PACE4 Expression in Mouse Basal Keratinocytes Results in Basement Membrane Disruption and Acceleration of Tumor Progression

Research Article

Daniel E. Bassi,1,3 Ricardo Lopez De Cicco,1 Jonathan Cenna,1 Samuel Litwin,2 Edna Cukierman,2 and Andres J.P. Klein-Szanto1,3

Departments of 1Pathology and 2Biomathematics and Biostatistics, and 3Tumor Cell Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania

Abstract and studying the in vivo effects after overexpressing genes, such as h Collagen type IV degradation results in disruption and transforming growth factor- (3), epidermal growth factor receptor breakdown of the normal basement membrane architecture, (4), and insulin-like growth factor (5). a key process in the initiation of tumor microinvasion into the Proprotein convertases constitute a family of serine proteases that activate their cognate substrates by limited proteolysis at consensus connective tissue. PACE4, a proprotein convertase, activates # membrane type matrix metalloproteinases (MT-MMPs) that in sequence RXR/KR (6). Many of these, such as insulin-like growth turn process collagenase type IV. Because PACE4 is overex- factor-I (7) and its receptor (8), membrane-type matrix metal- pressed in skin carcinomas and in vitro overexpression of loproteinase (MT-MMP; refs. 9, 10), and integrins (11–14) have direct PACE4 resulted in enhanced invasiveness, we investigated roles in tumor progression and metastasis. Although proprotein whetherornotin vivo PACE4 expression leads to the convertases recognize and cleave a specific sequence, variations in the amino acid sequences that surround this motif, as well as acquisition of invasiveness and increased tumorigenesis. Two transgenic mouse lines were designed by targeting PACE4 to preferences in K or R preceding the COOH-terminal R, account for the epidermal basal keratinocytes. Transgenic keratinocytes differences in the efficiency of substrate cleavage (15). Furin, a well- showed increased processing of MT1-MMP and MT2-MMP studied member of the proprotein convertase family, has been resulting in collagenase IV activation and collagen type IV associated with enhanced invasion and proliferation in head and degradation. Higher collagenolytic activity partially disrupted neck (16), breast (17), and lung cancers (18). Conversely, inhibition of normal basement membrane architecture favoring epithelial its activity by the inhibitor PDX, resulted in reduced proliferation endophytic growth into the dermis and accelerating invasion and invasion in human squamous cell carcinomas (SCC; ref. 19), and metastasis after chemical carcinogenesis. PACE4 over- colon adenocarcinoma (20), and astrocytoma cell lines (21). PACE4, expression resulted in enhanced susceptibility to carcinogen- a secreted extracellular proprotein convertase, shares many esis and tumor progression pointing to a new target for biochemical properties with furin (22, 23). PACE4 is overexpressed blocking tumor cell invasiveness. (Cancer Res 2005; 65(16): 7310-9) in aggressive mouse skin spindle cell carcinomas but not in low- grade SCC (24). Moreover, in vitro experiments showed that the sole overexpression of PACE4 conferred tumorigenic properties to Introduction papilloma-derived murine cells lines (25). Actively proliferating cells have a higher probability of accumu- In order to study the role of PACE4 in tumor progression in vivo, lating mutations leading to cancer. Because of their high rate of we developed transgenic mice that express PACE4 in the epidermal renewal or turnover, stratified epithelia are more likely to undergo basal layer. Although transgenic mice did not seem to have gross malignant transformation. Epidermal basal cells constitute a abnormalities, a more in-depth analysis revealed a weakened and proliferation-proficient cell layer that, after successive steps in less prominent epidermal basement membrane. In agreement with differentiation, will replenish the more superficial layers that are these observations, MT1-MMP and MT2-MMP were processed with constantly shed. Basal cells can also initiate a burst of cell division in increased efficiency in transgenic epidermal keratinocytes. Acute response to injuries or chemical stimuli. Among the latter, topical applications with 12-O-tetradecanoylphorbol-13-acetate environmental carcinogens are able to produce mutations that (TPA) led to increased proliferative response. Moreover, these together with enhanced cell division result in neoplastic develop- animals developed more chemically induced SCC than their wild- ment. The majority of non–melanoma skin cancers arise from the type counterparts. basal keratinocytes. Basal cells express keratin 5 (K5). The K5 promoter is silenced as soon as they commit to differentiation. This Materials and Methods specific localization of K5 expression in the basal layer of the Materials. TPA and 7,12-dimethylbenz(a)anthracene (DMBA) were epidermis and outer root sheath (1) has been used for targeting purchased from Sigma-Aldrich (St. Louis, MO). FVB/N mice, 6 to 8 weeks different cancer-related genes to epidermal basal keratinocytes (2) of age, were purchased from Taconic (Germantown, NY). Generation and identification of K5.PACE4 mice. The 4 kb full-length wild-type rat PACE4 cDNA (26) was excised from its parental pCIneo vector Note: Supplementary data for this article are available at Cancer Research Online using SalI and EcoRI. The fragment was blunt-ended by treatment with the (http://cancerres.aacrjournals.org/). Klenow fragment of DNA polymerase and ligated into the SnaBI site Requests for reprints: Andres J.P. Klein-Szanto, Department of Pathology, Fox between the rabbit h-globin intron and polyadenylation sequences from a Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: 215-728- vector described previously and inserted into the K5 expression vector (2). 3154; Fax: 215-728-2899; E-mail: [email protected]. I2005 American Association for Cancer Research. Orientation and integrity of the insert were confirmed by restriction doi:10.1158/0008-5472.CAN-05-1213 analysis and sequencing. The K5.PACE4 transgene was microinjected into

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. PACE4 Expression Enhances Tumor Progression the pronuclei of mouse embryos obtained from either ICR female mice mass is formed by keratinizing cells; (c) grade 3 SCC or poorly differentiated mated to FVB male mice or FVB female mice mated with FVB male mice. tumors, containing <25% tumor mass showing evidence of keratinization; DNA was extracted from clipped tails as described (27). Mice were and (d) grade 4 SCCs, very poorly differentiated tumors or spindle cell genotyped by PCR analysis of tail DNA utilizing primers specific for an carcinomas containing very little or no histologic evidence of keratinization. internal fragment rat PACE4 and the junction 3V-PACE4/SV40 polyadeny- Papilloma and carcinoma were photographed at a magnification of 2.5 lation signal (see Table 1). The sequences and the expected size of the (NA 0.08) and 10 (NA 0.45), respectively. amplicon are shown in Table 1. Throughout this study, all transgenic mice Culture of newborn keratinocytes. Primary epidermal keratinocytes used were homozygous. from newborn mice were used to determine expression levels of PACE4 and Southern blot and determination of copy number. Ten micrograms related proteases because of their suitability for in vitro growth and further per lane of spleen or liver DNA was digested with KpnI and HindIII, and molecular analyses. Primary epidermal keratinocytes were established then subjected to electrophoresis on 0.8% agarose and transferred to in vitro as described (30). Briefly, 2- to 3-day-old mice were killed, then Hybond N+ (Amersham, Buckinghamshire, United Kingdom). The blot was washed in a 1:10 solution of betadine, rinsed twice in sterile distilled water hybridized with random primed-32P-dCTP-labeled probe from the trans- and twice in 70% alcohol. The skin was removed and floated overnight on 2 gene. The transgene band to be detected is a f7 kb DNA fragment mL of dispase (Dispase II, 25 units/mL; BD Biosciences, Bedford, MA). The generated by HindIII digestion of chromosomal DNA. As a standard for epidermis was separated from the dermis, minced and incubated with 2 mL determining the copy number, we digested the K5-PACE4 plasmid used for of 0.05% trypsin and 0.01% EDTA for 20 minutes at 37jC. DNase I (100 the microinjections with HindIII, separated 1% agarose gels and the units) was added and the cells were mechanically dissociated by vigorous corresponding 7 kb band was excised and purified. The amount of controls pipetting followed by filtration through a 40 Am cell strainer (BD were calculated as described in http://www.med.umich.edu/tamc/ Biosciences). The cells were washed in DMEM containing 10% FCS and spike.html. plated in low-Ca2+ keratinocyte growth medium (KGM/Ca2+-free KGM 1:2, Estimation of the copy number of the transgene was done by Cambrex, Walkersville, MD). densitometry, comparing the intensity of the signals generated by Protein analysis and zymography. Samples of skin or tumors were hybridization of the 32P-dCTP-labeled probe with DNA extracted from homogenized in PBS, centrifuged, and resuspended in RIPA lysis buffer transgenic animals with those generated by controls containing 0, 1, 2, 5 (1 PBS solution, 0.1% SDS, 0.5% Na deoxycholate, and 1% Nonidet P40) and 10 copies of the 7 kb band per genome. with protease inhibitors (aprotinin, 100 mmol/L phenylmethylsulfonyl

Tumor induction experiments. A single 100-nmol initiating dose of fluoride, and 100 mmol/L Na3VO3). The suspension was incubated at 4jC DMBA in 0.2 mL acetone was applied topically to the shaved dorsal skin of for 30 minutes to extract the proteins. The preparation was centrifuged 6- to 8-week-old female mice. One week after DMBA treatment, TPA (4 and the supernatants were used as cell lysates. The protein concentration nmol) in 0.2 mL of acetone or acetone alone was applied twice weekly to the was measured with a Bio-Rad protein assay system (Bio-Rad Laboratories, skin for the duration of the experiment (30 weeks). Tumor incidence and Richmond, CA). These lysates were fractionated by SDS-PAGE, transferred multiplicity were observed weekly starting at 8 weeks of TPA promotion. to a nitrocellulose membrane and immunoblotted with specific primary The number of mice per group was as follows: DMBA + TPA, 25 wild-type antibodies. For PACE4 detection, the primary antibody was JH1475, a mice, 24 transgenic mice (line #1) and 35 PACE4 transgenic (line #2); DMBA rabbit polyclonal antibody against amino acids 570 to 656 of PACE4 + acetone (initiation only), or acetone + TPA (promoter only) or acetone (kindly provided by Dr. R. Mains, University of Connecticut Health Center, alone, each treatment, 20 wild-type mice, 20 transgenic mice from each Farmington, CT; ref. 31). For MT1-MMP and MT2-MMP detection, the animal line. Carcinomas were recorded by gross observation as infiltrating monoclonal antibodies RP1MMP14 (Triple Point, Forest Grove, OR) and/or ulcerating lesions and confirmed by histologic analysis (see below). MAB3320 (Chemicon International, Temecula, CA) were used as the Autopsies of carcinoma-bearing mice were done and metastasis in axial primary antibody. The membranes were incubated with the appropriate lymph nodes, lung, liver, and spleen were recorded. All tumors were secondary antibody (anti-rabbit or anti-mouse linked horseradish analyzed histologically. Papillomas were classified either as regular peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ). The mem- pedunculated papillomas that had little or no dysplastic changes without brane was washed and developed using Amersham enhanced chemilumi- an endophytic component (type A) or as papillomas with an endophytic nescence reagent. A monoclonal antibody directed against h-actin (I-19, component characterized by moderate or advanced dysplastic changes Santa Cruz, Santa Cruz, CA) was used as loading control. Cell lysates from (type B; ref. 28) as well as by areas of epithelial down-growth into the the fibrosarcoma cell line HT-1080 were used as MT1-MMP-positive papillary dermis (endophytic component). SCC were classified according to controls (32). histopathologic grade (29). Most SCCs were endophytic growths that Zymography was done in 10% polyacrylamide gels that had been cast in invaded the dermis and s.c. tissue. The differentiation patterns defining the the presence of gelatin (Invitrogen, Carlsbad, CA). Briefly, samples histopathologic grade were: (a) grade 1 SCC, very well differentiated with containing 10 Ag proteins were mixed with 2 loading buffer, and without most of the tumor containing keratinizing cells and horny pearls; (b) grade prior denaturation were run on a 10% SDS-polyacrylamide gel containing 2 SCC, moderately differentiated tumors in which up to 50% of the tumor 0.5 mg/mL gelatin. After electrophoresis, gels were washed to remove SDS

Table 1. Sequences of the primers used to amplify PACE4 or GAPDH

Gene Sequence Size*

PACE4 internal sense 5V-CCCTCTGGAACCAAGTCTCAACTT-3V 1,000 bp (48) antisense 5V-TGAAGCCAGCTTTACATCTGCTGC-3V Junction PACE4-PolyA sense 5V-CATATGTCCTTCGAAGTGAGAGA-3V 450 bp antisense 5V-TTCTACCCGGAGATGCCT-3V GAPDH (mouse) sense 5V-GTA GAC AAA ATG GTG AAG GTC-3V 350 bp antisense 5V-GTT GTC ATA TTT CTC GTG GTT-3V

*The size of the amplicon is indicated.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research and incubated for 18 hours at 37jC in a renaturing buffer (50 mmol/L Tris, segment of either 450 bp corresponding to the PACE4-PolyA 5 mmol/L CaCl2, 0.02% NaN3, 1% Triton X-100). Gels were subsequently boundary or 1,000 bp internal to rat PACE4 discriminating stained with Coomassie brilliant blue G-250 and destained in 30% methanol endogenous from transgenic PACE4 genes. A representative and 10% acetic acid (v/v). genotyping experiment is shown in Fig. 1B, Two founders, referred RNA extraction and RT-PCR. Total RNA from epidermis or cultured to as animal lines #1 and #2 were selected to perform subsequent newborn keratinocytes was extracted using TRIZOL Reagent (Life Technologies/Invitrogen) and reverse-transcribed by Superscript One-Step experiments. RT-PCR (Invitrogen). Rat PACE4 and murine glyceraldehyde-3-phosphate Transgene copy number in the two animal lines was assessed by dehydrogenase (GAPDH, housekeeping gene used as a control for the RT- Southern blot analysis and densitometry. The results showed that PCR reaction) were amplified with the primers described in Table 1. animal line #1 contained four to five transgene copies whereas line Analysis of epidermal thickness and cell proliferation following #2 had two copies (Fig. 1C). Expression at the RNA level was treatment with TPA. Groups of mice (n = 5) were treated with two weekly assessed using RNA extracted from cultured newborn keratinocytes applications of TPA (5 nmol) or the acetone vehicle and sacrificed 48 hours (Fig. 1D). Both lines showed RNA expression. The relative level of after the last treatment. Paraffin sections were stained with H&E and the PACE4 protein expression was analyzed by Western blot. As a skin thickness was measured with a micrometer. For analysis of cell protein source, we used either skin lysates or conditioned medium proliferation, mice treated as described above were injected i.p. with from keratinocyte culture obtained from newborn mice. The bromodeoxyuridine (BrdUrd, 100 Ag/g body weight; Sigma-Aldrich) in PBS, 2 hours prior to sacrifice. Treated skin was then fixed in formalin, relative amount of PACE4 expressed by the wild-type and embedded in paraffin, sectioned at 4 Am, stained with H&E and anti-BrdUrd transgenic lines was analyzed by densitometry and was normalized antibody (Roche), and then treated with biotinylated anti-mouse IgG and to h-actin expression. In agreement with the respective transgene horseradish peroxidase-conjugated ABC reagent (Vector Laboratories, Inc., copy number, PACE4 protein levels were twice as high in animal Burlingame, CA). Epidermal cell proliferation (presented as the labeling line #1 as in line #2 (Fig. 1E). Wild-type animals did not show index) was determined as follows: a minimum of 500 basal cells from five expression of PACE4 in total skin or keratinocyte cultures. mice per group were counted, and the percentage of BrdUrd-positive cells PACE4 enhances induced epidermal proliferation and was calculated. Slides were mounted and observed with a Nikon Optiphot activates matrix metalloproteinases in vivo. Both transgenic with a Plan/Apo objective 20, NA, 0.75, Nikon eyepiece 10, final animal lines resemble the wild-type animals in major phenotypic magnification 200. Micrographs were obtained with a Magnafire characteristics. However, as PACE4 is a proprotein convertase Optronics digital camera. Immunofluorescence. Frozen sections (5 Am) from skins were fixed in associated with increased proliferation and invasive ability, we acetone, and incubated with an anti-mouse anticollagen IVantibody (AB756P, examined the biochemical and microstructural alterations of the Chemicon International) for 1 hour at room temperature. FITC-labeled epidermis. First, the effect of PACE4 expression on basal keratino- antigoat IgG antibody (The Jackson Laboratory, West Groove, PA) was used cyte proliferation was evaluated by measuring epidermal thickness as secondary antibody. Imaging was done in a Nikon Eclipse TE 2000-U in H&E-stained specimens from wild-type and transgenic mice skin. microscope using a magnification of 60 (NA = 1.4). Samples were mounted The measurements indicated that epidermal thickness was unal- in fluorescent mounting media (Prolong Gold antifade reagent, Molecular tered by the expression of the transgene (Fig. 2A). However, Probes, Eugene OR) and visualized with Hoechst H 33342 (Calbiochem, La increased susceptibility to the hyperplasiogenic and proliferative Jolla, CA) for nuclei staining and FITC (collagen IV) by excitation at 364 and effects of TPA was observed after acute topical treatment. The 520 nm, respectively. Pictures, representing a stack of Z planes, were obtained epidermis from transgenic mice showed a 30% to 40% increase in with Roper Scientific CoolSNAP HQ camera, the images were processed using Metamorph software. Briefly, images were two-dimensionally deconvoluted, thickness compared with their wild-type counterparts after 2 weeks three-dimensionally reconstructed using tilt angles between 0 and of TPA treatment (2 Ag twice a week; Fig. 2A and B). Proliferation was 180 degrees. For quantitative measurements of collagen IV fluorescence, also evaluated by BrdUrd incorporation rates. Similar to results the ratio between the fluorescence associated with collagen IV and the observed by measuring epidermal thickness, control skin from wild- stratum corneum (considered as the internal control) was calculated. type or transgenic animals did not show significant differences in Statistical analysis. Statistical significance was determined by calculat- BrdUrd incorporation. Nevertheless, after TPA treatment, BrdUrd ing P values. P values corresponding to the curves of carcinoma incorporation to both transgenic lines increased about 25%, above m2 development were determined by Kruskal-Wallace test. The differences the wild-type levels in agreement with the changes observed in skin between the number of endophytic papillomas per animal were analyzed by thickness (Fig. 2C and D). comparison of frequencies using a normal approximation to the Poisson Cell invasiveness is another biological process affected by PACE4 distribution. In all other studies, P values were determined using Fisher two-tailed exact test. expression (25). In this context, the main substrates for PACE4 are MT-MMPs, metalloproteinases responsible for activation of collagenase IV (MMP-2), a major extracellular matrix–degradating enzyme. Results In order to evaluate whether PACE4 expression influenced the Generation and characterization of transgenic mice. To activation of MT-MMPs, and consequently, the activation of MMP-2, study the effects of PACE4 expression on the highly proliferative we evaluated MT1-MMP and MT2-MMP processing by Western blot epidermal basal cells, the full-length rat PACE4 cDNA was placed in cell lysates from cultured keratinocytes obtained from wild-type under the control of the K5 promoter targeting PACE4 expression or transgenic newborn mice. As shown in Fig. 3, both MT1-MMP to the basal skin keratinocytes. The construct contains the bovine (Fig. 3A) and MT2-MMP (Fig. 3B) were more efficiently processed in K5 promoter (Fig. 1A), followed by the first intron from rabbit h- transgenic lines than in wild-type animals. This enhanced activation globin to enhance the efficiency of transcription, the full-length rat of MT-MMPs correlated with enhanced activation of MMP-2 as PACE4 cDNA and, finally the polyadenylation signal from SV40. assessed by zymography of whole-skin lysates and conditioned Five founders were produced; two of them were selected to expand medium from cultured keratinocytes. Levels of pro-MMP-2 were the animal line because they showed approximately two to four almost undetectable in transgenic mice. Conversely, a band transgene copies (data not shown). Founders were genotyped by corresponding to the unprocessed form of this metalloproteinase PCR of genomic DNA using two set of primers amplifying a DNA was clearly observed in wild-type animals (Fig. 3C).

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PACE4 disrupts basement membrane structure. Because recorded. In agreement with the measurements of collagen type IV increased MMP-2 activation results in enhanced degradation of fluorescence intensity, the number of disruptions was 2 to 2.5 times collagen type IV, one of the main components of the basement higher in the transgenics than in the wild-type animals. These results membrane, we hypothesized that the structure of transgenic indicate that constitutive expression of PACE4 is associated with basement membrane could be altered. In order to evaluate in vivo increased collagen type IV degradation, resulting in basement type IV collagen degradation, we analyzed the integrity of the membrane alteration such as membrane thinning and localized basement membrane collagen component of wild-type and trans- disruptions. Collagen type IV immunofluorescence of TPA-treated genic epidermis. Collagen type IV indirect immunofluorescence, skins pointed in the same direction. Transgenic animals showed done on frozen sections from skin derived from wild-type animals, increased collagen type IV disruptions and altered basement revealed a well-defined and continuous basement membrane with a membrane structure. Nevertheless, measurements of the basement uniform thickness (Fig. 4A, top). Conversely, in line #1, many points membrane’s collagen type IV component showed that both of discontinuity and areas in which the basement membrane transgenic and wild-type animals exhibited similar levels collagen type IV was markedly thinner than in its wild-type of fluorescence intensity, attributable to TPA-driven stimulation of counterpart were seen (Fig. 4A, middle). The phenotype observed collagen type IV synthesis (ref. 33; Fig. 4E). However, transgenic in the skin of animal line #2 was less pronounced (Fig. 4A, bottom), animals showed increased number of collagen type IV disruptions suggesting that this effect may be dependent on gene dosage (see Fig. 4D, insets). The number of disruptions per micron was three (Fig. 4C). Collagen type IV disruption was quantified by measuring or two times higher in transgenic lines #1 or #2, respectively, than in the intensity of fluorescence associated with this protein within the wild-type epidermis (Fig. 4F). These data suggest that even after TPA collagen IV component of the basement membrane (Fig. 4). treatment, transgenic animals were still able to degrade collagen Transgenic lines #1 and #2 showed 2.5 and 1.4 times the reduction type IV and exhibited significant basement membrane structural relative to wild-type in the collagen type IV fluorescent intensity, alterations. Alteration in basement membrane structure can be respectively (Fig. 4B). In addition, the number of disruptions in the observed online, under supplemental materials, videos V#1, control collagen type IV layer per micron of linear basement membrane was skins, and V#2 TPA-treated skins.

Figure 1. Generation of PACE4 transgenic mice. A, K5-PACE4 wild-type construct. B, PCR from DNA extracted from tails, amplified with either primers A or B and h-globin as control. C, determination of the number of copies of the transgene by Southern blot analysis. Note the higher intensity of the band in line #1. D, RNA expression of the transgene. RNA was extracted from cultured keratinocytes and analyzed for PACE4 expression by RT-PCR. GAPDH was added as control. E, Western blot and densitometry showing differential expression of PACE4 protein in transgenic lines. Note the complete absence of PACE4 expression in the wild-type mice. www.aacrjournals.org 7313 Cancer Res 2005; 65: (16). August 15, 2005

Figure 2. Skin of wild-type, transgenic line #1, and transgenic line #2 were treated either with acetone or TPA. A, note the enhanced susceptibility to TPA-induced effects (increased thickness) on transgenic mice (P < 0.001). B, H&E staining of TPA-treated skin. Note the increase in skin thickness in transgenic animals as compared with wild-type after TPA treatment. C, proliferative response to TPA. There is a 30% to 40% increase in nuclear labeling of transgenic epidermis (P < 0.001). D, BrdUrd immunostaining of TPA-treated skin. Note the higher number of stained nuclei in transgenic tissue. A and C, data represent an average of five mice; magnification, 20; bar, 50 Am.

PACE4 increases susceptibility to skin cancer. In order to papillomas. Wild-type animals also showed these lesions in determine the effects of PACE4 overexpression and enhanced reduced numbers. As this feature constituted a distinctive MMP activation on SCC development, a two-stage carcinogenesis phenotype between wild-type and transgenic animals, we protocol was used. During the carcinogenesis experiment, examined the lesions at the completion of the experiments transgenic animals revealed numerous lesions resembling small (30 weeks after DMBA initiation). Histologic examination confirmed that the lesions were papillomas that could be classified into two distinct types (Fig. 5B and C). Typical exophytic cauliflower-like pedunculated papillomas (type A, Fig. 5B) and papillomas containing an endophytic component that grew towards the dermis (type B, Fig. 5C). Most of the papillomas (>70-80%) were type B in both transgenic lines. In contrast, papillomas in wild-type mice showed a higher proportion of type A papillomas. The ratio between the number of endophytic papillomas per mouse in transgenic lines to wild-type mice was recorded (Fig. 5A). Transgenic lines harbored at least three times more (P < 0.001) endophytic papillomas per animal than the wild- type animals. On the other hand, line #2 developed twice as many exophytic papillomas as line #1, highlighting the more moderate phenotype of line #2 mice expressing lower levels of PACE4 (supplemental material S1). SCC development was remarkably enhanced in transgenic animals (Fig. 5C). The incidence of carcinoma showed a clear difference between the transgenic and wild-type animals (see supplemental material S2). Transgenic mice developed SCCs faster that the wild-type mice and the number of malignant lesions was Figure 3. Activation of metalloproteinases by wild-type and transgenic epidermal keratinocytes. Western blot MT1-MMP (A), MT2-MMP (B) and zymography higher at any time point in agreement with the proposed role of from conditioned medium of cultured newborn keratinocytes depicting MMP-2 PACE4 as a facilitator of malignant conversion (ref. 25; Fig. 5D). activity (C). Note increased MT1-MMP and MT2-MMP activation in transgenic Rates of SCC formation also differed strikingly between wild-type keratinocytes as well as increased MMP-2 activity. Pro, proenzyme; i MT1-MMP, inactive MT1-MMP. As a marker to position MT1-MMP species (Pro, mature and (0.04 carcinomas per animal per week) and transgenic animals inactive enzyme), we used a cell lysate from the fibrosarcoma cell line HT-1080. (0.092 carcinomas per animal per week; Fig. 5D).

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Conversion of papillomas into SCC, measured as the ratio PACE4 induces higher levels of matrix metalloproteinases between the number of carcinomas and the number of papillomas, and tumors of advanced malignant phenotype. In order to was much higher in transgenic lines than in wild-type mice (see confirm that tumors from transgenic mice expressed PACE4 and supplemental material S3). Moreover, the number of metastases showed increased activation of MT-MMPs and MMP-2, we observed in transgenic mice was dependent on PACE4 expression. analyzed cell lysates from wild-type and transgenic tumors by Although the formation of metastasis is a relatively rare event at 30 Western blot (Fig. 5E). All transgenic tumors exhibited PACE4 weeks of TPA treatment, the number of metastatic foci in axial expression. In contrast, only a small fraction (f20%) of tumors lymph nodes and lung was higher in line #1 than in wild-type mice from wild-type animals expressed this proprotein convertase (see supplemental material S4). Line #2 showed only a marginal (Fig. 5E, top). PACE4-mediated proteolysis resulted in activation increment in the number of metastasis when compared with wild- of MT-MMP’s, the principal proprotein convertase substrates type probably due to its lower expression of PACE4. Neither involved in tumor cell invasiveness (34, 35). Lysates of tumors control untreated transgenic mice nor control wild-type mice obtained from PACE4 transgenic animals showed increased ability exhibited spontaneous skin tumors during the 18-month observa- to cleave MT1 and MT2-MMP, the active form being the only one tion period. detectable in some cases (Fig. 5E, middle). In contrast, these

Figure 4. Collagen IV immunofluorescence. Sections from acetone (A-C) or TPA (D-E)–treated animals were immunostained with a collagen IV antibody. Slides from untreated (A) and TPA-treated (D) skin are shown. Collagen IV immunofluorescence was quantified measuring the integrated absorbance using the Metamorph software. Note the decreased fluorescence associated with collagen IV in transgenic animals (Fisher, two-tailed exact test, P < 0.05; B). TPA treatment did not result in major differences in collagen IV immunofluorescence (E). Collagen IV disruptions were counted and the number of these disruptions per unit (Am) of basement membrane length were recorded. C, nontreated; F, TPA-treated. Note the increased number of basement membrane disruptions in the transgenic samples. Differences in the number of disruptions between wild-type and transgenic mice were highly significant (Fisher two-tailed exact test, P < 0.001). Magnification, 60; bar, 10 Am. www.aacrjournals.org 7315 Cancer Res 2005; 65: (16). August 15, 2005

Figure 5. Responsiveness of wild-type and transgenic mice to two-stage carcinogenesis protocol (DMBA/TPA). A, relative number of endophytic papillomas in transgenic compared with wild-type mice. Notice the higher proportion of endophytic papillomas in transgenic animals (*, ** P < 0.005; normal approximation of Poisson distribution). Examples of exophytic (B) and endophytic (C) papillomas. Magnification, 2.5; bar, 250 Am. D, carcinoma development. Note increased number of carcinomas in transgenic compared with wild-type animals (P < 0.05). E, biochemical characterization of tumors. Proteins were extracted from tumors. PACE4 Western blot (top), MT1- and MT2-MMP Western blot analysis showing activation of the proform into the mature form (middle), and zymogram for the evaluation of MMP-2 activation (bottom). Notice the higher processing activity in tumors derived from transgenic mice.

MT-MMPs were cleaved with lower efficiency in wild-type tumors overexpressing transgenic animal lines (#1 and #2). Southern blot (Fig. 5E, middle). This enhanced activation of the MT-MMPs was analyses showed that animal line #1 contained four to five copies of reflected in enhanced activation of its substrate, MMP-2, as shown the transgene and line #2 showed only two. This difference in copy by zymography (Fig. 5E, bottom). number was reflected in the higher protein expression (twice as In order to confirm that PACE4 expression alters the malignant high in line #1 than in line #2). phenotype, we evaluated and graded the tumors. Histologic analysis The enhanced PACE4 expression was reflected in higher levels of of tumors from wild-type and transgenic mice showed notorious activated MT1- and MT2-MMP. Increased MT1-MMP activation differences. In wild-type animals and transgenic line #2 mice (which drives increased type IV collagenolytic activity (37, 38). Constitutive showed lower expression of PACE4), >50% SCC were well- PACE4 activation may be responsible for the premature activation of differentiated, grade 1 tumors (Fig. 6A and C). Grade 2 accounted MMP-2, allowing early disruption of the basement membrane for 25% and the rest (f20%) were more advanced grades 3 and 4. In collagen IV component. Sole activation of MT1-MMP may also be contrast, histology of tumors from transgenic line #1 showed a responsible for basement membrane degradation. MT1-MMP was greater number of more aggressive phenotypes (Fig. 6B). Figure 6D recently identified as a major proteinase necessary for cell shows the distribution of tumors according to their grade. invasiveness of either normal or cancer cells (35). Moreover, animal models targeting the expression of this MT-MMP to the mammary gland resulted in increased induction of adenocarcinomas (39). Discussion Collagen type IV, a major basement membrane structural protein, PACE4 is a serine protease overexpressed in high-grade murine provides a scaffold that binds other basement membrane proteins SCC (24). Its activity results in proteolysis of substrates leading to such as laminins, entactins, and proteoglycans (40). Hence, collagen activation of many cancer-related proteins (36). In order to show a type IV degradation may disrupt the integrity of the basement causal relationship between PACE4 expression and increased membrane creating an invasion-permissive environment that susceptibility to carcinogenesis in vivo, we studied two PACE4 contrasts with the invasion-restraining environment provided by

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. PACE4 Expression Enhances Tumor Progression an intact basement membrane (41). PACE4- overexpressing mice tumor promoters resulted in a decisively magnified response during showed disruption of the collagen IV component of the basement carcinogenesis (Fig. 7). membrane. Moreover, the magnitude of this effect paralleled the The skin of transgenic animals showed numerous flat lesions that levels of transgene expression. The intensity of basement membrane were not as prominent in wild-type animals. Interestingly, these collagen type IV was reduced by 85% and 50% in lines #1 and #2, lesions proved to be endophytic papillomas. As these mice showed respectively. Further evidence that transgenic mice had a higher increased collagen type IV degradation, it may be argued that due to collagenolytic activity was the increased number of basement thinning or weaker basement membrane collagen type IV compo- membrane collagen type IV disruptions. nent, the developing tumor cells grow unopposed directly into the Degradation of collagen type IV might allow direct contact dermis (Fig. 7). This feature resembles the behavior observed during between keratinocytes and stroma, generating signals that would complete carcinogenesis, consisting of repetitive treatments with a not take place in the absence of direct contact (42). Early complete carcinogen such as benzo(a)pyrene (46). In this protocol, degradation of collagen type IV also predisposes the epithelial cells few exophytic (type A) papillomas develop, whereas most of the to accelerated invasion bypassing the need for both attachment and SCCs are preceded by in situ flat precursor lesions and endophytic additional degradation of the basement membrane (40, 43) by lesions (47). This observation points to a close association between directly migrating into the dermis. In this sense, before a normal cell increased PACE4 expression and enhanced susceptibility to is initiated/promoted, PACE4 transgenic basal keratinocytes already chemical carcinogens, i.e., increased PACE4 expression in animals possess the necessary conditions to start migration and invasion treated with a two-stage protocol results in early aggressive tumors (Fig. 7). In summary, these results suggest that increased efficiency in preceded by papillomas with endophytic growth mimicking the MT-MMPs activation results in acceleration of tumor development response vis-a`-vis complete carcinogen protocols (29). and progression due, in part, to increased collagenase type IV After the 30 weeks of treatment with the two-stage carcinogen- activity. Early activation of collagen-degrading enzymes, mainly MT- esis protocol, the number of carcinomas per animal in both MMPs and also MMP-2, produce premature changes in the biology transgenic animal lines was twice as high as in wild-type mice. of the extracellular matrix, i.e., basement membrane disruption (44) Moreover, the incidence of malignant tumors, which is defined as that may result in an early interaction between fibroblasts and the percentage of animals harboring carcinomas, was consistently epithelial cells. This early encounter may trigger a plethora of pro- higher in transgenic mice: 65% to 70% of transgenic animals had proliferative effects via fibroblast growth factors, cytokines, and/or tumors. In contrast, only 45% of wild-type animals developed matrix proteases contributing to premature increase in epithelial tumors (see supplemental material S2). The rate of carcinoma proliferation and invasion (45). These features of accelerated growth showed remarkable differences between transgenic and basement membrane degradation and increased susceptibility to mice, 0.092 versus 0.04 carcinomas/animal/wk, respectively. The

Figure 6. Histology of representative SCC. Wild-type (A) and transgenic line #2 (C) developed mostly well-differentiated tumors (grades 1 and 2). Line #1 (B) generated poorly differentiated tumors (grades 3 or 4) SCC. Magnification, 10; bar, 50 Am(C). D, relative number of grades 3 to 4 SCCs in wild-type and transgenic animals.

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Figure 7. Proposed model for the mechanisms involved in PACE4-driven acceleration of tumor progression. MMP activation by PACE4 results in premature disruption of the collagen IV component of the basement membrane (BM, red). This leads to early epidermal down-growth into the stroma and development of endophytic papillomas. The latter, earlier gave rise to more advanced SCC that had the extra advantage of metastasizing.

data pointed to the pivotal role of PACE4 in accelerating or number of endophytic papillomas growing towards the dermis, predisposing the rate of tumor development and progression. Line providing an extra advantage to tumor development, and #1, which expressed high levels of PACE4, developed more eventually invading sooner and deeper into the dermis, finally malignant tumors and metastasis than line #2, which expressed producing distant metastases (Fig. 7). only modest levels of PACE4. Moreover, line #2 resembled wild-type animals in both tumor grade (mostly grades 1 or 2) and the relative Acknowledgments low number of metastases. Received 4/7/2005; revised 5/26/2005; accepted 6/7/2005. In summary, PACE4 expression in vivo resulted in premature Grant support: NIH grants CA75028, CA06927, and by an appropriation from the disruption of the component of the basement membrane, allowing Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page early down-growth of carcinogen-transformed cells to the under- charges. This article must therefore be hereby marked advertisement in accordance lying structures. This early behavior resulted in an increased with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. PACE4 Expression in Mouse Basal Keratinocytes Results in Basement Membrane Disruption and Acceleration of Tumor Progression

Daniel E. Bassi, Ricardo Lopez De Cicco, Jonathan Cenna, et al.

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