1,25-dihydroxyvitamin D₃ inhibits phosphorylation of AKT and synergizes with pharmacological PI3K/AKT inhibitors to suppress prostate cancer cell growth

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1 alpha, 25-dihydroxy vitamin D₃ (1,25(OH)₂ D₃) demonstrates strong antiproliferative effects in a variety of cancer cell types including prostate cells while the PI3K/AKT pathway presents a pro-survival pathway implicated in prostate cancer. The antiproliferative effect of 1,25(OH)₂ D₃ almost universally involves upregulation of p21 and/or p27, while loss of PTEN and activation of PI3K/AKT can downregulate p21 and p27 levels by a number of mechanisms. We hypothesized that 1,25(OH)₂ D₃ signaling is antagonized by PI3K/AKT signaling. To test this we isolated Pten WT mouse prostatic epithelial cells (MPECs) from Pten floxed, Cre-negative mouse prostates and Pten null MPECs from the Pten lox/lox prostates where Pten was deleted by probasin-driven Cre-recombinase. We found that while Pten WT MPECs were significantly growth-inhibited by 1,25(OH)₂ D₃, Pten null MPECs were completely unresponsive to 1,25(OH)₂ D₃. By shRNA knockdown and by in vitro knockout of Pten in WT MPECs we then showed that Pten loss itself is insufficient to antagonize 1,25(OH)₂ D₃-mediated growth suppression. To evaluate the ability of PI3K/AKT to antagonize 1,25(OH)₂ D₃-mediated growth inhibition we used an inhibitor of PI3K/AKT - LY294002 and an inhibitor of AKT - SH-5. Pre-treatment of insensitive Pten-/- MPECs with LY294002 or SH-5 partially restored the sensitivity to and synergized with the antiproliferative actions of 1,25(OH)₂ D₃. We extended the study to the human prostate cancer cell line LNCaP which is only moderately responsive to 1,25(OH)₂ D₃. LY294002 and 1,25(OH)₂ D₃ treatment demonstrated statistically significant synergistic inhibition of LNCaP cell growth (CI=0.171 at 0.2μM LY294002 and 1nM 1,25(OH)₂ D₃). Interestingly, LY294002 sensitized DU145 cells to 1,25(OH)2 D3, a cell line that is insensitive to 1,25(OH)₂ D₃-mediated growth inhibition, leading to a strong statistically significant synergism. While neither 2.5μM LY294002 nor 10nM 1,25(OH)₂ D₃ affected the growth of DU145 cells, the combination of the two led to a 40% reduction in the number of viable cells. Rapamycin did not synergize with 1,25(OH)₂ D₃ in the growth inhibition suggesting that the mTOR pathway is not involved in the mechanism. Immunoblot analysis showed that 1,25(OH)₂ D₃ alone was able to decrease AKT phosphorylation in prostate cancer cells. In DU145 cells LY294002 and 1,25(OH)₂ D₃ cooperatively inhibited the phosphorylation of AKT. Taken together we conclude that inhibition of the PI3K/AKT pathway synergizes with the antiproliferative actions of 1,25(OH)₂ D₃ in prostate cancer cell lines possibly by additive reduction of AKT phosphorylation. The findings from the present study provide the rationale for the development of improved combinational therapeutic interventions utilizing 1,25(OH)₂ D₃ or its analogs combined with inhibition of PI3K/AKT for the treatment of prostate cancer.