CADM4 associated with 4.1B, while CADM1 interacted with 4.1N in normal human kidney. Western blotting showed that CADM4 expres- sion was lost or markedly reduced in 7 of 10 (70%) RCC cell lines and 14 of 20 (70%) primary RCCC. Treatment of 5’-aza CdR to 786-O cells restored CADM4 expression. Finally, introduction of CADM4 into 786-O cells suppressed tumor formation in nude mice. CONCLUSIONS: CADM4 cascade of cell adhesion is important in the proximal uriniferous tubules and appears to act as a tumor suppressor in RCCC. Source of Funding: None 358 EPIGENETIC UPREGULATION OF SECRETED FRIZZLED-RELATED PROTEIN 1 IN METASTATIC RENAL CELL CARCINOMAS: IMPLICATIONS ON INVASION AND METASTASIS Sharanjot Saini*, Jan Liu, Shahana Majid, Soichiro Yamamura, Kazumori Kawakami, Hiroshi Hirata, Yuichiro Tanaka, Peter R. Carroll, Rajvir Dahiya, San Francisco, CA INTRODUCTION AND OBJECTIVES: Dysregulation of Wing- less-type (Wnt) signaling pathway is common in a variety of human malignancies. Secreted Frizzled related proteins (SFRPs) are a family of secreted glycoproteins that act as modulators of Wnt signaling. The Secreted frizzled related protein 1 (SFRP1) is a Wnt antagonist that has been associated with various malignancies, including renal cell carci- nomas (RCC). However, the functional significance of SFRP1 has never been investigated in metastatic RCC. Here we investigated the role of this molecule in kidney cancer progression and metastasis. METHODS: RNA was extracted from a normal renal cell line (HK-2), primary RCC (A498, Caki2) and metastatic RCC cell lines (Caki1, ACHN and Hs891.T) and Wnt pathway-focused cDNA expres- sion profiling was done to identify dysregulated components of Wnt signaling. SFRP1 expression was confirmed by quantitative real-time PCR and immunostaining in renal tissues. We explored the molecular mechanisms underlying SFRP1 upregulation by analyzing DNA meth- ylation and histone modification patterns on SFRP1 promoter by bisul- fite sequencing and Chromatin immunoprecipitation (ChIP) assay, re- spectively. Further, to understand the functional significance of SFRP1 overexpression in metastatic RCC with regard to tumorigenesis, we used siRNA-mediated approach to knockdown the gene in metastatic RCC cell lines followed by functional assays for cellular proliferation, apoptosis and metastatic behavior. RESULTS: This study identified that SFRP1 is upregulated in metastatic RCC. We found that this gene is unmethylated/hypomethy- lated and enriched in activating histone modifications in metastatic RCC. Apoptosis increased upon attenuation of SFRP1 expression. Also SFRP1 depletion decreased the invasive potential of the meta- static RCC cell line suggesting that the overexpression of this Wnt antagonist may be related to invasiveness and metastatic behavior in RCC. We investigated the molecular basis of the role of SFRP1 in invasion and metastasis and found that matrix metalloproteinase MMP10 is regulated by SFRP1. CONCLUSIONS: Our data suggests that SFRP1 plays a role in the metastatic potential of RCC. Source of Funding: Grants RO1CA130860 and T32DK007790 from NIH 359 ONCOGENIC FUNCTIONS OF SECRETED FRIZZLED-RELATED PROTEIN 2 (SFRP2) IN HUMAN RENAL CANCER Soichiro Yamamura*, Kazumori Kawakami, Hiroshi Hirata, Koji Ueno, Sharanjot Saini, Shahana Majid, Peter Carroll, Rajvir Dahiya, San Francisco, CA INTRODUCTION AND OBJECTIVES: The secreted Frizzled- related proteins (sFRPs) are modulators of the Wnt signaling pathway which is involved in embryonic development and tumor progression. The functions of secreted frizzled-related protein 2 (sFRP2) have not been studied in renal cancer. Therefore, we investigated the functions of sFRP2 in renal cancer cells. METHODS: We studied expression level of sFRP2 using hu- man renal cancer tissue microarray. To study the functions of sFRP2 gene in renal carcinoma cells, we established A498 renal cancer cell lines which stably expressed sFRP2. To characterize the stable sFRP2 cell lines, we performed assays of in vitro and in vivo cell proliferation, apoptosis, cell cycle, cell invasion and Western blot. RESULTS: Human renal cancer tissue microarray revealed that the level of sFRP2 expression was high in normal kidney, low in primary renal cancer tissues and high in metastatic renal cancer tissues, suggesting different roles of the sFRP2 gene in different grades of renal cancer. Stably expressed sFRP2 significantly promoted cell prolifera- tion in vitro and in vivo tumor growth. The stably expressed sFRP2 cells were also found to reduce UV-induced apoptosis and increase in the G2 phase of the cell cycle. In the stable sFRP2 cell lines, expression of c-Fos, Bcl2, Bcl-w, cyclinB2 and cyclinE2 genes was significantly increased and p53 expression was decreased. In metastatic Caki1 cells, sFRP2 knockdown inhibited invasion and reduced expression of CD146, MMP2 and Rac1/2/3 genes. This is the first report documenting that sFRP2 modulates proliferation and invasion pathways by altering various genes in renal cancer cells. CONCLUSIONS: We found that the level of sFRP2 expression was high in normal kidney, low in primary renal cancer tissues and high in metastatic renal cancer tissues, suggesting different roles in renal cancer. Overexpressed sFRP2 in renal cancer cells promoted cell growth and cell invasion by evoking diverse signalling cascades. This data may provide better strategies for the management of renal cancer through regulation of sFRP2 pathways. Source of Funding: This study was supported by Grants RO1CA130860 (NIH), T32DK007790, VA Research Enhancement Award Program (REAP) and Merit Review grants. 360 ENHANCER OF ZESTE HOMOLOG 2 IS OVEREXPRESSED IN RENAL CELL CARCINOMA POSSIBLY DUE TO DOWNREGULATION OF MICRORNA-101 Toshihiko Sakurai*, Vladimir Bilim, Kaori Yuuki, Masaaki Tsukigi, Yamagata, Japan; Andrei Ougolkov, Chicago, IL; Yoshihiko Tomita, Yamagata, Japan INTRODUCTION AND OBJECTIVES: Enhancer of zeste ho- molog 2 (EZH2), a subunit of polycomb repressive complex 2(PRC2), is commonly associated with gene silencing. It has already been reported that EZH2 is overexpressed in various cancer, including prostate, breast and pancreatic. It was also reported that EZH2 tran- scripts levels were higher in renal cell carcinoma (RCC) than in normal kidney. Database search returned EZH2 as a highly conserved target of micro RNA miR-101. Our objectives were to determine the expres- sion pattern of EZH2 in RCC and to search for possible mechanism of its dysregulation. Furthermore, we tried to search genes which are donwregulated by EZH2 in RCC resulting in higher malignant character of the tumors. METHODS: Immunohistochemistry and cytosolic/nuclear frac- tionation were performed to determine the expression pattern of EZH2. We used quantitative RT-PCR to examine the expression levels of miR-101 in RCC cell lines, and RCC tissues. RNA interference, West- ern blotting, were applied to study the effect of EZH2 genetic depletion on downstream genes products and renal cancer cell proliferation. RESULTS: We detected nuclear overexpression of EZH2 in all 8 RCC cell lines and in 53 of 58 (91%) cases of human RCC whereas no nuclear accumulation was detected in normal kidney. MiR-101 expression was drastically decreased in all cell lines and tumor tissues compared with normal kidney. When we transfected cell lines with siRNA targeting EZH2 or miR-101 precursor, we observed EZH2 downregulation, decreased H3 methylation and upregulation of p27kip1. This was accompanied with diminished cellular viability. e142 THE JOURNAL OF UROLOGY Vol. 183, No. 4, Supplement, Sunday, May 30, 2010