Nodularin-R Synergistically Enhances Abiraterone Against Castrate- Resistant Prostate Cancer via PPP1CA Inhibition

Abstract

Clinically, most prostate cancer (PCa) patients inevitably progress to castration-resistant prostate cancer (CRPC) with poor prognosis after androgen deprivation therapy (ADT), including abiraterone, the drug of choice for ADT. Therefore, it is necessary to explore the resistance mechanism of abiraterone in depth. Genome-wide CRISPR/Cas9 knockout technology was used to screen CRPC cell line 22Rv1 for abiraterone-resistant genes. Combined with bioinformatics, a key gene with high expression and poor prognosis in CRPC patients was screened. Then, the effects of target gene on abiraterone-resistant 22Rv1 cell function were explored by silencing and overexpression. Further, a natural product with potential targeting effect was identified and validated by molecular docking and protein expression. Molecular dynamics simulations revealed potential mechanism for the natural product affecting target protein expression. Finally, the combined anti-CRPC effects of the natural product and abiraterone were validated by cellular and in vivo experiments. Five common resistance genes (KCNJ3, COL2A1, PPP1CA, MDH2 and EXOSC5) were identified successfully, among which high PPP1CA expression had the worst prognosis for disease-free survival. Moreover, PPP1CA was highly expressed in abiraterone-resistant 22Rv1 cells. Silencing PPP1CA increased cell sensitivity to abiraterone while promoting apoptosis and inhibiting clone formation. Overexpressing PPP1CA exerted the opposite effects. Molecular docking revealed the binding mode of the natural product nodularin-R to PPP1CA with a dose-dependent manner for inhibition. Mechanistically, nodularin-R attenuates the interaction between PPP1CA and USP11 (deubiquitinating enzyme), potentially promoting PPP1CA degradation. Additionally, combination of 2.72 μM nodularin-R and 54.5 μM abiraterone synergistically inhibited the resistant 22Rv1 cell function. In vivo experiments also revealed that combination therapy significantly inhibited tumour growth and reduced inducible expression of PPP1CA. PPP1CA is a key driver for abiraterone resistance, and nodularin-R enhances the anti-CRPC effects of abiraterone by inhibiting PPP1CA.

Keywords: CRISPR/Cas9; PPP1CA; abiraterone; castration‐resistant prostate cancer; nodularin‐R.

FIGURE 1

Genome‐wide CRISPR/Cas9 screening for abiraterone‐resistant genes in 22Rv1 cells. (A) Schematic of abiraterone‐resistant 22Rv1 cell construction for high‐throughput sequencing analysis. (B) Number of up‐ (n = 969) and down‐ (n = 629) regulated DEGs in surviving cells. (C) Top 10 genes in each of the up‐ and down‐regulated DEGs. (D) Number distribution of independent sgRNAs in up‐regulated DEGs. (E) KEGG pathway enrichment analysis for up‐regulated DEGs. (F) GO functional enrichment analysis for up‐regulated DEGs.

FIGURE 2

Identification for key resistance genes by bioinformatics. (A) Volcano diagram illustrating DEGs between CRPC and CSPC patients in the GSE221961 dataset. (B) Venn diagram illustrating the interaction of up‐regulated DEGs (n = 77) in CRISPR/Cas screening with up‐regulated DEGs (n = 1172) in CRPC patients. (C) Heat map illustrating the expression of five key resistance genes between CRPC and CSPC patients. (D) DFS of PCa patients with low (n = 239) and high (n = 245) KCNJ3 expression (p = 0.28, HR = 0.8). (E) DFS of PCa patients with low (n = 244) and high (n = 245) COL2A1 expression (p = 0.64, HR = 0.91). (F) DFS of PCa patients with low (n = 246) and high (n = 246) PPP1CA expression (p = 0.017, HR = 1.7). (G) DFS of PCa patients with low (n = 246) and high (n = 246) MDH2 expression (p = 0.13, HR = 1.4). (H) DFS of PCa patients with low (n = 246) and high (n = 246) EXOSC5 expression (p = 0.14, HR = 1.4). (I) Expression of PPP1CA in normal (n = 152) and PCa (n = 492) tissues from the PRAD cohort.

FIGURE 3

Effects of PPP1CA on abiraterone‐resistant 22Rv1 cell function. (A) IC₅₀ of 22Rv1 cells with different resistance degrees. (B) Expression of PPP1CA in 22Rv1 cells with different resistance degrees. (C) Expression validation for PPP1CA silencing and overexpression in moderate‐resistant (Res‐M) cells. (D) Effects of PPP1CA silencing and overexpression on IC₅₀ of Res‐M cells. (E) and (F) Effects of PPP1CA silencing and overexpression on apoptosis in Res‐M cells. (G) and (H) Effects of PPP1CA silencing and overexpression on clone formation in Res‐M cells (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 4

(A) Molecular docking result for nodularin‐R with PPP1CA. (B) IC₅₀ of nodularin‐R on Res‐M cells. (C) Effects of different doses of nodularin‐R on PPP1CA expression in Res‐M cells. Molecular dynamics simulations of USP11‐PPP1CA‐Nodularin‐R complex (D) and USP11‐PPP1CA complex (E). Amino acids involved in hydrogen bonds from USP11 complex A (pink) and complex B (brown), as well as PPP1CA (grey), are shown in three‐letter code followed by their ordinal position in each chain. Nodularin‐R is represented by a green surface.

FIGURE 5

Anti‐tumour effects of nodularin‐R in combination with abiraterone in vitro. (A) IC₅₀ of abiraterone on Res‐M cells after nodularin‐R pre‐treatment. (B) and (C) Effects of the combination for nodularin‐R and abiraterone on Res‐M cell viability. (D) and (E) Effects of the combination for nodularin‐R and abiraterone on apoptosis in Res‐M cells. (F) and (G) Effects of the combination for nodularin‐R and abiraterone on clone formation in Res‐M cells (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 6

Anti‐tumour effects of nodularin‐R in combination with abiraterone in vivo. (A) Overall observation of tumour size in nude mice. (B) Monitoring of body weight changes in nude mice. (C) Monitoring of tumour volume changes in nude mice. (D) Observation (100 ×) of tumour histopathological changes by HE staining. (E) Observation (200 ×) of PPP1CA protein changes in tumour tissues by IHC staining. (F) WB assay for PPP1CA protein expression in tumour tissues (*p < 0.05, **p < 0.01, ***p < 0.001).