BRCA1 expression is epigenetically repressed in sporadic ovarian cancer cells by overexpression of C-terminal binding protein 2

Abstract

Introduction: Ovarian cancer is the leading cause of mortality from gynecological malignancy despite advancements in novel therapeutics. We have recently demonstrated that the transcriptional co-repressor C-terminal binding protein 2 (CtBP2) is overexpressed in epithelial ovarian carcinoma.

Materials and methods: Reverse-transcribed cDNA from CtBP2 wild-type and knockdown ovarian cancer cell lines was hybridized to Affymetrix Gene 1.0 ST microarrays, and differentially expressed genes were studied. Immunohistochemical analysis of CtBP2 and BRCA1 staining of ovarian tissues was performed. Chromatin immunoprecipitation (ChIP) and luciferase assays were carried out. The effect of the drugs 4-methylthio-2-oxobutyric acid (MTOB) and poly(ADP-ribose) polymerase (PARP) inhibitor Olaparib on CtBP2 wild-type and knockdown cell lines was examined using methylthiazol tetrazolium assays and an xCELLigence System.

Results: Eighty-five genes involved in DNA repair, mitotic checkpoint, nucleosome assembly, and the BRCA1 network were differentially regulated by CtBP2 expression. ChIP and luciferase reporter assays using a BRCA1 promoter-regulated luciferase construct indicated that the CtBP2 complex binds the BRCA1 promoter and represses BRCA1 transcription. Immunohistochemistry illustrated a significant inverse CtBP2 and BRCA1 expression in a panel of malignant ovarian tumor tissues. The CtBP2 inhibitor MTOB suppressed ovarian cancer cell survival in a CtBP2-dependent manner. Ovarian cancer cells with CtBP2 knockdown did not display increased sensitivity to the PARP inhibitor Olaparib.

Conclusion: CtBP2 is an ovarian cancer oncogene that may play a significant role in epigenetically silencing BRCA1 function in sporadic epithelial ovarian cancer. CtBP2-specific inhibitors, such as MTOB, may be effective adjunct therapies in the management of patients with CtBP2-positive ovarian carcinoma.

Figure 1

Gene expression profiling to identify differentially expressed gene signatures in the CtBP2 knockdown cell lines. (A) A novel BRCA1 DNA damage response pathway identified by Ingenuity Pathway Analysis program. (B) Western blot analysis to show that BRCA1 protein expression in the MCAS cells corresponds to cellular CtBP2 expression. β-Actin was used as a loading control.

Figure 2

CtBP2 binds to BRCA1 promoter and epigenetically represses BRCA1 expression. (A) ChIP assay using anti-CtBP2 antibody. (B) ChIP assay using anti-acetyl histone H3K9 antibody. (C) ChIP assay using anti-acetyl histone H3K14 antibody. (D) ChIP assay using anti-dimethyl histone H3 antibody. (E) ChIP assay using anti-RNA Pol II antibody. All the ChIP data are shown relative to the controls (defined as 1). (F) Luciferase reporter assay to determine the BRCA1 promoter activity. Results shown are averages of both MCAS and SKOV3 cancer cell lines and all significant values are relative to control. Significance of differences was determined using two-tailed t test. *P < .01; **P < .05.

Figure 3

IHC to investigate the relationship between CtBP2 and BRCA1 protein expression in clinical ovarian tumors. (A) Representative images of malignant ovarian carcinomas that show negative association of CtBP2 and BRCA1 staining. Scale bar represents 50 µm. (B) A scatterplot to show the negative relationship between CtBP2 and BRCA1 protein staining.

Figure 4

Sensitivity of ovarian cancer cell lines with changes of CtBP2 expression to (A) PARP inhibitor Olaparib and (B) CtBP2 inhibitor MTOB. *P < .05 relative to the control cell lines.