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

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 cont rol of gene ex pression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-S eq) 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.

Fine-tuning the metabolic rewiring and adaptation of translational machinery during an epithelial-mesenchymal transition in breast cancer cells

“…(iii) Our work analyzed just the final state after 48 h of the induction of MKL1 variants expression. However, the results we present here supported by the use of two different control cells, together with the studies reported in Jehanno and Fernández-Calero et al [29], allow us to validate the current cell model to deepen studies on EMT processes.…”

“…Similarly, a number of genes involved in kinase cascades and signal transduction are upregulated, consistent with the induction of an EM-like transition [30]. We also found an underrepresentation of genes involved in epigenetic marks such as chromatin remodeling and DNA methylation, which indicates cell cycle arrest, consistent with the induction of an EM-like transition (reviewed in [29,30]).…”

“…Furthermore, the motile invasion properties of MCF7 MKL1 ΔN200 cells suggested by GO analysis (Supplementary File S5 and S7), were illustrated by a 3D spheroid invasion assay on Matrigel. Only MCF7 cells expressing MKL1 ΔN200 variant showed invasive capability of the Matrigel (Supplementary Figure S5) which was previously quantified [29]. Interestingly, these cells also increased their size but decreased their viability after MKL1 ΔN200 expression with tetracycline.…”

“…As expected, for several epithelial markers like EPCAM, KRT8, KRT18, TJP3, and GATA3, expression levels were reduced in the MKL1 ΔN200 cells compared to control cell lines. Similarly, gene expression changes associated with breast EMT and cancer progression were detected, as the reduction of ERa and ERBB2 [29] and the increase of NOTCH1 and WNT5B [31,32]. In contrast, no decrease in E-cadherin levels was observed in MKL1 ΔN200.…”

“…Notably, MKL1 ΔC301 and MCF7 control cells showed less than 500 differentially expressed genes (Supplementary Files S2 and S3). To test data consistency, we compared log2 fold changes from total RNA differential expression analysis to log2 fold changes from microarray data recently published [29]. We found a significant correlation between both data sets (correlation coefficient 0.84, p < 2.2 e−16).…”