; p<0.001), which was independent of TNM stage and nuclear grade on multivariate analysis (HR1.8 [1.1-3.0]; p¼0.02). This was confirmed on cohort 4. CONCLUSIONS: RCC intra-tumoural protein heterogeneity is significantly greater than methylation and gene-expression ITH. While methylation ITH exists, its occurrence is significantly less than interpatient variance. This led us to investigate methylation associated prognostic markers in RCC and we found promoter methylation of NEFM is associated with poorer cancer specific survival independent of tumour stage, grade and node status. Source of Funding: Medical Research Council, The RCSEd Robertson’s Trust and The Melville Trust