Linc-RoR promotes MAPK/ERK signaling and confers estrogen-independent growth of breast cancer

Sources of primary antibodies were: pERα (16 J4), ERα (D6R2W), pERK1/2 (D13.14.4E), ERK1/2(137F5), pMEK1/2(41G9), MEK1/2(D1A5) and MKP-2 (D9A5) from Cell Signaling (Danvers, MA, USA); MKP-1(D-3) and MKP-3 (F-12) from Santa Cruz (Dallas, TX, USA); DUSP7/MKP-X(26910–1-AP) and GAPDH (60004–1-Ig) from Proteintech (Rosemont, IL, USA). Secondary antibodies conjugated with IRDye 800CW or IRDye 680 were purchased from LI-COR Biosciences (Lincoln, NE, USA). PCR primers were purchased from IDT (Coralville, IA, USA). U0126 and NSC95397 were purchased from Cell Signaling and Santa Cruz (Dallas, TX, USA), respectively. TAM was obtained from Sigma-Aldrich (St. Louis, MO, USA).

MCF-7 parental, gRNA control and RoR KO cells were maintained in RPMI 1640 medium supplemented with 10% FCS. For estrogen deprivation treatment, these cells were cultured in phenol red-free RPMI medium containing 5% charcoal stripped FCS (E2-free medium). All the medium contain 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

For lncRNA profiling, we used Human Disease-Related LncRNA Profiler, as described previously [20]. Total RNA was extracted using Direct-zol™ RNA MiniPrep (Zymo Research) and cDNA synthesis was carried out using RevertAid™ Reverse Transcriptase (Thermo Fisher) with random primers. Delta-delta Ct values (regular medium versus E2-free medium) were used to determine their relative expression as fold changes, as previously described [21].

Cells were harvested, and proteins were extracted and quantified as previously described [5]. Protein samples were separated in a polyacrylamide SDS gel before transferring to PVDF membrane. After probing with a primary antibody, the membrane was incubated with a secondary antibody labeled with either IRDye 800CW or IRDye 680. Finally, signal intensity was detected by the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The intensities of band were quantified by imageJ software (version 1.6.0, Windows, NIH).

PCR was performed using a standard SYBR Green method (Bio-Rad, Hercules, CA, USA) as previously described [22]. We used primers linc-RoR-RT-5.1A and linc-RoR-RT-3.1A; pS2-RT-5.1 and pS2-RT-3.1; CXCL12-RT-5.1 and CXCL12-RT-3.1; c-Myc-RT-5.1 and c-Myc-RT-3.1; cyclin D1-RT-5.1 and cyclin D1-RT-3.1 to detect the mRNA level of these genes (Additional file 1: Table S1). GAPDH was used as an internal control. Delta-delta Ct values were used to determine their relative expression.

We used a dual gRNA approach to knock out exon 4 of linc-RoR (RoR E4) by CRISPR/Cas9 system [23, 24]. Briefly, dual gRNA and donor vector were co-transfected into MCF-7 cells. One week later, the transfected cells were subject to puromycin (1 μg/ml) selection; and surviving cells were sorted by FACS based on GFP signal into 96-well plates and then expanded in 12-well plates. Potential clones were further verified by genomic PCR and qRT-PCR.

Cell proliferation assays were carried out by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, as previously described [25].

Correlations between breast cancer patient survival and DUSP7 expression (probe: 214793_at) was analyzed by KM plotter (http://​kmplot.​com). Auto select best cutoff was chosen in the analysis. A total of 3951 breast cancer samples were split in high and low groups according to the cutoff value, respectively. The hazard ratio with 95% confidence intervals and log rank P value was calculated and significance was set at P < 0.05. The expression of DUSP7 in breast cancer grouped by ER status was identified from TCGA databases in Oncomine (Compendia Biosciences, www.​oncomine.​org) using the analysis of “Dataset view” as previously reported [26].

Estrogen-independent growth of ER+ cells is one of important factors that leads to the failure of endocrine therapy [27]. To determine whether lncRNAs play a role in endocrine resistance, we asked whether any of lncRNAs is induced by estrogen deprivation. Therefore, we first treated MCF-7 cells with E2-free medium for 6 days, and then detected ERα activation at Ser118 by western blot (Fig. 1a). Meanwhile, we analyzed the expression of pS2, CXCL12 (two widely used genomic target of ERα); c-Myc and cyclin D1 (two widely used non-genomic target of ERα) by qRT-PCR (Fig. 1b). The statistically significant downregulation of pS2 and CXCL12 mRNA levels were observed after 6 days treatment with E2-free medium (Fig. 1b, left panel), while a dramatic upregulation was found in c-Myc and cyclinD1 mRNA level (Fig. 1b, right panel). These results suggest that ER signaling may be activated by non-genomic action mechanism and then modulating downstream target genes under 6 days E2-free condition. Therefore, we then chose E2-free culture for 6 days for profiling experiments and identified 7 lncRNAs with over a 5-fold of induction by estrogen deprivation (Fig. 1c). We were particularly interested in linc-RoR because a previous report suggests its potential oncogenic role in metastasis TNBC [19].

To further confirm the effect of estrogen deprivation on linc-RoR expression, we treated MCF-7 cells for different time points in E2-free medium (1, 2, 4 and 6 days) and then determined linc-RoR mRNA level by qRT-PCR. Linc-RoR expression was significantly induced by estrogen deprivation in a time-dependent manner (Fig. 1d). In addition, we also examined linc-RoR expression in response to E2 stimulation. The significant increase of linc-RoR was detected in 24 h E2-free treatment group but not in E2-free and E2 combined group (Additional file 1: Figure S1A), suggesting the negative regulation of linc-RoR by estrogen signal. Next, we asked whether linc-RoR expression is affected by tamoxifen (TAM). After MCF-7 cells were treated with 4 μM TAM for 1, 2 and 4 days, we detected ~8-fold increase in linc-RoR level at day 4 day, similar to the induction by estrogen deprivation (Fig. 1e). Collectively, the above results suggest that linc-RoR is important to estrogen-independent growth of MCF-7 cells. Therefore, linc-RoR was selected for further characterization of its role in estrogen-independent growth and tamoxifen resistance of breast cancer.

To determine the significance of linc-RoR in estrogen-independent growth, we took advantage of CRISPR/Cas9 system [23, 24] to knockout (KO) linc-RoR in MCF-7 cells since RNAi was not effective. Colony formation assays revealed that linc-RoR KO suppressed cell proliferation in E2-free condition (Fig. 2a, bottom left). Of interest, the level of linc-RoR was correlated with the colony number. For instance, there was little growth in KO#21. On the other hand, there was about 50% colony formation for KO#78. To our surprise, there was no difference in colony formation between vector control and linc-RoR KO in regular medium (Fig. 2a, up left), suggesting a role of linc-RoR in estrogen deprivation. Furthermore, MTT assays revealed that linc-RoR KO significantly enhanced the cell sensitivity to TAM as compared with vector control cells (Fig. 2b).

To further obtain the insight into the role of linc-RoR in estrogen-independent growth and tamoxifen resistance, we performed a rescue experiment, that is, re-expression of linc-RoR in KO cells. As expected, estrogen deprivation- and tamoxifen-mediated cell growth inhibition was alleviated by linc-RoR re-expression in KO cells (Fig. 2c and d), further supporting a critical role of linc-RoR in estrogen-independent growth.

In contrast to the relatively well-characterized estrogen-dependent activation of transcription, ERα can be activated in absence of cognate hormone by processes referred to as ligand-independent activation [28]. In this case, phosphorylation at Ser118 facilitates ligand-independent activation of ER, and thus confers endocrine resistance [29, 30]. We thus sought to test the possibility that linc-RoR might have any role in ligand-independent activation of ER at Ser118. First, we examined the phosphorylation of ER at Ser118 in gRNA control and linc-RoR KO cells under regular and E2-free condition. Not surprisingly, knockout of linc-RoR dramatically suppresses the activation of ER under E2-free condition, but had no effect on phosphorylation of ER under regular condition (Fig. 3a). We next analyzed the expression of c-Myc and cyclin D1, the two non-genomic ER target genes that are upregulated by E2-free treatment (Fig. 1b), in gRNA and linc-RoR KO cells after E2-free treatment. As shown in Fig. 3b, linc-RoR KO significantly decreased the expression of those two non-genomic target genes. These results suggest that linc-RoR KO abrogates estrogen-independent activation of ER signaling. In addition, rescue experiment further confirmed the role of linc-RoR in ligand-independent activation of ER. Western blot also indicated that phosphorylation of ER was restored by linc-RoR (Fig. 3c); consistent with these findings, we also found that re-expression of linc-RoR increased the levels of c-Myc and cyclin D1, as detected by qRT-PCR (Fig. 3d).

It is known that activation of the MAPK/ERK signaling cascade pathway can promote phosphorylation of ER at Ser118 [31, 32]. Thus, we asked whether linc-RoR contributes to estrogen-independent activation of ER through MAPK/ERK signaling pathway. First, we studied the response of ERK to estrogen deprivation in parental and gRNA control cells by Western blot. As shown in Fig. 4a, estrogen deprivation remarkably activated ERK. By contrast, inhibition of ERK by U0126 abrogated estrogen deprivation-induced ERK phosphorylation and activation of ER (Fig. 4b), suggesting the role of MAPK/ERK in estrogen-independent activation of ER signaling. Next, we tested the role of linc-RoR in estrogen deprivation-induced activation of MAPK/ERK pathway. The induction of ERK by estrogen deprivation was significantly repressed in linc-RoR KO cells (Fig. 4c and Additional file 1: Figure S1B). Rescue experiments further confirmed that re-expression of linc-RoR restored ERK activation in E2-free medium (Fig. 4d). Together, these results suggest that linc-RoR plays a role in estrogen deprivation-induced activation of ER via MAPK/ERK pathway.

Given that ERK activity is activated by the upstream kinase MEK, we determined the expression and activation of MEK1/2 in response to estrogen deprivation. As expected, the induction of MEK1/2 was detected after estrogen deprivation in parental MCF-7 and vector control cells (Fig. 5a). We then examined the activation of MEK1/2 in response to estrogen deprivation in gRNA and KO cells. Surprisingly, the difference of MEK1/2 activation was not detected between gRNA and linc-RoR KO cells (Fig. 5b), suggesting that additional mechanism might be involved in linc-RoR-mediated ERK activation by estrogen deprivation.

It is well known that MAPK/ERK signaling is negatively regulated by mitogen-activated protein kinase phosphatases (MKPs). Thus, we employed MKP inhibitor NSC95397 to study the possible involvement of MKPs in this apsect. Linc-RoR KO cells were cultured in E2-free medium for 6 days, and then different concentrations of NSC95397 (1, 2, 4, 5, 8, 10, 20 μM) treated for 3 h before harvesting for Western blot. The dramatic restoration of ERK activation was observed in linc-RoR KO cells at 10 and 20 μM (Fig. 5c). The dramatic upregulation of ERK phosphorylation was also observed in gRNA control cells (Additional file 1: Figure S1C). These results indicate that MKPs play roles in the requirement for linc-RoR in estrogen deprivation-induced ERK activation. MKP family consist of six distinct groups based on their physiological functions; among them, groups 1 ~ 4 are involved in dephosphorylation of ERK [33]. Thus, we selected one member each from these four groups and they were DUSP1/MKP-1, DUSP4/MKP-2, DUSP6/MKP-3 and DUSP7/MKP-X. The expression of MKP-1 was detected in MCF-7 cells under regular condition, and there was a decrease of MKP-1 after E2-free treatment (Additional file 1: Figure S2A and B). However, there was no difference in the MKP-1 level between gRNA and linc-RoR in regular or E2-free condition (Additional file 1: Figure S2A and B). We also examined the expression of MKP-2 and MKP-3; none of them were detectable in MCF-7 cells in regular or E2-free condition (Additional file 1: Figure S2C and D). Finally, we identified DUSP7/MKP-X as a potential candidate. For instance, DUSP7/MKP-X was detected both in gRNA and linc-RoR KO cells under regular condition. Furthermore, there was a decrease of DUSP7 in both gRNA control or linc-RoR KO cells after E2-free treatment. More importantly, the expression of DUSP7 in gRNA was lower than in linc-RoR KO cells (Fig. 5d). These results suggest that DUSP7 might be involved in linc-RoR-mediated ERK activation in response to estrogen deprivation.

To better understand the role of linc-RoR in DUSP7 expression, we examined the half-life of DUSP7. After addition of protein synthesis inhibitor cycloheximide (CHX), we found that DUSP7 degraded much faster in gRNA cells than in linc-RoR KO cells (Fig. 5e). For instance, the half-life of DUSP7 was ~8 h in gRNA cells, whereas it was more than 12 h in linc-RoR KO cells. Together, these results suggest that linc-RoR promotes ERK activation by facilitating DUSP7 degradation.

To determine the clinical relevance of DUSP7 in breast cancer, we interrogated the TCGA breast cancer database at Oncomine and found that DUSP7 was lower in ER+ breast cancer samples (n = 225) than in ER- samples (n = 87) (Fig. 6a). Importantly, Kaplan-Meier plotter analysis indicated that the low level of DUSP7 was associated with poor RFS among ER+ patients (Fig. 6b). For example, the upper quartile survival of low DUSP7 expression cohort is 60 months, whereas the upper quartile survival of high DUSP7 expression cohort is 78.48 month. Together, these results highlight the clinical significance of DUSP7 in breast cancer.