Fibroblast-derived exosomal microRNA regulates NKX3-1 expression in androgen-sensitive, androgen receptor-dependent prostate cancer cells.

Androgen deprivation therapy (ADT) targeting androgen production and androgen receptor (AR) signaling is the primary antihormonal therapy in the treatment of advanced prostate cancer (PCa). However, no clinically established molecular biomarkers have been identified to predict the effectiveness of ADT before starting ADT. The tumor microenvironment of PCa contains fibroblasts that regulate PCa progression by producing multiple soluble factors. We have previously reported that AR-activating factor-secreted fibroblasts increase the responsiveness of androgen-sensitive, AR-dependent PCa cells to ADT. Thus, we hypothesized that fibroblast-derived soluble factors may affect cancer cell differentiation by regulating cancer-related gene expression in PCa cells and that the biochemical characteristics of fibroblasts may be used to predict the effectiveness of ADT. Here, we investigated the effects of normal fibroblasts (PrSC cells) and three PCa patient-derived fibroblast lines (pcPrF-M5, -M28, and -M31 cells) on the expression of cancer-related genes in androgen-sensitive, AR-dependent human PCa cells (LNCaP cells) and three sublines showing different androgen sensitivities and AR dependencies. The mRNA expression of the tumor suppressor gene NKX3-1 in LNCaP cells and E9 cells (which show low androgen sensitivity and AR dependency) was significantly increased by treatment with conditioned media from PrSC and pcPrF-M5 cells but not from pcPrF-M28 and pcPrF-M31 cells. Notably, no upregulation of NKX3-1 was observed in F10 cells (AR-V7-expressing, AR-independent cells with low androgen sensitivity) and AIDL cells (androgen-insensitive, AR-independent cells). Among 81 common fibroblast-derived exosomal microRNAs that showed 0.5-fold lower expression in pcPrF-M28 and pcPrF-M31 cells than in PrSC and pcPrF-M5 cells, miR-449c-3p and miR-3121-3p were found to target NKX3-1. In only LNCaP cells, the NKX3-1 mRNA expression was significantly increased by transfection of an miR-3121-3p mimic but not that of the miR-449c-3p mimic. Thus, fibroblast-derived exosomal miR-3121-3p may be involved in preventing the oncogenic dedifferentiation of PCa cells by targeting NKX3-1 in androgen-sensitive, AR-dependent PCa cells.

Heterogeneous induction of an invasive phenotype in prostate cancer cells by coculturing with patient-derived fibroblasts.

Prostate cancer (PCa) cells frequently invade the surrounding stroma, leading to heterogeneous formation of structural atypia. The surrounding stroma contains multiple functionally diverse populations of fibroblasts that trigger numerous changes in PCa cells including motility. Thus, we hypothesized that direct or indirect contact of PCa cells with fibroblasts determines an invasive phenotype in PCa cells. We investigated the effects of 10 different patient-derived fibroblast lines on the three-dimensional (3D) morphogenesis of PCa cells growing on a viscous substrate in vitro. When grown alone, all 10 patient-derived fibroblast lines clumped on the viscous substrate, whereas the human androgen-sensitive PCa cell line LNCaP did not. Cocultures of LNCaP cells with seven of the patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M7, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a thick fibroblast layer that resembled human prostate stromal structures. In contrast, cocultures of LNCaP cells with the remaining three fibroblast lines (NPF-M13, pcPrF-M10, and pcPrF-M26) did not form a thick fibroblast layer. Of the seven fibroblast lines that caused thick layer formation, four patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) induced an invasive phenotype in LNCaP cells with a cord-like infiltrating growth pattern, whereas the other three fibroblast lines (pcPrF-M7, pcPrF-M23, and pcPrF-M24) induced no or a very weak invasive phenotype. Using cell culture inserts, none of the four patient-derived fibroblast lines that induced an invasive phenotype (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) affected CDH1 mRNA expression in LNCaP cells; yet, two patient-derived fibroblast lines (pcPrF-M5 and pcPrF-M28) increased CDH2 mRNA expression in LNCaP cells, whereas the other two fibroblast lines (PrSC and pcPrF-M31) did not. These results suggest that the existence of multiple functionally diverse populations of fibroblasts in PCa tissue may be responsible for the diversity in PCa cell invasion, leading to heterogeneous formation of structural atypia.