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431 MOLECULAR PATHWAYS REGULATED BY TUMOR SUPPRESSIVE MICRORNAS IN RENAL CELL CARCINOMA BASED ON MICRORNA EXPRESSION SIGNATURE
Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012
cordance with primary metastatic tumors. Specific expression in metastases might be caused by a specific microenvironment at the metastatic site. MiR-30c regulates cellular processes involved in metastasis. MiRNA target identification can provide an insight into signal pathways deregulated in metastasis. Thus, several candidate target proteins of miR-30c and miR-451 were identified which have to be validated in this ongoing study. Source of Funding: Dr.-Robert-Pfleger-Stiftung
431 MOLECULAR PATHWAYS REGULATED BY TUMOR SUPPRESSIVE MICRORNAS IN RENAL CELL CARCINOMA BASED ON MICRORNA EXPRESSION SIGNATURE Hideo Hidaka*, Hirofumi Yoshino, Hideki Enokida, Takeshi Yamasaki, Shuichi Tatarano, Kagoshima, Japan; 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. It is needed to find molecular mechanisms based on recent genome analysis in RCC oncogenesis and metastasis. Growing evidences suggested that microRNAs (miRNAs) contribute to the initiation, development and metastasis of various types of cancer. Some lower expressed miRNAs could function as tumor suppressors by negatively regulating oncogenes. In this study, we identified down-regulated miRNAs based on RCC expression signature and investigated the functional significance of these down-regulated miRNAs. Furthermore, molecular pathways regulated by tumor suppressive miRNAs were identified. METHODS: The expression levels of 667 human mature miRNAs were examined using real-time quantitative PCR (Taq Man MicroRNA Assay; Human Panel v2.0) of 10 paired RCC and the normal tissues. Cell proliferation assay was performed to investigate functional significance of top 20 down-regulated miRNAs by RCC signature using mature miRNA transfectant RCC cells. Genome-wide gene expression analysis was performed to identify the molecular targets of miR-1285 by microarray technique. RESULTS: We identified 104 down-regulated miRNAs based on miRNA expression signature. Among the down-regulated miRNAs, transient transfection of miR-1285, miR-206, miR-1, miR-200c, miR135a, miR-429, and miR-133b inhibited cell growth in cancer cell lines by XTT assay (each, P⬍0.0001). In this study, we focused on miR1285 which was most inhibited cell proliferation. Genome-wide molecular targets search showed that seven genes (LHPP, TGM2, NF2, CERCAM, SYNPO, LYPLA2 and AHNAK) were down-regulated more than 4.0-fold in miR-1285 transfectants compared to the controls, and they have putative target sites on their 3▫fUTR region. We also found that the expression level of TGM2 was elevated in RCC specimens compared with matched normal tissues (P⬍0.0037). CONCLUSIONS: We identified tumor suppressive miRNAs (miR-1285, miR-206, miR-1, miR-200c, miR-135a, miR-429 and miR133b) according to the expression signature and functional screening in RCC. Genome-wide analysis revealed that tumor suppressive miR1285 regulates molecular pathways involved in RCC development. Tumor suppressive miRNA mediates novel molecular pathways provide new insights of molecular mechanisms of RCC oncogenesis. Source of Funding: None
THE JOURNAL OF UROLOGY姞
e177
432 A NOVEL TUMOR-ASSOCIATED ANTIGEN, DNAJB8 IS HIGHLY EXPRESSED IN CANCER STEM-LIKE CELLS/CANCER-INITIATING CELLS OF RENAL CELL CARCINOMA, AND IS POTENT TARGET FOR IMMUNOTHERAPY. Satoshi Nishizawa*, Wakayama, Japan; Yoshihiko Hirohashi, Toshihiko Torigoe, Sapporo, Japan; Nagahide Matsumura, Takeshi Inagaki, Yasuo Kohjimoto, Wakayama, Japan; Noriyuki Sato, Sapporo, Japan; Isao Hara, Wakayama, Japan INTRODUCTION AND OBJECTIVES: Cancers are supposed to contain a small population of highly tumorigenic cells expressing stem cell-like markers, called Cancer stem-like cells/Cancer-initiating cells (CSCs/CICs). We found that tumor-associated antigen, DNAJB8 was highly expressed in CSCs/CICs. We evaluated usefulness of CSCs/CICs-targeted immunotherapy. METHODS: CSCs/CICs in mouse renal cell cancer cell line (RenCa) were isolated as side population (SP) cells with Hoechst33342 staining. The expression of DNAJB8 was assessed by flowcytometric analysis in SP and main population (MP). The expression of DNAJB8 was enhanced by gene transduction. DNAJB8 immunization was done with subcutaneous inoculation of DNA plasmid vaccine including DNAJB8 cDNA. Cytotoxic T lymphocytes (CTL) induced by immunization was evaluated by IFN-gamma Elispot assay. In vivo anti-tumor effect of DNAJB8 vaccination was assessed by inoculation of RenCa into immunized Balb/c mice. RESULTS: SP cells of RenCa exhibited stem cell-like features and higher tumorigenicity than MP cells, suggesting enriched with CSCs/CICs population. DNAJB8 was preferentially expressed in SP cells compared with MP cells. The enhanced expression of DNAJB8 by gene transduction into RenCa significantly increased the population of SP and greater tumorigenicity in vivo. DNA plasmid vaccine coding DNAJB8 CTL induced specific IFN-gamma secretion of CD8⫹ T cells. DNAJB8 immunization by plasmid DNA significantly inhibited the tumor growth comparing with other type of tumor antigen, Survivin, which was expressed in both SP and MP cells equivalently (fig). CONCLUSIONS: DNAJB8 is highly expressed in CSCs/CICs of RenCa. DNAJB8 might be a powerful target for immunotherapy against CSCs/CICs.
Source of Funding: None
433 SPERM ASSOCIATED ANTIGEN 4 IS A NOVEL BIOMARKER FOR RENAL CELL CARCINOMA Takumi Shiraishi*, Naoki Terada, Yu Zeng, Steven Mooney, Sayuri Takahashi, Baltimore, MD; Natsuki Takaha, Tsuneharu Miki, Kyoto, Japan; Robert Getzenberg, Prakash Kulkarni, Baltimore, MD INTRODUCTION AND OBJECTIVES: Loss of the von HippelLindau (VHL) tumor suppressor gene is associated with a variety of proliferative disorders, especially renal cell carcinoma (RCC). In the
cordance with primary metastatic tumors. Specific expression in metastases might be caused by a specific microenvironment at the metastatic site. MiR-30c regulates cellular processes involved in metastasis. MiRNA target identification can provide an insight into signal pathways deregulated in metastasis. Thus, several candidate target proteins of miR-30c and miR-451 were identified which have to be validated in this ongoing study. Source of Funding: Dr.-Robert-Pfleger-Stiftung
431 MOLECULAR PATHWAYS REGULATED BY TUMOR SUPPRESSIVE MICRORNAS IN RENAL CELL CARCINOMA BASED ON MICRORNA EXPRESSION SIGNATURE Hideo Hidaka*, Hirofumi Yoshino, Hideki Enokida, Takeshi Yamasaki, Shuichi Tatarano, Kagoshima, Japan; 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. It is needed to find molecular mechanisms based on recent genome analysis in RCC oncogenesis and metastasis. Growing evidences suggested that microRNAs (miRNAs) contribute to the initiation, development and metastasis of various types of cancer. Some lower expressed miRNAs could function as tumor suppressors by negatively regulating oncogenes. In this study, we identified down-regulated miRNAs based on RCC expression signature and investigated the functional significance of these down-regulated miRNAs. Furthermore, molecular pathways regulated by tumor suppressive miRNAs were identified. METHODS: The expression levels of 667 human mature miRNAs were examined using real-time quantitative PCR (Taq Man MicroRNA Assay; Human Panel v2.0) of 10 paired RCC and the normal tissues. Cell proliferation assay was performed to investigate functional significance of top 20 down-regulated miRNAs by RCC signature using mature miRNA transfectant RCC cells. Genome-wide gene expression analysis was performed to identify the molecular targets of miR-1285 by microarray technique. RESULTS: We identified 104 down-regulated miRNAs based on miRNA expression signature. Among the down-regulated miRNAs, transient transfection of miR-1285, miR-206, miR-1, miR-200c, miR135a, miR-429, and miR-133b inhibited cell growth in cancer cell lines by XTT assay (each, P⬍0.0001). In this study, we focused on miR1285 which was most inhibited cell proliferation. Genome-wide molecular targets search showed that seven genes (LHPP, TGM2, NF2, CERCAM, SYNPO, LYPLA2 and AHNAK) were down-regulated more than 4.0-fold in miR-1285 transfectants compared to the controls, and they have putative target sites on their 3▫fUTR region. We also found that the expression level of TGM2 was elevated in RCC specimens compared with matched normal tissues (P⬍0.0037). CONCLUSIONS: We identified tumor suppressive miRNAs (miR-1285, miR-206, miR-1, miR-200c, miR-135a, miR-429 and miR133b) according to the expression signature and functional screening in RCC. Genome-wide analysis revealed that tumor suppressive miR1285 regulates molecular pathways involved in RCC development. Tumor suppressive miRNA mediates novel molecular pathways provide new insights of molecular mechanisms of RCC oncogenesis. Source of Funding: None
THE JOURNAL OF UROLOGY姞
e177
432 A NOVEL TUMOR-ASSOCIATED ANTIGEN, DNAJB8 IS HIGHLY EXPRESSED IN CANCER STEM-LIKE CELLS/CANCER-INITIATING CELLS OF RENAL CELL CARCINOMA, AND IS POTENT TARGET FOR IMMUNOTHERAPY. Satoshi Nishizawa*, Wakayama, Japan; Yoshihiko Hirohashi, Toshihiko Torigoe, Sapporo, Japan; Nagahide Matsumura, Takeshi Inagaki, Yasuo Kohjimoto, Wakayama, Japan; Noriyuki Sato, Sapporo, Japan; Isao Hara, Wakayama, Japan INTRODUCTION AND OBJECTIVES: Cancers are supposed to contain a small population of highly tumorigenic cells expressing stem cell-like markers, called Cancer stem-like cells/Cancer-initiating cells (CSCs/CICs). We found that tumor-associated antigen, DNAJB8 was highly expressed in CSCs/CICs. We evaluated usefulness of CSCs/CICs-targeted immunotherapy. METHODS: CSCs/CICs in mouse renal cell cancer cell line (RenCa) were isolated as side population (SP) cells with Hoechst33342 staining. The expression of DNAJB8 was assessed by flowcytometric analysis in SP and main population (MP). The expression of DNAJB8 was enhanced by gene transduction. DNAJB8 immunization was done with subcutaneous inoculation of DNA plasmid vaccine including DNAJB8 cDNA. Cytotoxic T lymphocytes (CTL) induced by immunization was evaluated by IFN-gamma Elispot assay. In vivo anti-tumor effect of DNAJB8 vaccination was assessed by inoculation of RenCa into immunized Balb/c mice. RESULTS: SP cells of RenCa exhibited stem cell-like features and higher tumorigenicity than MP cells, suggesting enriched with CSCs/CICs population. DNAJB8 was preferentially expressed in SP cells compared with MP cells. The enhanced expression of DNAJB8 by gene transduction into RenCa significantly increased the population of SP and greater tumorigenicity in vivo. DNA plasmid vaccine coding DNAJB8 CTL induced specific IFN-gamma secretion of CD8⫹ T cells. DNAJB8 immunization by plasmid DNA significantly inhibited the tumor growth comparing with other type of tumor antigen, Survivin, which was expressed in both SP and MP cells equivalently (fig). CONCLUSIONS: DNAJB8 is highly expressed in CSCs/CICs of RenCa. DNAJB8 might be a powerful target for immunotherapy against CSCs/CICs.
Source of Funding: None
433 SPERM ASSOCIATED ANTIGEN 4 IS A NOVEL BIOMARKER FOR RENAL CELL CARCINOMA Takumi Shiraishi*, Naoki Terada, Yu Zeng, Steven Mooney, Sayuri Takahashi, Baltimore, MD; Natsuki Takaha, Tsuneharu Miki, Kyoto, Japan; Robert Getzenberg, Prakash Kulkarni, Baltimore, MD INTRODUCTION AND OBJECTIVES: Loss of the von HippelLindau (VHL) tumor suppressor gene is associated with a variety of proliferative disorders, especially renal cell carcinoma (RCC). In the