Effect Of Inhibition Of The Pi3k/Akt/Mtor Pathway On Ar Splicing And Downstream Targets

101 Background: Increased androgen receptor (AR) and PI3K/Akt/mTOR signaling drive androgen-independent (AI) prostate cancer (PC) progression. AR splice variants (SV) lacking the ligand-binding domain activate ligand-independent transcriptional programs that support castration resistant proliferation and survival. Treatment of the androgen-dependent PTEN null LNCaP (AD-cells), its AI derivative (AI-cells) and wt PTEN RV1 cells with the dual PI3K/mTOR inhibitor BEZ235 (E) combined with the histone deacetylase inhibitor Panobinostat (PAN) induced synergistic growth inhibition and cell death in vitro and in vivo. The effect of these treatments on AR splicing is unknown.LNCaP AD, AI and RV1 cells were treated with PAN 20nM, E 350nM, rapamycin (Rapa) 20nM and PI3Ki BKM120 (K) 1.5µM for 24h. Total (t) AR, SV's and AR targets mRNA were measured by qtPCR and western. RV1 xenografts received 3 weeks E 35mg/kg/d or PAN 10mg/kg/QOD or PAN+E.Baseline levels of tAR mRNA in AI were higher than AD and RV1 cells while SV1, SV7 and SV5-7 were higher in RV1 than AD and AI cells. Treatment with E, Rapa and K increased both tAR and SV's mRNA's and AR and PSA proteins. In contrast, treatment with PAN reduced tAR and SV's levels in AD and AI with no effect on RV1. PAN reduced the induction of tAR by Rapa or K in AD, AI and RV1 but did not abrogate induction of SV's. PAN inhibition was sufficient to abrogate the effect of E and decrease tAR and SV's levels as well as AR protein and AR canonical and non-canonical targets (PSA, TMPRSS2 and UBE2C) and PSA protein below baseline levels in all cells treated with PAN+E. Similarly, RV1 tumors treated with E had a 50% increase in tAR and 20% in SV5-7 but no induction of CDK1 or UBE2C and a 35% reduction in PSA while RV1 tumors treated with PAN+E showed 40% reduction in both SV's 1 and 7 and downstream targets CDK1 and UBE2C.Inhibition of PI3K/mTOR with E increases overall levels of AR transcription that is more pronounced on SV's and uncouples AR's transcriptional activity on canonical downstream targets in vivo. PAN can abrogate the stimulation of E by decreasing both SV's expression and AR transcriptional activity on non-canonical targets. These effects may underlie their synergistic growth inhibition.