Combined N‐terminal androgen receptor and autophagy inhibition increases the antitumor effect in enzalutamide sensitive and enzalutamide resistant prostate cancer cells

Abstract

Introduction and Objectives 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 compounds, including EPI‐001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD‐AR treatment may upregulate autophagy and that a combination of NTD‐AR and autophagy inhibition might therefore increase antitumor effects. Methods AR‐expressing prostate cancer cell lines (LNCaP, LNCaP‐EnzR) were treated with different concentrations of EPI (10, 25, 50 μM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 μM) or 3‐methyladenine (3‐MA, 5 mM). Cell proliferation was assessed by WST‐1‐assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3‐I/II and p62. Results Treatment with EPI resulted in a dose‐dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP‐EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3‐II expression and a decreased p62 expression in all EPI‐treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines. Conclusions Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP‐EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.

Inhibition of CAMKK2 impairs autophagy and castration-resistant prostate cancer via suppression of AMPK-ULK1 signaling

“…Although autophagy has context-dependent roles in cancer ( 16 , 61 – 64 ), our data support a pro-cancer role for this cellular process in prostate cancer. These findings are consistent with our previous work ( 23 , 24 ) and the work of others in the field ( 25 , 26 , 29 , 31 , 63 , 65 – 67 ). As presented in our previous reports, blocking autophagy by molecular or pharmacological approaches resulted in decreased androgen-mediated prostate cancer cell growth ( 23 , 24 ).…”

“…In prostate cancer, studies from our laboratory and others using cell lines, xenografts and genetic mouse models indicate that autophagy can promote disease progression ( 23 – 30 ). This included various studies demonstrating that chloroquine, a compound known to block autophagic flux, could inhibit prostate cancer progression ( 23 , 26 , 29 , 31 ). These preclinical data provided the rationale for a series of clinical trials ( NCT04011410 , NCT00726596 , NCT00786682 , NCT03513211 , NCT01828476 , NCT02421575 , NCT01480154 ) that tested the efficacy of chloroquine derivatives such as hydroxychloroquine in men with prostate cancer ( 32 ).…”

Apalutamide in combination with autophagy inhibitors improves treatment effects in prostate cancer cells

“…In addition, we have shown that combined N-terminal AR binding and autophagy inhibition increases the antitumor effect in Enzalutamide-sensitive and -resistant prostate cancer cells 6 .…”

The role of autophagy in resistance to targeted therapies

“…Treatment with EPI-001 (EPI), an androgen receptor inhibitor, reduces cell growth and increases apoptosis in PC cells. Also, EPI-treated cells showed increased autophagosome formation [121]. The combination of EPI with autophagy inhibitors further reduces cell viability significantly.…”

“…The combination of EPI with autophagy inhibitors further reduces cell viability significantly. Therefore, this combination may offer a strategy to overcome resistance mechanisms in advanced PC [121].…”