Abstract A1: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a noncoding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line has been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analyzed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. This growth inhibition was specific for DD3 expressing cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. Besides, DD3 mRNA expression was mainly detected in nuclei and ribosome cell compartments, indicating that in this cell niches DD3 ncRNA exert its main functional roles. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These .ndings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR prosurvival signal and their target genes. Citation Format: Luciana Bueno Ferreira, Antonio Palumbo, Kivviduarte de Mello, Felipe Leite, Cinthya A. Sternberg, Mauricio Caetano, Adriana Freitas Neves, Luiz Ricardo Goulart, Etel Rodrigues Pereira Gimba. DD3/PCA3 noncoding RNA regulates prostate cancer cell survival and modulates AR signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A1.