miR-489 expression is lost in tamoxifen resistance, predicts breast cancer aggressiveness and is regulated by estrogen/ERα axis
To specifically identify miRNAs that are clinically relevant in endocrine resistance, we analyzed miRNA screening datasets of endocrine resistant models previously published by three independent laboratories including our own (Fig. 1A)(11, 12, 16). Since, all three models are derived through completely different processes, they represent independent tamoxifen resistant models with different resistant mechanisms. MCF7-HER2 cell line represents de novo resistance while MCF7-TAM and MCF7:2A represent acquired resistance. miRNAs that are dysregulated in all three cell lines suggest their potential roles in regulation of multiple mechanisms involved in tamoxifen resistance, which therefore may be more clinically relevant with promising therapeutic promise to address tamoxifen resistance. Although, many miRNAs were dysregulated in these cell lines we only found few miRNAs that were aberrantly expressed in all three cell lines (Fig. 1B). Out of these miRNAs, miR-135b, miR-33b and miR-505 showed opposite expression pattern among these cell lines. miR-378a-3p and miR-218 were significantly upregulated in all three cell lines while miR-342-5p and miR-489 were significantly downregulated. Intriguingly, miR-489 was one of the top downregulated miRNAs in all three datasets, suggesting its role in tamoxifen resistance. We validated these results using qRT-PCR and indeed found significant downregulation of miR-489 in both resistant cell lines (Fig. 1C). To determine whether the expression of miR-489 was associated with endocrine resistance in patient cohorts, we examined miR-489 expression in hormone-therapy treated ER + breast cancer patients. We observed a statistically significant association between lower miR-489 expression and poorer overall survival in these patients. In fact, miR-489 expression remained as an independent prognostic factor in hormone-therapy treated breast cancer patients obtained from two independent datasets GSE19783 and METBRIC (Fig. 1D). These results suggest that loss of miR-489 may promote tamoxifen resistance.
Previously we have observed that average expression of miR-489 was notably higher in luminal cells compared to basal cells (13, 16). Furthermore, analysis of miR-489 expression on 13 different breast cancer cell lines also demonstrated that it was expressed at a higher level in hormone positive luminal breast cancer cell lines (Fig. 1E) compared to the cells lines from other subtypes. Similarly, analysis of primary breast tumors revealed that miR-489 along with its host gene CALCR was positively correlated with expression of estrogen receptor and its responsive genes such as PGR and TFF1 (Fig. 1F), suggesting miR-489 and its host gene CALCR expression may be regulated by estrogen signaling. To examine this hypothesis, we stimulated three ER+ breast cancer cell lines (T47D, MCF7 and BT474) with estrogen or ethanol for indicated time periods and measured the expression of miR-489 and its host gene CALCR. We found significant upregulation of miR-489 and CALCR in all three cell lines treated with estrogen (Fig. 1G). Estrogen regulation of miR-489 was further investigated in complete media or estrogen deprived media. As expected, depletion of estrogen drastically reduced the expression of miR-489 and CALCR like other classical ER target genes such as trefoil factor 1 (TFF1), progesterone receptor (PGR), and C-X-C motif chemokine ligand 12 (CXCL12) (Fig. 1H). In summary, these data strongly suggest that miR-489 is an estrogen regulated miRNA in breast cancer and may play a regulatory role in tamoxifen resistance.
miR-489 restoration overcomes acquired and de novo tamoxifen resistance.
Since miR-489 was lost in tamoxifen resistant tumors and cell lines, we hypothesized that restoration of miR-489 would sensitize the resistant cell lines. Our previous studies have shown that overexpression of miR-489 inhibited breast cancer cells proliferation(13, 16). We first tested whether Tamoxifen-resistant cells were still sensitive to miR-489 mimics. Notably, we observed that both acquired and de novo resistant cell lines were equally sensitive to miR-489 mimics as their sensitive counterpart as opposed to their different sensitivities to tamoxifen (Fig. 2A, 2B). These results further bolstered a possibility that miR-489 might target pathways involved in resistance and could potentially sensitize these resistant cell lines to tamoxifen. Indeed, forced expression of miR-489 significantly sensitized both resistant cell lines to tamoxifen. Tamoxifen failed to cause any significant death in both resistant cell lines at 5 µM while combination with miR-489 led to around 40% death in MCF7-TAM (Fig. 2C) and 30% death in MCF7-HER2 cells (Fig. 2D). To directly assess the role of miR-489 in tamoxifen resistance, we inhibited endogenous miR-489 in tamoxifen sensitive cells MCF7-Vec and MCF7-WT cells. As expected, inhibition of miR-489 significantly increased tamoxifen resistance in both sensitive cell lines. At the highest concentration tested, inhibition of miR-489 increased the survival by 25% and 40% in sensitive counterpart of MCF7-Vec and MCF7-WT, respectively (Fig. 2E, 2F). Similarly, colony formation assays also revealed that forced expression of miR-489 significantly reduced the survival and colony forming ability in resistant cell lines while inhibition of endogenous miR-489 enhanced the survival of sensitive counterpart and promoted tamoxifen resistance. (Fig. 2G, 2H).
miR-489 acts as an endogenous negative feedback regulator to balance estrogen signaling
To elucidate the underlying pathways targeted by miR-489 to induced tamoxifen sensitization, we re-examined the gene expression profiles of T47D cells transfected with miR-489 mimics and scrambled RNA(13). Interestingly, gene expression analysis revealed enrichment of multiple pathways involved in estrogen signaling and tamoxifen resistance (Fig. 3A, 3B). We observed enrichment of ErBB signaling pathway and several stress associated pathways including endoplasmic reticulum (ER)-stress and lysosomal pathways. All of these pathways have been previously reported to be involved in tamoxifen resistance (9, 17–21) (4). Meanwhile, these results are also in accordance with our previous studies showing role of miR-489 in HER2 singling and metabolic stress and autophagy regulation (13, 14, 16). Interestingly, we noticed significant enrichment of estrogen dependent gene expression and ESR-mediated signaling. Upon further analysis, we found substantial downregulation of estrogen responsive genes (Fig. 3C). This data suggested that estrogen regulated miR-489 might function as a negative regulator of estrogen signaling. To determine the clinical significance of these data, we applied miR-489 gene expression signature to a gene expression profiling obtained from patient datasets. Consistent with our microarray results, we found strong inverse correlation between estrogen responsive gene signature and miR-489 signature (p value < 0.0001) (Fig. 3D) and between PI3K-ERBB2 signature and miR-489 signature (p value < 0.0001) (Fig. 3E). In addition, we also noticed that low miR-489 expression in ER + breast cancer was indicative of worse overall survival (Fig. 3F), further supporting essential tumor suppressive role of miR-489 in ER + breast cancer.
To examine how miR-489 negatively regulated estrogen signaling, we measured transcriptional activity of estrogen receptor by performing Luciferase reporter assay in T47D-ERE-Luc reporter cell line. We observed the inhibition of estrogen receptor transcriptional activity upon miR-489 overexpression and increased activity upon inhibition of endogenous miR-489 (Fig. 3G). We then validated the microarray results by performing qRT-PCR analysis on ER + and ER- cell lines. As expected, miR-489 caused down regulation of estrogen responsive genes only in ER + cell lines including T47D, MCF7 and BT474 cells but did not affect or in some instances increased the expression of these genes in ER- cell lines such as AU565 and HCT116 cells (Fig. 3H-I). These results suggest that miR-489 regulates the expression of these ERα-downstream genes by inhibiting estrogen signaling.
miR-489 inhibits ER-induced cell proliferation and cancer stem cells expansion
Our previous studies have shown that miR-489 inhibits proliferation of all breast cancer cell lines including ERα + cell lines (13, 16). However, it remains unknown whether miR489-medieated growth inhibition in ER + breast cancer cells is due to its effects on ER signaling. To this end, we examined the effects of miR-489 on estrogen-induced cell proliferation in MCF7 and T47D cells. Both cell lines showed poor proliferation when treated with vehicle (Ethanol) while estrogen treatment enhanced proliferation by more than 2-fold and 4-fold in MCF7 and T47D cell lines respectively. Restoration of miR-489 completely abolished estrogen induced proliferation in both cell lines while inhibition of endogenous miR-489 further increased estrogen induced proliferation by more than 2 fold in MCF7 and more than 3 fold in T47D cell lines (Fig. 4A-B). Estrogen treatment has been previously shown to enhance population of stem-like in ER α cell lines(22). These so-called cancer stem cells have been thought to be the responsible population for tumor relapse(23). We hypothesized that miR-489 may inhibit estrogen induced population of cancer stem like cells by its effect on estrogen signaling. Therefore, we studied the effect of miR-489 on estrogen induced cancer stem cell population using colony formation assay, mammosphere assay and flow cytometry. Consistent with previous results, estrogen treatment significantly increased colony formation ability of both MCF7 and T47D cells. Forced expression of miR-489 almost completely inhibited estrogen induced colony formation of both cells. Interestingly, inhibition of endogenous miR-489 drastically enhanced estrogen mediated colony formation (Fig. 4C). We then examined the effect of miR-489 on cancer stem like cells by measuring CD44+CD24− population using flow cytometry and by measuring mammosphere forming efficiency (MFE) analysis. Consistent with previous results, estrogen increased cancer stem like cell population (CD44+CD24−) by 3-fold in MCF7 cells and 10-fold in T47D cells (Fig. 4D). As observed in clonogenic assay, inhibition of endogenous miR-489 increased cancer stem like cell population by more than 3-fold in MCF7 cells and by more than 11-fold in T47D cells (Fig. 4D). Similarly, we also observed increased MFE upon estrogen treatment and miR-489 inhibition not only increased MFE but also increased the mammosphere size. As observed in colony formation assay, forced expression of miR-489 almost completely prevented mammosphere formation (Fig. 4E). Together, these results suggest that estrogen regulated miR-489 as a feedback regulator to confine estrogen induced tumor cell growth and inhibit population of cancer stem like cells.
miR-489 inhibits estrogen induced signaling by inhibiting p38 MAPK, PI3K-AKT and MAPK-ERK pathways
We further seek to elucidate the molecular mechanism responsible for miR-489 mediated inhibition of estrogen-ERα axis. Multiple mechanisms have been identified for regulation of estrogen-ERα mediated gene expression(24, 25). Direct inhibition of ERα or its co-factors, inhibition of kinases that activates ERα, and inhibition of estrogen induced nuclear localization of ERα have been previously reported to regulate estrogen induced gene transcription (26). We first examined if miR-489 exerted its effects by affecting localization of estrogen receptor. Interestingly, forced expression of miR-489 strongly promoted translocalization of estrogen receptor from nucleus to cytoplasm in MCF7 and T47D cell lines (Fig. 5A-B). In contrast, inhibition of endogenous miR-489 resulted in increased nuclear localization (Fig. 5A-B). Searching through factors which have been reported to regulate localization of estrogen receptor, we found that one of these factors, p38 MAPK, is a potential miR-489 target (26) (Fig. 5C). Interestingly, forced expression of miR-489 significantly downregulated total p38 MAPK (Fig. 5D). We then performed 3’UTR assay to examine if p38 MAPK is a direct target of miR-489. Forced expression of miR-489 significantly reduced luciferase activity of wild type construct while it did not affect luciferase activity of construct with mutant miR-489 binding site (Fig. 5E). This result confirms that p38 MAPK is a direct target of miR-489. Next, we examined the effect of p38 MAPK inhibitor SB203580 can phenocopy the effect of miR-489 on estrogen receptor localization. We transfected control siRNA or miR-489 mimic for 72hrs or treated with DMSO or 10 µM SB203580 for 24hrs in hormone starved cells followed by treatment with estrogen to examine estrogen induced nuclear localization of estrogen receptor. Indeed, cells treated with p38 MAPK inhibitor phenocopied the effect of miR-489 on ER localization in both cell line (Fig. 5F). These results indicate miR-489 affects nuclear translocation of ERα by at least partially by downregulating p38 MAPK. Considering that phosphorylation of ERα protein is also a critical step for its transcription activity, we investigated changes in their expression upon modulation of miR-489 expression. Western blot analysis of total ERα protein and its phosphorylated forms suggest that forced expression of miR-489 reduced the phosphorylation of ERα at both S118 and S167 sites (Fig. 5G). This data suggests that miR-489 may further regulate ligand dependent activation of ERα through inhibition of kinases that phosphorylates ERα at residues S118 and S167 residue. MAPK and AKT have been known to regulate ERα phosphorylation at S118 and S167 respectively. Indeed, miR-489 restoration significantly reduced the activated form of these kinases while its inhibition enhanced their activation (Fig. 5G).
All three kinase pathways have been shown to prompt the tamoxifen resistance. We examined the effect of miR-489 modulation in MCF7-HER2 and its isogenic cell line MCF7-Vec on estrogen-independent growth. We found that miR-489 inhibition alone was sufficient to promote estrogen-independent growth while miR-489 restoration prevented estrogen independent growth in MCF7-HER2 cell line (Fig. S1A, additional file 2). Western blot analysis confirmed the upregulation of HER2, pAKT and pERK in MCF7-HER2 cell line compared to MCF7-Vec. Consistent with previous results, MCF7-HER2 cell line showed increased estrogen independent phosphorylation of ERα at S118 and S167 residue and restoration of miR-489 abolished this hyperactivation and ERα phosphorylation (Fig. S2B, additional file 2). In summary, these results suggest that miR-489 regulates tamoxifen resistance by targeting multiple kinase signaling pathways and therefore could potentially be used as a therapeutic sensitizer to treat resistant patients.
p38 MAPK, PI3K-Akt and MAPK inhibitors phenocopy the effect of miR-489 in ER + breast cancer cell lines
Next, we examined whether inhibition of all three responsible kinase signaling pathways phenocopies the effect of miR-489. We inspected estrogen induced transcription and proliferation after inhibition of all three kinases. To a variable extent, we observed significant inhibition of transcriptional activity of ERα using ERE-reporter cell line (Fig. 6A). qRT-PCR analysis also showed downregulation of estrogen responsive genes (Fig. 6B). Consistently, these data were also supported by diminished estrogen induced proliferation upon inhibition of all three kinases (Fig. 6C, 6D). However, downregulation of endogenous miR-489 was partially able to rescue cells from the growth inhibitory effect of kinase inhibitors (Fig. 6E). This data suggests that miR-489 may exert its inhibitory effect on estrogen signaling partially, if not completely, by simultaneously inhibiting p38 MAPK, AKT and ERK signaling pathways.
Intriguingly, we observed that p38 MAPK inhibition had significant inhibitory effect only in the presence of estrogen and did not affect estrogen independent growth (Fig. 6F) indicating p38 MAPK activation may be estrogen dependent. Therefore, we tested if estrogen activates p38 MAPK and then mediates nuclear translocation. Since estrogen induced ERα nuclear translocation occurs within 5–30 minutes(27), we performed time course of estrogen treatment on MCF7 and T47D cell lines. We observed a sharp increase in phospho-p38 MAPK and its downstream target phosphor-ATF2 upon estrogen treatment in both cell lines (Fig. 6G). These results are consistent with previous studies that showed estrogen mediated activation of p38 MAPK in various tissues(26) (28).This provided an evidence of positive feedback loop between E2-ERα axis and p38 MAPK in mammary cells. Binding of E2 leads to activation of p38 MAPK and activation of p38 MAPK leads to nuclear translocation of ERα which is necessary for its function as transcription factor (Fig. 6H). This data together with ERα nuclear localization data, demonstrated that estrogen may cause p38 MAPK activation upon binding to ERα and this activated p38 MAPK in turn increases nuclear localization of ERα. Indeed, we observed that increased p38 MAPK phosphorylation starting as early as 5 min of estrogen treatment (Fig. 6G). This mutual activation of E2-ERα and p38 MAPK was previously demonstrated in endometrial cell lines (26). As p38 MAPK activation was estrogen dependent, we therefore suspected p38 MAPK inhibition might have a pronounced effect in pre-menopausal women as compared to post-menopausal women. Indeed, we observed higher p38 MAPK gene signature score in pre-menopausal luminal patients compared to post-menopausal luminal patients (Fig. 6I). Furthermore, luminal pre-menopausal patients with higher tumor grade also showed higher p38 MAPK gene signature (Fig. 6J). Additionally, high p38 MAPK expression in pre-menopausal patients predicted poor survival more significantly compared to post-menopausal patients. Our correlation analysis of miR-489 and p38 MAPK signature showed statistically significant inverse correlation in pre-menopausal patients. However, this correlation was reduced in post-menopausal patients (Fig. 6K). In summary, these results suggest that miR-489 regulates tamoxifen resistance by targeting multiple kinase signaling pathways and therefore could potentially be used as a therapeutic sensitizer to treat resistance patients.
miR-489 knock out cells possess increased sensitivity to estrogen induced signaling and proliferation
To systematically investigate whether loss of miR-489 potentiated the estrogen signaling and estrogen-dependent growth of ER + breast cancer cells, we utilized CRISPR/ Cas9 gene editing to create a miR-489 knock out cell line (Additional file 2, Fig. S2). We validated these knock out cells using genotyping and sequencing to ensure deletion of miR-489. We characterized these cells using western blot analysis of previously established miR-489 targets. We then carried out functional assays to examine the effect of miR-489 knock out on these cells. As expected, we noticed that these cells exhibited increased growth rate (Fig. 7A, 7B). Consistent with above results, loss of miR-489 showed increased sensitivity to estrogen stimulated growth and colony forming ability (Fig. 7C). We also noticed more than 6-fold increase in estrogen induced cancer stem cell expansion (Fig. 7D, 7E). To confirm loss of miR-489 enhanced estrogen signaling, we examined gene expression level of estrogen responsive genes and found increased upregulation of these genes upon estrogen stimulation in knock out cells compared to wild type cells (Fig. 7F). Our western blot analysis also showed increased phosphorylation of ERα at Ser 167 and Ser 118 along with increased activity of respective kinases AKT and ERK (Fig. 7G). We also noticed significant increase in total p38 MAPK and observed dramatic increase in nuclear localized estrogen receptor (Fig. 7H). Hyperactivity of all three kinases have been reported to increase estrogen dependent proliferation. To test their contribution in increased estrogen dependent proliferation of knock out cells, we stimulated WT and KO cells with E2 in presence of their inhibitors. Knock out cells demonstrated resistance towards these inhibitors to different extent with almost complete resistance to p38 MAPK and AKT inhibitors (Fig. 7I-J). These knockout cells also showed significant resistance to tamoxifen evidenced by MTT based cell viability assay and colony formation assay (Fig. 7K, 7L). These results provide direct evidence of miR-489 as an auto-regulatory loop of estrogen signaling and in tamoxifen resistance.