Charly Jehanno

Tamara Fernández-Calero

Denis Habauzit

Stéphane Avner

Frédéric Percevault

Emmanuelle Jullion

Pascale Le Goff

Marie May Coissieux

Simone Muenst

Mónica Marín

Tamara Fernandez-Calero

Frederic Percevault

Monica Marin

Denis Michel

Raphaël Métivier

Dr Gilles Flouriot
email: gilles.flouriot@univ-rennes1.fr

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

Keywords: Breast cancer, Endocrine resistance, Estrogen receptor, MKL1, Gene regulation, Cistrome coupled to high -throughput sequencing, megakaryoblastic leukemia 1, selective estrogen receptor modulators, SRF: serum response factor, TNBC: triple-negative breast cancer

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Introduction

Breast cancers exhibit strong heterogeneity from genetic and phenotypic features to clinical behavior [START_REF] Skibinski | The origin of breast tumor heterogeneity[END_REF]. Based on global gene expression profiles, at least four major molecular subtypes have been identified, including luminal A, luminal B, HE R2-enriched and basal-like tumor [START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sørlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF]. 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 [START_REF] Platet | Estrogens and their receptors in breast cancer progression: a dual role in cancer proliferation and invasion[END_REF][START_REF] Dahlman-Wright | International Union of Pharmacology. LXIV. Estrogen receptors[END_REF]. This specificity makes ERα an ideal target for endocrine therapies that use selective estrogen rec eptor modulators (SERM) such as tamoxifen and/or aromatase inhibitors to block estrogen-dependent cell proliferation [START_REF] Osborne | Steroid hormone receptors in breast cancer management[END_REF][START_REF] Wickerham | The use of tamoxifen and raloxifene for the prevention of breast cancer, Recent Results Cancer Res. Fortschritte Krebsforsch[END_REF]. Unfort unat ely, a significant number of ERα positive breast tumors fail to initially respond to endocrine therapy or stop benefiting from such treatments and ac quire resistance [START_REF] Jordan | Selective estrogen-receptor modulators and antihormonal resistance in breast cancer[END_REF][START_REF] Clarke | Endocrine resistance in brea st cancer--An overview and update[END_REF]. Endocrine resistance often relies on changes in the functional properties of ERα, whose activity may shift from ligand-dependent to ligand-independent activity [START_REF] Clarke | Endocrine resistance in brea st cancer--An overview and update[END_REF]. 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 [START_REF] Musgrove | Biological determinants of endocrine resistance in breast cancer[END_REF][START_REF] Gajulapalli | Oestrogen receptor negativity in breast cancer: a cause or consequence?[END_REF]. 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 [START_REF] Carroll | Genome -wide analysis of estrogen receptor binding sites[END_REF][START_REF] Welboren | ChIP-Seq of ERalpha and RNA polymerase II defines gene s differentially responding to ligands[END_REF][START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF]. Data highlighted that ERα binds to several thousand of sites across the genome and that most of t hese sites are locat ed far away from t he promoter regions. The genomic regions bound by ERα are enriched with cis-regulatory elements bound by pioneer factors, notably FOXA1 and GA TA3 whose expression is correlated with luminal subt ype [START_REF] Jozwik | Pioneer factors in hormone-dependent cancers[END_REF]. ERα binding to chromatin still occurs in breast cancer cell lines and tumors that are resistant to antiestrogen therapies [START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF]. However, these cells do present a modified cistrome of E Rα, 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 GA TA 3 motifs. In addition, the ERα cistrome in drug resistant cell lines and tumors is characterized by an inc rease d average ERα binding signal intensity likely resulting from a constitutively active estrogen receptor. The transcript omic signature of thes e cells remains associated with ERα-positive luminal subtype [START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF]. More aggressive in nature, wit h higher proliferation and metastasis potential than luminal subtypes, basal-like tumors often have a triple ERα/progesterone receptor (PR)/ HER2 negative phenotype [START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Badve | Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists[END_REF]. Consequently, these tumors are not amenable to conventional target ed therapies. Recent studies suggest that basal-like breast cancers originat e from luminal cells rather than a mammary stem cell (MaSC) [START_REF] Skibinski | The origin of breast tumor heterogeneity[END_REF][START_REF] Lim | Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers[END_REF][START_REF] Molyneux | BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells[END_REF]. Up to 30% of initially E Rα-positive tumors that have developed resistance to antiestrogen therapies lose part of ERα expression [START_REF] Kuukasjärvi | Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy[END_REF]. 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 t he myocardin-related transcription factor (MRTF) family, whose members are coactivators of the serum response factor (S RF) [START_REF] Miralles | Actin dynamics control SRF activity by regulation of its coactivator MAL[END_REF][START_REF] Pipes | The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis[END_REF]. Its main role is to sense the degree of actin polymerization controlled by Rho GTPas es and to subsequently integrate this information at the gene expression level through a nuclear translocation [START_REF] Miralles | Actin dynamics control SRF activity by regulation of its coactivator MAL[END_REF]. 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 [START_REF] Pipes | The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis[END_REF]. In mammary gland, MKL1 is essential for the basal/myoepithelial cell differentiation and function [START_REF] Li | Requirement of a myocardin -related transcription factor for development of mammary myoepithelial cells[END_REF][START_REF] Sun | Acute myeloid leukemia -associated Mkl1 (Mrtfa) is a key regulator of mammary gland function[END_REF]. During tumorigenesis, MKL1 is required for tumor cell invasion and metastasis mediating t he adaptive changes in cell shape, adhesion, and migration linked to the actin cytoskeleton [START_REF] Medjkane | Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis[END_REF]. 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][START_REF] Gurbuz | SAP domaindependent Mkl1 signaling stimulates proliferation and cell migration by induction of a distinct gene set indicative of poor prognosis in breast cancer patients[END_REF][START_REF] Lindström | Genome-wide association study identifies multiple loci associated with both mammographic density and breast cancer risk[END_REF]. 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 [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF]. 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 E Rα 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.

Materials and Methods

Cell culture and transfection

Stably transfected MCF7 T-Rex sub-clones (T-Rex system, Invitrogen), MCF7-control (with empty pcDNA 4/ TO expression vector) and MCF7-MKL1ΔN200 (wit h MKL1ΔN200 pcDNA4/ TO expression vector), were previously described [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF][START_REF] Flouriot | The actin/MKL1 signalling pathway influences cell growth and gene expression through large -scale chromatin reorganization and histone post-translational modifications[END_REF]. MCF7 T-Rex sub-clones as well as MCF10A, T47D, ZR-75-1, MCF7 AKT+ [express myr-akt1 (activated) plasmid (Upstate cell signaling solutions)], MDA-MB-468, MDA -MB-231 and SUM159P T cell lines were routinely maintained in DMEM (Invit rogen) supplemented with 10% fetal calf serum (FCS ) (Biowest) and antibiotics (Invitrogen) at 37ºC in 5% CO 2 . Before any experiments, cells were grown in phenol red-free DMEM (Invitrogen) containing 2.5% charc oal-stripped FCS (Biowest) for at least 72 hours. In order to induce the expression of the constitutively active mutant of MKL1 (MK L1Δ N200), MCF7 cells were treat ed the last 48 hours with 1 µg/mL tetracycline. Tetracycline treatment was systematically performed on both MCF 7-control and MCF7-MKL1Δ N200 cells. 17β-estradiol (E2, Sigma-Aldrich), 4hydroxytamoxifen (OHT, Sigma-Aldrich) and ICI 182,780 (ICI, Sigma-Aldrich) were used at a final concentration of 1 or 10 nM, 1000 nM and 100 nM, respectively. Jasplakinolide (Abcam) and Erlotinib (Sigma-Aldrich) were used at the final conc entration of 0.5 g/ml and 100 M, respectively. Transient transfection experiments were performed with the reporter gene C3 (complement 3) -Luc, the pCR-ERα expression vector, p3Xflag-MKL1 ΔN200 and the CMV-βgal internal control as previously described [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF][START_REF] Huet | Repression of the estrogen receptor-alpha transcriptional activity by the Rho/megakaryoblastic leukemia 1 signaling pathway[END_REF].

Matrigel invasion assay

3D spheroids of breast cancer cell lines were formed in untreated plastic petri dishes by constant gyratory shaking at 60 r. p.m. as previously described [START_REF] Flouriot | Monolayer and aggregate cultures of rainbow trout hepatocytes: long-term and stable liver-specific expression in aggregates[END_REF]. Matrigel solution was prepared in culture medium at the final concentration of 1mg/ml. Resuspended in Matrigel solution, spheroids were then seeded in on the top of a matrigel cushion already formed in 96-well plates. To monitor cell invasion of Mat rigel, images were taken every 24 hours by microscopy (DMIRB-Leica) and the size of the cellular spreading zone was calculated using Image J software.

Protein extraction and western blotting

Whole-cell extracts were directly prepared in 3X Laemmli buffer. Following sonication, the protein extracts were denatured for 5 min at 95°C, separated on 10 % SDS polyacrylamide gels, and trans ferred to poly vinylidene difluoride membrane (Millipore). Western blots were performed as previously described Journal Pre-proof J o u r n a l P r e -p r o o f [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF][START_REF] Flouriot | The actin/MKL1 signalling pathway influences cell growth and gene expression through large -scale chromatin reorganization and histone post-translational modifications[END_REF] using the primary antibodies against MKL1 (ab14984) from Abcam, ERα (sc-543) and p-E RK (sc-7383) from Santa Cruz Biotechnology, HER1 (SAB1405746) and  actin (A1978) from Sigma-Aldrich and p-Akt (4060), Akt (9272) and ERK 1/2 (4695) from Cell signaling technology.

Immunofluorescence and immunohistochemical analysis

For cell lines, cells were grown on 10 mm-diameter coverslips in 24-well plates. After treatment, cells were fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PAF) for 10 min and then permeabilized in PBS containing 0.3% Triton X-100 for 10 min. Incubation with the primary antibody (1/1000) was performed overnight (ON) at 4°C. Primary antibodies against N-cadherin (N19, sc-1502), P-cadherin (H105, sc-7893), cytokeratin 14 (LL02, sc-58724), cytokeratin 18 (C-04, sc-51582), E Rα (C-terminal, HC-20, sc-543), GA TA3 (sc-22206) and HE R1 (1005, sc-03) were purchased from Sant a Cruz Biotechnology.

Antibodies directed against E-cadherin (ab15148), alpha smooth muscle actin (ab5694), MKL1 (ab14984), FOXC1 (ab5079), FOXA1 (ab23738), P300 (ab14984) and HER2 (ab16901) were obtained from A bcam.

Anti-vimentin (clone V9) antibody was purchased from Sigma-Aldrich Primary antibody against ERα (Nterminal, 6F11) was purchased from Thermo Fisher Scientific. Dye-conjugated secondary antibodies (Abcam) were incubated 1h at room temperature. The cover slides were mounted in Duolink II mounting medium with DAP I (Sigma-Aldrich), images were obtained with an ApoTome Axio Z1 Imager microscope (Zeiss) and processed with Axio Vision Software. Fluorescence was quantified with ImageJ software from images obtained with identical exposure times. Immunofluorescence was scored for at least 20 cells per image on n images obt ained from diverse experiments. The means obtained for every image were t hen averaged.

For MKL1 and ER co-labeling experiments on tissue sections, paraffin-embedded tissues were cut at 4 µm, mounted on positively charged slides and dried at 58°C for 60 minutes. Immunohistology staining was performed on the Discovery XT A utomated IHC stainer using the Ventana detection kit and Discovery Rhodamine and FAM kits (Ventana Medical Systems, Tucson, Ariz ). Primary antibodies were anti-ERα (6F11, Thermo Fisher Scientific) and anti-MKL1 (HPA030782, Sigma-Aldrich).

For the immunohistchemical analysis, MKL1 staining on tissue microarrays (TMA) from breast cancer was performed using a Ventana Benchmark Discovery instrument (Roche Diagnostics). 4uM sections of the TMA blocks were pret reat ed with CC1 for 40 min. Then, slides were incubated with MKL1 primary antibody (ab14984, Abcam ) at a dilution of 1:50 for 1h, followed by secondary antibody incubation and det ection.

Hematoxylin II and bluing reagent (Ventana, Roche diagnostics) were us ed for c ounterstaining. Samples were scored as positive when the number of cells per sample displaying nuclear localization of MKL1 was superior to 30% or as low positive/negative when this number was below 30%. The blinded analysis of MKL1 expression was performed two times on two independent MKL1 staining of the TMA slides. For immunohistochemistry sections, images were captured using a Nikon Ni -e upright microscope coupled to a PRIOR slide loader. Acquisitions were performed with a 10x AIR objective with a DS -Fi3 camera using NIS software. Written informed consent was obtained from patients and the institutional ethics committee approved the study.

RNA extraction, gene expression array and RT-PCR assays

RNA purifications were performed immediately after a treatment of MCF7 cells with 10 nM E2 or ethanol (vehicle control) for 4h. RNAs were extracted with the RNeasy kit (Quiagen) and the quantity and quality of RNAs were assessed using a Nanodrop 1000 spectrophotometer (Nanodrop technology) and an Agilent Bioanalyser. RNAs were then reverse -transcribed and the obtained c DNA were amplified and Cy3-labelled according to the manufacturer's instructions (Agilent Technologies). Gene expression profiling was carri ed out on A gilent Whole Human Genome 8x60K Microarrays (Agilent Technologies) at the Biosit Rennes facility. Four independent RNA samples per condition (n=4) were randomly examined. Data analysis was performed using 'R' (R Foundation for Statistical Comput ing, Vienna, Austria), mainly through packages included in the Bioconductor suite [START_REF] Gentleman | Bioconductor: open software development for computational biology and bioinformatics[END_REF]. Probes were filtered considering saturation, signal above background and uniformity. Only those with no replicate flagged in at least one condition were taken into account for data analysis. Samples were normalized using 75 -percentile shift normalization. Gene expression values were computed as the median intensity values of all filtered probes design for each particular gene. Differential expression was assayed using the limma software package [START_REF] Wettenhall | limmaGUI: a graphical user interface for linear modeling of microarray data[END_REF]. When we compared MCF7-MKL1ΔN200 and MCF7-cont rol cells, genes were considered differentially expressed when the multiple testing adjusted P-value was <0.01 and the absolute value of fold change greater than 4. When we analyzed E2 transcriptional regulation, genes were considered differentially expressed when the multiple testing adjusted P-value was <0.05 and the abs olute value of fold change greater than 1.8. Gene Ontology analysis and Kegg pathways enrichment analysis were conducted with clusterP rofiler [START_REF] Yu | clusterProfiler: an R package for comparing biological themes among gene clusters[END_REF]. Up-regulated and downregulated genes were used independently in these analyses. Quantitative RT-P CRs were performed as previously described [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF]. Primers for RT-PCR are provided in Supplementary Materials (Table S1).

Chromatin immunoprecipitation (ChIP)

MCF7 cells were treated with 10 nM E2 or ethanol (vehicle control) during 50 min, was hed twic e with PBS and cross-linked for 10 min with 1.5% formaldehyde (Sigma). The cross -link reaction was stopped with 0. 125 M glycine for 1 min and c ells were was hed twice with PBS, scraped int o 500 µl PBS with protease inhibitors (Complet e Inhibitors, Roche), spun 2 min at 3000 g and snap frozen to -80°C. Aft er cell lysis in 300 μl of lysis buffer [10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 1% SDS, 0.5% Empigen BB detergent], ChIP was performed as previously described [START_REF] Quintin | Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities[END_REF][START_REF] Palierne | Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment[END_REF]. Antibodies against ERα (HC-20, sc-543, S anta Cruz Biotechnology), H3K27ac (ab4729, Abcam) and H3K4me2 (07 -030, Merk Millipore) were used in this assay. DNA was purified on NucleoSpin columns (Macherey-Nagel) using NTB buffer. ChIP experiments were performed from at least five biological independent replicates. Primers used for real-time PCR are provided in Supplementary Materials (Table S1).

ChIP-sequencing (ChIP-Seq) and ChIP-Seq data analysis

We pooled DNA originating from at least 18 different ChIP experiments conducted as described above. The ChIP DNA was prepared into libraries and sequenced using an Illumina HiSeq apparatus at the GenomEast platform (Institut de Génétique et de Biologie Moléculaire et Cellulaire; Strasbourg, France). Pooled control inputs DNA were processed in parallel. Reads were aligned onto the indexed chromosomes of the human hg19 (GRCh37) genome using bowtie 0.12.7 [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF], allowing at most two mismatches (parameters -n 2; -l 28;

-m 1 with --best and --strata options). Sequencing statistics are given in Table S2. Samtools 0.1.12a [START_REF] Li | Genome Project Data Processing Subgroup, The Se quence Alignment/Map format and SAMtools[END_REF] implemented under galaxy (https://usegalaxy.org/) were used to generate signal files. Duplicate reads were removed using the rmDupB am tool, and GCbias were subseq uently corrected by the compute/correct GC bias algorithms included in the DeepTools package. MACS 2.1.1.2016 [START_REF] Feng | Identifying ChIP -seq enrichment using MACS[END_REF] was then us ed for converting signal into .wig files and peak calling using low/up mfold bound adjusted to 5 and 50 res pectively and at different P-values cutoff, as previously performed [START_REF] Quintin | Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities[END_REF]. Input control file was used as reference for these peak-callings. The obtained .bed files corresponding to the genomic coordinates of identified ERα BSs were subsequently filtered against the lists of repetitive sequences obtained from the UCS C (blacklist; http://genome.ucsc.edu/cgi-bin). Regions ex hibiting high background signal generated by a poor normalization to input due to excessive sequenc es overrepresentation (consecutive of the highly rearranged genome of MCF7 cells) were also removed. ER-bound identified genomic regions are provided in the Table S3. For alignment and calculations of mean signals at precise subset of genomic regions, we corrected the bias of diverging sequencing depths bet ween different samples by normalizing the signal intensities of a given .wig to the one wit h the highest sequencing depth. Motifs analyses were performed using th e CentDist (http://biogpu.ddns.comp.nus.edu.sg/~chipseq/ webs eqtools2) and SeqPos (http://cistrome.org/ap/ [START_REF] Liu | Cistrome: an integrative platform for transcriptional regulation studies[END_REF]) algorithms. All other integrative analyses of the ChIP -Seq data were performed using home-made scripts and algorithms from the cistrome web-platform (http://cistrome.org/ap/).

Availability of data

The microarray and ChIP -Seq data generat ed in this study have been submitted to the NCB I Gene Expression Omnibus website (http://www. ncbi.nlm.nih. gov/ geo/) [START_REF] Barrett | NCBI GEO: archive for high-throughput functional genomic data[END_REF] under accession No. GSE 107924 and GSE 107476, respectively.

Results

Nuclear accumulation of MKL1 favors invasiveness and impairs transactivation efficiency of ERα

We previously showed that breast cancer cell lines exhibit different MKL1 activity [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF]. As shown in Fig 1A

and Although we were unable to measure the invasive capability of the MDA -MB-468 due to their inability to form spheroids, this cell line is known to be highly invasive in the literature. Transactivation efficiency of the estrogen recept or on an E RE-driven reporter gene was evaluat ed in parallel through transient trans fection experiments in these different cell lines. Importantly, we observed that the transactivation activity of ERα was the strongest in the ERα-positive breast cancer cell lines and dramatically lower in the nuclear MKL1-positive invasive cell lines MDA-MB-468, MDA-MB-231 or SUM159P T (Fig. 1C). These correlative data suggest a close link between MK L1 nuclear translocation, invasiveness and impaired ERα transactivation efficiency. To shift from correlation to causality, we investigated whether nuclear accumulation of MKL1 in ERα-positive breast cancer cell lines, using a MKL1 mutant or a drug inducing MKL1 translocation, could impact invasiveness and E Rα trans activation efficiency. We previously established a MCF7 cell line expressing a Nterminal deleted mutant of MKL1 (MKL1 ΔN200) devoid of the RPEL motifs (actin binding sites), allowing permanent translocation and constitutive activity of MKL1 into the nucleus [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF][START_REF] Flouriot | The actin/MKL1 signalling pathway influences cell growth and gene expression through large -scale chromatin reorganization and histone post-translational modifications[END_REF] (Fig. 1D). As shown in

Nuclear accumulation of MKL1 is associated with endocrine resistance in breast cancers.

Above results associates the nuclear localization of MKL1 in breast cell lines with de-differentiation and invasive processes. To evaluate the physiopathological relevance of this finding, we next extended our study to healthy and cancerous human breast tissues. As illustrated in Fig. 2A, immunofluorescence experiments confirm that the transcriptional coactivator MKL1 is active and expressed in the nucleus of basal/myoepithelial cells while ERα is present in a small number of luminal cells in healthy breast tissues [START_REF] Platet | Estrogens and their receptors in breast cancer progression: a dual role in cancer proliferation and invasion[END_REF][START_REF] Li | Requirement of a myocardin -related transcription factor for development of mammary myoepithelial cells[END_REF][START_REF] Sun | Acute myeloid leukemia -associated Mkl1 (Mrtfa) is a key regulator of mammary gland function[END_REF]. Nuclear co-localization of the two proteins was not observed in healthy human mammary gland. In contrast, a nuclear co-localization of E Rα and MKL1 prot eins was detected in E Rα-positive malignant tumors where s ome E Rα-positive cells begin to express high level of nuclear MKL1 (Fig. 2A). In light of these results, we performed an immunohistochemical analysis of MKL1 protein expression on a tissue microarray encompassing 130 breast cancer patient samples and covering every breast tumor subtypes (Fig. 2B and Table S4). We quantified the perc entage of cells displaying nuclear localization of MKL1 in each sample. We first detected a significant increase in the number of MKL1 nuclear positive (NP) cells in tumor tissue compared to adjacent non-tumor tissue, except for the ER+/PR+/HE R2 -subtype (Fig. 2C). Then we aimed Journal Pre-proof to evaluate whether ER positive breast tumors clinically described as hormone therapy refractory could be discriminated based on the their abundance in cells showing nuclear MKL1. Samples were split in two groups, MKL1 high or MKL1 weak/negative. In the 41 cases for which t he status hormone therapy status was available, we detected a significant increase of MKL1-high tumors (Fig. 2D), indicating that the nuclear localization of MKL1 is associated with hormone resistance in our cohort.

Nuclear accumulation of MKL1 in MCF7 cells induces a mixed luminal/basal phenotype

In order to better understand how MKL1 could promot e endoc rine resistance, we aimed at studying the consequences of MK L1 activation in ERα -positive breast cancer cells using unbias ed approaches. We previously showed that actin-cytoskeleton remodeling and nuclear accumulation of MKL1 using the MKL1 ΔN200 mutant, abolishes the E2-dependent proliferation of MCF7 cells and impairs estrogen mediated regulation of few ER-target genes [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF]. To further explore the changes in gene signature induced by the MKL1 mutant, we analyzed the gene expression profiles of MCF7 MKL1 ΔN200 and MCF7 cont rol cells through microarray experiments. We defined a set of differentially expressed genes setting an arbitrary cutoff of adjusted P-value < 0.01 and a fold change > ±4 (Table S5). This threshold res ulted in a set of 1, 016 up-regulated and 976 down-regulated genes in MCF7 MKL1 ΔN200 as compared to MCF7 control cells.

Functional analysis revealed an overrepresentation of genes involved in cell adhesion, ECM -receptor interactions, actin cytoskeleton organization and migration as well as genes involved in kinase cascades and signal trans duction upon MK L1 ΔN200 signaling (Fig. 3A and Table S6), functions that are consistent with the induction of EMT [START_REF] Lamouille | Molecular mechanisms of epithelial -mesenchymal transition[END_REF]. Furthermore, we found an underrepresentation of genes involved in epigenetic modulations such as chromatin remodeling or DNA methylation, processes that have been also associated with EMT [START_REF] Stadler | Linking epithelial-to-mesenchymal-transition and epigenetic modifications[END_REF][START_REF] Tam | The epigenetics of epithelial -mesenchymal plasticity in cancer[END_REF].

Because patterns of gene ex pression allow classifying breast tumors according to major molecular subtypes, we next investigated the expression of subsets of genes that are distinctive of clinically relevant breast cancer subgroups. We selected 48, 37 and 11 genes which have been associated to basal-like, luminal and HE R2-overexpressing tumors, respectively [START_REF] Sørlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF][START_REF] Bernardo | FOXA1 represses the molecular phenotype of basal breast cancer cells[END_REF] (Table S7), and performed a gene set enrichment analysis. The analysis revealed a significant association of MCF7 control and MKL1 ΔN200 cells to the luminal and to the basal markers gene set, respectively (Fig. S2A). No significant association was observed to the HER2 markers gene set.

Henc e, the microarray analysis described above evokes a partial transition of MCF7 MKL1 ΔN200 cells from a luminal to a basal-like phenotype. To further strengthen this observation, we performed immunocytology staining of certain basal-like biomarkers such as cytokeratin 14, P-cadherin, N-cadherin, vimentin, alpha actin and the human epidermal growth factor 1 (HER1), and confirmed their up-regulation in MKL1 ΔN200 cells (Fig. 3B and Fig. S2B). Moreover, FOXC1, the main transcription factor regulating EMT in basal-like breast cancers [START_REF] Ray | FOXC1 is a potential prognostic biomarker with functional significance in basal -like breast cancer[END_REF], was also strongly up-regulated in these cells. Among luminal biomarkers, cytokeratin 18 staining was not affected, E-cadherin exhibited a disrupted net work and the luminal pioneer factor GA TA3 was drastically repressed. The expression level of ERα dropped by 60%, reaching a level comparable to the one observed in control MCF7 cells treat ed with E2. Interestingly, the pioneer transcription factor FOXA 1 exhibited a different pattern with a clear increase of its expression in MCF7 MKL1 ΔN200 cells. Finally, the expression level of HER2 was inhibited by the constitutively active form of MKL1.

Altogether, these results clearly show that an increased nuclear-t ransloc ation and activity of MKL1 shift the phenotypical features of MCF7 cells from a luminal to luminal/basal hybrid phenotype.

MCF7 cells with a nuclear translocation of MKL1 lose E2 transcriptional regulation

In order to monitor estrogen signaling c hanges after MKL1 nuclear translocation, we used a microarraybased transcriptome analysis to identify E2-regulated genes in MCF7 MKL1 ∆N200 versus control cells treated for 4h with E2 or ethanol as vehicle control. We performed a differential gene expression (DE) analysis and selected an arbitrary cutoff of adjusted P-value < 0.05 and fold change > ±1.8 to define DE genes (Table S8). Differential gene expression analysis evidenc ed 225 estrogen-regulated genes in control cells when treated with E2 (Fig. 4A; 185 up-regulated and 40 down-regulated). Ontology and pathway enrichment analysis of these genes revealed some terms and pathways previously described as E2regulated in MCF7 cells, such as localization, cell communication and pathways in cancer [START_REF] Frasor | Profiling of estrogen up-and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype[END_REF] (Table S9).

Strikingly, only 2 up-regulated genes were found in E2 -treated MCF7 MKL1 ∆N200 and none were repressed by E2 (Fig. 4A). In general, the average of the fold-changes of the 185 up-regulated genes in control cells dropped from 2.3 to 1.2 in MCF7 MK L1 ∆N200 cells, while the one of the 40 down -regulated genes increased from 0.5 to 0.87. These data indicate that MCF7 MKL1 ∆N200 almost lose E2 transcriptional regulation.

We next addressed among the 225 E 2-regulated genes whether some of them have modified basal expression due to MKL1 ΔN200 nuclear translocation and whether this could be correlated to the loss of their estrogenic response. Among these genes, 109 present ed changes in their expression in the absence of ligand, including approximately half of the E 2 up-regulated and E2 down-regulated genes. We categorized these 109 mRNAs in 4 categories depending upon the fold changes of their basal expression and of t heir response to E2 in control MCF7 cells. As shown within Fig. 4B and4C, the E2 up-regulated genes in control cells were distributed equally between those whose basal expression is down-regulated and thos e whose basal expression is up-regulat ed in MCF7 MKL1 ΔN200 cells (C2 and C4 categories in Fig. 4B and4C). In contrast, the basal expression of the majority of the E 2 down-regulated genes in cont rol cells (C1 and C3) were also down-regulated in the presence of MKL1 ΔN200, which might indicat e a correlation between E2 and MKL1 effects for the E2 down-regulated genes only.

The expression profile of some E2-regulated genes from the clusters C1, C2 and C4 in MCF7 MKL1 ∆N200 and cont rol cells was confirmed by quantitative RT-PCR (Fig. S3). Furt hermore, these experiments show that MKL1 ΔN200-induced c hanges in basal expression of the genes were insensitive to antiestrogen treatments (Fig. S3). It should be noted that both antiestrogens used, the 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), inhibit residual E2-induced activity of GREB1 gene in MCF7 MKL1 Δ N200 cells, demonstrating the pres ervation of their antiestrogenic activity in these cells. Similar results were obtained on an ERE -driven reporter gene in t ransient trans fection experiments (Fig. S3C). To confirm the link between MKL1 localization and these change in the expression of E2-regulated genes in different ERα-positive breast cancer cell lines, we used a complementary approach, in which MCF7 and T47D cells were treated with jasplakinolide drug leading to a nuclear translocation of MKL1 (Fig. 1

and S1). Results clearly show similar

Journal Pre-proof expressions between the two cell lines and reproducible regulations regardless of how MKL1 nuclear translocation is induced. However, the amplitude of the effects was less marked after jasplakinolide treatment than after MKL1 ΔN200 expression (Fig. S4).

Finally, gene ontology and pathway enrichment analysis of the categories of genes could not be achieved due to the limited number of genes included in each subgroup. However, it should be noted that some genes generally considered as hallmarks of hormone -resistant breast cancer cells exhibited changes in expression, which could be expected to lead to such a cell phenotype. This is for instance the case of genes encoding the transcription factor FOXC1 and the growth factor AREG (amphiregulin) included in the C2 category of genes whose expression is up -regulated both by E2 and MKL1 ΔN200 and the chemokine CXCL12 in the category of genes (C4) in whic h MKL1 ΔN200 strongly counteracts the stimulating effect of E2.

Nuclear accumulation of MKL1 in MCF7 cells impacts ERα cistrome

We next sought to determine whether the abrogation of gene responses to E2 observed in MCF7 cells expressing the MKL1 ΔN200 protein was correlated to an alt ered mobilization of ERα to its binding-sites. To this purpos e, we treated control and MKL1 ΔN200 expressing cells with E2 or ethanol vehicle for 50 min, a time point adequate for determining representative direct sites of ERα binding to chromatin genom e-wide [START_REF] Ovaska | Integrative analysis of deep sequencing data identifies estrogen receptor early response genes and links ATAD3B to poor survival in breast cancer[END_REF], and subjected the prepared chromatin to ChIP-S eq as previously [START_REF] Palierne | Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment[END_REF]. Bioinformatic treatment of these data included a filtration of the identified ERBSs against regio ns referenced as repetitive and sources of ChIP-Seq biases as well as against regions heavily duplicated/ remodeled within MCF7 cells genome (see the Materials and Methods section as well as Fig. S5). The number of ERBSs retained before and after these filtering steps is given wit hin Table S10. To ascertain the elimination of a maximum of false-positive regions, we measured the mean enrichment of ERα ChIP-S eq signal at ERBSs det ermined at different Pvalues, and subsequently selected those for which the signal/noise exceeded a 2-fold ratio (see Fig. S5).

The Venn diagrams presented within Fig. 5A illustrate the overlap between the different set of ERBSs identified in each cell lines in the presence of absence of E2 at these optimal P-values. Mean ChIP -Seq signals measured on each specific subset of ERBSs are illustrated on the bottom of eac h Venn and confirm their selectivity (Fig. 5B).

In bot h cell lines, the number of common ERBSs found in EtOH and E2 conditions was relatively low (less than 5% or 1% of the E RBSs bound by ERα in the presence of E2 in control or MKL1 ΔN200 MCF7 cells, respectively). Interestingly, ERα cistromes included 6,237 or 2,658 E RBSs in E2-treated and 1, 439 and Journal Pre-proof J o u r n a l P r e -p r o o f 20 454 E RBSs in absence of hormone in control or MKL1 ΔN200 MCF7 cells, respectively. Th ese observations indicate that the expression of the constitutive MKL1 ΔN200 protein alters ERα cistrome and induces a massive drop in the number of E RBSs, regardless of the presence of ligand. In addition, the overlaps between the cistromes of ERα in each cell line are relatively poor (Fig. 5A): only 1 ERBS in the absence of ligand and 367 in the presence of E 2. This indicates that some reprogramming of ER binding on the genome had furt her occurred following the expression of the MKL1 ΔN200 protein. Importantly, using cistromes determined at less or more stringent P-values raised similar conclusions towards these overlaps (see Fig. S6). Hence, a maximum of 2% of the E RBSs identified in EtOH -treated control cells were common with those identified in similarly treated MK L1 ΔN200 MCF7 cells. The overlap reached 32% at low-stringency for E2 treated cells (see Fig. S6). To stress the robustness of our analysis, we performed ChIP -qP CR experiments on subsets of categorized ERBSs. Res ults obtained from these experiments are summarized in part of Fig. 6, and confirm that 100 % of the 4 lost and 4 gained tested ERBS recapitulate the expected profiles of enrichment. Interestingly, we also observed that the mobilization of ERα on common ERBSs located at the vicinity of E2 regulated genes such as GREB1 was also strongly reduced in MKL1 ΔN200 MCF7 cells. Similarly, these ChIP -qPCR experiments evidenced a reduced or absent recruitment of ERα on distant enhancers establishing chromatin loops (as extracted from the ChiA -PE T data from Fullwood et al., 2009 [50] ) with the promoter of the two genes which are still regulated by E Rα in MCF7 c ells expressing the constitutive version of MKL1: GPR68a and IFITM10a.

To assess whether this reprogramming of ERα binding has a direct functional influence on MCF7 cell biology, we checked whether genes putatively regulat ed by these ERBSs share specific or similar annotations. We therefore applied GREA T on our data (http://great.stanford. edu/ ) [START_REF] Mclean | GREAT improves functional interpretation of cis-regulatory regions[END_REF], and found a number of associations of the E Rα cistrome in MKL1 ΔN200 MCF7 c ells with genes involved in E GF and RAS pathways. This was especially striking in absence of hormone (see Fig. S7) and congruent with our previous observations that MAP kinase signaling pathway is boosted in MKL1 ΔN200 MCF7 cells [START_REF] Kerdivel | Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen -responsive breast cancer cell lines[END_REF] (Fig. S1B).

Therefore, in regard of these results, we analyzed by quantitative RT-PCR the expression profile of some E2-regulated genes from the clusters C1, C2 and C4 in MCF7 MKL1 ∆N200 and control cells after inhibition of HE R1 pathway by the erlotinib drug. As shown in Fig. S8, the expression of the tested genes of cluster C2 (EGR3 and AREG) was clearly inhibited in both cell lines after erlotinib treatment. However, it should be noted that erlotinib does not restore E2-dependent gene regulation and sensitivity to antiestrogen in MKL1 ∆N200 MCF7 cells.

We next investigated whet her these s pecific cistromes could reflect the engagement of E Rα on different type of DNA sequences. As visible from the motifs analysis summarized within Table S11, EREs are the first motifs found in each cistromes except within sites bound by ERα in the absence of E2 in MKL1 ΔN200 MCF7 cells. This again points to the conclusion that the very limited cistrome of ERα in this condition has probably specific properties. Accordingly, whilst the overlap of our ERα cistromes with the bindin g sites of other transcription factors determined in MCF7 cells (references in Table S12) did not evidence the existence of a preferential cofactor for t he specific ERBSs from MKL1 ΔN200 MCF7 cells. Motifs recognized by proteins allowing an indirect recruitment of ERα such as AP1 (JUN/FOS) were also identified in these cells. However, the ERα cistrome in these cells in the absence of E2 was found particular as it did not cluster with the others (Fig. 5C). It can be noted that the ERBSs of these untreated cells present a depletion of GATA 3 binding sites and the maintenanc e of enrichment in FOXA 1. We further evaluated the overlap of the ERBSs specifically lost or created in MKL1 ΔN200 MCF7 cells with the cistromes of other transcription factors and some chromatin marks determined by others in MCF7 cells. Interestingly, these analyses summarized within the Fig. S9 indicated that the novel E RBSs may exhibit a relative open chromatin conformation as revealed by a little accessibility to DNase I digestion in MCF7 c ells. However, these sites may not be fully functional since they are not overlapping with FOXA1 or GATA 3 cistromes (Fig. S9). Since MKL1 is a coactivator of SRF, we envisioned that the specific ERBSs from MKL1 ΔN200 MCF7 cells could be enriched in SRF binding sites. However, our motif analysis and search using known PSSM matric es (data not shown) showed that it is not the case.

These conclusions led us to hypothesize that the loss of any estrogenic regulation in the MK L1 ΔN200 cells could be a consequence of the impacted mobilization of ERα onto chromatin in the abs ence of ligand.

We therefore interrogat ed the ERα cistromes characterized in control and MKL1 ΔN200 MCF7 cells to determine whether they may correlat e with spec ific variations of gene basal activities preventing their estrogenic response. To do so, we first identified the closest ERBS from the TSS of the genes included in the 6 categories previously defined (Fig. 4B and4C). As shown within Fig. 5D, there was no obvious correlation between the variations in the basal transcriptional activity of a given gene category and either the conservation/gain or loss of their most proximal ERBS. For instance, genes with increas ed (C1 and C2) or decreased (C3 and C4) basal activity did not present coordinated changes in E RBS proximity significantly different from control genes with no changes in basal activity (C5 and C6). On the other hand, genes from the C2 and C4 categories of genes up-regulated by E2 in control cells seemed to conserve their more proximal E RBS in MKL1 ΔN200 MCF7 cells more frequently than the other categories. In the opposite, down-regulat ed genes (C1, C3 and C6) have a tendency to gain an ERBS at their vicinity. These observations led us to check whether the levels of mobilization of E Rα rather than the existence of the E RBS in itself could correlate better with the transcriptional activity. We therefore retrieved the mean ChIP -Seq signal from the conserved proximal ERBSs of the categorized genes, as well as the lost ones as a control. These analyses, summarized within Fig. 5E indicate t hat the mobilization of ERα on all cons erved E RBSs from all categories is significantly reduced in the presenc e of E 2 in MKL1 ΔN200 MCF7 cells. This is also the case in the absence of E2, except in the C6 category.

In summary our comparative analysis of E Rα cistrome in MCF7 and MCF7 MKL1 Δ N200 cells indicates that ERα binding on the MCF7 cell's genome is strongly impaired by the cellular context imposed by the expression of the mutant MKL1 protein. This reprogramming involves a massive loss of ERBSs and a diminished recruitment of E Rα on conserved ERBSs that may explain at least partly the loss of estrogenic transcriptional regulations. This is certainly partly associated with the loss of ERα protein amounts in MKL1 ΔN200 cells, but novel ERBSs also appear in these cells although their association with a particular physiological function remains unclear except for the EGF and RAS pathways. Columns with different superscripts differ significantly (p<0.05, Student 's t-test). NA: n too low.

Journal Pre-proof

ERBSs enrichment in H3K27ac is altered in MCF7 MKL1 ∆N200 cells

We reasoned that the novel ERBSs identified in MCF7 MKL1 ΔN200 might not be functional in terms of chromatin remodeling required for transcriptional modulations. We therefore determined whet her these ERBSs and variations in ERα mobilization on E RBSs in MCF7 MKL1 ∆N200 cells were associated with changes in chromatin structure around enhancers. We investigat ed the enrichment of ERBSs in dimethylated H3K4 (H3K 4me2) and ac etylated H3K27 acetylation (H3K27ac) in response to estrogenic treatment. Both chromatin marks are present at active enhancers and promoters, and H3K 27ac is a major histone modification deposited at nucleosomes flanking enhancer elements by coactivators with histone acetyl activity (HA T) recruited by ERα and other transcription factors [START_REF] Calo | Modification of enhancer chromatin: what, how, and why?[END_REF]. ChIP -qPCR experiments were performed on the same subs ets of categorized ERBSs and with the sam e chromatin samples previously used to confirm ERα recruitment. As illustrated within the heatmap in Fig. 6A, the enrichment of all tested ERBSs in H3K4me2 was found independent of E2, as expected, in both cell lines. Importantly, the H3K4me2 levels were significantly lower in MCF7 MKL1 ∆N200 cells on the tested lost ERBSs (see Fig. 6B for the mean signals and statistics). In contrast, gained E RBSs did not present a differential enrichment in H3K4me2 depending upon the cell and/or treatment. This suggests that the gained ERBSs are genomic regions which are already poised for functionality in the absenc e of E Rα, at least for those we tested. This was in line with the observed little overlap of these novel E RBSs with regions determined as having an open chromatin in native MCF7 cells (Fig. S9). H3K27ac enrichment at ERBS was extremely different between both cell lines with a clear decrease in common ERBSs, which was amplified in lost ERBSs. Importantly, although already present in MCF7 control cells within gained ERBSs there was a significant increase in the amounts of H3K27ac at these regions in MCF7 MKL1 ∆N200 cells. Interestingly, although insensitive to E2 in tested lost and gained E RBSs, 6 of the 7 common tested ERBSs showed an increased enrichment in H3K27ac in MCF7 MKL1 ∆N200 cells following E2 treatment, leading to a partial rescue of the levels of its enrichment when compared to control MCF7 cells (Fig. 6A and6B).

Altogether, these data indic ate that gained ERBSs are, for the ones tested, already in a poised/prepared state and that the nuclear accumulation of the constitutively active form of MKL1 disturbs the H3K27 acetylation of ERBSs in a close association with the provoked changes in ERα mobilization.

Discussion

Thirty to 50% of recurrent tumors that arise from ERα-positive primary breast cancer patients fail to respond to endoc rine therapy [START_REF] Jordan | Selective estrogen-receptor modulators and antihormonal resistance in breast cancer[END_REF][START_REF] Clarke | Endocrine resistance in brea st cancer--An overview and update[END_REF]. While the majority of these breast tumors retain ERα expression, high reduction or loss of ERα expression represents a non-negligible step in the progression from endocrine sensitive to resistance [START_REF] Kuukasjärvi | Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy[END_REF]. Notably, luminal B breast cancers, which generally express lower level of ERα, exhibit worse response to endocrine therapy. Understanding the underlying mechanisms of this progression is a major challenge towards the design of efficient hormone therapy -based treatments of breast cancers with reduced or ideally no relapses. We previously examined the processes controlling the tissue specific activity of ERα and identified the Rho/actin/MKL1 signaling pathway as a main actor, able to strongly inhibit the transcriptional capability of the receptor [START_REF] Huet | Repression of the estrogen receptor-alpha transcriptional activity by the Rho/megakaryoblastic leukemia 1 signaling pathway[END_REF]. MKL1 is a master regulat or of actin dynamic and cellular motile functions in many processes [START_REF] Miralles | Actin dynamics control SRF activity by regulation of its coactivator MAL[END_REF][START_REF] Pipes | The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis[END_REF]. In breast tissue, MKL1 is vital in sustaining differentiation and function of mammary myoepithelial cells, accountable for ejection of the milk during lactation [START_REF] Li | Requirement of a myocardin -related transcription factor for development of mammary myoepithelial cells[END_REF][START_REF] Sun | Acute myeloid leukemia -associated Mkl1 (Mrtfa) is a key regulator of mammary gland function[END_REF]. During tumor development, MLK1 can also promote malignancy by enhancing tumor cell invasion and metastatic dissemination [START_REF] Medjkane | Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis[END_REF]. We demonstrate in the present study that while MK L1 remains mainly cytoplasmic in estrogen-responsive, ERα-positive breast cancer cell lines, its nuclear localization is associated with basallike phenotype in breast cancer cell lines and with endocrine resistance in a c ohort of breast canc ers. We further show that a provoked nuclear accumulation of MKL1 in ERα-positive breast cancer cells results in a genetic and phenotypic reprograming of luminal cancer cells to a mixed luminal/basal phenotype, conferring changes in ERα activity and the development of hormonal resistance.

As expected, the expression of a constitutively active form of MKL1 in MCF7 cells modulat ed the expression of numerous genes whose ontologies are associated with pathways involved in the regulation of actin cytoskeleton, focal adhesion, ECM-recept or interaction and cell migration. Importantly, the evaluation of the expression of subset of genes associated wit h specific breast cancer subgroup gene signature showed that the MKL1 ∆N200 expression triggered a partial shift from a luminal t o a basal-like gene expression Journal Pre-proof J o u r n a l P r e -p r o o f profile. Notably, the expression of the pioneer factor GA TA 3 and ERα were down-regulated whilst that of the transcription factor FOXC1, a pivotal diagnostic marker for bas al-like breast cancer [START_REF] Ray | FOXC1 is a potential prognostic biomarker with functional significance in basal -like breast cancer[END_REF], was induced. Of interest, FOXC1 is localized to the basal/myoepit helium in normal breast tissue and is particular ly enriched in the luminal progenitor cell population, which is thought to be at the origin of bas al -like tumors [START_REF] Sizemore | FOXC1 is enriched in the mammary luminal progenitor population, but is not necessary for mouse mammary ductal morphogenesis[END_REF]. Markedly, the ectopic expression of FOXC1 was shown to induce a progenitor -like phenotype in differentiated mammary epithelial cells. Likewise, FOXC1 expression was associated wit h dec reased or undet ectable E Rα expression in recurrent tumors from ERα-positive primary breast cancers, which were treated with endocrine therapy [START_REF] Yu-Rice | FOXC1 is involved in ERα silencing by counteracting GATA3 binding and is implicated in endocrine resistance[END_REF]. Amongst the possible mechanisms involved in t his silencing of ERα expression, FOXC1 was shown to counteract GATA 3 binding on ERα promoter region [START_REF] Yu-Rice | FOXC1 is involved in ERα silencing by counteracting GATA3 binding and is implicated in endocrine resistance[END_REF]. The switch between GA TA3 and FOXC1 hence appears to be a good indicator of breast cancer luminal to basal-like reprograming [START_REF] Yu-Rice | FOXC1 is involved in ERα silencing by counteracting GATA3 binding and is implicated in endocrine resistance[END_REF][START_REF] Tkocz | BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal -like breast cancers[END_REF]. Unlike GATA 3 and ERα, FOXA1 not only remained expressed but even increased in MKL1 ∆N200 MCF7 cells. This may appear paradoxical because FOXA1 is generally associated, as GATA3 and ERα, with the luminal subtype [START_REF] Jozwik | Pioneer factors in hormone-dependent cancers[END_REF]. However, recent studies evidenced an overexpression of FOXA1 in breast cancer metastases that are resistant to endocrine therapy, suggesting an altered role of FOXA 1 in disease progression [START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF]. The presence of FOXA1 might also be related to the fact that the transition of MKL1 ∆N200 MCF7 cells from a luminal to a basal -like phenotype is not complete. Accordingly, we observed that MKL1 ∆N200 MCF7 cells still express some luminal markers such as cytokeratin 18. Interestingly, recent studies show that some luminal cell populations in normal human breast express myoepithelial/basal-like markers, whic h makes classification even more difficult [START_REF] Santagata | Taxonomy of breast cancer based on norm al cell phenotype predicts outcome[END_REF].

Our microarray data further showed that the expression of MKL1 ∆N200 in MCF7 cells almost abolishes all E2-mediated transcriptional modulations. Of the 225 E2 -regulated genes identified in MCF7 control cells, only two retained an E2-regulation in MKL1 ∆N200 MCF7 cells. The loss of E2-regulation was concomitant with a major overhaul in the basal expression of genes. For 28% of these genes, the change in their basal activity was in the same direction as the E2 -response. Among these genes, we found AREG (amphiregulin), the main growth factor mediating E2 -driven epithelial proliferation in a parac rine fashion [START_REF] Mcbryan | Amphiregulin: role in mammary gland development and breast cancer[END_REF].

AREG was also shown to be involved in breast cancer progression, contributing to cell motility and invasion.

The sharp increase of its expression in MKL1 ∆N200 MCF7 cells in a constitutive and E2-independent manner, associated with a higher ex pression of HE R1 obviously contributes to the hormonal escape of these cells. For other E2-responsive genes such as the chemokine C XCL12, the changes in their basal expression were opposite to their normal E 2-regulation. Here again, a shutdown of CX CL12 ex pression associated with a high expression of its receptor CXCR4, as observed in MKL1 ∆N200 MCF7 cells, is known to favor metastasis, cancer cells migrating in vivo to organs that express high level of CXCL12 [START_REF] Luker | Functions of CXCL12 and CXCR4 in breast cancer[END_REF]. Importantly, we did not observe any obvious correlation between the directions of changes of basal gene expression and the directions of E2-responses (up vs. down) for 72% of the genes that lose their responsiveness to the hormone. This point allows us to almost exclude the hypothesis that an increased ligand-independent activity of E Rα on these genes might be the cause for a loss of their regulation by the hormone. Furthermore, we showed that changes in the basal expression of E2 target genes in MK L1 ∆N200 MCF7 cells were also insensitive to tamoxifen and ICI treatments, demonstrating unambiguously that the presence of a constantly active MKL1 in MCF7 induces ER-independent endocrine resistance.

ChIP-Seq experiments performed on endocrine resistant breast cancers and cell lines that retained ERα expression showed a clear increase in both number of ERBSs and the intensity of ER binding to these genomic regions [START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF]. Notably, the highest ERα chromatin binding signal intensities were observed in metastatic samples, suggesting a correlation with disease progression [START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF]. The estrogen responsive element (E RE) was the main DNA motif enriched in the E RBSs. It should be noticed that ligand -free constitutively activated mutant forms of ERα are often reported in endocrine resistant breast cancer metastases [START_REF] Jordan | Estrogen receptor mutations found in breast cancer metastases integrated with the molecular pharmacology of selective ER modulators[END_REF]. Because of the high ERα expression, these endocrine resistant cancer cells still remain in luminal breast cancer subtypes. Our data show that endocrine resistant breast cancer cells , which result from a massive MKL1 nuclear translocation, exhibit in contrary a reduced number of ERBSs generally associated with lower ERα-binding intensity. Besides a reduced overlap between the E Rα cistromes identified in control and MKL1 ∆N200 MCF7 cells, we also identified novel ERBSs in cells expressing the MKL1 ΔN200 protein. This was particularly true in untreated cells for which the overlap was very weak. Interestingly, a number of genes located in the vicinity of these new ERBSs were associated with growth factor pathways, notably in untreated MKL1 ΔN200 MCF7 cells. This could be related to the sharp increase of expression of members of the EGF family (AREG as mentioned above, but also TGFα and HB-EGF) and their recept or in t hese cells. Interestingly, we s how that the HE R1 inhibitor erlotinib down regulates AREG expression suggesting the existence of a positive self-regulation loop in MCF7 between HER1 and its ligand, which becomes constitutive and E2-unregulated after nuclear translocation of MKL1 in the cells. Ross-Innes et al [START_REF] Ross-Innes | Differential oestrogen receptor binding is associated with clinical outcome in breast cancer[END_REF] previously showed that ERα positive breast cancer cell lines treated with a mitogenic cocktail present novel ERBSs at chromatin sites already bound by FOXA 1 or to which FOXA 1 was recruited in response to mitogenic stimulus. This phenomenon was also observed in ERα positive cancers associated with poor prognosis or in ERα positive metastases. This supports the hypothesis that FOXA1 might mediate ERα-binding reprogramming in advanced dis eases. Partially corroborating the hypothesis, the comparison of the new ERBSs identified in untreated MKL1 ΔN200 MCF7 cells with FOXA1 binding sites determined by ChIP-Seq in MCF7 show maintenanc e of FOXA1 enrichment. These gained ERBSs were als o found to be contained within genomic regions exhibiting histone marks of chromatin poised for functionality. In contrast, depletion in GA TA3 binding to chromatin was observed at these sites. Importantly, the reprogramming of ERBSs in untreated MKL1 ΔN200 MCF7 cells is therefore closely associated with the changes obs erved in the expression level of the two pioneer factors in these cells: down-regulation of GATA3 and up-regulation of

  Fig S1A, MKL1 is almost cytoplasmic and inactive in luminal ERα-positive breast cancer cell lines such as MCF7, T47D or ZR-75-1, which exhibit a well-differentiated epithelial phenotype. In the non-t umorigenic epithelial cell line MCF10A, the vast majority of cells exhibit a cytoplasmic localization of MKL1. Only cells on islet periphery present a nuclear translocation of MKL1. Similarly, a slight onset of nuclear accumulation of MKL1 is observed in the endoc rine resistant ERα-positive breast cancer cell line expressing a constitutively active form of AKT (MCF7 AK T+). Finally, all cell lines having an epithelial-mesenchymal transition (EMT) phenotype such as MDA -MB-468, MDA-MB-231 or SUM159P T display MKL1 nuclear localization (Fig. 1A). While MKL1 has a different localization in these different cell lines, its expression level measured by Western blot remains similar (Fig S1B). The invasive capability of the different cell lines was then tested on 3D cell culture using a Matrigel Invasion assay (Fig. 1B). Results clearly show a positive correlation between the nuclear localization of MKL1 and invasiveness. As expected, the basal-like cell lines MDA-MB-231 and SUM159P T strongly invade the Mat rigel while ERα -positive breast cancer cell lines are noninvasive.

Fig. 1E and

 1E Fig. 1E and Fig S1C, the expression of this MKL1 Δ N200 mut ant triggered M atrigel invasion by MCF7 cells.This invasion was partly slowed down in presenc e of E2. Finally, transactivation efficiency of ERα was measured in MCF7 and T47D after either transiently expressing of MKL1 ΔN200 mutant or treating cells with

Fig. 1 .

 1 Fig. 1. Nuclear accumulation of MKL1 favors invasiveness and impairs the transactivation efficiency of E Rα. Were analyzed the non-tumorigenic epithelial cell line MCF10A, the ERα-positive breast cancer cell lines MCF7, T47D and ZR-75-1 (luminal A subtype), the ERα-positive breast cancer cell line MCF7 expressing a constitutively active form of AK T (endocrine resistant cell line) and the triple negative breast cancer cell lines MDA-MB-468, MDA-MB-231 and SUM159P T (basal-like subtype). (A) MKL1 expression and localization were analyzed by immunofluorescence in the different cell lines and the percent age of cells with nuclear MKL1 staining was then quantified (mean values from at least 30 images ± SEM). Columns with different superscripts differ significantly (p< 0.01, student 's t-test). (B) A 3D spheroid cell invasion assay in Matrigel

Fig. 2 .

 2 Fig. 2. Nuclear accumulation of MKL1 is associated with endocrine resistance in breast cancers. (A) Detection of E Rα and MKL1 proteins by immunoflorrescence in healthy and E R-positive grade III breast cancer cells. In healthy tissue, ERα is expressed in few luminal cells while MKL1 is located in myoepithelial cells. In the tumor, some breast cancer cells co-expressed ERα and MKL1 proteins. Nuclei were stained with DAPI. (B) Representatives images of MKL1 immunohistochimical staining across different human tumor samples. (a) refers to as a MKL1 negative sample, (b) shows a cytoplasmic localization of MKL1, therefore considered as MKL1 nuclear negative staining (c) shows a weakly fraction of MKL1 nuclear positive (NP) cells, therefore also considered as MKL1 negative, (d) shows a MK L1 nuclear positive staining. (C) Quantification of MKL1 NP cells per breast cancer subtype, compared to adjacent non tumor tissue. *p -value <0.05, with a student t-test compared to the control, ns: non-significant. (D) MKL1 nuclear staining according to hormone therapy responsiveness status. ER𝛼+ PR+/-(HER2 -) tumor samples were subdivided into two categories according to MKL1 staining. Samples harboring more than 30% of MKL1 NP cells were considered as MKL1 high whereas samples harboring less than 30% of MKL1 NP cell s were considered as low or negative. Hormone refractory status comprises both innate and acquired lack of responsiveness to hormone therapy. *p-value <0.05, with a Chi-square test.

Fig. 3 .

 3 Fig. 3. Nuclear accumulation of MKL1 in MCF7 cells induc es a mixed luminal /basal phenotype. The transcriptional profiles of control and MKL1 ΔN200 MCF7 cells were compared through microarray analysis. (A) Enriched K egg pat hways in our dat aset are shown alongside the differentially expressed genes [showing P<0.01 and abs(FC)> 4] involved. Genes are colored according to their log2FC expression values. (B) Immunofluorescence pictures of FOXC1, ERα, GATA3, FOXA1 and P300 after 48 hours of tetracycline treatment of control and MKL1 ΔN200 MCF7 cells. Densitometry quantification of the immunofluorescence expressed as percentage of the intensity measured in control MCF7 cells is shown on the left sid e of the panel. Error bars represent SEM (n ranges from 10 to 20; P<0.01, Student 's t-test).

Fig. 4 .

 4 Fig. 4. E2 modulation of transcription is abrogated in MK L1 Δ N200 MCF7 cells. E2-regulat ed genes were identified in control and MKL1 ΔN200 MCF7 cells through microarray analysis. Cells were treated for 4h with 10 nM E 2 or EtOH as a control. (A) Venn diagram of E 2-regulated genes [showing P< 0.05 and abs(FC)> 1.8] in control and MKL1 ΔN200 MCF7 cells. (B) The 225 E2-regulated genes [P<0.05 and abs(FC)> 1.8] were classified into 6 clusters (C1 to C6) according to the variations in their basal activity in MKL1 ΔN200 MCF7 cells [P<0.01 and abs(FC)> 4]. Box plots represent the average expression level of the genes for each category in cont rol and MKL1 ΔN200 MCF7 cells, treated or not with E2. (C) For the 109 genes that lose their res ponse to E2 and presented significant variations in their basal activity in MKL1 ΔN200 MCF7 cells, we represented their changes in expression against their fold change in expression by E2 in control cells (C1-C4).

Fig. 5 .

 5 Fig. 5. ERα cistrome is affected by the expression of MKL1 ΔN200 protein in MCF7 cells. (A) Venn diagrams summarizing the overlaps of genomic regions bound by ERα in control MCF7 cells or MCF7 cells expressing the MKL1 ΔN200 following a 50 min treatment with either E2 or ethanol (EtOH) as vehicle control, as determined from ChIP -Seq experiments. (B) The ERα ChIP-S eq signals were aligned and averaged wit hin a -2.5/+2.5 kbp window centered on E RBSs belonging t o categories illus trated in the Venns on the upper panel (A). Upper and lower panels correspond to specific ERBSs to each of the two populations analyzed on the venn diagrams while the middle panel corresponds to overlap genomic regions of ERα binding. NA stands for non-applicable, since only one ERBS is common between the ERα cistromes determined in the two cell lines in the absence of E2. (C) Hierarchical clustering of the % of overlap of E Rα cistromes in control and MKL1 ΔN200 MCF7 cells with the binding sites of other transcription factors or enriched region of histone marks previously determined in MCF -7 cells (see references in Table S 11). (D) To correlate this change of ERα cistrome and our transcriptome analysis, we first identified the closest ERBSs from the TSSs

Fig. 6 .

 6 Fig. 6. Chromatin status of ERBSs in c ontrol and MK L1 Δ N200 MCF7 c ells. (A) Heatmap summarizing data obtained in ChIP-qP CR experiments that evaluated ERα recruitment and H3K4me2 and H3K27ac enrichments on 7 genomic regions bound by ERα in both control and MKL1 ΔN200 MCF7 cells as well as on 4 lost and 4 gained E RBSs in MKL1 ΔN200 MCF7 cells. Amongst the common ERBSs, we evaluated ERα binding and chromatin modifications occurring at regions located within the promot er of the model estrogenresponsive GREB1 gene (GREB1 p) and two enhanc ers regulating GPR68A or IFITMA genes. Cells were

FOXA1.

  E2 treatment allows MKL1 Δ N200 MCF7 cells to regain enrichment profiles closer to those observed in c ontrol cells. The exact functional consequence of E RBS reprogramming in cells expressing the constitutively active form of MK L1 remains however poorly understood due to the lack of E2 transcriptional regulation in these cells. Collectively, our study offers new mechanistic insights into ERα functional changes engaged during breast cancer progression particularly involving a nuclear accumulation of MKL1. Our work also implicates the targeting of the nuclear location of MKL1 as a potential therapeutic strategy for the treatment of endocrine-resistant and recurrent breast cancer.

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Acknowledgements

We thank the biosit Health genomics and H2P 2-Histo pat hology High precision core facilities of biogenouest and the IGBMC Micrroarray and Sequencing plat form at Illkrich. This work was supported by the Uni versity of Rennes 1, Inserm, CNRS, and the Ligue Contre le Cancer.

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Competing interests

The authors declare that they have no competing interests.

Suplementary data

Supplementary figures and part of supplementary tables: