Abstract
To identify novel oncogenic E3 ubiquitin ligases as anticancer targets, we screened an E3 ubiquitin ligase siRNA library containing siRNA pools against 555 individual E3s using the sulphorhodamine B assay in the MDA-MB-231 breast cancer cell line and the PC3 prostate cancer cell line. RNF126 was identified and validated as a candidate from this screening. Knockdown of RNF126 dramatically decreased cell viability in these cancer cell lines. Consistently, RNF126 knockdown delayed cell-cycle G(1)-S progression and decreased cell proliferation. Using protein array analysis we found that RNF126 silencing increased cell-cycle dependent kinase inhibitor p21(cip) protein levels in both MDA-MB-231 and PC3. Knockdown of RNF126 stabilized the p21 protein rather than increased p21 mRNA levels. We showed that RNF126 interacts with p21 and RNF126 overexpression increased p21 protein ubiquitination in an E3 ligase activity-dependent manner. RNF126 knockdown induced loss of cell viability in MDA-MB-231 and PC-3 can be partially rescued by depletion of p21. RNF126 stable knockdown in PC3 inhibited tumor growth in SCID mice. Finally, we found that RNF126 is highly expressed in a subset of breast cancer cell lines and negatively correlated with p21 expression levels. These findings suggest that RNF126 promotes cancer cell proliferation by targeting p21 for ubiquitin-mediated degradation. RNF126 could be a novel therapeutic target in breast and prostate cancers.
Highlights
Protein ubiquitination regulates a multitude of cancerrelated cellular processes, including the cell cycle [1]
To identify novel oncogenic E3 ligases for cancer treatment, we screened a siRNA library against 555 E3 ubiquitin ligases by the SRB assay using the MDA-MB-231 breast cancer cell line and the PC-3 prostate cancer cell line
Ring finger protein 126 (RNF126) can ubiquitinate itself in E3 ligase activity dependent manner in the presence of E1, UbcH5b (E2), ATP, and Ub (Fig. 4A). These results show that RNF126 is an E3 ubiquitin ligase
Summary
Protein ubiquitination regulates a multitude of cancerrelated cellular processes, including the cell cycle [1]. Protein ubiquitination is typically sequentially mediated by 3 enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) that controls substrate specificity [2]. Ubiquitin E3 ligases are considered the wave of molecules for targeted therapy [3,4,5,6]. Authors' Affiliations: 1Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology; 2The Department of Pathology of the 1st affiliated Hospital of Kunming Medical University; 3The Department of Breast of the 3rd affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China; and 4The Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York.
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