Abstract B53: RANK- and AR-mediated signaling axes in prostate cancer metastasis

Introduction and Objective: The development and progression of prostate cancer (PCa) is strongly associated with androgen receptor (AR)-mediated signaling. In our lab we found in addition to AR, Receptor Activator of NF-κB (RANK) plays a coordinated role in driving PCa progression. PCa cells are initially androgen-sensitive (AS) and respond to androgen-deprivation therapy (ADT). With time, while some PCa cells remain AR positive others are AR-negative, but they exhibit a common androgen-insensitive (AI) phenotype with increased invasiveness and metastatic potential. Our lab showed activation of RANK ligand (RANKL)-RANK signal network in human PCa specimens correlated with poor prognosis of PCa patients. The objectives of this study are: 1) to compare RANK- and AR-mediated signaling axes in a PCa cell line and to study their coordinated regulation with growth under 2-D versus 3-D cultures; 2) to investigate the behaviors of PCa cells with both activated RANK- and AR- signaling axes; 3) to determine if RANKL-RANK signal network activates integrin expression and promotes the attachment of PCa cells to type 1 collagen (Col1), rich in bone matrix. Methods: Stable RANKL-expressing LNCaP cells, LN-RANKL, were obtained and their AR expression were compared under 2-D versus 3-D cultures. Expression of AR and its functionality, at day 7, was further confirmed using AR promoter- and PSA promoter-luciferase activities. RANKL-RANK-mediated expression of cell adhesion, mediated by integrins, is determined. AR and RANK-mediated adhesion will be assessed in the presence and absence of isotype-specific integrins and extracellular matrices. Results: Using the suspension 3-D growth model, we found that a functional AR expression is restored in LNCaP-RANKL cells when compared to their growth under 2-D condition. We observed a downregulation of AP4 transcription factor, as possible mechanism negatively regulating AR gene transcription. We showed that down-regulation of AP4 using sh- or si-RNA in 2-D culture also restored functional AR expression and increased vimentin expression. It did not, however, affect the expression of stem cell markers such as CD133, CD44, and CD49f. We further investigated metastatic behavior of these cell lines in suspension model. LNCaP-RANKL cells grown on osteoblastic matrices travelled much faster and longer distance compared to LNCaP-RANKL cells grown on 2-D or the control LNCaP-Neo cells. LNCaP-RANKL cells grown in suspension showed higher and faster adhesion to Col1 coated plate in comparison with their parental cells. Cell adhesion was similar between the two cell lines on plastic- and fibronectin-coated plates. This observation is in agreement with higher expression of α2β1, which was confirmed by FACS and qRT-PCR, and a higher propensity for bone metastasis in mice. Conclusions: 3-D culture systems are established to study changes in RANK- and AR-mediated signal network and results are correlated with tumor growth and metastasis in mice. Our data suggest, in 3-D suspension model, AR expression is restored through AP-4 downregulation and increased aggressiveness of PCa. RANK-mediated signal also increased PCa cells to adhere to Col1 and exhibit increased cell motility on collagen matrices. Given that RANKL and AR are co-expressed in clinical human PCa specimens, our model allows us better define RANK- and AR-signal axes at a molecular level in human PCa. Citation Format: Shabnam Ziaee, Leland W.K Chung. RANK- and AR-mediated signaling axes in prostate cancer metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B53. doi:10.1158/1538-7445.CHTME14-B53