Cystathionine beta-synthase (CBS) contributes to advanced ovarian cancer progression and drug resistance

Abstract

Background: Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer.

Methods/principal findings: Using patient tissue microarray (TMA), in vitro and in vivo studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The in vitro effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H2S producing chemical Na2S. Silencing CBS in a cisplatin resistant orthotopic model in vivo by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics.

Conclusion: The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer.

Figure 1. Expression and phenotypic effects of CBS in vitro.

(A) Immunohistochemical staining of a tissue microarray of epithelial ovarian cancer samples. Representative images are shown of none (i), weak (ii), moderate (iii), and (iv) strong staining. (B) Expression of CBS and CSE in various ovarian cell lines as determined by immunoblotting. α-tubulin is used as the loading control. (C) RT-PCR data showing the expression of CBS mRNA in various ovarian cell lines. (D) RT-PCR data showing the expression of CSE mRNA in various ovarian cell lines. (E) Effect of CBS knockdown on the proliferation of OV202, SKOV3, A2780 and A2780/CP-70 cells and (F) Immunoblotting data to determine the extent of siRNA-mediated knockdown. (G) Effect of CBS inhibition on the proliferation of OV202, SKOV3 and A2780 cells by AOAA (24 h treatment) determined through MTS assay.

Figure 2. Effect of CBS knockdown on metabolite levels.

(A) Change in Hcy levels in the cell lysates of Sc-siRNA and CBS siRNA treated A2780 cells measured by mass spectrometry. (B) Hcy overdose experiment to demonstrate the effect of Hcy (24 h treatment) on the proliferation of OSE, A2780 and SKOV3-ip cells by MTS assay. (C) H₂S levels in OV202, SKOV3 and A2780 cells after chronic inhibition with different doses of AOAA for 3 h. (D) Na₂S rescue experiment after knockdown of CBS in A2780 cells. The cells were treated with different doses of Na₂S for 24 h and the cell viability was determined by MTS assay. (E) Change in total glutathione level 48 h post-transfection with scrambled control and CBS siRNA in OV202, SKOV3 and A2780 cells. (F) GSH rescue experiment after knockdown of CBS in A2780 cells. The cells were treated with various doses of GSH for 24 h and the cell viability was assessed by MTS assay.

Figure 3. Silencing of CBS increases oxidative stress and sensitizes to Cisplatin chemotherapy.

(A) Effect of CBS knockdown (48 h post-transfection) on total ROS level determined by DCF assay and quantified by flow cytometry. (B) Immunoblotting data exemplifying the effect of CBS silencing on the expression of p53 and NF-κB p65 in A2780 cells. β-actin serves as the loading control. (C) Luciferase reporter assay showing the effect of CBS knockdown on the activity of NF-κB in A2780 cells transfected with Firefly luciferase with multiple NF-κB binding site and scrambled control siRNA or CBS siRNA. wt-renilla luciferase was transfected to normalize firefly luciferase activity. (D) CBS silencing enhances the activity of cisplatin in A2780 cells. The (•) and (О) shows the effect of cisplatin on scrambled control siRNA and CBS siRNA treated cells. The cell viability was determined after treatment with increasing doses of cisplatin for 24 h through MTS assay and the cell viability was expressed as a percentage ratio of treated cells to the untreated controls. The IC₅₀ values were obtained by non-linear regression analysis.

Figure 4. Localization and effect of silencing CBS on mitochondrial function.

(A) Localization of CBS in A2780 cells determined by immunofluorescence using confocal microscopy. Nuclear stain with DAPI (blue channel), CBS (red channel), MitoTracker green (green channnel) was used to label mitochondria. Scale bar is 10 µm. (B) MitoSOX staining in live A2780 cells showing the buildup of mitochondrial superoxide upon silencing CBS. Scale bar is 30 µm. (C) Measurement of citrate synthase activity in AOAA-treated A2780 cells following 3 h of treatment. (D) Rate of oxygen consumption in scrambled control and CBS siRNA treated A2780 cells under resting condition and upon uncoupling with FCCP. (E) Rate of oxygen consumption in control (vehicle treated only) and 3 h AOAA treated A2780 cells under resting condition and upon uncoupling with FCCP.

Figure 5. Effect of CBS silencing on OXPHOS.

(A) Fold change in NAD/NADH ratio in CBS silenced A2780 cells measured 48 h after transfection (B) Fold change in NAD/NADH ratio in AOAA-treated A2780 cells measured after 3 h of treatment. (C) Fold change in total ATP levels in AOAA-treated A2780 cells with respect to scrambled control siRNA treated cells after 3 h treatment (D) Percent change in ADP/ATP ratio in AOAA-treated A2780 cells measured after 3 h of treatment. (E) Fold change in total ATP levels in CBS siRNA treated A2780 cells with respect to scrambled control siRNA treated cells measured 48 h after transfection (F) Percent change in ADP/ATP ratio in CBS siRNA treated A2780 cells with respect to scrambled control siRNA treated cells measured 48 h after transfection.

Figure 6. Effect of CBS knockdown on orthotopic chemoresistant ovarian cancer growth.

(A)To assess the effects of siRNA therapy on tumor growth, treatment was initiated 1 wk after i.p. injection (1.0×10⁶ A2780/CP20) of tumor cells. Mice were divided into four groups (n = 10 mice per group): (i) control siRNA-DOPC (150 µg/kg i.p. twice weekly), (ii) control siRNA-DOPC (150 µg/kg i.p. twice weekly)+cisplatin (160 µg/mouse i.p. weekly), (iii) CBS siRNA-DOPC (150 µg/kg i.p. twice weekly), and (iv) CBS siRNA-DOPC (150 µg/kg i.p. twice weekly)+cisplatin ((160 µg/mouse i.p. weekly). Treatment was continued until 4 weeks after tumor inoculation before sacrifice. (A) Mouse and tumor weights and (B) the number of tumor nodules for each group were compared using Student's t test (for comparisons of two groups). A two-tailed P≤0.05 was deemed statistically significant. (C) Immunoblotting of tumor samples for confirmation of CBS knockdown. One animal from each group was selected for immunoblotting analysis. Lane 1 and lane 4 are from different blots. (D) Representative histology of tumors from mice xenografts of A2780/CP-20 cells with Ki67 expression (middle row) and CD31 expression (lower panel) acquired at 20X magnification. Scale bar represents 100 µm. (E) and (F) Quantification of Ki-67 staining and CD31 staining in the mouse xenografts respectively (n = 4). Statistical analysis was determined using One-way ANOVA with *P<0.05 and **P<0.01.