High-dose-androgen-induced autophagic cell death to suppress the Enzalutamide-resistant prostate cancer growth via altering the circRNA-BCL2/miRNA-198/AMBRA1 signaling

Abstract

Androgen deprivation therapy (ADT) is a gold standard treatment for advanced PCa. However, most patients eventually develop the castration-resistant prostate cancer (CRPC) that progresses rapidly despite ongoing systemic androgen deprivation. While early studies indicated that high physiological doses of androgens might suppress rather than promote PCa cell growth in some selective CRPC patients, the exact mechanism of this opposite effect remains unclear. Here we found that Enzalutamide-resistant (EnzR) CRPC cells can be suppressed by the high-dose-androgen (dihydrotestosterone, DHT). Mechanism dissection suggested that a high-dose-DHT can suppress the circular RNA-BCL2 (circRNA-BCL2) expression via transcriptional regulation of its host gene BCL2. The suppressed circRNA-BCL2 can then alter the expression of miRNA-198 to modulate the AMBRA1 expression via direct binding to the 3'UTR of AMBRA1 mRNA. The consequences of high-dose-DHT suppressed circRNA-BCL2/miRNA-198/AMBRA1 signaling likely result in induction of the autophagic cell death to suppress the EnzR CRPC cell growth. Preclinical studies using in vivo xenograft mouse models also demonstrated that AMBRA1-shRNA to suppress the autophagic cell death can weaken the effect of high-dose-DHT on EnzR CRPC tumors. Together, these in vitro and in vivo data provide new insights for understanding the mechanisms underlying high-dose-DHT suppression of the EnzR CRPC cell growth, supporting a potential therapy using high-dose-androgens to suppress CRPC progression in the future.

Fig. 1. DHT can inhibit Enzalutamide-resistant PCa cells growth.

A–D DHT could affect MTT assays, therefore colony formation assays were performed to show EnzS1-C4-2 (A) EnzS4-C4-2B (B), EnzR1-C4-2 (C), and EnzR4-C4-2 (D) cells growth with different concentrations of DHT. E, F Cell counting assays to show cell proliferation in EnzS vs EnzR cells (C4-2 and C4-2B) treated with EtOH or 50 nM DHT. Data are presented as means ± SD. *p < 0.05, **p < 0.01.

Fig. 2. High-dose-DHT suppresses EnzR PCa cell growth via induction of autophagic cell death.

A BCL2 mRNA expression in EnzR1-C4-2 and EnzR4-C4-2B cell lines treated with/without (w/wo) 50 nM DHT. B BCL2 protein expression in EnzR1-C4-2 and EnzR4-C4-2B cell lines treated w/wo 50 nM DHT. C, D Western blot assays were performed on EnzR1-C4-2 (C) and EnzR4-C4-2B (D) cell lines to show that caspase-dependent apoptosis marker is decreased with 50 nM DHT treatment. E, F Western Blot was performed on EnzR1-C4-2 and EnzR4-C4-2B cell lines to show that autophagy markers LC3 (LC3-I and LC3-II) are increased (E) and p62 is decreased (F) with 50 nM DHT treatment. G, H GFP-LC3 plasmid transfection was performed using Lipofectamine 3000 transfection reagent on EnzR1-C4-2 (G) and EnzR4-C4-2B (H) cells to show that GFP-LC3 puncta positive cells increased with 50 nM DHT treatment. The fluorescence images were captured by microscopy, scale bar = 40 µm. Data are presented as means ± SD. *p < 0.05.

Fig. 3. Increasing BCL2 could not block, while increasing circBCL2 could reverse, the DHT inhibition effect on the EnzR PCa cell growth.

A The qRT-PCR was performed to show quantification of BCL2 overexpressing (OE) BCL2 (OE-BCL2) in EnzR1-C4-2 and EnzR4-C4-2B cell lines with/without (w/wo) OE-BCL2. B Western blot assays show BCL2 expression in EnzR1-C4-2 and EnzR4-C4-2B cell lines w/wo OE-BCL2. C, D Colony formation assays show cell growth with 50 nM DHT in EnzR1-C4-2 (C) and EnzR4-C4-2B (D) cells. E The circBCL2 expression in EnzR1-C4-2 and EnzR4-C4-2B cell lines with 50 nM DHT. F The qRT-PCR was performed to show quantification of circBCL2 w/wo OE-circBCL2 treated w/wo 50 nM DHT. G, H Colony formation assays show cell growth with 50 nM DHT in EnzR1-C4-2 and EnzR4-C4-2B cell lines w/wo OE-circBCL2. Data are presented as means ± SD. *p < 0.05, **p < 0.01, NS not significant.

Fig. 4. Mechanism dissection of how DHT/circRNA-BCL2 induces autophagic cell death in EnzR PCa cells: via regulating the AMBRA1 expression.

A, B RNase R assay to determine the sensitivity of circBCL2 by RNase R digestion in EnzR1-C4-2 (A) and EnzR4-C4-2B (B) cell lines. C Venn diagram for prediction of the potential miRNAs using two miRNA related websites (miRDB and Regrna2.0). D Seven miRNA candidates can physically interact with circRNA-BCL2 and showed up after screening EnzR-C4-2 cells by biotin-circRNA-BCL2 pull-down assay. E The qRT-PCR was performed to show quantification of circRNA-BCL2 related miRNAs expression in EnzR-C4-2 cells after 50 nM DHT treatment. F Argonaute2 (AGO2) IP assay to detect 14 candidate genes mRNA in AGO2 complex and results revealed that 8 genes have less miRNAs binding in EnzR-C4-2 cells after 50 nM DHT treatment. G, H Western blot assays were performed to show candidates genes expression in EnzR-C4-2 cells after 50 nM DHT treatment. I, J Western blot assays show AMBRA1, LC3, and p62 expression after oe-circRNA-BCL2 in EnzR1-C4-2 (I) and EnzR4-C4-2B (J) cell line with 50 nM DHT. K, L Western blotting show AMBRA1, LC3 and p62 expression after sh-AMBRA1 in EnzR1-C4-2 (K) and EnzR4-C4-2B (L) cell line with 50 nM DHT. M, N Colony formation assays show cell growth with 50n M DHT when sh-AMBRA1 is transduced in EnzR1-C4-2 (M) and EnzR4-C4-2B (N) cell lines. Data are presented as means ± SD. *p < 0.05, **p < 0.01, NS not significant.

Fig. 5. Mechanism dissection of how DHT/circRNA-BCL2 alters the AMBRA1 expression in EnzR PCa cells: via regulating the miRNA-198 expression.

A, B Western blot assays show AMBRA1, LC3 and p62 expression after OE-miRNA-198 (OE-miR-198) transduced in EnzR1-C4-2 (A) and EnzR4-C4-2B (B) cell lines with 50 nM DHT. C, D Colony formation assays show cell growth with 50 nM DHT when OE-miR-198 transduced in EnzR1-C4-2 (C) and EnzR4-C4-2B (D) cell lines. E, F The miR-198 stability was measured with the addition of 5 μM Actinomycin D (ActD) in EnzR1-C4-2 (E) and EnzR4-C4-2B (F) cells with/without knocked-down circRNA-BCL2. G The circRNA-BCL2 with and without miR-198 binding sites (CircNet). H The qRT-PCR was performed to show quantification of circRNA-BCL2 after OE-mutantcircBCL2. I Colony formation assay shows decreased cell growth with 50 nM DHT and OE-mutant circRNA-BCL2 in EnzR1-C4-2 cell line. J The wild type (WT) and mutant (Mut) AMBRA1 luciferase constructs. K, L Luciferase activity after transfection of WT or Mut AMBRA1 3’-UTR with 50 nM DHT treatment in EnzR1-C4-2 (K) and EnzR4-C4-2B (L) cell lines. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant.

Fig. 6. Preclinical study using In vivo mouse model to confirm the role of DHT and AMBRA1 in EnzR cell growth.

A Equal numbers of EnzR1-C4-2 cells (5 × 10⁶ cells) were subcutaneously injected into each mouse to establish the CRPC xenograft model. Mice were injected with Enz (10 mg/kg/week, twice weekly). Bipolar androgen therapy treatment was conducted by injection of Testosterone (200 μg/kg/week, twice weekly) or EtOH at the 3th/5th/7th week. Mice were euthanized after 8 weeks and tumors were removed for studies. B, C Tumor weights (B) were shown and presented as means ± SD (C). D Representative IHC images of AMBRA1 expression in tumor tissue samples from the 4 groups, ×200 magnification. *p < 0.05.

Fig. 7. A mechanistic diagram.

A mechanistic diagram for DHT effect in CRPC cells.