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