Preclinical studies using cisplatin/carboplatin to restore the Enzalutamide sensitivity via degrading the androgen receptor splicing variant 7 (ARv7) to further suppress Enzalutamide resistant prostate cancer

Abstract

The FDA-approved anti-androgen Enzalutamide (Enz) has been used successfully as the last line therapy to extend castration-resistant prostate cancer (CRPC) patients' survival by an extra 4.8 months. However, CRPC patients eventually develop Enz-resistance that may involve the induction of the androgen receptor (AR) splicing variant ARv7. Here we found that Cisplatin (Cis) or Carboplatin, currently used in chemotherapy/radiation therapy to suppress tumor progression, could restore the Enz sensitivity in multiple Enz-resistant (EnzR) CRPC cells via directly degrading/suppressing the ARv7. Combining Cis or Carboplatin with Enz therapy can also delay the development of Enz-resistance in CRPC C4-2 cells. Mechanism dissection found that Cis or Carboplatin might decrease the ARv7 expression via multiple mechanisms including targeting the lncRNA-Malat1/SF2 RNA splicing complex and increasing ARv7 degradation via altering ubiquitination. Preclinical studies using in vivo mouse model with implanted EnzR1-C4-2 cells also demonstrated that Cis plus Enz therapy resulted in better suppression of EnzR CRPC progression than Enz treatment alone. These results not only unveil the previously unrecognized Cis mechanism to degrade ARv7 via targeting the Malat1/SF2 complex and ubiquitination signals, it may also provide a novel and ready therapy to further suppress the EnzR CRPC progression in the near future.

Fig. 1. Cis/Carboplatin degrades AR wild type, mutant, and ARv7 expression and cleaved PARP in CRPCs.

We established 1 EnzR PCa cell line in C4-2 cells (named as EnzR1_C4-2) and obtained EnzR4_C4-2B from another research group. a–d The a EnzS1_C4-2, b EnzR1_C4-2, c EnzS4_C4-2B, and d EnzR4_C4-2B cells were treated with increasing dosages of Cis for 24 h for fAR/ARv7 expression using Western blot (WB). e EnzR3_CWR22Rv1 and f PC3-AR-F876L cells were treated with increasing dosages of Cis for 24 h for fAR/ARv7 expression using WB. g EnzR4_C4-2B cells were treated with Carboplatin for 24 h for protein expression levels of fAR and ARv7 using WB.

Fig. 2. Cisplatin restores Enzalutamide sensitivity in the EnzR CRPC cells.

a–c Cell survival rates of EnzR1_C4-2 cells treated with a DMSO or 20 μM Enz, b 1 μg/ml Cis or Cis (1 μg/ml) + Enz (20 μM), and c IC50 of Cis. d–f Survival rates of EnzR4_C4-2B treated with d DMSO or Enz (20 μM), e Cis (1 μg/ml) or Cis (1 μg/ml) + Enz (20 μM), and f IC50 of Cis, g–i Survival rate of EnzR1_C4-2 cells under g DMSO or Enz (20 μM), h Carboplatin (5 μg/ml) or Carboplatin + Enz (20 μM) and i IC50 of Cis. j–l Protein levels of cleaved PARP in j EnzR1_C4-2 cells and in k EnzR4_C4-2B cells that were treated with Cis for 24 h, and in l EnzR4_C4-2B cells that were treated with Carboplatin for 24 h. m Cell survival rates in EnzR1_C4-2 cells transfected with pWPI vector or OEARv7 and treated with/without Enz 10 μM + 1 μg/ml Cis (upper left), DMSO (upper right), 10 μM Enz (lower left) and Enz 10 μM + 1 μg/ml Cis (lower right). All the MTT results were performed in triplicate. Data presented as Mean ± S.D. *P < 0.05, ***P < 0.001 or n.s=not significant.

Fig. 3. Cisplatin postpones/delays Enzalutamide-resistance development.

a–c Cell survival rates of EnzS1_C4-2 cells treated with a DMSO, b 10 μM Enz or c 10 μM Enz + 0.2 μg/ml Cis for 2 months, then challenged with 20 μM Enz for 6 days for MTT assays. d–f Cell survival rates of EnzS4_C4-2B cells treated with d DMSO, e 10 μM Enz or f 10 μM Enz + 0.2 μg/ml Cis for 2 months, then challenged with 20 μM Enz for 6 days for MTT assays. Enz sensitivity was analyzed by measuring proliferation rates using MTT assays. g Schematic of PCa cells responses under different concentration of Cis and Carbopltin treatment. All the MTT results were performed in triplicate. Data presented as Mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. Cisplatin degrades ARv7 expression through increasing AR ubiquitination in EnzR C4-2 cells.

a Western blot and b quantification of ARv7 in EnzR1_C4-2 cells which were treated with 1 μg/ml cisplatin and 1 μg/ml cisplatin for 6 h plus MG132 for another 6 h. c Ubiquitination levels of GFP /AR in 293 T cells transfected with Ubi-GFP and AR, which were treated with/without 1 μg/ml Cisplatin for 6 h and treated with MG132 for another 4 h. Protein was extracted and analyzed using Western blot. d Quantification of Ubi-flAR/ARv7.

Fig. 5. Cisplatin suppress Malat1/ARv7 pathway.

EnzR1_C4-2 cells were treated with different Cis concentrations (0–5 μg/ml) for 24 h. a, b RNA level of Malat1 (a) and ARv7 (b) In EnzS1_C4-2 cells, the RNAs expression level of Malat1 (c) and ARv7 (d) after treated with (DMSO, Cis 0.3 μg/ml, 5 μM Enz, Cis 0.3 μg/ml + 5 μM Enz) for 2 weeks. In EnzS4_C4-2B cells the RNAs expression level of Malat1 (e) and ARv7 (f) after treated with (DMSO, Cis 0.3 μg/ml, 5 μM Enz, Cis 0.3 μg/ml + 5 μM Enz) for 2 weeks. g Quantified PCR results of ARv7 at CRPC patients after Cis treatment. All Quantified PCRs were performed in triplicate. A-F multiple comparison was analyzed by One-way ANOVA. QPCR results presented as Mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. Cisplatin restores Enzalutamide sensitivity and inhibits the growth of EnzR tumors in pre-clinical mouse PCa model.

a Survival rate of EnzR3_22Rv-1 cells with (w) or without (w/o) 1 μg/ml Cis treatment for 24 h and 20 μM Enz treatment for 6 days. b Schematic for athymic mice xenograft model treatment. c Tumor growth curve after 20 days in mice treated with DMSO (CTRL), 3.5 mg/kg Cis, 30 mg/kg Enz, or 3.5 mg/kg Cis + 30 mg/kg Enz (each group n = 5). d Box plot for day 20 tumor growth ratio. Comparison between multiple groups using One-way ANOVA. e Immunohistology staining result of ARv7 in different groups. Data presented as Mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7

Schematic of ARv7/AR-F876L changes in EnzR cells under Cisplatin and Carboplatin treatment.