tpl2 is an oncogenic driver in keratocanthoma and ......jun-han lee1, joo-hyung lee1, sang hyuk...

Tumor and Stem Cell Biology

TPL2 Is an Oncogenic Driver in Keratocanthomaand Squamous Cell CarcinomaJun-Han Lee1, Joo-Hyung Lee1, Sang Hyuk Lee2, Sung-Im Do2, Sung-Dae Cho3,Ola Forslund4, Kyung-Soo Inn5, Jeong-Sang Lee6, Fang-Ming Deng7,Jonathan Melamed7, Jae U. Jung8, and Joseph H. Jeong1

Abstract

Squamous cell carcinoma (SCC) and keratoacanthoma (KA;SCC/KA) research has been hampered mainly by our lack ofunderstanding the underlying genetic and epigenetic altera-tions associated with SCC/KA development, as well as the lackof animal models that faithfully recapitulate histopathologicfeatures of human SCC/KA. Here, we show that TPL2 over-expression induced both cell transformation in immortalizedhuman keratinocytes and SCC and KA-like cutaneous SCC(cSCC) development in mice. Mechanistically, activation ofTPL2 downstream signaling pathways such as MEK/ERK MAPK,

mTOR, NF-kB, and p38 MAPK leads to TPL2-mediated celltransformation in immortalized human keratinocytes andtumorigenesis in mice. Most importantly, TPL2 overexpressionis required for iTPL2 TG–driven SCC and KA-like cSCC tumormaintenance, validating TPL2 as a possible drug target for thetreatment of SCC/KA. Finally, we verified that TPL2 is over-expressed in human cutaneous metastatic SCC and KA clinicalspecimens compared with normal skin. Taken together, ourresults establish TPL2 as an oncogenic driver in SCC/KA devel-opment. Cancer Res; 76(22); 6712–22. �2016 AACR.

IntroductionSquamous cell carcinoma (SCC) is the second most common

type of skin cancer with an estimated 700,000 new annual cases(1).Most SCCcases are benign tumors, but about 20%of themareaggressively invasive and metastatic, in particular to the lymphnodes (2, 3).

Although it resembles SCC, keratoacanthoma (KA) is a benignsquamous cell proliferation with unique clinical features of rapidgrowth and spontaneous regression (2, 3). KA itself is commonand a considerable amount of dermatologists' time and effort isdevoted to the diagnosis and treatment ofKA. Because there are noclear criteria to differentiate KA from SCC, KA is referred to as"squamous cell carcinoma, keratoacanthoma-type" (SCC, KA-type; refs. 2, 3). Interestingly, "SCC, KA-type" develops in more

than 30% of melanoma patients treated with vemurafenib, aBRAF (V600E) inhibitor, making "SCC, KA-type" one of the mostcommon side effects of this agent (4). Recently, a study identifieda genetic alteration, HRAS (Q61L) mutation, as the causativemolecular mechanism for the development of "SCC, KA-type"in patients treated with BRAF inhibitors (5).

Many genetic and epigenetic alterations and dysregulated sig-naling pathways associated with SCC initiation and progressionhave been identified, including the downregulation of p53 andNotch 1 and the upregulation of EGFR, FYN,MYC, ATF-3, STAT-3,andRAS (6–15). In particular, these genetic and epigenetic factorsappear to be disturbed by UV exposure, a well-known environ-mental factor for the development of cSCC. Recently, accumu-lating results suggest that tumor progression locus 2 (TPL2) playsa role in promoting cSCCdevelopment (16, 17).However, severalother studies have also reported increased cSCC development inTPL2-deficient mice using the two-stage skin carcinogenesis mod-el (18, 19), so TPL2 appears to have double-sided functions incSCC development, tumor promotion versus tumor suppression.

TPL2 is a serine/threonine MAP kinase kinase kinase 8(MAP3K8), and regulates diverse signaling pathways associatedwith inflammation and cell growth. TPL2 was also called "COT"(cancer Osaka thyroid) because it was initially cloned as a trans-forming kinase gene from a human thyroid carcinoma cell linewith a deletion of its C-terminal region (20). Structurally, thecentral TPL2 kinase domain is flanked by the N-terminal and C-terminal regions (21). In particular, the C-terminus plays aninhibitory role on its own kinase activity and is also associatedwith direct interaction of TPL2 with its negative regulator, the NF-kB-inhibitory protein NF-kB1 (p105; refs. 22–24). Therefore, theC-terminal–deleted TPL2 has a higher specific kinase activity thanwild-type TPL2 (TPL2 wt) and cannot be sequestered by itsnegative regulator NF-kB1 (p105). In this regard, for TPL2 wt tobe activated, it firstly should be released from NF-kB1 (p105)–mediated negative sequestration, either by overexpressing TPL2

1Department of Urology, Dermatology, and Biochemistry, MedicalCollege of Wisconsin, Milwaukee, Wisconsin. 2Kangbuk SamsungHospital, Sungkyunkwan University School of Medicine, Seoul, Korea.3Department of Oral Pathology, School of Dentistry and Institute ofOral Bioscience,ChonbukNational University, Jeonju, Korea. 4Depart-ment of Medical Microbiology, Lund University, Malm€o, Sweden.5Department of Pharmaceutical Science, College of Pharmacy, KyungHee University, Seoul, Korea. 6Food Industry Research Institute,Department of Bio and Functional Food, College of Medical Science,JeonjuUniversity, Jeonju, Korea. 7Department of Pathology, NewYorkUniversity LangoneMedicalCenter,NewYork,NewYork. 8Departmentof Molecular Microbiology and Immunology, Keck School of Medicine,University of Southern California, Los Angeles, California.

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

Corresponding Author: Joseph H. Jeong, Laboratory of Developmental Biologyand Genomics, College of Veterinary Medicine, Seoul National University andKorea Mouse Phenotyping Center, Seoul, Republic of Korea, 08826. Phone:82-02-885-8397; Fax: 82-02-885-8397; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-3274

�2016 American Association for Cancer Research.

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wt to overcome the threshold of inhibitory binding of NF-kB1(p105) or by activating signaling pathways, including IKKcomplex, to break the inhibitory interaction (25, 26). Then,the released inactive TPL2 proteins are transformed into anactive form by additional phosphorylation (27). Consequently,the released and activated TPL2 induces ERK, JNK, p38, and NF-kB signaling pathways in both stimulus- and cell type–specificmanners (28).

Functionally, TLP2 plays important roles in both the innateimmune system and the adaptive immune system. TPL2 is acti-vated by diverse proinflammatory stimuli, including bacteriallipopolysaccharides (LPS), TNFa, cluster of differentiation 40(CD40), and IL1 beta (IL1b) to induce innate immune responses(29–35). However, under certain circumstances, TPL2 also inhi-bits innate immune responses by suppressing the production ofproinflammatory cytokines as demonstrated in TPL2 knockoutmouse studies (36–39). In the adaptive immune system, TPL2activation either positively or negatively contributes to severaldiseases by modulating functions of B and T lymphocytes in astimuli-specific manner. For example, TPL2 ablation either ame-liorated TNF-inducedCrohn's-like inflammatory bowel disease orpromoted intestinal inflammation and polyp development in theApcmin mouse model (39, 40). Recently, it was also shown thatTPL2 plays an important role in an autoimmune disease. TPL2knockout mice were refractory to experimental autoimmuneencephalomyelitis (EAE) induction by inhibiting the IL17-medi-ated signaling pathway (41, 42).

The increased level of TPL2 in many different types of humancancers, including breast cancer, prostate cancer, and lymphoma,also suggests that TPL2 plays important roles in tumorigenesis ofvarious tumor types (43–45). Here, to investigate the possibleroles of TPL2 overexpression in tumorigenesis in vivo, we gener-ated a genetically engineered mouse (GEM) model expressingeither TPL2 wt or a constitutively activated form of TPL2 (TPL2DC) in all tissues in an induciblemanner as an unbiased study. Toour surprise, these mice developed SCC and KA-like cSCC on theskin,mouth, paws, and the genital area. In addition,we confirmedthe clinical relevance of TPL2 overexpression in human SCC/KAspecimens.

These data indicate that TPL2 overexpression is another caus-ative molecular mechanism for the development of SCC/KA. Inthe future, this model will serve as an in vivo model system toevaluate TPL2 as a target for future therapeutic interventions andto evaluate the therapeutic efficacy of TPL2 inhibitors for thetreatment of this disease. Finally, we carefully suggest that thisstudy lays the groundwork for combinational therapeutic inter-vention strategies that target both the oncogenic BRAF (V600E)mutant and TPL2 to treatmelanoma and to prevent the unwantedside effect of "SCC, KA-type" development in the patients withmelanoma.

Materials and MethodsCell lines

To establish stable cell lines overexpressing either full-lengthTPL2wt or a kinase-inactive form of TPL2with a substitution of Dto A at TPL2 270 a.a. (TPL2-IN) in the pBabe-hygro retroviralvector, we used previously immortalized human keratinocytesectopically overexpressing two proteins, CDK4 and hTERT [Ker-CT; this cell line was initially established, authenticated, andkindly provided (in 2013) by Dr. Jerry W. Shay at the University

of Texas Southwestern Medical Center, Dallas, TX; ref. 46]. Formore detailed information on virus production, please checkSupplementary Material.

Cell growth/viability assayCells were trypsinized, counted, and plated at 3� 103 cells per

well in a 96-well tissue culture plate in 4 replicates. Photomicro-graphs were taken every 2 hours using the Incucyte Live CellImager (Essen Bioscience), and confluence of the cultures wasmeasuredusing Incucyte software (EssenBioscience) for 66hours.The number of dead cell was counted via YoYo-1 (Invitrogen)staining using an Incucyte live cell imager. The numbers ofabsolute and relative viable cells were determined via VybrantDye Cycle Green (Invitrogen) staining using Incucyte software at72hours. Cytotoxic indexwas determined as the number of YoYo-1–positive objects divided by the total number of DNA-contain-ing objects. For experiments using inhibitors, 10 or 20 mmol/LU0126 (Cell Signaling Technology), 1 or 5 mmol/L SB203580(Cell Signaling Technology), and 20 or 100 hmol/L rapamycin(Cell Signaling Technology)were added into eachwell at 24hoursafter the cell plating, and cell confluences were measured by theIncucyte software for 40 hours.

Western blotProteins were extracted from cells by incubating RIPA (Milli-

pore) supplemented with protease inhibitor (Roche) and phos-phatase inhibitor (Sigma) for 30 minutes. The lysate was soni-cated for 10 seconds on ice and centrifuged at 4�C for 10 minutesat 14,000 rpm. The supernatant was taken and used to measureprotein concentration with the Pierce BCA Protein Assay Kit(Thermo Scientific). Proteins (20 mg) were resolved by SDS-PAGEand transferred to PVDF membrane (Bio-Rad). Membranes wereblockedwith 5% skimmilk (Bio-Rad) in Tris-Buffered SalinewithTween 20 for 1 hour, and incubated with primary antibody at 4�Covernight. Primary antibodieswerepurchased fromCell SignalingTechnology (Erk1/2, cat. #4695; P-ERK, cat. #4370; p38, cat.#9212; P-p38, cat. #9211; p70S6K, cat. #9202; P-p70S6K, cat.#9205; P-p65, cat. #3033; JNK, cat. #9258; P-JNK, cat. #4669; P-MK2, cat. #3316; and MK2, cat. #3042) and Santa Cruz Biotech-nology (b-actin, cat. #sc-47778; p65, cat. #sc-372; AKT1/2/3, cat.#sc-8312; and P-AKT1/2/3, cat. #sc-33437).

Histology and immunohistochemistryAll the tissue sampleswerefixed in 10% formalin for 2 days and

embedded in paraffin. For antigen retrieval, slides were heated in0.01 mol/L citrate buffer (pH 6.4) in the microwave, withoutboiling the samples, four times at 4 minutes each time. Theantibodies and dilutions are: TPL2, 1:200 (cat. #sc-720, SantaCruz Biotechnology); TPL2 Nt, 1:200 (we generated this TPL2antibody through the GenScript); TPL2 Cen, 1:200 (we generatedthis TPL2 antibody through the GenScript); TPL2 Ct, 1:200 (wegenerated this TPL2 antibody through the GenScript); P-ERK,1:250 (cat. #4695, Cell Signaling Technology); P-p38, 1:100 (cat.# 9211, Cell Signaling Technology); Ki-67, 1:2,000 (cat. #15580,Abcam). Apoptotic cell death was detected using the ApopTagPlus Peroxidase In Situ Apoptosis Detection Kit (EMDMillipore).We purchased a tissuemicroarray (TMA) composed of 80 cases oflymph node metastatic SCC (LY802; US Biomax Inc.) and a TMAcomposed of 40 cases of SCC and 8 cases of normal human skin(SK483; US Biomax Inc.). TMAs containing 64 KA cases wereobtained from Lund University, Lund, Sweden (Dr. Ola

TPL2 Is an Oncogenic Driver for SCC and KA-Like cSCC

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Forslund). The staining was reviewed and the expression of TPL2was scored for intensity and proportion by Drs. Fang-Ming Dengand Jonathan Melamed at NYU and Dr. Sung-Im Do at theKangbuk Samsung Hospital.

Foci formation assayKer-CT stable cells expressing either TPL2-WT or TPL2-IN,

along with vector control cells, were seeded at 6 � 104 cells perwell in 6-well tissue culture plate. Media were changed every2 days for 4 weeks. Cells were fixed with 10% formalin andstained with Giemsa staining solution (EMD Millipore) atroom temperature for 5 minutes. After Giemsa staining, plateswere washed with running water. Plates were dried at roomtemperature overnight. Images of foci were taken by a Bio-RadMolecular Imager ChemiDoc XRSþ.

Generation of inducible TPL2 transgenic (iTPL2 TG) miceAll animal experimental protocols were approved by the

Medical College of Wisconsin (MCW) Committee for AnimalWelfare and by the Institutional Animal Care and Use Com-mittees of MCW (AUA#5031). Also, all animal experimentswere performed in accordance with guidelines and regulationsof the approved animal protocol (AUA#5031) under the super-vision of the MCW Committee for Animal Welfare and theInstitutional Animal Care and Use Committees of MCW. Aschematic diagram of DNA constructs used for generatinginducible TPL2 transgenic mice (Supplementary Fig. S1A).N-terminal HA-tagged either wild-type TPL2 (HA-TPL2 wt) ora constitutively activated form of TPL2 with a deletion of 70amino acids in C-terminus (HA-TPL2 DC) was cloned intopTRE-Tight vector (Clontech). The constructs were injectedinto oocytes derived from B6D2F1 mice at the USC NorrisCancer Center Transgenic Core Facility. A total of 8 founders,including 5 independent founders for HA-TPL2 wt (#831,#998, #982, #100, and #102) and 3 independent founders forHA-TPL2 DC (#316, #876, and #205), were generated (Sup-plementary Fig. S1C). Each founder was maintained by back-crossing to B6 (inbred strain) so each subsequent generationhad more and more B6 genomic DNA and less DBA2 (D2)genomic DNA. We crossed these TPL2 transgenic mice (2nd to4th generations of backcrossing) with the ROSA26-M2 rtTAmice (the B6.Cg-Gt(ROSA)26Sortm1(rtTA�M2)Jae/J strainfrom the Jackson Laboratory) in order to express transgenesin all mouse tissues. Compound mice were administered doxy-cycline [2 g doxycycline (BioWorld) in 1 L water, along with 4 gSplenda] in drinking water, just after being weaned at the age of3 weeks.

TPL2-MEK1 kinase assaysNormal mouse skin samples or tumor samples from iTPL2 wt

TGmice (ONDOX)were lysed in kinase lysis buffer [1%Triton X-100, 10 mmol/L b-glycerophosphate, 50 mmol/L Tris (pH 7.5),150 mmol/L sodium chloride, 1 mmol/L EDTA, 5 mmol/Lsodium pyrophosphate, 1 mmol/L sodium orthovanadate, 100nmol/L okadaic acid, 50 mmol/L sodium fluoride, and 0.1%b-mercaptoethanol] containing complete protease inhibitor(Roche). To remove NF-kB1/p105 in both lysates, eitherp105C antibody (provided byDr. Steven C. Ley) or normal rabbitcontrol IgG (Santa Cruz Biotechnology, cat. # sc-2027) was usedfor preclearing, and protein amounts were measured using thePierce BCA Protein assay Kit. For immunoprecipitation, the

lysateswere incubatedwith goat polyclonal TPL2 antibody (SantaCruz Biotechnology, cat. # sc-1717) at 4�C for 4 hours to pulldown endogenous TPL2. After incubation, 25 mL of protein A/Gbeads (Santa Cruz Biotechnology) were added, and the mixtureswere incubated at 4�C for 3 hours. TPL2-immune complexeswere washed four times in kinase lysis buffer and twice in kinasebuffer (50 mmol/L Tris, pH 7.5, 5 mmol/L b-glycerophosphate,100 nmol/L okadaic acid, 1 mmol/L DTT, 10 mmol/L MnCl2,and 0.03% Brij35). Beads were re-suspended in a total volumeof 50 mL kinase buffer supplemented with 1 mmol/L ATP and500ngof recombinantMEK1 (inactive) substrate (Invitrogen; cat.# p3093), and kinase reactions were performed for 30 minutes at30�C. Then, precipitated TPL2 protein was eluted from the beadscomplexes using 0.2 mol/L glycine (pH 2.5). Both kinasereaction supernatant and eluted TPL2 protein were subjectedto Western Blot. TPL2, p105, phospho-MEK1 (inactive), andMEK1 (inactive) were assessed by Western blot using 70-merTPL2 (provided by Dr. Steven C. Ley), p105 (Cell SignalingTechnology, cat. # 13586), phospho–MEK1/2 (Cell SignalingTechnology, cat. #2338), and MEK1/2 (Cell Signaling Technol-ogy, cat. #4694) antibodies, respectively.

ResultsiTPL2 TG mice develop SCC and KA-like cSCC

To investigate the roles of TPL2 overexpression in tumorigen-esis in vivo, we generated a GEMmodel. In these mice, N-terminalHA-tagged TPL2wt or a constitutively activated form of TPL2witha deletion of 70 amino acids inC-terminus (TPL2DC) is expressedunder the control of a modified Tet response element (TRE)–responsive promoter through the activation of the reverse tetra-cycline-controlled transactivator (rtTA) in a doxycycline (DOX)-dependent manner (Supplementary Fig. S1A–S1C). We crossedthese TPL2 transgenicmicewithROSA26-M2 rtTAmice inorder toexpress transgenes in all mouse tissues. The compound mice ofiTPL2 wt (#100) [TPL2 wt (line #100)::ROSA26-M2-rtTA] (n ¼14), iTPL2 wt (#102) [TPL2 wt (line #102)::ROSA26-M2-rtTA] (n¼ 8), and iTPL2 DC (#205) [TPL2 DC (line #205)::ROSA26-M2-rtTA] (n ¼ 20) developed cutaneous lesions on the dorsal andventral skin [11/14 mice for iTPL2 wt (#100), 14/20 mice foriTPL2 DC (#205), and 2/8 mice for iTPL2 wt (#102)], the mouth[2/14mice for iTPL2 wt (#100), 15/20mice for iTPL2 DC (#205),and 4/8 mice for iTPL2 wt (#102)], the paw [2/14 mice for iTPL2wt (#100), 7/20mice for iTPL2DC (#205), and 7/8mice for iTPL2wt (#102)], and the genital area [0/14mice for iTPL2wt (#100), 8/20 mice for iTPL2 DC (#205), and 0/8 mice for iTPL2 wt (#102)]only after DOX administration in the drinking water (ON DOX)with 100%penetration (Fig. 1A, i–iv and Supplementary Fig. S2).No lesions were observed in compound mice without DOXadministration (OFF DOX; Supplementary Fig. S2). The averagelatency for skin lesion development ONDOX was approximately82 days, 81 days, and 46 days for iTPL2 wt (#100), iTPL2 wt(#102), and iTPL2 DC (#205), respectively (Supplementary Fig.S2). iTPL2DC(#205)miceONDOXdeveloped cutaneous lesionswith significantly decreased average latency in comparison withiTPL2 wt (#100) and iTPL2 wt (#102) mice [Log-rank test: �P ¼0.0018 for iTPL2 DC (#205) ON DOX vs. iTPL2 wt (#100) ONDOX and �P¼ 0.0493 for iTPL2 DC (#205) ONDOX vs. iTPL2 wt(#102) ON DOX; Supplementary Fig. S2].

Histologically, the cutaneous lesions showed papilloma-likestructures with extensive keratinization, moderate to severe

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atypical epidermal hyperplasia with occasional focal areas ofpearl formation, and indications of early invasion (Fig. 1A, v–xii). Therefore, these lesions are representative of early epider-moid carcinoma, grade 1. In particular, some of the cutaneouslesions on the dorsal and ventral skin are likely to be KA,presenting characteristic exo-endophytic lesions with severalcentral keratotic horns. However, these iTPL2 TG–driven KA-likelesions did not spontaneously regress unlike the spontaneousregression observed in approximately 50% of human KA cases.Thus, based on these histopathologic characteristics of iTPL2TG–driven cutaneous lesions on the dorsal and ventral skin, wedefined the lesions as early-stage cutaneous SCC with KA-likehistologic features, but without spontaneous resolution, referredto as KA-like cutaneous SCC (KA-like cSCC). The other lesionsdeveloped on the mouth, on the paw, and on the genital area of

iTPL2 TG mice ON DOX are defined as early-stage SCC withextensive keratinization. Most of the other tissues, includingliver, gallbladder, central nervous system, colon, kidney, spleen,intestine, stomach, pancreas, bladder, and salivary gland, in thecompound mice ON DOX showed no remarkable abnormalities(data not shown).

Next, to determine if these observedphenotypes are truly due totransgene TPL2 expression, we examined the expression of TPL2transgenes in iTPL2 TG–driven SCC and KA-like cSCCONDOX atthe protein level using immunohistochemistry (IHC). IHC anal-ysis revealed high levels of transgene TPL2 expression only intumor cells of iTPL2 TG–driven SCC and KA-like cSCC ON DOX(Fig. 1B, TPL2 and Supplementary Fig. S8).We also confirmed thetransgene expression in tumor cells of iTPL2 TG–driven SCC andKA-like cSCCONDOX using three different TPL2 antibodies that

Figure 1.

iTPL2 TGmice develop SCC and KA-likecSCC. A, gross photos of SCC (i,mouth;iii, penis; iv, paw) and KA-like cSCC (ii,skin horn) developed in iTPL2 TG micewith doxycycline administration to turnon transgene expression (ONDOX; top)and their histologic examinations usinghematoxylin and eosin staining[original magnification, �40 (middle)and �200 (bottom): v and ix, mouth;vi and x, skin horn; vii and xi, penis;viii and xii, paw). Scale bars, 300 mmfor v to viii and 100 mm forix to xii. B, representative photos forhistologic and IHC analyses of an iTPL2TGmouse-drivenKA-like cSCCONDOXand wild-type mouse normal skin.Hematoxylin and eosin (H&E) staining(original magnification, �100 and�400) and IHC staining for transgeneexpression (TPL2), TPL2 downstreamsignaling molecules [phospho-ERK,P-ERK; NF-kB1 p105 (cytoplasmiclocalization)/p50 (nuclear localization);and phospho-p38, P-p38], mTORpathway (phospho-S6 ribosomalprotein, P-S6), and a cell proliferationmarker (Ki-67). Scale bars in blue are200 mm and those in black are 50 mm.






we generated (Supplementary Fig. S3B), as well as a HA-tagantibody (Supplementary Fig. S3A). Therefore, it appears thattransgene TPL2 proteins are more stabilized in tumor cells ofiTPL2-driven SCC and KA-like cSCC than cells in the other tissuesdespite the ubiquitous transcriptional activity of the ROSA26locus. To confirm the functional integrity of transgene TPL2protein expressed in the tumors, we examined the activationof TPL2 downstream signalingmolecules, such asMEK/ERK, JNK,p38, and NF-kB. The tumors showed strong activations of theMEK/ERK pathway (evaluated by IHC for phospho-ERK staining;Fig. 1B, P-ERK and Supplementary Fig. S8), the p38 pathway(evaluated by IHC for phospho-p38 staining; Fig. 1B, P-p38 andSupplementary Fig. S8), and the NF-kB pathway (evaluated byIHC for nuclear localization of NF-kB1/p50; Fig. 1B, NF-kB1p105/p50 and Supplementary Fig. S8). Of note, NF-kB1/p105 islocalized in the cytoplasm in resting cells, while upon stimulationby proinflammatory signals for the activation of NF-kB pathway,NF-kB1/p105 is degraded to NF-kB1/p50with consequent nucle-ar localization of the NF-kB1/p50. We could not detect theactivation of the JNK pathway in the tumors (data not shown).Interestingly, we detected activation of the mTOR pathway (eval-uated by IHC for phospho-S6 ribosomal protein staining; Fig. 1B,P-S6 and Supplementary Fig. S8) in the tumors. In addition,we confirmed the expression of transgene TPL2 (both TPL2 wtin iTPL2 wt TG–driven cSCC and TPL2 DC in iTPL2 DC TG-drivencSCC) and the activation of its downstream signaling pathwaysusing Western blot (Supplementary Fig. S5). As a functionalconsequence of transgene TPL2 overexpression, these tumorsshowed strong cell proliferation (evaluated by IHC for Ki67staining; Fig. 1B, Ki-67 and Supplementary Fig. S8). Taken togeth-er, these observations indicate that TPL2 activation by overexpres-sing either wild-type or a constitutively activated form of TPL2 issufficient for the development of SCC and KA-like cSCC in mice.

TPL2 overexpression is required for iTPL2 TG–driven tumormaintenance

To determine whether constitutive TPL2 activation by over-expressing either TPL2 wt or TPL2 DC is required for the main-tenance of iTPL2 TG–driven tumors, we turned off the expres-sion of TPL2 transgene in tumor-bearingmice [n¼5 for iTPL2wt(#100), n ¼ 4 for iTPL2 wt (#102), and n ¼ 5 for iTPL2 DC(#205)] by withdrawing doxycycline from the drinking water(OFF DOX). Ten days after OFF DOX, tumors remarkablyregressed (Fig. 2A, i–ii and v–vi). When the recovered mice wereadministered doxycycline (ONDOX) again, themice developedtumors again (Fig. 2A, iii and vii). We repeated these ON DOXandOFFDOX cycles twice, and the tumor growth and regressionresponded to the ON andOFFDOX cycles every time (Fig. 2A, ivand viii). We confirmed significantly decreased cell proliferation(Fig. 2B, Ki-67) and increased apoptotic cell death in the tumorsOFF DOX in comparison with iTPL2 TG–driven tumors ONDOX (Fig. 2B, TUNEL), as the expression of TPL2 transgene (Fig.2B, TPL2) and the activation of TPL2 downstream signalingmolecules of ERK (Fig. 2B, P-ERK), p38 (Fig. 2B, P-p38), NF-kB(Fig. 2B, nuclear localization of NF-kB1/p50), and mTOR (Fig.2B, P-S6) in the iTPL2 TG–driven tumors OFF DOX significantlydecreased at 10 days after OFF DOX (Fig. 2B). Thus, theseobservations indicate that TPL2 overexpression is required forthe maintenance of established iTPL2 TG–driven tumors, sug-gesting that TPL2 may serve as a therapeutic intervention targetfor the treatment of SCC/KA.

TPL2 overexpression transforms immortalized humankeratinocytes through activation of ERK MAPK, mTOR, NF-kB,and p38 MAPK pathways

To investigate the molecular mechanisms underlying TPL2-mediated SCC and KA-like cSCC development in iTPL2TG mice, we established stable cell lines with immortalizedhuman keratinocytes, expressing either TPL2 wt or a kinase-inactive form of TPL2 (TPL2-IN). The immortalized humankeratinocytes were previously established by ectopically over-expressing two proteins, CDK4 and hTERT (46). The effects ofTPL2 overexpression in immortalized human keratinocyteson cell growth, foci formation (cell growth without cell-to-cellcontact inhibition), and cytotoxicity were analyzed (Fig. 3).Overexpression of TPL2-WT in the cells significantly increasedcell growth in comparison with vector control cells, whereasTPL2-IN overexpression significantly suppressed cell growth(Fig. 3A). In addition, only the stable cells overexpressingTPL2-WT showed significantly decreased cell death (Fig. 3B)and foci formation in comparison with both the vector controlcells and the stable cells overexpressing TPL2-IN (Fig. 3C).Therefore, the overexpression of TPL2-WT alone is sufficientfor the cell transformation of immortalized human keratino-cytes. Next, to identify TPL2 downstream signaling pathwaysthat contribute to the TPL2-WT–mediated cell transformation,we assessed the activation of TPL2 downstream signaling path-ways in the cells using Western blot. Overexpression of TPL2-WT in the immortalized cells induced activation of MEK/ERKMAPK (evaluated by phospho-ERK, P-ERK), mTOR (evaluatedby phospho-p70S6K, P-p70S6K), NF-kB (evaluated by phos-pho-p65, P-p65), and p38 MAPK (evaluated by phospho-p38,P-p38) pathways without any changes in JNK and AKT path-ways (Fig. 3D). To confirm the functional contributions of theseactivated TPL2 downstream signaling pathways to cell growth,we treated stable cell lines with individual specific inhibitorsand measured cell growth. Treatment of either U0126 (a MEKinhibitor), rapamycin (a mTOR inhibitor), or SB203580 (ap38 MAPK inhibitor) abolished TPL2-WT–mediated increasedcell growth in the immortalized human keratinocytes (Fig. 3E).In particular, the inhibition of TPL2-WT–mediated activationof the MEK pathway by U0126 treatment also abrogated themTOR pathway in the same cells, suggesting that TPL2 acti-vates the mTOR pathway through the MEK pathway (Supple-mentary Fig. S4A, ratios both in red and in blue with U0126treatment). We also confirmed the efficacy of each inhibitortreatment at different concentrations in those cell lines usingWestern blot (Supplementary Fig. S4A, in red). Therefore, theoverexpression of TPL2-WT in immortalized human keratino-cytes increased cell growth, decreased apoptotic cell death, andinduced cell transformation by mechanistically activating itsdownstream signaling pathways ERK MAPK, mTOR, NF-kB,and p38 MAPK.

TPL2 is upregulated in human metastatic SCC and KAspecimens

To validate the clinical relevance of TPL2 overexpressionin human SCC/KA development, we examined the level ofTPL2 expression in TMAs containing 8 human normal skins,40 human SCC specimens, 65 human metastatic SCC speci-mens, and 64 human KA specimens using IHC with an anti-body against TPL2 (Fig. 4A). The overall mean/median stainingscores of TPL2 were 71.25/55.0 for normal skins, 63.5/60.0 for

Lee et al.





Figure 2.

TPL2 overexpression is required for iTPL2 TG–driven tumor maintenance. A, representative gross photos of changes in tumor size and tumor morphology forKA-like cSCC on the head (Skin horn) and SCC on the paw (Paw) from two iTPL2 TG mice with repeated ON DOX (transgene expression ON) and OFF DOX(transgene expression OFF) cycles. Two tumor-bearing iTPL2 TGmice of an iTPL2DC TGmouse (#835) and an iTPL2wt TGmouse (#362) were put into the repeatedcycle of OFF and ON DOX twice, and changes in tumor size were monitored. Red arrows, time periods of ON DOX just before OFF DOX; yellow arrows,changes in tumor size during the OFF DOX period at 1 day and at 10 days of each ON/OFF cycle. B, left, representative photos to compare IHC features of aniTPL2 TG–driven KA-like cSCCONDOXwith those of an iTPL2 TG–driven KA-like cSCCOFFDOX [transgene expression, TPL2; TPL2 downstream signalingmolecules[phospho-ERK, P-ERK; NF-kB1 p105 (cytoplasmic localization)/p50 (nuclear localization); and phospho-p38, P-p38,mTORpathway (phospho-S6 ribosomal protein,P-S6), a cell proliferation marker (Ki-67), and apoptotic cell death (TUNEL)]. Scale bars in blue are 200 mm and those in black are 50 mm. Right, quantificationof each stainingwith three tumor-bearingmice for eachON andOFFDOXgroup in terms of the staining intensity (TPL2 and P-S6) and the number of positive nuclearstaining among 100 cells [P-ERK, P-p38, Ki-67, NF-kB1 (p50), and TUNEL]. Statistically significant differences (t test), � , P < 0.05. a.u., arbitrary unit.






Figure 3.

TPL2 overexpression transforms immortalized human keratinocytes through activations of ERK MAPK, mTOR, NF-kB, and p38 MAPK pathways. A, cell growthof immortalized human keratinocytes expressing the indicated TPL2 constructs was measured using the Incucyte Live Cell Imager. Stable cells expressingwild-type TPL2 (TPL2 wt) showed significantly increased cell proliferation than control vector cells, whereas stable cells expressing a kinase-inactive form of TPL2(TPL2-IN) showed significantly decreased cell proliferation than control vector cells. Statistically significant differences (t test): � , P < 0.05; ��, P < 0.001.B, cell viability of the indicated stable cell lines above was also measured by the Incucyte Live Cell Imager after 66 hours. Cells expressing TPL2-WT showedsignificantly decreased cell death than vector control cells (t test, � , P ¼ 0.0042). Cytotoxicity index, # of dead cells/# of total cells. C, foci formation assay withGiemsa staining of the indicated stable cells. Only stable cells expressing TPL2-WT showed foci formation after confluency.D,Western blot analyses of the indicatedcell lines for TPL2 signaling pathways [exogenous TPL2 overexpression, TPL2; TPL2 downstream signaling molecules (P-ERK, NF-kB, P-p65, P-p38, and P-JNK;mTOR pathway, P-p70S6K; and AKT, P-AKT]. The ratio of phosphorylated form to total form for each indicated signaling molecule was calculated by theImage J software. E, cell growth of the indicated stable cell lines with the treatments of the indicated inhibitors [U0126 (a MEK inhibitor), rapamycin (a mTORinhibitor), or SB203580 (a p38MAPK inhibitor)] wasmeasured using the Incucyte Live Cell Imager. Red arrows, time points for starting treatmentswith the indicatedinhibitors. Significantly decreased cell growth comparedwith cells treatedwith DMSO control at 30 hours after treatmentswasmarked (�, P

SCC, 103.9/110.0 for metastatic SCC, and 110.1/100.0 for KA(Fig. 4B). Although the overall mean/median staining scores formetastatic SCC were also higher than those for normal skins,only KA showed statistically significant increases in overallmean/median staining scores compared with normal skinsamples (Fig. 4B, F test: �P ¼ 0.0429). In addition, the overall

mean/median staining scores for SCC specimens did not showany significant changes compared with those for normal skinsamples. Thus, these TMA data suggest that TPL2 expressionis upregulated in human metastatic SCC and KA specimens,and is statistically significantly increased in KA specimens inparticular.

Figure 4.

TPL2 is upregulated in humanmetastatic SCC and KA specimens. A,representative photos of TPL2 stainingwith a tissue microarray containing 8human normal skins, 40 human SCCspecimens, 65 human metastatic SCCspecimens, and 64 humanKA specimensusing IHCwith an antibody against TPL2.Two samples for each group (F6 and F8for human normal skin samples, A6 andB5 for human SCC, G3 and G8 forhuman metastatic SCC, and K1 and K4for human KA) were selected asrepresentative photos for each group,along with their corresponding averageintensity scores that were determinedindependently by two pathologistsin a blinded manner. Areas in reddotted boxes are shown with highmagnification. Scale bars, 200 mm (left)and 50 mm (right). B, statistical analysesof TPL2 expression between a group ofhuman normal skins and a group ofeither human SCC specimens, humanmetastatic SCC specimens, or humanKAspecimens in terms of medians forstaining score (black long line) and thequartiles (red line). Statisticallysignificant difference in medianstaining scores between two groupsin dotted line is marked witha � (� , P < 0.05). Total sample numbersfor each group are indicated. Stainingintensity was scored independently bytwo pathologists in a blinded mannerwith 0 (no staining), 100 (weak staining),200 (intermediate staining), or 300(strong staining) for each sample, andthe average of two scores for eachsample is shown in the graph. Thecorresponding average staining scoresfor the representative photos (A) arealso indicated in the graph.






DiscussionWhile genetic alterations associated with the initiation and

progression of skin cancers, in particular melanoma, have beenwell studied, those underlying SCC and KA (SCC/KA) arerelatively less well understood. Therefore, the identification ofTPL2 as an oncogenic driver for SCC/KA development in thisstudy is important. Most notably, iTPL2 transgenic mice devel-op both KA-like cSCC and SCC in as early as a few weeks. This isexceptional. Given that most genetically engineered mousemodels develop tumors approximately 6 months or later,tumor development within 2 weeks in iTPL2 TG mice issurprising. Therefore, this mouse model will enable moreefficient research by saving a tremendous amount of time spentwaiting for tumor development.

The finding of TPL20s role in the maintenance of both KA-likecSCC and SCC using this inducible mouse model is also signif-icant, because it may be able to provide a scientific basis fortargeting TPL2 to treat both KA-like cSCC and SCC. As previouslymentioned, "SCC, KA-type" is one of the most common sideeffects of the antimelanoma drug vemurafenib (4). Therefore, thisstudy will also provide a framework for future combinationaltreatment strategies for melanoma patients using one drug,vemurafenib, to treat melanoma and another, a TPL2 inhibitor,to prevent or treat possible "SCC, KA-type" development.

RAS mutations were identified in approximately 21% ofhuman SCCs, among which 9% are HRAS, 7% are NRAS, and5% are KRAS mutations (47). A transgenic mouse model expres-sing oncogenicHRAS (V12) in the skin using the promoter regionof the suprabasal keratin 10 gene developed skin hyperkeratosisand papilloma formation (48). These phenotypes occurredmain-ly at injured areas resulting from biting or scratching, suggesting a"secondary hit" of a wound stimulus is required for tumorinduction in this HRAS (V12) transgenic mouse model (48).However, although iTPL2 TG–driven SCC and KA-like cSCC werehistologically similar to HRAS (V12)-driven skin hyperkeratosisand papilloma, iTPL2 TG–driven tumor development was notsimply limited to specific injured areas. Therefore, we questionedwhy iTPL2TGmicedevelopedKA-like cSCConly in the dorsal andventral skin and SCC in the mouth, genital area, and paw, giventhat the ROSA26 locus promoter used for TPL2 expression in thisiTPL2 TG mouse model is constitutively and ubiquitously activein most mouse tissues at similar levels.

In general, TPL2 is sequestered and stabilized as an inactiveform in the cytoplasm through its interaction with an inhib-itory binding partner, NF-kB1 (p105), in resting cells. Uponstimulation, elevated IKK activity dissociates TPL2 from NF-kB1 (p105)–mediated sequestration and subsequently releasedTPL2 activates its downstream signaling pathways in a cell typeand stimulus-specific manner before its rapid degradation (22,24, 26, 28). Of note, we did not find significantly dysregulatedlevels of NF-kB1 (p105) expression in the cytoplasm of iTPL2TG–driven tumors compared with that in normal wild-typeskin in spite of the increased nuclear translocation of NF-kB1(p50; Fig. 1B, NF-kB1 p105/p50 and Supplementary Figs. S4Band S8). Consistently, immortalized human keratinocytesexpressing TPL2-WT showed significantly increased levels ofNF-kB1 (p50) than immortalized human keratinocytes expres-sing either vector control or TPL2-IN without any changes in thelevel of NF-kB1 (p105) protein expression (Supplementary Fig.S4B). Therefore, it is possible that TPL2 overexpression induces

the degradation of NF-kB1 (p105) to generate NF-kB1 (p50) inkeratinocytes in particular as suggested by Belich and collea-gues (22). Alternatively, some intrinsic factors in keratinocytes,such as high basal IKK activity in keratinocytes, induce thedegradation of NF-kB1 (p105) to release TPL2 from its seques-tration. If these possibilities were true, levels of NF-kB1 (p105)-free transgene TPL2 wt proteins in the cytoplasm wouldincrease in the tumors. We confirmed increased levels of NF-kB1 (p105)-free transgene TPL2 wt proteins in the cytoplasmof iTPL2 TG–driven cSCC using both an in vitro kinase assay andcoimmunofluorescence (Supplementary Figs. S6 and S7). Theseresults suggested that increased levels of NF-kB1 (p105)-freetransgene TPL2 wt proteins by currently unknown intrinsicfactors in keratinocytes might contribute to acceleratingTPL2-mediated transformation in the skin.

Finally, the activation of the mTOR pathway by TPL2 over-expression and the suppression of cell proliferation with thetreatment of rapamycin in immortalized keratinocytes (Fig.3DandE; Supplementary Fig. S4) are intriguing in termsof clinicalrelevance, because rapamycin is routinely used for the treatmentof SCC (49, 50). Previously, it was suggested that the MEK/ERKMAPK pathway activates the mTOR pathway. Treatment ofimmortalized keratinocytes expressing TPL2-WT with MEK inhi-bitors (U0126) suppresses the mTOR pathway and abolishesTPL2-WT-mediated increased cell growth, suggesting that TPL2activates the mTOR pathway through the MEK/ERK MAPK path-way. Therefore, these data provide a possible mechanistic expla-nation for the positive response of SCC to rapamycin.

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

Authors' ContributionsConception and design: J.-H. Lee, K.-S. Inn, J.-S. Lee, J.U. Jung, J.H. JeongDevelopment of methodology: J.-H. Lee, J.H. JeongAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-H. Lee, J.-H. Lee, S.H. Lee, O. Forslund, F.-M. Deng,J. MelamedAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-H. Lee, J.-H. Lee, S.H. Lee, S.-I. Do, S.-D. Cho,J.-S. Lee, F.-M. Deng, J.H. JeongWriting, review, and/or revision of the manuscript: J.-H. Lee, S.H. Lee,K.-S. Inn, F.-M. Deng, J.H. JeongAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.H. LeeStudy supervision: S.H. Lee, J.-S. Lee, J.H. Jeong

AcknowledgmentsWe thank Stacy Lee for her manuscript proofreading and Nathan E. Duncan

for his tissue preparation.

Grant SupportThis studywas supported by J.H. Jeong's award for the Advancing aHealthier

Wisconsin Endowment (AHW) Cancer Cell Biology (CCB) research program(#5520240) in theMedical College ofWisconsin (MCW)Cancer Center and theAmerican Cancer Society Institutional Research Grant Pilot Project "Seed"Funds (#ACS-IRG-58-007-48). Jun-Han Lee and Joo-Hyung Lee received sup-port from the AHW CCB (#5520240).

The costs of publication of this articlewere defrayed inpart 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 December 16, 2015; revised May 9, 2016; accepted June 27, 2016;published OnlineFirst August 8, 2016.


Lee et al.




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Lee et al.




2016;76:6712-6722. Published OnlineFirst August 8, 2016.Cancer Res Jun-Han Lee, Joo-Hyung Lee, Sang Hyuk Lee, et al. Cell Carcinoma

Is an Oncogenic Driver in Keratocanthoma and SquamousTPL2

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