pad2 overexpression in transgenic mice promotes...

Tumor and Stem Cell Biology

PAD2 Overexpression in Transgenic Mice PromotesSpontaneous Skin Neoplasia

John L. McElwee1, Sunish Mohanan2, Sachi Horibata3, Kelly L. Sams3, Lynne J. Anguish3, Dalton McLean3,Iva Cvita�s4, Joseph J. Wakshlag5, and Scott A. Coonrod3

AbstractPeptidylarginine deiminase 2 (PAD2/PADI2) has been implicated in various inflammatory diseases and, more

recently, cancer. The goal of this study was to test the hypothesis that PAD2 promotes oncogenesis using atransgenic mouse model. We found that about 37% of transgenic mice overexpressing human FLAG-PAD2downstream of the MMTV-LTR promoter develop spontaneous neoplastic skin lesions. Molecular and histo-pathologic analyses of the resulting lesions find that they contain increased levels of markers for invasion,inflammation, and epithelial-to-mesenchymal transition (EMT) and that a subset of the lesions progress toinvasive squamous cell carcinoma (SCC). We then stably overexpressed FLAG-PAD2 in the human SCC cell line,A431, and found that the PAD2-overexpressing cells were more tumorigenic in vitro and also contained elevatedlevels of markers for inflammation and EMT. Collectively, these studies provide the first genetic evidence thatPAD2 functions as an oncogene and suggest that PAD2 may promote tumor progression by enhancinginflammation within the tumor microenvironment. Cancer Res; 74(21); 6306–17. �2014 AACR.

IntroductionThe peptidylarginine deiminase (PADI or more commonly

referred to as PAD) family of posttranslational modificationenzymes converts positively charged arginine residues onsubstrate proteins to neutrally charged citrulline. This activity,alternatively called deimination or citrullination, has beenshown to havewide-ranging effects on target protein structure,function, and protein–protein interactions. Increasingly, thedysregulation of PAD activity is associated with a range ofdiseases, including rheumatoid arthritis, multiple sclerosis,ulcerative colitis, neural degeneration, chronic obstructivepulmonary disorder (COPD), and cancer (1–3). While thepresumptive function of PAD activity inmost diseases is linkedto inflammation, the role that PADs play in cancer progressionis still under investigation (4–7). Our recent studies suggesta role for PAD2 in the oncogenic progression of breast cancer(8–11) and provide preclinical evidence showing that the PAD

inhibitor, Cl-amidine, could be used as a therapeutic agent forthe treatment of tumors in vivo (11). To further investigate theinvolvement of PAD2 in the oncogenesis of epithelial tumors,we generated FVB/N mice expressing FLAG-PAD2 under thecontrol of the mammary tumor virus (MMTV) promoter. Aswith previous studies (12, 13), we found that the MMTVpromoter drives transgene expression in a range of tissues,including themammary gland, salivary gland, ovaries, and skin.Results from our spontaneous tumor study show that about37% of the PAD2 transgenic mice developed skin lesions withina period of 4 to 12 months after birth. These tumors expressedhigh levels of transgenic human PAD2 and display markers ofincreased invasiveness and epithelial-to-mesenchymal transi-tion (EMT). Furthermore, a subset of these tumors displays thehallmarks of malignant progression to highly invasive squa-mous cell carcinomas (SCC). The findings identify a novelgenetic mouse model of skin neoplasia and define a role forPAD2 in cancer progression.

Materials and MethodsGeneration of MMTV-FLAG-PAD2 mice

To generate the MMTV-FLAG-PAD2 construct, humanPAD2 cDNA was subcloned from pcDNA3.1-FLAG-PAD2 (14)into theEcoRI sites of theMMTV-SV40-Bssk plasmid (Addgeneplasmid #1824), originally generated in the laboratory of Dr.Philip Leder at Harvard Medical School (Boston, MA; ref. 15).The linear MMTV-FLAG-PAD2 construct was purified andmicroinjected into the pronuclei of fertilized embryos fromsuperovulated FVB/N (FVB/NJ, JAX) mice, and 2-cell stageembryos were transferred to pseudopregnant mothers. Themicroinjection and embryo transfer were performed by theStem Cell and Transgenics Core at Cornell University's Collegeof Veterinary Medicine (Ithaca, NY). Mice were genotyped for

1Department of Nutritional Sciences, College of Human Ecology, CornellUniversity, Ithaca, New York. 2Lilly Research Laboratories, Indianapolis,Indiana. 3Baker Institute for Animal Health, College of Veterinary Medicine,Cornell University, Ithaca, New York. 4Faculty of Veterinary Medicine,University of Zagreb, Zagreb, Croatia. 5Department of Clinical Sciences,College of Veterinary Medicine, Cornell University, Ithaca, New York.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J.L. McElwee and S. Mohanan contributed equally to this article.

Corresponding Author: Scott A. Coonrod, Baker Institute for AnimalHealth, College of Veterinary Medicine, Cornell University, Ithaca, NY14853. Phone: 607-319-4698; Fax: 607-256-5608; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-0749

�2014 American Association for Cancer Research.

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the presence of an integrated human PAD2 transgene by PCRwith the primers hPAD2-cds-F/R, andmouse Pad4was used asgenomic control (see Supplementary Table S1). All mouseexperiments were reviewed and approved by the InstitutionalAnimal Care and Use Committees (IACUC) at CornellUniversity.

Stable FLAG-PAD2 expression in A431 cellsThe human SCC A431 cell line was obtained from ATCC

(CRL-1555) in 2013 and cultured according to manufacturer'sdirections for 2 weeks before the generation of stable lines.Authentication was performed by ATCC using short tandemrepeat (STR) DNA fingerprinting (16). To generate A431 cellsoverexpressing FLAG-tagged PAD2, two separate plasmidswere generated by subcloning FLAG-PAD2 into the pIRES2-EGFP vector (Clontech) and pLenti-PGK-GFP-Puro plasmid(Addgene #19070), followed by transfection and transduction,respectively (see Supplementary Methods).

Immunohistochemistry and immunofluorescenceImmunohistochemistry (IHC) and immunofluorescence

experiments were carried out using a standard protocol aspreviously described (9). Primary antibodies are as follows:anti-PAD2 (12110-1-AP, ProteinTech), anti-FLAG-M2 (F1804,Sigma), anti-pan-Citrulline (ab6464, Abcam), anti-Ki67(ab15580, Abcam), and anti-IL6 (ab6672, Abcam). Negativecontrols were either normal rabbit or mouse IgG.

Western blottingWestern blotting was carried out as previously described (9).

Primary antibodies against PAD2 (12110-1-AP, ProteinTech)and FLAG-M2 (F1804, Sigma) were incubated overnight at 4�C.To confirm equal protein loading, membranes were strippedand reprobed with anti-b-actin (ab8227, Abcam).

RNA isolation, semiquantitative, and quantitative real-time PCRRNAwas purified using the Qiagen RNeasy Kit, including on-

column DNase treatment, and reverse-transcribed using theApplied BiosystemsHigh Capacity RNA-to-cDNA kit accordingto the manufacturer's protocol. See Supplementary Methodsfor a more detailed description of semiquantitative and quan-titative real-time PCR (qPCR) and Supplementary Tables S1–S3 for primer sequences.

Assay for cellular malignancy and invasionCollagen-coated inserts for 24-well plate wells (Falcon BD

Fluoroblok; #351152) were used to conduct Transwell migra-tion assays as previously described (17) and further detailed inthe Supplementary Methods. Focus formation assays werecarried out as previously described (18).

Statistical analysisAll experiments were independently repeated at least three

times unless otherwise indicated. Values were expressed as themean� SD and analyzed using Student t test or ANOVA (withTukey post hoc analysis) where three or more groups werecompared with one another using continuous data. Incidence

of spontaneous lesions in MMTV-FLAG-PAD2 mice wasmeasured 4 to 12 months post-birth, along with age-matchedFVB/N mice from the corresponding founder line. Data wereanalyzed using a c2 test with Yates correction for continuity.Significance was set at an a of 0.05.

ResultsGeneration of MMTV-FLAG-PAD2 transgenic mice

To assess the potential role of PAD2 in the oncogenesis ofepithelial tissue, we generated a mouse model in which thehuman PAD2 gene is overexpressed under the control of thehormone-responsive MMTV-LTR promoter. The transgenicconstruct consists of an MMTV-LTR promoter placedupstream of the human FLAG-tagged PAD2 cDNA, followedby an SV40 splice/polyadenylation site (Fig. 1A).MMTV-FLAG-PAD2mice were generated, and seven potential founders weretested for the presence of transgene by PCR. Four founders(4807, 4853, 4680, and 4863) were identified (indicated inred, Fig. 1B) that carried germline transmission of theFLAG-PAD2 transgene. We note that 4807 was not includedin subsequent analyses due to poor breeding. Using semiquan-titative RT-PCR, we confirmed the presence of FLAG-PAD2transcript in the skin, salivary gland, mammary gland, andovary of the transgenic mice (Fig. 1C). These four tissues werechosen for analysis because they have previously been shownto express high levels of MMTV-LTR–driven transgenes (13).qPCR analysis of the same tissues demonstrated that whilePAD2 expression was highest in salivary andmammary glands,significant levels of transgenic PAD2were also found in the skinof all three founders (P < 0.01; Fig. 1D). We note here that whiletransgenic expression of PAD2 was observed in the mammaryglands of both virgin (Supplementary Fig. S1A) and multipa-rous mice (Supplementary Fig. S1B), we did not detect anygross abnormalities or any observable phenotype in the mam-mary glands of thesemice. The potentialmechanisms thatmaybe blocking PAD2-mediated oncogenesis in the mammarygland are currently being investigated.

PAD2 transgenic mice develop skin lesions with thepotential to advance to invasive SCCs

Analysis of spontaneous neoplastic growth in experimentalmice found that about 37% of the mice across all founderlines developed skin lesions between 4 and 12 months of age(Table 1). When compared with age-matched wild-type (WT)FVB/N mice, the increased rate of neoplastic growth in FLAG-PAD2 transgenic mice was highly significant (x2 ¼ 30.65;P < 0.001), suggesting that the PAD2 transgene is likely pro-moting oncogenesis. The percentage of males developingtumors was about 44%, whereas females developed tumors ata rate of about 56%,whichwas not significantly different acrossgenders. These lesions occurred on both the dorsum andventrum of transgenic mice and are characterized by grossabnormalities, such as alopecia, multifocal epidermal ulcera-tion often covered in serocellular crust, dysplasia, and thick-ening of the adjacent epidermis (Fig. 2A).

Histologic evaluation of skin lesions from PAD2-overexpres-sing mice by hematoxylin and eosin reveals that these sites

PAD2 Overexpression Promotes Skin Neoplasia

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contain highly neoplastic tissue, having features consistentwith invasive SCC. We found nests of neoplastic cells thatappeared to originate from the epidermis and to invade intothe dermis and subcutis (Fig. 2B, i and ii); moreover, theepidermis overlying the neoplasm was extensively ulceratedand frequently adjacent to hyperplastic epidermal layers (Fig.2B, i). In addition, we observed that concentric layers of keratinsurrounded tumor cells forming keratin pearls, which arecharacteristic of SCC (Fig. 2B, ii). We found that tumor borderswere poorly defined, often with highly anaplastic tumor cellsinvading the deeper dermis and subcutis, and that neoplastic

cells frequently infiltrated and separated skeletal myofibers(Fig. 2B, iii). The nests and islands of neoplastic cells within thesubcutis showed a high degree of anisokaryosis and anisocy-tosis, with the nuclei often containing one to two prominentnucleoli (Fig. 2B, iv). Clusters of neoplastic cells were found tobe surrounded by loose collagenous stroma with amarked lossof adnexal structures, consistent with desmoplastic response(Fig. 2B, ii–iv). Finally, we observed that these lesions oftencontained carcinoma cells budding from the primary neo-plasm (Fig. 2B, iv), again indicating an invasive componentto these tumors.

Figure 1. MMTV-FLAG-PAD2transgenic construct. A, schematicof the linearized MMTV-FLAG-hPAD2 transgene, containingFLAG-tagged human PAD2 cDNAcloned between the EcoRI sites ofthe MMTV-SV40-Bssk plasmid(15). The construct used forgeneration of FLAG-hPAD2-transgenic mice (herein FLAG-PAD2) consists of FLAG-PAD2under the control of the hormone-responsive MMTV-LTR promoterenhancer with an SV40 splice-polyadenylation signal (SV40pA).B, PCRscreeningofDNAextractedfrom mouse tails for the presenceof integrated human PAD2transgene (TG: hPAD2). Fourfounders were identified (red);primers for mouse Pad4were usedas a WT (mPad4) control foramplification. Primer details can befound in Supplementary Table S1.C, semiquantitative RT-PCR wasperformed on tissues known tohave high expression in MMTV-LTR transgenic mice: skin, salivarygland (SG), mammary gland (MG),and ovary. Relative mRNA levelswere measured for the transgenichuman PAD2, along withendogenous mouse Pad2. MouseGapdh was used as the loadingcontrol. D, qPCR for the humanPAD2 transgene was performedacross the same tissues, usingWT (FVB/N) skin as the reference,with mouse Gapdh normalization(�, P < 0.01).

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Recombinant PAD2 is expressed in the skin and tumortissue of PAD2 transgenic miceTo test whether the observed skin neoplasia in the trans-

genic mice was driven by overexpression of PAD2, we nextinvestigated PAD2 expression levels in these mice by IHC. Weprobed these tissues with an anti-human PAD2 antibody andfound that PAD2 protein was observed in the hyperplasticepidermis, neoplastic islands, and in the hair follicular epithe-lium (Fig. 2C, i). In addition, we observed high PAD2 expressionin the neoplastic epithelium surrounding keratin pearls(Fig. 2C, ii and iii). PAD2 was also expressed in the invasive,budding nests of carcinoma cells, which appeared to invadestromal tissue adjacent to the primary neoplasm (Fig. 2C, iv).To test whether PAD2 overexpression correlated withincreased deimination activity, we next probed sections fromWT skin and transgenic PAD2 lesions with a pan-citrullineantibody that has been previously shown to specifically detectcitrullinated proteins (19, 20). Results show that the skinlesions from MMTV-FLAG-PAD2 mice appear to haveincreased pan-citrulline staining when compared with WTskin (Fig. 2D), with the most intense levels of citrullinationappearing to occur in the hyperplastic regions. In addition, wefound strong nuclear anti-pan-citrulline staining in theselesions (Fig. 2D, inset bottom image), which is in line withour previous studies showing that PAD2-mediated histonedeimination regulates gene expression (9, 10, 14, 21). Becausethe anti-human PAD2 antibody likely cross-reacts with endog-enous mouse PAD2, we also stained these tissues with an anti-FLAG antibody to specifically stain the PAD2 fusion protein.We observed a similar expression pattern within the prolifer-ating basal layers of hyperplastic/neoplastic epidermis, sug-gesting that much of the PAD2 signal originates from over-expression of the transgene (data not shown). We confirmedthis prediction using indirect immunofluorescence to co-stainfor FLAG and PAD2 expression in the skin lesions of transgenicmice (Fig. 3). Both PAD2 and FLAG were seen to colocalize tothe neoplastic epithelial cells (Fig. 3A, i–iv). While PAD2 showsstrong staining in multiple layers of the epidermis, we found

that FLAG-PAD2 expression is slightly more restricted to thebasal layer, which is known to be more stem cell–like andhighly proliferative (22). Interestingly, we also see an apparentslight increase in the proliferative marker, Ki67, within theFLAG staining section of the SCC lesion (Fig. 3B, ii–iv). Thisobservation supports the hypothesis that PAD2 overexpressionin the skin promotes epidermal proliferation, which is furthersupported by the identification of a subset of lesions that arehighly proliferative and characteristic of invasive SCC. We alsonote that control staining with rabbit IgG was negative (Sup-plementary Fig. S2) and that the anti-FLAG antibody did notstain epidermal tissue fromWTmice (Fig. 3C, i–vi) or adjacentnormal skin from transgenic mice (data not shown).

Enhanced inflammation marker expression is observedin the skin of transgenic mice when comparedwith WT skin and with the mammary gland oftransgenic animals

Previous reports have implicated PAD2 in a wide range ofinflammation-associated diseases (23–25), including ourrecent work where we reported that PAD2 appears to promotecancer progression in vitro and in vivo using a xenograft modelof breast cancer (11). Therefore, in an effort to identify poten-tial pathways involved in the progression of MMTV-FLAG-PAD2 skin lesions, we next investigated the expression of asubset of inflammation and invasion-associated genes in thetransgenic PAD2 lesions. Interestingly, the lesions containinghigh levels of transgenic PAD2 (Fig. 4A–C, lesions 2 and 4) alsoexpressed high levels of Ki67, Il6, and the functional murineorthologs for human IL8; Cxcl1 (KC; ref. 26), Cxcl2 (MIP-2;ref. 27), and Cxcl5 (LIX; ref. 28; P < 0.001; Fig. 4D). These lesionsalso expressed high levels of the inflammatory mediator, Cox2(Fig. 4E), which has previously been shown to induce Il6expression (29). In addition, these tumors also displayedelevated levels of the EMT markers, vimentin (Vim) and theE-cadherin repressor, Snail (Snai1, or more commonly referredto as Snail), along with decreased levels of E-cadherin (Cdh1, orhereafter Ecad; P < 0.01) and Gsk3b (Fig. 4E), which has been

Table 1. Incidence of spontaneous skin lesions in MMTV-FLAG-PAD2 mice

MMTV-FLAG-PAD2 WT FVB/Nb

Founder line Lesions/mice Incidence (%) Male/female (%) Lesions/mice Incidence (%) Male/female (%)

4853 4/20 20.0 25/75 1/18 5.6 0/1004860 20/46 43.5 55/45 2/43 4.7 50/504863 9/24 37.5 33/67 0/33 0.0 0/0Total 33/90a 36.7c 45/55 3/94a 3.2 44/56

NOTE: Transgenic (MMTV-FLAG-PAD2) and age-matchedWT FVB/Nmice were observed over a period of 4 to 12 months post-birth,and spontaneous lesions were characterized by gross abnormalities, such as alopecia, multifocal epidermal ulceration (covered inserocellular crust), dysplasia, and thickening of the adjacent epidermis. Data were analyzed using a c2 test with Yates correction forcontinuity), c2 ¼ 30.65, and P � 0.001.aMice were observed over 1 year, with lesions appearing spontaneously between 4 and 12 months.bWT FVB/N mice are from the corresponding founder lines and age-matched to transgenic mice.cc2 analysis (with Yates correction), P � 0.001.






previously shown to be downregulated in SCCs of the tongue(30) and mouse models of skin tumorigenesis (31).

Our finding that PAD2 overexpression in the skin, but notthemammary gland, leads to neoplastic growthwas somewhatsurprising, and we speculated that the lack of tumor growth inthe mammary gland may have been due to a reduced ability of

FLAG-PAD2 to promote inflammatory and/or EMT markerexpression in this tissue. To test this hypothesis, we nextexamined inflammatory/EMT marker expression in skin andmammary gland from WT and transgenic mice. Results showthat, in large part, while PAD2 overexpression enhancedinflammatory marker expression in the skin, similar levels of

Figure 2. Transgenic FLAG-PAD2 expression in the epidermis of mice leads to the development of skin lesions. A, representative gross lesions showingthe skin phenotype, which includes alopecia, multifocal epidermal ulceration often covered in a serocellular crust, and thickening of the adjacentepidermis. B, histologic evaluation of the skin sections by hematoxylin and eosin (H&E) reveals features consistent with invasive SCC. i, nests ofneoplastic cells arising from the epidermis are shown invading the deep dermis and subcutis, with the epidermis overlying the neoplasm extensivelyulcerated. i and ii, adjacent epidermal layers are hyperplastic, with the loss of adnexal structures and increased desmoplastic response. iii, tumorborders are poorly defined, with highly anaplastic tumor cells invading the deeper dermis and subcutis, often infiltrating and separating the skeletalmyofibers. iv, carcinoma cells are frequently found budding off from the primary neoplasm. C, representative IHC analysis of FLAG-PAD2 proteinexpression in skin neoplasms from transgenic mice. i, lower magnification (�4) image showing PAD2 protein expression in the hyperplastic epidermis,neoplastic islands, and hair follicular epithelium. ii and iii, PAD2 protein expression in neoplastic cells surrounding concentric layers of keratin (arrow,keratin pearl). iv, representative image showing high levels of PAD2 protein expression in budding nest of carcinoma cells (broken arrow). D, comparisonof citrulline levels in WT skin epithelium and lesion from FLAG-PAD2 transgenic mouse (TG). Rabbit IgG served as negative control. Magnification forimages: B, i (�4), ii (�10), iii (�20), and iv (�40); C, i (�4), ii (�20), iii (�20), and iv (�40); D, all �5 (inset, �40).

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induction were not observed in the mammary gland (Supple-mentary Fig. S3). These observations supported the hypothesisthat PAD2 overexpression selectively promotes inflammatorymarker expression and tumorigenesis in the skin but not themammary gland. To test this hypothesis, we probed tissuesections with a well-validated anti-IL6 antibody. Resultsshow that protein levels of Il6 appeared to be higher in thetransgenic skin lesions than in the transgenic mammarygland and in WT skin (Fig. 4F). Taken together, thesefindings suggest that, in the skin, PAD2 overexpression mayenhance tumor progression by selectively promoting inflam-mation and by altering the invasive properties of epithelialcells. We also evaluated the effect of PAD2 overexpression oninflammatory and EMT marker expression in the salivarygland and found that EMTmarker expression appeared to bedampened, whereas inflammatory marker gene expressionwas not noticeably affected when compared with skin (Sup-plementary Fig. S3). Interestingly, there does not appear tobe a direct PAD2 gene dosing effect with respect to inflam-matory mediator/EMT expression in the different tumorsand we predict that this discrepancy may be due to opposing

and/or synergistic interactions between PAD2 and other PADfamily members within individual tumors. In support of thisprediction, we found that expression levels of PAD1–4 varieddramatically between tumors (Supplementary Fig. S4B andS4C). While speculative, it is possible that the ratio of PADisozyme expression levels could modulate inflammatory/EMTexpression levels and tumor growth and/or invasiveness tovarying degrees.

Overexpression of PAD2 in human SCC A431 cellsincreases invasiveness and malignancy

Outcomes from our transgenic MMTV-FLAG-PAD2 mousestudy show that ectopic expression of PAD2 is sufficient todrive tumorigenesis in epithelial cells and that this expressioncorrelates with an increase in the expression of markers ofinflammation and invasion. To test whether PAD2might play asimilar role in humans, we next investigated whether PAD2overexpression in the human skin cancer cell line, A431, mightlead to a similar phenotype. This cell line functions as a modelsystem for human SCC and was derived from a vulvularepidermoid carcinoma (32). To investigate the oncogenic

Figure 3. Confocalimmunofluoresence analysis ofPAD2, FLAG, and Ki67 expressionin neoplastic skin lesions of theFLAG-PAD2 transgenic mouse. A,representative image showing co-staining (iv) of FLAG (red, ii) andPAD2 (green, iii) in the neoplasticepithelial cells of an SCC from atransgenic mouse. Nuclei werestained with 40,6-diamidino-2-phenylindole (DAPI; blue, i).Endogenous PAD2 shows strongpositive staining in two to threelayers of epidermal cells, whereasFLAG-PAD2 is predominantlylocalized to the basal cell layer.Scattered tumor cell islandsunderlying the epidermis showhighlevels of PAD2 expression. B,representative image showing co-staining (iv) of the proliferativemarker FLAG (red, ii) and Ki67(green, iii) in neoplastic epithelialcells of SCC. Tumor islands that arestrongly positive for FLAG-PAD2expression levels contain anincreased number of Ki67-positivecells (iv). Nuclei were stained withDAPI (blue, i). Normal skin fromWTmice (FVB/N) is absent of transgeneexpressionandhas low levels of theproliferation marker, Ki67. C,representative image showing co-staining (vi) of FLAG (red, iii) andKi67 (green, iv) in normal skinsections from a WT mouse. Bright-field image (ii and v). Both FLAGandPAD2 protein expression areabsent, concomitant with reducedKi67 levels. Nuclei were stainedwith DAPI (blue, i). Magnification inimagesA–C,�40.Scalebar, 50mm.






Figure 4. Transgene expression in the skin lesions ofMMTV-FLAG-PAD2mice. A, semiquantitative RT-PCRof four representative skin lesions from transgenicmice (lesions 1–4). Relative mRNA levels were measured for the transgenic human PAD2, along with endogenous mouse Pad2. Mouse Gapdh wasused as the loading control. (Continued on the following page.)

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potential of PAD2 in these cells, we first transiently expressedFLAG-PAD2usingmammalian vectors (pcDNA3.1 andpIRES2)and found that these cell lines expressed high levels of theFLAG-PAD2 protein (Fig. 5A and B). In addition, we found thatPAD2 overexpression suppressed E-cadherin and increasedvimentin protein expression when compared with A431 cellsthat had been transfected with an empty vector (Fig. 5A). Wenext tested whether PAD2 overexpression altered the expres-sion of inflammation and EMTmarkers and found that, similarto what was seen in transgenic mice, IL6 and IL8 gene levelswere significantly increased in the PAD2-overexpressing cells,along with the inflammatory mediators COX2 andNFkB/RELA.Moreover, in addition to increasing the proliferation markerKi67, we found that transient PAD2 overexpression alsoincreased SNAIL and SLUG levels while suppressing E-cadherinlevels (P < 0.05; Fig. 5C). Finally, we tested the effects of PAD2overexpression on the expression of two proliferative onco-

genes H-Ras (33, 34) and c-Myc (35), which are known to beupregulated in skin carcinomas. Results show that levels ofthese oncogenes were unchanged in the PAD2-overexpressingcells, suggesting that the observed effects of PAD2 on onco-genesis are more direct in nature.

Given the data from our transiently transfected A431 cells,along with our data from the transgenic mice, we decided toinvestigate whether stable overexpression of FLAG-PAD2might have an effect on the cellular malignancy and/or inva-siveness of A431 cells. Using both lentiviral transduction ofFLAG-PAD2 and traditional transfection, we created twostable PAD2-overexpressing lines: (i) pLenti-FLAG-PAD2(pLenti-FP2), which expresses low levels of FLAG-PAD2 and(ii) pIRES2-FLAG-PAD2 (pIRES-FP2), which expresses highlevels of FLAG-PAD2 (Fig. 6A and B). Next, we tested theinvasive properties of these cell lines bymeasuring their abilitytomigrate through a collagenmatrix. For both cell lines, we see

(Continued.) B, qPCR (TaqMan) for the human PAD2 transgene was performed across the same lesions, using normal WT skin as the reference, alongwith mouse Gapdh normalization (�, P < 0.01). C, Western blot analysis of skin from WT mice, along with transgenic lesions (2 and 4). Whole-cell lysateswere probed for FLAG expression, with a predicted protein size of about 75 kDa. b-Actin was used as a loading control. D and E, skin lesions fromMMTV-FLAG-PAD2 transgenic mice express markers of inflammation (D; �, P < 0.001) and EMT (E; �, P < 0.01). Relative mRNA levels for the representativegenes (see Supplementary Table S2) were determined by qPCR (SYBR) using normal WT skin as the reference, along with b-actin (Actb) normalization. F,representative IHC image of anti-Il6 staining in WT skin (bottom, left), FLAG-PAD2 transgenic (TG) mammary gland (bottom, middle), and TG skin lesion(bottom, right). Rabbit IgG served as negative control. Magnification, �40. Scale bar, 20 mm.

Figure 5. Transient overexpressionof FLAG-PAD2 increases markersof inflammation and EMT in thehuman SCC A431 cell line. A,human SCC cells (A431) weretransiently transfected with FLAG-PAD2 (pcDNA3.1-FP2 or pIRES2-FP2) or empty vector (pcDNA3.1-empty). Western blot analysis ofprotein expression for EMTmarkers, E-cadherin and vimentin,along with PAD2, was performedon transfected A431 cells. b-Actinwas used as a loading control. B,PAD2 levels (�, P < 0.001) for theFLAG-PAD2–transfected A431cells, relative to empty vectorcontrol (ACTB normalized), wereanalyzed by qPCR (SYBR). C,additional genes known to beinvolved in EMT and inflammationwere also analyzed (�, P < 0.05).See Supplementary Table S3 forprimer sequences.






a significant increase in cellular migration after 24 hours(P < 0.01), with the pIRES2-FP2 cell line also showing asignificant increase in migration after only 4 hours (P < 0.01;Fig. 6C). These results suggest that PAD2 dosage might cor-relate with invasion. Finally, we wanted to examine the stableA431-pIRES2-FP2 cells for any increase in cellular malignancy.Assaying for focus formation, we show a significant increase inthe FLAG-PAD2–overexpressing A431 cells compared with theempty vector control (P < 0.001; Fig. 6D). In addition, we findthat the morphology of these cells also displays an elongated,fibroblast-like shape, indicative of cells that have undergoneEMT (Fig. 6E).

DiscussionIn the present study, we demonstrate that transgenic over-

expression of PAD2 in the epidermis promotes carcinomaformation in the skin. In addition, we found that these lesionshave the potential to advance to invasive SCC. These in vivofindings were supported by our in vitro studies, which showedthat stable overexpression of PAD2 in the human SCC line,A431, resulted in increased cellular invasiveness andmalignan-cy. At amoremechanistic level, we also demonstrate that PAD2may promote carcinogenesis by enhancing the production offactors that promote inflammation and an EMT phenotype.

Figure 6. A431 cells stablyoverexpressing PAD2 are moretumorigenic in vitro. A431 cellswere stably transfected withpIRES2-FLAG-PAD2 (pIRES2-FP2) or infected with lentivirusexpressing FLAG-PAD2-EGFP(pLenti-FP2). Control vectors onlyexpress the EGFP gene (pIRES2-GFP and pLenti-GFP). A, Westernblot analysis of PAD2 proteinexpression levels. b-Actin wasused as a loading control. B, PAD2expression in FLAG-PAD2–transfected A431 cells wasanalyzed by qPCR (SYBR). GFP-only vector was used as a control(ACTB normalized). Expressionlevels were analyzed using the2 �DDC(t) method, and dataexpressed as the mean � SD fromthree independent experiments(�, P < 0.001). C, Transwellmigration assay through a collagenmatrix—cells were seeded andallowed to migrate through acollagenmatrix. After 4 or 24 hours,migrating cells were counted underamicroscope at�40magnification(�, P < 0.01). D and E, A431 skincancer cells overexpressing FLAG-PAD2 show increased malignancyand EMT morphology. D, focusformation assay. Cells stablyoverexpressing FLAG-PAD2(A431-FP2) or EGFP control(A431-GFP) were grown for 4 daysin a 6-well plate, fixed with 4%paraformaldehyde, and stainedwith crystal violet for subsequentfocus formation analysis.Absorbance levels were measuredat 600 nm (�, P < 0.001). E,representative morphology ofA431 cells overexpressingFLAG-PAD2.

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A number of mouse models, including models of advancedSCC, have documented a role for inflammation in cancerprogression (36). In humans, the links between inflammationand cancer are now so strong that inflammation is consideredto be the seventh hallmark of cancer (36, 37). Two key med-iators of inflammation-driven cancers are the cytokines, IL6and IL8, which promote cancer progression by stimulatingtumor growth and angiogenesis (38–48). Interestingly, a num-ber of previous studies have documented a role for PAD2-mediated deimination in regulating inflammatory activitiesduring disease progression (23, 24). In addition, PAD activityhas also been found to regulate tissue inflammation via directmodification of IL8 (25). Given these observations, we hypoth-esized that PAD2 may promote tumorigenesis by regulatingthe expression of IL6 and IL8. We tested this hypothesis andfound that Il6 and Il8 ortholog expression is, in fact, increasedin the skin lesions of PAD2-overexpressing mice and that IL6and IL8 expression is elevated in PAD2-overexpressing cells.In this study, we also show that PAD2 overexpression in

transgenic mice, in addition to human cell lines, increasedmarkers of invasiveness and EMT. An early critical molecularfeature of EMT is the downregulation of E-cadherin (Cdh1), acell adhesion molecule present in the plasma membrane ofmost normal epithelial cells. As cells progresses from anepithelial-like to mesenchymal-like state, E-cadherin down-regulation is accompanied by the increased expression of Snail(Snai1) and the intermediate filament vimentin (Vim)(reviewed by Kaluri and colleagues; ref. 49). In our study, wefound that PAD2 overexpression in mice induced an EMT-likephenotype in the resulting tumor cells as evidenced by thereduction in E-cadherin expression, along with an increase inSnail and vimentin expression. This molecular characteriza-tion of an EMT transition matches well with the invasivehistology of the PAD2-overexpressing lesions.A number of previous studies have shown that inflammatory

cytokines, such as IL8, appear to promote cancer progressionby inducing an EMT in tumor cells (41). Thus, it is possible thatPAD2 overexpression promotes cell invasiveness and an EMTphenotype by upregulating cytokine production in tumor cells.Interestingly, however, we recently reported that another PAD,PAD4, plays an important role in regulating an EMT in breastcancer cells via regulation of GSK3b–TGFb signaling (50). Inthat study, we found that PAD4 targetsGSK3b for citrullinationand that PAD4 depletion dramatically reduces levels of nuclearGSK3b, promotes EMT, and increases tumor invasiveness. Inthe current study, we found that overexpression of PAD2suppressed Gsk3b expression, promoted an EMT phenotypeand tumor growth. Together, these results raise the possibilitythat PAD2 andPAD4mayoppose or counterbalance each otherin the maintenance of epithelial identity and in oncogenesis.A surprising outcome of our project was that neoplastic

growth appeared to be limited to the skin of PAD2 transgenicmice and was not observed in the mammary glands. While theultimate explanation for this outcome is likely manifold, wefound that the PAD2-mediated induction of inflammatorymediator expression was suppressed in the mammary glandsof transgenic mice when compared with both the normal skinand the hyperplastic lesions from transgenic mice. These

results suggest that, as opposed to the skin, a dampeningmechanism may exist within the mammary gland that pre-vents FLAG-PAD2 from inducing inflammatory cytokineexpression, thereby suppressing tumorigenesis. A potentialexplanation for this dampening effect may be derived fromour finding that FLAG-PAD2 overexpression in vivo appears tosignificantly modulate the expression of other PAD familymembers in both normal and tumor tissue (SupplementaryFig. S4). Therefore, while purely speculative, it is possible thatthe relatively low levels of FLAG-PAD2 that we observed in theskin promotes the expression of a specific ratio of PAD familymembers that is conducive to inflammation and tumorigen-esis, whereas the relatively higher level of FLAG-PAD2 in themammary gland alters this ratio, leading to reduced inflam-matory mediator expression and an absence of neoplasticgrowth. Regarding the potential mechanisms by which PAD2overexpression regulates gene activity, we have recently foundthat PAD2 functions as a transcription factor co-factor andmodulates target gene expression via histone deimination attranscription factor–binding sites (9, 10, 14, 21). Therefore, weare currently testing the hypothesis that PAD2 regulates theexpression of target genes via similar mechanisms.

In conclusion, this study demonstrates that overexpressionof human PAD2 in mice and in human cell lines promotescarcinogenesis. Furthermore, we show that a subset of tumorsin mice display hallmarks of malignant progression from skinlesions to highly invasive SCCs. In addition, we demonstratethat PAD2 overexpression in vivo and in vitro enhances theexpression of inflammatory cytokines and mediators of theEMT. Collectively, these studies provide functional and mech-anistic evidence establishing PAD2 as a potential novel onco-gene in the initiation and progression of epidermalcarcinomas.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.L. McElwee, S. Mohanan, S. Horibata, S.A. CoonrodDevelopment of methodology: J.L. McElwee, S. Mohanan, S. Horibata,L.J. Anguish, S.A. CoonrodAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.L. McElwee, S. Mohanan, S. Horibata, K.L. Sams,L.J. Anguish, D. McLean, S.A. CoonrodAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.L. McElwee, S. Mohanan, S. Horibata, L.J. Anguish,D. McLean, J.J. Wakshlag, S.A. CoonrodWriting, review, and/or revision of the manuscript: J.L. McElwee,S. Mohanan, J.J. Wakshlag, S.A. CoonrodAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Horibata, K.L. SamsStudy supervision: J.L. McElweeOther (technical and veterinary assistance in vivo and in vitro experi-ments): I. Cvita�s

Grant SupportThis work was supported in part by funding through the DOD Era of Hope

Award W871XWH-07-1-0372 to S.A. Coonrod and through an NIH GraduateFellowship (grant T32HD057854) to J.L. McElwee.

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

Received March 14, 2014; revised July 23, 2014; accepted August 19, 2014;published OnlineFirst September 11, 2014.






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2014;74:6306-6317. Published OnlineFirst September 11, 2014.Cancer Res John L. McElwee, Sunish Mohanan, Sachi Horibata, et al. Skin NeoplasiaPAD2 Overexpression in Transgenic Mice Promotes Spontaneous

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