Login to Access Video or Poster Abstract: MP83-07
Sources of Funding: none

Introduction

Multiple androgen receptor (AR) dependent and independent resistance mechanisms limit the efficacy of current castration resistant prostate cancer (CRPC) treatment modalities. EPI-001 is a novel N-terminal domain (NTD) binding AR targeting component with the promising ability to block constitutively active splice variants; a major resistance mechanism in CRPC. Autophagy a conserved lysosomal degradation pathway, is a 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, which can be targeted by a combination therapy with autophagy inhibitors.

Methods

The human prostate cancer cell line LNCaP was cultured in steroid-free medium. Cells were treated with different concentrations of EPI-001 (10, 25, 50uM) and in combination with autophagy inhibitors chloroquine (CHQ, 20uM) or 3-methyladenine (3MA, 5mM). Viability was assessed by WST-1-assays after 1, 3 and 7 days. AnnexinV and Propidium Iodide were used to measure apoptosis and necrosis on day 7 after treatment. Autophagic activity was monitored on protein levels by western blot (WB) and immunocytochemistry for the expression of LC3-I/II, Atg5 and Beclin1. In addition, autophagosome increase was detected by Autodot staining.

Results

Treatment with EPI-001 resulted in a dose dependent reduction of cell viability up to 50% with 50uM EPI-001 on day 7. At the same time apoptosis increased by 11.53% and necrosis by 3.74% compared to the control. Combination of 50 uM EPI-001 with autophagy inhibitors led to a further significant reduction of cell viability up to 40% for CHQ and 28% for 3MA. Assessment of autophagy levels in EPI-001 treated cells by WB showed an increase of LC3-II in a dose dependent manner. No Change in Beclin1 expression was seen. Immuncytochemistry detected a significant increase of Atg5 and pronounced LC3-II punctuation in EPI-001 treated cells. This was supported by an increase in autophagosome punctuation observed by Autodot staining.

Conclusions

Our data demonstrate that the treatment with EPI-001 leads to increased autophagic activity in LNCaP prostate cancer cells. Combination of N-terminal androgen receptor blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI-001 in vitro. This may offer a promising strategy to overcome resistance mechanisms in castration resistant prostate cancer.

Funding

none