PACE4 Expression in Mouse Basal Keratinocytes Results in Basement Membrane Disruption and Acceleration of Tumor Progression
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Research Article PACE4 Expression in Mouse Basal Keratinocytes Results in Basement Membrane Disruption and Acceleration of Tumor Progression 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 Cancer Res 2005; 65: (16). August 15, 2005 7310 www.aacrjournals.org 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 2.5Â lation signal (see Table 1). The sequences and the expected size of the (NA 0.08) and 10Â (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 (1Â PBS solution, 0.1% SDS, 0.5% Na deoxycholate, and 1% Nonidet