Myosin II Isoform Switching Mediates Invasiveness After TGF-Β–Induced Epithelial–Mesenchymal Transition

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. 1B). lation of S1943 and S1916 may have a role in enhancing the MHC IIC was expressed in all four luminal cell lines but was motile behavior of basal-like cells. absent in two of the three basal lines (Fig. 1B). In addition, the Myosin Heavy Chain Isoform Switching During EMT. Given the pre- ferential expression of myosin IIB and the elevated MHC IIA phosphorylation in basal-like lines, we asked whether luminal- like mammary epithelial cells, induced to undergo EMT by TGF- β treatment, would drive similar changes in myosin II expression. To test this possibility, we used the normal murine mammary gland (NMuMG) cell EMT model (24), which under normal tissue culture conditions forms a classic epithelial sheet with a “cobblestone” morphology (Fig. S1A). Upon treatment with TGF-β for 24 h, the cells convert to a spindle-shaped mesenchymal morphology (Fig. S1A). This morphological switch is accom- panied by extensive transcriptional and phenotypic changes (25), including the induction of a number of classic mesenchymal cell markers, such as vimentin (Fig. 2A). Whereas MHC IIA showed a small but significant twofold increase at the mRNA and protein level during EMT (Fig. 2 A and D), MHC IIB protein expression was up-regulated dramatically at the 24-h time point (Fig. 2A). Quantitative PCR analysis showed that this is due to an ≈3,000- fold increase in mRNA level (Fig. 2D). Accompanying the dra- matic increase in MHC IIB expression, an opposing decrease in mRNA abundance (approximately fivefold) and protein expression of MHC IIC was observed (Fig. 2 A and D). Multiple efforts have been made to identify targets of TGF-β treatment using expression microarrays. In agreement with our data, MHC IIA and MHC IIC reside within a list of genes that are up-regulated and down-regulated, respectively, during EMT (26– 29). MHC IIB was previously included in a list of genes up-regulated in a lung epithelial cell EMT model (28). The present work shows that MHC IIB expression is also regulated in mammary epithelial cell EMT driven by TGF-β.Wealsoconfirm these expression changes at the protein level.

MHC IIA Phosphorylation Is Elevated During EMT. To further inves- tigate myosin regulation during EMT, we analyzed the same TGF-β–treated NMuMG time course for MHC IIA phosphory- Fig. 1. MHC isoforms are differentially expressed in normal mouse mam- lation. Untreated NMuMG cells maintain low levels of MHC mary gland and breast epithelial cell lines. (A) Normal mouse mammary ﬁ Top IIA phosphorylation at S1916 and undetectable phosphorylation tissue was xed, sectioned, and immunostained for MHC IIA ( , red), MHC at S1943. However, upon treatment with TGF-β there is a dra- IIB (Middle, green), or MHC IIC (Bottom, red). The sections were coimmu- Top Bot- matic increase in MHC IIA phosphorylation, especially at S1943 nostained with smooth muscle actin to mark the basal layer ( and A tom, green) or keratin-8 to mark the luminal layer (Middle, red). (Scale bar, (Fig. 2 ). Interestingly, no increase in MHC IIA phosphoryla- 100 μm.) (B) Whole-cell lysates of the indicated cell lines were subjected to tion was observed until 12 h after the initiation of TGF-β Western blot analysis with the indicated antibodies. treatment, with a more robust increase occurring after 24 h. The

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1106499108 Beach et al. Downloaded by guest on September 27, 2021 To determine whether changes in myosin expression and phosphorylation observed in TGF-β–treated NMuMG cells are downstream of E1, we analyzed cells with abrogated E1 expression (E1-shRNA) and cells overexpressing E1 (E1++) during aTGF-β treatment time course. E1-shRNA cells showed the same profile of myosin expression and phosphorylation as NMuMG cells treated with TGF-β, namely strikingly increased MHC IIB expression, increased MHC IIA phosphorylation, and decreased MHC IIC expression (Fig. 2B). The effect of E1 shRNA on MHC IIB expression was confirmed using two additional E1 shRNA target sequences (Fig. S1B). This myosin profile was not altered upon treatment with TGF-β, supporting previous data demonstrating that E1 mediates the EMT-inducing effects of TGF-β. In contrast, E1++ cells displayed a similar myosin isoform expression profile to the parental NMuMG cells, and this profile was not affected by TGF-β treatment (Fig. 2C). These data demonstrate that the myosin isoform switching and phosphorylation changes seen in TGF-β–treated cells are downstream of E1 and that manipulation of E1 expression has profound effects on myosin isoform and phosphorylation patterns independent of TGF-β addition. Given the large increase in MHC IIB transcript level observed upon TGF-β treatment (Fig. 2D)or in E1-shRNA cells (Fig. 2E), we suggest that changes in MHC IIB expression are mediated by transcriptional events downstream of E1, rather than via direct silencing of MHC IIB mRNA by E1. This contrasts with the translational repression seen in previously reported direct targets of E1 (7). To quantify MHC II isoform switching downstream of E1, we CELL BIOLOGY performed mass spectrometric analysis to determine the relative molar abundance of MHC IIA, IIB, and IIC in E1++ and E1- shRNA cell lines. In E1++ cells, ≈99% of the MHC II detected was the IIA isoform, and ≈1% was the IIC isoform. No MHC IIB was detected. In contrast, in E1-shRNA cells, MHC IIC peptides Fig. 2. MHC regulation during TGF-β and E1-shRNA treatment of NMuMG were not detected by mass spectrometry, and the remaining cells. (A) NMuMG, (B) E1-shRNA, and (C)E1++ cells were untreated or treated MHC II peptides detected revealed a ratio of ≈90% MHC IIA with TGF-β for the indicated time. Whole-cell lysates were collected, nor- and ≈10% MHC IIB. These data are consistent with mRNA malized, and probed via Western blot with the indicated antibodies. levels in the cells (Fig. 2E). On the basis of the relatively low Vimentin represents a positive control for EMT, and actin and GAPDH are levels of MHC IIC present in the epithelial state and the rela- shown as loading controls. (D) NMuMG cells were treated with TGF-β for the tively greater fold induction of MHC IIB expression upon EMT, indicated time, after which RNA was collected and quantitative PCR was we focused our analysis on the roles of MHC IIB in post-EMT performed for the indicated myosin isoform. Data are the mean ± SEM from four independent experiments. *P < 0.05, Student’s t test, relative to the mesenchymal phenotypes. untreated. (E) RNA was collected from NMuMG, E1-shRNA, and E1++ cells and quantitative PCR was performed for the indicated myosin isoform. For MHC IIB shRNA Inhibits Transmigration and Invasion. In 2D migra- each cell line, relative isoform transcript level is expressed as a percentage of tion assays, E1-shRNA cells are ≈50% more migratory com- ++ the total transcript abundance of all three isoforms. Data are the mean ± pared with E1 cells (Fig. S2 A and B). Additionally, whereas SEM. n =3. E1++ cells migrate as epithelial sheets, the E1-shRNA cells display properties more characteristic of mesenchymal cell migration, with little or no apparent cell–cell contacts and in- kinetics of these phosphorylation changes suggest regulation dividual cells often emerging at the leading edge. In contrast to through TGF-β–mediated transcriptional or translational events, the 2D migration assay in which the E1-shRNA cells are slightly as opposed to immediate signaling downstream of TGF-β. The more migratory than the E1++ cells, transwell assays revealed kinetics also correlate with previously reported times for in- that E1-shRNA cells have a nearly 40-fold higher propensity for creased migratory and invasive behavior by the post-EMT mes- transmigration (Fig. S2C). Thus, E1-shRNA treatment only enchymal cells (30). Taken together these data further support slightly increases the rate at which epithelial cells migrate in 2D the concept that expression of myosin IIB, and possibly elevated but dramatically increases their ability to squeeze through small MHC IIA tail phosphorylation, are consistent features of a more pores, a surrogate for invasive behavior. Taken together these motile phenotype, possibly relevant to the invasive behavior of data suggests that E1-shRNA cells recapitulate a mesenchymal- cells that have undergone EMT. like phenotype and can be used as a model to further our understanding of the mechanics of mesenchymal cell invasive behavior. E1 Modulation Alters Myosin Expression and Phosphorylation in To determine whether the increased expression of MHC IIB is NMuMG Cells. Recent work has revealed E1 as a critical down- important for the increased migration and invasion that occurs stream effector of TGF-β treatment in NMuMG cells (7). E1 is after E1 silencing, we used lentiviral-mediated shRNA to sup- a3′UTR mRNA binding protein that represses translation of its press MHC IIB expression in E1-shRNA cells (Fig. S3). targets. shRNA-mediated knockdown of E1 induces a mesen- Knockdown of MHC IIB did not affect mesenchymal morphol- chymal state, whereas overexpression of E1 locks cells in an ogy, significantly alter E1 shRNA cell migration in 2D, or alter epithelial state that is resistant to TGF-β–stimulated EMT (7) expression of other EMT markers (Fig. 3A and Fig. S3). How- [Fig. 2 B and C (see vimentin) and Fig. S1A]. E1 thus seems to be ever, MHC IIB shRNA did significantly reduce the cells ability a master regulator of many downstream EMT events. to migrate through 8-μm pores (Fig. 3B). Similar results were

Beach et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 in transmigrating cells, we fixed, immunostained, and imaged E1- shRNA cells as they were migrating through a transwell membrane. Confocal z-stacks were collected from individual cells and imaged from each side of the transwell membrane. 3D re- construction of cells actively undergoing transmigration shows that myosin IIB is highly enriched in the rear of the cell (Fig. 4 B and C, blue arrows, and Fig. S6), especially around the nucleus that is just beginning to enter the pore. We hypothesize, on the basis of this posterior and perinuclear localization, that myosin IIB may specifically facilitate squeezing the cytoplasm and nucleus through tight spaces, an essential step in both intravasation and extravasation. In contrast, although myosin IIA appears more assembled in the anterior regions of the cell that has al- ready passed through the pore (Fig. 4B, yellow arrowheads), quantitative assessment of myosin IIA localization suggests that it is not polarized in this assay (Fig. S6). These data show that myosin IIB, unlike myosin IIA, remains polarized during transmigration, with enrichment specifically in perinuclear regions. Discussion The studies presented here report myosin II isoform expression patterns in normal mammary gland tissue, and demonstrate that isoform expression patterns in breast epithelial cell lines parallel the expression patterns in native luminal vs. basal tissue layers (e.g., expression of myosin IIC in luminal tissues and cell lines and expression of myosin IIB in basal tissue and cell lines). Considering the biophysical properties of myosin II isoforms, it is Fig. 3. MHC IIB shRNA inhibits transmigration. (A and B) 2D migration and intriguing that the myosin IIB isoform is preferentially expressed transwell assays were performed on uninfected, scramble shRNA cells or MHC in the myoepithelial cell layer, which also expresses many pro- IIB shRNA cells. Data were normalized to the mean of scramble shRNA cells. teins involved in smooth muscle cell function. Of the three Data points are the mean ± SEM. For B, n ≥ 28 fields of view from three in- nonmuscle myosin II isoforms, myosin IIB has the highest duty dependent experiments. For C, n ≥ 11 fields of view from three independent ratio (34) and the highest affinity for ADP (35), suggesting that it P < C experiments. * 0.001 relative to scramble shRNA cells. ( ) Scramble shRNA may be tuned for prolonged force production and minimal en- and IIB shRNA cells were tested for their ability to invade a collagen gel in the presence (black bars) or absence (gray bars) of BAC macrophages. MDA-MB- ergy consumption. Previous work has shown that myosin IIB 231 cells were used as a positive control. For D, n ≥ 12 regions analyzed from contributes to force maintenance in aortic smooth muscle (36). five experiments performed on three independent dates. *P < 0.001 relative The myoepithelial layer of mammary gland tissue is responsible to invasion in the absence of BACs. Data points are the mean ± SEM. for the contractile events during milk secretion (37) and has been suggested to be critical for proper branching architecture (38), an essential component of mammary gland development. We obtained when MHC IIB expression was suppressed in MDA- suggest that myosin IIB may be selectively expressed in the MB-231 cells using an shRNA construct with a different target myoepithelial layer to contribute to the prolonged contractile sequence (Fig. S4). MHC IIB abrogation also inhibited E1- events that are involved in milk secretion and branching mor- shRNA cells’ ability to invade through Matrigel-coated transwell phogenesis. chambers (Fig. S5). To complement these results, we examined We suggest that up-regulation of MHC IIA phosphorylation the requirement for MHC IIB in a macrophage-induced invasion during EMT may have a role in the enhanced motility and in- assay. As previously reported (31), MDA-MB-231 invasion into vasiveness of mesenchymal cells. We favor a model in which a collagen matrix can be potently enhanced via a macrophage- heavy chain phosphorylation permits redistribution of myosin II dependent CSF–EGF paracrine loop (Fig. 3C). Coculture of E1- from posterior to anterior regions, where it can facilitate adhe- shRNA cells with macrophages increased the number of invasive sion stabilization and maturation, a concept supported by our tumor cells by ≈3.5 fold, whereas knockdown of MHC IIB recent studies using site-directed mutants of these target sites completely abolished macrophage-stimulated invasion (Fig. 3C). (18). Given the potential role for integrin-based adhesions in These results demonstrate a critical role for myosin IIB in 3D transmigration, it is logical that modulation of MHC IIA phos- invasion in multiple settings. phorylation could alter cell invasion capability. Future studies To dissect the mechanistic roles of myosin IIB in 3D migra- are needed to explore this possibility. Additionally, MHC IIB tion, we immunostained migrating E1-shRNA cells for MHC IIA phosphorylation has established roles in modulation of filament and MHC IIB in the 2D setting (Fig. 4A) and in the 3D-like assembly in prostate cancer cells (39). Further studies directed at setting of transwell migration (Fig. 4 B and C). Whereas myosin understanding MHC IIB phosphorylation events in the context IIA appears preferentially localized to the anterior lamellar of EMT and invasive behavior will be of great interest. regions in 2D (Fig. 4A, yellow arrowheads), myosin IIB is en- In our system, the up-regulation of MHC IIB mRNA levels riched in the posterior retraction zones (Fig. 4A, blue arrows). after TGF-β treatment or E1-shRNA treatment suggests that This staining pattern is in agreement with previous studies in there are transcription factors downstream of E1 that directly other cell types reporting a tendency for myosin IIB to reside in regulate MHC IIB expression. To date, few studies have ana- the posterior region of polarized cells relative to myosin IIA lyzed the promoter region of MHC IIB in great detail. Those during 2D migration (32). However, the forces required for studies that have addressed this subject suggest possible regula- transmigration and 3D migration are quite different from the tion through the transcription factors KLF5 (40) and Hex (41), forces required for 2D migration (10, 33) and may result in al- both of which have been shown to be downstream effectors of tered myosin isoform localization that is not seen in 2D migra- TGF-β family signaling (42, 43). Therefore, although there are tion. To determine the localization of myosin IIA and myosin IIB interesting candidates for regulation of MHC IIB expression in

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Fig. 4. MHC IIB localizes to the rear and perinuclear regions of migrating and transmigrating E1-shRNA cells. (A) E1-shRNA cells migrating in 2D were ﬁxed and immunostained for MHC IIA (Top, green) and MHC IIB (Middle, red). The white arrow in the lower image indicates the direction of migration. (B)E1- shRNA cells actively moving through a transwell pore were ﬁxed and immunostained for MHC IIA (green) and MHC IIB (red) and counterstained with DAPI (blue). Confocal z-stacks were collected. Images are orthogonal maximum intensity projections. Bottom: Merge 2 is a merge of DAPI, MHC IIA, and MHC IIB. Merge 1 excludes DAPI. The dotted line in merge 2 represents the position of the transwell membrane. (C) Same dataset used in Fig. 4B was separated into images above the membrane (Upper) and below the membrane (Lower). Each separated dataset was then averaged into a single image. (Scale bars, 20 μm.)

this system, the precise molecular mechanism by which TGF-β earlier studies implicating myosin II as contributing to nuclear and E1 regulate MHC IIB mRNA requires further investigation. translocation when cells are migrating through narrow spaces (33, TGF-β–stimulated EMT involves induction of a large number 11, 10). If this model proves valid with further studies, myosin IIB of genes (26), many of which contribute to the acquisition of may prove to be an outstanding therapeutic target for intervention invasive behavior. Our work demonstrates induction of MHC IIB in invasive metastatic behavior of basal-like carcinomas. as a critical component of this transition. Interestingly, depletion Taken together, our data demonstrate a clear role for myosin of myosin IIB in the mesenchymal E1-shRNA cells has no effect IIB as contributing to invasive potential in mammary epithelial on 2D migration, a moderate effect on transmigration through cells that have undergone EMT. Our data also demonstrate 8-μm pores, and a very strong negative impact on migration a consistent induction of myosin IIA heavy chain phosphoryla- through dense (5.8 mg/mL) collagen gels in the macrophage- tion in post-EMT NMuMG cells and elevated phosphorylation stimulated migration assay. We suggest that induction of myosin of the same sites in basal-like breast epithelial lines. We hypo- IIB when cells undergo EMT enhances their force-generating thesize that MHC IIA phosphorylation and myosin IIB expres- capacity for invasive migration and that this effect becomes more sion may both be critical components in the set of global changes apparent the greater the resistance of the external environment is. that induce cellular invasion during EMT. Further studies are This study localized myosin IIA and IIB isoforms imaged in needed to test the roles of MHC IIA phosphorylation in invasive migration. We suggest that ﬂuxes in myosin II expression and a setting where cells are migrating in a 3D-like setting in which phosphorylation may also have roles in breast epithelial cell the cell is experiencing signiﬁcant mechanical resistance from its metastasis and possibly in developmental settings where EMT is environment. We suggest that the posterior localization of my- essential for proper organogenesis. osin IIB in this setting may be critical for helping squeeze the nucleus through the transwell pore and that migration through Methods dense collagen gels represents an even more demanding migra- Cell Culture and Treatments. NMuMG, E1-shRNA, and E1++ cells were main- − tion setting, such that depletion of myosin IIB by shRNA com- tained in DMEM with 10% FBS, 10 μgmL1 insulin, and antibiotic/anti- pletely abrogates invasiveness. This model is consistent with mycotic and, where indicated, were treated with 5 ng mL−1 of TGF-β2.

Beach et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 MDA-MB-231 cells were maintained in RPMI with 10% FBS and antibiotic/ stained with DAPI. Fields of view were randomly imaged, and the number of antimycotic. A list of shRNA target sequences is provided in Table S1. cells present was quantified using ImageJ software. Additional details for imaging cells during transmigration can be found in SI Methods. Mammary Gland Immunohistochemistry. Mammary glands were isolated from female wild-type C57BL/6 mice at ≈5 wk of age, fixed, embedded in paraffin, Collagen Invasion Assay. The collagen invasion assay was performed as de- sectioned, and immunostained with the indicated antibodies. scribed previously (31). Additional methods details can be found in SI Methods. 2D Migration Assays. Migration assays were performed as described previously (18). ACKNOWLEDGMENTS. We thank Dr. Belinda Willard for mass spectrometry analysis and Dr. Judith Drazba for imaging advice. This work was supported Transwell Migration Assays. Cells were plated in 12-well 8-μm-pore PET in part by National Institutes of Health Grants GM 50009 and GM 077224 (to transwell membranes (Falcon) or CytoSelect 96-well Cell Invasion Assay Kit T.T.E.), CA100324 (to A.R.B.), and CA090398 (to R.A.K.). T.E.M. was supported (Cell Biolabs), allowed to migrate for 18 h or 24 h, respectively, fixed and by National Institutes of Health Training Grant T32-GM07250.

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