Apalutamide and autophagy inhibition in a xenograft mouse model of human prostate cancer

Abstract

Background Apalutamide (APA) is a next-generation androgen receptor antagonist for the treatment of advanced prostate cancer. We have previously shown that upregulation of autophagy is one of the mechanisms by which prostate cancer (PC) cells survive APA anti-tumor treatment in vitro. Therefore, we investigated the characteristics of the autophagic response to APA treatment, alone and in combination with autophagy inhibition, in an in vivo model. Methods Tumor cells were injected into previously castrated nude mice. Four groups of mice bearing LNCaP xenografts were treated with daily intraperitoneal (i.p.) injections of vehicle (control), APA (10 mg/kg), APA (10 mg/kg) + Chl (Chloroquine, 10 mg/kg) or Chl (10 mg/kg). The animals of each treatment group (3/treatment) were kept for the duration of 2 and 3 weeks. At the end of the experiments, the animals were sacrificed and all samples assessed for tumor weight and size, histological analysis, immunoblotting (WES) and immunofluorescence. Results The tumor weight was significantly reduced in mice treated with APA + Chl (203.2 ± 5.0, SEM, P = 0.0066) compared to vehicle control (380.4 ± 37.0). Importantly, the combined treatment showed a higher impact on tumor weight than APA (320.4 ± 45.5) or Chl (337.9 ± 35) alone. The mice treated with the combination of APA + Chl exhibited a reduced expression of ATG5 (autophagy-related five protein), Beclin 1 and LC3 punctuations and an increase in P62 as visualized by immunofluorescence and WES. In addition, Ki-67 nuclear staining was detected in all samples however reduced in APA + Chl (58%) compared to vehicle control (100%). The reduction in Ki-67 protein was associated with an increase in caspase 3 and endothelial CD31 protein expression. Conclusion These data demonstrate that a treatment with APA + Chl leads to reduced autophagy levels and to tumor suppression compared to the APA monotherapy. Hence, the increased antitumor effect of APA in combination with autophagy inhibitors might provide a new therapeutic approach potentially translatable to patients.

Making the Case for Autophagy Inhibition as a Therapeutic Strategy in Combination with Androgen-Targeted Therapies in Prostate Cancer

“…These results from Eberli et al [78,79] strongly suggest the cytoprotective autophagy is being induced by apalutamide in LNCaP cells either in vitro, or after being implanted in mice models, highlighting the possible targeting of autophagy to increase the effectiveness of apalutamide. Although promising, a major limitation for these studies, as well as that of Boutin et al [55], is the utilization of a single cell line, LNCaP cells, which may limit the generalization of these results to other prostate cancer cell lines and the clinical situation.…”

“…However, some patients do not respond to this therapy, and eventually become resistant. Recently, Eberli et al [78,79] studied the potential targeting of autophagy to increase the effectiveness of and possibly overcome resistance to apalutamide both in vitro and in vivo.…”

“…In vivo, Eberli et al [79] studied the possibility of autophagy targeting in combination with apalutamide, using LNCaP-injected castrated nude mice models. The apalutamidetreated mice showed signs of autophagy induction, as evidenced by the increased expression of ATG5 and a punctuated pattern for LC3 by immunofluorescence.…”

CAPN2 promotes apalutamide resistance in metastatic hormone-sensitive prostate cancer by activating protective autophagy

“…PCa cells use autophagy as a survival mechanism in response to apalutamide treatment. This induction of autophagy is associated with increased expressions of Beclin1 and ATG5 proteins, and with the autophagy-related conversion of the microtubule-associated protein light chain 3 (LC3I to LC3II), the main regulators of autophagy in PCa [ 15 ]. Classic studies have indicated that at least two mechanisms underlie ADT-stimulated autophagy: one in which ADT induces local vasculature degeneration and hypoxia; and another in which, upon energy deficiency caused by ADT, AMP-activated protein kinase (AMPK) activation leads to mTOR pathway suppression and the promotion of autophagy [ 16 ].…”

Evolution of Models of Prostate Cancer: Their Contribution to Current Therapies

“…Determining efficacy and safety is critical during the initial phases of drugs discovery and development, and ultimately for approval and commercialization. Some of the drugs approved by the FDA or EMA, and listed above, were tested in rodent models (92)(93)(94)(95)(96)(97)(98)(99)(100)(101)(102).…”