Decreased miR26a/b and increased HuR expression post-transcriptionally upregulates ERBB2 to mediate acquired tamoxifen resistance in ER+ breast cancer cells

Tamoxifen-resistant ER-positive breast cancer is characterized by elevated ERBB2 expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the tamoxifen resistant (TAMR) cells remains poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein HuR, both of which associate with the 3’UTR of the ERBB2 transcripts. We demonstrated that http://www.jbc.org/cgi/doi/10.1074/jbc.M117.780973 The latest version is at JBC Papers in Press. Published on June 21, 2017 as Manuscript M117.780973 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jne 4, 2017 hp://w w w .jb.org/ D ow nladed from Post-transcriptional regulation of ERBB2 by miR-26 and HuR 2 miR26a/b inhibits the translation of ERBB2 mRNA, while HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributed to an increase in HuR mRNA isoform with shortened 3’UTR, which exhibit increased translational activity. This shortening of the HuR mRNA 3’UTR via alternative polyadenylation was observed to be CSTF2 (CSTF64)-dependent, with CSTF2 expression being upregulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally upregulates ERBB2 expression in TAMR ER+ breast cancer cells. ________________________________________ Approximately 70% of human breast cancer is estrogen receptor (ER)-alpha-positive (1,2). The use of tamoxifen (a selective oestrogen receptor modulator) has been shown to improve overall survival in ER+ early breast cancer and achieve a substantial objective response rate in ER+ metastatic breast cancer (3). However, many patients whose tumors are tamoxifen responsive eventually develop resistance accompanied with tumor recurrence (4). Therefore, an improved understanding of the molecular basis of the acquisition of tamoxifen resistance and development of new strategies to improve the long term efficacy of tamoxifen are required (4). The human epidermal growth factor receptor 2 (ERBB2/HER-2), a receptor tyrosine kinase, has been suggested as a critical mediator of resistance to hormonal therapies including tamoxifen (3-6). ER+ breast cancers that overexpress ERBB2 showed reduced tamoxifen sensitivity (7-9). Furthermore, the acquisition of tamoxifen resistance in ER+ breast cancers is characterized by elevated ERBB2 levels (10,11). Previous studies have shown that ERBB2 is regulated at the transcriptional (12,13) and post-transcriptional levels (14-17). However, the underlying mechanisms leading to increased expression of ERBB2 in tamoxifen resistant breast cancer remains poorly understood. The post-transcriptional regulation of gene expression involves the regulation of mRNA stability and translation by non-coding RNAs, including microRNAs (miRNAs), and RNA-binding proteins (18). Increasing evidence has established the importance of miRNAs in the initiation, promotion, and progression of various human cancers (19,20). The dysregulation of miRNAs has been demonstrated to contribute to breast cancer development and progression through promoting cell proliferation, survival, and metastasis [5, 6]. In particular, several studies have reported the downregulation of miR-26a and/or miR-26b in breast cancer, thus implicating them as tumor suppressor miRNAs (21-25). Interestingly, an increasing number of studies have also demonstrated that miRNAs are regulated by tamoxifen and are involved in tamoxifen resistance (26-28). Hu-antigen R (HuR) is an RNA-binding protein that belongs to the Hu/ELAV family (29). It selectively binds to AU-rich regions in the 3’UTR of mRNAs and serves to increase the stability of the transcripts by gest on Jne 4, 2017 hp://w w w .jb.org/ D ow nladed from Post-transcriptional regulation of ERBB2 by miR-26 and HuR 3 (30). Increasing evidence have supported the critical roles of HuR in breast cancer initiation, progression, metastasis and drug resistance through its modulation of the stability of relevant transcripts (29). Importantly, the elevated expression of HuR has been reported to contribute to tamoxifen resistance (31,32). In addition, alternative polyadenylation (APA) of the mRNA 3’UTR also plays an important role in post-transcriptional regulation (33). Approximately 70% of human genes are characterized by multiple polyA sites that produce distinct transcript isoforms with variable 3’UTR (34-37). More recent studies have shed light on the importance of APA in human cancers that favor expression of transcripts with shortened 3’UTRs, resulting in the activation of several proto-oncogenes (33,38-40). Herein, we report that ERBB2 is post-transcriptionally upregulated in tamoxifen resistant breast cancer cells attributed to the decreased expression of miR-26a/b and increased expression of HuR. Forced expression of miR-26a/b and/or depletion of HuR, which decreased ERBB2 expression, enhanced the response of the tamoxifen resistant breast cancer cells to tamoxifen. We further elucidated a novel mechanism leading to upregulated HuR expression in tamoxifen resistant breast cancer cells, in which higher levels of CSTF2 (CSTF64) mediates increased production of HuR mRNA isoform with shorter 3’UTR through alternative polyadenylation (APA), thus resulting in higher HuR protein expression. RESULTS ERBB2 is post-transcriptionally upregulated in tamoxifen resistant (TAMR) ER+ breast cancer cells— ER+ breast cancer cells (MCF7 and T47D) with acquired tamoxifen resistance were generated by chronic treatment with tamoxifen (Figure 1A) (47,48). These TAMR cells exhibited elevated ERBB2 protein (Figure 1B) and mRNA levels (Figure 1C), which is consistent with existing literatures implicating increased ERBB2 expression and the ERBB2-ERα crosstalk as a key mechanism in driving tamoxifen resistance in ER+ breast cancer (3-5,12,47,49,50). It has previously been reported that ERBB2 is transcriptionally upregulated in tamoxifen resistant breast cancer (47). To determine whether the increased expression of ERBB2 in tamoxifen resistant breast cancer also occurs in a post-transcriptional manner, we analyzed the ERBB2 3’UTR-driven luciferase reporter gene activities in the control and TAMR MCF7 and T47D cells. The full-length 3’ UTR of ERBB2 was cloned downstream of the Renilla luciferase gene in the psiCHECK2 vector, and the resulting luciferase reporter constructs were transfected into the cells. The TAMR MCF7 and T47D cells exhibited increased ERBB2 3’UTR-driven luciferase reporter gene activities as compared to the respective control cells (Figure 1D). Hence, post-transcriptional regulation at ERBB2 3’UTR results in increased ERBB2 expression in TAMR ER+ breast cancer cells. ERBB2 expression is decreased post-transcriptionally by miR-26a/b binding to its 3’UTR—We went on to determine whether ERBB2 is regulated post-transcriptionally by miRNAs targeting its 3’UTR. We have previously demonstrated that miR-26a and miR-26b are repressed by estrogen, leading to increased estrogen-stimulated cell proliferation in ERα+ breast cancer cells (25). Importantly, low levels of miR-26 has previously been reported to be associated with tamoxifen resistance (51). In our MCF7/TAMR and T47D/TAMR cells, the levels of miR-26a/b were significantly decreased as by gest on Jne 4, 2017 hp://w w w .jb.org/ D ow nladed from Post-transcriptional regulation of ERBB2 by miR-26 and HuR 4 compared to their respective control cells (Figure 2A). The forced expression of miR-26a/b decreased ERBB2 protein levels in MCF7/TAMR and T47D/TAMR cells (Figure 2B). Similarly, the forced expression of miR-26a/b in the TAMR cells decreased the protein levels of enhancer of zeste homolog 2 (EZH2), which is a validated target of miR-26 (52), and is thus chosen as a positive control (Figure 2B). Despite the observed miR-26a/b-mediated decrease in ERBB2 protein levels, the forced expression of miR-26a/b did not significantly decrease ERBB2 mRNA levels in MCF7/TAMR and T47D/TAMR cells (Figure S1A). This suggests that ERBB2 is expression is regulated post-transcriptionally by miR-26a/b. To determine whether ERBB2 is a direct target of miR-26a/b, we first performed bioinformatics analysis. A putative miR-26 binding site in the 3’UTR of the ERBB2 transcript was predicted by RNAhybrid (53) (Figure 2C). To further study whether miR-26a/b regulates ERBB2 expression through this predicted miR-26a/b binding site in the ERBB2 3’UTR, we introduced mutations into this site (Figure 2C) and compared the luciferase reporter activities controlled by either the wildtype or mutant ERBB2 3’UTR in the presence of miR-26a/b. The forced expression of miR-26a/b markedly reduced the activity of luciferase reporter gene fused to wild type ERBB2 3’UTR by more than 50%, but did not affect the luciferase reporter activity when the predicted miR-26a/b target site in the ERBB2 3’UTR was mutated (Figure 2D). In addition, to verify the direct interaction of miR-26 and ERBB2 mRNA, we performed RNA pull-down assay using biotin-labeled miR-26a or miR-26b (Figure 2E). ERBB2 and EZH2 (positive control) mRNAs were both markedly enriched in the pull-down fractions of biotin-miR-26a or biotin-miR-26b, but not in the pull-down fraction of control scrambled miRNA (Figure 2F). The association of biotinylated miR-26a/b with the ERBB2 and EZH2 mRNAs was