RNF6 Promotes The Migration And Invasion Of Breast Cancer By Promoting The Ubiquitination And Degradation Of MST1

Background and Objective: Breast cancer(BC)is one of the most common cancers worldwide and is the main cause of mortality of female patients with cancer.The 5-year survival rate of early BC is about 83%,but the 5-year survival rate of those with metastatic disease remains poor.Therefore,investigating the molecular mechanism of BC metastasis may be helpful for the formulation of treatment strategies for breast cancer.Ring finger protein 6(RNF6),a member of E3 ubiquitin ligases,regulates a number of target genes that are important for cell growth and survival.Recent accumulating evidence indicated that RNF6 could promote the degradation of its target protein by a ubiquitin proteasome.Recently,a number of studies confirmed that RNF6 is a carcinogenic gene in numerous types of cancer,including prostate cancer,oesophageal squamous cell carcinoma and lung adenocarcinoma.For instance,the expression levels of RNF6 are elevated in colorectal cancer,and high RNF6 levels is correlated with poor prognosis.However,the effect of RNF6 on the invasion and metastasis of BC and its potential mechanisms remain to be elucidated.Hippo signalling is an evolutionarily conservative signalling pathway that plays a key role in controlling the invasion and migration of tumour cells.At the core of the Hippo pathway is a kinase cascade that activates mammalian STE20-like protein kinase(MST)1/2 phosphorylation and activates large tumour suppressor kinase 1/2,which phosphorylates and inhibits the two major downstream effectors of the Hippo pathway,YES-associated protein(YAP)and transcriptional co-activator with PDZ-binding motif(TAZ).Dephosphorylated YAP and TAZ enter the nucleus and induce target gene expression,which promotes cell migration.Studies demonstrated that YAP is overexpressed in some tumours,and sustained expression of YAP promoted growth and tumour occurrence,suggesting that the Hippo pathway plays a key role in tumourigenesis,However,the mechanism of YAP expression regulation in BC is still unclear.In the present study,we first found that RNF6 was significantly upregulated in breast cancer tissues compared with the adjacent normal tissues.We then studied the expression and function of RNF6 in breast cancer cells.We also identified RNF6 as a possible E3 ligase of MST1(also known as S100A4),as it interacted with and promoted the degradation of MST1.These data might highlight the role of RNF6 in BC,which could serve as a valuable prognostic indicator and potential therapeutic target for patients with BC.Methods: 1.The expression of RNF6 gene m RNA and protein in 146 breast cancer tissues and matched adjacent tissues were detected by qRT-PCR,Western blot and immunohistochemistry.The correlation between RNF6 gene expression and clinical pathological characteristics of patients and its influence on clinical prognosis were analyzed.2.qRT-PCR and Western blot experiments were used to detect the m RNA and protein expression of RNF6 gene in human normal breast MCF10 cells and 4 breast cancer cell lines(BT547,MCF7,MDA-MB-453 and MDA-MB-231).Select breast cancer MDA-MB-231 and MDA-MB-453 cells with high expression of endogenous RNF6 gene to transfect RNF6 sh RNA interference plasmids to down-regulate RNF6 expression in the two cells respectively,and detect the interference effect by qRT-PCR and Western blot.Transwell experiment was used to detect the influence of the expression of RNF6 protein on the invasion and migration of MDA-MB-231 and MDA-MB-453 cells in breast cancer.3.The luciferase reporter gene and Western blot were used to detect the effect of downregulation of RNF6 expression in breast cancer cells on the protein expression,exogenous introduction and endogenous YAP transcription activity of YAP and its downstream target genes.In addition,the expression of RNF6 in breast cancer cells was down-regulated while the Hippo-YAP pathway was activated and the Hippo-YAP pathway was blocked while the expression of RNF6 was up-regulated.The protein expression levels of RNF6 and YAP were determined by Western blot experiment.The changes of invasion and migration ability of breast cancer cells were analyzed by Transwell experiment and nude mouse lung metastasis experiment model.4.After transfecting MST1 sh RNA interfering plasmid and p-MST1 overexpression plasmid into breast cancer MDA-MB-231 and BT549 cells respectively to change the expression of MST1,the expression changes of MST1 and YAP proteins were detected by Western blot experiment.Similarly,RNF6 sh RNA interference plasmid and p-RNF6 overexpression plasmid were transfected into the above-mentioned breast cancer MDA-MB-231 and BT549 cells respectively to change the expression of RNF6.The protein expression changes of RNF6,YAP and MST1 were determined by Western blot experiment.In addition,the protein expression changes of RNF6,YAP and MST1 were determined by Western blot after downregulating the expression of RNF6 and MST1 simultaneously in breast cancer cells,and the changes of invasion and migration ability of breast cancer cells were analyzed by Transwell experiment and nude mouse lung metastasis experiment model.5.Cellular immunofluorescence and immunocoprecipitation experiments were used to detect whether RNF6 protein and MST1 protein in breast cancer cells are directly combined with each other.After the proteasome inhibitor MG132 were added to block protein degradation while RNF6 was down-regulated and up-regulated in breast cancer,And after the cycloheximide(CHX)were added to block protein synthesis while RNF6 was down-regulated,the expression changes of RNF6 and MST1 proteins were detected by Western blot.Immunocoprecipitation experiment was used to observe the effect of downregulation of RNF6 on the ubiquitination level of MST1.Results: 1.qRT-PCR results showed that the m RNA expression of RNF6 gene in breast cancer tissues was significantly increased compared with that in the adjacent tissues(P<0.01).The results of Western blot experiment showed that the RNF6 protein was highly expressed in breast cancer tissues than in the adjacent tissues(P<0.01).Immunohistochemical results showed that RNF6 protein was significantly overexpressed in 65.75%(96 /146)of breast cancer tissues,while only a few of matched adjacent tissues were highly expressed(P <0.01).Statistical analysis of RNF6 gene expression and clinical pathological characteristics of patients shows that the high expression of RNF6 in breast cancer tissues is closely related to TMN staging(P <0.001)and lymph node metastasis(P=0.004).Kaplan-Meier analysis showed that the overall survival rate and disease-free survival rate of patients with higher RNF6 level were lower than those with lower RNF6 expression level(P <0.05).In addition,multivariate Cox regression analysis showed that high expression level of RNF6 was an independent predictor of low survival rate of breast cancer patients(P <0.05).2.The m RNA and protein expression of RNF6 in breast cancer cells were significantly higher than those in normal human breast cells(P<0.05).After breast cancer MDA-MB-231 and MDA-MB-453 cells downregulated the expression of RNF6 gene,qRT-PCR and Western blot tests showed that the m RNA and protein expression of RNF6 were significantly down-regulated after transfecting RNF6 sh RNA interfering plasmids respectively(P <0.05).And Transwell experiment found that the migration and invasion of breast cancer cells were significantly inhibited(P <0.01).In addition,lung metastasis experiments in nude mice further found that downregulating the expression of RNF6 gene in breast cancer MDA-MB-231 and MDA-MB-453 cells could reduce distant lung metastasis of breast cancer in nude mice(P <0.05).3.After downregulating RNF6 gene expression in breast cancer cells,it was found that YAP protein expression,exogenous introduction,endogenous YAP transcription activity and downstream target genes Cyr61,CTGF and cyclin E protein expression were significantly inhibited.Western blot test results showed that the protein expression of YAP could be obviously restored after activating Hippo-YAP pathway while downregulating the expression of RNF6 in breast cancer cells,and Transwell test results showed that the invasion and migration ability of breast cancer cells were restored,while the distant lung metastasis of nude mice breast cancer was significantly increased(P <0.01).In addition,Western blot test results showed that the protein expression of YAP could be significantly inhibited after upregulating the expression of RNF6 and inhibiting Hippo-YAP pathway in breast cancer cells,and Transwell test results showed that the invasion and migration ability of breast cancer cells were significantly inhibited,while the distant lung metastasis of nude mice breast cancer was significantly reduced(P <0.01).4.Western blot experiment results showed that YAP protein expression also decreased and increased after up-regulating and down-regulating the expression of MST1 protein in breast cancer MDA-MB-231 and BT549 cells respectively.Western blot experiment also showed that YAP and MST1 protein expression also increased and decreased after up-regulating and down-regulating the expression of RNF6 protein in breast cancer MDA-MB-231 and BT549 cells respectively.In addition,Western blot experiment showed that YAP protein expression was significantly increased after the expression of RNF6 and MST1 were simultaneously down-regulated in breast cancer cells,and Transwell experiment showed that invasion and migration ability of breast cancer cells were restored,and distant lung metastasis of nude mice breast cancer was significantly increased(P <0.01).5.Cell immunofluorescence and immunocoprecipitation experiments found that RNF6 and MST1 were directly combined in breast cancer MDA-MB-231 cells.After breast cancer cells were treated with proteasome inhibitor MG132,the expression of endogenous MST1 protein gradually accumulated,indicating that MST1 protein was degraded by ubiquitin proteasome system in breast cancer cells.When actinomycin(CHX)was added to block protein synthesis,while RNF6 was down-regulated in breast cancer MDA-MB-231 cells,the degradation rate of MST1 was obviously decreased.And after the proteasome inhibitor MG132 were added to block protein degradation while RNF6 was down-regulated and up-regulated in breast cancer MDA-MB-231 cells,there was no obvious change in the expression of MST1 protein,indicating that RNF6 participated in the degradation process of MST1 protein.Finally,after reducing the expression of RNF6 in breast cancer cells,we found that the ubiquitination level of MST1 protein decreased significantly,which indicated that RNF6 affected the expression of MST1 protein by affecting the ubiquitination level of MST1.Conclusion: In conclusion,RNF6 is highly expressed in breast cancer tissues,and is closely related to clinical features such as tumor lymphatic metastasis and poor prognosis of patients.It reveals the specific mechanism of RNF6 regulating invasion and metastasis of breast cancer through mediating the MST1/YAP axis,and provides scientific theoretical basis for prognosis indicators and therapeutic targets of breast cancer treatment.