Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis

// Changlin Li 1 , Wencong Jiang 1, 2 , Qingting Hu 1, 3 , Long-cheng Li 4 , Liang Dong 1 , Ruibao Chen 1 , Yinghong Zhang 1 , Yuzhe Tang 1 , J. Brantley Thrasher 1 , Chang-Bai Liu 3 , Benyi Li 1, 2, 3 1 Department of Urology, University of Kansas Medical Center, Kansas City, KS 66160, USA 2 Department of Urology, The Affiliated Hospital, Guangdong Medical University, Zhanjiang 524001, China 3 Institute of Cell Therapy, China Three Gorges University, Yichang 443002, China 4 Laboratory of Molecular Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100073, China Correspondence to: Benyi Li, e-mail: bli@kumc.edu Keywords: DPYSL3, CRMP4, metastasis, RNAa, saRNA Received: January 25, 2016      Accepted: February 23, 2016      Published: March 23, 2016 ABSTRACT To explore a novel strategy in suppressing tumor metastasis, we took the advantage of a recent RNA activation (RNAa) theory and used small double-strand RNA molecules, termed as small activating RNAs (saRNA) that are complimentary to target gene promoter, to enhance transcription of metastasis suppressor gene. The target gene in this study is Dihydro-pyrimidinase-like 3 ( DPYSL3 , protein name CRMP4), which was identified as a metastatic suppressor in prostate cancers. There are two transcriptional variants of DPYSL3 gene in human genome, of which the variant 2 is the dominant transcript (DPYSL3v2, CRMP4a) but is also significantly down-regulated in primary prostate cancers. A total of 8 saRNAs for DPYSL3v1 and 14 saRNAs for DPYSL3v2 were tested in multiple prostate cancer cell lines. While none of the saRNAs significantly altered DPYSL3v1 expression, 4 saRNAs showed a strong enhancing effect on DPYSL3v2 expression, resulting in reduced cell mobility in vitro . To achieve a prostate cancer-specific delivery for in vivo testing, we conjugated the most potent sa V2-9 RNA molecule with the prostate-specific membrane antigen (PSMA)-targeting aptamer A10-3.2. The conjugates successful increased DPYSL3v2 gene expression in PSMA-positive but not PSMA-negative prostate cancer cells. In nude mice bearing orthotopic xenograft of prostate cancer, a 10-day consecutive treatment with the sa V2-9 conjugates significantly suppress distal metastasis compared to the control saRNAs. Analysis of xenograft tissues revealed that DPYSL3v2 expression was largely increased in sa V2-9 conjugate-treated group compared to the control group. In conclusion, DPYSL3v2 promoter-targeted saRNA molecules might be used as an adjunctive therapy to suppress prostate cancer metastasis.