Cullin-5, a ubiquitin ligase scaffold protein, is significantly underexpressed in endometrial adenocarcinomas and is a target of miR-182

Abstract

Altered expression of cullin-5 (CUL5), a member of the cullin-RING E3 ubiquitin ligase family, has been implicated in a number of types of cancers including breast, cervical and hepatocellular cancers. In the present study, we found that CUL5 expression was significantly decreased in both endometrioid and serous endometrial adenocarcinomas with the more aggressive serous type displaying a higher reduction (-4.3-fold) than the less aggressive endometrioid type (-2.9-fold). Overexpression of CUL5 mRNA and protein in Ishikawa H endometrial cancer cells resulted in decreased cell proliferation and in a reduction in CUL5-RING E3 ligase downstream clients JAK2 and FAS-L. Finally, we demonstrated for the first time that CUL5 is a direct target of miR-182 that we previously showed to be significantly overexpressed in endometrial adenocarcinomas and we provided evidence that increased miR-182 expression is, at least in part, a result of demethylation of its upstream promoter. These data suggest a cascade in which miR-182 expression is epigenetically increased leading to decreased CUL5 expression and increased cellular proliferation. The final step in the cascade may be operating through a decrease in ubiquitination of pro-growth CUL5 ubiquitin ligase clients. This cascade offers a series of potential interventional steps involving epigenetic modification, miRNA and/or gene targeting and ubiquitination.

Figure 1

CUL5 mRNA expression in endometrial endometrioid adenocarcinomas (EEA) and endometrial serous adenocarcinomas (ESA) relative to benign endometrium (BE). In both cases underexpression in the tumors was statistically significant. ^*p<0.001 EEA or ESA vs. BE.

Figure 2

miR-182 targets CUL5 in endometrial cancer cells. (A) Evolutionary conservation of the miR-182 binding sites in the 3′-UTR of CUL5. Position 1 conservation covers >350,000,000 years while position 2 conservation covers 180,000,000 years. (B) Relative CUL5 mRNA in Ishikawa H and Hec50co endometrial cancer cells transiently transfected with a miR-182 mimic compared with mock-transfected Ishikawa H and Hec50co cells; ^*p<0.01.

Figure 3

The effect of CUL5 protein overexpression on cell proliferation and on other proteins. (A) A ³H-thymidine uptake assay comparing mock-transfected Ishikawa H cells with the stable CUL5-overexpressing Ishikawa H subcell line CUL5wt35. (B) Western blot analyses of CUL5 and possible client proteins in the mock-transfected Ishikawa H cells and the stable CUL5-overexpressing Ishikawa H subcell line CUL5wt35; ^*p<0.05.

Figure 4

Methylation-specific PCR (MSP) of the miR-182 promoter in five endometrial cancer (EC) cell lines. MSP of the miR-182 promoter (top) showed that all five EC cell lines lacked methylation in the promoter while all five cell lines were methylated in the control miR-181c promoter (bottom). In both amplifications MSP primers were designed (mir-182) or validated (miR-181c) (16,24) using MethPrimer (15). Un, unmethylated; Me, methylated; M, 100-bp ladder (TrackIt^®; Thermo Fisher).