Study On The Mechanisms And Clinical Significance Of The E3 Ubiquitin Ligase TRIM4 Regulating Tamoxifen Resistance In Breast Cancer

BackgroundBreast cancer is the most common type of cancer among women worldwide,and it is also one of the most important factors for cancer death in female.Among the molecular subtypes of breast cancer,estrogen receptor(ER-α)positive breast cancer accounts for about 70%of all the cases.The over-activation of estrogen/ER-α signaling pathway plays an important role in the occurrence and development of this subtype of breast cancer.Therefore,the endocrine therapy that antagonizes this pathway is the critical adjuvant treatment for ER-α positive breast cancer.Tamoxifen is a selective ER modulator that can competitively bind to ER,blocking the function of the estrogen/ER-α signaling pathway.Researches show that tamoxifen can significantly reduce the recurrence rate and death risk of ER-α positive breast cancer patients,and it is the first choice of adjuvant endocrine therapy for premenopausal female patients.However,about 30-50%of patients will face the problem of primary or acquired resistance to tamoxifen.Endocrine therapy resistance is one of the main factors of recurrence and metastasis in positive breast cancer patients.Previous studies have revealed that decreased or absent expression of ER-α is an important mechanism leading to tamoxifen resistance,while the key factors and mechanisms that regulate ER-α expression are unclear.In recent years,the roles and potential mechanisms of E3 ubiquitin ligase family proteins in the carcinogenesis and progression of cancer have gradually become a research hotspot in the field of cancer.TRIM4 belongs to the E3 ubiquitin ligase TRIM(tripartite motif)family.Studies have shown that TRIM4 plays important roles in regulating various biological processes such as antiviral innate immunity,oxidative stress,and mitochondrial homeostasis in human cells.However,its function in breast cancer progression remains unclear.In this study,we firstly revealed that TRIM4 was an important regulator on ER-α expression and tamoxifen resistance in breast cancer,and its potential mechanisms and clinical values were explored.Research methods and resultsThis study is divided into three parts.The research methods and results of each part are as follows:Part Ⅰ:Functional study on TRIM4 regulating tamoxifen sensitivity in breast cancer Research Methods1.We compared the expression of TRIM4 in tamoxifen resistant cell lines MCF7/TR and T47D/TR with that of their parent cell lines by reverse transcription quantitative real time PCR(RT-qPCR)experiments,and further verified by Western blot.2.The regulatory roles of TRIM4 overexpression or knockdown on the sensitivity of ER positive breast cancer cells to tamoxifen and the expression levels of related signaling pathways were detected by IC50 experiment,MTT assay,clone formation assay,flow cytometry,Western blot,RT qPCR and other in vitro experiments.3.The regulatory effects of TRIM4 on the sensitivity of breast cancer to tamoxifen were further verified by breast cancer xenograft models,immunohistochemistry(IHC),etc.Research Results1.The expression of TRIM4 in tamoxifen resistant breast cancer cell lines was significantly lower than that in parental cell lines.2.In vivo and in vitro experiments showed that overexpression of TRIM4 significantly enhanced the sensitivity of breast cancer cells to tamoxifen,while knockdown of TRIM4 expression could lead to increased drug resistance to tamoxifen.In addition,knockdown of TRIM4 expression can activate PI3K-AKT and MAPK signaling pathways,and promote the formation of estrogen independent growth phenotype of breast cancer cells.Part Ⅱ:The mechanism of TRIM4 regulating tamoxifen sensitivity in breast cancer Research Methods1.The expression levels of TRIM4 in TNBC,Luminal A,Luminal B,HER-2 positive breast cancer subtypes were explored using the TCGA public database;The relationship between the expression levels of TRIM4 mRNA and that of ESR1,PGR,HER-2 mRNA was investigated.The relationship between the expression level of TRIM4 and that of ESR1 and PGR was verified by RT-qPCR and Western blot experiments at the RNA and protein levels.2.The potential binding proteins of TRIM4 were explored through co-immunoprecipitation assays(co-IP)and mass spectrometry analysis.3.The interaction between TRIM4 and the target protein was verified through co-IP and immunofluorescence experiments.4.We determined the binding domains of TRIM4 and the target protein through construction of protein truncates and co-IP experiments.5.Western blot and RT-qPCR experiments were used to explore the regulatory effect of TRIM4 on the expression level of downstream target proteins,and the effect of TRIM4 on the stability of target proteins was investigated through the cycloheximide experiment.6.We explored the effect of TRIM4 on the level of ubiquitination modification on targeted proteins through co-IP experiments,and identified the key ubiquitination sites of the targeted proteins.The mediating role of the target protein in TRIM4 affecting tamoxifen sensitivity and ER-α expression in breast cancer was validated through rescue experiments.Research Results1.The expression of TRIM4 in Luminal breast cancer subtype was significantly higher than that in TNBC subtype;The expression of TRIM4 was significantly positively correlated with the expression of ESR1 and PGR,but not with the expression of HER-2.The results of RT-qPCR and Western blot experiments showed that knocking down TRIM4 significantly reduced the expression levels of ESR1 and PGR mRNA and protein.2.TRIM4 can bind to SET protein,which is the downstream targeted protein of TRIM4.3.The binding of TRIM4 to SET is mediated by the RING and B-box domains of TRIM4 and the carboxyl terminal of SET.4.TRIM4 can promote the degradation of SET protein,but has no effect on the expression of SET mRNA.5.TRIM4 can promote the K48-linked ubiquitination of the SET protein,and the lysines 150 and 172 of the SET protein are the key sites for its ubiquitination.The rescue experiments further confirmed that TRIM4 can promote P53 protein mediated ESR1 transcriptional activation and PP2A protein mediated ESR1 mRNA stabilization through the degradation of SET proteins,thereby enhancing the expression of ESR1 and the sensitivity of breast cancer to tamoxifen treatment.Part Ⅲ:Clinical values of TRIM4 in predicting tamoxifen sensitivity and prognosis of ER positive breast cancer patientsResearch Methods1.We explored the relationship between the expression level of TRIM4 and the overall survival(OS),relapse and metastasis free survival(RFS),progression free survival(PPS),and distant metastasis free survival(DMFS)of breast cancer patients using the KM-PLOTTER public database.The relationship between the expression level of TRIM4 and the prognosis of ER positive breast cancer patients who received endocrine therapies after surgery were also analyzed.2.The expression levels of TRIM4 and SET protein in ER-α positive breast cancer tissues were detected by IHC assays,and the staining index of TRIM4 and SET according to the staining intensity and the proportion of positive cells was calculated for each slide.3.According to the staining index,the receiver operating characteristic curve(ROC)was used to determine the optimal cut-off value for distinguishing between high and low expression of TRIM4.To explore the relationship between the expression level between TRIM4 and SET,spearman correlation analysis was performed;Chi square test was used to verify the relationship between the expression levels of TRIM4 or SET proteins and the clinicopathological characteristics of ER-α positive breast cancer patients;Log rank test was used to explore the correlation between the expression levels of TRIM4 and SET and the prognosis of ER-α positive breast cancer patients.Research Results1.Public database data show that the higher expression of TRIM4 is significantly related to the favorable prognosis of breast cancer patients.In the ER positive breast cancer patients who received endocrine therapy after surgery,the lower expression of TRIM4 was significantly associated with poor prognosis.2.TRIM4 is negatively correlated with the expression of SET protein.The expression of TRIM4 protein was positively correlated with the expression of ER-α protein,while the expression of SET protein was negatively correlated with the expression of ER-α protein.At the same time,higher expression of TRIM4 protein was positively correlated with smaller tumor volume and fewer lymph node metastasis,while higher expression of SET protein was positively correlated with larger tumor volume and more lymph node metastasis.3.IHC assay and prognosis analysis showed that the OS and RFS of ER-α positive breast cancer patients with higher TRIM4 expression were significantly improved,while the RFS of patients with higher SET expression was significantly worse than those with lower SET expression.Research Conclusion1.TRIM4 was significantly downregulated in tamoxifen resistant cell lines.Besides,the expression level of TRIM4 in Luminal breast cancer is significantly higher than that in TNBC.2.In vitro and in vivo experiments confirmed that the higher expression of TRIM4 was positively related to the sensitivity of breast cancer to tamoxifen treatment.3.Knocking down the expression of TRIM4 can reduce the expression of ER-α and PGR.4.TRIM4 can promote K48-linked polyubiquitination of SET,and the lysine sites 150 and 172 of SET protein are the key ubiquitination sites.5.TRIM4 can promote the ubiquitination degradation of SET protein and further promote the expression of TP53 and PP2A mediated ER-α protein.6.Higher expression levels of TRIM4 protein is positively correlated with smaller tumor volume and less lymph node metastasis,while the overexpression of SET protein is positively correlated with larger tumor volume and more lymph node metastasis.The high expression of TRIM4 protein is a predictor of favorable prognosis of ER-α positive breast cancer patients,while higher expression of SET protein is associated with poor prognosis of the patients.