Deubiquitination And Stabilization Of HER2 By Ubiquitin-Specific Protease 7 Promotes Breast Tumorigenesis

Background:In the global cancer statistics of 2020,breast cancer ranks first among female tumors in terms of morbidity and mortality,and HER2-positive breast cancers account for 20-25%in the category of breast cancer.Human epidermal growth factor receptor 2(HER2)is an important biomarker and therapeutic target for breast cancer.Targeted therapy for HER2 or combination therapy has greatly improved the survival rate of patients,but drug resistance and adverse events are still inevitable,which makes the clinical treatment of HER2-positive breast cancer more difficult.Therefore,it is urgent to find a novel HER2-targeting therapy.Recent studies have shown that the ubiquitin proteasome system is closely related to the occurrence and development of tumors,also plays an important role in the occurrence and development of diseases,such as cellular DNA damage and repair,immune response,epigenetic control and tumor progression.Deubiquitinases(DUBs)in UPS is considered as a new target for cancer treatment.Studies have shown that HER2 protein in cells can be degraded by the ubiquitin proteasome system,but the involvement of the deubiquitination enzyme in the ubiquitination of HER2 is rarely reported.In this study,a TCGA database search found that ubiquitin-specific protease 7(USP7)was expressed at a higher level in breast cancer,and clinical samples further showed that USP7 was expressed at a higher level in cancer tissues than in the paracancer tissues.Next,we demonstrated that USP7 regulates ubiquitination of HER2 and affects the progression of HER2-positive breast cancer cells.The current study aims to explore the role and molecular mechanism of USP7 in HER2-positive breast cancer,and provide a new idea for the treatment of HER2-positive breast cancer.Methods1.Cell viability assay:MTS reagent was used for cell viability detection.2.Clone formation experiment:the experimental treated cells were observed by crystal violet staining for the colony formation.3.EDU cell proliferation experiment:the proliferating cells after staining were photographed by fluorescence microscope and calculated the ratio of the number of EDU positive cells to the total number of cells.4.Cell cycle and apoptosis detection:After digestion and collection,cell cycle and apoptosis were detected by flow cytometry.5.SiRNA transfection technology:two USP7 sites were knocked down by SiRNA transfection reagents and detected the knockdown efficiency.6.Western blot experiment:Cell protein lysates were collected and the expression of target proteins was detected by western blot assay.7.Immunofluorescence experiment:SK-BR3 cells were treated and the intensity of HER2 and co-location of USP7 and HER2 were detected by immunofluorescence.8.Real-time quantitative fluorescent polymerase chain reaction(RT-qPCR):Total RNA was extracted from the cells after experimental treatment with RNAisoplus,and cDNA was obtained after reverse transcription.After amplification,the target genes were detected to obtain their Cq values.Results1.USP7 expression is positively correlated with HER2 and predicts poor prognosis in HER2-positive breast cancer.TCGA database analysis showed that the mRNA level of the deubiquitination enzyme USP7 was highly expressed in breast cancer,and USP7 was positively correlated with HER2.HER2-positive breast cancer patients with high expression of USP7 showed worse survival rate,suggesting that USP7 could influence the prognosis of breast cancer patients as a oncogenic factor.2.USP7 interacts with HER2Immunoprecipitation and immunofluorescence showed that USP7 and HER2 could interact in cytoplasm.3.USP7 regulates the expression of HER2 in breast cancer cellsWestern blot and immunofluorescence results showed that inhibition or knockdown of USP7 down-regulated HER2 protein expression.RT-qPCR and CHX tracing experiments showed that USP7 regulated HER2 expression through the protein degradation pathway rather than the transcription way.4.USP7 inhibition and knockdown promote the ubiquitination and degradation of HER2Cells treated with the 20S proteasome inhibitor MG132 in combination with P5091 showed increased the expression of HER2,suggesting that USP7 regulates HER2 expression through the ubiquitin proteasome pathway.Immunoprecipitation assay results showed that both inhibition and knockdown of USP7 increased endogenous ubiquitination levels of HER2.5.USP7 mediates the proliferation of HER2-positive breast cancer cellsMTS activity assay showed that cell viability decreased with time concentration gradient after inhibition and knockdown of USP7.Clonal formation and EDU proliferation assay also showed that inhibition or knockdown of USP7 inhibited the growth of breast cancer cells.6.USP7 mediates cell cycle and apoptosis to regulate the process of breast cancer cellsFlow cytometry results showed that inhibition of USP7 blocked the transformation of G0/G1 phase from S phase,and knocking down USP7 promoted the apoptosis of breast cancer cells.Western blotting showed the same result,pro-cycle molecule expression was down-regulated,anti-cycle molecule expression was upregulated,apoptotic molecule PARP was cleaved.7.Overexpression of HER2 rescues USP7 inhibition-induced cell cycle arrestWe inhibited USP7 on SK-BR3 cells and then overexpressed HER2.Flow cytometry and western blot assay showed that USP7 inhibiting blocked the cycle progression of breast cancer cells depending on the expression of HER2 protein.Conclusion1.The deubiquitination enzyme USP7 is highly expressed in HER2-positive breast cancer and shows poor prognosis;2.USP7 can acts as a deubiquitination enzyme of HER2,which blocks the degradation of HER2 by removing the ubiquitin chain on HER2,stabilizes the expression of HER2 protein and promotes the growth of breast cancer cells,providing a new idea and direction for the treatment of patients with HER2-positive breast cancer.