The S100A4 Metastasis Factor Regulates Cellular Motility Via a Direct Interaction with Myosin-IIA

Research Article

The S100A4 Metastasis Factor Regulates Cellular Motility via a Direct Interaction with Myosin-IIA

Zhong-Hua Li and Anne R. Bresnick

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York

Abstract orthotopic models, overexpression of S100A4 in nonmetastatic S100A4, a member of the Ca2+-dependent S100 family of mammary tumor cells confers a metastatic phenotype (6, 7), proteins, is a metastasis factor that is thought to regulate the whereas inhibition of S100A4 expression suppresses the metastatic motility and invasiveness of cancer cells. Previously, we capacity of tumor cells (8, 9). S100A4 itself is not tumorigenic as showed that S100A4 specifically binds to nonmuscle myosin- transgenic mice that overexpress S100A4 do not develop tumors IIA and promotes the unassembled state. S100A4, thus, (10); however, in a tumorigenic background, S100A4 expression in provides a connection between the actomyosin cytoskeleton the tumor cells facilitates the development of highly aggressive and the regulation of cellular motility; however, the step primary tumors and the formation of metastases (10, 11). In or steps in the motility cycle that are affected by S100A4 addition, the progeny from mice expressing the polyoma virus expression have not been investigated. To examine how the middle T antigen crossed with mice carrying null alleles for biochemical properties of S100A4 affect cell motility, we S100A4 display a significant decrease in metastases (12). Alto- gether, these observations suggest that S100A4 is not simply a determined the effect of S100A4 expression on protrusive behavior during chemoattractant-stimulated motility. Our marker for metastatic disease but rather has a causal role in studies show that S100A4 modulates cellular motility by mediating this process. affecting cell polarization, with S100A4 expressing cells The transition from benign tumor growth to malignancy is described by the three-step hypothesis of metastasis, which displaying few side protrusions and extensive forward involves (a) attachment to the extracellular matrix, (b) local protrusions during chemotaxis compared with control cells. proteolysis, and (c) subsequent migration (13). Of particular To establish a direct link between S100A4 and the regulation relevance to the role of S100A4 in promoting a metastatic of myosin-IIA function, we prepared an antibody to the phenotype, several studies suggest that S100A4 expression levels S100A4 binding site on the myosin-IIA heavy chain that has correlate strongly with cell motility. For instance, fibroblasts and comparable effects on myosin-IIA assembly as S100A4. epithelial tumor cells that overexpress S100A4 display increased Microinjection experiments show that the antibody elicits migratory properties (14), and S100A4 expression is associated with the same effects on cell polarization as S100A4. Our studies mesenchymal cell morphology and motility (15). Conversely, show for the first time that S100A4 promotes directional ablation or reduction of S100A4 expression in tumor cells motility via a direct interaction with myosin-IIA. These correlates with decreased cellular motility (9, 16). A consideration findings establish S100A4 as a critical regulator of myosin-II of all these data suggests that S100A4 may regulate the motility of function and metastasis-associated motility. (Cancer Res 2006; tumor cells. 66(10): 5173-80) In vitro studies show that S100A4 specifically binds to nonmuscle myosin-IIA and inhibits the assembly of myosin-IIA Introduction monomers into filaments and promotes the disassembly of S100A4 (mts1) belongs to the S100 family of Ca2+-binding preexisting myosin-IIA filaments in a calcium-dependent manner proteins (1). These proteins typically form homodimers (10-12 kDa (17). The S100A4 binding site maps to residues 1909 to 1924 in the per subunit) that are characterized by their solubility in saturated COOH-terminal end of the coiled coil region of the myosin-IIA 100% ammonium sulfate (2). There are >20 known members, most heavy chain (17, 18). Although this segment contains a protein of which are expressed in a highly tissue specific manner (1, 3). kinase C (PKC) phosphorylation site at Ser1917, S100A4 binding is Importantly, most S100 family members display a high degree of not affected by PKC phosphorylation. Rather, phosphorylation on target specificity, suggesting that individual S100 proteins regulate Ser1944 by casein kinase 2 (CK2), which is located 20 residues specific cellular processes. downstream of the S100A4 binding site, inhibits S100A4 binding Elevated S100A4 expression levels are associated with meta- and protects against S100A4-induced destabilization of myosin-IIA static cancers, and S100A4 has emerged as a prognostic marker filaments (19). Thus, heavy-chain phosphorylation on the CK2 site for poor patient survival in a number of cancers (4, 5). In addition, and calcium binding to S100A4 modulate the S100A4/myosin-IIA animal studies have provided evidence supporting the involve- interaction. ment of S100A4 in tumor progression and metastasis. In To examine how the biochemical properties of S100A4 affect cell motility, we determined the effect of S100A4 expression on protrusive behavior during chemoattractant-stimulated motility. In addition, we prepared an antibody to the S100A4 binding site on Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). the nonmuscle myosin-IIA heavy chain that has comparable effects Requests for reprints: Anne R. Bresnick, Department of Biochemistry, Albert on myosin-IIA assembly as S100A4. Our studies show that S100A4 Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718- promotes directional motility via a direct interaction with myosin- 430-2741; Fax: 718-430-8565; E-mail: [email protected]. I2006 American Association for Cancer Research. IIA and establish S100A4 as a critical regulator of myosin-II- doi:10.1158/0008-5472.CAN-05-3087 mediated motility. www.aacrjournals.org 5173 Cancer Res 2006; 66: (10).May 15, 2006

Materials and Methods Tris-HCl (pH 8.8); 100 mmol/L sodium pyrophosphate; 100 mmol/L NaF; 250 mmol/L NaCl; 10 mmol/L EGTA; 5 mmol/L EDTA; 1 mmol/L Cell lines. HeLa cells were maintained in DMEM containing 10% fetal phenylmethylsulfonyl fluoride; 5 Ag/mL each of chymostatin, leupeptin, bovine serum (FBS). The BAC-1.2F5 macrophage cell line was cultured in and pepstatin; 1% NP40; and 10 mmol/L ATP. The soluble fraction a -MEM containing 10% FBS and 10 ng/mL colony-stimulating factor-1 containing myosin-II was recovered by centrifugation at 105,000 Â g for j (CSF-1). All cells were grown at 37 C in a humidified atmosphere of 5% CO2. 15 minutes at 4jC in a Beckman TL-100 ultracentrifuge. Total protein The rat mammary nonmetastatic adenocarcinoma cell line MTC-GFP (20) concentrations were determined using the Bio-Rad Detergent-Compatible a was cultured in -MEM containing 5% FBS. Protein assay. Before the immunoprecipitation, myosin-IIA S100A4 binding MTC-GFP cells stably expressing S100A4 were established as follows. The site antibodies were bound to protein A-Sepharose in PBS containing rat S100A4 cDNA was amplified from an expressed sequence tag (American 1 mg/mL bovine serum albumin (BSA); 0.5 mg of supernatant was Type Culture Collection, Manassas, VA) and subcloned into the HindIII/ incubated with the antibody-protein A-Sepharose for 2 hours at 4jC. HpaI sites of the retroviral expression vector pLHCX (Clontech, Palo Alto, Immune complexes were collected by centrifugation; the remaining CA). The S100A4 sequence was confirmed by DNA sequencing. The supernatant was saved for analysis, and the immune complexes were recombinant vector and vector without insert were transfected into Phoenix washed thrice with extraction buffer and then boiled in Laemmli sample packaging cells to produce recombinant virus and unrecombinant control buffer. Immunoprecipitates and recovered supernatant were separated by virus, respectively, using standard protocols (21). MTC-GFP cells were 5% SDS-PAGE. For immunoblots of HeLa whole-cell lysates and myosin-II infected for 24 hours with virus-containing supernatant in the presence of rods, proteins were separated by 8% SDS-PAGE. Proteins were transferred to A j 8 g/mL polybrene at 32 C. After 2 days, stably expressing cells were nitrocellulose membranes and reacted with antibodies to the myosin-IIA A selected by the inclusion of 300 g/mL of hygromycin in the medium. After S100A4 binding site, myosin-IIA, or myosin-IIB. Immunoreactive proteins 7 to 10 days of selection, the clones infected with the control pLHCX virus were detected as described above. were pooled together (MTC-VC). S100A4-pLHCX virus–infected clones were Myosin-IIA assays. Myosin-IIA rod assembly and disassembly assays subjected to subcloning by limiting dilution into two 96-well plates in were done as described previously (17) using 1.5 Amol/L myosin-IIA rods A medium containing 300 g/mL hygromycin. All MTC-S100A4 clones and and 0.75 to 1.5 Amol/L S100A4 dimer or 0.75 to 4.5 Amol/L of the myosin-IIA a the MTC-VC cell line were maintained in -MEM containing 5% FBS and S100A4 binding site antibody. 300 Ag/mL hygromycin. For ATPase assays, human myosin-IIA at a concentration of 0.228 mg/mL Antibodies. A rabbit polyclonal antibody to the S100A4 binding site on was phosphorylated with 10 nmol/L rabbit smooth muscle myosin light myosin-IIA was generated using a synthetic peptide to residues 1907 to 1916 chain kinase (MLCK) in the presence of 1 Amol/L calmodulin in a buffer

(TADAMNREVS) of the human myosin-IIA sequence. Rabbit polyclonal containing 20 mmol/L Tris-HCl (pH 7.5), 15 mmol/L KCl, 5 mmol/L MgCl2, antibodies to myosin-IIA and myosin-IIB were prepared using synthetic 0.4 mmol/L CaCl2, 0.02% NaN3, 0.5 mmol/L DTT, and 0.5 mmol/L ATP for peptides corresponding to the COOH termini of the two proteins, 45 minutes at 30jC. The reactions were stopped by the addition of EGTA to GKADGAEAKPAE (residues 1950-1961) and SDVNETQPPQSE (residues a final concentration of 0.5 mmol/L. Monophosphorylation of the myosin-II 1965-1976), as described previously (22). A rabbit polyclonal antibody to regulatory light chain was confirmed by glycerol PAGE using the method of S100A4 was prepared using recombinant human S100A4. All antibodies Perrie and Perry (26). Before the start of the ATPase assay, the myosin-IIA were produced using standard methodologies (Covance Research Products, S100A4 binding site antibody or S100A4 was incubated with the Denver, PA). Antibodies to synthetic peptides (myosin-IIA S100A4 binding phosphorylated myosin-IIA for 45 minutes at room temperature in the site, myosin-IIA, and myosin-IIB) were purified by affinity chromatography presence of 0.3 mmol/L CaCl2. Actin-activated ATPase assays were done at on a Sulfolink column (Pierce, Rockford, IL) coupled with the synthetic room temperature and contained 0.05 Amol/L phosphorylated myosin-IIA, peptide via a COOH-terminal cysteine residue. The S100A4 antibodies were 10 Amol/L filamentous actin (F-actin), 0.5 to 0.8 Amol/L myosin-IIA S100A4 purified by affinity chromatography on an Aminolink column (Pierce) binding site antibody or 0.4 to 0.8 Amol/L S100A4 dimer in 20 mmol/L coupled with the human S100A4. The specificities of the myosin-IIA, Tris-HCl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.1 mmol/L EGTA, myosin-IIB, and myosin-IIA S100A4 binding site antibodies were assessed 0.3 mmol/L CaCl2, and 1 mmol/L ATP. Phosphate release was measured for by immunoblot and immunoprecipitation analysis using HeLa cell lysates 20 to 30 minutes using the EnzChek Phosphate Assay kit (Molecular Probes, and purified recombinant myosin-IIA and myosin-IIB rods. The specificity Eugene, OR). of the S100A4 antibody was evaluated by immunoblot analysis of NIH Collagen invasion assay. The collagen invasion assay was done as 3T3 and MDA-MB-231 cell lysates and purified recombinant S100A4 (data described previously (27). In brief, 80,000 MTC-VC or MTC-S100A4 cells not shown). were plated on 35-mm glass-bottomed dishes (MatTek Corp., Ashland, MA) Purified rabbit IgG was purchased from Sigma (St. Louis, MO). The in the presence or absence of 400,000 BAC1.2F5 cells in a-MEM containing phosphotyrosine monoclonal antibody was from Covance Research 10% FBS and 10 ng/mL CSF-1. After 18 to 24 hours, cells were overlaid with Products. The h-actin monoclonal antibody was from Sigma. a 750- to 1,000-Am layer of 6.22 mg/mL collagen I (BD Biosciences, San Jose, Protein purification. Rabbit skeletal muscle actin was purified from CA), which was allowed to gel for 90 minutes before adding 1 mL of the acetone powder by the method of Spudich and Watt (23). Recombinant medium. After 24 hours, the cells were fixed for 30 minutes with 4% human S100A4 and myosin-IIA and myosin-IIB rods were purified as formaldehyde and analyzed by confocal microscopy on a Bio-Rad Radiance described previously (17). Full-length myosin-IIA was prepared from out- 2000 Laser Scanning Confocal Microscope. Optical z sections were taken dated human platelets according to the method of Daniel and Sellers (24). every 5 Am, starting from the base of the dish to 50 Am into the collagen gel. Immunoblots and immunoprecipitation. HeLa whole-cell lysates were To quantify the invasion of the cells, the green fluorescent protein (GFP) prepared by washing monolayers with cold PBS and placing the culture fluorescence of the z sections from 20 to 50 Am were added and divided by plate on dry ice for 10 minutes. The cell lysate was prepared by directly the sum of the GFP fluorescence from all the z sections. Two to three scraping the frozen cells into 2Â Laemmli sample buffer (25) containing different fields of cells from each dish were imaged. All data were collected 5 Ag/mL each of chymostatin, leupeptin, and pepstatin. Whole-cell lysates from two to three independent experiments. of MTC-S100A4 clones and MTC-VC cells were prepared as described Micropipette assay. For light microscopy experiments, 4,000 cells were above. Lysates for each cell line were separated on a 12% Tricine SDS- plated on poly-L-lysine-coated glass-bottomed dishes (MatTek). After f24 polyacrylamide gel system. Proteins were transferred to a polyvinylidene hours, cells were starved in L15 medium (Life Technologies, Gaithersburg, difluoride membrane and reacted with antibodies to S100A4 or h-actin, MD) supplemented with 0.35% BSA (starvation medium) for 3 to 4 hours. A and immunoreactive proteins were detected using the SuperSignal West Femtojet micromanipulator and pump (Eppendorf-Brinkman Instruments, Pico chemiluminescent detection system (Pierce). Hamburg, Germany) were used to control the position of the micropipette For myosin-II immunoprecipitations, HeLa cell lysates were prepared by and the pressure required for the chemoattractant flow. To induce the scraping frozen monolayers into an extraction buffer containing 25 mmol/L formation of protrusion, an Eppendorf femtotip II micropipette was filled

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. S100A4 Regulates Cell Polarization with 50% FBS and was placed f40 Am from the edge of a quiescent cell, and a pressure of 16 hPa was exerted to induce flow. Time-lapse series were taken for 10 minutes using a Â20 objective, numerical aperture (NA) of 0.30, and a 12-bit Cooke Sensicam QE cooled CCD camera. Images were analyzed, and protrusion was measured using ImageJ as described by Mouneimne et al. (28). For studies examining the effects of blebbistatin on protrusive activity, the s-(À) active isomer or the s-(+) control isomer (Toronto Research Chemicals, Inc., North York, Ontario, Canada) were dissolved in 100% DMSO to make a 10 mmol/L stock solution. MTC-VC cells were starved for 2.5 hours and then treated with 50 Amol/L blebbistatin in starvation medium for 10 to 30 minutes before stimulation with FBS. Treatment of MTC-VC cells with the inactive isomer produced protrusive activity that was similar to untreated cells (data not shown). Microinjection. For microinjection experiments, cell were plated on poly-L-lysine-coated glass dishes as described above and starved for 1.5 to 2 hours. The myosin-IIA S100A4 binding site antibody or control rabbit IgG were mixed with rhodamine-dextran (10,000 molecular weight; Molecular Probes) to obtain final needle concentrations of 5 mg/mL antibody and 0.5 mg/mL dextran. We estimated a 10-fold dilution of the antibody upon microinjection to yield a final intracellular concentration of 0.5 mg/mL or 3.12 Amol/L antibody, which is comparable with the S100A4 concentration in clone 7. Injected cells were identified by their fluorescence. Micro- injections were done using an Eppendorf semiautomatic microinjection system and a micropipette with a 0.5-Am inner diameter and 1.0-Am outer diameter pulled on a P-97 Flaming/Brown micropipette puller (Sutter Instrument, Inc., Novato, CA). Injected cells were allowed to recover for 1 hour after the microinjection and were then subjected to the micropipette assay. Immunofluorescence. Cells examined in the micropipette assay were Figure 1. S100A4 expression promotes invasion into a three-dimensional fixed at specific times following the addition of chemoattractant with 4% collagen matrix. A, immunoblot analysis of S100A4 expression in MTC-VC and formaldehyde in PBS for 15 to 30 minutes and then permeabilized for MTC-S100A4 clones 6, 7, and 21. h-Actin immunoreactivity was used as a 10 minutes with 0.1% Triton X-100 in PBS. Fixed cells were incubated loading control. B, the proportion of invasive MTC cells (above 20 Am) was j quantified in the absence and in the presence of BAC1.2F5 macrophages. overnight at 4 C with an antibody against the COOH terminus of myosin- Columns, mean for at least two to three independent experiments; bars, SE. IIA (2 Ag/mL), with the S100A4 antibody (2 Ag/mL) or a phosphotyrosine antibody (1:100 dilution). The samples were incubated with Cy3-conjugated secondary antibodies for 1 hour at room temperature, rinsed with PBS, invasion (27). When cultured alone, <9% of the virus control and mounted using Pro-Long Anti-Fade (Molecular Probes). Images were and S100A4-expressing MTC cells invade the collagen gel (Fig. 1B). acquired using IPLab Spectrum software (Scanalytics, Inc., Rockville, MD) f with a CoolSNAP HQ interline 12-bit, cooled CCD camera (Roper Scientific, In the presence of the BAC-1.2F5 macrophages, 7% of the Tucson, AZ) mounted on an Olympus IX70 microscope with a PlanApo Â60, MTC-VC cells and 26% of the MTC-S100A4 clones invade at 1.4 NA objective (Olympus, Melville, NY), and HiQ bandpass filters (Chroma least 20 Am into the collagen. These data show that in the Technology Corp., Rockingham, VT). IPLab images were processed using presence of macrophages, S100A4 expression promotes the Photoshop (Adobe Systems, San Jose, CA). invasion of carcinoma cells into a three-dimensional collagen matrix. To evaluate whether the invasive behavior exhibited by S100A4- Results expressing carcinoma cells was due to alterations in cell motility, To examine the regulation of cellular motility by S100A4, we used we used a micropipette chemotaxis assay (28). A micropipette, a variant of the MTC cell line derived from the 13762NF rat containing FBS, was placed near quiescent cells, and membrane mammary adenocarcinoma tumor, which displays low metastatic protrusion was monitored by video microscopy for 10 minutes potential (29, 30) and stably expresses GFP (20). The parental MTC (Fig. 2E). The MTC-S100A4 cells displayed a strong front protrusion cells infected with the empty vector express very low levels of oriented towards the micropipette, which was observed within S100A4 (Fig. 1A). S100A4-expressing MTC cells were established 2 to 3 minutes after the introduction of the microneedle (Fig. 2A-C; by retroviral infection, and the expression level of S100A4 in clone see Supplementary Video S1), whereas the sides of the MTC- 7 was determined to be 3.54 Amol/L, which is comparable with S100A4 cells did not show any significant protrusion. For clones the expression levels observed in CSML100 cells (10.14 Amol/L), expressing S100A4, forward protrusions were enhanced by 22% to a metastatic mouse adenocarcinoma cell line that normally 34% over side protrusions (Fig. 2A-C), which is consistent with expresses S100A4 (31). Clones 21 and 6 displayed reduced levels the protrusive activity observed in other highly chemotactic and of S100A4 expression compared with clone 7. metastatic carcinoma cells (28). In contrast to S100A4-expressing To determine whether S100A4 expression is associated with cells, the front and side regions of MTC-VC cells protruded nearly invasive behavior, we examined the three MTC-S100A4 clones for to the same extent (Fig. 2D; see Supplementary Video S2). their abilities to invade a collagen I gel. Recent studies indicate Retraction of the back edge was similar in MTC-S100A4 and that carcinoma cells and macrophages are codependent for MTC-VC cells. Altogether, these observations suggest that S100A4 invasion in vivo (32), and that a paracrine loop between these allows cells to assume a more polarized morphology during two cell types is both necessary and sufficient for tumor cell directed motility by affecting the localization of protrusions. www.aacrjournals.org 5175 Cancer Res 2006; 66: (10).May 15, 2006

the supernatant, consistent with our previous observations (17). At a molar ratio of 1 or 2 antibody molecules per myosin-IIA rod, the S100A4 binding site antibody inhibited the assembly of f47% and 84% of the myosin-IIA rods, respectively (Fig. 3C). We also assessed whether the S100A4 binding site antibody could destabilize myosin-IIA filaments in a similar manner to S100A4. At a molar ratio of 1 S100A4 dimer per myosin-IIA rod, we observed significant disassembly of the filaments with f70% of the myosin-IIA in the supernatant (Fig. 3D), which is comparable with the extent of disassembly we observed previously in the presence of S100A4 (17). The S100A4 binding site antibody also promoted the disassembly of myosin-IIA filaments, with 55% and 65% of the myosin-IIA in the supernatant at molar ratios of 2 or 3 antibody molecules per myosin-IIA rod, respectively. These biochemical studies show that the S100A4 binding site antibody and S100A4 have comparable effects on myosin-IIA assembly. S100A4 has been reported to inhibit the actin-activated ATPase activity of myosin-II (34); thus, we examined whether the S100A4 binding site antibody also effects myosin-IIA motor activity. Myosin-IIA phosphorylated on Ser19 had an actin-activated ATPase of 0.40 F 0.14 mol Pi/mol myosin head second (Fig. 3E). At a molar ratio of 8:1 and 10:1 (S100A4 or antibody to full-length myosin-IIA), the actin-activated ATPase activity was inhibited by 50%. To determine if the S100A4 binding site antibody elicits the same effects on protrusive activity as S100A4, MTC-VC cells were microinjected with the antibody or control IgG, and membrane protrusion was evaluated in the micropipette assay. The overall pattern of protrusive activity observed in MTC-VC cells injected with the S100A4 binding site antibody was similar to that observed in the MTC-S100A4 cells. Cells injected with the S100A4 binding Figure 2. S100A4 expression effects cell polarization in response to chemoattractant. Membrane protrusion was quantified for three MTC-S100A4 site antibody exhibited a predominant front protrusion, which clones (A) clone 7, n = 14 cells; (B) clone 21, n = 17 cells; (C) clone 6, n =11 began almost immediately after the introduction of the micropi- cells; and (D) MTC-VC cells, n = 15 cells. Black circles, front protrusion pette (Fig. 4A and B). Furthermore, the extent of the forward (protrusion along the front axis formed between the cell centroid and the tip of the pipette); blue squares, back protrusion (the axis that forms a 180-degree protrusions of cells injected with the S100A4 binding site antibody angle with the front axis); red diamonds and green triangles, side protrusion was enhanced by f10% compared with cells injected with control (the axes that form a 90-degree angle with the front axis). Points, mean for IgG. This is similar to the enhancement observed in forward cells from at least three independent experiments; bars, SE. E, response of a clone 7 cell to a micropipette containing chemoattractant. White triangle, position protrusions in S100A4-expressing cells compared with nonexpress- of the micropipette tip; white arrows, direction of protrusion. Bar, 10 Am. ing cells (Fig. 2A-C). However, forward protrusions formed more rapidly in cells injected with the S100A4 binding site antibody than in S100A4 expressing cells. In contrast to the differences detected Previous studies have shown that antibodies recognizing in forward protrusions, the extent of the side protrusions and epitopes near the COOH terminus of the myosin-IIA heavy chain retraction of the back edge was similar for cells injected with either shift the monomer-polymer equilibrium to the monomeric state antibody. Interestingly, treatment of MTC-VC cells with the and inhibit the actin-activated ATPase activity of myosin-IIA (33). nonmuscle myosin-II ATPase inhibitor blebbistatin ablated all These effects are remarkably similar to the activity of S100A4, protrusive activity in response to chemoattractant (Fig. 4C; see which binds to the COOH-terminal tip of the myosin-IIA a-helical Supplementary Video S3), suggesting that the observed effects on coiled-coil and has comparable effects on myosin-II assembly and protrusive activity in S100A4-expressing cells and cells injected motor activity (17, 34). To test if alterations in the directed motility with the S100A4 binding site antibody does not occur via inhibition of MTC-S100A4 cells are due to the regulation of myosin-IIA of myosin-II motor activity. function by S100A4, we generated an antibody to the S100A4 To determine if the distribution of myosin-IIA is altered in binding site on the myosin-IIA heavy chain. The antibody S100A4 expressing versus nonexpressing cells, MTC-VC or MTC- recognizes a single polypeptide of f200 kDa on immunoblots of S100A4 cells were fixed 7.5 minutes following the addition of HeLa cell lysates and only reacts with purified, recombinant chemoattractant in the micropipette assay and stained with a myosin-IIA rods (Fig. 3A). In addition, under native conditions, the myosin-IIA antibody. In both MTC-S100A4 and MTC-VC cells, antibody specifically immunoprecipitates myosin-IIA from whole- myosin-IIA was observed in the leading edge (Fig. 5B and D). cell homogenates of HeLa cells (Fig. 3B). However, we observed significant myosin-IIA staining in side Next, we examined the ability of the S100A4 binding site protrusions of MTC-VC cells, whereas in MTC-S100A4 cells the antibody to modulate myosin-IIA function. At physiologic salt myosin-IIA was concentrated in the front protrusion that was concentrations, f90% of the myosin-IIA rods assembled into oriented towards the micropipette. S100A4 also concentrated in the filaments (Fig. 4C). In the presence of S100A4 (1:1 ratio of 1 S100A4 leading edge but had a broader distribution than that observed for dimer to myosin-IIA rod), f72% of the filaments were present in myosin-IIA (Fig. 5J). We also compared the localization of focal

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. S100A4 Regulates Cell Polarization contacts in the two cell lines with a phosphotyrosine antibody. injected with the S100A4 binding site antibody do not. Additionally, Although qualitatively similar, focal contacts were observed in both we found that the protrusive activity observed in cells injected cell types, in MTC-S100A4 cells, focal contacts predominated at the with the S100A4 binding site antibody occurs more rapidly in leading edge; whereas in MTC-VC cells, we observed focal contacts response to chemoattractant than S100A4-expressing cells. These along the entire periphery of the cell (Fig. 5F and H). differences in cellular behavior between antibody injected and S100A4-expressing cells likely results from the different regulatory Discussion controls modulating the function of these proteins, as the interaction of S100A4 with myosin-IIA is calcium dependent The motility cycle of migrating cells involves lamellipod (17, 34), whereas binding of the antibody to myosin-IIA is not extension, attachment of the leading lamella to the extracellular regulated. In addition, the calcium dependency of the S100A4/ matrix, the formation of focal contacts, and finally the production myosin-IIA interaction will allow for regional activation of S100A4 of contractile force that causes retraction of the tail towards that is independent of myosin-II concentration, whereas the the leading lamella. Several studies have shown that S100A4 expression levels correlate strongly with motility (14, 15), which is a central element of the metastatic cascade. Moreover, the regulation of myosin-IIA by S100A4 provides a direct link between the actomyosin cytoskeleton and the modulation of cellular motility by S100A4; however, the step or steps in the motility cycle that are affected by S100A4 expression have yet to be examined. Our results show that S100A4 regulates cell polarization during directed motility by affecting the localization of protrusions through interactions with myosin-IIA, with S100A4 expressing cells displaying few side protrusions and extensive forward protrusions during chemotaxis compared with control cells. Consistent with this finding, our localization studies show that during chemotaxis, both S100A4 and myosin-IIA localize primarily to the leading edge of forward protrusions with some staining detected in side extensions. These observations are in agreement with previous studies showing enrichment of S100A4 and myosin-IIA at the leading edge of highly polarized cells (35). In addition, the distribution of focal contacts in S100A4 expressing versus nonexpressing cells differed, consistent with the different protrusive activity of these cells. We show here that S100A4 modulates cellular motility by affecting cell polarization during chemotaxis. To determine if the observed effects of S100A4 expression on protrusive activity are due to the direct modulation of myosin-IIA function, we generated an antibody to the S100A4 binding site on the myosin-IIA heavy chain that mimics the effects of S100A4 binding on myosin-IIA assembly and motor activity. Notably, we found that the antibody elicits comparable effects on cellular protrusive activity as S100A4. Similar to S100A4, the S100A4 binding site antibody enhances forward protrusions. However, cells expressing moderate to high levels of S100A4 also suppress side protrusions, whereas cells

Figure 3. The S100A4 binding site antibody specifically recognizes myosin-IIA (MIIA) and elicits the same biochemical effects as S100A4 on myosin-IIA assembly and ATPase activity. A, immunoblots of a HeLa whole-cell lysate and purified myosin-IIA and myosin-IIB (MIIB) rods. The S100A4 binding site antibody reacts with a single band in HeLa lysates and only recognizes the myosin-IIA rods. B, a HeLa cell lysate was treated with protein A-Sepharose coupled with the S100A4 binding site antibody. Samples of the immunoprecipitate and unbound flow through (FT) were examined by immunoblot analysis with antibodies to myosin-IIA or myosin-IIB. The S100A4 binding site antibody immunoprecipitates only myosin-IIA. C, the S100A4 binding site antibody inhibits the assembly of myosin-IIA monomers. At physiologic salt concentrations, the assembled myosin-IIA rods pellet, whereas the unassembled monomers (f10%) remain in the supernatant. At 2 mol antibody per mol of myosin-IIA rod, the S100A4 binding site antibody strongly inhibits the assembly of the myosin-IIA rods in a similar manner to S100A4. D, the S100A4 binding site antibody promotes the disassembly of preexisting myosin-IIA filaments. At a molar ratio of 3 antibody molecules per myosin-IIA rod, f64% of the myosin-IIA rods are recovered in the supernatant, which is comparable with the effects of S100A4 on filament destabilization. E, the S100A4 binding site antibody inhibits the actin-activated ATPase activity of myosin-IIA. At a molar ratio of 10 antibody molecules per myosin-IIA, the ATPase activity decreased by 50%. Columns, mean for at least three independent experiments; bars, SE. www.aacrjournals.org 5177 Cancer Res 2006; 66: (10).May 15, 2006

with our findings that the antibody primarily effects protrusive activity at the front of the cell. Lastly, potential differences could also result from the interaction of S100A4 with other proteins, as S100A4 has been proposed to bind several target molecules, such as F-actin, tropomyosin, and p53 (36–38). The apparent correlation between increased directional protrusions and the regulation of myosin-IIA is consistent with previous observations. Kendrick-Jones et al. showed that a monoclonal antibody directed against the tip of the avian myosin-IIA tail, which has the same biochemical effects on myosin-IIA assembly and motor activity as S100A4, induces the formation of extensive lamellae and increased cellular motility in primary fibroblasts (33, 39). The mechanisms by which these antibodies and S100A4 increase protrusive activity and promote cellular motility are not well understood, but the observation that S100A4 can inhibit the actin-activated ATPase of myosin-IIA (34) suggests that S100A4 could function as a myosin-IIA inhibitor in vivo. However, the inhibition of myosin-IIA motor activity by S100A4 is likely an indirect effect of the ATPase assay and is related to the actin cross- Figure 4. The S100A4 binding site antibody effects cell polarization in response linking properties of myosin-II itself. In an actin-activated ATPase to chemoattractant. Membrane protrusion was quantified for MTC-VC cells assay, full-length myosin-II forms filaments that cross-link actin injected with (A) the S100A4 binding site antibody, n = 10 cells or (B) control IgG, n = 11 cells. Black circles, front protrusion; blue squares, back protrusion; filaments, thus decreasing the apparent dissociation constant red diamonds and green triangles, side protrusion. Points, mean for cells from (Kapp) for actin and increasing the actin-dependent ATPase activity at least three independent experiments; bars, SE. C, response of a MTC-VC at subsaturating actin concentrations. Under conditions in which cell to a micropipette containing chemoattractant following treatment with blebbistatin. White triangle, position of the micropipette tip. Bar, 10 Am. the myosin-II filaments are partially disassembled, there is decreased actin filament cross-linking and thus an apparent decrease in the actin-activated ATPase activity, which is due to a antibody will accumulate in subcellular compartments that are rich lower local F-actin concentration rather than a direct biochemical in myosin-IIA (i.e., the leading edge). Interestingly, our localization effect on the myosin-II motor (40–43). Moreover, our observations studies show that myosin-IIA is enriched in the leading edge but that blebbistatin suppresses protrusive activity, whereas S100A4 not the sides of locomoting cells. This observation is consistent enhances protrusive activity, suggests that S100A4 does not elicit

Figure 5. Distribution of myosin-IIA, S100A4, and focal complexes in MTC-S100A4 and MTC-VC cells. Following stimulation with chemoattractant for 7.5 minutesin the micropipette chemotaxis assay, cells were fixed and stained with a myosin-IIA antibody (B and D), with a phosphotyrosine antibody (F and H), or with an S100A4 antibody (J). Corresponding phase-contrast and fluorescence micrographs of MTC-S100A4 clone 7 cells (A, B, E, F, I, and J) and MTC-VC cells (C, D, G, and H). Insets, phase-contrast micrographs of the cells before stimulation. White triangles, positions of the micropipette tips. Bar, 10 Am.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. S100A4 Regulates Cell Polarization its biochemical effects through inhibition of the myosin-IIA motor regulated proteins at the leading edge of motile cells is necessary for but primarily via the regulation of myosin-IIA assembly. Given all localized control of protrusive activity. Moreover, the transient of the above considerations, we propose a model in which S100A4 nature of the calcium waves detected at the leading edge provides a enhances directed motility by promoting the turnover of myosin- mechanism for regional activation of S100A4 and MLCK during IIA filaments at the leading edge. directed motility and would allow for the cycles of protrusion, An examination of calcium levels in migrating cells has shown adhesion, and retraction needed for migration. Future studies that the intracellular calcium concentration is high at the rear of directed at examining how S100A4 affects the assembly of myosin- the cell and low at the leading edge (44, 45), a finding that is not IIA at the leading edge will provide insight into S100A4-mediated consistent with the regulation of myosin-IIA by S100A4 at the front regulation of normal cell physiology and the pathologies contrib- of locomoting cells. However, more recent studies have reported uting to tumor cell invasion and metastasis. a locally high calcium wave near the plasma membrane that originates at the leading edge and propagates along the cell periphery (46, 47). These findings provide a mechanism for the Acknowledgments activation of S100A4 at the leading edge of migrating cells and are Received 8/29/2005; revised 3/6/2006; accepted 3/16/2006. consistent with the observation that a number of calcium-regulated Grant support: NIH grant GM069945. The costs of publication of this article were defrayed in part by the payment of page proteins (e.g., MLCK and gelsolin) are active in the leading edge of charges. This article must therefore be hereby marked advertisement in accordance motile cells (48–50). Interestingly, inhibition of MLCK, a dedicated with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. Richard Stanley (Albert Einstein College of Medicine, Bronx, NY) for myosin-II kinase, results in multiple protrusions and a loss of cell CSF-1; Drs. Jeff Segall and John Condeelis (Albert Einstein College of Medicine, Bronx, polarity (49). These observations suggest that MLCK and S100A4 NY) for the MTC-GFP cell line; Dr. Jim Stull (University of Texas Southwestern Medical may act in concert to promote a highly polarized morphology in Center, Dallas, TX) for the rabbit smooth muscle myosin light chain kinase; Dr. Erik Sahai and Ghassan Mouneimne for assistance with the three-dimensional invasion response to chemoattractant and indicate that tight regulation of and micropipette assays, respectively; and Dr. Steven Almo for critical reading of the myosin-II motor function and filament assembly by calcium- article.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. The S100A4 Metastasis Factor Regulates Cellular Motility via a Direct Interaction with Myosin-IIA

Zhong-Hua Li and Anne R. Bresnick

Cancer Res 2006;66:5173-5180.

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