Expression of the a7b1 Laminin Receptor SuppressesMelanoma Growth and Metastatic Potential1

Barry L. Ziober,2 Yao Qi Chen, Daniel M. Ramos,Nahid Waleh, and Randall H. Kramer3

Departments of Stomatology [B. L. Z., Y. Q. C., D. M. R., R. H. K.] andAnatomy [R. H. K.], University of California San Francisco, SanFrancisco, California 94143; and SRI International, Menlo Park,California 94025 [N. W.]

AbstractThe a7b1 integrin is a laminin-binding receptor thatwas originally identified in melanoma. Here, we showthat, in clonally derived mouse K1735 melanomavariant cell lines with high (M-2) and low (C-23)metastatic potential, elevated expression of a7correlates with reduced cell motility, metastasis, andtumor growth. Both cell lines showed similar b1integrin-dependent adhesion to laminin-1 and the E8laminin fragment. However, the highly metastatic M-2cells rapidly migrated on laminin, whereas thenonmetastatic C-23 cells were minimally motile.Laminin-binding integrin profiles showed that the M-2cells expressed moderate amounts of a1 and abundanta6 but low or undetectable levels of a2 and a7. Bycontrast, C-23 cells expressed low or undetectablelevels of a1, a2, and a6 but had up-regulated levels ofa7. Consistent with the protein data, Northern blotanalysis showed that levels of a7 mRNA were highestin the poorly metastatic variant cells, whereas a6message was not detected; in contrast, a6 mRNA waselevated in the highly metastatic cells, whereas a7message was not detected. Forced expression of a7 inthe M-2 cells suppressed cell motility, tumor growth,and metastasis. Collectively, these results indicatethat, during melanoma progression, acquisition of ahighly tumorigenic and metastatic melanomaphenotype is associated with loss of the a7b1 lamininreceptor.

IntroductionThe process of melanoma tumor growth and disseminationinvolves specific interactions with tumor cell surface adhe-

sion receptors and multiple adhesive components of theECM.4 For example, stationary cells form stable focal adhe-sions with the ECM, whereas motile cells form weak andtransient contacts. The receptors that mediate the cellularadhesive interactions with the ECM are derived from a largefamily of heterodimeric molecules referred to as the integrins(1). An abundance of evidence has shown that alterations inintegrin expression are linked to tumorigenicity and metas-tasis (2, 3). In melanoma, several integrins, including the b1integrins and avb3, have been implicated in the diseaseprocess (2, 4–6).

Some of the most pronounced changes in melanoma in-tegrin expression occur in the laminin-binding receptors.Laminin contains multiple sites for cell attachment, which ismediated by several b1 integrins. The major laminin recep-tors are a1b1, a2b1, a3b1, and a6b1 and are typically up-regulated during conversion to malignant melanoma (7–12).Increasing evidence indicates that integrin-laminin interac-tions not only promote cell attachment but can also stimulatecell migration, tumor growth, metastasis, angiogenesis, andprotease production (13–16). Overall, the laminin integrinreceptors appear to play some active role in the processesthat lead to melanoma invasion and metastasis.

Previously, we reported the identification and character-ization of the integrin complex designated a7b1 (17–19).More recently, we showed that this integrin complex is alaminin receptor that adheres to laminin-1 and laminin-2/4(20). Although originally identified as a laminin receptor inhuman and murine melanoma, a7b1 integrin has been pri-marily characterized in muscle (17, 21–24). In skeletal andsmooth muscle, a7 expression is regulated in a differentia-tion specific manner, being highly expressed in the terminaldifferentiated nonproliferated state (17, 22–24). The functionof a7 in melanoma has yet to be defined.

The murine K1735 melanoma model is a well-character-ized tumor system that is composed of several clones thatdiffer in tumorigenicity and metastatic potential (25, 26). Inthis study, we examined the role of laminin-binding integrinsin metastatic and nonmetastatic cell lines derived from theparental K1735 cells. We show that, in the K1735 melanomavariants, there is an inverse correlation between expressionof the a7 subunit and metastatic potential. Transfection ofthe a7 subunit into a7-null metastatic cells resulted in a cellline that was less motile and metastatic and formed smallertumors. Our results suggest that loss of a7b1 during mela-noma progression contributes to the tumorigenic and meta-static phenotype.

Received 1/25/99; revised 5/24/99; accepted 5/27/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 This work was supported by NIH Grants DE 13479 and DE 11912 and bya University of California Individual Investigator Research Award(to B. L. Z.).2 Present address: Department of Otorhinolaryngology, University ofPennsylvania, Philadelphia, PA 10104.3 To whom requests for reprints should be addressed, at, Department ofStomatology, University of California San Francisco, San Francisco, CA94143-0512. Phone: (415) 476-3274; Fax: (415) 476-4204; E-mail: randyk@itsa.ucsf.edu.

4 The abbreviations used are: ECM, extracellular matrix; FACS, fluores-cence-activated cell sorting; HRP, horseradish peroxidase; AP, alkalinephosphatase.

479Vol. 10, 479–490, July 1999 Cell Growth & Differentiation

ResultsAdhesion and Migration of Melanoma Cell Variants onLaminin-1 and E8 Fragment. In adhesion assays, wefound that both the highly tumorigenic and metastatic M-2and the less tumorigenic, poorly metastatic C-23 cellsadhered well to laminin-1 (Fig. 1, A and B). Both cell linesreached maximal binding at ;30 mg/ml laminin. In addi-tion, both M-2 and C-23 cells adhered to the E8 fragment,which represents the long arm of the laminin molecule.However, C-23 cells bound the E8 fragment more effi-ciently at lower coating concentrations than they boundlaminin (Fig. 1A), and they bound more efficiently than didthe M-2 cells (Fig. 1B). Blocking antibodies specific to b1integrins and to the E8 fragment could separately blockadhesion of C-23 cells to laminin and E8, respectively (Fig.

1C). M-2 cell binding to laminin and E8 was also inhibitedwith anti-b1 blocking antibodies, but anti-E8, althoughable to disrupt binding to E8, only partially blocked bindingto laminin (Fig. 1D). Thus, in both cell lines, b1 integrins, inparticular those interacting with the E8 fragment of lami-nin, are responsible for adhesion to laminin-1.

In contrast to adherence, migration on laminin-1 was sub-stantially different for M-2 and C-23 cells. The highly meta-static M-2 cells migrated efficiently over a wide range oflaminin-1 concentrations, whereas C-23 cells migratedpoorly or not at all (Fig. 1E). For both cell lines, optimalmigration occurred at coating concentrations of 10–30 mg/ml; at higher concentrations, migration was inhibited (Fig.1E). The E8 fragment as ligand was also able to induce adifferential motility response, with the M-2 cells moving more

Fig. 1. Adhesion and migrationof K1735 melanoma cell lines. Aand B, dose response of adhe-sion of C-23 (2 3 104/well; A) andM-2 cell (2 3 104/well; B) to lami-nin-1 (f) and the E8 fragment oflaminin (F). C and D, C-23 cells(2 3 104/well; C) and M-2 cells(2 3 104/well; D) were tested foradhesion to laminin-1 (at 10 mg/ml; M) or laminin E8 (at 5 mg/ml;u), as described in “Materialsand Methods.” Blocking anti-bodies to b1 (AIIB2) or to the E8fragment of laminin or controlIgG were preincubated with cellsat 10 mg/ml. For all adhesion as-says, cells bound to collagentype I (at 100 mg/ml) were usedto indicate 100% adhesion. Ad-herence of cells in 1% BSA-coated wells was treated asbackground binding and sub-tracted. A–D, data are presentedas percentages of the total cellsadded to each well. Data points(A and B) and columns (C and D),means of triplicate wells; bars,SD. E, dose response of M-2 (F)and C-23 (f) cell migration onlaminin-1-coated surfaces. F,migration of M-2 (f) and C-23 (o)cells on surfaces coated withlaminin-1 (10 mg/ml), the lamininE8 fragment (5 mg/ml), and fi-bronectin (20 mg/ml). Migrationwas measured using the micro-screen assay, as described in“Materials and Methods.” Thearea covered by out-migratingcells, from the fixed diameter ofthe microscreen, was measuredby computer-assisted imageanalysis (NIH Image). Data points(E) and columns (F), means of atleast five individual measure-ments in pixel units (3 103) andrepresent “relative migration”;bars, SD.

480 a7 Integrin Suppresses Tumorigenicity and Metastasis

rapidly on E8 than the C-23 cells (Fig. 1F). On fibronectin, themigration efficiencies were reversed: the M-2 cells migratedslightly more slowly than the C-23 cells (Fig. 1F), indicatingthat the poor migration of C-23 cells on laminin-1 is not dueto a general defect in their migratory activity. Thus, the factthat M-2 and C-23 cells require b1 integrins for adhesion onlaminin-1 and E8 suggests that these same ECM receptorsare also responsible for the contrasts in cell migration.

Integrin Cell Surface Profile. To determine which b1 in-tegrins were responsible for adhesion and, thus, migration on

laminin-1, we performed Western immunoblotting. We pre-viously showed that a7b1 is expressed in several melanomacell lines and that it binds primarily to laminin-1 (4, 18, 19). ByWestern blot analysis, the two poorly metastatic cell lines,C-23 and C-19, expressed high levels of the a7 subunit (Fig.2A). In contrast, the M-2 cells expressed little to no a7. Theseinitial results suggested that differentially expressed integrinreceptors may be responsible for the adhesive and migratoryproperties displayed by these cells on laminin. Therefore, weanalyzed the integrin profiles of M-2 and C-23 cells further.

We used FACS analysis to examine the cell surface inte-grin profile for M-2 and C-23 cells (Table 1). Both cell linesexpressed very little a2. In M-2 cells, a6 and a1 were themost prominent laminin-1-binding integrin subunits ex-pressed. Little a7 was detected in M-2 cells. In contrast,C-23 cells expressed high levels of a7 and very little a6 or a1.Thus, in the highly metastatic M-2 cells, a6 and a1 appear tobe the major laminin-1 receptors, whereas a7 is the majorlaminin-1 receptor in the nonmetastatic C-23 cell line. Theseresults also suggest that, during conversion to the highlytumorigenic/metastatic melanoma phenotype, a7 expres-sion is down-regulated and that of a1 and a6 is up-regulated.We, therefore, hypothesized that the a7 subunit may play arole in regulating cell motility and, thus, metastatic potential.

Gene Transcription. To identify a mechanism responsi-ble for the differential expression of the a6 and a7 proteins inM-2 and C-23 cells, we performed Northern blot analysis. Wescreened total RNA from a panel of K1735 melanoma celllines with differing metastatic potential, using cDNA probesfor a6 and a7. The mRNA levels for a6 were consistentlyelevated in cells of high metastatic potential (C-26, M-2, andM-4) but low or undetectable in cell lines that were poorlymetastatic (C-10, C-19, and C-23; Fig. 2B). In contrast,mRNA levels for a7 reflected those seen for protein expres-sion: high in poorly metastatic cell lines like C-23 and unde-tectable in the highly metastatic cell lines like M-2 (Fig. 2B).By densitometric analysis, the relative level of a7 transcriptsin C-23 cells was at least 10-fold that of the M-2 cells (datanot shown). Thus, the highly metastatic cells lacked expres-sion, at both the protein and mRNA levels, of the laminin-binding integrin complex a7b1.

Forced Expression of a7 in Metastatic Cells. To directlydetermine whether a7b1 plays a role in regulating melanomamotility and metastatic potential, we transfected the full-length a7 cDNA into M-2 cells by retroviral infection. Afterselection with G418, a population of a7-expressing M-2 cells(designated M-2-a7) were isolated after two rounds of FACSanalysis (Fig. 3). Expression of a7 in M-2 parental and

Fig. 2. Expression of a7 mRNA in K1735 cell variants. A, equal quantitiesof cellular lysates from M-2 (Lane 1), C-19 (Lane 2), and C-23 (Lane 3) cellswere processed for SDS-PAGE in a 7.5% polyacrylamide gel. Followingtransfer, the nitrocellulose membrane was probed with anti-a7 (1211)antiserum and the position of the a7 subunit was determined using thecolor development detection system for AP. The position of the a7 subunitis indicated. The band at Mr ;70,000 is a proteolytic fragment of the a7subunit (20, 24). B, total RNA was isolated as described in “Materials andMethods” from nonmetastatic (C-10, C-19, and C-23) and highly meta-static (C-26, M-2, and M-4) K1735 cell lines and analyzed by Northernblotting using a 32P-labeled murine a6 cDNA probe (a). The blot wasstripped and then reprobed with an a7 cDNA probe (b). Positions of the;6.0-kb a6 mRNA and the ;4.1-kb a7 mRNA transcripts are indicated.Equal loading was assessed by ethidium bromide staining.

Table 1 Flow cytometry-determined expression levels of laminin-binding integrin subunits in K1735 M-2, C-23, and M-2-a7 cell lines

Cell lineIntegrin subunit expression levela

a1 a2 a6 a7

M-2 24.8 6.3 53.4 13.3C-23 6.6 3.8 4.0 62.1M-2-a7 10.8 4.9 22.0 129.3

a Flow cytometry peak area (arbitrary units). Shown is a representativeexperiment that was performed three independent times.

481Cell Growth & Differentiation

M-2-a7 cells was analyzed by Western blotting (Fig. 4). Thelevel of a7 in M-2-a7 cells exceeded that expressed by C-23.Upon reduction, a7 expressed in M-2-a7 cells displayed thecharacteristic light chain (Mr 37,000, representing the cyto-plasmic domain; Ref. 20; Fig. 4). Western blotting detectedlittle to no a7 expression by M-2 parental cells (Fig. 4).However, some expression of a7 could be seen in the M-2parental cells when the more sensitive method of FACSanalysis was used (Table 1). This agrees with our observationthat a7 protein can be seen by Western blots in M-2 cellsonly after prolonged exposure to X-ray film or protein over-loading (data not shown). M-2 cells expressed at least 5-foldless a7 than the nonmetastatic C-23 cells, as determined byFACS (Table 1). In contrast, a7 levels in M-2-a7 cells werenearly 10-fold higher than levels in the M-2 parental cells and2-fold higher than C-23 cells. Thus, the transfected M-2 cellsnow expressed a7 at levels similar to those of the nonmeta-static C-23 cell line. Furthermore, the FACS analysis con-firmed that this expression was located at the cell surface.

Because alterations in integrin expression are linked tomotility, differentiation, cell attachment, tumor growth, and

metastasis (2, 3), we asked what changes in integrin expres-sion occurred as a consequence of a7 expression in M-2cells. Forced expression of a7 resulted in no changes in a2and only a modest decrease in a1 expression (;2-fold re-duction; Table 1). The most dramatic changes were seen inthe expression levels of a7 and a6 (above and Table 1). Thus,expression of a7 in M-2 cells resulted in a cell surface inte-grin profile that now more closely resembled that of thenonmetastatic C-23 cell line (Table 1), in that a7 levels werehigh and a6 levels were reduced.

Adhesion to Laminin-1. We next compared the laminin-1-binding ability of the M-2-a7 cells with that of M-2 parentaland C-23 cell lines. All three cell lines adhered to laminin-1,but the M-2-a7 cells adhered more readily (Fig. 5). Anti-a2antibodies did not block laminin-1 adhesion in any of the celllines tested (data not shown). This probably reflects the factthat M-2, M-2-a7, and C-23 cells express little a2 (Table 1).Addition of blocking antibodies to a1 only slightly reducedthe binding of any of the cell lines to laminin-1. When block-ing antibodies to a6 were added alone, the adhesion of M-2cells was reduced (Fig. 5A), indicating that the primary lami-nin receptor in the M-2 cell line is a6. In contrast, adhesion ofC-23 and M-2-a7 cells was essentially unaffected by block-ing antibodies to a6 or by a mixture of anti-a1 and -a6 (Fig.5, B and C). Blocking antibodies to a7 slightly blocked M-2cells but nearly completely inhibited M-2-a7 cells (Fig. 5D).Finally, when blocking antibodies to a7 were added to theanti-a1 and -a6 mAb mixture, adhesion to laminin-1 wascompletely inhibited for all cell lines. Thus, these resultsindicated that the contribution of a3b1 to laminin-1 adhesionis minimal, if at all, in these cell lines, which is consistent withthe laminin-5 isoform specificity of this integrin (27–29). Moreimportantly, these results demonstrate that M-2-a7 cells, incontrast to M-2 cells, use a7b1 as their primary laminin-1receptor.

Tumor Growth in Vivo. Previous work has shown that,when M-2 cells are injected s.c. into syngeneic mice, theyform aggressively growing tumors that are highly metastatic(25, 30, 31). In contrast, the a7-expressing C-23 cell line

Fig. 3. Analysis of a7 cell surface expression in M-2, M-2-a7, and Mockcell lines. Flow cytometry analysis of the cell-surface expression levels inM-2, M-2-a7, and Mock cell lines was performed with optimal concen-tration of mAb CY8 (anti-a7), followed by incubation with FITC-labeledgoat antirat IgG. The retrovirally transfected cell lines M-2-a7 and Mockwere sorted for expression of a7 and lack of a7 expression, respectively.As a control, cells were stained with secondary antibody only (2° Anti-body). a7 expression peaks for each cell line are indicated.

Fig. 4. Expression of a7 in M-2, M-2-a7, and C-23 cells. Equal quantitiesof cellular lysates from M-2 (Lanes 1 and 4), M-2-a7 (Lanes 2 and 5), andC-23 (Lanes 3 and 6) cells were processed for SDS-PAGE in a 7.5%polyacrylamide gel under nonreducing (Lanes 1–3) and reducing (Lanes4–6) conditions. Following transfer, the nitrocellulose membrane wasprobed with anti-a7 (1211) antiserum and the position of the a7 subunitwas determined by ECL. When reduced, the cytoplasmic domain of the a7subunit is released (20). The positions of the a7 subunit and the releasedcytoplasmic domain are indicated.

482 a7 Integrin Suppresses Tumorigenicity and Metastasis

forms slower-growing tumors that metastasize at lower effi-ciency, if at all (25, 30, 31). Furthermore, a7 expression isassociated with the terminally differentiated nonproliferatingstate in muscle. We, therefore, investigated whether in-creased expression of a7 in M-2 cells can affect in vivo tumorgrowth. M-2 parental and M-2-a7 cells were injected s.c. intosyngeneic C3H/HeN mice. In all mice injected with M-2 pa-rental cells, rapidly growing tumors were observed (Table 2and Fig. 6). In contrast, mice injected with M-2-a7 cellsshowed reduced tumor take, and the slow-growing tumorsthat did form were, on average, at least 2-fold smaller, bysize, than the M-2 parental cell tumors (Table 2 and Fig. 6).For example, by day 34, tumors produced from the M-2 cellswere .41 mm in diameter or 38.8 3 103 mm3 in volume,whereas tumors derived from M-2-a7 cells did not exceed 23mm in diameter or 6.4 3 102 mm3 (Table 2). Growth ofM-2-a7 tumors started to plateau at day 29, a pattern thatwas more pronounced by day 34. In contrast, tumors estab-lished from M-2 parental cells were in logarithmic growth atday 29 and continued in logarithmic growth even up to day34 (Table 2 and Fig. 6). At termination of the experiment,tumors originating from M-2 cells weighed ;20.0 g, on av-erage, whereas those originating from M-2-a7 cells averagedonly ;9.0 g. Expression of a7 was detectable, by Westernblot analysis, in tumors derived from the M-2-a7 cells but notfrom tumors produced by M-2 cells (Fig. 7). Tumors origi-nating from both cell lines were similar histologically.

As an added control, tumors derived from a population ofretrovirally infected G418-resistant M-2 cells that do notexpress a7 (designated Mock; Fig. 3) were compared withtumors generated by M-2-a7 cells (Table 3). The results werevery similar to those obtained with M-2 parental and M-2-a7cells (Table 2). Mock M-2 tumors were faster-growing andnearly 2-fold larger than tumors generated by the a7-trans-

Fig. 5. Adhesion of M2, M-2-a7, and C-23 cell lines to laminin-1. Paren-tal M-2 (A), C-23 (B), and M-2-a7 (C) cells and parental M-2 (f) andM-2-a7 (o) cells (D; 2 3 104/well) were tested for adhesion to laminin-1 (at15 mg/ml) alone or in the presence of anti-integrin antibodies as describedin “Materials and Methods.” Ln, adhesion to laminin-1 only. Blockingantibodies to a1 (Ha 31/8), a6 (GoH3), and a7 (CY8) were preincubatedwith cells at 10 mg/ml. Cells bound to collagen type I (at 100 mg/ml) wereused to indicate 100% adhesion. Adherence of cells in 1% BSA-coatedwells was treated as background binding and subtracted. Data are pre-sented as percentages of the total cells added to each well. Columns,means of triplicate wells; bars, SD.

Table 2 Tumorigenicity of K1735 M-2 and M-2-a7 cell lines injected s.c.

Growth of tumors in mice after s.c. injection of each cell line wasmonitored and tumor diameters were measured.

DayaM-2b M-2-a7b

Tumor takec Sized Tumor takec Sized

C3H/HeN micee

Experiment I20 3/3 18.0 6 5.0 2/3 11.0 6 2.829 3/3 25.3 6 4.0 3/3 12.0 6 2.4

Experiment II16 6/6 15.3 6 5.2 5/6 6.3 6 4.023 6/6 18.0 6 5.3 5/6 10.1 6 6.029 6/6 30.5 6 5.6 6/6 19.5 6 5.434 6/6 41.1 6 5.2 6/6 23.0 6 5.9

Athymic nude micee

14 6/6 24.5 6 4.1 6/6 12.6 6 3.216 6/6 27.3 6 4.0 6/6 13.2 6 2.4

a Days postinjection when tumor size was measured.b Cells (2 3 106) injected s.c.c No. of mice with tumors/no. of mice given injection.d Mean 6 SD of the diameter of tumor in mm.e By Student’s t test mean tumor diameter of M-2 vs. M-2-a7: for exper-iment I, P 5 0.19; for experiment II, P 5 0.015; and for athymic nude miceresults, P 5 0.05.

483Cell Growth & Differentiation

fected M-2 cells. In particular, by day 21, tumors derivedfrom Mock M-2 cells were .21 mm in diameter, whereastumors from M-2-a7 cells did not exceed 12.5 mm in diam-eter (Table 3).

Although the retroviral vector pLNCX-4 used to infect theM-2 cells was designed not to express any viral genes, so asto be usable for gene therapy, it was still possible that acti-vation of the host immune system in the C3H/HeN mice wasinterfering with growth of the M-2-a7-generated tumors (25,32). To determine whether augmented antigenicity was re-sponsible for the observed differences in tumorigenicity ofthe M-2 parental and M-2-a7 cells, we repeated the exper-iments above using athymic nude mice (Table 2). Except forthe observation that all tumors grew faster in the athymicmice, the results were very similar to those obtained in thenormal syngeneic mice. M-2 parental cells tumors were fast-er-growing and, on average, 2-fold larger than tumors gen-

erated by the a7-transfected M-2 cells. In particular, by day16, tumors derived from M-2 cells were .27 mm in diameter,whereas tumors from M-2-a7 cells did not exceed 14 mm indiameter (Table 2). At termination of the experiment, tumorsoriginating from M-2 cells weighed 3.9 g whereas thoseoriginating from M-2-a7 cells averaged only 1 g.

Reduced Migration and Metastasis. Because migrationis the hallmark of an invading and metastasizing melanomacell, we asked whether transfection of a7 into M-2 cells hadany effect on their highly migratory phenotype (33, 34).Transfection of a7 reduced migration of M-2-a7 cells onlaminin-1, as compared to the nontransfected M-2 cells, bynearly 2-fold. However, M-2-a7 cells still migrated 2-foldbetter on laminin-1 than the poorly migratory C-23 cells (Fig.8A). The diminished migratory phenotype shown by theM-2-a7 cells suggested that transfection of a7 may also alterthe cells’ metastatic potential.

It was previously shown that C-23 cells produce very fewexperimental pulmonary metastases, averaging only fivelung nodules, whereas M-2 cells produce, on average, atleast 250 experimental pulmonary metastases when 2 3 105

cells are injected (25, 31). To determine whether expressionof a7 affected the metastatic potential of the M-2 cells, weinjected 1 3 105 M-2 or M-2-a7 cells i.v. into syngeneicC3H/HeN mice. After 2 weeks, the mice were sacrificed, andthe number of lung-tumor colonies was determined. All an-imals injected developed experimental metastases. How-ever, on average, M-2-a7 cells produced .3-fold fewer lungtumors than the M-2 parental cells (Fig. 8B). Together, theseresults demonstrated that M-2-a7 cells were now interme-diate between M-2 and C-23 cells with respect to reducedlaminin motility and in vivo tumor growth and decreasedmetastatic potential.

Growth Properties. Because M-2-a7 cells showed de-creased in vivo tumor growth, we next asked whether trans-fection of a7 into M-2 cells had any effect on the in vitrogrowth properties of these cells. We first determined whetherthere was any difference in the growth of M-2 and M-2-a7cells when seeded on plastic as compared to laminin-1. Theresults (Fig. 9A) showed that the growth rates of M-2 andM-2-a7 cells on each substrate were similar. However, theirgrowth rates on laminin-1 were slightly slower than their

Fig. 6. Growth of M-2 and M-2-a7 cells in the subcutis of C3H/HeNmice. Tumor cells (4 3 105) in HBSS were injected s.c. into syngeneicC3H/HeN mice. Tumors produced from M-2 (F) and M-2-a7 cells (f) weremonitored at the indicated days as described in “Materials and Methods.”Ordinate, average tumor diameter (mm), means of five to six s.c. tumors;bars, SD.

Fig. 7. Expression of a7 in tumors derived from M-2 and M-2-a7 cells.Equal quantities of cellular lysates from tumors derived from M-2 (Lane 1)and M-2-a7 (Lane 2) cells were processed for SDS-PAGE in a 7.5%polyacrylamide gel. Following transfer, the nitrocellulose membrane wasprobed with anti-a7 (1211) antiserum and the position of the a7 subunitwas determined by ECL. The position of the a7 subunit is indicated (a-7).

Table 3 Tumorigenicity of K1735 Mock and M-2-a7 cell lines injecteds.c.

Growth of tumors in C3H/HeN micea after s.c. injection of each cell linewas monitored and tumor diameters were measured.

DaybMockc M-2-a7c

Tumor taked Sizee Tumor taked Sizee

Experiment III16 4/4 14.4 6 2.7 4/4 7.0 6 2.222 4/4 21.0 6 5.0 4/4 12.5 6 1.3

a By Student’s t test mean tumor diameter of Mock vs. M-2-a7: forExperiment III, P 5 0.03.b Days postinjection when tumor size was measured.c Cells (2 3 106) injected s.c.d No. of mice with tumors/no. of mice given injection.e Mean 6 SD of the diameter of tumor in mm.

484 a7 Integrin Suppresses Tumorigenicity and Metastasis

growth rate on plastic, but there was no difference in growthbetween M-2 and M-2-a7 cell lines.

Next, we asked whether transfection of a7 into the M-2 cellline altered the cells’ dependence on serum growth factors.Growth in 0.5 or 10% serum had no effect on the proliferativecapacity of M-2 as compared to M-2-a7 cells, except thatboth cell lines grew more slowly when plated in reducedserum (Fig. 9B). Finally, we found that there was no signifi-cant difference in growth rate when the transfected cellswere grown on other ECM ligands, collagen and fibronectin(data not shown). Together, these results indicate that trans-

fection of a7 into M-2 cells had no detectable effect on thein vitro growth properties of these cells.

DiscussionIn this study, we examined the role of laminin-binding integrinreceptors in regulating the invasive behavior of melanomacells. The well-characterized murine K1735 melanomamodel, originally derived from a primary tumor, is composedof a mixture of phenotypically different clones with meta-static heterogeneity (26). Our findings show that a7 integrinis poorly expressed in a set of highly tumorigenic and met-astatic cell lines but is strongly expressed in nonmetastaticcells. This initial result suggested that a7 may function inregulating cell motility and metastatic potential. To define thefunction of a7 in metastatic melanoma, we overexpressed

Fig. 8. Production of experimental lung metastasis by M-2 and M-2-a7cells. A, migration of M-2 (o), M-2-a7 (f), and C-23 (M) cells on laminin-1-coated surfaces. Migration was measured using the microscreen assay asdescribed in “Materials and Methods.” The area covered by out-migratingcells, from the fixed diameter of the microscreen, was measured by com-puter-assisted image analysis (NIH Image). Columns, means of at least fiveindividual measurements in pixel units (3 103) and represent “relative migra-tion”; bars, SD. B, M-2 (o) and M-2-a7 (f) cells (1 3 105) were injected intothe lateral tail vein of syngeneic C3H/HeN mice (six mice per cell line perexperiment). After 2 weeks, mice were sacrificed, and the number of exper-imental lung metastases was determined as described in “Materials andMethods.” Columns, mean number of tumors per mouse; bars, SD. Theresults for M-2-a7 cells are significantly different from those for the nontrans-fected M-2 parental cell line by Student’s t test (P 5 0.025).

Fig. 9. In vitro proliferation properties of M-2 and M-2-a7 cells. A, pro-liferation rates of M-2 (F, M-2 on laminin-1; Œ, M-2 on plastic) and M-2-a7(f, M-2-a7 on laminin-1; �, M-2-a7 on plastic) cells when plated either onplastic or laminin-1-coated dishes. For laminin-1, microtiter plates (96-well Immulon plates) were precoated with 10 mg/ml of laminin-1 in PBS for1 h at 37°C in a humidified atmosphere. Tissue culture-treated microtiterplates were used for growth on plastic. Cell lines were plated at a densityof 50 cells per well. Data points, average cell number from triplicate wells,determined on the indicated days; bars, SD. O.D., absorbance. B, prolif-eration rate of M-2 (M) and M-2-a7 (o) cells when plated in DMEM H16containing either 0.5% or 10% FBS as described in “Materials and Meth-ods.” Columns, average proliferation rates from triplicate dishes, deter-mined after 5 days of incubation at 37°C; bars, SD.

485Cell Growth & Differentiation

the receptor in the M-2 cell line. Our results show that forcedexpression of a7 in M-2 cells diminished the highly migratoryphenotype and suppressed tumor growth and metastaticpotential. This is the first reported example of reduced in vivogrowth and decreased metastatic potential by transfection ofa laminin-binding integrin receptor in metastatic melanoma.

The highly metastatic M-2 cells migrated efficiently onlaminin-1, whereas the poorly metastatic C-23 cells exhibitedlittle motility on the same ligand. Laminin-1 induced a stronghaptotatic response in the metastatic M-2 cells but not in theC-23 cells, and a relatively narrow range of coating concen-trations promoted locomotion. Palecek et al. (35) recentlyshowed that cell migration is dependent on several factors,including substratum ligand levels, ligand-binding affinity,and cell integrin expression levels. These conclusions areconsistent with our results described here; that is, a mini-mum density of laminin-1 was needed for attachment andsubsequent motility, but, at higher concentrations, move-ment was suppressed. These migration-dependent factorsmay also account for the reduced migratory phenotype seenwhen a7 was overexpressed in M-2 cells. Whether reducedmigration was due to differences in laminin-1 affinity be-tween a6 and a7 or to an increase in the total laminin-1-binding receptors is unknown. However, we previouslyshowed that a7 binds with more avidity to laminin-1 affinitycolumns than does a6 (4, 18, 19, 36).

A connection between laminin receptor expression andmetastatic potential is illustrated by the cells’ ability to mi-grate. M-2 cells, which are both highly migratory and meta-static, use a6b1 as their primary laminin receptor. In con-trast, C-23 cells, which are nonmetastatic and poorlymigratory, use a7 as their main laminin receptor. When theprimary laminin receptor in M-2 cells was switched froma6b1 to a7b1 by overexpression of the a7 subunit, themigratory phenotype and metastatic potential were reversed.Importantly, the M-2-a7 cells still expressed relatively mod-erate levels of a1 and a6 laminin-binding receptors, butthese integrins were unable to affect the motility-reducingproperties of a7. These results indicate that, in murine mel-anoma, high-level expression of a7 can regulate the cells’migratory properties. However, Echtermeyer et al. (37) re-ported that low-level expression of transfected a7 in a hu-man melanoma cell line slightly increased laminin migration.In addition, we have seen that a7b1 expression in other celltypes, including smooth muscle cells, skeletal myoblasts,and a7-transfected MCF-7 breast carcinoma cells, can pro-mote cell migration on laminin-1 substrates (20–22). Whetherthese differences in a7-mediated cell motility are due to thetotal number of laminin-1 receptors expressed, the level ofa7 expression, or cell type-specific factors has not beendetermined.

Support for the idea that a7 reduces melanoma cell mi-gration and metastasis comes from the differential ability ofa7 and a6 to bind immobilized laminin. We previouslyshowed that a6 is eluted from a laminin-Sepharose columnwith 50 mM NaCl (4, 18, 19, 36). In contrast, a7 is stronglybound to laminin-Sepharose columns and can usually beeluted only by chelation of divalent cations (4, 18, 19, 36). Wefavor the possibility that melanoma cells expressing a6 as

the major laminin receptor may bind laminin less efficiently,thereby facilitating motility and invasion. In contrast, thosetumor cells expressing high levels of a7 may bind laminin soefficiently that motility and invasion are suppressed. It isinteresting that, in somatic cell hybrid analyses, fusion of M-2cells and a7-expressing C-23 cells resulted in hybrids thatwere dramatically less metastatic (31). Whether this is due toretention of the a7 subunit in the M-2/C-23 fusion clone or toother factors has yet to be determined. Finally, we recentlyestablished cell lines from rare lung tumors derived fromC-23 cells. In these cell lines, little a7 expression was de-tected, supporting the notion that loss of a7 is required formetastasis (data not shown).

An important question arising from this study is why forcedexpression of a7 in the highly metastatic cells decreasestumor growth. A partial explanation may be related to thenormal tissue expression pattern of this subunit. High a7levels are primarily observed in skeletal, cardiac, and smoothmuscle, where its expression is linked to the differentiationprogram (17, 21, 23, 24, 38). At present, little is known aboutthe regulation of a7 during cardiac muscle differentiation.However, in the terminally differentiated, nonproliferativestate of skeletal and smooth muscle, a7 is highly expressed(17, 21, 23, 24, 38). In contrast, during proliferation of skeletalmuscle precursors or dedifferentiation of smooth muscle, a7expression is lost (17, 21, 23, 24, 38). These observationssuggest that expression of a7 either is associated with orinduces a nonproliferative, terminally differentiated state.Thus, in the low serum and plasma environment of themouse, a7 may alter the proliferative or differentiation pro-gram in M-2-a7 cells, slowing their growth. Preliminary workhas indicated that, when M-2-a7 cells are maintained in aserum-free environment, they fail to proliferate. In contrast,M-2 cells continue to proliferate but at a much reduced rate.Finally, when the mitogen-activated protein kinase pathway,which is primarily involved in cell proliferation, is inhibited inB16 melanoma cells, cell differentiation follows (39). Whetherexpression of a7 in melanoma regulates the mitogen-acti-vated protein kinase pathway or activates an antagonistpathway that may moderate cell proliferation or differentia-tion remains to be determined.

Several laminin integrin receptors, including a2b1, a3b1,and a6b1, have been implicated in melanoma cell progres-sion (7–12). Increasing evidence suggests that a6b1 is amajor laminin receptor in metastatic melanoma. Severalstudies have reported that a6 is frequently up-regulated inmetastatic melanoma lesions (4, 9, 11). Furthermore, recentwork has shown that addition of laminin peptides can stim-ulate melanoma invasion in vitro and in vivo (40–42). Inparticular, Nakahara et al. (42) showed that laminin peptidesthat interact with a6b1 can induce invasion independently ofa6’s adhesive functions. The interaction of a6b1 with lamininappears to be required for the stimulation of invadopodia andextravasation during hematogenous metastasis; blockingantibodies to the a6 receptor can abolish experimental pul-monary metastasis (11, 42–44). Other studies have shownthat, when ligated to laminin, the a6b1 receptor can stimulatemitogenic activity that is independent of growth factor asso-ciation (45). We have shown here that, in M-2 cells, a6 plays

486 a7 Integrin Suppresses Tumorigenicity and Metastasis

a prominent role in adhesion to laminin. In addition, the M-2parental cells form faster-growing tumors than M-2 cellsoverexpressing a7. Together, the results presented here andthe observations above suggest that expression of the a6laminin-binding receptor correlates not only with melanomatumor growth but also with an invasive and metastatic phe-notype.

In addition to a6, both a2 and a3 integrins are frequentlyup-regulated in melanoma. However, in K1735 cells a2b1was barely detectable and did not contribute to laminin ad-hesion. a3b1 has been described as a receptor for severalECM ligands, including laminin-1, fibronectin, collagen, andthrombospondin (30, 46–48). More recent studies indicatethat a3 is a receptor specific for laminin-5 (30, 49). In case ofthe a3b1 receptor, our data do not support an important rolefor it in murine melanoma adhesion to laminin-1.

It is well established that tumor formation and acquisitionof the metastatic state require a number of complicatedprocesses and factors. Integrins appear to be important forseveral components of metastasis, including growth, inva-sion, and vascular dissemination. For example, several stud-ies have implicated integrins in cell cycle progression andregulation of apoptosis (50, 51). Whether a7 has any effectson these processes in regard to regulating in vivo growth andmetastasis is unknown. It has recently been suggested thatthe reduced tumor growth that follows transfection of a3 intorhabdomyosarcoma cells (52) may be the consequenceof several altered parameters, including changes in ECM-ligand-binding interactions, secreted proteases (53), and/orresponsiveness to growth factors (54). Any of these factorsmay contribute to the decreased tumor growth and meta-static potential we observed in the M-2-a7 cells. Finally, weshowed previously that a7 has the greatest amino acid ho-mology with a3 and a6 (17). Both a3 and a6 have beenshown to associate with members of the tetraspan family ofmolecules (55, 56). Such associations can alter the adhesive,proliferative, and migratory properties of these a subunits(55–60). Any involvement of a7 with members of the tetras-pan family has not been reported.

The development of metastases, a highly selective proc-ess, is dependent on the existence of tumor cell variantsubpopulations; that is, certain subsets of cells acquire aphenotype enabling them to survive the rigors of the meta-static cascade and successfully establish secondary foci. Asuccessful phenotype would likely have a suitable array ofintegrin receptors that directly determine the cell’s adhesiveand migratory activity. In the K1735 variant cell lines, theexpression pattern of the a6b1 integrin receptor was oppo-site that for a7b1, in that the a6b1 receptor was expressedstrongly in the highly metastatic tumorigenic cell lines M-2,M-4, and C-26 and poorly in the nonmetastatic cells (25, 26).Cells that retained expression of a7 (for example, C-23,C-19, and C-10) or had forced expression of a7 (for example,M-2-a7), were less tumorigenic and less motile and metas-tasized infrequently (25, 26). Northern blot analysis demon-strated a good correlation between the level of a6 and a7protein and the corresponding RNA transcripts. It is possiblethat high levels of a7 expression may directly regulate thelevels of a6. However, indirect mechanisms could also ac-

count for the reduction in a6 expression when a7 is present.Finally, the parental K1735 cell line, which was establishedfrom a primary tumor, is composed of a mixture of pheno-typically different clones with metastatic heterogeneity (25,26, 30, 33). It is not surprising that the integrin profile of theparental cell line represents a composite of that displayed bythe metastatic and nonmetastatic cell types. In conclusion,our results suggest that during tumor progression, there isselection of cells with an invasive phenotype that includes anintegrin repertoire enriched in specific receptors that bindlaminin transiently (e.g., a6) but lacking others that appear tobind laminin more efficiently (e.g., a7). Further work is re-quired to determine the factors regulating a7 expression andhow, once expressed, this laminin-binding receptor governsthe growth and migratory properties of melanoma.

Materials and MethodsCell Culture and Materials. The murine melanoma K1735 highly meta-static and low to nonmetastatic clones were obtained from Dr. I. J. Fidler(M. D. Anderson Cancer Center, Houston, TX). Cells were maintained inDMEM H16 with 10% fetal bovine serum, sodium pyruvate, nonessentialamino acids, L-glutamine, and penicillin/streptomycin. Laminin-1 was pu-rified from mouse Engelbreth-Holm-Swarm tumor as described previously(36). Type I collagen was obtained from Collagen Biomaterials (Palo Alto,CA).

Antibodies against integrin subunits included mouse monoclonal anti-bodies to a1b1 (Ha 31/8), b1 (Ha 2/11; Ref. 61), a5b1 (CD49E; PharMin-gen, San Diego, CA), a6b1 (GoH3; PharMingen), b1 (AIIB2; kindly pro-vided by Dr. C. Damsky, University of California San Francisco), and a7b1(CY8; Ref. 20). The rabbit polyclonal anti-a7 antibody 1211 was preparedin this laboratory as described previously (62), and the polyclonal anti-laminin-E8 antibody was a kind gift from Dr. P. D. Yurchenco (63). Goatantirabbit IgG conjugated to HRP and the ECL kit were purchased fromAmersham (Arlington Heights, IL).

Specific pathogen-free 6–8-week-old inbred C3H/HeN and athymicBalb/C mice were obtained from the Simonsen Laboratories, Inc. (Gilroy,CA). Mice were housed under specific pathogen-free conditions.

Cell Adhesion Assay. Microtiter plates (96-well Immulon plates; Dy-natech) were coated with ECM proteins at the indicated concentrations inPBS for 1 h at 37°C in a humidified atmosphere. Plates were washed withPBS and incubated with medium containing 0.1% BSA for 60 min in a CO2

incubator to block nonspecific adhesion. Single-cell suspensions wereprepared in DMEM with 0.1% BSA at 4 3 105 cells/ml, added in triplicateto 96-well plates, and then incubated for 30–60 min at 37°C. Nonadherentcells were removed by shaking on a titer plate shaker and washed withPBS. Cells were fixed with 1% formaldehyde, stained with 1% crystalviolet, and solubilized in 2% SDS; absorbance was then read at 562 nm.Binding of cells to collagen (10 mg/ml) on a separate plate was used torepresent 100% attachment. Background cell adhesion to 1% BSA-coated wells was subtracted from all readings. The effect of specificblocking antibodies was tested by preincubating the cells with the indi-cated dilutions of purified antibodies on ice for 30 min prior to the assay.

Migration Assay. Cell migration on laminin-1-coated surfaces wasmeasured using the microscreen assay (64). Briefly, sheets of virginpolystyrene were assembled in a 96-well dot blot apparatus (Schleicherand Schuell, Keene, NH); the wells were coated with laminin-1 for 1 h atthe indicated concentrations. After washing, the apparatus was reassem-bled with the laminin-1-coated sheet and a polished stainless steel screencontaining 0.9-mm-diameter perforations. Cells were seeded onto theexposed surface of the polystyrene sheet (1 3 105/ml per well). After a 1-hincubation at 37°C to permit cell attachment, the screen was removed, theapparatus was reassembled, and the cells were incubated for 8 h. Cells onthe sheet were then processed by fixation with 0.5% formaldehyde andstained with hematoxylin. The area covered by out-migrating cells, fromthe fixed diameter of the microscreen, was measured by computer-assisted image analysis (NIH Image); the data were expressed as themean and SD of at least five individual measurements in pixel units (3 103)and represent “relative migration.”

487Cell Growth & Differentiation

Western Blot. Retrovirally infected cell lines, parental cells, and tu-mors derived from each were solubilized with SDS-solubilization buffer[50 mM Tris (pH 7.5), 0.5% Triton X-100, 1 mM MgCl2, 2 mM phenylmeth-ylsulfonyl fluoride, and 1 mM N-ethylmaleimide]. Equal amounts of proteinwere separated by SDS-PAGE on 7.5% polyacrylamide gels under non-reducing and reducing conditions (using 2-mercaptoethanol as reducingagent), transferred to a polyvinylidene difluoride membrane (Millipore,Bedford, MA), and incubated with anti-a7 polyclonal antibody 1211 fol-lowed by goat antirabbit IgG-HRP or IgG-AP. Migration of the a7 subunitwas determined, where indicated, by using an ECL detection system forHRP (Amersham) or the color development detection system for AP(Promega, Madison, WI).

Flow Cytometry. After detachment with a brief treatment with 0.25%trypsin, single-cell suspensions of 106 cells/ml were incubated with opti-mal concentrations of primary antibodies in wash buffer (2% normal goatserum in PBS) for 1 h on ice. Cells were washed three times and incubatedwith secondary fluorescein-labeled antibodies for 30 min on ice. Afterthree more washes, the cells were stained with propidium iodide (1 mg/ml)to identify nonviable cells. Flow cytometry was performed on a FACScanflow cytometer (Becton Dickinson, San Jose, CA). Control samples con-sisted of cells with or without secondary antibody binding. Nonviable cellsstained with propidium iodide were eliminated from the analysis. The cellpopulations, Mock and M-2-a7, were obtained by FACS sorting.

RNA Isolation and Northern Blot. Total RNA was isolated by theguanidium isothiocyanate/phenol method and analyzed by Northern blot,as described previously (65). Briefly, RNA samples were electrophoresedin a 1.2% agarose gel containing formaldehyde. Then the RNA wastransferred to nylon membranes by capillary blotting and fixed to the filterby exposure to UV light. The RNA was hybridized with an a6 or an a7cDNA fragment labeled with 32P. Hybridizations were carried out at 42°Cin 50% formamide, 53 SSC, 53 Denhardt’s solution, 0.1% SDS, and 300mg/ml salmon sperm DNA. Filters were washed twice in 13 SSC-0.1%SDS at room temperature and once at 65°C in 0.13 SCC-0.1% SDS.Filters were exposed to X-ray film at 280°C with intensifying screens.

Transfection. The construction of a7-X2B cDNA has been describedpreviously (62). The murine a7-X2B cDNA was ligated into the retroviralexpression vector pLNCX-4 (32). Retroviral particles were obtained aftertransient transfection of the ecotropic packing cell line c2 (obtained fromthe American Type Culture Collection, Manassas, VA). For transduction,culture medium was removed from M-2 cells and replaced with viralsupernatant containing 8 mg/ml polybrene. After overnight incubation, theviral supernatant was removed and replaced with culture medium con-taining 800 mg/ml G418. After selection with G418, the retrovirally trans-fected cell populations M-2-a7 and Mock were FACS-sorted for a7 ex-pression and lack of a7 expression, respectively. Expression of a7 wasverified by Western blot analysis using polyclonal antibody 1211 (62).

Tumorigenicity Studies. Subconfluent cultures of M-2, Mock, andM-2-a7 cells were harvested by a brief incubation with a solution of 0.25%trypsin and 0.02% EDTA. Cells were washed in M-2 medium containing10% FBS, centrifuged at 4°C, and resuspended in Ca21- and Mg 21-freeHBSS at a concentration of 1 3 106 cells/ml. Only single-cell suspensionsof .90% viability, as determined by trypan blue exclusion, were used.Groups of six C3H/HeN and six athymic mice were given s.c. injectionscontaining 4 3 105 tumor cells. The growth rate of the s.c. tumors wasmonitored at the indicated times by examination of the mice and meas-urement of the tumors with calipers.

Experimental Metastasis. Unanesthetized syngeneic C3H/HeN micewere given injections in the lateral tail vein with Ca21- and Mg 21-freeHBSS containing 1 3 105 tumor cells. Mice were sacrificed 2 weeks aftertumor cell injection and necropsied. The lungs were removed, rinsed indistilled water, and fixed in Bouin’s solution (25). The number of surfacetumor nodules was determined with the aid of a dissecting microscope.

In Vitro Growth Assays. The growth rates of M-2 and M-2-a7 cells inmedium supplemented with high and low concentrations of serum werecompared to determine whether serum dependence was altered upontransfection of the a7 subunit. M-2 and M-2-a7 cells (104) were plated in60-mm tissue culture dishes in 3 ml of medium containing 10% FBS. Afterincubation for 24 h, the medium was aspirated, and the monolayers werewashed twice with serum-free DMEM H16 and then refed with DMEM H16supplemented with either 10 or 0.5% FBS. After 5 days of incubation at37°C, proliferation rates from triplicate dishes were determined.

Proliferation rates of M-2 and M-2-a7 cells were also compared todetermine whether transfection of the a7 subunit affected the growth ofthese cells when plated on plastic versus laminin-1. For growth on lami-nin-1, microtiter plates (96-well Immulon plates) were precoated with 10mg/ml of laminin-1 in PBS for 1 h at 37°C in a humidified atmosphere. Forgrowth on plastic, tissue culture-treated microtiter plates were used. Celllines were plated at a density of 50 cells per well. On the indicated days,cell numbers from triplicate wells were determined.

Changes in proliferation of M-2 and M-2-a7 cells under the conditionsabove were detected by using the CellTiter 96 AQueous one-solution cellproliferation assay according to the manufacturer’s instructions (Pro-mega).

AcknowledgmentsWe thank Dr. Lucia Belviglia for assistance with the tumorigenicity and

metastasis studies and Evangeline Leash for editorial assistance.

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490 a7 Integrin Suppresses Tumorigenicity and Metastasis