MP31-15 TUMOR SUPPRESSIVE MICRORNA-224 INHIBITS CANCER CELL MIGRATION AND INVASION VIA TARGETING TPD52 IN PROSTATE CANCER Yusuke Goto*, Rika Nishikawa, Satoko Kojima, Shinichi Sakamoto, Koji Kawamura, Takashi Imamoto, Chiba, Japan; Takeshi Chiyomaru, Hideki Enokida, Kagoshima, Japan; Takashi Kinoshita, Yukio Naya, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan; Tomohiko Ichikawa, Naohiko Seki, Chiba, Japan INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs), a class of small noncoding RNAs, regulate protein-coding gene expres- sion by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to prostate cancer (PCa) progression, development, and metastasis. Understanding the molecular mechanism underlying castration-resistant PCa could significantly improve diagnosis, therapy and disease prevention. Our recent studies of miRNA expression signature revealed that microRNA-224 (miR-224) was significantly downregulated in PCa and was putative tumor suppressive miRNA in PCa. Our aim in this study was to investigate the functional significance of miR-224 in cancer cells and to identify novel miR-224-mediated cancer pathways and responsible genes in PCa oncogenesis and metastasis. METHODS: Gain-of-function studies using mature miR-224 was performed to investigate cell proliferation, migration and invasion in PCa cell lines (PC3 and DU145). To identify the molecular pathways potentially regulated by the miR-224, we applied gene expression data and in silico analysis using TargetScan database [http://www. targetscan.org/ ] and GENECODIS software. Loss-of-function assays were performed to investigate the functional significance of miR-224 target genes. RESULTS: Firstly, we evaluated the expression levels of miR- 224 in normal prostate tissues (n ¼ 29) and PCa tissues (n ¼ 39). The expression level of miR-224 was significantly lower in PCa tissues compared to normal tissues (P < 0.0001). Restoration of miR-224 in PCa cell lines revealed significant inhibition of cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that miR-224 modulated the several pathways such as 0 Pathway in Cancer 0 , 0 MAPK signaling pathway 0 and 0 Ubiquitin mediated proteolysis 0 . More- over, TPD52 gene was candidate target of miR-224 regulation. Silencing of TPD52 was significantly inhibited cell migration and inva- sion in cancer cells. Up-regulation of TPD52 was detected in PCa specimens. CONCLUSIONS: Down-regulation of miR-224 was frequent events in PCa. The miR-224 acted as tumor suppressors, especially contributes to cancer cell metastasis and directly targeted miR-224- TPD52 signalling. Recognition of tumor suppressive miRNA-mediated cancer pathways provides new insights into the potential mechanisms of PCa oncogenesis and metastasis and suggests novel therapeutic strategies for the disease. Source of Funding: none MP31-16 LAMININ BINDING GLYCAN DEPLETION ON a-DYSTROGLYCAN IN PROSTATE CANCER CELLS PROMOTES EPITHELIAL-MESENCHYMAL TRANSITION AND ENHANCES TUMOR FORMATION Tohru Yoneyama*, Shingo Hatakeyama, Yuki Tobisawa, Takuya Koie, Chikara Ohyama, Hirosaki, Japan; Minoru Fukuda, La Jolla, CA INTRODUCTION AND OBJECTIVES: a-dystroglycan (a-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelial cell-BM interaction. a-DG mediated epithelial cell-BM interaction is often impaired in aggressive prostate cancer (PCa), yet roles and underlying mechanisms in PCa progression remain unclear. We report here a suppressor function of laminin binding (LB) glycan on a-DG in PCa progression. METHODS: PC3 cells express varying amounts of LB glycans attached to a-DG. After 2 consecutive cell sorting, PC3 cells were separated into LB glycan expressing substantial (PC3-H) and minimal (PC3-L) amount of LB glycans. Epithelial-mesenchymal transition (EMT) related gene expression profiles of PC3-H and PC3-L were analyzed by Affymetrix GeneChip human Gene1.0 ST Array. Expression of EMT related proteins were also analyzed immunoblot- ting. To evaluate LB glycan function in PCa progression, we per- formed cell motility, matrigel invasion assay and orthotopic tumor formation assay. RESULTS: We found that LB glycan depletion caused cell shape change. PC3-L has a mesenchymal shape (spindle-like) and downregulated E-cadherin while upregulated N-cadherin and vimentin. PC3-H cells reverted to an epithelial shape (rounded) and expressed epithelial cell markers. Matrigel invasion assay showed PC3-L cells invaded much faster than PC3-H cells. Strikingly, PC3-L cells produced much bigger tumor after being inoculated in the orthotopic prostate of SCID mice and much metastasis to the draining lymph nodes compared with PC3-H and parental PC3 cells. ERK/AKT phosphorylation is also increased PC3-L tumor compare with PC3-H. CONCLUSIONS: These results indicate that the LB glycan depletion on prostate cancer cells promotes EMT and correlated to the progression of PCa. Source of Funding: none MP31-17 NOTCH 4 REGULATED, LEF1 MEDIATED PROSTATE CANCER METASTASIS AND ANDROGEN INDEPENDENCE Yirong Li, New York, NY; Liang Chen, Los Angeles, CA; Yang Yang, Hongying Huang, Jinhua Wang, Valerio Zhang, Garrett Daniels, new york, NY; A. de las Morenas, Boston, MA; Hebert Lepor, Ross Basch, Xinyu Wu, Fei Ye, David Zhang, Peng Lee*, new york, NY INTRODUCTION AND OBJECTIVES: The mechanism of Notch and Wnt on prostate cancer progression remains elusive. Our previous in vitro studies indicated that lymphoid enhancing factor 1 (LEF1) was highly expressed in androgen-independent prostate cancer cells. Furthermore, LEF1 regulated AR expression and, subsequently, growth and invasion of prostate cancer cells. In this study, we further assess the role of Notch and Wnt signaling in prostate cancer pro- gression with human prostate cancer cells in vitro, coupled with xeno- graft mice model and human prostate cancer samples in vivo. METHODS: Retrovirus system was used to construct stable cell lines overexpressing LEF1 (LNCaP-LEF1) in LNCaP or knockdown of LEF1 (LNCaP-AI-LEF1shRNA) in LNCaP-AI cells. Cell proliferation, invasion and migration were analyzed with WST-1 assay, matrigel assay and transwell assay in vitro. Subrenal capsule xenograft mice e328 THE JOURNAL OF UROLOGY â Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014