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Targeting a Tumor-Specific Laminin Domain Critical for

Human Carcinogenesis

Mark Tran,1,2Patricia Rousselle,

3Pasi Nokelainen,

1,2Sruthi Tallapragada,

1,2

Ngon T. Nguyen,1,2Edgar F. Fincher,

1,2and M. Peter Marinkovich

1,2

1Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California; 2Dermatology Service, VA Palo Alto HealthCare System, Palo Alto, California; and 3IFR128 BioSciences Lyon-Gerland, Institut de Biologie et Chimie des Proteines, UMR 5086,Centre National de la Recherche Scientifique, Univ. Lyon1, Lyon, France

Abstract

Laminin-332 is critical for squamous cell carcinoma (SCC)tumorigenesis, but targeting it for cancer therapy has beenunachievable due to key role of laminin-332 in promotingtissue integrity. Here, we show that a portion of laminin-332,termed G45, which is proteolytically removed and absent innormal tissues, is prominently expressed in most human SCCtumors and plays an important role in human SCCtumorigenesis. Primary human keratinocytes lacking G45(#G45) showed alterations of basal receptor organization,impaired matrix deposition, and increased migration. AfterSCC transformation, the absence of G45 domain in #G45cells was associated with deficient extracellular signal-regulated kinase and phosphotidylinositol 3-kinase (PI3K)pathway activation, impaired invasion, deficient metallopro-teinase activity, and absent tumorgenicity in vivo . Expressionof G45 or activated PI3K subunit in #G45 cells reversedthese abnormalities. G45 antibody treatment induced SCCtumor apoptosis, decreased SCC tumor proliferation, andmarkedly impaired human SCC tumorigenesis in vivo withoutaffecting normal tissue adhesion. These results show aremarkable selectivity of expression and function forlaminin-332 G45 in human SCC tumorigenesis and implicateit as a specific target for anticancer therapy. [Cancer Res2008;68(8):2885–94]

Introduction

Squamous cell carcinoma (SCC) is a prevalent (1) invasiveneoplasm arising in many tissues and causing significantmorbidity and mortality. SCC is the most common cancercapable of metastasis and is second in frequency only to basalcell carcinoma. The incidence of SCC seems to be rising and ismore frequently affecting younger individuals (2). SCC tumors canshow a high risk of recurrence, and those derived from mucosalsites often invade neighboring tissues and can also metastasize tothe lymph nodes, lung, and other distant sites. Chemotherapy forSCC has not been shown to significantly affect long-term survival,and most patients with advanced disease die despite currentlyavailable therapies (3). Results from the use of epidermal growthfactor receptor inhibitory agents in clinical trials of advanced

head and neck SCC in combination with conventional chemo-therapy have been only modestly encouraging (4). All of thesefactors have led to the search for new and more specific agents inthe treatment of SCC.Laminins are a family of trimeric extracellular glycoproteins

associated with the basement membrane zone (BMZ; ref. 5).Laminins interact with cell surface receptors and other BMZcomponents to provide cells with an interface to communicatewith their surrounding extracellular environment (6). Laminin-332,a large molecule consisting of a3, h3, and g2 chains (7), showswidespread expression in many epithelial tissues, as well as inthe tumor microenvironment of many carcinomas (8). Inparticular, several recent findings suggest that laminin-332 playsa crucial role in human SCC progression. For example, laminin-332 is required for tumorigenesis in a well-characterized in vivomodel of human SCC (8, 9). In addition, laminin-332 expression inSCC tumors arising from a number of tissues (10) correlates bothwith tumor invasiveness and patient prognosis (11, 12). Solubleand insoluble laminin-332, furthermore, have been observed toinduce the phosphotidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase pathways, which are known to mediatecarcinogenesis (13).Due to its critical role in SCC tumorigenesis, laminin-332 would

represent an excellent candidate as an antitumor target were itnot for the equally critical role of laminin-332 in maintainingepithelial-mesenchymal cohesion across a broad range of normaltissues (14). For example, in the inherited disorder Herlitz’sjunctional epidermolysis bullosa (JEB), absence of laminin-332expression due to laminin-332 gene mutations leads to wide-spread blistering and erosions and usually death during infancy(15). Therefore, any anticancer strategy targeting laminin-332would need to address how to selectively disrupt protumorigenicfunction of laminin-332 without affecting its procohesive functionin normal tissues.Towards this end, we focused on the a3 chain of laminin-332,

which undergoes proteolytic cleavage shortly after secretion. Thisproteolytic event, which takes place in the large COOH terminalglobular (G) domain near the junction of the third and fourthepidermal growth factor (EGF)–like repeats termed G3 and G4(Fig. 1A), converts the laminin a3 chain from a 200-kDa precursorto a 165-kDa processed product. The 37-kDa precursor region ofthe laminin a3 chain, containing two EGF-like repeats G4 and G5(G45), is removed during processing and, as a result, is absent innormal mature tissues (16, 17). Any postnatal expression ofunprocessed laminin a3 chain/G45 is only detectable transientlyat healing wound edges (17). There are many parallels between theprocess of wound healing and tumorigenesis, including activesynthesis of BMZ components, proliferation, and cell migration.Because of these parallels and the absence of unprocessed laminin

Note: M. Tran and P. Rousselle contributed equally to this work.Requests for reprints: M. Peter Marinkovich, Program in Epithelial Biology,

Stanford University School of Medicine, Room 2145, 269 Campus Drive, Stanford, CA94305. Phone: 650-498-5425; Fax: 650-723-8762; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-6160

www.aacrjournals.org 2885 Cancer Res 2008; 68: (8). April 15, 2008

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a3 in normal mature tissues, we focused on the role of the laminina3 G45 in human SCC. In this study, we show that, whereas G45 isundetectable in normal mature tissues, it shows widespreadexpression in human SCC tumors, where it plays an importantrole in SCC tumorigenesis. Furthermore, we show that laminin a3G45 can be selectively targeted in vivo by antibodies to inhibithuman SCC tumorigenesis without disrupting normal tissues.

Materials and Methods

Cell lines. Primary human keratinocytes isolated from normal skin and a

patient with junctional dystrophic epidermolysis bullosa lacking laminin-332

expression due to LAMA3 mutations (18) were cultured in a 1:1 mix of

defined keratinocyte serum-free medium (SFM; Life Technologies) andMedium 154 (Cascade Biologics) at 37jC in a humidified 5% CO2 incubator.

Modified human 293 PHOENIX cells (gift from Dr. G. Nolan) were cultured

in DMEM supplemented with 10% FCS, 100 IU/mL penicillin, and 100 Ag/mL

streptomycin at 37jC in a humidified 10% CO2 incubator.

Complementary DNA constructs. Human laminin a3 chain (ref. 19;

Genbank NM227.2) is physiologically processed at residues 1337 to 1338,

according to NH2 terminal sequencing studies (20). As there are no known

mutations in laminin-332 a3 G45 domain in JEB patients (21), threecDNAs encoding HuLAMA3 were produced: the first one comprising the

200-kDa full-length a3 wild-type (WT) chain, coding for residues 1 to

1713; the second comprised the 165-kDa a3 chain truncated at the

physiologic processing site comprising residues 1 to 1337 (DG45); and thethird comprised the 37-kDa cleaved G45 (G45), residues 1338 to 1713.

These were generated by PCR, verified by direct sequence analysis, and

cloned into the retroviral vector backbone LZRS (22) containing theencephalomyocarditis virus–IRES and blasticidin-resistance sequences (23)

and a GATEWAY (Invitrogen) destination site (pLZRS-GATEWAY). The

BM40 signal sequence was incorporated upstream and in-frame of the

mutant HuLAMA3 for directing expression. Retroviral expression vectorsencoding either activated Ha-Ras, InBa (9), or activated PI3K p110-CAAX

(24) have been previously characterized. Amphotropic retroviral superna-

tant production and retroviral keratinocyte transduction were performed

as described (23).

Figure 1. Laminin-332 G45 is present in human SCC tumors but absent in normal skin. A, schematic diagram of laminin-332 a3, h3, and g2 chains, highlighting a3chain domain structure (black ), proteolytic cleavage, and epitopes for BM165 mAb and G45 pAb. B, immunofluorescence microscopic analysis of frozen sections ofhuman SCC (top ) and neonatal skin (bottom ) using BM165 mAb (green ) and G45 pAb (red ). Merged images with nuclear Hoechst staining (blue ) are shown(right ). Images are representative of four frozen SCC and skin samples tested. Scale bar, 50 Am. C, results of analysis of 75 cases of paraffin-embedded humancutaneous SCC using G45 pAb by immunohistochemical analysis. Top, left, number and percentage of samples which showed negative, moderate, or strongexpression. Bottom and right, representative examples of moderate/strong expression and negative skin control. Scale bar, 50 Am. D, left, representative samples of56 cases of paraffin-embedded human extracutaneous SCC from various tissues using G45 pAb by immunohistochemical analysis showing moderate to strongexpression. Scale bar, 50 Am. Right, results of extracutaneous SCC tissue survey, showing tissues of origin, and intensity of staining for G45 pAb antibody.

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Cancer Res 2008; 68: (8). April 15, 2008 2886 www.aacrjournals.org



Statistical analysis. Student’s t test was used to ascertain significantdifference between data sets using Microsoft Excel. P values are listed in

figure legends.

WT and DG45 cDNA forward primer, 5¶-AAAAAAGCTAGCATGGGTTGG-CTTATA-3¶;a3WT cDNA reverse primer, 5¶-CCCCCCGGGCCCGCGGCCGCTTACAG-GTCCTCCTCGCTAATCAATTTTTGCTCCTGGTCAGGACAACCATTCAG-

ACTGAC-3¶;DG45 reverse primer, 5¶-AAAAAACCAGGTTTAACAAGACCAAGACTTTT-CGTATCAACCTGCTGTTGCTGGCCACACCAGTGGCCTCCCCTAG-

GAGC-3¶;G45 forward primer, 5¶-TTATGCTAGCGGACACACCAGT-3¶;G45 reverse primer, 5¶-TATTCTCGAGTTACTGGTCAGGAC-3¶.

Antibodies. Anti–laminin a3 chain mouse monoclonal antibody (mAb)BM165 (14) and G45 rabbit polyclonal antibody (pAb; ref. 25), anti–laminin-332

rabbit pAb and laminin h3 chain mouse mAb K140 (16, 26) were previously

characterized. We commercially obtained anti–phosphorylated extracellular

signal-regulated kinase (ERK) and phosphorylated Akt (Ser473; CellSignaling), anti-a6 integrin mAb G0H3 (Chemicon), Ki67 mAb (LabVision),

paxillin mAb (BD Biosciences PharMingen), phalloidin pAb (Invitrogen),

and h-actin mAb (Sigma).

Protein analysis. Laminin-332 deposition among WT, DG45, and DG45 +G45 cells were studied over a 24-h period. Proteins from whole-cell lysates,

conditioned media, and matrix deposition were extracted and quantified by

immunoblot, as previously described (26). K140, an anti-laminin h3 mAb,was used for laminin deposition comparisons, which controlled for cell

density by normalizing laminin h3 to actin bands. For measurement of AKT

phosphorylation, near-confluent cells were extracted in radioimmunopre-

cipitation assay buffer (RIPA) with protease and phosphatase inhibitors.Ratio of phosphorylated AKT to total AKT was calculated through

densitometry. For ERK phosphorylation, Ras-InBa (or Ras-InBa + PI3K)

transformed cells were growth factor starved for 24 h and then stimulated

with 10 ng/mL EGF for 2 min before lysis in RIPA buffer for immunoblotanalysis. Densitometric analysis was shown as phosphorylated with ERK1/2

normalized to untreated WT controls. NIH ImageJ software was used for

densitometric analysis.Immunofluorescence microscopy. Confocal analysis of cell adhesion

proteins was performed exactly as previously described (27). Assaying ERK

activation of keratinocytes after pulsing with 10 ng/mL EGF for 2 min has

been described previously (28). Briefly, representative images from JEBnull

keratinocytes expressing the indicated laminin a3 constructs were

visualized by immunofluorescence microscopy using phosphorylated ERK

antibody. Nuclear localization of phosphorylated ERK was quantified as

percentage of phosphorylated ERK staining nuclei over total nuclei. Imagingwas carried out with a Zeiss LSM 510 confocal laser scanning microscope.

Immunohistochemistry. For immunoperoxidase staining, 5 Am paraffin

sections of SCC tissue microarrays from skin (SK802) and multiple organs

(BC00019) obtained from U.S. Biomax, Inc., were deparaffinized, rehydrated,

and antigen unmasked by boiling in 50 mmol/L Tris-HCl (pH 9.5) for

15 min. Sections were then incubated with G45 rabbit pAb followed by

biotin-conjugated secondary and 3,3¶-diaminobenzidine detection (Lab-

Vision). Tissues were counterstained with hematoxylin. G45 staining was

graded by two independent blinded observers according to the percentage

of number of tumor cells positive with staining: >75% (strong), 25% to 75%

(moderate), and <25% (negative). For immunofluorescence, 5-Am cryosec-

tions were incubated with antibodies listed above and Hoescht-counter-

stained. Images were taken using a Zeiss Axiovert-100 microscope.

Cell adhesion, migration, and invasion assays. G45 adhesion studieswere carried out by coating purified LG4/5 fragment (10 Ag/mL overnight at

4jC; ref. 25). Ras/InBatransformed normal primary human keratinocytes

were detached in PBS containing 10 mmol/L EDTA and rinsed in serum-free medium. After washing with PBS and saturation of the wells with 1%

bovine serum albumin (BSA), the cell adhesion assays were performed in

serum-free medium, as described (29). Adhesion was determined after

fixation with 1% glutaraldehyde in PBS and staining with 0.1% crystal violet

by absorbance at 570 nm using a MR5000 ELISA reader. A blank valuecorresponding to BSA-coated wells was subtracted. Adhesion inhibition

with G4/5 antibody (20 Ag/mL) or heparin (10 Ag/mL; Sigma) took place

60 min before, as well as during, the cell adhesion experiment.

Cell detachment assays were carried out as described (30). Briefly, 2 �104 cells/cm2 were incubated for 24 h at 50% confluence. Detached cells

were quantified at increasing time intervals after incubation in a 1:70

dilution of trypsin/EDTA in PBS (BioWhittaker). Each adhesion/detach-

ment experiment was performed in triplicate.Cell monolayer scratch assays (27) were performed by plating 106 cells

into 60-mm tissue culture plates and incubating cells in SFM for 24 h. Media

was changed to SFM without additives for 16 h. Fresh mitomycin-C (Sigma)

was added at 10 Ag/mL, and cells were incubated 3 h on ice. Cells werewashed twice with SFM/WA and scratched with a 1-mm cell scraper. Plates

were washed thrice with SFM/WA and marked areas photographed using a

Zeiss Axiovert 25 microscope (50� magnification). Migration wasquantified by calculating percentage change in the area between migrating

cell sheets using NIH image software and more than three repeats per data

point.

The in vitro invasion assays (31) were performed as previously described;briefly, assays were performed in triplicate using chambers containing a

polycarbonate membrane with eight micron pores, coated with Matrigel

(Becton Dickinson). After 24 h, invasive cells in the bottom chamber were

lysed and quantified using CyQuant GR dye. Invasion index was quantifiedrelative to percentage invasion by JEBnull keratinocytes.

Zymography. One million keratinocytes were starved for 24 h and

incubated in SFM. Conditioned media were recovered and concentrated80�. Samples were dissolved in nonreducing sample buffer (6% glycerol, 1%

SDS, and 0.004% bromophenol blue), incubated in 37jC water bath for

10 min, and loaded on a 10% gelatin gel for detection of matrix

metalloproteinase 2 (MMP2) and MMP9 and 12% casein gel for detectionof MMP1 (Invitrogen). The gel was run in Tris/glycine buffer for 2 h and

then incubated in 2.5% Triton X-100 solution for 15 min twice to remove

SDS. To detect MMP activity, the gel was incubated in reaction buffer

containing 50 mol/L Tris-HCl (pH 7.4), 0.2 mol/L NaCl, 5 mmol/L CaCl2, and1 Amol/L ZnCl2 overnight at 37jC. Protease activity was detected as

translucent area in a Coomassie blue–stained gel. The scanned results of

gels were calculated using NIH ImageJ software.Tumorigenicity assay. Tumorgenicity assay was performed as previ-

ously described (9). Briefly, keratinocytes were incubated with LZRS-IRES-

Blasticidin/H-Ras or LZRS-IRES-Blasticidin/InBaM retroviral titer. Gene

transfer was verified by immunoblotting of cell lysates. One million Ras/InBaM transformed cells suspended in 200 AL Matrigel (Beckton-Dickenson)

were injected s.c. to the dorsal flank of 6-wk-old nude mice; five mice were

used per each condition. Tumors were measured on a weekly basis and

analyzed at the end of 4 wk. All animal studies were conducted inaccordance with protocols approved by Stanford Animal Use Committee.

In some experiments, affinity purified G45 pAb, affinity purified mAb K140,

or control rabbit IgG (Sigma) were injected i.p. on a weekly basis at a dose

of 500 Ag/mouse/wk, which has been previously shown to maintain highcirculating antibody levels (9, 31). Apoptotic tumor cells were detected

using Roche’s in situ cell detection kit. Proliferating SCC cells were detected

with Ki67 and 4¶,6-diamidino-2-phenylindole immunofluorescent staining.Proliferation and apoptosis were quantified as the ratio of staining of

nuclear Ki67 and TUNEL, respectively, to total nuclear staining. NIH image

software was used to quantify the subset of apoptotic or proliferating tumor

cells in five representative low-power fields on each tumor.

Results

Widespread accumulation of laminin-332 G45 in humanSCC tumors. In an effort to determine whether unprocessedlaminin a3 G45 domain was accumulated in human SCC tumors,we examined frozen sections of four normal skin and fourcutaneous SCC specimens obtained by Moh’s surgery usingimmunofluorescence microscopy. Using an antibody specific to

Targeting a Tumor-Specific Laminin Domain




the G45 domain and an antibody (BM165) which recognized theprocessed laminin-332 trimer (Fig. 1A), we found that G45, whileconsistently undetectable in normal skin, was abundantly presentand showed colocalization with mAb BM165 in human cutaneousSCC tumors from each of the four patient samples tested (Fig. 1B).In a more extensive survey of 75 cutaneous (Fig. 1C) and 56extracutaneous (Fig. 1D) paraffin-embedded SCC tumors, over 75%showed moderate to strong G45 expression. All G45-positivetumors also stained positively with total laminin-332 pAb, whereasall but one of the G45 negative tumors were also negative for totallaminin-332 expression (not shown). Thus, G45 domain accumu-lation correlated well with total laminin-332 expression in SCCtumors.Laminin-332 G45 domain facilitates the organization and

function of matrix receptor complexes. To investigate laminin-332G45 in SCC, we produced three laminin-332 a3 chain constructs(Fig. 2A), a full-length WT chain, a mutant lacking G45 (DG45), andG45 (G45) itself. These constructs were stably expressed in laminin-332 null keratinocytes derived from a patient with JEB (JEBnull)with underlying LAMA3 gene mutations (18). As will be shownbelow, G45 separately expressed from the rest of the laminin-332molecule (DG45 + G45) performed many of the same functions,albeit less efficiently (detailed below), as G45 synthesized as partof the laminin-332 molecule (WT). JEBnull keratinocytes expressingG45 without the DG45 construct, like untransduced JEBnull

keratinocytes, did not adhere to culture surfaces and were notanalyzed further in vitro .

DG45 JEBnull cells synthesized, assembled, and secreted tri-meric laminin-332 as shown by nonreduced immunoblot analysis(Fig. 2B, left) and the DG45 chain was of equivalent apparentmolecular weight compared with processed WT a3 chain (a3p), asshown by reduced immunoblot (Fig. 2B, center). The G45 constructwas expressed in DG45 cells at levels similar to WT cells, as clearlyseen in conditioned cell medium (Fig. 2B, right), as well as celllysate (not shown). We compared laminin-332 in these cellsisolated from culture medium or extracted from culture matrix, aspreviously described (26), using actin from cell lysates as a control.Normally, keratinocytes deposit more laminin-332 into their matrixthan they secrete into their media, but DG45 cells secreted morelaminin-332 into medium than matrix (Fig. 2C), consistent withprevious observations (17). G45 synthesis in DG45 cells led to themajority of laminin-332 being deposited into matrix, indicatingthat G45 promoted laminin-332 deposition.This was further examined by confocal microscopy (Fig. 3A).

Analysis of laminin-332 antibody staining confirmed that theabsence of G45 in DG45 cells correlated with a reduction indeposited laminin-332, which was again improved with theexpression of G45 in DG45 cells (Fig. 3A, top). Additional effectsof G45 on basally located cell receptor complexes were also noted.WT cells showed characteristic peripheral focal adhesions con-taining paxillin and central stable adhesions (32) containing a6h4integrin (ref. 33; Fig. 3A, second panel). However stable adhesions inDG45 cells were abnormally peripheral, adjacent to focal adhesions(Fig. 3A, third panel). G45 expression in DG45 cells restored some

Figure 2. Laminin-332 G45 expression and function in human keratinocytes. A, schematic of the laminin a3 cDNA constructs used in this study. WT, WT full-lengthlaminin a3 construct; DG45, laminin a3 chain truncated at amino acid 1337 (major processing site; ref. 20); G45, portion of the laminin a3 chain G domain removedduring processing. B, retroviral expression of laminin a3 cDNA constructs in JEBnull keratinocytes. Left, nonreduced immunoblot of conditioned keratinocytemedium using laminin-332 pAb, position of laminin-332 trimer, and molecular weight markers (kDa; left). NK, normal human keratinocytes. Center, reduced immunoblotof extracted keratinocyte matrix using laminin-332 pAb, positions of molecular weight markers (kDa; left) and positions of individual laminin-332 chains (right ).Right, nonreduced immunoblot analysis of conditioned keratinocyte medium using G45 pAb, position of G45 (left). C, quantification of cell layer (Matrix + Lysate) andconditioned media fractions from laminin a3 null keratinocytes expressing the indicated laminin a3 constructs by immunoblot using anti-laminin h3 antibody K140 oranti-actin antibody followed by densitometric analysis showed as integrated density of the ratio of laminin h3 to actin bands. Position of laminin h3 band (right).

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stable adhesions to their normal central location. G45 colocalizedwith a6h4 integrin in stable adhesions in WT and DG45 cells(Fig. 3A, bottom ), suggesting interaction of G45 with stableadhesions. In accordance with the analysis of stable adhesion for-mation, DG45 cells showed increased sensitivity to trypsin-induceddetachment (Fig. 3B), which was corrected with G45 expression,suggesting that G45 induced laminin-332 deposition and stableadhesion formation led to increased stable cell adhesion. Previousstudies have shown an inverse relationship between the rate of kera-tinocyte migration and laminin-332 deposition (34). In accordancewith this, DG45 keratinocytes, with decreased laminin-332 deposi-tion, migrated faster into scratches placed in confluent monolayersthan WT cells with normal laminin-332 deposition (Fig. 3C).Expression of G45 construct in DG45 cells partially reversed theseeffects. We next determined whether these adhesion and migrationabnormalities and these changes in extracellular matrix depositionand organization correlated with changes in tumorigenic potential.Laminin-332 G45 promotes tumor invasion and metal-

loproteinase activity. SCC tumorigenesis was examined byretroviral transduction of primary human keratinocytes with

oncogenic Ras and the nuclear factor-nB inhibitor InBa, whichproduces transformed cells that generate human epidermal tumorsindistinguishable from human SCC upon transfer to immunode-ficient mice (9). After Ras/InBa transformation, DG45 cells showedimpaired invasion into Matrigel compared with WT cells which waspartially corrected through G45 retroviral transduction (Fig. 4A).Carcinoma invasion has been linked to metalloproteinase activity,and DG45 cells showed a striking deficiency of MMP-9 and MMP-1,which have been associated with SCC invasion (ref. 35; Fig. 4B).Metalloproteinase deficiencies in DG45 cells were reversed throughexpression of G45, although not to the levels of WT cells. Thisdeficient MMP expression explains why, despite their increasedmigration in the untransformed state, transformed DG45 cellsinvaded Matrigel more poorly than transformed WT cells.Activation of PI3K and ERK pathways by laminin-332 G45.

PI3K pathway activation is critical for SCC invasion (36) and DG45cells showed decreased AKT phosphorylation, suggesting thatG45 was essential in promoting PI3K pathway activation (Fig. 4C).G45 expressed as part of the laminin-332 molecule was moreefficient at promoting AKT phosphorylation compared with G45

Figure 3. Laminin-332 G45 influences matrix receptor organization and function. A, effects of G45 on keratinocyte matrix deposition and adhesion complex formation.Keratinocytes expressing the indicated a3 chain constructs were analyzed by triple-label confocal microscopy. Color of secondary antibodies and staining aredesignated by the color of the text listing the primary antibody. FA, focal adhesion; SA, stable adhesion. Scale bar, 10 Am. B, G45 promotes resistance totrypsin dissociation. Established keratinocyte cultures expressing the indicated laminin a3 constructs were subjected to diluted trypsin at the indicated intervals andpercentage of cells dissociated was quantified. Columns, results of triplicate experiments; error bars, SD. *, P < 0.05 compared with WT cells, #, P < 0.05compared with DG45 cells. C, G45 modulates keratinocyte migration during in vitro wound healing assay. Confluent monolayers of keratinocytes expressing indicatedlaminin constructs were tested for their ability to migrate into 1-mm scratches over the course of 24 h and percentage of closure of scratch was quantified.Columns, results of triplicate experiments; error bars, SD. *, P < 0.05 compared with WT cells, #, P < 0.01 compared with DG45 cells.





expressed separate from laminin-332 (DG45 + G45). We also foundan impairment of ERK activation in transformed DG45 cells, whichwas restored partially through overexpression of G45 but fullythrough overexpression of activated PI3K p110 catalytic subunit(Fig. 4C). In addition, DG45 cells showed defective nuclear translo-cation of activated ERK, which was corrected partially through G45expression but fully through activated p110 expression (Fig. 4D).Laminin-332 G45 is required for in vivo Ras-driven SCC

tumorigenesis. We next examined G45 in human SCC tumori-genesis in vivo . After transfer to immunodeficient mice, trans-formed DG45 cells showed strikingly impaired tumorigenesis(Fig. 5A, top row), This was slightly improved when G45 wasexpressed in DG45 cells and tumor growth was more fully restoredin DG45 cells through activated p110 expression. TransformedJEBnull cells overexpressing G45 alone produced no detectabletumors 4 weeks after injection, similar to what has been previouslyshown for transformed JEBnull cells alone (ref. 9; not shown).Whereas invasion into underlying muscle was consistently noted intumors expressing WT laminin a3 chain, DG45 tumors showed aconspicuous invasive defect (Fig. 5A, second row). However,invasion into muscle was noted in DG45 cell tumors afterexpression of G45 or activated p110 subunit. Widespread apoptosiswas present in DG45 tumors (Fig. 5A, third row and B). G45

expression diminished, and p110 expression completely inhibitedapoptosis in DG45 tumors. DG45 tumors showed deficientproliferation, which was modestly increased with G45 domainexpression and fully restored to WT levels with p110 expression(Fig. 5A, fourth row and C). Expression of G45 promoted laminin-332 deposition in the tumors, as did activated p110 subunitexpression (Fig. 5A, fifth row), suggesting a possible link betweenG45 function, PI3K pathway activation, and laminin-332 depositionduring SCC tumorigenesis.Laminin-332 G45 antibody disrupted SCC tumorigenesis

in vivo without affecting normal tissue integrity. Over thecourse of 4 wk of treatment, G45 antibodies dramatically inhibitedin vivo human SCC tumorigenesis (Fig. 6A). G45 antibody–treatedtumors produced both inhibition of proliferation and pronouncedapoptosis compared with control antibody–treated tumors(Fig. 6B) similar to the G45 genetic deletion experiments describedabove. Affinity purified G45 antibody was shown to specificallyinhibit transformed keratinocyte adhesion to recombinant G45protein (Fig. 6C). Interestingly heparin also disrupted thisinteraction, suggesting that the heparan-binding properties ofG45 were involved in its cellular interactions. Given its inhibitoryeffects on SCC tumor growth, we recognized the potential of theG45 antibody as an anticancer agent and looked for possible toxic

Figure 4. Laminin-332 G45 drives human SCC tumorigenesis and invasion by increasing activation of MMP, ERK, and PI3K pathways, promoting proliferation, andinhibiting apoptosis. Ras-InBa transformed JEBnull keratinocytes expressing the laminin constructs indicated in Fig. 2 (and in some instances, activated p110 PI3Ksubunit) were analyzed under the following conditions. A, keratinocytes expressing G45 showed increased cellular Matrigel invasion. Data represents triplicateindependent experiments F SD and are quantified as a percentage of basal invasion by JEBnull cells alone. *, P < 0.05 compared with WT cells; #, P < 0.05 comparedwith DG45 cells, B, G45 expression promotes MMP activity. Conditioned medium was analyzed by gel zymography, and intensity of digestion of bands by indicatedmetalloproteinases was quantified by densitometry, as shown above the zymogram. C, G45 expression promotes PI3K pathway and ERK activation. Top, celllysates were analyzed by immunoblot using phosphorylated AKT (p-AKT ) and total AKT antibodies. Ratio of phosphorylated AKT to total AKT staining was quantifiedusing densitometry. All cells studied were Ras/InBa transformed except where indicated (no Ras ). Bottom, lysates from growth factor–starved cells were analyzedby immunoblot before or after EGF treatment, using antibodies to phosphorylated ERK, total ERK, and actin. Densitometric analysis is shown at the bottom asphosphorylated ERK1/2 normalized to untreated WT controls and total ERK1/2 expression. D, G45 promotes phosphorylated ERK nuclear translocation. Left,representative images from JEBnull keratinocytes expressing the indicated laminin a3 constructs were visualized by immunofluorescence microscopy usingphosphorylated ERK antibody. Graph on right shows quantification of the effects of G45 on phosphorylated ERK nuclear translocation. Data represents triplicateindependent experiments F SD and are quantified as a percentage of phophorylated ERK-staining nuclei over total nuclei. *, P < 0.001 compared with WT cells; #,P < 0.01 compared with DG45 cells.

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side effects of the antibodies in a survey of normal tissues.Interestingly, although the G45 pAb specifically recognized nativemurine laminin-332, as shown by nonreduced immunoblot analysisof conditioned mouse keratinocyte culture medium (Fig. 6C), wefound no blistering epithelial-mesenchymal separation or othermorphologic abnormalities observed in laminin-332–expressingtissues of mice treated with G45 antibodies, even after 4 weeks ofantibody injections (Fig. 6D). Transmission electron microscopy ofmouse skin after 4 weeks of antibody treatment showed novesiculation or BMZ abnormalities (Fig. 6D).

Discussion

This study clearly indicates, through genetic and antibody-mediated inhibition, a key role for laminin-332 G45 in SCCtumorigenesis. One of the striking aspects of this study was the

stark contrast between undetectable G45 in normal mature tissuesand prevalent accumulation of G45 in a wide array of SCCs.Over 75% of both cutaneous and noncutaneous SCCs expressedlaminin-332 and G45. Laminin-332 G45 was present in nearly 100%(all but one) of human SCC tumors positive for total laminin-332expression. As has been previously shown in clinical studies,laminin-332 expression correlates with carcinoma invasivenessand a poor prognosis in SCC patients. As G45 persistence alsocorrelates closely with laminin-332 expression in SCC tumors but isabsent in normal tissues, G45, through additional clinicalcorrelative studies, may prove to be an extremely useful markerto identify invasive SCC tumors. Also, as this study only looked attumors at the carcinoma stage, the question of whether theinitiation of detectable G45 coincides with invasion or begins at anearlier stage of in situ or premalignant carcinoma development

Figure 5. G45 promotes human SCC tumorigenesis in vivo. A, representative photos in the top show a lack of tumor growth in cells lacking G45 expression (DG45, top ),but a partial restoration when G45 was separately expressed (DG45 +G45), and a near-complete restoration of tumorigenesis when activated p110 (DG45 + PI3K).Histologic invasion of underlying muscle (second panel ) was absent in tumors lacking G45 expression (arrow). Apoptosis was greatly increased in tumors lacking G45(third panel ) as shown by TUNEL assay of frozen tumor sections. Inset, Hoechst nuclear stain. Proliferation was greatly reduced in tumors lacking G45 (fourth panel )shown by Ki67 antibody analysis of frozen tumor sections. Inset, Hoechst nuclear stain. Laminin-332 deposition (fifth panel ) was greatly reduced in the absence ofG45 subunit, but was partially rescued by expression of G45 and fully rescued by activated p110 (PI3K) expression as shown by immunofluorescence microscopyof frozen tumor sections using laminin-332 pAb. G45 construct produced levels of G45 expression in tumor sections equivalent to WT construct as assessed byimmunofluorescence microscopy of frozen tumor sections using G45 pAb (bottom ). Scale bar, 100 Am. B, G45 promotes human SCC tumorigenesis. Tumor growth wasmeasured 4 wk after s.c. transfer of indicated cells to immunodeficient mice. Error bars, SD. *, P < 0.05 compared with WT cells; #, P < 0.05 compared with DG45 cells.C, G45 promotes proliferation and protection from apoptosis. TUNEL assay and Ki67 staining as assessed by immunofluorescence microscopy of frozen tumorsections was quantified as a percentage of staining of total nuclei. Error bars, SD. *, P < 0.05 compared with WT cells; #, P < 0.05 compared with DG45 cells.





remains to be tested. Whereas laminin-332 is not extractable fromtissue without the use of proteases (16), which complicates theability to analyze tumor samples by immunoblot analysis, it ispossible that the G45 domain may persist in a soluble state aftercleavage. Thus, clinical studies examining serologic detection of theG45 domain in carcinoma patients as a potential diagnostic orprognostic tool would be a worthwhile future consideration.Although laminin-332 G45 is undetectable in mature tissues, it is

transiently detectable at the leading edges of wounds (17). SCCinvasion shares similarities with wound healing, as both areprocesses of epithelial proliferation and extension, which requirethe active synthesis and deposition of new BMZ components.These two processes differ in significant ways, however. In wounds,laminin-332 G45 becomes undetectable after closure, whensynthesis of new BMZ components subsides and full processingand BMZ assembly is completed. With SCC tumor invasion, thesynthesis of BMZ components is not regulated by a closure event,such as in wound healing, and thus, the synthesis of BMZ

components continues, without allowing for processing andmaturation of the BMZ to occur to completion. This may accountfor the poor ultrastructural BMZ organization in invasivecarcinomas compared with normal tissues (37).Why G45 accumulates at SCCs and at the leading edges of

healing wounds may simply be a reflection of higher levels of totallaminin-332 expression. One possibility is that laminin-332 isproduced in wounds and tumors at a rate exceeding the ability ofthe processing enzyme to remove the G45 domain, resulting in asteady-state persistence of unprocessed laminin-332. The enzymeswhich process laminin a3 chain include plasmin (38) and theC-proteinase family of enzymes, especially mammalian tolloid andbone morphogenic protein 1 (26, 39). Alternatively, there may beother mechanisms that control the rate of laminina3 G45processing, such as the tissue plasminogen proteolytic cascade(38). In addition, a group of enhancer proteins, which modulateC-proteinase activity, has also been described, and while one,termed PCPE-1, has not been shown to influence laminin-332

Figure 6. Laminin a3 G45 pAb inhibited human SCC tumorigenesis without disrupting normal epithelial adhesion. A, G45 pAb blocked human tumorigenesis. Left,representative photos of Ras/InBa-transformed SCC tumors in immunodeficient mice after 4 wk of treatment with G45 pAb or control IgG. Right, quantification oftumor growth during weekly tumor volume measurements in mice treated with G45 pAb, laminin h3 mAb K140, or control IgG. B, after 4 wk of G45 pAb antibodyinjection, proliferation was reduced in tumors as shown by analysis of frozen tumor sections using immunofluorescence microscopy and Ki67 antibody. Left, proliferationwas significantly reduced and apoptosis was greatly increased with G45 pAb treatment, as shown by Ki67 staining and TUNEL assay of frozen tumor sections;insets, Hoechst nuclear stain. Scale bar, 100 Am. Right, quantification expressed as number of cells with Ki67 or TUNEL staining, respectively, as a percentage of totalnuclei. All error bars, SD. *, P < 0.05 compared with control antibody. C, left, Ras/InBa-transformed normal human epidermal keratinocytes (NHEK ) wereplated on dishes coated with recombinant laminin a3 G45 domain in the presence of 20 Ag/mL control or affinity purified G45 IgG or heparin for 1 h, and thenattached cells were analyzed by colorimetery and quantified as percentage of control. Right, laminin a3 G45 pAb reacted with native mouse laminin-332 as shown bynonreduced immunoblot of conditioned mouse keratinocyte medium using laminin-332 pAb (pLam), G45 pAb (G45), or nonimmune rabbit IgG (control ). *, P < 0.01compared with control. D, top, representative micrographs of G45 antibody–treated mouse tissues known to express laminin-332 show no evidence of epithelialdetachment or other histologic defects. Scale bar, 200 Am. Bottom, transmission electron microscopy of antibody-treated mouse skin revealed no vesiculation ordisruption of BMZ ultrastructure in representative samples. Left bar, 5 Am; right bar, 100 nm.

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processing, other members of this family, including PCPE-2, remainto be evaluated (40). As the G45 seems to have potent pro-tumorigenic effects, factors that influence its proteolytic removalmay have important bearing on SCC tumor progression.Major protumorigenic effects of G45 include its ability to

enhance laminin-332 deposition in SCC tumors. It is possible thatG45 promotes laminin-332 deposition in SCC directly by anchoringthe laminin-332 molecule to the matrix during the process of BMZassembly. Laminin-332 G45 domain (41) has heparin-bindingproperties (42) and may have the ability to bind with extracellularheparan sulfate proteoglycan BMZ components, such as perlecan(43) or dystroglycan (44). However, even in the absence of G45,laminin-332 deposition was shown to be restored to near WT levelsin SCC tumors through activation of the PI3K pathway. Thissuggests that, rather than acting by anchoring, G45 likely promoteslaminin-332 deposition by a signaling mechanism, perhaps inducedthrough interaction with another cell surface receptor. As weshowed our tumor cell interactions with G45 could be blocked withheparin, one possible candidate that deserves further study issyndecan-1, a transmembrane heparan sulfate proteoglycanreceptor which is expressed in epidermal cells and can directlybind the laminin a3 G45 domain (25). We previously showed thatRas/InBa transformed keratinocytes expressing deletion ofdomains IV to III on the short arm of the h3 chain of laminin-332also showed impaired tumorigenesis associated with increasedapoptosis (36); however, these cells showed no significant impair-ment of proliferation or laminin-332 deposition. Thus, the G45domain seems to provide unique protumorigenic functions,including laminin deposition and proliferative stimulation, whichare not provided by the laminin h3 chain short arm.While G45 is not known to directly bind a6h4 integrin, a number

of observations suggest that G45 and a6h4 integrin functions inpromoting tumorigenesis may be related. First, we noted that G45and a6h4 integrin localized together in basal keratinocyte receptorcomplexes termed stable adhesions. While a6h4 integrin is wellknown to play a key role in the formation of these complexes (32),here, we also noted a role for G45 in stable adhesion formation.Specifically, the absence of G45 disrupted the localization of stableadhesion complexes, changing them from central to peripherallocalization. In addition to colocalizing to and playing a role in theformation of stable adhesions, a6h4 integrin and G45 also showedinteresting parallels of relevance to SCC tumorigenesis. G45 wasnoted for its promotion of cellular invasion and its activation ofPI3K and ERK signaling pathways, leading to protection fromapoptosis and increased proliferation. It is also well known thata6h4integrin, like G45, promotes carcinoma invasion, PI3K

pathway activation (45), and nuclear ERK translocation, whichleads to increased proliferation (28). Furthermore, a6h4 integrinwas shown to perform these functions in a laminin-dependentmanner through its substrate domain. Deletion of this substratedomain led to phosphorylated ERK, which accumulated in thecytoplasm (28), similar to DG45 cells in our study. Thus, it is likelythat a6h4 integrin and signaling from its substrate domain isinvolved in the function of the laminin a3 G45 domain. It shouldalso be noted that, in our studies, G45 promoted protumorigenicfunctions best when it was expressed as part of the laminin a3chain, suggesting G45 may need to be associated with the rest ofthe laminin-332 trimer for optimal function. It is possible that theclose proximity of the G45 domain to the a6h4 integrin (G1-G3)binding site on the unprocessed laminin a3 chain (Fig. 1A) plays arole in this process.G45 domain clearly modulates the expression of MMP-9 and

MMP-1, two metalloproteinases known to play important roles incarcinoma invasion. Our observation extends earlier findings of therole of G45 domain in up-regulating MMP-1 (46) and MMP-9 inkeratinocytes to squamous carcinoma cells (47). These results areconsistent with other studies showing the role of extracellularmatrix molecules changing MMP expression and activities (48).Overexpression of PI3K has been previously shown to promoteMMP-9 expression in carcinoma cells (49, 50), and it is possible thatthe ability of G45 to promote activation of the PI3K pathway may berelated to its function in promoting MMP-9 activity in human SCC.While proadhesive function of G45 may play a role in vivo , either

in healing wound edges or SCC tumors during periods of activelaminin-332 synthesis, G45 is not required for maintenance ofnormal tissue cohesion, as no epithelial-mesenchymal disruptionwas seen in normal tissues repeatedly treated with G45 inhibitoryantibodies. This is not surprising, as G45 is completely proteolyt-ically removed from normal mature tissues (16, 17). Overall, itsstriking protumorigenic activity, its prevalent accumulation inhuman SCC tumors, and its absence and lack of function in normaldeveloped tissues collectively make G45 an attractive anticancertarget.

Acknowledgments

Received 11/8/2007; revised 12/4/2007; accepted 12/17/2007.Grant support: NIH grants AR44012 and AR47223, Office of Research and

Development, Palo Alto VA Health Care System (M.P. Marinkovich), and Associationpour la Recherche sur le Cancer grant 3848 (P. Rousselle).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Paul Khavari and Anthony Oro for helpful advice.



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2008;68:2885-2894. Cancer Res Mark Tran, Patricia Rousselle, Pasi Nokelainen, et al. Human CarcinogenesisTargeting a Tumor-Specific Laminin Domain Critical for

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