THE HYALURONAN RECEPTORS CD44 AND RHAMM (CD168) FORM COMPLEXES WITH ERK1,2, WHICH SUSTAIN HIGH BASAL MOTILITY IN BREAST CANCER CELLS

Sara R. Hamilton1, Shireen F. Fard1*, Frouz F. Paiwand2*, Cornelia Tolg1*, Mandana Veiseh4,

Chao Wang2, James B. McCarthy3, Mina J. Bissell4, James Koropatnick1 and Eva A. Turley1**

1London Regional Cancer Program, London Health Sciences Centre/The University of Western Ontario (London, ON Canada); 2Department of Cardiovascular Research, Hospital for Sick Children (Toronto, ON, Canada); 3Department of Laboratory Medicine and Pathology and

University of Minnesota Comprehensive Cancer Center (Minneapolis, MN, USA); 4 Division of Life Sciences, Lawrence Berkeley National Laboratories, Berkeley CA

Running Title: Rhamm and CD44 coordinate in breast cancer motility. **Address correspondence to: Eva A. Turley, London Regional Cancer Program, Cancer Research Laboratories, Room A4-931, 790 Commissioners Road E, London ON, Canada N6A 4L6, Tel. 519 685-8600 ext. 53677; Fax: 519 685-8616; E-mail: eva.turley@lhsc.on.ca CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a non-integral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture and its expression is strongly upregulated in diseases like arthritis and aggressive cancers. Here we describe an autocrine mechanism utilizing cell surface Rhamm/CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines. This mechanism requires endogenous hyaluronan synthesis and the formation of Rhamm/CD44/ERK1,2 complexes. Motile/ invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit elevated basal activation of ERK1,2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm and ERK1,2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Rapid motility of the invasive cell lines requires interaction of hyaluronan with cells, activation of ERK1,2 and the participation of both cell surface CD44 and Rhamm. Combinations of anti-CD44, anti-Rhamm antibodies and a MEK1 inhibitor (PD098059) have less-than-additive blocking effects, suggesting action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent, autocrine mechanism to coordinate sustained signaling through ERK1,2 leading to high

basal motility of invasive breast cancer cells. Since CD44/Rhamm complexes are not evident in less motile cells, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, in this case cell surface Rhamm.

Breast cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment (1,2). Motogenic signaling in tumor cells can be stimulated by both paracrine and autocrine factors: the latter decrease the requirement of invasive carcinomas for stromal support and is often associated with tumor progression (3-6). Hyaluronan (HA, an anionic polymer of repeating units of glucuronic acid and N-acetylglucosamine) is one stromal ECM component that is associated with breast cancer progression (7,8). In culture, HA stimulates breast cancer cell motility (9-11), pointing to a possible important role of this glycosaminoglycan in breast cancer cell invasion in vivo.

CD44 is a broadly expressed, type I integral cell surface membrane glycoprotein that participates in cell-cell and cell-matrix adhesions, and in particular binds to HA (12,13). It is encoded by a single gene but exists as multiple isoforms that are generated by alternative splicing of 10 variable exons, as well as through post-translational modifications (12). The most commonly expressed CD44 isoform (the standard form or CD44std) is an 85kDa protein that contains none of the variable exons. Originally

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http://www.jbc.org/cgi/doi/10.1074/jbc.M702078200The latest version is at JBC Papers in Press. Published on March 28, 2007 as Manuscript M702078200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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described as the principal cell surface receptor for HA (14), CD44 has since been shown to bind multiple ligands including fibronectin (15) and osteopontin (16), although, in general, the ligands for many of the CD44 variants are not yet known. However, a role for CD44std as a mediator of HA-promoted motility in breast cancer cell lines is well established on tissue culture plastic (2D cultures) (7,17-19). CD44 isoforms containing variable exons v6 and v9 are also involved in HA-mediated signaling [e.g. in activated T cells (20)] and expression of isoforms containing variable exons v4-v7 enhances HA binding of a rat pancreatic adenocarcinoma cell line (21). These results suggest that in addition to CD44std, at least some variants of CD44 isoforms can bind HA. CD44 binds to HA via an extracellular domain while the cytoplasmic tail activates intracellular signaling pathways that regulate the association of signaling complexes with the cortical actin cytoskeleton (13,17,22,23).

Evidence suggesting that CD44 has motogenic/invasive functions in 2D cultures motivated numerous histopathological evaluations of CD44 expression in breast cancer. Although several groups report that CD44std expression positively correlates with disease-related survival whereas expression of CD44 variants correlates with poor prognosis (Gotte M and Yip G 2006), other studies contradict these results (24-27). Furthermore, analysis of breast cancer progression in a CD44-/- mouse background (where there is an absence of all CD44 isoforms) indicates that loss rather than gain of CD44 expression is associated with increased metastasis (13,27). These observations predict a potential for CD44 to act as both as a tumor progression enhancer and a tumor suppressor [e.g. (28,29)]. The basis for an association of CD44 with different outcomes in breast cancer patients or in animal models of this disease is not well understood. One possibility is that differential expression/function of CD44 isoforms in tumor cell subsets, including progenitors, may affect clinical outcome (30-32). However, CD44 is also known to associate with, and facilitate, signaling through such tumor cell-associated proteins/receptors as metalloproteinases (MMPs) (33,34), c-met and EGFR (35,36); therefore, the consequences of CD44 expression to tumor cell behavior and its signaling properties may be modified by proteins it associates with, and vice versa. For example, co-expression of CD44v4 with one of its cell surface binding partners (MMP-9) correlates with node-positivity in breast cancer patients, while the expression of CD44v4 alone

does not (34). Furthermore, hyper-expression of other CD44-binding partners including specific hyaluronan synthases and hyaluronidases (e.g., HAS-2 and hyal-2), together with CD44 (but not CD44 alone) correlate with the degree of invasiveness of human breast cancer cell lines (10).

Cell surface Rhamm (CD168) is an HA-binding protein/receptor that is not highly expressed in normal tissues but is commonly over-expressed in many advanced cancers (18,19), including breast cancer (37,38). Rhamm was first identified as an HA-dependent motility cell surface receptor that can transform fibroblasts when overexpressed (39,40). Rhamm is also a cytoplasmic and nuclear protein that interacts with interphase microtubules, centrosomes and the mitotic spindle, suggesting that it performs multiple functions in a number of cell compartments (41-43). Importantly, spindle-associated functions of Rhamm are blocked by the breast/ovarian tumor suppressor gene BRCA1 (43). This functional link between Rhamm and BRCA1, a factor in hereditary forms of breast cancer, as well as evidence that hyper-expression of Rhamm predicts poor clinical outcome and increased risk of sporadic breast cancer metastasis (37) suggest it may be influential in both inherited and non-inherited forms of breast cancer. However, the relevance of its multiple intracellular vs. extracellular functions to human breast cancer aggressiveness has not been fully established yet. We recently showed that the motility defects of Rhamm-/- wound fibroblasts could be rescued by soluble recombinant Rhamm protein linked to sepharose beads. Rescue required a concomitant surface display of CD44 (42) but did not require expression of intracellular Rhamm forms. These results suggest that at least some of the functions regulated by intracellular vs. extracellular Rhamm are distinct.

As a consequence of its ability to bind to HA, cell surface Rhamm activates multiple motogenic signaling pathways that have been implicated in breast cancer progression. These include Ras (40), pp60-c-src (44) and ERK1,2 (37). Cell surface Rhamm is required for sustained activation and intracellular targeting of ERK1,2 in dermal wound fibroblasts (45) suggesting that the extracellular Rhamm form could potentially function in tumor progression to increase the intensity and duration of signaling pathways associated with tumor invasion/motility. Importantly, cell surface Rhamm can additionally perform motogenic/invasive functions similar to CD44 and can even replace CD44 (46). These observations have

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raised the possibility that cell surface Rhamm may partner with CD44 to “unleash” its motogenic potential (45,46).

Although cell-autonomous tumor progression events can clearly contribute to the aggressiveness of breast cancer cells, such cells still remain sensitive to some exogenous factors in their microenvironment [for review see (47)], including cytokines/growth factors and extracellular matrix components such as HA (48,49). Indeed, the accumulation of HA within breast tumors or peritumor stroma is an indicator of poor prognosis in breast cancer patients (50). ECM factors such as HA act coordinately with activating mutations in critical signal transduction pathways to modify tumor cell behavior (51). ECM-mediated activation of the Ras/Raf/MEK1,2/ERK1,2 cascade (52-54) is one motogenic pathway associated with breast tumor progression. Invasive breast cancer cell lines such as MDA-MB-231 have higher basal ERK1,2 activity than less invasive cell lines (including MCF7) and sustained ERK1,2 activity is required for the increased motility/invasion of the aggressive breast cancer cell lines (54). The molecular mechanisms driving sustained ERK1,2 activation and the consequent effects on tumor motility/invasion are poorly understood (55-58).

In this study we show that invasive breast cancer cells (MDA-MB-231 and Ras-MCF10A) sustain elevated levels of ERK1,2 activation upon growth factor/motogenic stimulation when cell surface CD44 and Rhamm are co-expressed and co-associate with each other. In contrast, less invasive (MCF7) and non-malignant (MCF10A) cell lines express lower levels of either HA receptor at the cell surface despite the fact that Rhamm is expressed as a cytoplasmic protein, and can only transiently activate ERK1,2. We demonstrate also that CD44 and Rhamm co-associate with ERK1,2 as complexes in aggressive breast tumor cell lines. Finally, we show that these CD44/Rhamm/ERK1,2 complexes are required for rapid basal motility of the more invasive cell lines. These results are consistent with a model in which HA, CD44, Rhamm and activated ERK1,2 are physically and functionally linked in a biological complex to establish an autocrine mechanism for promoting motility in breast cancer cells.

Experimental Procedures

Reagents (Antibodies, Growth factors, Hyaluronan and Peptide Inhibitors) – Medical grade HA prepared

from bacterial fermentation (provided by Hyal Pharmaceutical Co., Mississauga, ON) was free of detectable proteins, DNA or endotoxins. The MW range was 250-300kDa. The following primary antibodies were obtained commercially or as gifts: ERK1 and non-immune IgG (Santa Cruz); phospho-ERK1,2 (Cell Signaling); p21Ras (Oncogene Science, Cambridge, MA); CD44 (IM7, Pharmingen); CD44 (Hermes-3, kind gift of Dr. Sirpa Jalkanen, University of Kuopio, Finland). Anti-CD44 antibodies used were raised against sequence in CD44std form and therefore detect all CD44 isoforms. Polyclonal Rhamm antibodies used in this study were prepared (Zymed, San Diego, CA) against the following human Rhamm sequences: Antibody-1 (Ab-1): KSKFSENGNQKN (aa150-162), antibody-2 (Ab-2): VSIEKEKIDEKS (aa217-229), and antibody-3 (Ab-3): QLRQQDEDFR (aa543-553) (59,60). Specificity of Rhamm and CD44 antibodies were determined using Rhamm-/- and CD44-/- lysates, respectively, and with Rhamm peptide competition. The following secondary antibodies were purchased: for western blot detection, horseradish peroxidase (HRP)-conjugated anti-mouse (Bio-Rad Laboratories, Hercules, CA), anti-rabbit (Amersham, Oakville, ON), and anti-rat (Santa Cruz); for immunofluorescence analysis, anti-rabbit Alexa 555 and anti-rat Alexa 433 (Molecular Probes). The MEK1 inhibitor, PD098059 (2-[2’-amino-3’-methoxyphenyl]-oxanaphthalen-4-one]), was purchased from Calbiochem Biosciences (Mississauga, ON). An HA binding peptide (YKQKIKHVVKLK), which blocks macrophage migration, was synthesized (61). A scrambled peptide, YLKQKKVKKHIV was used as a control. Cell Culture - Human breast carcinoma cell lines MDA-MB-231 and MCF7 were obtained from American Type Culture Collection (Manassas, VA) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, Burlington, Ontario) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Hyclone Laboratories Inc., Logan, UT) and 10mM HEPES (Sigma Chemical Co., St. Louis, MO), at pH 7.2. The immortalized human breast epithelial cell line MCF10A, transfected with the empty pH106 plasmid containing the neomycin resistance gene and MCF10A cells transfected with the human mutant H-Ras oncogene (mutated at G12-V12) were a kind gift of Dr. Channing Der (North Carolina) and were grown as previously described (62,63). Briefly, the cells were grown in DMEM/F-12 (1:1) supplemented with 5% equine serum, 0.1µg/mL

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cholera toxin, 10µg/mL insulin (Gibco BRL), 0.5µg/mL hydrocortisone (Sigma) and 0.02µg/mL epidermal growth factor (Collaborative Research Inc., Palo Alto, CA). All cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C. Western Immunoblotting - Cells plated at 50% subconfluency for 12 hours were washed with ice-cold phosphate buffered saline (PBS) and lysed in ice-cold RIPA buffer (25mM Tris-HCl, pH 7.2, 0.1% SDS, 1% Triton-X-100, 1% sodium deoxycholate, 0.15M NaCl, 1mM EDTA, and 50nM HEPES [pH 7.3]) containing the protease and phosphatase inhibitors leupeptin (1µg/mL), phenylmethylsulfonyl fluoride (PMSF, 2mM), pepstatin A (1g/mL), aprotinin (0.2TIU/mL) and 3,4-dichloroisocoumarin (200mM), sodium orthovanadate, and 1mM NaF (Sigma Chemical Col, St. Louis, MO). Cell lysates were then micro-centrifuged at 13,000 x g for 20 minutes at 4°C (Heraeus Biofuge 13, Baxter Diagnostics, Mississauga, ON) after standing for 20 minutes on ice. Protein concentrations of the supernatants were determined using the DC protein assay (Bio-Rad). 10µg of total protein from each cell lysate was loaded and separated by electrophoresis on a 10% SDS-PAGE gel together with prestained molecular weight standards (Gibco BRL). Following electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad) in a buffer containing 25mM Tris-HCl (pH 8.3), 192mM glycine and 20% methanol using electrophoretic transfer cells (Bio-Rad) at 100V for 1.5 hour at 4°C. Additional protein binding sites on the membrane were blocked with 5% defatted milk in TBST (10mM Tris base (pH 7.4), 150mM NaCl, and 0.1% Tween-20 (Sigma)). The membranes were incubated with the primary antibodies for Rhamm, CD44, Ras or ERK1,2 (all diluted at 1:1000 or 1µg/mL in 1% defatted milk in TBST) for 2 hours at room temperature. The membranes were washed three times at 15 minute intervals with 1% defatted milk in TBST. Immunodetection was performed using secondary antibodies conjugated to HRP (diluted 1:5000 or 1mg/mL) in 1% defatted milk in TBST for 1 hour at room temperature followed by three washes with TBST. Blotting was visualized by the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. Quantification of optical densities of the reactive protein bands was performed on a Bio-Rad Video Densitometer. To account for variations in loading, parallel SDS gels were carried out with each

experiment in which equal amounts of the protein lysates were run. These gels were then stained with Coommassie blue dye in order to confirm equal loading. The densitometric results were presented as a mean of three experiments ± standard deviations and are presented as a percentage of the protein of interest normalized to a dominant 60kDa marker band on the Coommassie stained gels. Analysis of EGF stimulated ERK1,2 activation – 5 x 104 MCF7 and MDA-MB-231 cells were plated in complete growth medium (DMEM, 10% FCS) on 6 cm cell culture plates and allowed to attach for 4 hours. The growth medium was replaced by defined medium (DMEM, 4μg/mL insulin, 8μg/mL transferrin). After overnight culture, cells were stimulated with 20ng/mL EGF (Sigma) in defined medium. For antibody blocking experiments, cell surface Rhamm and/or CD44 function was blocked by pre-incubating cells for 30 minutes in the presence of either anti-CD44 antibody (IM-7, 10μg/mL), anti-Rhamm antibody (10μg/mL), IgG (10μg/mL) or a combination of anti-CD44 and anti-Rhamm antibodies prior to EGF stimulation. EGF stimulation, protein isolation and SDS-PAGE were performed as described above. The densitometric results were presented as a mean of triplicate samples ± standard deviations. Levels of phospho-ERK1,2 protein were normalized to total ERK1,2 protein and were presented as fold changes. Measurement of HA production - Cells were plated at sub-confluence in DMEM + 10% FCS for 12 hours, followed by serum free medium replacement for another 24-48 hours. HA released into the medium was collected and assayed using an ELISA assay (Amersham Pharmacia Biotech, Piscataway, NJ) as per the manufacturer’s instructions. Fluorescence Activated Cell Sorting (FACS) - Cells were grown to 50% subconfluence on 15cm culture plates in growth media and were rinsed with Ca2+-free Hank’s Buffered Saline Solution (HBSS)/20mM HEPES, pH 7.3 hours later. Cells were harvested with non-enzymatic HBSS-based cell dissociation solution (Sigma) and resuspended in 5mL cold PBS and centrifuged at 1200 rpm for 3 minutes. Cells were washed in another 5mL cold PBS and then blocked in cold 10% FCS/HBSS/HEPES (FACS buffer) for 30 minutes. The viability of released cells was between 85% and 95%, by Trypan blue exclusion. For detection of cell surface Rhamm (64), an aliquot of 2 x 106 cells was incubated with anti-Rhamm antibody

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(1:100, 1µg/µL) in a total volume of 200µl of FACS buffer for 30 minutes on ice, and washed three times in cold FACS buffer. Rabbit IgG (1:100 of 1µg/mL) was used as a negative control for each cell line. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:300 dilution, Sigma) in FACS buffer was then added and incubated for 30 minutes in the dark on ice. The cells were washed again and examined with a flow cytometer (Beckman Coulter) using FACS Calibur with Cell Quest acquisition and analysis software (Becton Dickinson, Lincoln Park, NJ). For detection of cell surface CD44 (65,66), 1 x 106 cells were incubated with 1μg anti-CD44 antibody (clone IM7, Pharmingen) or 1μg rat IgG antibody (Santa Cruz) in PBS/2% BSA for 1 hour on ice after which time they were washed with cold PBS/2% BSA. Cells were then incubated with rabbit anti-rat Alexa 488 (diluted 1:100, Molecular Probes) in PBS/2% BSA for 1 hour on ice. Cells were washed in cold PBS/2% BSA. Cells were resuspended in 1mL fresh, cold PBS/2% paraformaldehyde (Sigma) and were stored overnight at 4˚C. Before flow cytometric analysis, samples were filtered (cell strainer caps, Becton Dickinson Labware). Data was collected using a Beckman Coulter flow cytometer. Viable cells were gated based on forward and side scatter to eliminate dead aggregates and debris, and then the distribution of fluorescence intensity was calculated. Immunoprecipitation assays - Co-immunoprecipitation analyses were performed using 400µg of protein from each cell lysate mixed with 5µg of either anti-Rhamm, anti-CD44, anti-ERK1, anti-rabbit IgG, anti-mouse IgG antibodies or anti-rat IgG antibodies. After 12 hours of incubation at 4°C on a rotator, 25µl of a 50% suspension of protein A/G-Sepharose beads (Gibco BRL) was added to each tube and the samples were mixed end-over-end for another 4 hours at 4°C. The beads were pelleted by brief centrifugation at 7000 x g and washed three times with cold 0.5% Triton-X-100/PBS. Bound proteins were released from the beads by heating the samples in 25µl of 2X Laemmli buffer for 5 minutes. Protein samples were subjected to 12% SDS-PAGE and immunoblotted as described above. Pull down binding assays - In vitro pulldown binding assays were performed using recombinant Rhamm protein (63kDa and 43kDa isoforms) that was purified as a GST fusion protein as previously described (67). Briefly, 1mg of cellular lysate was incubated with recombinant Rhamm-GST or recombinant GST

protein on glutathione Sepharose beads (Amersham) overnight at 4oC. Beads were then pelleted by centrifugation and washed five times with 1mL of cold lysis buffer. Bound proteins were released from the beads by heating the samples in 25µl of 2X Laemmli buffer for 5 minutes. Protein samples were subjected to 10% SDS-PAGE and immunoblotted as described above. Time-Lapse Cinemicrography - Motility analyses were performed to quantify the effect of function blocking Rhamm and CD44 antibodies, HA binding peptide, hyaluronan, and the MEK1 inhibitor, PD098059 on cell motility. PD098059 MEK1 inhibitor was used to test the involvement of the MAP kinase pathway in the motility of these cells. Cells were seeded on T-25 flasks (Costar, Cambridge, MA) at 1 x 105 cells/flask. Cells were incubated with anti-Rhamm antibody (30µg/mL), anti-CD44 antibody (30µg/mL), a mixture of anti-Rhamm (30µg/mL) and anti-CD44 (30µg/mL) antibodies, HA binding peptide (1µg/mL) or its scrambled control (1µg/mL), and/or 50µM PD098059 for 30 minutes prior to filming. Alternatively, cells were stimulated with 50µg/mL of HA immediately prior to filming. As a control, a mixture of mouse or rat and rabbit IgG (30µg/mL each), DMSO (for PD098059) or PBS (for HA) were used. Cell locomotion was monitored for a period of 6 hours using a 10X modulation objective (Zeiss, Germany) attached to a Zeiss Axiovert 100 inverted microscope equipped with Hoffman Modulation contrast optical filters (Greenvale, NY) and a 37°C heated stage. Cell images were captured with a CCD video camera module attached to a Hamamatsu CCD camera controller. Motility was assessed using Northern Exposure 2.9 image analysis software (Empix Imaging, Mississauga, Ontario). Nuclear displacement of 20-30 cells was measured and data were subjected to statistical analysis (see below). Each experiment was repeated at least three times. The results of motility analyses were expressed as means (µm/4 hour) means + standard deviations, unless otherwise indicated. Confocal Microscopy - MCF7 and MDA-MB-231 cells were plated sparsely (approximately 5000 cells/well) on coverslips in a 24-well dish. The cells were incubated overnight in DMEM supplemented with 10% FCS. Cells were rinsed briefly with 1%BSA/TBS and were then fixed in fresh 3.7% paraformaldehyde in TBS for 10 minutes at room temperature. Cells were rinsed with 1%BSA/TBS and were then permeabilized with 0.5% Triton X-100 in

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1%BSA/TBS for 15 minutes at room temperature. Cells were again rinsed in 1%BSA/TBS and blocked in 5%FCS in 1%BSA/TBS for 1 hour at room temperature. For the phospho-ERK1,2/CD44 double staining, cells were incubated with anti-phospho p44/p42 MAP kinase (Thr202/Tyr204) and anti-CD44 (IM7) antibodies, each diluted 1/100 in 1%BSA/TBS, for 1 hour at room temperature. For the Rhamm/CD44 double staining, cells were first incubated with an anti-Rhamm (59,60) antibody, diluted 1/100 in 1%BSA/TBS, overnight at 4oC. After the overnight incubation, cells were further incubated with the anti-CD44 antibody (IM7), diluted 1/100 in 1%BSA/TBS, for 1 hour at room temperature. After incubation with primary antibodies, cells were rinsed four times for 10 minutes each in 1%BSA/TBS. Primary antibodies were then visualized by incubating cells with anti-rabbit Alexa 555 and anti-rat Alexa 488, diluted 1/150 in 1%BSA/TBS, for 1hour at room temperature. After incubation with secondary antibodies, cells were rinsed four times for 10 minutes each in 1%BSA/TBS. Cells were briefly incubated with DAPI (4’,6-Diamidino-2-phenylindole) diluted in 1%BSA/TBS and then mounted (IF mounting medium, DAKO) on slides. A Zeiss LSM510 Meta Multiphoton confocal microscope equipped with LSM 5 imaging software was used to visualize the cells (Dept. Anatomy and Cell Biology, UWO). Green and red images were captured individually and were merged using the LSM 5 Imaging software. Image Analysis – Colocalization was identified using the co-localization finder plug in of Image J software (https://rsh.info.nih.gov/ij/). This software allows identification of co-localization based on a correlation diagram of two (red and green) images. Pixels that contain information from both channels are considered co-localization and are converted to white on the image. This is therefore an enhancement tool used to more easily visualize co-localization and should not be considered quantitative. Statistical analysis - Statistically significant (p<0.05) differences between means were assessed by the unpaired, two-tailed Student’s t-test method. Cell motility was based on means of at least 30 cells per experiment and western blot quantification was based on triplicate samples, unless otherwise indicated. Hyaluronan production was reported as a mean of 10 separate cultures.

RESULTS

Invasive breast tumor cell lines produce endogenous HA that sustains rapid motility. HA, a motogenic factor that is synthesized in large amounts by aggressive breast cancers and that correlates with clinical progression of breast cancer (10), promotes both ERK1,2 activation and breast tumor cell motility/invasion (7,10,17,18). Consistent with these previous reports, HA levels in the medium of cultured cells were significantly greater in the aggressive MDA-MB-231 cells than in less aggressive MCF7 cells (Fig. 1A); similar differences were observed also between Ras-MCF10A and MCF10A cells in culture (data not shown). We therefore quantified the reliance of breast tumor cell lines on endogenous HA production for maintaining motility in culture using an HA binding peptide that blocks cell-HA interactions (61). The HA-binding peptide (YKQKIKHVVKLK) significantly reduced motility of MDA-MB-231 cells but had no effect on the MCF7 cell line (Fig. 1B). A scrambled control peptide (YLKQKKVKKHIV), which does not bind to HA or affect HA-mediated macrophage motility (61), did not block motility of either cell line. A motogenic response of breast cancer cell lines to exogenous HA was also quantified. Cell cultures were first serum-starved for 24-48 hours to reduce endogenous levels of HA production (<1ng/mL) (Fig. 1C), then rescue of motility with the addition of 50µg/mL exogenous HA was quantified. Using these conditions, the exogenous HA significantly stimulated motility of MDA-MB-231 but not MCF7 cells (Fig. 1C). Collectively, these results show that invasive breast tumor cell lines such as MDA-MB-231 have established an HA-dependent autocrine mechanism in the presence of growth factors and/or serum that is required for rapid rates of motility. By contrast, poorly invasive breast cancer cells (e.g., MCF7) lack this mechanism since they produce low levels of HA and do not respond to exogenous HA provided in their microenvironment.

Invasive breast tumor cells display both cell surface CD44 and Rhamm. Since the invasive breast cancer cell lines can utilize HA to promote motility, we determined if HA receptors such as CD44 and Rhamm are expressed on these cells. CD44 and Rhamm protein expression was quantified by western blot, flow cytometry and fluorescent confocal analysis. Western blot and flow cytometry analysis indicated that levels of total CD44std (Fig. 2A) and cell surface CD44 (Fig. 2B) were greater in MDA-MB-231 than in MCF7 cells. Confocal analysis confirmed high CD44 expression in MDA-MB-231 cells and lower

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expression in MCF7 cells (Fig. 5A, panels b and f; see below). Similar differences in CD44 were observed also between Ras-MCF10A cells and their parental, non-transformed counterparts (Suppl. Fig. Ia and data not shown).

Like CD44, Rhamm has been reported to exist as several protein isoforms (18,68): these include N-terminal truncations of full length Rhamm (40) that are transforming in fibroblasts. To characterize expression of protein isoforms in breast cancer cell lines, peptide-specific polyclonal antibodies were generated against sequence in the amino- (aa150-162, Ab-1) and carboxyl termini (aa542-553, Ab-3) as well as an internal region (aa217-229, Ab-2) of the human full-length Rhamm sequence (Fig. 3A). An 85kDa protein corresponding to full-length Rhamm was expressed to a greater extent in MDA-MB-231 than in MCF7 cells and, as expected, was detected by all three antibodies (Fig. 3A, and data not shown). Shorter Rhamm forms (64 and 43kDa) expressed by the breast cancer cell lines were detected by Ab-2 (Fig. 3A) and Ab-3 (data not shown) but not Ab-1 indicating they are N-terminal truncations of the full length Rhamm protein. In particular, the 63kDa Rhamm protein was abundant in MDA-MB-231 cells and corresponds to the oncogenic Rhamm isoform that is expressed in aggressive human tumors (18,69,70). This isoform is transforming in fibroblasts as well (40). Intriguingly, this molecular weight also corresponds to a cell surface form of Rhamm expressed by macrophages (68). FACS analysis showed that MDA-MB-231 cells expressed more cell surface Rhamm than MCF7 cells (Fig. 4) although both cell lines expressed intracellular Rhamm, detected by confocal microscopy (Fig. 5A, panels a and e). Intracellular Rhamm occurred in the cytoplasm and on cytoskeletal structures in MCF7 cells (most likely the interphase microtubules, Fig. 5A, panel a). In contrast, Rhamm was primarily detected in cell processes and the perinuclear region of MDA-MB-231 cells (Fig. 5A, panel e). Similar quantitative and qualitative differences in Rhamm expression were observed also when Ras-MCF10A and parental MCF10A cells were compared (Suppl. Fig. Ib and data not shown). Thus, aggressive breast cancer cell lines are characterized by high levels of both cell surface CD44 and Rhamm while less aggressive cell lines express only low levels of these two proteins at the cell surface.

Confocal analyses were next done to look at the distribution of CD44 and Rhamm in the MDA-MB-231 and MCF7 cells. Fig. 5A shows Rhamm

[panels a (MCF7) and e (MDA-MB-231)] and CD44 staining in both cell lines [panels b (MCF7) and f (MDA-MB-231)], as well as the overlay of Rhamm and CD44 staining [panels c (MCF7) and g (MDA-MB-231)]. IgG was used as control [panels d (MCF7) and h (MDA-MB-231)]. These images showed that CD44 and Rhamm co-distributed in cell processes and in the perinuclear region of MDA-MB-231 cells (Fig. 5A, panel g; panel gi shows higher magnification images of MDA-MB-231 Rhamm and CD44 co-localization) but not in MCF-7 cells (Fig. 5A, panel c). Co-localization of Rhamm and CD44 can readily be seen in high-resolution images of MDA-MB-231 cells (panel gi). This co-association of Rhamm and CD44 was confirmed using immunoprecipitation (Fig. 5B, C) and “pull-down” assays (Fig. 5D). Anti-Rhamm antibodies co-immunoprecipitated two major CD44 isoforms in MDA-MB-231 and Ras-MCF10A cells, including an 85kDa species (CD44std) and an additional 116kDa isoform, which likely represented a splice variant or post-translationally modified form of CD44 (Fig. 5B). However, the same antibodies did not co-immunoprecipitate detectable CD44 protein in MCF7 and MCF10A cells (Fig. 5B). In reciprocal IP assays (e.g. detection of Rhamm proteins in CD44 immunoprecipitates), CD44 antibody co-immunoprecipitated the 85kDa full length Rhamm protein in all breast cell lines (Fig. 5C). The 63kDa N-terminal truncated form was co-immunoprecipitated in all the cancer cells lines (MDA-MB-231, Ras-MCF10A, and MCF7) but not the non-transformed MCF10A cells. Greater amounts of both the 85kDa and 63kDa Rhamm proteins were co-immunoprecipitated with CD44 in the invasive (MDA-MB-231 and Ras-MCF10A) vs. non-invasive (MCF7) breast tumor cell lines (Fig. 5C). The 43kDa Rhamm protein was not detected in the IP assays. An association of 63kDa Rhamm protein, which resembles the transforming and cell surface isoform, with CD44std and the 116kDa isoform was confirmed in MDA-MB-231 cells by pull-down assays using a 63kDa recombinant Rhamm protein as bait (Fig. 5D). In contrast, no CD44 isoforms were pulled down from the MCF7 lysates (data not shown). These results suggest that cell surface display and co-association of both CD44 and Rhamm are linked to HA responsiveness and an aggressive tumorigenic phenotype.

In order to be invasive, tumor cells must acquire the ability to migrate (1,2). CD44 was shown previously to promote motility of breast cancer cell

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lines and Rhamm was shown to promote motility of fibroblasts and immune cells (9,45,65,71,72). Therefore, we compared the relative roles played by these HA receptors in the motility of the fibroblast-like MDA-MB-231 and Ras-MCF10A tumor cells to the epithelial MCF7 and MCF10A cells, using function blocking antibodies specific to each of these receptors.

CD44 and cell surface Rhamm are necessary for motility of invasive but not non-invasive breast tumor cell lines. We confirmed that the MDA-MB-231 and Ras-MCF10A aggressive breast cancer cell lines are more motile than either MCF7 or MCF10A cells as previously reported (63,73) (Fig. 6A). To determine the extent to which motility is coordinated through HA receptor interaction, we blocked receptor function with inhibitory antibodies against CD44 and Rhamm, added alone or in combination. The motility of MDA-MB-231 and Ras-MCF10A cells was significantly inhibited by either anti-CD44 or anti-Rhamm antibodies (Fig. 6B). The addition of both antibodies together had no additive inhibitory effect on motility (Fig. 6B). Antibodies had only minor effects on the motility of the non-invasive MCF7 and MCF10A cell lines (data not shown). These results indicate that both cell surface CD44 and Rhamm contribute to the rapid motility rates of the invasive breast cancer cell lines and that they appear to act coordinately on the same motogenic pathway. However, these receptors are much less important for the motility of poorly invasive breast tumor cell lines.

Sustained activation of ERK1,2 motogenic pathways has previously been reported as an important factor in promoting invasive and metastatic behavior of aggressive breast cancer cell lines (52-54). We and others have shown that both CD44 and cell surface Rhamm regulate ERK1,2 activity (60,74). We then determined their role in coordinating ERK1,2 motogenic signaling.

CD44 and Rhamm complex with ERK1,2, and these complexes are required for motility in invasive breast cancer cell lines. Both MDA-MB-231 and Ras-MCF10A cells expressed more total ERK1,2 protein than MCF7 and MCF10A cells (Fig. 7A), consistent with previous reports (63,75). Under standard culture conditions, MDA-MB-231 and Ras-MCF10A cells exhibited significantly higher constitutively active (phospho) ERK1,2 than MCF7 or MCF10A cells, consistent with the expression of mutant active Ras (H-Ras) in the invasive cell lines (data not shown). MDA-MB-231 and MCF7 tumor cells also differed in their ability to activate ERK1,2 in response to EGF, a

growth factor linked to breast cancer progression. MDA-MB-231 cells, which have been reported to express high endogenous levels of EGF (76), maintained significantly greater levels of ERK1,2 activity than MCF7 cells (Fig. 7B, time 0). As expected from the endogenous EGF production, the addition of EGF did not increase ERK1,2 activity further in MDA-MB-231 cells while it transiently increased levels in MCF7 cells, which always maintained significantly less active ERK1,2 in any case (Fig. 7B). These differences correlated with distinct patterns of CD44/Rhamm/ERK1,2 co-localization in the two cell types as observed using confocal analyses (Fig. 8A). Fig. 8A shows CD44 [panels a (MCF7) and e (MDA-MB-231)] and phospho-ERK1,2 staining in both cell lines [panels b (MCF7) and f (MDA-MB-231)], as well as the overlay of Rhamm and phospho-ERK1,2 staining [panels c (MCF7) and g (MDA-MB-231)]. IgG was used as control [panels d (MCF7) and h (MDA-MB-231)]. These images showed, for example, that active ERK1,2 and CD44 co-localized in MDA-MB-231 cells as perinuclear vesicular structures and, to a more limited extent, in the nucleus (Fig. 8A, panel g; panel gi shows higher magnification images of phospho-ERK1,2 and CD44 co-localization in MDA-MB-231 cells). The inserts in panels c and g have been “enhanced” (refer to methods) to show all red/green co-localization as white. These images reveal extensive co-localization of ERK1,2 with CD44 in MDA-MB-231 (panel g) but not in MCF7 (panel c) cells. These results suggest that the majority of CD44/Rhamm/activated ERK1,2 complexes occur as vesicles near the nucleus of MDA-MB-231 cells. In contrast, levels of both active ERK1,2 and CD44 were low and limited co-localization was observed in MCF7 cells.

To confirm the association between ERK1,2 and Rhamm, we co-immunoprecipitated ERK1,2 in all cell lines using anti-Rhamm antibodies. As expected, there were greater amounts of ERK1,2 in immunoprecipitates from MDA-MB-231 and Ras-MCF10A than in MCF7 and MCF10A cells (Fig. 8B). The converse experiment using anti-ERK1,2 antibodies, also co-immunoprecipitated larger amounts of Rhamm proteins in the aggressive cell lines (Fig. 8C). Intriguingly, the 85kDa (full-length) Rhamm protein form was not detected in any of the ERK1,2 immunoprecipitates (Fig. 8C); the 63kDa protein was the predominant Rhamm form present. We confirmed the ability of both the 63kDa and 43kDa Rhamm

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proteins to associate with ERK1,2 in pull down assays using 63 or 43kDa N-terminally truncated recombinant Rhamm, which correspond to the endogenous Rhamm isoforms, as bait (Fig. 8D). ERK1,2 was present in both pull-downs confirming that, unlike the full length Rhamm form, the smaller Rhamm protein forms can associate with ERK1,2. Since the 63kDa Rhamm expression, in particular, is greater in the invasive breast tumor cell lines, it may be responsible for the high ERK1,2 activity observed in the invasive breast cancer cell lines.

Rhamm proteins that are smaller than the full length form have been reported to be predominant at the cell surface (68). We determined the role of cell surface Rhamm, as well as CD44, in activating ERK1,2 in MDA-MB-231 cells after exposure to either anti-Rhamm, anti-CD44 or both antibodies (Fig. 9A). Exposure to isotype matched non-immune IgG served as a control. ERK1,2 activity was significantly reduced by anti-Rhamm antibodies (Fig. 9A). Anti-CD44 antibodies also appeared to reduce ERK1,2 activity although the effect did not reach a significance level of p<0.05. Addition of both inhibitory antibodies together had no greater inhibitory effect on ERK1,2 activation than either antibody alone (Fig. 9A). These results show that ERK1,2 associates with Rhamm/CD44 complexes and that both HA receptors are required for activation of these MAP kinases.

Since ERK1,2 activity is essential for motility and invasion of MDA-MB-231 cells, we asked whether these HA receptors mediate motility also via an ERK1,2-dependent pathway. The addition of PD098059, a MEK1 inhibitor (Fig. 9B), or anti-Rhamm antibodies (data not shown) significantly reduced the basal motility rate of MDA-MB-231 cells. Again, the addition of these two reagents together had no additive inhibitory effect (Fig. 9B). Furthermore, neither the MEK1 inhibitor nor the anti-Rhamm antibodies, individually or in combination, had a significant inhibitory effect on MCF7 cell motility (Fig. 9B). Similar results were observed when using anti-CD44 antibodies alone or in combination with the MEK1 inhibitor (data not shown). These combined results suggest that Rhamm, CD44, and ERK1,2 activity are required for rapid basal motility of the aggressive cell lines and that they act collectively on the same motogenic pathway. Results suggest further that although Rhamm can associate with both ERK1,2 and CD44, even in the less aggressive breast cancer cell lines, the subcellular localization of these complexes and the consequent effects on the kinetics

of ERK1,2 activity as well as motogenic signaling are limited in the cell lines that express little to no cell surface CD44 or Rhamm.

DISCUSSION

We have identified an autocrine motility

mechanism by which aggressive breast cancer cell lines maintain rapid basal rates of motility. This mechanism requires HA production, ERK1,2 activity and cell surface display of CD44 and Rhamm (CD168). It is associated with the formation of signaling complexes composed of cell surface CD44, Rhamm, and active ERK1,2 that are most abundant in the aggressive, highly motile breast cancer cell lines. Although previous reports have demonstrated that either CD44 or cell surface Rhamm are required for HA-mediated motility of tumor cells including MDA-MB-231 cells, this is the first report documenting both a functional and physical interaction between these two HA receptors. It is also the first to demonstrate coupling of this complex to a motogenic signaling pathway through ERK1,2 in aggressive tumor cells. Our results raise the possibility that the association of cell surface Rhamm with CD44, and their subsequent association with ERK1,2, may modify tumor suppression by CD44 to favor its latent tumor promoter functions. This is consistent with the strong association amongst elevated HA accumulation, ERK1,2 activity, Rhamm expression and aggressive forms of breast carcinoma (31,37).

Hyaluronan was originally proposed to be an autocrine motility factor for mesenchymal cells (77). Indeed, high endogenous production of HA has since been shown to provide autocrine motility signals in embryonic cells (78,79), hematopoietic progenitor cells (80), and a variety of other human tumor cells (17,18). The possibility that HA is an autocrine motility factor that is produced by and required for the motility of aggressive breast cancer cells is attractive given the close relationship of HA production and HAS expression (7,17,81), and co-localization of HA with CD44 in later stages of breast and other cancers (27). Our data also fit well with the prognostic value of elevated HA in peri-tumor stroma or tumors themselves as a marker of poor outcome in this disease (82).

HA has consistently been demonstrated to activate motogenic signaling through Ras (40,78,79) and to require activation of ERK1,2 and PI3-kinase/AKT for this function (17,49,83). Although the

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HA receptors, CD44 and cell surface Rhamm, have been shown to mediate HA regulation of a Ras-controlled motogenic pathway, the majority of studies have focused on the exclusive role of CD44. An overwhelming number of these support a major role for CD44 in promoting aggressive breast cancer behavior, including cell motility in culture and in experimental tumor models as demonstrated by use of CD44 antibodies, blocking soluble CD44 recombinant protein, or small HA fragments, and genetic deletion or knockdown of this HA receptor (17,81,84). Nevertheless, reports have documented also that increased motility or invasion of breast and other tumor cells is associated with either shedding of CD44 [e.g. (85)], genetic loss or blocking of CD44 functions (83,86). These experimental discrepancies, which reveal a capacity of CD44 for both promoting and inhibiting motility and invasion, mirror the dual relationship of CD44 expression to clinical outcome of breast cancer patients. In breast cancers, over-expression of the standard or specific variant forms has not consistently been demonstrated to relate to outcome parameters (25,28,29,87). One conclusion from these studies is that it is CD44 variant forms, which are expressed at different stages of breast tumor progression (13,27), that perform distinct functions from CD44std in tumor progression. For example, different CD44 protein forms may act as tumor suppressors during early stages of breast cancer: CD44std expression positively correlates with disease-related survival in node negative invasive breast carcinoma (50), but as enhancers of metastases during later stages (88). Our results support a role for CD44 in aggressive functions of breast tumor cells when it is partnered with other motogenic proteins such as cell surface Rhamm (45).

Rhamm is structurally unrelated to CD44, yet cell surface Rhamm can perform similar functions to CD44 including mediating motogenic signaling by HA (46). Since cell surface Rhamm is not an integral protein, it must partner with other receptors that are able to take over these functions of CD44 in its absence. To our knowledge, the identities of such additional proteins have not yet been reported.

In addition to its location at the cell surface, Rhamm also occurs in several intracellular compartments. In contrast to cell surface Rhamm, intracellular forms affect centrosome and mitotic spindle formation/integrity (41,43) and are not likely involved in motogenic functions. In support of this conclusion, we have shown that cell surface Rhamm is

sufficient to rescue ERK1,2 dependent motility in Rhamm-/- fibroblasts that do not express intracellular Rhamm forms (45). Previous studies have suggested that the presence of cell surface Rhamm is required for HA-mediated activation of ERK1,2 in endothelial cells (89) and fibroblasts (60), and for the motility and invasion of endothelial cells (90) when CD44 is co-expressed. Also, we have shown recently that genetic deletion of Rhamm blunts both ERK1,2 activity and motility of dermal fibroblasts even though they express CD44 protein (45). This study further showed that one function of cell surface Rhamm is to promote cell surface display of CD44: in the absence of cell surface Rhamm, CD44 is retained within intracellular vesicles (40). In contrast, in breast cancer cells (e.g. MCF7), the low levels of cell surface CD44 appear to result from its reduced transcription (data not shown) rather than from defects in a Rhamm-regulated display mechanism.

ERK1,2 kinases are ubiquitous and homologous MAP kinases that mediate proliferation, differentiation and motility via growth factor and ECM receptor activation. Over-expression and elevated activation of these kinases are common in human tumors. The importance of increased ERK1,2 activity in breast cancer is demonstrated by the anti-tumor effects of a specific inhibitor of the upstream kinase activators MEK1,2 (PD184352) in breast cancer patients (91). Although these MAP kinases are amongst the most common effectors in growth factor and ECM-regulated signaling pathways, a variety of temporal, spatial and quantitative cues confer a specificity of functional outcome resulting from their activation (55,92,93). For example, sustained activation of ERK1,2 is required to initiate motility of breast cancer cells (94). The duration of ERK1,2 activity is determined by many factors including concentration of the stimulus, receptor dimerization, presence of co-receptors that modify the rate of receptor internalization and the expression/compartmentalization of intracellular scaffolding/accessory proteins (92,93). The mechanisms by which CD44 and Rhamm sustain elevated basal ERK1,2 activity remains unclear but confocal analysis suggests that these HA receptors co-localize with phospho-ERK1,2, predominantly in the perinuclear area of cells where these proteins appear as vesicles. Although internalization of some receptor/ERK1,2 complexes terminate ERK1,2 activity, internalization of ERK1,2 with other receptors (i.e. Protease-activated Receptor-2 [PAR-2])

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results in sustained ERK1,2 activation (95). We propose that internalization and trafficking of CD44/Rhamm/ERK1,2 complexes to the perinuclear area promotes sustained ERK1,2 activity in the cytoplasm. By this type of mechanism, active ERK1,2 would be available to traffic to key cytoplasmic

compartments such as focal adhesions or the nucleus, sites where its activity is required for cell motility/invasion (52,54). Further experimentation is needed to assess this possibility.

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FOOTNOTES

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Funding for this study was provided by CIHR (grant to E.A.T., MOP-57694; grant to J.K., MOP-62836; fellowship to S.R.H., UST-63811), the Breast Cancer Translational Unit at the London Regional Cancer Program [Breast Cancer Society of Canada salary support (E.A.T).; (fellowships to C.T. and S.R.H.)], the DOD (JBM/EAT, DOD-PC050959), BCRP-CDMRP (E.A.T. and M.J.B., BC044087), and the USDOE (M.J.B., Office of the Biological and Environmental Research), USNCI (M.J.B. with Dr. Z. Werb). M.J.B. is the recipient of an Innovator award from the USDOD Breast Cancer Program and a USDOE Distinguished Scientist Fellowship. The abbreviations used are: 2D, two-dimensional; Ab, antibody; BSA, bovine serum albumin; CD44std, CD44 standard form; CD44v, CD44 variant; DMEM, Dulbecco’s modified Eagle’s Medium; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK1,2, Extracellular Regulated Kinase 1,2; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; FCS, fetal calf serum; HA, hyaluronan; HAS, hyaluronan synthase; HRP, horse radish peroxidase; IB, immunoblot; IP, immunoprecipitate; kDa, kilodalton; MAPK, Mitogen Activated Protein kinase; MEK1, Mitogen Activated Kinase 1; MMP, matrix metalloproteinase; MW, ,olecular weight; PBS, phosphate buffered saline; Rhamm, Receptor for Hyaluronic Acid Mediated Motility; TBS, tris buffered saline.

FIGURE LEGENDS

Fig. 1. MDA-MB-231 invasive breast tumor cells produce greater levels of HA compared to MCF7 non-invasive breast tumor cells and rely more strongly upon HA to promote basal motility. A, MDA-MB-231 cells produce significantly higher levels of HA than MCF7 cells. Values represent the Mean and S.E.M., n=10 from 1 of 3 similar experiments. B, A 12-mer HA binding peptide significantly reduces the motility of the MDA-MB-231 cells compared to controls, whereas it has no effect on the motility of MCF7 tumor cells. Values represent the Mean and S.E.M., n=30 cells from 1 of 3 similar experiments. C, Exogenous HA (25-50µg/mL) significantly stimulates the motility of serum-starved MDA-MB-231 cells; MCF7 cells do not respond to HA. Values represent the Mean and S.E.M., n=30 cells from 1 of 3 similar experiments. Fig. 2. Cell surface and total cellular CD44 protein levels are increased in MDA-MB-231 breast cancer cells relative to MCF7 cells. A, Western blot analysis and densitometric quantification of CD44std protein levels in breast cancer cell lines. Values represent the Mean and S.E.M., n=3 experiments. Percent expression was determined by normalizing the densitometric value of CD44std protein to that of a 60kDa dominant marker protein on parallel Coommassie stained gels. B, Flow cytometry analysis of cell surface CD44 expression in MDA-MB-231 and in MCF7 breast cancer cell lines. Values are from 1 of 3 similar experiments. Fig. 3. Rhamm isoforms (63 and 85kDa) are more highly expressed in MDA-MB-231 than in MCF7 breast cancer cell lines. A, Diagram showing the location of the sequences to which the three anti-Rhamm antibodies were raised (Ab-1, Ab-2 and Ab-3). Three protein isoforms are expressed in MDA-MB-231 cells and their sequence is predicted based upon reactivity with these anti-peptide antibodies in western blots. Full-length Rhamm isoform (85kDa) reacts with all three antibodies while shorter Rhamm forms (43 and 63kDa) react only with Ab-2 and Ab-3 (data not shown), suggesting the latter are N-terminal truncations of the full-length protein. Detectable Rhamm isoforms expressed in MCF7 cells are the 85kDa (full-length) and 43kDa isoforms (Ab-2). B, Quantification of Rhamm protein expression was determined by calculating the densitometric ratios of each of the protein isoforms / total Rhamm protein (obtained by totaling the densitometric values of each Rhamm immunoreactive band recognized by Ab-2). Values represent the Mean and S.E.M., n=3 experiments. Fig. 4. Cell surface Rhamm expression in breast cancer cell lines. Flow cytometry analysis shows cell surface Rhamm expression is increased in MDA-MB-231 compared to MCF7 breast tumor cells. Cells were gated such that M1 refers to the negative population while M2 refers to the positive population. Values represent 1 of 3 similar experiments.

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Fig. 5. Subcellular distribution of CD44 and Rhamm differs in invasive and non-invasive breast cancer cell lines. A, Confocal analysis shows that MCF7 cells express Rhamm as a cytoplasmic protein (red fluorescence; a, arrow, c) but express little or no CD44 protein (green fluorescence; b,c). In contrast, MDA-MB-231 cells express both CD44 (f,g) and Rhamm (e,g) and these co-localize (yellow) in perinuclear vesicular structures (g, arrow). Panel gi shows higher magnification images (2X) of CD44 and Rhamm co-localization in MDA-MB-231 cells. Non-immune IgG serves as a control (d,h). DAPI (blue fluorescence) is used to detect the nuclei. Representative micrographs are from 1 of 4 similar experiments. B, Anti-Rhamm antibodies immunoprecipitate two CD44 isoforms (approximately 85 and 116kDa) in MDA-MB-231 and Ras-MCF10A cells but do not immunoprecipitate detectable CD44 in MCF7 and MCF10A breast cells. n=3 experiments. C, Anti-CD44 antibodies immunoprecipitate the 85kDa Rhamm isoform in all cell lines whereas the 63kDa isoform was associated only with CD44 in MDA-MB-231, Ras-MCF10A, and MCF7 cells but not in the non-transformed parental MCF10A cells. Greater levels of these Rhamm isoforms are detected in the invasive MDA-MB-231 and Ras-MCF10A cells than in the less invasive MCF7 and MCF10A cells. n=3 experiments. D, Recombinant Rhamm-GST (63kDa isoform) protein pulls down CD44std (85kDa) and a variant or post-translationally modified CD44 isoform (116kDa) from MDA-MB-231 lysates. GST recombinant protein is used as control. n=4 experiments. Fig. 6. High basal motility of MDA-MB-231 and Ras-MCF10A tumor cells is CD44 and Rhamm-dependent. A, Basal motility rates of MDA-MB-231 and Ras-MCF10A are significantly increased over MCF7 and MCF10A cells. Values are the Mean and S.E.M., n=30 cells from 1 of 3 similar experiments. B, Both anti-Rhamm and anti-CD44 antibodies significantly reduce motility of MDA-MB-231 and Ras-MCF10A breast cancer cells but have no effect on MCF7 or MCF10A breast cells (data not shown). Addition of the two blocking antibodies together does not have an additive inhibitory effect on motility of MDA-MB-231 or Ras-MCF10A cells. Values represent the Mean and S.E.M., n=30 cells from 1 of 3 similar experiments. Fig. 7. ERK1,2 is activated in response to EGF in MDA-MB-231 cells A, Constitutive expression of total ERK1,2 protein is significantly increased in MDA-MB-231 and Ras-MCF10A cells compared to MCF7 and MCF10A cells. Values represent the Mean and S.E.M., n=3 experiments. Percent expression was determined by normalizing the densitometric value of expressed total ERK1,2 to that of a 60kDa dominant marker protein on parallel Coommassie stained gels. B, Basal levels of phospho-ERK1,2 are significantly higher in MDA-MB-231 than in MCF7 cells (0 minutes). Levels of phospho-ERK1,2 remain elevated in MDA-MB-231 cells and are not further increased in response to 20ng/mL EGF, whereas EGF transiently increases phospho-ERK1,2 levels in MCF7 cells, albeit not to the levels observed in MDA-MB-231 cells. Values represent the Mean and S.E.M., n=3 from 1 of 3 similar experiments. Densitometric values for phospho-ERK1,2 were normalized to total ERK1,2 and fold changes were calculated by arbitrarily setting the ratio of phospho-ERK1,2 to total ERK1,2 in unstimulated MCF7 cells to 1. Fig. 8. CD44 and Rhamm co-localize and co-immunoprecipitate with active (phospho-) ERK1,2. A, Confocal analysis shows that MCF7 cells do not express detectable CD44 (a,c), as shown also in Fig. 5, and only low levels of phospho-ERK1,2 (b,c) using non-immune IgG as a control (d,h). In contrast, MDA-MB-231 cells express more CD44 (e,g) and phospho-ERK1,2 (f,g) and these co-localize (yellow; white enhancement in inset panels) in perinuclear vesicular structures (g, arrow) and, to a more limited extent, in the nucleus (g). Panel gi shows higher magnification images (2X) of CD44 and phospho-ERK1,2 co-localization in MDA-MB-231 cells. DAPI (blue fluorescence) is used to detect the nuclei. Micrographs are from 1 of 4 similar experiments. B, Anti-Rhamm antibodies immunoprecipitate ERK1,2 in all cell lines but a greater amount of ERK1,2 is present in Rhamm immunoprecipitates of MDA-MB-231 and Ras-MCF10 compared to MCF7 or MCF10A cells. n=3 experiments. C, Anti-ERK1,2 antibodies co-immunoprecipitate the 63kDa Rhamm isoform. As expected, greater amounts of this Rhamm protein form is present in the ERK1,2 immunoprecipitates of MDA-MB-231 and Ras-MCF10A than in MCF7 or MCF10 cells. The 85kDa full-length Rhamm form is not detected in ERK1,2 immunoprecipitates. Non-specific reactive bands (see IgG lane) migrating at 43kDa obscured detection of the smallest Rhamm protein form (43kDa) expressed by these cells. n=3 experiments. D, Recombinant Rhamm-GST protein (63kDa and

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43kDa isoforms) pull down ERK1,2 protein from MDA-MB-231 lysates, confirming an interaction of the shorter Rhamm protein forms with these MAP kinases. GST recombinant protein is used as control. n=3 experiments. Fig. 9. Anti-Rhamm and anti-CD44 antibodies reduce ERK1,2 activity and ERK1,2-dependent motility of MDA-MB-231 cells. A, Anti-Rhamm or anti-CD44 antibodies reduce levels of phospho-ERK1,2 in MDA-MB-231 but have no effect on MCF7 cells (data not shown). Combining the antibodies does not increase inhibition further. Values are the Mean and S.E.M., n=3 experiments. Densitometric values for phospho-ERK1,2 were normalized to total ERK1,2 and fold changes were calculated by arbitrarily setting the ratio of phospho-ERK1,2 to total ERK1,2 in IgG treated cells to 1. B, Addition of the MEK1 inhibitor, PD098059, significantly reduces MDA-MB-231 cell motility but has no effect on MCF7 cell motility. A mixture of PD098059 and anti-Rhamm antibodies does not increase inhibition of MDA-MB-231 cell motility further. Values represent the Mean and S.E.M., n=30 cells from 1 of 3 similar experiments. Supplemental Fig. I. Total cellular CD44 and Rhamm protein expression is greater in Ras-MCF10A breast tumor cells than in the parental MCF10A cell line. a, Western blot analysis and densitometric quantification of total cellular CD44std protein levels shows a elevated constitutive expression of CD44std protein in Ras-MCF10A cells relative to parental MCF10A cells. Values represent the Mean and S.E.M., n=3 experiments. Percent expression was determined by normalizing the densitometric value of CD44std protein to that of a 60kDa dominant marker protein on parallel Coommassie stained gels. b, Constitutive expression of total Rhamm protein, and of each isoform (85, 63 and 43kDa) is greater in Ras-MCF10A cells than MCF10A cells. Quantification of Rhamm protein isoform expression was determined by calculating the densitometric ratios of each of the protein isoforms / total Rhamm protein (obtained by totaling the densitometric values of each Rhamm immunoreactive band recognized by Ab-2). Values represent the Mean and S.E.M., n=3 experiments.

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A. Hyaluronan Production by MDA-MB-231 and MCF7 Cells

B. Effect of Hyaluronan Binding Peptide on Breast Cancer Cell Motility

C. Effect of Exogenous Hyaluronan on Breast Cancer Cell Motility

Figure 1 (Hamilton, Fard et al)

MCF7MDA-MB-231

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Figure 2 (Hamilton, Fard et al)

B. Cell Surface CD44 Expression in MDA-MB-231 and MCF7 Cells

A. Total CD44 Protein Expression in MDA-MB-231 and MCF7 Cells

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Figure 3 (Hamilton, Fard et al)

A. Rhamm Protein Isoforms expressed by MDA-MB-231 and MCF7 Cells

B. Densitometry Analysis of Rhamm Protein Isoform Expression

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Cell Surface Rhamm Expression in MDA-MB-231 and MCF7 Cells

Figure 4 (Hamilton, Fard et al)

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Figure 6 (Hamilton, Fard et al)

A. Basal Motility Rates of Breast Cancer Cell Lines

B. Effect of Anti-Rhamm and Anti-CD44 Antibody Combinations on Breast Cancer Cell Motility

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Figure 7 (Hamilton, Fard et al)

A. Total ERK1,2 Protein Expression in Breast Cancer Cell Lines

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Figure 8 (Hamilton, Fard et al)

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Figure 9 (Hamilton, Fard et al)

A. Effect of Anti-Rhamm and / or Anti-CD44 antibodies on ERK1,2 Activation in Breast Cancer Cell Lines

B. Effect of PD098059 and / or Anti-Rhamm Antibodies on Breast Cancer Cell Motility

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Chao Wang, James B. McCarthy, Mina J. Bissell, James Koropatnick and Eva A. TurleySara R. Hamilton, Shireen F. Fard, Frouz F. Paiwand, Cornelia Tolg, Mandana Veiseh,

which sustain high basal motility in breast cancer cellsThe hyaluronan receptors CD44 and RHAMM (CD168) form complexes with ERK1,2,

published online March 28, 2007J. Biol. Chem. 

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