Androgen Plays a Carcinogenic Role in EOC via the PI3K/AKT Signaling Pathway in an AR-Dependent Manner

Abstract

Background: Epithelial ovarian cancer (EOC) is one of the most common gynecological cancers with the highest mortality rate. Studies indicate that androgens contribute to initiation or progression of EOC through poorly understood mechanisms, however, in the phase II clinical studies of antiandrogen therapy for EOC, neither flutamide nor bicalutamide showed good antitumor effects. Based on the contradictions, the purpose of this study was to explore the role of androgen receptor (AR) in the androgen pathogenesis of EOC and the possible mechanism, and further to find an indicator to screen the anti-androgen therapy sensitive cases. Methods: In this study, 70 EOC biopsies and 17 para-cancerous tissues with complete medical information were collected and analyzed. The expression of the androgen receptor (AR) was detected by immunohistochemistry. In addition, ovarian cancer cell lines were used for in vitro studies to further explore the role of androgen in cell proliferation and the possible mechanisms. Results: The results showed that the expression of AR in ovarian cancer tissues was significantly elevated compared to the para-cancerous tissues, particularly in low-grade EOC, and the presence of high AR expression often suggested a worse prognosis. The in vitro study indicated that testosterone promoted the proliferation of the AR-positive SKOV3 cell line, which could be blocked by flutamide, but not in the AR-negative A2780 cell line. Next, we showed that testosterone-promoted proliferation in SKOV3 cells was abolished after we knocked out the AR. The mechanism studies revealed that the p-AKT expression in the ovarian cancer tissue was increased compared to the para-cancerous tissues, following a pattern similar to the increase of AR expression. Furthermore, the deletion and overexpression of SKOV3 cells' ARs lead to corresponding changes in the p-AKT levels. In addition, the BEZ235, an inhibitor of the PI3K/AKT signaling pathway blocked the proliferative effect of testosterone in SKOV3 cells. Conclusion: We showed that testosterone was able to promote the proliferation of ovarian cancer cells through activating the PI3K/AKT signaling pathway in an AR dependent manner and AR may be a screening indicator for anti-androgen therapy sensitive cases of EOC.

Keywords: PI3K/AKT signalling pathway; androgen receptor (AR); ovarian carcinoma; proliferation promotion; tissue microarray (TMA).

Figure 1

Protein levels of AR in ovarian epithelial tissue and the clinical significance of AR. (A) Tissue microarray immunohistochemical staining of AR: representative positive and negative IHC staining of AR (b, d) and corresponding HE staining in ovarian cancer tissues (a, c); Representative positive and negative IHC staining of AR (f, h) and corresponding HE staining in ovarian para-cancerous tissues (e, g). As a negative control, the primary antibody was omitted. Scale bar represents 100 μm. (B) Semiquantitative analysis of AR in ovarian cancer tissues and ovarian para-cancerous tissues. Immunostaining is expressed as the median with interquartile range. The analysis of the numeric data relied on non-parametric tests. *p<0.05; **p<0.01; ***p<0.001. (C) Kaplan-Meier plot analysis of EOC patients. Comparison of overall survival in AR positive vs. negative cases revealed that AR positivity is strongly associated with a shortened overall survival (p=0.009).

Figure 2

Testosterone stimulated the growth of AR-positive ovarian cancer cells. (A) Immunofluorescence detection of AR in ovarian cancer cell lines. Scale bar represents 50 μm. (B) Semiquantitative analysis of AR in the two ovarian cancer cells. (C) Western blotting analysis was performed to detect AR in SKOV3 and A2780 cell lines. Protein AR was detected as bands with a molecular mass of 100 kDa. (D) Analysis of the effects of different concentrations of testosterone on cell proliferation revealed that 10 nM and 100 nM testosterone significantly promote SKOV3 cell proliferation (p=0.000) in the 24h group, but there was no significant difference between the two concentrations (p=0.653). All different concentrations of testosterone had no effect on proliferation in the A2780 cell line (24h: p=0.481; 48h: p=0.982). (E) Flutamide blocked the proliferative effects of testosterone on the SKOV3 cell line (p=0.01). *p<0.05; **p<0.01; ***p<0.001.

Figure 3

Testosterone effects on proliferation in SKOV3 cells is mediated by AR. (A) Changes in AR-mRNA after the plasmid knockout and overexpression of AR. (B) Changes in the AR protein after the plasmid knockout and overexpression of AR. (C) Knockout of AR reduced the proliferation effect of testosterone on SKOV3 cells (p=0.043). (D) Overexpression of AR increased the proliferation effect of testosterone on SKOV3 cells (p=0.001). (E) Overexpression of AR had no effect on the proliferation effect of testosterone on A2780 cells (p=0.456). *p<0.05; **p<0.01; ***p<0.001.

Figure 4

Testosterone and AR promote growth in SKOV3 cells via the PI3K/AKT signalling pathway. (A) Tissue microarray immunohistochemical staining of AKT, p-AKT, and AR and corresponding HE staining in ovarian cancer tissues and ovarian para-cancerous tissues. As a negative control, the primary antibody was omitted. Scale bar represents 100 μm. (B) Semiquantitative analysis of AKT, p-AKT and the value of p-AKT/ AKT. The scores of AKT and p-AKT are expressed as the median with interquartile range ( M(QR) ) and the analysis of the numeric data relied on non-parametric tests. The value of p-AKT/ AKT is expressed as mean with standard deviation(± S) and the analysis of the numeric data relied on t-test. (C) Western blotting analysis of AKT and p-AKT was performed in the SKOV3 and A2780 cell lines. Protein levels of AKT and p-AKT were detected as bands with a molecular mass of 60 kDa. (D, E and F) Expression of AKT and p-AKT in the cells after the knockout and overexpression of AR. (G) BEZ235 concentration vs inhibition rate curve (IC50=187 nM), and (H) BEZ235 blocked the proliferative effects of testosterone in the SKOV3 cell line (p<0.001).

Figure 5

Bioinformatics analyses based on the TCGA database. (A) Expression of AR based on patient`s age (p>0.05). (B) Expression of AR based on tumor grades (p>0.05). (C) Expression of AR based on individual stages (p>0.05). OV: Ovarian serous cystadenocarcinoma.