Combined N-terminal androgen receptor and autophagy inhibition increases the anti-tumor effect in enzalutamide-sensitive and enzalutamide-resistant LNCaP cells
Multiple androgen receptor (AR) dependent and independent resistance mechanisms limit the efficacy of current castration resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR targeting components including EPI-001 have the promising ability to block constitutively active splice variants; a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway and acts as survival mechanism in cells exposed to anti-cancer treatment. We hypothesized that also a promising NTD-AR treatment may lead to up-regulation of autophagy and can be targeted by a combination treatment with autophagy inhibitors.
Prostate cancer cell lines LNCaP and LNCaP-EnzR were cultured in steroid-free medium and treated with different concentrations of EPI-001 (EPI: 10, 25, 50uM) and in combination with autophagy inhibitors chloroquine (CHQ, 20uM) or 3-methyladenine (3MA, 5mM). Cell growth was assessed by WST-1-assays after 1 and 7 days. Etidium bromide and AnnexinV were used to measure viability and apoptosis on day 7 after treatment. Autophagosome increase was detected by Autodot staining. In addition, autophagic activity was monitored by western blot (WB) and immunocytochemistry for the expression of LC3-I/II, Atg5 and Beclin1.
Treatment with EPI resulted in a dose dependent reduction of cell growth and increase of apoptosis on day 7 in both cell lines. Combination of 25uM EPI with autophagy inhibitors led to a further reduction of cell viability up to 17% for CHQ, 15% for 3MA in LNCaP and up to 24% for CHQ, 36% for 3MA in LNCaP-EnzR. Assessment of autophagy levels in EPI treated cells by WB showed an increase of LC3-II and no change in Beclin1 expression in both cell lines. Immunocytochemistry detected a significant increase of Atg5 and pronounced LC3-II punctuation in EPI-001 treated LNCaP and LNCaP-EnzR. This was supported by an increase in autophagosome punctuation observed by Autodot staining.
Our data demonstrate that N-terminal androgen receptor inhibition with EPI-001 leads to increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. Combination of N-terminal androgen receptor blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI even in enzalutamide-resistant cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.