The Regulation of OCT4 Ubiquitination by E3 Ligase CHIP that Effects on Tumorigenesis of Breast Cancer

Abstract

Breast cancer has been considered to be hierarchically organized and it contains cancer stem cells (CSCs), which do self-renewal and relapse after therapy. Therefore, characterization and targeting CSCs are an issue recently. Because the role of protein stability and homeostasis against initiation and maintenance of CSCs was not elucidated, after preparation of mammospheres using breast cancer MDA-MB231 cells and MCF-7 cells, I performed DNA microarray analysis and observed that the expression of CHIP E3 ubiquitin ligase was down-regulated in both cell-made mammospheres. Ablation of CHIP increased mammosphere formation, whereas overexpression of CHIP reversed these effects. I searched the interactomes by mass-spectrum analysis and found that CHIP directly interacted with OCT4. Overexpression of CHIP reduced OCT4 stability by proteosomal degradation. K30A chaperone-binding mutant CHIP showed less interaction with OCT4, suggesting that CHIP interacts with OCT4 by chaperone-dependent manner. Whereas CHIP induced ubiquitination of OCT4, H260Q catalytic mutant CHIP couldn’t effect it. Interestingly, I determined 284 lysine of OCT4 is an ubiquitination site by CHIP. Overexpression of CHIP couldn’t degrade K284R mutant OCT4. Overexpression of CHIP reduced cell proliferation and side population of breast cancer cells, however co-overexpression of K284R mutant OCT4 and CHIP didn’t reduce them. Moreover, only 1,000 cells with CHIP depleted or OCT4 overexpressed could generate breast tumors and metastasis in xenografted mice model. Breast cancer patients with low-expressed CHIP E3 ligase have poor survival probability. Taken together, ubiquitination of OCT4 by CHIP plays a role in breast tumorigenesis and I propose that regulation of OCT4 stability could be a potent approach in breast cancer therapy.