Stromal AR inhibits cancer epithelial cells

We have observed and previously demonstrated by co-culture experiments using well characterized stromal cell lines, both in vitro and in vivo that, in the presence of androgen, stromal cells expressing AR decrease the growth and invasive ability of prostate cancer epithelial cells. It was hypothesized that this distinct effect of AR in stromal cells is due to the involvement of paracrine factors/mechanisms regulated by both the epithelial and stromal cells.

The analysis was established [21] by using a well characterized prostate stromal cell line morphologically similar to the tumor stroma. We constructed an immortalized stromal cell line from prostate with BPH, termed as PShTert, stably expressing the human telomerase catalytic subunit - hTert. Morphologically and ultra structurally, the cells expressed typical characteristics of myofibroblasts. IHC showed diffuse, strongly positive stain for Vimentin with a strong SMA staining in 25% of cells, and negative staining for Desmin. Together these data support the myofibroblastic phenotype of the PShTert stromal cells. Western blot analysis showed the absence of AR in these cell lines. We transduced this cell line with pBabeAR retroviral vector and selected stable clonal cell lines expressing AR, termed as PShTertAR. Functionality of the ectopic AR was confirmed by in vivo dual luciferase assay eliciting ligand dependent transcriptional activation in the presence of androgens.

For in vitro analysis, transwell indirect co-culture assays using these two stromal cell lines with PC3 cells were performed. In the presence of androgen, co-culture with PShTertAR resulted in inhibition of PC3 cell proliferation compared to PC3 cell growth when cultured alone (p = 0.045). In contrast, co-culture with AR negative PShTert cells resulted in enhancement of growth rate of PC3 cells compared to PC3 cells grown alone (p = 0.03). Flow cytometric analysis revealed that PC3 cells co-cultured with PShTertAR showed 20% S-phase cells, decreased from the 27% S-phase cells measured inPC3 cells co-cultured with PShTert cells. We examined the expression of cell cycle genes, including cyclin A, cyclin B, p21 and p27, and the expression of Skp2, and all were decreased in PC3 cells co-cultured with PShTertAR compared with PC3 cells co-cultured with PShTert cells.

However, with co-cultures in androgen free media, both PShTert and PShTertAR cells stimulated the growth of PC3 cells. Similarly in vivo analysis by co-injecting PC3 cells with PShTert subcutaneously in the flank region of nude male mice resulted in development of tumors twice as large as when PC3 was injected alone. On the other side, co-injection of PC3 cells and PShTertAR cell line resulted in statistically significant reductions of tumor growth and size.

There were two important observations drawn from the analysis. Firstly, both AR negative and AR positive stromal cells promote growth of prostate cancer epithelial cells in the absence of androgen by secretion of a paracrine factor which is independent of AR. Secondly, AR positive stromal cells secrete another paracrine factor which is growth inhibitory for prostate cancer epithelial cells and is dependent on the presence of androgen and AR.

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