Regulation Of P53Level By UBE4B In Breast Cancer

Objective:p53is possibly the most important mammalian tumor suppressor and it is mutated or lost in more than half of all human cancers. The Ub chain assembly factor (E4), a new class of ubiquitylation enzyme, has recently been discovered and is shown to physically interact with p53and Hdm2and to negatively regulate p53stability and function. Our results indicate that UBE4B promoted the degradation and ubiquitination of p53to inhibit the apoptosis of cancer cells and promote tumorigenesis and could be a viable target for developing new therapeutic strategies for breast cancer treatment.Methods:Breast cancer tissue samples were obtained from patients who underwent surgical resection. We detected UBE4B protein expression in normal breast tissues and breast cancer tissues and then examined the protein expression of UBE4B and p53in the breast cancer tissues using western blot analysis. Breast cancer cell lines MCF-7and SK-BR3was used to carry out the in vivo ubiquitylation assay, co-immunoprecipitation, apoptosis assays and the nude mouse experiments. We showed that UBE4B physically interacts with p53and negatively regulates p53stability and function in breast cancer finally.Results: 1. UBE4B and p53are expressed in breast cancer tissues and UBE4B enhances p53ubiquitinationWestern blot results showed that UBE4B expression was higher in breast cancer tissues than in normal breast tissues. We then examined the protein expression of UBE4B and p53in the breast cancer tissues. The levels of p53and UBE4B mainly showed an opposite state. In MCF-7cells, the p53protein was heavily ubiquitinated in the presence of Hdm2or UBE4B and was more heavily ubiquitinated by the coexpression of Hdm2and UBE4B. The level of p53ubiquitination was evidently decreased when p53and UBE4B-siRNA were coexpressed. The same condition was observed in the SK-BR-3cells.2. Interactions of UBE4B with Hdm2and with p53in vivoIn MCF-7cells, co-immunoprecipitation results showed that UBE4B interacts with p53, similar experiments were conducted to determine whether UBE4B interacts with Hdm2. The results proved that UBE4B interacts with both Hdm2and p53in breast cancer.3. UBE4B negatively regulates p53and promotes p53degradationP53-/-MCF-7cells (p53KO) were cotransfected with Flag-UBE4B or with Myc-Hdm2and p53. The amount of p53protein was reduced. On the contrary, we transfected MCF-7cells with UBE4B siRNA or Hdm2siRNA, the amount of p53protein was raised. Increasing amounts of UBE4B were transfected into WT p53-expressing MCF-7cells, which showed that UBE4B-induced p53degradation is dose dependent. We transfected MCF-7with UBE4B siRNA or the control siRNA to determine whether UBE4B is crucial for Hdm2-mediated p53degradation in breas cancer cells.4. UBE4B inhibits p53-dependent apoptosis and promotes tumorigenesisTo verify this conclusion, we carried out the apoptosis assays. The amount of p53protein was reduced and the decrease in breast cancer cells apoptosis induced by UBE4B overexpression in contrast to that of p53 indicated that UBE4B inhibits cancer cell apoptosis. Coexpression of UBE4B and Hdm2more significantly prevented apoptosis than cotransfected with UBE4B-siRNA and Hdm2-siRNA. To test the in vivo tumorigenic efficacy of UBE4B, we established a xenograft animal model in nude mice. We subcutaneously injected MCF-7expressing vector encoding UBE4B or an empty vector into the mice. We found that UBE4B-overexpressing tumors have significantly faster growth rates than tumors with empty vector expression.Conclusion:In this research, we first identified the physiological significance of UBE4B in p53degradation and promote the Hdm2-mediated ubiquitination of p53in breast cancer. These new findings proved further that UBE4B is an oncogene and may promote the formation of breast cancer tumors. UBE4B will ultimately provide the most effective way to take advantage of the ever-increasing list of new biological therapies for breast cancer.