Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition Jordan R. Beacha,b, George S. Husseyc, Tyler E. Millera, Arindam Chaudhuryd, Purvi Patele, James Monslowa, Qiao Zhenga, Ruth A. Kerif, Ofer Reizesa, Anne R. Bresnicke, Philip H. Howec, and Thomas T. Egelhoffa,1 aDepartment of Cell Biology, Cleveland Clinic, Cleveland, OH 44195; Departments of bPhysiology and Biophysics and fPharmacology, Case Western Reserve University, Cleveland, OH 44106; cHollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425; dDepartment of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030; and eDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461 Edited by Robert Adelstein, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board September 27, 2011 (received for review April 22, 2011) Despite functional significance of nonmuscle myosin II in cell gratory modes have been suggested, including amoeboid and migration and invasion, its role in epithelial–mesenchymal transition mesenchymal, and some studies have suggested that cells can (EMT) or TGF-β signaling is unknown. Analysis of normal mammary switch between migration modes depending on the extracellular gland expression revealed that myosin IIC is expressed in luminal environment (9). Recent work indicates that nuclear translocation cells, whereas myosin IIB expression is up-regulated in myoepithelial can be a rate-limiting step during amoeboid 3D migration (10, 11). cells that have more mesenchymal characteristics. Furthermore, TGF- Others studies have shown that contraction of the cell rear is ab- β induction of EMT in nontransformed murine mammary gland ep- solutely necessary for cancer cell invasion (12). Nonmuscle myosin ithelial cells results in an isoform switch from myosin IIC to myosin II has been suggested to be involved in both of these processes. IIB and increased phosphorylation of myosin heavy chain (MHC) IIA The myosin II holoenzyme consists of two myosin heavy chains fi on target sites known to regulate lament dynamics (S1916, S1943). (MHC), two essential light chains, and two regulatory light chains. These expression and phosphorylation changes are downstream of In mammals, three different genes encode nonmuscle MHC II heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of proteins, which are named MHC IIA (Myh9), IIB (Myh10), and TGF-β signaling. E1 knockdown drives cells into a migratory, inva- IIC (Myh14). These isoforms have both unique and redundant sive mesenchymal state and concomitantly up-regulates MHC IIB CELL BIOLOGY expression and MHC IIA phosphorylation. Abrogation of myosin roles in vivo (13). MHC phosphorylation has clear roles in regulating filament IIB expression in the E1 knockdown cells has no effect on 2D migra- Dictyostelium discoideum tion but significantly reduced transmigration and macrophage-stim- assembly in the model organism (14). ulated collagen invasion. These studies indicate that transition However, the contribution of heavy chain phosphorylation to fi between myosin IIC/myosin IIB expression is a critical feature of mammalian myosin lament assembly remains less well un- EMT that contributes to increases in invasive behavior. derstood. MHC IIA is phosphorylated on S1916, a putative PKC target (15), and S1943, a putative casein kinase II target (16). In vitro studies with recombinant MHC tail domains show that ormal mammary tissue consists of a branched multilayer fi Nductal network residing in an expanse of adipocytes. The heavy chain phosphorylation shifts the monomer/ lament equi- inner luminal epithelial layer is a cuboidal epithelium that is sur- librium into the monomeric, disassembled state, suggesting a rounded by an outer myoepithelial or basal cell layer that displays potential inhibitory role for heavy chain phosphorylation (17). mesenchymal-like features, including a spindle-shaped morphol- However, recent studies in live cells suggest a model in which ogy and expression of markers such as α-smooth muscle actin heavy chain phosphorylation is required for myosin IIA recycling (SMA) and vimentin. During transition to a tumor state, breast from distal to anterior regions of the lamellum (18), where it can epithelial cells characteristically exhibit features of either luminal contribute to focal adhesion stability and maturation (19). Other or basal cell types. Thus, basal-like breast carcinomas are defined studies have suggested that myosin IIA heavy chain phosphory- by having a gene expression profile similar to basal, or myoepi- lation increases breast cancer cell migration rates (20). Despite β thelial cells (1). In addition, basal-derived tumors are typically myosin II having roles in migration and invasion, and TGF- more invasive and metastatic than luminal-derived carcinomas (2). treatment clearly leading to a more migratory and invasive Tumor cell metastasis is a process that includes migration to phenotype in the context of EMT, the regulation of myosin II and intravasation of the vasculature, followed by extravasation expression or phosphorylation by TGF-β signaling has not been and migration into the distant tissue to form a secondary tumor. examined. One of the initial steps in metastasis is thought to be the process In this study we show that myosin IIB expression and myosin of epithelial–mesenchymal transition (EMT). During EMT, a IIA heavy chain phosphorylation are dramatically increased after polarized epithelial cell breaks down E-cadherin–based cell–cell TGF-β–induced EMT in mammary epithelial cells. Inhibition of contacts and acquires migratory and invasive properties, together myosin IIB expression in post-EMT mesenchymal cells reduces with changes in gene and protein expression patterns (3). TGF-β transmigration and invasion. These data indicate that shifts in is a known inducer of EMT (4) that signals through both Smad myosin II isoform expression and possibly MHC IIA phosphor- (5) and non-Smad pathways, including PI3K/Akt (6). Recent work has shown heterogeneous nuclear ribonucleoprotein-E1 (hnRNP-E1; hereafter referred to as E1) to be a downstream Author contributions: J.R.B., R.A.K., O.R., A.R.B., and T.T.E. designed research; J.R.B., effector of the TGF-β–Akt2 pathway (7). E1 regulates trans- G.S.H., T.E.M., A.C., P.P., J.M., Q.Z., and A.R.B. performed research; G.S.H., A.C., J.M., Q.Z., R.A.K., O.R., and P.H.H. contributed new reagents/analytic tools; J.R.B., T.E.M., Q.Z., R.A.K., lation of a number of critical EMT transcripts, including Dab2 O.R., A.R.B., P.H.H., and T.T.E. analyzed data; and J.R.B. and T.T.E. wrote the paper. ILEI and (7). Attenuation of E1 expression in epithelial cells The authors declare no conflict of interest. induces EMT and increases metastatic capability (8). This article is a PNAS Direct Submission. R.A. is a guest editor invited by the Editorial Although the metastatic process can be separated into in- Board. dividual steps, one accepted theme is that tissue invasion requires 1To whom correspondence should be addressed. E-mail: [email protected]. cytoskeletal force generation. How cytoskeletal forces drive the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mechanical process of invasion is not understood. Multiple mi- 1073/pnas.1106499108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106499108 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 ylation are essential for mediating mammary epithelial cell mi- two cell lines that lack MHC IIC are “basal B” lines (MDA-MB- gration and invasion. 231 and MCF-10A), which are more consistently mesenchymal in their transcript profiles than the “basal A” line (MBA-MB- Results 468) that has mixed basal/luminal properties (Fig. 1B) (22). Myosin Heavy Chain Isoforms Are Differentially Expressed in Normal Overall, these expression patterns generally match the distribu- Mouse Mammary Gland and Breast Epithelial Cell Lines. To in- tion profile observed in the native tissue, suggesting that MHC vestigate the expression pattern of the myosin II isoforms in IIC expression may be a marker for the luminal layer of ductal native mammary gland we immunostained sections of mouse tissue and that MHC IIB expression is a part of the basal or mammary gland with markers for the luminal layer (cytokeratin myoepithelial gene expression program. 8, or K8) or myoepithelial layer (SMA). Myosin IIA and myosin IIC expression was primarily restricted to the luminal cell layer MHC IIA Phosphorylation Patterns in Breast Epithelial Cell Lines. (Fig. 1A, Top and Bottom, respectively), whereas myosin IIB MHC IIA expression did not seem to correlate strongly with showed minimal expression in luminal cells but was prominently either luminal or basal breast cancer cell lines. To determine expressed in the myoepithelial layer (Fig. 1A, Middle). whether MHC IIA phosphorylation is differentially regulated in Given the observed differences in isoform distribution in the luminal vs. basal cell lines, we took advantage of our recently fi normal mammary gland, we sought to determine whether this developed phospho-speci c antibodies directed against two phos- correlated with differential isoform expression in breast cancer phorylated residues in the MHC IIA tail region, S1916 and S1943 and breast epithelial cell lines that display luminal vs. basal (23). Western blot analysis revealed strongly elevated MHC fi fi phosphorylation at both target sites in all three basal-like cell characteristics as de ned by transcriptome pro ling (21). Anal- B ysis of a set of seven breast epithelial cell lines revealed elevated lines, relative to the luminal lines (Fig. 1 ). Given earlier anal- yses demonstrating that S1943 phosphorylation regulates myosin MHC IIB expression in three lines that have been previously IIA filament dynamics at the leading edge of cells during po- defined as “basal” and very low levels of in MHC IIB expression larized migration (18, 20), we suggest that elevated phosphory- in four lines defined in the same studies as “luminal” (Fig.