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

Abstract

Background: The conversion from estrogen-dependent to estrogen-independent state of ER+ breast cancer cells is the key step to promote resistance to endocrine therapies. Although the crucial role of MAPK/ERK signaling pathway in estrogen-independent breast cancer cell growth is well established, the underlying mechanism is not fully understood.

Methods: In this study, we profiled lncRNA expression against a focused group of lncRNAs selected from lncRNA database. CRISPR/Cas9 was employed to knockout (KO) linc-RoR in MCF-7 cells, while rescue experiments were carried out to re-express linc-RoR in KO cells. Colony formation and MTT assays were used to examine the role of linc-RoR in estrogen-independent growth and tamoxifen resistance. Western blot and qRT-PCR were used to determine the change of protein and lncRNA levels, respectively. The expression of DUSP7 in clinical specimens was downloaded from Oncomine ( www.oncomine.org ) and the dataset from Kaplan-Meier Plotter ( http://kmplot.com ) was used to analyze the clinical outcomes in relation to DUSP7.

Results: We identified that linc-RoR functions as an onco-lncRNA to promote estrogen-independent growth of ER+ breast cancer. Under estrogen deprivation, linc-RoR causes the upregulation of phosphorylated MAPK/ERK pathway which in turn activates ER signaling. Knockout of linc-RoR abrogates estrogen deprivation-induced ERK activation as well as ER phosphorylation, whereas re-expression of linc-RoR restores all above phenotypes. Moreover, we show that the ERK-specific phosphatase Dual Specificity Phosphatase 7 (DUSP7), also known as MKP-X, is involved in linc-RoR KO-induced repression of MAPK/ERK signaling. Interestingly, linc-RoR KO increases the protein stability of DUSP7, resulting in repression of ERK phosphorylation. Clinical data analysis reveal that DUSP7 expression is lower in ER+ breast cancer samples than that in ER- breast cancer. Moreover, downregulation of DUSP7 expression is associated with poor patient survival.

Conclusion: Taken together, these results suggest that linc-RoR promotes estrogen-independent growth and activation of MAPK/ERK pathway of breast cancer cells by regulating the ERK-specific phosphatase DUSP7. Thus, this study might help not only in establishing a role for linc-RoR in estrogen-independent and tamoxifen resistance of ER+ breast cancer, but also suggesting a link between linc-RoR and MAPK/ERK pathway.

Keywords: Breast cancer; DUSP7; ERK; Estrogen-independent growth; Linc-RoR.

Fig. 1

Identification of linc-RoR as an estrogen deprivation-induced lncRNA. a Detection of ER activation after 6 days E2-free treatment by Western blot. The intensity value is relative to GAPDH as 1. b Expression of ER genomic and non-genomic target genes after 6 days of E2-free treatment. c LncRNA profiling after 6 days of E2-free treatment. d Linc-RoR is induced by E2-free treatment in a time-dependent manner. e Induction of linc-RoR by TAM treatment. Error bars represent S.E.M., n = 3, **P < 0.01

Fig. 2

Linc-RoR KO suppresses estrogen-independent growth. a Linc-RoR KO suppresses estrogen-independent growth ad determined by clonogenic assays. b Linc-RoR KO sensitizes MCF-7 cells to TAM, as determined by MTT assay. c Re-expression of lincRoR partially restores the growth suppression by estrogen deprivation in linc-RoR KO cells. d Re-expression of lincRoR partially restores the TAM resistance in linc-RoR KO cells. Error bars represent S.E.M., n = 3, **P < 0.01

Fig. 3

Linc-RoR KO abrogates ligand-independent activation of ER. a Linc-RoR KO suppresses estrogen deprivation-induced activation of ER. The intensity value is relative to GAPDH as 1. b Linc-RoR KO causes a significant reduction of ER non-genomic target genes mRNA level under E2-free condition. c Re-expression of linc-RoR restores activation of ER. d Linc-RoR is required for ER non-genomic target gene expression. Re-expression of linc-RoR confirms the capability of estrogen-independent activation, n = 3, *P < 0.05, **P < 0.01

Fig. 4

Linc-RoR promotes ligand-independent activation of ER via MAPK/ERK pathway. a ERK is activated after 6 days estrogen deprivation. b U0126, the MEK inhibitor, abolishes the activation of ERK and ER in response to estrogen deprivation. gRNA control cells were cultured in E2-free medium for 6 days, and then cells were treated with 20 μM U0126 by 8 h before harvesting for Western blot. c Linc-RoR KO suppresses the activation of ERK induced by estrogen deprivation. d Re-expression of linc-RoR restores the phenotype of ERK activation

Fig. 5

Linc-RoR activates ERK by promoting the degradation of DUSP7. a MEK is activated in response to estrogen deprivation. b Detection of MEK activity in gRNA and RoR KO cells in E2-free condition. Note that there was no difference in MEK activity between gRNA and RoR KO. c Suppression of MKP activity by NSC97397 restores ERK activation in linc-RoR KO cells under E2-free condition. d Linc-RoR KO prevents DUSP7 from dagradation under E2-free conditon. e Linc-RoR KO increases protein stability of DUSP7. MCF-7 gRNA and linc-RoR KO cells were treated with CHX (cycloheximide) at 20 μg/ml and then were harvested for western blot at indicated time points. Half-life curve is on the right

Fig. 6

The clinical relevance of DUSP7 in breast cancer. a Oncomine data shows DUSP7 mRNA expression in ER negative (ER-) breast cancer tissue (n = 87) vs ER positive (ER+) breast cancer tissue (n = 225), **P < 0.01. b Kaplan-Meier survival curves suggests poor relapse-free survival (RFS) with low expression of DUSP7 compared with those in the high DUSP7 expression group

Fig. 7

The working model of present study. Linc-RoR promotes estrogen-independent growth (EIG) of ER+ breast cells through conferring the activation of MAPK/ERK pathway. See text for explanation