Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance

Highlights

• MKL1 is a master regulator of actin dynamic and cellular motile functions.

• Nuclear translocation of MKL1 is associated with endocrine resistance.

• Nuclear translocation of MKL1 induces a mixed luminal/basal phenotype.

• Nuclear translocation of MKL1 suppresses estrogen-mediated control of gene expression.

• Nuclear translocation of MKL1 induces a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs).

Abstract

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

Introduction

Breast cancers exhibit strong heterogeneity from genetic and phenotypic features to clinical behavior [1]. Based on global gene expression profiles, at least four major molecular subtypes have been identified, including luminal A, luminal B, HER2-enriched and basal-like tumor [2,3]. Estrogen receptor-alpha (ERα) expression defines the luminal subtypes. More than two-thirds of breast cancers overexpress ERα allowing most of them to depend on estrogen to proliferate [4,5]. This specificity makes ERα an ideal target for endocrine therapies that use selective estrogen receptor modulators (SERM) such as tamoxifen and/or aromatase inhibitors to block estrogen-dependent cell proliferation [6,7]. Unfortunately, a significant number of ERα positive breast tumors fail to initially respond to endocrine therapy or stop benefiting from such treatments and acquire resistance [8,9]. Endocrine resistance often relies on changes in the functional properties of ERα, whose activity may shift from ligand-dependent to ligand-independent activity [9]. Actually, luminal A breast cancers generally express higher level of ERα, exhibit better response to endocrine therapy and have better prognosis than luminal B subtype [10,11]. These last years, estrogen signaling in luminal breast cancer cells was deeply explored through gene expression and genome-wide chromatin binding profiling using both cell lines and human tumors [[12], [13], [14]]. Data highlighted that ERα binds to several thousand of sites across the genome and that most of these sites are located far away from the promoter regions. The genomic regions bound by ERα are enriched with cis-regulatory elements bound by pioneer factors, notably FOXA1 and GATA3 whose expression is correlated with luminal subtype [15]. ERα binding to chromatin still occurs in breast cancer cell lines and tumors that are resistant to antiestrogen therapies [14]. However, these cells do present a modified cistrome of ERα, having lost a number of ERα binding sites (ERBSs) but also harboring specific ones. Interestingly, these novel ERBSs found in antiestrogen-resistant cells, are still bound by FOXA1 but are depleted in GATA3 motifs. In addition, the ERα cistrome in drug resistant cell lines and tumors is characterized by an increased average ERα binding signal intensity likely resulting from a constitutively active estrogen receptor. The transcriptomic signature of these cells remains associated with ERα-positive luminal subtype [14]. More aggressive in nature, with higher proliferation and metastasis potential than luminal subtypes, basal-like tumors often have a triple ERα/progesterone receptor (PR)/HER2 negative phenotype [2,16]. Consequently, these tumors are not amenable to conventional targeted therapies. Recent studies suggest that basal-like breast cancers originate from luminal cells rather than a mammary stem cell (MaSC) [1,17,18]. Up to 30% of initially ERα-positive tumors that have developed resistance to antiestrogen therapies lose part of ERα expression [19]. Therefore, endocrine resistance may also rely on a dedifferentiation process leading luminal cancers to switch to an ERα-negative phenotype.

MKL1, also called MRTFA, MAL or BSAC, is a member of the myocardin-related transcription factor (MRTF) family, whose members are coactivators of the serum response factor (SRF) [20,21]. Its main role is to sense the degree of actin polymerization controlled by Rho GTPases and to subsequently integrate this information at the gene expression level through a nuclear translocation [20]. As a master regulator of cellular motile and contractile functions, MKL1 exerts important roles in vascular smooth muscle cell and cardiac myocyte differentiation, and neuronal migration [21]. In mammary gland, MKL1 is essential for the basal/myoepithelial cell differentiation and function [22,23]. During tumorigenesis, MKL1 is required for tumor cell invasion and metastasis mediating the adaptive changes in cell shape, adhesion, and migration linked to the actin cytoskeleton [24]. Recent genome-wide association studies have identified MKL1 locus as a susceptible risk factor for breast cancer and especially triple-negative breast cancer (TNBC) [[25], [26], [27]]. We previously showed that the activation of MKL1 in estrogen sensitive breast cancer cell lines leads to hormonal resistance and reduced expression of ERα, PR and HER2, recalling the triple negative phenotype [28]. In the present study, we show that nuclear accumulation of MKL1 is associated with endocrine resistance in a cohort of breast cancers. Using genome wide analysis of gene expression and ERα chromatin binding, we demonstrate that, upon the expression of a constitutively active form of MKL1, initially ERα-positive breast cancer cells initiate a dedifferentiation process associated with a profound reprogramming in ERα cistrome leading to hormonal escape.