Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells

UNCORRECTED PROOF

ORIGINAL PAPER

Cystatin M suppresses the malignant phenotype of human MDA-MB-435S

cells

Ravi Shridhar1, Jun Zhang2, Jin Song2, Blake A Booth3, Christopher G Kevil3,4, GeorgiaSotiropoulou5, Bonnie F Sloane1,6 and Daniel Keppler*,1,2,4,6

1Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA; 2Department of CellularBiology & Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA; 3Department of Pathology,Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA; 4Feist-Weiller Cancer Center, Louisiana StateUniversity Health Sciences Center, Shreveport, LA 71130, USA; 5Department of Pharmacy, University of Patras, Rion 26500,Patras, Greece; 6Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA

Proteases are involved in many aspects of tumorprogression, including cell survival and proliferation,escape from immune surveillance, cell adhesion andmigration, remodeling, and invasion of the extracellularmatrix. Several lysosomal cysteine proteases have beencloned and shown to be overexpressed in cancer; yet,despite the great potential for development of noveltherapeutics, we still know little about the regulation oftheir proteolytic activity. Cystatins such as cystatin M arepotent endogenous protein inhibitors of lysosomal cysteineproteases. Cystatin M is expressed in normal andpremalignant human epithelial cells, but not in manycancer cell lines. Here, we examined the effects of cystatinM expression on malignant properties of human breastcarcinoma MDA-MB-435S cells. Cystatin M was foundto significantly reduce in vitro: cell proliferation, migra-tion, Matrigel invasion, and adhesion to endothelial cells.Reduction of cell proliferation and adhesion to anendothelial cell monolayer were both independent of theinhibition of lysosomal cysteine proteases. In contrast, cellmigration and matrix invasion seemed to rely onlysosomal cysteine proteases, as both recombinant cysta-tin M and E64 were able to block these processes. Thisstudy provides the first evidence that cystatin M may playimportant roles in safeguarding against human breastcancer.Oncogene (2003) 0, 000–000. doi:10.1038/sj.onc.1207340

Keywords: cystatins; cathepsins; lysosomal proteases;protease inhibitors; tumor progression

Introduction

Lysosomal cysteine proteases are involved in manybiological processes such as intracellular protein cata-bolism, pericellular matrix remodeling, antigen proces-sing, virulence, and programmed cell death (for recentreviews, see Chapman et al., 1997; Villadangos et al.,1999; Bromme and Kaleta, 2002). Impaired regulationof expression and activity of lysosomal cysteine pro-teases has been implicated in cancer progression(Keppler and Sloane, 1996; Roshy et al., 2003). Severalgroups have hypothesized that the increased proteolyticactivities observed in malignant tumors are due toupregulation of proteases and/or downregulation oftheir endogenous inhibitors (Liotta and Stetler-Steven-son, 1991; Brunner et al., 1994; DeClerck and Imren,1994). Cystatins are considered physiological inhibitorsof lysosomal cysteine proteases (Turk and Bode, 1991;Abrahamson, 1994). They control the catalytic functionof target proteases by forming reversible, high-affinitycomplexes (Barrett et al., 1986; Alvarez-Fernandez et al.,1999).Cystatin M was first identified by differential display

as a transcript that was downregulated in a metastaticbreast cell line when compared to a matched primarytumor cell line (Sotiropoulou et al., 1997). The sameinhibitor was independently cloned from embryoniclung fibroblasts and named cystatin E (Ni et al., 1997).Here, we will refer to cystatin E/M as cystatin M.Cystatin M is a cell-secreted cystatin of 121 amino acidsthat exists in both glycosylated (17 kDa) and unglyco-sylated (14.4 kDa) forms (Ni et al., 1997; Sotiropoulouet al., 1997). Since cystatin M is expressed in normal andpremalignant breast epithelial cells but not in metastaticbreast cancer cell lines, it was hypothesized to be a novelmetastasis suppressor (Sotiropoulou et al., 1997).Metastasis is a multi-step process in which tumor cells

escape from the primary tumor and establish colonies atlocal and distant sites (Poste and Fidler, 1980).Metastatic tumor cells must detach, acquire motilefunction, and penetrate the basement membranes andother connective tissues. Thus, degradation of thesesupportive and confining structures has been hypothe-

Journal: ONC Disk used Despatch Date: 22/11/2003Article : NPG_ONC_6988 Pages: 1–11 OP: Chandra ED: Pouly

Gml : Ver 6.0Template: Ver 6.1

Received 29 September 2003; revised 5 November 2003; accepted 7November 2003

*Correspondence: D Keppler, Department of Cellular Biology &Anatomy, LSUHSC, School of Medicine, 1501 Kings Highway, POBox 33932, Shreveport, LA 71130-3932, USA;E-mail: [email protected]

Oncogene (2003) 00, 1–11& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00

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sized to be a critical component of the metastatic process(Liotta and Stetler-Stevenson, 1991). Studies haveshown that lysosomal cysteine proteases can degradecomponents of connective tissues and basement mem-branes in vitro, including various collagens, elastin,tenascin, laminin, and fibronectin (Maciewicz et al.,1990; Buck et al., 1992; Guinec et al., 1993; Brommeet al., 1996; Sameni et al., 2000; Mai et al., 2002).Secretory cystatins, like cystatin M, could potentiallyregulate the function of exocytosed cysteine proteases(Linebaugh et al., 1999; Hulkower et al., 2000). Loss ofexpression of cystatin M could therefore lead toproteolysis of the glycoprotein scaffolds that maintaintissue architecture, and this could facilitate invasion andmetastasis of cancer cells.The long-term goal of our studies is to determine if

cystatin M is a bona fide metastasis suppressor. As a firststep towards that aim, we have stably transfected thehighly tumorigenic and metastatic human breast cancerMDA-MB-435S cell line with a cystatin M expressionvector and studied the effects of cystatin M expressionon the malignant properties of these cells. We report forthe first time that ectopic expression of cystatin Mreduced cell proliferation, migration, matrix invasion,and tumor-endothelial cell adhesion. Surprisingly, how-ever, when mock-transfected cells were treated with abroad-spectrum inhibitor of lysosomal cysteine pro-teases (E64), no effect was seen on cell proliferation andtumor-endothelial cell adhesion, while the same agentinhibited both cell migration and matrix invasion. Weconclude from these studies that cystatin M may havedistinct functions in the safeguard against malignantprogression.

Results

Expression pattern of cystatins C and M in human cancercell lines

Cystatin M expression has been analysed before invarious normal human tissues as well as in neoplasticskin disorders and established breast cancer cell lines (Niet al., 1997; Sotiropoulou et al., 1997; Zeeuwen et al.,2002). Little is known about its expression pattern inother cancers. Previously, we had shown that anothersecreted cystatin, cystatin C, was quite ubiquitouslyexpressed in human cancer cells (Keppler et al., 1994).Therefore, we compared by semiquantitative RT–PCRanalysis the levels of expression of cystatin C andcystatin M mRNAs in a number of different humancancer cell lines (breast: BT-20, BT-549, and MDA-MB-435S; prostate: DU-145, LNCaP, and PC-3; colon:CaCo-2 and HT-29; glioma: U-87MG; and melanoma:WM-164, WM-793, WM-852, WM-902B, WM-1205Lu,and WM-1341D). The diploid, spontaneously immorta-lized and nontumorigenic, human breast epithelialMCF-10A cell line served as a ’normal’ counterpart inthis screen. The cystatin M mRNA was detected only ina few cell lines, including immortalized MCF-10A andthree cancer cell lines (BT-20, DU-145, and PC-3). Low

levels of expression were also detectable in BT-549,LNCaP, CaCo-2, HT-29, and U-87MG cells. As formost established human breast cancer cell lines such asMDA-MB-435S (Sotiropoulou et al., 1997), little or nocystatin M transcript could be detected in any of themelanoma cell lines used in this study (Figure 1, cyst.M). In contrast to the cystatin M mRNA, the cystatin CmRNA was detected in all cell lines analysed, with theexception of one, BT-20 (Figure 1, cyst. C). RT–PCRanalysis of the b2-microglobulin mRNA was performedto verify equal sampling of total RNA from one cell lineto another (Figure 1, 2-micro).

Transfection studies

MDA-MB-435S cells express cystatin C, but little or nocystatin M message (Figure 1, compare MDA-435S inthe middle and upper panels, respectively). These cellsare highly tumorigenic, invasive, angiogenic, and meta-static in immunocompromised mice (Price and Zhang,1990). Therefore, they seemed an appropriate model inwhich to test the antiproliferative, anti-invasive, anti-angiogenic, and/or antimetastatic properties of cystatinM. As a first step towards that aim, we transfectedMDA-MB-435S cells with the cystatin M cDNAexpression vector or the ‘empty’ control vector. Likethe cystatin M vector, the ‘empty’ control vector has anintact CMV-promoter, a transcriptional start site,followed some 300 bp downstream by a patent poly-adenylation signal. PCR analysis of genomic DNAisolated from individual Zeocin-resistant clones showedstable integration of the cystatin M cDNA in cystatinM-transfected clones (Figure 2a, CM-12, CM-13, CM-15, and CM-17). The 422-bp PCR product could not bedetected in parental (untransfected) or two mock-transfected control clones (Figure 2a; parental, mock-1, and mock-2). The bands of higher sizes (4422 bp),seen only with genomic DNA containing the transfectedcystatin M cDNA, most likely represent oligomers ofthe amplified product, as they are not seen in controlDNA.

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Figure 1 Standard RT–PCR analysis of cystatin M and cystatin Cexpression in human cell lines. Total RNA was isolated fromhuman cell lines and subsequently reverse-transcribed and analysedby PCR amplification for expression of cystatin M (upper panel),cystatin C (middle panel) and, as a loading control, b2-micro-globulin (lower panel). Cell lines are as follows: breast (MCF-10A,BT-20, BT-549, and MDA-MB-435S), prostate (DU-145, LNCaP,and PC-3), colon (CaCo-2 and HT-29), glioblastoma (U-87MG),and melanoma (WM-164–WM-1341D)

Cystatin M suppresses malignant phenotypeR Shridhar et al

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UNCORRECTED PROOFImmunoblot analysis of cell-conditioned media

showed that clones CM-12, CM-13, CM-15, and CM-17 expressed and secreted two forms of cystatin M(Figure 2b, upper panel, cyst. M). Two forms of cystatinM, that is, a 17-kDa glycosylated and a 14.4-kDaunglycosylated form, have been identified previously (Niet al., 1997; Sotiropoulou et al., 1997). Cystatin Mprotein could not be detected in the conditioned mediaof control clones (Figure 2b, upper panel, parental,mock-1, mock-2). Cystatin C is a closely related secretedinhibitor that has a similar molecular mass to cystatin Mand may have a partially overlapping inhibitory profile(Turk and Bode, 1991; Abrahamson, 1994). Immuno-blot analysis revealed that ectopic expression of cystatinM did not affect expression and secretion of cystatin C(Figure 2b, lower panel, cyst. C), suggesting that therewas not a compensatory downregulation of cystatin C incells that overexpress cystatin M.

Cystatin M reduced cell proliferation in vitro

Several studies have shown that cystatin C fromdifferent species can modulate cell proliferation (Sun,1989; Leung-Tack et al., 1990; Tavera et al., 1992;Taupin et al., 2000; Konduri et al., 2002). Therefore, wedetermined the effect of cystatin M on proliferation ofMDA-MB-435S cells. We observed a significantly(Po0.01) slower rate of proliferation of the cystatin Mtransfectants as compared to that of the mock transfec-tants (Figure 3a). If secreted cystatin M was responsiblefor the growth differences in these cells, then condi-tioned media from the cystatin M transfectants shouldalso slow the growth of mock-transfected cells. This wasindeed the case, as cystatin M-conditioned mediasignificantly (Po0.001) slowed the rate of proliferationof mock-transfected cells when compared to the same

cells grown in the presence of mock-conditioned media(Figure 3b). Our results are thus consistent with reducedproliferation in the cystatin M –transfectants being dueto secreted cystatin M.These results suggested that recombinant cystatin M

should also reduce cell proliferation. We tested thishypothesis with a GST-fusion protein in which GST isfused to the N-terminus of cystatin M (GSTCM).Recombinant GST was used as a control. Neitherrecombinant protein had any effect on proliferation ofthe cells (Figure 3c). This was surprising becauseGSTCM expressed in bacteria inhibited papain with asimilar nanomolar Ki (Figure 3d) as cystatin Mexpressed in insect Sf9 cells (Ni et al., 1997). The correctfolding of GSTCM was further substantiated by the factthat GST had no apparent papain-inhibitory potentialwhen tested under similar conditions (Figure 3d). Inaddition, the broad-spectrum inhibitor of lysosomalcysteine proteases, E64, or its membrane-permeant esterderivative, E64d, had no effect on cell proliferation(Figure 3e). Previously, we had established that the 10-mM dose of E64/E64d used was able to completelyinhibit lysosomal cysteine proteases present in asubconfluent 100-mm Petri dish. Therefore, lysosomalcysteine proteases do not seem to be involved inproliferation of MDA-MB-435S cells. Additionally, itmight suggest that either post-translational modifica-tions or an accessible amino-terminus might be requiredfor cystatin M to function as a regulator of cellproliferation.

Cystatin M reduced cell migration

One of the hallmarks of malignant cancers is theirability to invade/infiltrate the surrounding normaltissues. MDA-MB-435S cells are highly motile andinvasive (Thompson et al., 1992; Liu et al., 1997). To testwhether cell migration was affected by overexpression ofcystatin M, we performed Transwell migration assaysusing FBS as the chemoattractant. During the initialoptimization of this assay in our laboratory, wedetermined that 1% heat-inactivated FBS was maxi-mally stimulatory for MDA-MB-435S cell migration(data not shown). We observed a time-dependentaccumulation of migratory cells on the bottom surfaceof the 8-mm filters. Little or no migration was seen in theabsence of chemoattractant over a 13-h incubationperiod (data not shown). Migration of the cystatin M-overexpressing clone CM-13 was significantly (Po0.05)and X60% reduced in comparison to migration of theparental and mock-1 control clones (Figure 4a). Migra-tion of the mock-1 clone was reduced X70% in thepresence of either 200 nM of recombinant GSTCM or10 mM of E64 (Figure 4b). This was in contrast to whatwe observed with cell proliferation, which was notsensitive to either 2 mM of GSTCM or 10 mM of E64.These assays indicate that FBS-stimulated migration ofMDA-MB-435S cells required the participation of oneor several lysosomal cysteine proteases inhibited bycystatin M and/or E64.

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Figure 2 Characterization of MDA-MB-435S transfectants. Thecystatin M expression plasmid or the empty vector were transfectedinto MDA-MB-435S cells. (a) Several zeocin-resistant clones wereisolated and characterized by PCR for stable integration of thecystatin M cDNA into genomic DNA. The expected 422-bp PCRproduct was detected only in genomic DNA isolated from cystatinM clones (CM-12, CM-13, CM-15, and CM-17), but not fromcontrol clones (parental, mock-1, and mock-2). (b) The same cloneswere analysed by immunoblotting for constitutive secretion ofcystatin M (upper panel) and cystatin C (lower panel). Clones CM-12, CM-13, CM-15, and CM-17 expressed and secreted two formsof cystatin M, that is, the 14.4-kDa unglycosylated and the 17-kDaN-glycosylated form (Cyst. M, upper panel). The parental, mock-1,and mock-2 control clones produced no detectable cystatin M inthe media. As additional control, all clones were shown to secretesimilar levels of cystatin C (Cyst. C, lower panel)


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Figure 3 Effect of cystatin M on proliferation of MDA-MB-435S cells. (a) Comparison of growth rates between MDA-MB-435S cellclones stably transfected with either the empty vector (mock-1 and mock-2) or the cystatin M expression vector (CM-12, CM-13, andCM-17). Cells (500) of each clone were seeded in seven 96-well plates and fed every 48 h with DMEMþ 10% FBSþ 200mg/ml zeocin.Plates were collected at days 1, 3, 5, 7, 9, 11, and 13, and cell number measured with the CyQuant proliferation kit. Each pointrepresents the mean7s.e.m. of six wells. The experiment was repeated several times with similar results. Statistical analysis comparingmock-transfected and cystatin M-transfected clones was performed using one-way ANOVA (*Pp0.01). (b) Effect of cell-conditionedmedia on proliferation of mock-1. Conditioned media (24 h) were collected from parental and mock-transfected cells and pooled (mockmedium); the same was done with the media from CM-12, 13 and 17 cells (CM medium). Mock-1 cells were then seeded as above andgrown in either mock or CM medium (n¼ 6). Cells were fed with the pooled media and plates collected and processed as describedabove. Statistical analysis comparing the effect of mock and CM media was performed using two-factor ANOVA (*Pp0.001). (c)Effect of recombinant GSTCM on cell proliferation. Mock-1 cells were seeded as above, and grown in the presence of variousconcentrations of recombinant GSTCM or GST (n¼ 6). Cells were fed with fresh medium and recombinant protein every 48 h, andplates collected and processed as described above. Statistical analysis comparing control-treated and GSTCM/GST-treated mock-1cells was performed using one-way ANOVA. (d) Inhibition of papain activity by GSTCM. The ability of GSTCM to inhibit papainwas tested using various concentrations of recombinant GSTCM or GST (as control). Active site titrated papain (20 nM) waspreincubated with GSTCM/GST for 20min at room temperature. The enzyme was then incubated at 371C in the presence of 100mM ofZ-Phe-Arg-AMC, and initial reaction velocities recorded semicontinuously. Residual enzyme activity is expressed as relativefluorescence units (RFU)/min. (e) Effect of a broad-spectrum inhibitor of lysosomal cysteine proteases on cell proliferation. Mock-1cells were seeded as above and grown in the absence (control) or presence of an excess concentration (10mM) of the inhibitor E64 or itsmembrane-permeant derivative E64d (n¼ 6). Cells were fed with fresh medium and inhibitor every 48 h, and plates collected andprocessed as described above. Statistical analysis comparing control-treated and inhibitor-treated mock-1 cells was performed usingtwo-factor ANOVA


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Cystatin M blocked Matrigel invasion

To determine if cystatin M affected the ability of MDA-MB-435S cells to invade, we performed Matrigelinvasion assays (Hendrix et al., 1987). At 60- and 72-htime points, invasion of CM-13 cells that overexpressedcystatin M was significantly lesser (495%; Po0.001)than invasion of the parental and mock-1 control clones(Figure 5a). Invasion of the mock-1 clone was nearlycompletely suppressed by a single addition of 200 nM ofrecombinant GSTCM or 10 mM of E64 to the media inthe top well (Figure 5b). Our data therefore suggestedthat specific lysosomal cysteine protease(s) might play a

rate-limiting role in serum-stimulated migration andinvasion.

Cystatin M reduced tumor-endothelial cell adhesion

The ability of circulating tumor cells to adhere firmly tothe microvascular endothelium at a distant site isconsidered a crucial step in establishing clinicallysuccessful metastases (Liotta and Stetler-Stevenson,1991; Al-Mehdi et al., 2000). To test whether adhesionof MDA-MB-435S cells to endothelial cells was affectedby cystatin M, we performed in vitro adhesion assaysusing HUVEC monolayers. Adhesion of MDA-MB-435S cells overexpressing cystatin M (Figure 6, CysM)to HUVECs was significantly decreased (Po0.05)compared to adhesion of mock-1 cells (Figure 6, Ctrl).Adhesion of the mock-1 cells could be inhibited to asimilar extent (30%) as that of the CM-13 cells in the

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Figure 4 (a) Effect of cystatin M on cell migration. Parental,mock- and cystatin M-transfected cells (5� 104) in 500ml of serum-free DMEM were seeded into Transwell culture inserts on top of 8-mm filters. The bottom wells contained 750ml of DMEMsupplemented with 1% FBS as chemoattractant. Cells wereincubated for various times at 371C in this gradient of FBS. Allother conditions were as described in the Materials and methodssection. Cells that had migrated to the bottom surface of the filterwere counted on the whole surface using an inverted microscope at� 100 magnification. Results are expressed as mean7s.e.m. oftriplicate experiments for each clone (parental, mock-1, and CM-13) and for each time point (3, 6, 9, and 13 h). Little or nomigration was observed in the absence of the chemoattractant.Statistical analysis comparing migration of the cystatin M clone tothat of parental and mock control clones was performed using one-way ANOVA (*Pp0.05). (b) Effect of recombinant cystatin M andE64 on cell migration. Mock-1 cells were incubated as describedabove, in the absence (Ctrl) or presence of 200 nM of recombinantGST/GSTCM or 10mM of E64. Cell migration was assessed after13 h. *Indicates a highly significant difference with mock controland GST-treated mock cells (Pp0.001)

Figure 5 (a) Effect of cystatin M on Matrigel invasion. Theexperimental set up was the same as in the legend to Figure 4,except that (1) Matrigel-coated filter inserts were used instead ofuncoated filters (Matrigel was a preparation with reduced growthfactor content); (2) incubation times were much longer, that is, 13,24, 48, 60, and 72 h, because of the barrier function of Matrigel.*Indicates a highly significant difference with parental and mockcontrols (Pp0.001). (b) Effect of recombinant cystatin M and E64on Matrigel invasion. Mock-1 cells were incubated as describedabove, and in the absence (Ctrl) or presence of 200 nM ofrecombinant GST/GSTCM or 10 mM of E64. Invasion was assessedafter 72 h. *Indicates a highly significant difference with mockcontrol and GST-treated mock cells (Pp0.001)


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presence of 200 nM of recombinant GSTCM (Figure 6,GSTCM). In contrast, 10 mM of E64 did not inhibit theadhesion of mock-1 cells (Figure 6, E64). These resultsshow that – as for tumor cell proliferation – tumor celladhesion to endothelial cells in vitro does not seem torequire the participation of lysosomal cysteine pro-teases. In contrast to tumor cell proliferation, however,adhesion of MDA-MB-435S cells to HUVECs wassensitive to GSTCM. Cystatin M has been shown to be adouble-headed inhibitor capable of targeting bothpapain- and legumain-type lysosomal cysteine proteases(Alvarez-Fernandez et al., 1999). Further studies arerequired to determine whether or not a legumain-typecysteine protease(s) might be assisting tumor cells intheir adhesion to endothelial cells.

Discussion

Multiple genetic and epigenetic alterations are involvedin the development of a malignant tumor. Loss ofexpression of certain genes in tumor cells is often linkedto the potential of their gene products to act as tumor ormetastasis suppressors. Our overall working hypothesisis that cystatin M might be such a suppressor gene.Indeed, differential display of RNAs isolated from amatched pair of a primary human breast tumor and itsmetastatic lesion (Sager, 1997) identified cystatin M(CST6) (Sotiropoulou et al., 1997). Although cystatin Mis expressed in normal and premalignant breast epithe-lial cell lines, its expression is often lost in breast cancercell lines (Sotiropoulou et al., 1997). In this report, weanalysed the mRNA expression pattern of cystatin M

and cystatin C in a panel of human tumor cell lines ofdifferent degrees of differentiation and potential fortumor formation, tissue invasion, and metastasis. Whencompared to cystatin C, which is ubiquitously expressed(see also Keppler et al., 1994), cystatin M was found tobe abundantly expressed by only three out of 15 tumorcell lines. These comprised one breast cancer (BT-20)and two prostate cancer cell lines (DU-145 and PC-3).Regulation of the steady-state levels of the two cystatinmRNAs seemed therefore to be quite different. Togetherwith their quite different inhibition profiles towardslysosomal cysteine proteases (Abrahamson, 1994), thissuggested that cystatins C and M might likely playdifferent roles in tumor progression.In a recent study of a case of oropharyngeal

squamous cell carcinoma, cystatin M mRNA andprotein were found to be significantly increased in ametastatic lesion when compared to the primary tumor(Vigneswaran et al., 2003). Cystatin M immunostainingin the metastatic cervical lymph node lesion wasrestricted to a few focally keratinized areas. Therefore,downregulation of expression of cystatin M duringprogression of cancers may only be relevant for a fewcancers such as breast cancer and melanoma. Alterna-tively, immunostaining of cystatin M in a subpopulationof cells that have metastasized suggests that the cystatinM gene might be re-expressed in oropharyngeal tumorcells that are undergoing differentiation at the secondarysite. This would be in agreement with earlier studiesshowing that cystatin M is crosslinked to the stratumcorneum during terminal differentiation of keratino-cytes (Zeeuwen et al., 2001). Moreover, cystatin Mexpression was found to be increased 20-fold in humanhead and neck squamous carcinoma cells, upon induc-tion of cell differentiation by the low calcemic vitaminD3 analog EB1089 (Lin et al., 2002). Therefore, furtherstudies are needed to determine the extent of expressionand downregulation/re-expression of cystatin M innormal and tumor tissues, respectively.To address the potential biological significance of the

loss of expression of cystatin M in tumor cells, weconstitutively expressed cystatin M in the highlymalignant human breast cancer cell line MDA-MB-435S (Price and Zhang, 1990). The initial RT–PCRscreen had established that this cell line expressed littleor no cystatin M. Ectopic expression of cystatin M inMDA-MB-435S cells reduced their in vitro growth rate,migration, invasion through Matrigel, and adhesion toan endothelial cell monolayer. Reduced proliferation ofMDA-MB-435S cells was independent of the inhibitionof lysosomal cysteine proteases because neither thebroad-spectrum inhibitor E64 nor its membrane-per-meant ester derivative E64d were able to mimic theeffect of cystatin M expression. In addition, thebacterially expressed recombinant GSTCM fusionprotein was unable to reduce the proliferation ofMDA-MB-435S cells even at concentrations of 2.0 mM.The fusion protein, however, was fully functional as aninhibitor of cysteine proteases, since it inhibited papainwith a Ki (1.35 nM) very similar to that of the nativeinhibitor (0.39 nM) (Ni et al., 1997).

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Figure 6 Effect of cystatin M on tumor-endothelial cell adhesion.Mock-1 (Ctrl) and CM-13 (CysM) cells were labeled with 1-mMBCECF-AM, washed in HBSS, and added (3� 105 cells per well) toconfluent monolayers of HUVECs grown in 24-well plates.Transfected clones were allowed to adhere for 15min at 371C.Nonadherent cells were removed and endothelial monolayerswashed three times with HBSS to remove loosely bound tumorcells. Adherent cells were then lysed and fluorescence intensitydetermined. Adhesion of mock-1 cells to endothelial cells was alsostudied in the presence of 200 nM of recombinant GSTCM or 10 mMof E64, and compared to the adhesion of untreated Mock-1 cells(Ctrl). Results were expressed as mean7s.e.m. (n¼ 8) andcompared statistically using one-way ANOVA. Adhesion measure-ments were normalized to that of the mock clone to clearly identifythe differences in tumor cell adhesion


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The lack of effect of bacterially expressed cystatin Mon cell proliferation suggested that this effect coulddepend on post-translational modifications of theprotein. Indeed, several cystatins have been shown toundergo N- or O-glycosylation, Ser/Thr-phosphoryla-tion, dimerization, or to bind calcium ions (Bell et al.,1989; Laber et al., 1989; Ekiel et al., 1997; Merz et al.,1997; Taupin et al., 2000). Further studies are requiredto determine which of these modifications occur incystatin M and are important for its antiproliferativefunction. Others have shown that cystatin C is able toinduce DNA synthesis and mitosis in various cell typesincluding fibroblasts, mesangial cells, and neuronal stemcells (Sun, 1989; Leung-Tack et al., 1990; Tavera et al.,1992; Taupin et al., 2000). In rat neuronal stem cells,stimulation of cell proliferation by cystatin C was alsoshown to be independent of the inhibition of cysteineproteases (Taupin et al., 2000). Together with thepresent data, this suggests that cystatin C and cystatinM may have opposite effects on cell proliferation.The ability of cystatin M to inhibit migration and

invasion of MDA-MB-435S cells through Matrigel wasconsistent with previous reports on the inhibition ofmigration and invasion by stefin A (cystatin A), cystatinC, and E-64 (Boike et al., 1992; Kobayashi et al., 1992;Redwood et al., 1992; Sexton and Cox, 1997; Kolkhorstet al., 1998; Coulibaly et al., 1999; Konduri et al., 2002).However, the extent of inhibition of invasion by cystatinM observed in the present study, that is, X95%,suggested that cystatin M may target some cysteineprotease(s) that was rate-limiting in the proteolyticcascade, leading to matrix dissolution and invasion. Thepotential physiological target(s) of cystatin M could beone of the 11 papain-type cysteine proteases (Brommeand Kaleta, 2002; Mason et al., 2002; Puente et al.,2003), or it could be a legumain-type cysteine proteasesuch as Asn-endopeptidase (Alvarez-Fernandez et al.,1999). With a Ki of 1.6 pM, cystatin M indeed veryefficiently inhibits Asn-endopeptidase (¼mammalianlegumain) (Alvarez-Fernandez et al., 1999). This latterhighly selective protease has recently been shown to beable to activate the zymogen of matrix metalloprotei-nase-2 (proMMP-2 or progelatinase A) (Chen et al.,2001). Consistent with this view is also the fact thatoverexpression of Asn-endopeptidase in transformedhuman embryonal kidney HEK-293 cells leads toincreased migration, invasion, and proMMP-2 activa-tion (Liu et al., 2003). Preliminary studies from ourlaboratory using microarray and RT–PCR analysisshow that MDA-MB-435S cells did indeed expressAsn-endopeptidase. However, Asn-endopeptidase isnot sensitive to E64 even at concentrations of up to2mM (Kembhavi et al., 1993; Chen et al., 1997).Therefore, if the rate-limiting factor targeted by cystatinM is indeed Asn-endopeptidase, it would suggest thatcystatin M and E64 acted at two different levels withinthe same pathway. For example, Asn-endopeptidasemight be required for the activation of proMMP-2,which in turn is required to loosen up the extracellularmatrix, thereby stimulating endocytosis/phagocytosis

and digestion of extracellular matrix components bylysosomal proteases (Keppler et al., 1996).Cathepsin B, a papain-type cysteine protease, parti-

cipates in both pericellular and intracellular digestion ofmatrix proteins in breast cancer BT-20 and BT-549 cells,respectively (Sameni et al., 2000). Both E64 and thehighly selective cathepsin B inhibitor CA074 blockpericellular degradation of collagen-IV by BT-20 cellsand intracellular degradation of collagen-IV by BT-549cells by more than 50%. In addition, serine and matrixmetalloproteinase inhibitors blocked pericellular degra-dation of collagen-IV by B50%. This suggests thatcathepsin B, similar to Asn-endopeptidase, may parti-cipate in a pericellular proteolytic cascade. Indeed, otherstudies show that cathepsins B and L are involved in theconversion of pro-uPA into active uPA (urinary-typeplasminogen activator/urokinase), and that inhibition ofinvasion by E64 is due to interference with pro-uPAactivation at the tumor cell surface (Goretzki et al.,1992; Kobayashi et al., 1992; Guo et al., 2002).Inhibitors might thus regulate cysteine proteases in-volved in the activation of both MMP-2 and uPA.Besides the detachment from the primary tumor, the

dissolution of extracellular matrices, and the invasion ofsurrounding normal tissue, successful metastasis de-pends on a number of other critical steps. Metastatictumor cells also need to survive the shear stressencountered during hematogenous dissemination, con-tinuously escape immune surveillance, arrest at a distantsite, and, finally, initiate cell divisions within acompletely different microenvironment. Whereas mostof these steps cannot be analysed in vitro, we havenevertheless attempted to study the role of cystatin M inthe adhesion of MDA-MB-435S cells to vascularendothelial cells such as HUVECs. Ectopic expressionof cystatin M was found to reduce adhesion to thisendothelium by 30% when compared to control cells.Although HUVECs are by no means representative ofmicrovascular endothelial cells at sites of secondarycolonization, our data are the first to report on theinhibitory effect of a cystatin on tumor-endothelial celladhesion. Unlike cell proliferation and cell migration/invasion that were either not sensitive to exogenousadministration of E64 or GSTCM or respondedstrongly to both agents, respectively, tumor-endothelialcell adhesion showed a split response with E64 havingno significant effect and GSTCM reducing adhesion tothe same extent as ectopic expression of the inhibitor.Under physiological conditions of blood flow and shearstress, we can reasonably expect that the antiadhesiveeffect of ectopically expressed cystatin M will be muchmore pronounced.In summary, we have shown that ectopic expression

of cystatin M in the highly proliferative, invasive, andmetastatic MDA-MB-435S cells seriously compromisedtheir in vitro malignant properties. Some of the effects ofcystatin M, on cell proliferation in particular, werefound to be unrelated to the inhibition of lysosomalcysteine proteases, and suggest a novel mechanism ofaction. Future studies are now aimed at elucidating thismechanism. In order to clearly establish that cystatin M

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has a bona fide tumor- and/or metastasis-suppressingfunction, it will be crucial to analyse its role in tumorformation, invasion, angiogenesis, and metastasis invivo. Such in vivo studies in mice are currently inprogress in our laboratory.

Materials and methods

Cell culture and transfection conditions

BT-20, BT-549, MDA-MB-435S, CaCo-2, HT-29, and U-87MG cells were purchased from ATCC and maintainedaccording to their protocols. DU-145, PC-3, and LNCaP cellswere a kind gift from Dr Michael Cher (Wayne StateUniversity, Detroit, MI, USA). DU-145 cells were maintainedin DMEMþ 10% FBS. PC-3 and LNCaP cells were main-tained in RPMIþ 5% FBS. All media were supplemented with100U/ml of penicillin and 100mg/ml of streptomycin (Sigma,St Louis, MO, USA). MCF-10A, a human diploid breastepithelial cell line, was obtained from the Barbara AnnKarmanos Cancer Institute Cell and Tissue Core (WayneState University, Detroit, MI, USA). This cell line wasmaintained in DMEM/F12 containing 5% horse serum, 1mMinsulin (Sigma, St Louis, MO, USA) and 20 ng/ml EGF (BDCollaborative Research, Bedford, MD, USA). The humanmelanoma cell lines WM-164, WM-793, WM-852, WM-902B,WM-1205Lu, and WM-1341D were a generous gift from DrMeenhard Herlyn (Wistar Institute, Philadelphia, PA, USA).They were grown in MCDB-153/L-15 (Sigma, St Louis, MO,USA) supplemented with 2% FBS, 5mg/ml bovine insulin, and1.68mM CaCl2. Human umbilical vein endothelial cells(HUVECs) were isolated and cultured as previously reported(Kevil et al., 1998). HUVECs were seeded onto fibronectin-coated 24-well tissue culture plates in EGM media, accordingto the recommendations from Clonetics Cell Systems (Cam-brex, East Rutherford, NJ, USA), and cultured to confluency.All experiments were performed using passage 1 HUVECs.To eliminate a potential source of heterogeneity, the MDA-

MB-435S cell ‘line’ was first cloned by limiting dilution and aclone representative of the overall parental phenotype (e.g.,morphology and cystatin expression) isolated and used fortransfection experiments. Transfections were performed by theliposome method using Lipofectamine Plus (Gibco, Rockville,MD, USA). Briefly, 2 mg of DNA was transfected into 8� 105cells, according to the manufacturer’s instructions. At 48 hafter transfection, cells were split into two 100-mm dishes andthe selection drug Zeocin (Invitrogen, Carlsbad, CA, USA)was added at 400mg/ml. The media were changed every 48 h.After 2 weeks, colonies appeared. In all, 24 colonies from themock-transfected and cystatin M-transfected cells were ex-panded and maintained in DMEM supplemented with 10%FBS, antibiotics, and 200mg/ml Zeocin.

Semiquantitative RT–PCR analysis

RNA was isolated from human cell lines with Trizol (Gibco,Rockville, MD, USA) and reverse-transcribed. Briefly, 1mg ofDNase-treated total RNA was annealed with 0.5 mg oligo-dT15and reverse-transcribed in a 20ml volume containing 1�reverse transcription buffer, 0.1mg/ml BSA, 40U of RNasin(Promega, Madison, WI, USA), 1mM dNTPs, and 200U ofMMLV-reverse transcriptase (Promega, Madison, WI, USA)at 371C for 90min. A 2ml aliquot was used for subsequentPCR reactions using the Taq PCR Core kit from Qiagen(Valencia, CA, USA). For cystatin C (591C, 261-bp PCRproduct), cystatin M (611C, 422-bp PCR product), and b2-

microglobulin (561C, 150-bp PCR product), the amplificationconditions were as follows: 25 cycles of 941C for 30 s, 59, 61 or561C for 30 s, 721C for 3min, followed by a final 5-minextension at 721C. PCR products were resolved on a 2%agarose gel, stained with SYBR Green, imaged, and quantifiedusing a STORM 840 system from Molecular Dynamics. Theprimer sequences were as follows: cystatin C forward, 50-AGGAGGGTGT GCGGCGTG-30; cystatin C reverse, 50-GCCAAGGCAC AGCGTAGAT-30; cystatin M forward, 50-TGGTCGCATT CTGCCTCCTG-30; cystatin M reverse, 50-CTCGGGGACT TATCACATCT GC-30; b2-microglobulinforward, 50-TTAGCTGTGC TCGCGCTACT CTCTC-30; b2-microglobulin reverse, 50-GTCGGATGGA TGAAACCCAGACACA-30.

Cystatin M expression plasmids

Constitutive mammalian expression vector The full-lengthhuman cystatin M cDNA was PCR-amplified with thefollowing primers: forward, 50-ttaGGTACCATCATGGCGCG TTC-30 and reverse, 50-tttGAATTCGGACTTATCAC ATCTGC-30. The underlined sequencesindicate restriction enzyme sites for KpnI and EcoRI (NewEngland Biolabs, Beverly, MA, USA) in the forward andreverse primers, respectively. PCR was performed as follows:941C for 3min, then 30 cycles of 941C for 30 s, 601C for 30 s,and 721C for 1min, followed by a final extension of 721C for5min to produce a 475-bp product. The PCR product wasdigested with KpnI and EcoRI at 371C for 1 h, purified with aPCR purification kit (Qiagen, Valencia, CA, USA) and ligatedinto the mammalian expression vector pTracer-CMV2 (Invi-trogen, Carlsbad, CA, USA). This vector uses the CMVpromoter to drive expression of the inserted gene. Severalbacterial colonies were picked and plasmids were isolated andsequenced to verify the integrity of the construct.

Inducible bacterial expression vector The DNA sequenceencoding full-length mature human cystatin M was amplifiedby PCR and subcloned into the IPTG-inducible bacterialexpression vector pGEX2T (Amersham-Biosciences, Piscat-away, NJ, USA), as described previously (Sotiropoulou et al.,1997). IPTG-driven expression from this plasmid will generatea glutathione-S-transferase – cystatin M fusion protein(GSTCM). Several bacterial colonies were picked and plas-mids were isolated and sequenced to verify the integrity of thefusion construct.

Production and purification of recombinant cystatin M

The empty vector (pGEX2T) and the fusion construct(pGEX2T/cystatin M) were transformed into Origami (Nova-gen, Madison, WI, USA), an E. coli strain that has beenengineered to increase the likelihood of disulfide-bond forma-tion and correct the folding of recombinant proteins.Recombinant GST and GSTCM were expressed and purifiedas described earlier (Sotiropoulou et al., 1997). Correct foldingof recombinant GSTCM was assessed by titration of papainactivity on Z-Phe-Arg-AMC (Bachem, Torrance, CA, USA),as previously described (Keppler et al., 1997). A similartitration was performed with recombinant GST and served asa negative control. Before addition to cell cultures, purifiedrecombinant GST and GSTCM were filter-sterilized bypassage through a 0.22- mm pore-size Millex-GV low proteinbinding durapore PVDF membranes (Millipore Corp., Bed-ford, MA, USA).

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SDS–PAGE and immunoblot analysis

Parental, mock-transfected, and cystatin M-transfected clonesof MDA-MB-435S were seeded in 100-mm dishes. When 70–80% confluent, cells were washed with serum-free media andincubated in serum-free media for 24 h. Since cystatin M is asecreted protein (Ni et al., 1997; Sotiropoulou et al., 1997),conditioned media were collected and concentrated by TCAprecipitation of total proteins. Proteins were resuspended inLaemmli buffer, the volume of which was adjusted to cellnumber. Samples normalized this way were electrophoresed ona 15% SDS–PAGE gel, electroblotted onto nitrocellulosemembranes, and probed with rabbit polyclonal antibodiesraised against cystatin C (a kind gift from Dr MagnusAbrahamson, University of Lund, Sweden) (Abrahamsonet al., 1986) and cystatin M (Sotiropoulou et al., 1997). Thebound antibodies were visualized with an HRP-conjugatedsecondary antibody (Jackson ImmunoResearch Laboratories,West Grove, PA, USA), and enhanced chemiluminescence(Pierce, Rockford, IL, USA).

Quantitation of cell growth

A total of 500 cells of either mock-transfected or cystatin M-expressing clones were seeded in each of six wells in seven 96-well microtiter plates. Cells were fed every 48 h. Plates werecollected on days 1, 3, 5, 7, 9, 11, and 13. The media wereremoved and plates were stored at �701C until all plates werecollected. Cell number was determined with the CyQuantsystem (Molecular Probes, Eugene, OR, USA) on a TecanSpectraFluor Plus fluorescent plate reader, using an FITC-based filter set. Relative fluorescence units were converted tocell number using a calibration curve established with knowncell numbers.In separate experiments, 500 cells of the mock-transfected

clone were seeded in each of six wells in five 96-well microtiterplates containing either 24-h conditioned medium from mock-transfected cells or cystatin M-transfected cells. Cells were fedwith the appropriate medium every 48 h. Plates were collectedon days 1, 3, 5, 7, and 9. Cell number was determined asdescribed above. Experiments were repeated three times. Thegrowth of mock-transfected cells was also studied in thepresence and absence of various doses of recombinant GSTand GSTCM, or E64 and its membrane-permeant ester-derivative E64d (Buttle et al., 1992).

Migration and Matrigel invasion assays

Transwell culture inserts with their companion 24-well plates(BD Falcon, Bedford, MD, USA) were used for the assessmentof cell migration and extracellular matrix invasion. The cultureinserts consist of an 8-mm pore-size PET membrane uponwhich cells can be seeded and grown. In all, 50 000 cells in500ml of serum-free DMEM were seeded on top of the PETfilters. The bottom wells received 750 ml of DMEM supple-mented with 1% FBS as chemoattractant. Control wellsreceived DMEM only (no chemoattractant). After variousperiods of incubation at 371C in the CO2 incubator, cells in thetop well that had not migrated were wiped off the top surfaceof the PET membrane, using a cotton swab. Cells on thebottom surface of the filter, that is, cells that had migratedthrough the filter, were fixed in 2% paraformaldehyde andstained using Mayer’s hematoxylin and a 1% solution of eosinY (EMD Biosciences, Madison, WI, USA). Culture insertswere placed on top of a hemocytometer in a drop of immersion

oil and migrated cells counted using an inverted microscope at� 100 magnification. Triplicate experiments were performedfor each clone and each time point. Results are expressed asmeans7s.e.m. and compared statistically using ANOVA. Themigration of mock-transfected cells was also studied in thepresence and absence of various doses of recombinant GSTand GSTCM, or E64.For the Matrigel invasion studies, culture inserts with an 8-

mm pore-size PET membrane precoated with growth-factor-reduced Matrigel (BD Biocoat, Bedford, MD, USA) wereused. Matrigel, a reconstituted basement membrane, blocksnon-invasive cells from migrating through the membrane. Incontrast, invasive cells such as MDA-MB-435S cells are able todegrade and invade into Matrigel and eventually cross themembrane through the 8-mm pores (Hendrix et al., 1987).Matrigel was allowed to rehydrate for 1 h in serum-freemedium at 371C in the CO2 incubator. Culture inserts werethen transferred to a new 24-well plate and loaded with 50 000cells per well in serum-free medium. All other conditions wereas described for the migration assays.

Tumor-endothelial cell adhesion assay

Tumor cell adhesion to the vascular endothelium wasperformed as previously described for monocytes (Kevil et al.,2001). Briefly, mock-1 and CM-13 cells were labeled for 20minwith the fluorescent dye BCECF-AM (Molecular Probes,Eugene, OR, USA), at a final concentration of 1mM, in Hank’sbalanced salt solution (HBSS). After three washes in HBSS,tumor cell clones were added to HUVEC monolayers at 371Cfor 15min. Nonadherent cells were removed and endothelialmonolayers washed three times with HBSS to remove looselybound cells. Adherent cells were then lysed with 1-mM NaOHand fluorescent intensity determined using a Tecan GeniosPlus plate reader. Adhesion of mock-1 cells to HUVECs wasalso studied in the presence and absence of recombinantGSTCM (200 nM) or E64 (10mM), and compared to theadhesion of CM-13 cells. Eight experiments (n¼ 8) wereperformed for each clone and each treatment. Results wereexpressed as means7s.e.m. and compared statistically usingANOVA. Adhesion measurements were normalized to that ofthe mock clone to clearly identify differences in tumor celladhesion.

Statistical analyses

One-way ANOVA analyses were performed for the compar-ison of transfection clones. Two-factor ANOVA analyses wereperformed for the comparison of mock-transfected clonesgrown in the presence or absence of various compounds ormedia.

Acknowledgements

We wish to acknowledge the generous gift of humanmelanoma cell lines from Dr Meenhard Herlyn (The WistarInstitute, Philadelphia, PA, USA) and of cystatin C antibodiesfrom Dr Magnus Abrahamson (University of Lund, Lund,Sweden). Our warmest thanks are also due to Dr AvrahamRaz, for his lively input throughout this work. Part of thisstudy was supported by a Virtual Discovery Grant (DK) fromthe Barbara Ann Karmanos Cancer Institute and ResearchGrants CA91785 (DK) and CA36481 (BFS) from the NationalCancer Institute.

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Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells

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