Roles And Mechanisms Of CUL4B In Regulating Tamoxifen Resistance In Breast Cancer

BackgroundBreast cancer is one of the most common malignant tumors in women.According to the latest statistics in 2018,the morbidity and mortality of breast cancer are the highest among female malignant tumors,which seriously affects women's health.Abnormal estrogen metabolism has been identified as one of the hallmark features of breast cancer.Breast cancer can be divided into two subtypes according to the status of estrogen receptor-?(ER-?),ER-positive or ER-negative.Tamoxifen(TAM),a nonsteroidal antiestrogen,has long been used and still is the most commonly used endocrine therapy for treatment of estrogen receptor-positive breast cancer.However,despite the significant anti-neoplastic activity of TAM,about 40%-50%ER-positive breast cancer patients do not respond to TAM treatment by the time of diagnosis,which is primary resistance,and 17%-28%patients develop acquired TAM resistance during the treatment process.TAM resistance,including primary and acquired resistance,has become a serious problem that currently plagues TAM treatment of breast cancer.Estrogen receptor-?36(ER-?36),a novel estrogen receptor,is mainly localized near the plasma membrane and mediates membrane-initiated estrogen signaling.ER-?36 is able to mediate non-genomic estrogen signaling pathways such as PI3K/AKT and MAPK/ERK,and affects proliferation,migration and apoptosis of cells.It has been found that breast cancer patients with tumors expressing high concentrations of ER-a36 benefited less from TAM therapy than those with low concentrations of ER-a36.Further studies have demonstrated that the ER-?36-EGFR/HER2 positive regulatory loops are involved in TAM resistance.However,the molecular mechanism underlying the upregulation of ER-a36 in TAM resistance remains unknown.CUL4B,a member of Cullin(CUL)family,acts as a scaffold that assembles DDB1,RBX1 and substrate receptors to form Cullin4B-RING-based E3 ubiquitin ligases(CRL4B).CUL4B participates in the regulation of diverse physiologically and developmentally controlled processes.CUL4B is overexpressed in various solid tumors,and can repress many tumor suppressors through epigenetic mechanisms.It has been shown that CUL4B promote HER2 expression through transcriptionally repressing miR-125a,which furtherly affect the invasion and epithelial-mesenchymal transition of gastric cancer cells.It has also been confirmed there exit a positive regulatory loop between ER-?36 and HER2,which is involved in TAM resistance in breast cancer.Based on these studies,we hypothesized that CUL4B might be involved in breast cancer TAM resistance via upregulating of ER-?36.The present study focused on the role of CUL4B in TAM resistance,and the mechanism involved.ObjectiveTo examine the expression levels of CUL4B in TAM-resistant and TAM sensitive breast cancer cells;to examine the effect of up-or down-regulation of CUL4B on TAM sensitivity in breast cancer cells;to determine whether CUL4B regulates TAM resistance through upregulating ER-a36 expression;to investigate the regulatory mechanisms of CUL4B in TAM resistance.MethodsThe MTT assay was used to determine the TAM sensitivity of different breast cancer cells.The expression of genes and proteins was analysed by qRT-PCR or Western blot,respectively.TargetScan and microarray analysis were used in combination to determine the candidate miRNAs targeting to ER-?36.ChIP and qChIP were used to determine the binding of CUL4B complex to the promoter region.Results(1)CUL4B overexpression is associated with TAM resistance in breast cancer cells.To analyze the role of CUL4B in TAM resistance,we first examined the expression level of CUL4B in five breast cancer cell lines,and the sensitivity of these cells to TAM treatment.We found that the IC50 of MDA-MB-231,MDA-MB-436 and BT474 with high levels of CUL4B was significantly higher than that of MCF7 and T47D cells with lower levels of CUL4B.The expression level CUL4B in TAM-resistant cells was significantly higher than that of control cells.We also found that upregulation of CUL4B confers TAM resistance,while knockdown of CUL4B sensitized TAM-resistant cells to TAM.These results suggest that CUL4B is involved in the regulation of TAM resistance in breast cancer cells.(2)The expression of CUL4B was positively correlated with ER-?36 expression.To clarify whether CUL4B is involved in the regulation of TAM resistance by modulating ER-?36,we analyzed the expression levels of CUL4B and ER-?36 in different cells.We found that the expression levels of the two proteins were positively correlated.This positive correlation was also confirmed in tumor tissues from breast cancer patients.(3)The CUL4B complex represses miR-32-5p transcription.To investigate the mechanism by which CUL4B regulates ER-?36,we used software analysis combined with microarray analysis to screen for miRNAs targeting to ER-?36.Real-time PCR analysis showed that CUL4B negatively regulated miR-32-5p transcription.ChIP analysis revealed CUL4B could bind to the promoter of miR-32-5p.qChIP analysis demonstrated that knockdown of CUL4B significantly reduced the binding of CUL4B to the promoter of miR-32-5p.Consistently,CUL4B knockdown also significantly reduced the levels of H2AK119ubl,EZH2 and H3K27me3 at the promoters,but increased the enrichment of H3K4me3 at the promoter.These results indicates that the CUL4B complex represses miR-32-5p transcription through an epigenetic mechanism.(4)miR-32-5p regulates ER-?36 expression.We transfected mimics or inhibitor of miR-32-5p into MCF-7 cells,and examined ER-?36 expression levels,respectively.We found that overexpression of miR-32-5p significantly downregulated ER-?36 at protein level,while transfection with miR-32-5p inhibitors resulted in an increase of ER-?36 level.miR-32-5p could regulate the expression of the luciferase reporter vectors containing the wide-type 3'-UTR,but not that containing the mutant 3'-UTR of ER-?36.These results indicate that ER-?36 is a direct target of miR-32-5p in breast cancer cells.(5)The CUL4B complex induces ER-?36 expression through down-regulation of miR-32-5p.To examine whether CUL4B positively regulates ER-?36 by repressing miR-32-5p,we performed rescue experiments.We found that miR-32-5p mimics could block ER-?36 upregulation caused by CUL4B overexpression.In contrast,miR-32-5p inhibitor can restore the downregulation of ER-?36 caused by CUL4B knockdown.The analysis of clinical specimens also showed a negative correlation between miR-32-5p and CUL4B as well as ER-?36.Collectively,all these results indicate that CUL4B positively regulates ER-a36 expression through repressing miR-32-5p transcription.ConclusionThe present study demonstrated that CUL4B is involved in the regulation of TAM resistance in breast cancer cells.Mechanistic studies revealed that,CUL4B upregulates the expression of ER-?36 through repressing the transcription of miR-32-5p.