Cancer cell motility and invasiveness are fundamental characteristics of the malignant phenotype and are regulated through diverse signaling networks involving kinases and transcription factors. This study establishes an estrogen receptor (ERα)/MAPK (ERK5)/cofilin (CFL1) network that specifies the degree of breast cancer cell aggressiveness through coupling of actin reorganization and hormone receptor–mediated transcription. Using dominant negative and constitutively active forms, as well as small-molecule inhibitors of extracellular signal–regulated kinase (ERK)5 and MAP–ERK kinase (MEK)5, it was revealed that hormone activation of ERα determined the subcellular localization of ERK5, which functions as a coregulator of ERα-dependent gene transcription. Notably, ERK5 acted in concert with the actin remodeling protein, CFL1, and upon hormone exposure, both localized to active nuclear transcriptional hubs as verified by immunofluorescence and proximity ligation assays. Both ERK5 and CFL1 facilitated PAF1 recruitment to the RNA Pol II complex and both were required for regulation of gene transcription. In contrast, in cells lacking ERα, ERK5 and CFL1 localized to cytoplasmic membrane regions of high actin remodeling, promoting cell motility and invasion, thereby revealing a mechanism likely contributing to the generally poorer prognosis of patients with ERα-negative breast cancer. Thus, this study uncovers the dynamic interplay of nuclear receptor–mediated transcription and actin reorganization in phenotypes of breast cancer aggressiveness.
Implications: Identification of the ER/ERK5/CFL1 axis suggests new prognostic biomarkers and novel therapeutic avenues to moderate cancer aggressiveness. Mol Cancer Res; 12(5); 714–27. ©2014 AACR.
This article is featured in Highlights of This Issue, p. 637
Elucidation of the factors and networks that regulate cancer cell motility and invasiveness is fundamental to understanding the malignant phenotype and may also highlight biomarkers of disease and reveal opportunities for the development of novel targeted therapies to moderate cancer aggressiveness. The nuclear hormone receptor, estrogen receptor alpha (ERα), present in two thirds of human breast cancers, is a master regulator of the phenotypic properties of these cancers. It is considered the single most crucial predictor of breast cancer prognosis and is targeted by endocrine therapies, which are generally well tolerated and avoid the morbidity associated with radiation and chemotherapy (1). Molecular subtyping of breast cancers has revealed that ERα-containing tumors are generally less aggressive and that patients with ER-positive cancers have a better prognosis and overall survival. Although many studies have documented that ER directly regulates more than 3% of protein-encoding genes and indirectly regulates many more (2–6), the mechanisms by which this hormone-regulated transcription factor controls cell phenotype and reduces cell invasiveness remain unclear. To address this, we have examined the involvement of protein kinases in modulating ER activity.
The importance of kinases in cancer biology is well known, as increased kinase activity through phosphorylation, mutation, or elevated expression is often observed in tumors and is associated with a less good clinical outcome for patients with breast cancer (7–11). However, the cellular processes underlying the interplay between ERα and protein kinase pathways and the manner by which ERα maintains and controls these pathways and their phenotypic outcomes are poorly understood. Our previous genome-wide analyses revealed the importance of the mitogen-activated protein kinase (MAPK) signaling pathway and the involvement and direct binding of extracellular signal–regulated kinase (ERK)2 along with ERα at enhancers of many estrogen-regulated genes that control cell proliferation (12).
In the current studies, we have identified the protein kinase ERK5 and Cofilin (CFL1), an actin-severing protein required for actin cytoskeleton reorganization (13), as two interacting factors that are moved into the nucleus and recruited to the transcription start site (TSS) of estrogen-stimulated genes upon hormone treatment of ERα-containing breast cancer cells. Notably, in breast cancer cells that lack ERα, ERK5 and CFL1 remain outside the nucleus and increase cell motility and invasiveness. Thus, by eliciting nuclear localization of ERK5 and CFL1, thereby diminishing their colocalization to regions of high actin remodeling, ERα is playing a critical role in maintaining the lower metastatic activity characteristic of many ERα-positive breast cancers. These novel findings reveal a transcription factor–mediated regulatory mechanism that modulates cancer cell aggressiveness, through relocalization of two key factors, ERK5 and CFL1, highlighting the crucial cross-talk between ERα-driven nuclear events and the actin cytoskeleton and suggesting alternative opportunities for targeted therapies.
Materials and Methods
Cell culture, siRNA, adenovirus, and ligand treatments
MCF-7, BT474, T47D, MDA-MB-453, MDA-MB-468, and SKBR3 cells were obtained from and grown as recommended by the American Type Culture Collection. For experiments with 17β-estradiol (E2) treatment, cells were maintained in the corresponding phenol red–free medium for at least 5 days before use. Cell lines were authenticated by checking ERα expression by quantitative real-time PCR (qRT-PCR) and Western blot analysis, checking responsiveness to E2 in ERα(+) cell lines and monitoring morphology of the cells (compared with the images reported by ATCC). Recombinant adenoviruses were obtained from Applied Biological Materials and used to generate cells expressing dominant negative ERK5 (ERK5-DN) or dominant negative MEK5 (MEK5-DN). ERK5 knockdown used the smart pool of four siRNAs from Dharmacon and were performed as described (12) with 20 nmol/L siCtrl or siERK5 for 72 hours, and resulted in knockdown of ERK5 mRNA and protein by greater than 80%.
Western blotting, chromatin immunoprecipitation (ChIP), and ChIP-Re-ChIP assays
Western blot analysis used specific antibodies for ERα (HC-20; Santa Cruz Biotechnology); ERK2 (D-2; Santa Cruz Biotechnology); pERK5 (Upstate Biotechnology; 07-507); and total ERK5 (Bethyl Laboratories; A302-655A) and was performed as described (14).
ChIP was carried out as described (15, 16). Antibodies used were for pERK5 (Upstate Biotechnology; 07-507, sc-135760), phospho-PXSP motif (2325; Cell Signaling Technology), CFL1 (D3F9) rabbit (5175S; Cell Signaling Technology), pSer5 RNA Pol II (Santa Cruz Biotechnology; sc-47701), PAF1 (sc-74795), SPT5 (sc-28678), Cyclin T1 (sc-8127), and WDR61 (sc-100897). ChIP DNA was isolated using the Qiagen PCR Purification Kit and used for ChIP-seq analysis and qRT-PCR. ChIP-re-ChIP experiments were done following the same ChIP protocol. After the first pull down, immunoprecipitated material was recovered with 10 mmol/L dithiothreitol in immunoprecipitation buffer at 37°C for 30 minutes, diluted, and submitted to a second round of immunoprecipitation. qRT-PCR was used to calculate recruitment to the regions studied, as described before (16).
ChIP-seq analysis and clustering
The ChIP DNA was prepared into libraries according to Illumina Solexa ChIP-seq sample processing methods, and single-read sequencing was performed using the Illumina HiSeq 2000 (15). Sequences generated were mapped uniquely onto the human genome (hg18) by Bowtie2 (17). MACS (model-based analysis of ChIP-Seq) algorithm (18) was used to identify enriched peak regions (default settings) with a P value cutoff of 6.0e−7 and false discovery rate (FDR) of 0.01.
The seqMINER density array method with a 500-bp window in both directions was used for the generation of clusters, i.e., groups of loci having similar compositional features (15, 19). BED files for each cluster were used for further analysis with Galaxy cistrome (20) integrative analysis tools [Venn diagram, conservation, Cis-regulatory element annotation system (CEAS)].
GeneChip mRNA transcriptional profiling microarrays
Total RNA was used to generate cRNA, which was labeled with biotin as recommended by Affymetrix. The biotin-labeled cRNA was hybridized to Affymetrix U133 plus 2.0 GeneChips and analyzed using Affymetrix processing software. CEL files were processed using GeneSpring GX 11.0 software (Agilent) to obtain fold-change and P value with the Benjamini and Hochberg multiple test correction for each gene for each treatment relative to the vehicle control (15). We considered genes with fold-change >1.8 and P value < 0.05 as statistically significant, differentially expressed. Data are available from Gene Expression Omnibus (GEO) with the accession number GSE53394.
Motif and Gene Ontology category analysis
Immunofluorescence microscopy, proximity ligation assay, and data analysis
Cells treated with vehicle (0.1% EtOH) or 10 nmol/L E2 were washed in PBS, fixed on glass coverslips, and then incubated with antibodies against ERα (F10; Santa Cruz Biotechnology), CFL1 (Abcam; ab54532), pSer5 RNA Pol II (Santa Cruz Biotechnology; 47701), or PAF1 (sc-74795). Next day, cells were incubated with goat anti-mouse Alexa 568 or goat anti-rabbit Alexa 488 secondary antibodies. Slides were mounted and stained using Prolong Gold Antifade with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes) to identify the nuclei.
The proximity ligation assay (PLA) was performed using the Duolink In Situ kit (Olink Bioscience) according to the manufacturer's instructions. Briefly, cells were prepared for immunofluorescence analysis. Overnight incubation with primary antibodies was followed by hybridization with two PLA probes at 37°C for 1 hour, and then by ligation for 15 minutes and amplification for 90 minutes at 37°C. A cover slip was mounted on each slide and image acquisition and analysis were conducted. Samples were imaged using a 63×/1.4 Oil DIC M27 objective in a Zeiss LSM 700 or 710 laser scanning confocal microscope. Images were obtained in a sequential manner using a 488 Ar (10 mW) laser line for ERK5, pERK5, or PLA signal (500–550 nm emission) and 555 nm (10 mW) laser line for phalloidin, SC-35 or ERα (600–650 nm emission). The individual channels were obtained using a sequential scanning mode to prevent bleed-through of the excitation signal. Laser power, gain, and offset were kept constant across the samples and scanned in a high-resolution format of 512 × 512 or 1024 × 1024 pixels with 2/4 frames averaging. Further quantification of the images used Fiji software (http://fiji.sc/wiki/index.php/Fiji; ref. 22). Briefly, images were converted to 8 bit for segmentation for each channel, and images were background subtracted using a rolling-ball method, with a pixel size of 100 and segmented using the DAPI channel.
Cell proliferation, soft agar colony formation, migration, and invasion assays
For proliferation assays, cells were seeded at 1,000 cells per well in 96-well plates. On the second day, the cells were treated with Veh or 10 nmol/L E2 (day 0) in the presence or absence of 1 μmol/L XMD8-92 or 10 μmol/L BIX-02189 and again at day 2. Proliferation was assessed using WST-1 reagent (Roche) as described (23). Invasion assays used BD BioCoat Matrigel Invasion Chambers (BD Biosciences) with 10% FBS as chemoattractant in the lower chamber as described (23). Motility and soft agar colony formation assays were performed as described (7, 23).
Tumor data sets and data analysis
Log2 median-centered intensity expression values for signature genes were obtained from the Oncomine database. Hierarchical clustering of data was performed and displayed using Cluster3 and Java TreeView software for analysis and visualization. Patients were stratified according to average expression value of the genes in the signature and the top 30% and bottom 30% were used for computation of Kaplan–Meier curves by the Cox–Mantel log-rank test and the Gehan–Breslow–Wilcoxon test in GraphPad Prism.
ERK5 is a novel kinase recruited to the transcription start site of E2-regulated genes
To characterize MAPK signaling networks underlying ERα-mediated transcriptional regulation, we monitored MAPK activity on chromatin through ChIP assays by using an antibody that recognizes phosphorylated MAPK targets at the PXSP consensus motif (pMAPK substrates). Upon E2 treatment, pMAPK substrates were more highly enriched at the TSS of E2 target genes compared with the intragenic enhancers (Fig. 1A). Because we previously showed that ERK2 is responsible for activity at enhancers but not at the TSS (12), the data in Fig. 1A suggest that a different MAPK is responsible for phosphorylating targets at the TSS of E2-regulated genes.
ERK1/2, together with p38, c-jun-NH2-kinases (JNK), and ERK5 are members of the family of typical MAPKs. These kinases share high sequence homology at the N-terminal region (24, 25). Strikingly, however, ERK5 harbors a unique C-terminal transactivation domain and nuclear localization sequence (NLS; ref. 26; Fig. 1B). Therefore, we tested whether ERK5 was the kinase responsible for E2-mediated protein phosphorylation at the TSS of ER target genes by knocking down either ERK5 or ERK2 in MCF-7 cells. We did not observe any compensation between these two kinases after knockdown (Fig. 1C). ChIP assay demonstrated that, whereas ERK2 knockdown abrogated the increase in pMAPK substrates at the enhancer regions, ERK5 knockdown reduced them at the TSS of the LRRC54 gene (Fig. 1D and F, top). TFF1, which has a proximal promoter ERα binding site, showed decreased pMAPK substrate recruitment after knockdown of both kinases, consistent with the recruitment of ERα and RNA Pol II to this site (Fig. 1D and F, bottom).
We then compared the ChIP-seq cistromes for ERK5, ERK2, and pMAPK substrates after E2 treatment of cells to obtain a genome-wide characterization of the ERα/MAPK network (Fig. 1E). Nearly 30% of pMAPK substrate binding sites overlapped with pERK5 binding sites. Notably, less than 20% of pERK5 sites overlapped with ERK2 binding sites in the presence of E2 (Fig. 1E).
To obtain a better picture of the kinase landscape in the cells, we used a clustering approach using seqMINER (19), which identifies those binding sites that share a similar pattern of factor localization within a 1000-bp window (15). Comparison of pMAPK substrate, pERK5, RNA Pol II, ERK2, and ERα cistromes (Fig. 1G) revealed seven distinct clusters (C1–C7) showing different extents of conservation (Fig. 1H) and different genomic localization (Fig. 1I). For example, cluster 2 (C2) is characterized by increased pMAPK substrate, ERK5, and RNA Pol II recruitment, while lacking ERα and ERK2 binding; as expected, this cluster contained binding sites associated with the TSS of E2-regulated genes. Moreover, C2 had the highest conservancy score among the seven clusters.
As shown in Supplementary Fig. S1, we validated hormone-dependent recruitment of pERK5 to these binding sites by ChIP assays. Cluster C3, which exclusively contained ERα and ERK2 binding sites, mapped to enhancer regions of the E2-regulated genes and had the lowest conservation across vertebrates. Clusters C4 to C7 showed distinct patterns of factor binding. Thus, our clustering analysis revealed different modes of modulation of E2-mediated transcriptional events by ERK5 at the TSS and by ERK2 and ERα at enhancers.
ERK5 binds to the TSS of genes required for E2-mediated cell proliferation
To further elucidate ERK5 association with E2-mediated gene regulation, we compared the cistromes for pERK5, pMAPK substrates, and RNA Pol II in the presence and absence of E2, using the E2-induced pERK5 cistrome as the reference (Fig. 2A). This analysis revealed that there was minimal recruitment of pERK5, pMAPK substrates, and RNA Pol II before addition of hormone, suggesting that pERK5 marked E2-induced transcriptional events. Only at cluster C2 did we observe constitutive presence of RNA Pol II, possibly marking paused genes.
To understand the functional outcomes of ERK5 recruitment and its effect on E2-mediated cell processes, we performed gene expression microarray analysis in MCF-7 cells after ERK5 or control siRNA transfection. Hierarchical clustering of modulated transcripts revealed that E2 regulation of many genes was lost when ERK5 level was reduced (Fig. 2B). Of note, 296 genes that were stimulated with E2 treatment in siCtrl cells were no longer modulated when ERK5 was reduced, and by GO analysis these genes were associated with cell-cycle control (Supplementary Table S1 and Supplementary Fig. S2A, left). Interestingly, 277 new genes became regulated only in siERK5 cells, suggesting a repressive role for ERK5 in modulating transcription of these genes, which were associated with immune function and apoptosis (Supplementary Table S1). E2-mediated repression of 397 genes was lost with ERK5 knockdown, whereas 151 genes were now further repressed by E2 when ERK5 was knocked down (Supplementary Fig. S2A, right). We also generated scatter plots that showed that the direction of regulation by E2 did not change with depletion of ERK5, meaning that if a gene is regulated in both cell backgrounds, it will always change in the same direction (Supplementary Fig. S2B). Overall, this analysis revealed an activator role for ERK5 in control of genes regulating cell proliferation and a repressor role in processes associated with immune function and apoptosis.
To better correlate ERK5 binding with E2-mediated gene regulation, we generated BED files for the TSS of E2-regulated genes in siCtrl or siERK5 cells and monitored pERK5, pMAPK substrates, and RNA Pol II recruitment to these sites (Fig. 2C). Notably, ERK5 knockdown blocked regulation of 54% of E2-stimulated genes that had pERK5 binding at their TSS, emphasizing that recruitment and activity of ERK5 are good indicators of E2 target gene regulation.
Examination of ERK5-regulated genes and their association with breast cancer patient clinical outcome
To delineate the translational importance of ERK5-modulated genes, we monitored levels of these genes in large-scale breast tumor datasets. E2-upregulated genes that harbored pERK5 binding sites and that were blocked by ERK5 knockdown were associated with cell-cycle progression, specifically with the S phase of the cell cycle (Supplementary Table S1). Overexpression of these genes predicted a very poor outcome in 29 breast cancer datasets obtained from Oncomine. When we compared survival curves of patients with breast cancer stratified according to ERα status and expression of all the genes from our list (Supplementary Table S2), we observed that low expression of this gene signature predicted better outcome only in ERα-positive patients, consistent with the identification of these genes as being E2 modulated in MCF-7 cells. Analysis of expression of our ERK5 signature genes in two such large primary breast cancer datasets is shown in Fig. 2D. These findings emphasize the importance of ERK5 in E2-mediated regulation of genes in ERα-positive breast cancers and mark ERK5 transcriptional targets as a predictor of outcome in patients with ERα-positive breast cancers.
ERK5 signaling regulates hormone-dependent tumorigenicity of ERα-positive breast cancer cells
Because we observed association between ERK5 expression and E2-regulated genes linked to cell-cycle progression, we examined the impact of ERK5 on E2-stimulated cell proliferation in several ERα-positive breast cancer models. ERK5 was rapidly and transiently activated upon addition of E2 in all the three cell lines: MCF-7, T47D, and BT474 (Fig. 3A). Notably, the ERK5 small-molecule inhibitor (XMD8-92; ref. 27) and the upstream kinase MEK5 inhibitor (BIX-02189) completely blocked E2-mediated cell proliferation without affecting ERα protein levels (Fig. 3B and Supplementary Fig. S3). Moreover, in anchorage-independent growth assays, ERK5 inhibitor was very efficient in blocking E2-stimulated colony formation (Fig. 3C), suggesting a proproliferative role for ERK5 in ERα-containing breast cancer cells.
ERK5 kinase activity and activation by upstream MEK5 are required for the recruitment of ERK5 to chromatin and enrichment of pMAPK substrates at the TSS of hormone-regulated genes
To dissect the mechanism of ERK5 action, we knocked down ERK5 in MCF-7 cells and monitored the effect of this perturbation on the E2 stimulation of genes having ERK5 binding sites. ERK5 knockdown either completely abrogated or dampened E2 gene stimulation (Fig. 4A). To test the requirement of kinase activity of ERK5, or ERK5 activation by upstream MEK5 for kinase recruitment and gene activation, we infected MCF-7 cells with adenovirus containing a dominant negative ERK5 (ERK5-DN; Fig. 4B) or a dominant negative MEK5 (MEK5-DN; Fig. 4C) construct. With overexpression of either of these dominant negative kinases, we observed a dose-dependent decrease in gene stimulation, enrichment of pMAPK substrates, and pERK5 recruitment, implying that activation of ERK5 by MEK5 as well as ERK5 kinase activity are both required for chromatin-related functions of ERK5. Interestingly, overexpression of a constitutively active MEK5 was not sufficient to drive ERK5 to the TSS of E2-regulated genes or increase mRNA output of these genes, suggesting that ERα activation is required for ERK5 activity at the chromatin (Supplementary Fig. S4).
ERK5 localizes to transcription hubs in the nucleus upon cell treatment with E2
To further characterize the cross-talk between ERα and ERK5, we monitored by confocal microscopy the intracellular localization of total ERK5 and phosphorylated ERK5 in several ERα-positive breast cancer cell lines. In the absence of hormone treatment, ERK5 was localized throughout the cell, particularly outside the nucleus, but some nuclear-localized ERK5 appeared in a speckled pattern reminiscent of splicing factors and transcription hubs. This speckled pattern in the nucleus increased with time of E2 treatment, reaching maximum at 45 minutes (Fig. 5A). The same pattern was observed for pERK5, and it showed more clearly the E2-induced nuclear localization also peaking at 45 minutes (Fig. 5B). Because we observed a speckled nuclear pattern for both ERK5 and pERK5, we performed coimmunolabeling with antibodies for ERK5 and SC-35, a factor known to be part of splicing speckles and transcription hubs (28). Indeed, we observed that these two proteins colocalized in the nucleus after hormone treatment (Fig. 5C). In addition, we used a PLA (29) to assess interaction of ERK5 with several markers of transcriptional hubs. As seen in Fig. 5D, we detected an increase in PLA signal upon E2 treatment for interaction of ERK5 with SC-35, consonant with the colocalization findings, and also for ERK5 and pSer5-RNA Pol II, which is consistent with our ChIP-Seq data that showed overlap between RNA Pol II and ERK5 at the TSS of E2-regulated genes.
Nuclear localization of ERK5 is dependent on ERα, and CFL1 links the actin cytoskeleton to ERK5 and ER signaling in the nucleus
To test whether the presence of ERK5 in the nucleus is dependent upon ERα, we knocked down ERα and found that this abrogated the localization of pERK5 in the nucleus, supporting the idea that ERα is the main determinant of ERK5 nuclear localization in ERα-positive breast cancer cells (Fig. 6A and B and Supplementary Fig. S5). Of note, knockdown of ERα increased localization of pERK5 to actin bundles near the leading edge of MCF-7 cells, to the cell–cell interface in BT474 and to stress fibers in T47D cells, which are all actin-rich structures. These findings highlighted an interesting link between ERα, pERK5, and the actin cytoskeleton, and prompted us to investigate a possible role for actin reorganization modulator proteins in the ERα/ERK5 axis. We focused on CFL1, a member of the Cofilin/ADF family of actin-severing proteins, that is critical for formation of cell protrusions that adhere to the extracellular matrix (30, 31), define the direction of motility (31–33), and initiate cell crawling, leading to cell motility and increased invasiveness (34). We first monitored the intracellular localization of CFL1. PLA demonstrated an E2-dependent interaction of CFL1 and ERK5 (Fig. 6C). Immunofluorescence microscopy after treatment with the antiestrogen fulvestrant or ERK5 inhibitor XMD 8-92 showed nuclear localization of CFL1 to be ERα and ERK5 activity-dependent in ERα-positive cancer cells (Supplementary Fig. S6).
Very interestingly, ChIP assays demonstrated that active ERK5, active RNA Pol II, and CFL1 were all recruited to the TSS of E2 target genes with the same kinetics, and that this followed the pattern of enrichment of pMAPK substrates (Fig. 6D). ChIP-re-ChIP analysis showed that ERK5 and CFL1 are in the same complex that is recruited to the TSS but not to distal enhancers of ERα-regulated genes (Fig. 6E). PLA demonstrated that E2-mediated RNA Pol II interaction with ERK5, CFL1, or the transcription elongation factor PAF1 (polymerase associated factor-1) was dependent upon the presence of both CFL1 and ERK5 (Fig. 6F). Moreover, knockdown of CFL1 mimicked ERK5 knockdown in dampening E2 stimulation of target genes (Fig. 6G). To examine the function of ERK5 in E2-ERα–mediated transcriptional effects, we overexpressed ERK5-DN and compared recruitment of several factors involved in transcription (Supplementary Fig. S7). ChIP assays demonstrated that ERK5-DN overexpression caused a loss of E2-stimulated recruitment of PAF1 and an increase in chromatin recruitment of SPT5, a repressor of transcription elongation, whereas recruitments of RNA Pol II, WDR61, another component of the PAF1 complex, and the kinase Cyclin T1 were not affected. Thus, our findings uncover a novel mechanism for ERK5 and CFL1 function at chromatin, affecting transcription through recruitment of PAF1 and shedding of SPT5.
Expression of ERα in ER-negative cell lines induces nuclear localization of pERK5 and, coupled with ERK5 inhibition, abrogates cell migration and invasion
Because we observed an ERα-dependent switch of ERK5 intracellular localization in ERα-positive cells, we then performed the reverse experiments in ERα-negative cell lines. We introduced ERα by adenoviral infection into three ERα-negative breast cancer cell lines, MDA-MB-468, MDA-MB-453, and SKBR3. Introduction of ERα restored the nuclear localization of active ERK5 and localization of ERK5 to transcription factories in all three cell lines, while decreasing localization of ERK5 with F-actin in the cytoplasm (Fig. 7A and B and Supplementary Figs. S8 and S9). Moreover, we observed increased cell spreading, a cytoskeletal feature suggesting that ERα causes major rearrangements in the actin cytoskeleton. In monitoring the impact of ERα introduction and ERK5 inhibition on cell motility and invasiveness of these cells, we observed that ERα or ERK5-DN greatly impaired the migration and invasiveness of these breast cancer cells (Fig. 7C). Similarly, use of the ERK5 inhibitor XMD8-92 also blocked migration and invasion of all the three cell lines (Fig. 7D).
In this work, we have uncovered a previously unknown relationship between a nuclear hormone receptor (ERα) and the MAPK family member, ERK5, which, working along with cofilin, an actin remodeling protein, control cell migration and invasiveness. Intriguingly, our studies in cells containing or lacking ERα, revealed that hormone activation of ERα determines the intracellular localization of the kinase and cofilin, directing their binding at the transcription start site of estrogen-regulated genes to control gene expression and removing them from cytoplasmic regions of high actin remodeling, thereby reducing cancer cell motility and invasiveness. This dynamic interplay between nuclear receptor–mediated transcription and actin reorganization links these two key cell regulatory processes to phenotypes of cancer indolence or aggressiveness, and may underlie the generally lower metastatic potential and better prognosis characteristic of many ERα-containing breast cancers.
These findings are schematized in the model in Fig. 7E. In ERα-containing breast cancer cells, ERα is directly involved in modulating ERK5 and CFL1 intracellular localization, directing them for control of transcription of target genes together with basal transcription machinery and excluding them from their cytoplasmic prometastatic actions (Fig. 7E, left). This nuclear function of ERK5 and CFL1 thereby redirected and reduced their localization and activity at the actin cytoskeleton, decreasing the invasive potential of ERα-positive cells. In ERα-negative cells, which lack this regulatory mechanism, ERK5 and CFL1 are localized mainly to the actin cytoskeleton where they modulate actin turnover and increase cell invasiveness (Fig. 7E, right). Notably, we were able to reverse this phenotype by either reexpressing ERα, or by using ERK5 inhibitors, which could provide a new and potentially valuable therapeutic approach for reducing the aggressive and metastatic activity that is characteristic of many ERα-negative tumors. In this regard, it is of interest that inhibition of ERK5 has been reported to sensitize breast cancer cells to the action of therapeutic agents, such as anti-HER2 (35), suggesting that ERK5 represents a target of interest that might improve the efficacy of currently used cancer treatment agents. In addition, our findings suggest that monitoring the intracellular localization of ERK5 should prove to be a useful biomarker for disease prognosis and for predicting responsiveness to therapies.
ERK5, a member of the MAPK family, shows highest sequence homology to ERK1 and ERK2, yet it contains an extra C-terminal transactivation domain and a NLS, which renders this kinase a very attractive candidate for regulating both nuclear and cytoplasmic events (36). ERK5 is present in most human breast tumors and is overexpressed in 20% of them (35). We observed that high expression of a gene signature rich in genes regulated by ERα and ERK5 and possessing binding sites for ERK5 was strongly correlated with a shorter time to recurrence and with reduced survival in patients with ERα-positive breast cancers, consistent with clinical findings that overexpression of ERK5 is strongly associated with decreased disease-free survival (35).
Our observations present new insights into chromatin-tethered MAPKs (24) and, for the first time, document a role for ERK5 directly on chromatin, actively modulating hormone-regulated gene expression programs. In addition, we identified ERα as the key factor in regulating the dynamic nuclear versus cytoplasmic localization of this kinase and in linking CFL1, an actin-reorganizing protein, directly to ERK5 by showing that CFL1 was also recruited to the TSS of E2-regulated genes and that its nuclear localization was abrogated after treatment with ERK5 or ERα inhibitors.
As an actin-severing protein and regulator of actin dynamics, CFL1 is essential for the regulation of actin polymerization and depolymerization during cell migration and metastasis of tumor cells (37). Its expression is associated with the formation, stabilization, and maturation of invadopodia (31). Loss of the tumor suppressor PTEN, a frequent alteration in breast cancer, results in alterations in activation of CFL1, impacting the cell cytoskeletal phenotype (38). Hence, it is not surprising that there is accumulating evidence that CFL1 is a predictor of prognosis in several cancer types (39–42) and is a driver and biomarker of breast cancer metastasis (32, 33, 35). Our findings identified CFL1 as a requirement for ERK5 nuclear localization and for ERK5 and PAF1 interaction with RNA Pol II, and also documented CFL1 to be a crucial regulator of E2-mediated gene stimulation.
The paradigm we have observed for hormonal regulation by ERα, ERK5, and CFL1, and involvement of the actin cytoskeleton, may be used more generally in signaling induced by extracellular factors such as serum and DNA damage in a variety of cell types to alter the subcellular localization and functional activities of actin-regulated transcription factors. These include serum response factor (SRF) and its coregulators myocardin-related transcription factors (MRTF and MAL), and the junction-mediating and regulatory protein JMY, a transcriptional coactivator of p53 (43–46). Our study is the first to reveal the dynamic interplay between hormone receptor–mediated transcription and the actin cell skeleton to regulate cancer phenotypes. Our findings also uncover the pivotal roles of the protein kinase ERK5 and cofilin in estrogen-induced receptor regulation, contributing to the increasing evidence for involvement of actin-dependent mechanisms, and MAPKs, in enhancing coordinated transcription of target genes and signaling by nuclear receptors and their coregulators (12, 47–49).
In summary, our study identifies an ERα–ERK5–CFL1 network that modulates the degree of breast cancer cell aggressiveness through coupling of actin reorganization and nuclear hormone receptor–mediated transcription. Our findings highlight possible new biomarkers for disease state and for predicting therapy outcomes, and they suggest that targeting ERK5 in combination with endocrine therapies in ERα-positive breast cancers might provide more effective treatment. In particular, our work emphasizes the promise of combined reexpression of ERα along with ERK5 inhibition in ERα-negative tumors, thereby decreasing their invasive potential and hopefully improving patient clinical outcome. This novel paradigm for regulation of cell behavior might apply more broadly to other nuclear receptors and transcription factors controlling cancer cell aggressiveness, and could form a new conceptual basis for future research.
Accession numbers and data availability
Gene expression data are available from the GEO Database under accession number GSE53394. ChIP-Seq data for ERK5, PXSP motif-containing proteins, and RNA Pol II are given as BED files in Supplementary Table S3.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The contents of the article are solely the responsibility of the authors and do not necessarily represent the official views of the National Center for Complementary and Alternative Medicines (NCCAM), the Office of Dietary Supplements (ODS), the National Cancer Institute (NCI), or the National Institutes of Health (NIH).
Conception and design: Z. Madak-Erdogan, B.S. Katzenellenbogen
Development of methodology: Z. Madak-Erdogan, R. Ventrella, L. Petry, B.S. Katzenellenbogen
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z. Madak-Erdogan, R. Ventrella, L. Petry, B.S. Katzenellenbogen
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Madak-Erdogan, R. Ventrella, L. Petry, B.S. Katzenellenbogen
Writing, review, and/or revision of the manuscript: Z. Madak-Erdogan, R. Ventrella, L. Petry, B.S. Katzenellenbogen
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Z. Madak-Erdogan, B.S. Katzenellenbogen
Study supervision: Z. Madak-Erdogan, B.S. Katzenellenbogen
The authors thank Alvaro Hernandez, Mark Band, and the staff of the Roy J. Carver and W.M. Keck Biotechnology Centers at the University of Illinois at Urbana-Champaign for their assistance.
This research was supported by grants from The Breast Cancer Research Foundation (BSK) and by NIH grants T32 ES007326 (to Z. Madak-Erdogan) and P50AT006268 (to B.S. Katzenellenbogen) from NCCAM, ODS, and NCI.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.