Collagen and calcium-binding EGF domains 1 is frequently inactivated in ovarian cancer by aberrant promoter hypermethylation and modulates cell migration and survival

Abstract

Background: Collagen and calcium-binding EGF domains 1 (CCBE1) is an uncharacterised gene that has down-regulated expression in breast cancer. As CCBE1 maps to 18q21.32, a region frequently exhibiting loss of heterozygosity in ovarian cancer, the aim of this study was to determine the expression and function of CCBE1 in ovarian cancer.

Methods: Expression and methylation patterns of CCBE1 were determined in ovarian cancer cell lines and primary tumours. CCBE1 contains collagen repeats and an aspartic acid/asparagine hydroxylation/EGF-like domain, suggesting a function in extracellular matrix remodelling and migration, which was determined using small-interfering RNA (siRNA)-mediated knockdown and over-expression of CCBE1 in cell lines.

Results: CCBE1 is expressed in normal ovary, but is reduced in ovarian cancer cell lines and primary carcinomas. Pharmacological demethylation/deacetylation in ovarian cancer cell lines re-induced CCBE1 expression, indicating that epigenetic mechanisms contribute to its silencing in cancer. CCBE1 promoter hypermethylation was detected in 6/11 (55%) ovarian cancer cell lines and 38/81 (41%) ovarian carcinomas. siRNA-mediated knockdown of CCBE1 in ovarian cancer cell lines enhanced their migration; conversely, re-expression of CCBE1 reduced migration and survival. Hence, loss of CCBE1 expression may promote ovarian carcinogenesis by enhancing migration and cell survival.

Conclusions: These data suggest that CCBE1 is a new candidate tumour suppressor in ovarian cancer.

Figure 1

CCBE1 expression is lost or reduced in ovarian and breast cancer. (A) Real-time quantitative PCR showing CCBE1 mRNA expression in a panel of ovarian cancer cell lines as compared with immortalised HOSE 6.3 cells. CCBE1 mRNA was normalised to GAPDH mRNA expression levels. (B) Western blot analysis showing CCBE1 expression (51 kDa) in ovarian cancer cell lines as compared with HOSE 6.3 cells. A positive control protein lysate was extracted from IGROV1 cells transiently transfected with V5-tagged CCBE1. (C) Box plot of CCBE1 mRNA expression in NOSE (n=14) as compared with serous (n=64), endometrioid (n=5), clear cell (n=5) and mucinous (n=4) ovarian carcinomas. (D) Box plot of CCBE1 mRNA expression in low (grade 1; n=7) and high (grade 2,3, n=71) grade ovarian carcinomas. (E) Box plot of CCBE1 expression in FIGO stage I (n=10), stage II (n=10), stage III (n=51) and stage IV (n=7) ovarian carcinomas. (F) Kaplan–Meier survival curve for relapse-free survival time stratified by CCBE1 expression in all ovarian carcinomas.

Figure 2

Representative ISH for CCBE1 mRNA in normal ovary using (A) sense negative control and (B) antisense probes, respectively; (C) mucinous ovarian carcinoma; (D) serous ovarian carcinoma; (E) endometrioid ovarian carcinoma, all with antisense probe (magnification × 20).

Figure 3

CCBE1 is methylated in ovarian cancer cell lines and primary carcinomas. (A) Methylation-specific PCR analysis of bisulphite-treated DNA from HOSE 6.3 and ovarian cancer cell lines. Positive control is bisulphite-treated CpGenome universally methylated DNA; negative control is bisulphite-treated genomic unmethylated DNA. (B) Normalised expression of CCBE1 mRNA in HOSE 6.3 and five ovarian cancer cell lines after treatment with 5-AZA, TSA or in combination, as compared with untreated cells. (C) Kaplan–Meier survival curve for relapse-free survival time stratified by CCBE1 methylation status in all ovarian carcinomas.

Figure 4

Modulation of CCBE1 levels affects cancer cell line behaviour. (A) Cartoon depicting the structural domains of CCBE1, which predict a function in cross-talk between the ECM and NOSE. (B) Knockdown of CCBE1 expression by siRNA in CoLo316 cells increases cell migration. The graph shows the number of migrating cells expressed as a percentage of siGLO scrambled control from two independent experiments. Expression of CCBE1 72 h post-transfection was determined by western blotting. (C) Over-expression of CCBE1 in T-47D cells decreases cell migration. The graph shows the number of migrating cells expressed as a percentage of vector high GFP controls from three independent experiments (^*P<0.013, n=5). CCBE1 over-expression was confirmed by western blotting and was comparable with endogenous expression of CCBE1 in HOSE6.3 cells. (D) Over-expression of CCBE1 in T-47D cells decreases their colony-forming ability. The graph shows colony density expressed as a percentage of vector control cells expressing high levels of GFP from five independent experiments (^*P<0.01, n=15). All images show representative fields of view (magnification × 20).