Chromatin , Gene , and RNA Regulation NovelRoles forERK 5 andCo fi lin asCriticalMediatorsLinking ER a-Driven Transcription , Actin Reorganization , and Invasiveness in Breast Cancer

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 (ERa)/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 ERa determined the subcellular localization of ERK5, which functions as a coregulator of ERa-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 ERa, 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 ERa-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 newprognostic biomarkers and novel therapeutic avenues to moderate cancer aggressiveness. Mol Cancer Res; 12(5); 714–27. 2014 AACR. Introduction 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 (ERa), 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 ERa-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 ERa and protein kinase pathways and the manner by which ERa 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 ERa at enhancers of many estrogen-regulated genes that control cell proliferation (12). Authors' Affiliation: Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois at UrbanaChampaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, IL, 61801. Phone: 217-333-9769; Fax: 217-244-9906; E-mail: katzenel@illinois.edu doi: 10.1158/1541-7786.MCR-13-0588 2014 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 12(5) May 2014 714 on March 20, 2017. © 2014 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst February 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0588