Expression of MUS81 Mediates the Sensitivity of Castration-Resistant Prostate Cancer to Olaparib

Abstract

This project attempts to clarify the expression of MUS81 in castration-resistant prostate cancer (CRPC) and the effect on drug sensitivity to Olaparib. We collected clinical surgical samples of patients who were suffering from benign prostatic hyperplasia (BPH), common prostate cancer (PCa), and castration-resistant prostate cancer (CRPC) and detected the expression of MUS81 in healthy prostate epithelial cells, PCa cells, and androgen-independent PCa cells. We subsequently performed CCK-8 assays, flow cytometry, and Transwell invasion and migration assay to determine the proliferation, apoptosis, invasion, and metastasis abilities of transfected CRPC cells as well as drug toxicity of Olaparib to CRPC cells. The expression of MUS81 indicated marked upregulation in PCa and CRPC tissues, compared with the level of MUS81 in BPH tissues. MUS81 silencing inhibited the proliferation of CRPC cells and promoted their sensitivity to Olaparib. MUS81 silencing in CRPC cells remarkably accelerated cell apoptosis and greatly inhibited cell invasion and metastasis after Olaparib administration. MUS81 silencing in CRPC cells has significantly enhanced the sensitivity of cells to Olaparib, which provides evidence for the prediction of Olaparib resistance in CRPC cells by the MUS81 gene and is expected to become a promising gene target in CRPC therapy.

Figure 1

Immunohistochemical staining of MUS81 in BPH, PCa, and CRPC tissues. (a) IHC analysis of MUS81 in BPH (A and D), PCa (B and E), and CRPC (C and F) tissues. (b) Scores of IHC and analysis of MUS81 expression in BPH, PCa, and CRPC tissues. ^∗∗P < 0.01 and ^∗∗∗P < 0.001, versus the expression of MUS81 in BPH.

Figure 2

Differences in protein expression levels of MUS81 in PC3, RWPE-1, LNCaP, and DU145 cells. (a) The relative MUS81 expression in PC3, RWPE-1, LNCaP, and DU145 cells was detected via western blot. (b) MUS81 protein expression in PC3, RWPE-1, LNCaP, and DU145 cells was detected via western blot. ^∗P < 0.05 and ^∗∗∗P < 0.001, versus the expression of MUS81 in RWPE-1 cells.

Figure 3

After lentivirus transfection, the expression of MUS81 in DU145 and PC3 cells. (a) The relative levels of the MUS81 mRNA transcripts after silencing MUS81. (b) The relative levels of MUS81 protein after silencing MUS81. (c) The relative levels of the MUS81 mRNA transcripts after overexpressing MUS81. (d) The relative levels of MUS81 protein after overexpressing MUS81. ^∗∗∗P < 0.001 versus the no-load virus group (EV) cells and negative control group (BV) cells.

Figure 4

Effect of overexpression and MUS81 silencing on CRPC cell proliferation and its relationship with ATM protein levels. (a) CRPC cell proliferation. (b) The relative phospho-ATM and ATM protein expression. ^∗P < 0.05 and ^∗∗P < 0.01, compared with PC3 cells. ^∗∗∗P < 0.001.

Figure 5

Relationship between the MUS81 expression and the sensitivity to Olaparib. (a) CRPC cell viability after 80 μM Olaparib treatment. (b) Cell viability of PC3, PC3-LV, PC3-sh, DU145, DU145-LV, and DU145-sh cells at 3 × 10³ cells/well following Olaparib treatment at several concentrations for 48 h by CCK-8 assay. ^∗P < 0.05 and ^∗∗∗P < 0.001.

Figure 6

Effects of MUS81 silencing combined with Olaparib on apoptosis of CRPC cells. (a) Cell apoptosis of D: DU145, P: PC3.D-EV, D-sh, P-EV, and P-sh following 48 hours of either Olaparib or DMSO treatment by Annexin V-APC/7-AAD. (b) Apoptosis. (c) Expression of cleaved caspase-3, Bax, and Bcl-2 proteins in the CRPC cells by western blot. ^∗∗∗P < 0.001.

Figure 7

Effects of MUS81 silencing combined with Olaparib on the invasion and migration ability of CRPC cells. (a, b) The migration and invasion of D-EV, D-sh, P-EV, and P-sh following 48 hours of either Olaparib or DMSO treatment by Transwell assays. ^∗P < 0.05,^∗∗P < 0.01, and^∗∗∗P < 0.001, versus EV-treated group.