Loss of Trop2 Promotes Carcinogenesis and Features of ... · preferentially susceptible to transformation (11). These diverse epidemiologic and functional (12) data point to an important

Cancer Genes and Genomics

Loss of Trop2 Promotes Carcinogenesis and Features ofEpithelial to Mesenchymal Transition in Squamous CellCarcinoma

Jianbo Wang1, Kaihua Zhang1, Dorota Grabowska1, Aimin Li1, Yiyu Dong1, Ryan Day1,Peter Humphrey3, James Lewis3, Raleigh D. Kladney1, Jeffrey M. Arbeit4,5, Jason D. Weber1,2,5,Christine H. Chung6, and Loren S. Michel1,2,5

AbstractTrop2, an oncogenic cell surface protein under investigation as a therapeutic target, is commonly overexpressed

in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate therole of Trop2 in epithelial carcinogenesis, we generated Trop2�/� mice, which are viable and possess a normallifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2�/� keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT)and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf-null mice susceptible to theformation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components uponcarcinogen exposure in an otherwise skin cancer–resistant strain (C57BL/6). Immortalized keratinocytes derivedfrom Trop2�/�Arf�/�mice exhibit enhanced proliferative and migratory capacity as well as increased activation ofmitogen-activated protein kinase and Src prior to transformation. The clinical relevance of these findings wassupported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas.This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and totalloss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore,while previous studies have emphasized the potential importance of Trop2 gain of function, these results uncover arole forTrop2 loss in tumorigenesis and themesenchymal transdifferentiation observed in a subset of squamous cellcarcinomas. Mol Cancer Res; 9(12); 1686–95. �2011 AACR.

Introduction

Trop2 (TACSTD2, M1S1) is a single-pass transmem-brane protein expressed primarily in epithelial cells (1) and isoften overexpressed in various epithelial tumor types (2–6).We previously reported that Trop2 overexpression hastransforming activity, whereas loss of function in coloncancer cells suppresses their tumorigenicity (7). Morerecently, Trop2 overexpression was shown to stronglyinduce mitogen-activated protein kinase (MAPK) activityand metastasis in pancreatic cancer cells (8). As such, anti-

Trop2 antibodies are under investigation as cancer thera-peutics in the preclinical setting (9, 10). Intriguingly, Trop2expression was found to mark prostate stem cells that arepreferentially susceptible to transformation (11). Thesediverse epidemiologic and functional (12) data point to animportant role for Trop2 in tumorigenesis and as a possibletreatment target, yet its function(s) remains obscure. Studieson gelatinous drop-like corneal dystrophy (GDCD), a rareform of congenital blindness caused by Trop2 loss-of-func-tion mutations (1, 13, 14), have revealed a role for Trop2 inmodulating cell adhesion in the cornea. However, despitethis increasing interest in Trop2 as a therapeutic target, itsrole in normal biology or neoplastic progression is poorlyunderstood.The epithelial to mesenchymal transition (EMT) consists

of a series of cell biological and biochemical changes thatendow normal epithelial cells with the plasticity required forbody patterning and tissue differentiation. Acquisition of amesenchymal phenotype by epithelial cells is regulated atmultiple levels and is characterized by the loss of E-cadherin–mediated cell–cell adhesion, decreased cell–matrix adhesion,and gain of mesenchymal markers such as vimentin andfibronectin (15–18). AnEMT-like profile is considered to bea characteristic of aggressive cancers. However, definitive

Authors' Affiliations: 1Division of Oncology, Department of Internal Med-icine, Departments of 2Cell Biology and Physiology and 3Pathology, 4Divi-sion of Urology, and 5The Alvin J. Siteman Cancer Center, WashingtonUniversity School of Medicine, Saint Louis, Missouri; and 6Department ofOncology, The Johns Hopkins University School of Medicine, Baltimore,Maryland

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Loren S. Michel, Campus Box 8069, WashingtonUniversity School of Medicine, Saint Louis, MO 63110, Phone: 314-362-8965; Fax: 314-747-9320l; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0241

�2011 American Association for Cancer Research.

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identification of EMT in primary human tumors has beendifficult, possibly because the transition may be restricted–both spatially and temporally—to a subset of cells within thetumor mass (19). Notably, some epithelial tumors containareas of pure mesenchymal histology and possess the molec-ular features of EMT more uniformly. Sarcomatoid carci-noma is one specific example. These tumors are a distinctvariant of squamous cell carcinoma that frequently originatein the epithelia of the upper aerodigestive tract (12, 20),display spindle cell histology, and typically lose epithelialgene expression consistent with mesenchymal transdiffer-entiation. Notably, sarcomatoid carcinoma can be biphasicand exhibit discreet regions of squamous and spindle cellhistology. In addition, microarray analyses have resulted inthe identification of subtypes of epithelial tumors that do nothistologically appear as spindle cells yet possess a geneexpression signature suggestive of an epithelial to mesen-chymal transition (21, 22).Given the reported overexpression of Trop2 in various

epithelial tumors, we sought to examine its role in epithelialcarcinogenesis by deriving Trop2 knockout mice and reportthe generation of a viable Trop2-null strain. Contrary toexpectations, we show that Trop2 loss promotes the devel-opment of EMT during ras-mediated transformation ofprimary keratinocytes. In addition, Trop2 loss increasessusceptibility to the development of biphasic sarcomatoidcarcinomas of the skin in response to carcinogens. Accord-ingly, in head and neck squamous cell carcinomas(HNSCC), we find that Trop2 expression is decreased intumors possessing an EMT-like signature and completelyabsent in sarcomatoid variants. Collectively, these dataimplicate Trop2 loss in tumorigenesis in keratinocytes andin the promotion of EMT in a subset of squamous cellcarcinomas.

Materials and Methods

Generation of Trop2�/� miceBacterial artificial chromosome (BAC) DNA clone RP23-

23I10 containing Trop2 locus was obtained from CHORIand was used to construct targeting vector by PCR ampli-fication. The primer sequences used for 50 homology arm ofthe targeting construct were 50-GAT TGAGGA TCC AATCTC TCC CAG GTC ATA AAC-30 and 50-GAT TGAGGA TCC GGT GGG GTG GAG TAG AAT GGA-30.The primer sequences used for 30 homology arm of thetargeting construct were 50-GAT TGA GAT ATC CCATGG CCA AAG ACT TAA ACG GTT TGA AAT G-30and 50-GATTGAGCGGCCGCAGAGCCCCTCCTGCTT CTC ACA-30. The amplified 50 homology arm (2.6kb) and 30 homology arm (3.0 kb) were cloned into thep1338 vector. The targeting vector was linearized withNotI,and gene targeting was conducted following standard pro-cedures in C57BL/6SJ embryonic stem cell line B6/Blu,which was injected into a blastocyst. A pseudopregnantC57BL/6SJ mother was used as the recipient. Embryonicstem cell clones that underwent the correct integration wereidentified by Southern blotting with a 50 external probe and

30 external probe. The primer sequences used for the 50external probe were 50-CTC TAC TCC TAA CCC TAATCT GCC-30 and 50-TAC CAT CCC TAC CTT AGTCCA CC-30. The primer sequences used for the 30 externalprobe were 50-GTC CCT ACT CAA CTC CTT CTC CC-30 and 50-TAGCCAATTACAAAACAGTCATTCC-30.One embryonic stem cell clone was used to generate chi-meras by Washington University Murine Embryonic StemCell Core (St. Louis, MO).

Skin tumor inductionThe backs of 8-week-old male and female mice were

shaved and treated with a single application of 7,12-dimethylbenz(a)anthracene (DMBA; 20 mg in 200 mL ofacetone; Sigma) followed a week later by twice weeklyapplications of 12-O-tetradecanoylphorbol-l3-acetate (TPA,12.5 mg in 200 mL of acetone). The number and size ofpapillomas on each mouse were recorded every week for 36weeks. Animals were handled according to protocolsapproved by the Washington University Animal StudiesCommittee.

Mouse keratinocyte isolation and manipulationPrimary epidermal keratinocytes populations were har-

vested from newborn mice (1 day old). Skin was floateddermis down at 4�C overnight in dispase I (Invitrogen).Afterwards, skin pieces were placed with the dermis sideup, and the dermis was peeled off with a forceps. Kera-tinocytes were isolated by trypsinizing the tissues for anadditional 15 minutes at 37�C. Cells were collected,washed in PBS containing 0.5% FBS, and were resus-pended and seeded in Eagle's Minimal Essential Medium(EMEM; Lonza).

Antibodies and immunoblot analysisCells were lysed in 50 mmol/L Tris, pH 7.4; 150 mmol/L

NaCl; 0.5% sodium deoxycholate (DOC); and 0.5% NP-40. Fifty microgram of protein was resolved on a 4% to 15%Tris-HCl gel and transferred to polyvinylidene fluoridemembranes. Antibodies used in this study were againstTrop2 A650 and AF1122 (against human and mouse,respectively; R&D Systems), E-cadherin (BD TransductionLaboratories), vimentin (BD Pharmingen), p-Src (Tyr416;Calbiochem and Cell Signaling), tubulin (Abcam), Erk 1/2and p-ERK 1/2 (Thr202/Tyr204), p-FAK (Tyr397),p-p130Cas (Tyr165; Cell Signaling), and H-Ras (SantaCruz Biotechnology). Image quantification was carried outwith ImageJ software (NIH, Bethesda, MD).

Lentiviral gene transfer and tumorigenicity assaysLentiviruses expressing H-rasV12 or mouse Trop2 were

produced as previously described (7). Filtered supernatantsupplemented with 4-mg/mL polybrene was used to infectfreshly isolated mouse skin keratinocytes within 48 hourspostplating. Two days after infection, lentivirally transducedcells were harvested, and either 2 � 106 cells were injectedsubcutaneously into the flanks of nude mice. Tumor growthwas monitored every 3 days.

A Role for Trop2 Loss in Carcinogenesis

www.aacrjournals.org Mol Cancer Res; 9(12) December 2011 1687




Immunohistochemical analysisThe slides were deparaffinized and hydrated, treated in

citrate buffer (0.1 mol/L sodium citrate and citric acid, pH3.0) for 10 minutes in a pressure cooker, and quenched in3% hydrogen peroxide solution for 10 minutes. Sectionswere blocked with avidin solution and biotin solution afterwashing with TBS Tween 20 (TBST) thoroughly. Sec-tions were incubated with anti-Trop2 antibody for 1 hourat room temperature after TBST washing. Sections wereincubated with LSABþSystem-HRP solutions (Dako) perthe manufacturer's instructions. Color was developed byadding 3,30-diaminobenzidine (DAB) substrate solutionand stopped by washing with water. Mouse tumor slideswere counterstained in hematoxylin, dehydrated, cleared,and mounted in Cytoseal. The histology of keratinocytetumors was quantified by the Olympus Microsuite 5software package.

Statistical analysisAll statistical tests were 2-sided, and P values of less than

0.05 were considered statistically significant.Additional methods can be found in Supplementary

Material.

Results

Trop2 loss does not result in embryonic lethality and isnot required for development in miceTo gain insight into the effects of Trop2 loss of function,

we generated aTrop2-nullmouse strain by deleting the singleexon that encodes the open reading frame using homologousrecombination in embryonic stem cells (Fig. 1A–E). Trop2has been proposed to play a role in development because itsexpression is high in certain epithelial stem cell compart-ments (23, 24), and the levels fluctuate widely in response todevelopmental cues (25). In addition, EpCAM, the genemost closely related to Trop2 (49% homology) is essential inmice (26). However, we found that homozygous null miceare born at the normal expected Mendelian frequencies,display no overt developmental abnormalities, and have anormal life expectancy (Supplementary Figs. S1 and S2 anddata not shown).

Induction of tumors with spindle cell histology upontransformation of Trop2�/�-null keratinocytesTo model the effects of Trop2 loss of function on

epithelial transformation, we used the 2-hit transformationassay of primary mouse keratinocytes (27) in which raspathway activation in the context of Arf deletion drivesthe development of squamous cell carcinomas (28). Primarykeratinocytes were isolated from Trop2þ/þ:Arf�/� andTrop2�/�:Arf�/� mice, infected in vitro with an H-RasV12–expressing lentivirus (Supplementary Fig. S3), andthen grafted subcutaneously into the flanks of nude mice.Palpable tumors were noted in both genetic backgroundsafter 2 weeks when a cell dosage of 2 � 106 was injected.However, histopathologic examination of the tumors thatarose revealed that keratinocytes fromTrop2�/�:Arf�/�mice

displayed predominantly spindle with almost no squamouscell histology. In contrast tumors from Trop2þ/þ:Arf�/�

mice exhibited predominantly squamous cell histology andsome spindle cell components (Fig. 2A). Quantification ofthe percentages of spindle and squamous histology in eachtumor with image analysis software confirmed an increase inspindle cell conversion in tumors derived from Trop2�/�:Arf�/� cells compared with those arising from Trop2þ/þ:Arf�/� cells (Fig. 2B). Analysis of E-cadherin and vimentinin tumors arising from both genotypes revealed the retentionof E-cadherin and the absence of vimentin expression inareas of squamous cell histology, whereas spindle cells dis-played the opposite pattern, consistent with EMT (Fig. 2Cand Supplementary Fig. S4). Strikingly, in areas of spindlecell histology in nominally Trop2 wild-type tumors, Trop2expression was absent, and a gradient of decreasing expres-sion was evident mirroring the decline in E-cadherin andincrease in vimentin staining in transition zones borderingcells that had passaged through an EMT (Fig. 2C). Thesedata indicate that Trop2 deficiency facilitates EMT duringoncogenic transformation of keratinocytes in vivo. Further-more, Trop2 loss occurs during transformation associatedwith mesenchymal transdifferentiation in Trop2 positivecells.

Figure 1. Strategy for the generation and validation of a Trop2 knockoutmouse strain. A, schematic depicting the knockout vector and probes forSouthern blot analysis. B, Southern blot analysis showing knockout (KO)of the Trop2 locus in F1 offspring. C, reverse-transcriptase PCR of Trop2transcripts from RNA isolated from tails of the denoted genotypes. D,immunoblot analysis using anti-Trop2 antibodies on lysates fromprimarymouse keratinocytes. E, flow cytometric analysis of Trop2 on primarymouse keratinocytes. WT, wild-type.

Wang et al.

Mol Cancer Res; 9(12) December 2011 Molecular Cancer Research1688




Trop2 loss promotes tumorigenesis in miceThe ability of Trop2 loss tomodify the histologic outcome

of an identical set of transforming events at the cellular levelraised the question whether Trop2 loss contributes totumorigenesis. To investigate this possibility, we used a wellestablishedmodel of skin carcinogenesis (DMBA–TPA) thatinC57BL/6mice generatesH-Rasmutations and papillomasbut rarely invasive cancer (29). The absence of Trop2 did notalter the incidence or natural history of papilloma formationby this protocol (Supplementary Table 1A). However, giventhe cooperativity between Trop2 and Arf–ras pathwaydefects observed in keratinocytes, the effect of Trop2 losson skin cancer development was assessed in Arf-null animalsexposed to DMBA–TPA. Over a standard 36-week treat-ment period, both strains developed papillomas at equivalentrates and sizes (Supplementary Table 1B) and equivalentpercentages of mice survived the treatment protocol, somesuccumbing to the expected lymphoid and nonskin sarco-matoid appearing malignancies. However and most impor-tantly, Trop2 loss in this context was sufficient to promotethe formation of skin carcinomas (n ¼ 6/24 in Trop2�/�:

Arf�/� mice), whereas none of the Trop2þ/þ:Arf�/� mice(n¼ 0/25, P¼ 0.009, the Fisher exact test) developed cancer(Fig. 3A and B). All 6 tumors were invasive, 5 were classifiedas sarcomatoid carcinomas, and 1 was found to consist ofpoorly differentiated high-grade cancer. Interestingly, somesquamous cell histology was identified in 3 of the sarcoma-toid carcinomas, consistent with their epithelial origin.Accordingly, these squamous cell components expressedE-cadherin, whereas the spindle cells lacked this stainingbut were positive for vimentin (Fig. 3C). Thus, Trop2 loss,in collaboration with Arf inactivation, increases the suscep-tibility to skin carcinogenesis and promotes the developmentof tumors with mesenchymal features.

ImmortalizedTrop2-null keratinocytes exhibit increasedproliferative and migratory capacityTrop2-null mice display no overt phenotypic abnormal-

ities, yet Trop2 loss can contribute to tumor initiation and

Figure 2. Role of Trop2 loss in primary epithelial cell transformation.A, squamous histology in ras-transformed Trop2þ/þ:Arf�/� keratinocytesand spindle cell histology in ras-transformed Trop2�/�:Arf�/�

keratinocytes as shown by hematoxylin and eosin (H&E) staining.B, results of measurements of the percentages of spindle relative tosquamous cell histology in tumors from cells with the indicatedgenotypes with image analysis software to calculate the areas.�, P ¼ 0.008, the Student t test. C, H&E-stained section from a tumorderived from Trop2þ/þ:Arf�/� keratinocytes showing a region ofspontaneous progression from squamous to spindle cell histology (left).Indirect immunofluorescence staining reveals loss of E-cadherin (red)and gain of vimentin (green) as the tumor transitions from squamous tospindle cell histology (middle). Nuclei are stained with 40,6-diamidino-2-phenylindole (DAPI; blue). Indirect immunofluorescence staining of Trop2(right) shows loss of Trop2 coincident with the EMT observed in thenominally Trop2þ/þ tumor cells. Middle and right panel are from sectionscut adjacent to the H&E-stained section. sq, squamous; tran, area of lowE-cadherin; Trop2 representing a transition to spindle cells; sp, spindlecell. Bar, 100 mm.

Figure 3. Trop2 loss promotes the development of sarcomatoidcarcinomas in mice. A, enumeration of the incidence of carcinomaformation in Trop2þ/þ:Arf�/� and Trop2�/�:Arf�/� mice after 36 weeks ofDMBA–TPA exposure (P ¼ 0.009 based on the Fisher exact test). B, arepresentative Trop2þ/þ:Arf�/� mouse harbors multiple papillomas (lefttop). The box circumscribes a typical lesion. The associatedH&E-stainedsection is shown in the bottom panel. A representative large tumor (righttop, boxed in white) from a Trop2�/�:Arf�/� animal. The spindle cellhistology of the tumor is shown by H&E staining in the bottom. C, asection from a tumor arising in Trop2�/�:Arf�/� mice showing biphasichistology by H&E staining (left). Immunofluorescence staining of thecorresponding section (right) reveals that the spindle cell component (sp)is positive for vimentin (green), whereas the squamous component (sq) isE-cadherin positive. Bar, 100 mm.






drive progression towards spindle cell histology. Theseobservations suggest that nontransformed Trop2-null cellsare more likely to acquire characteristics that can facilitatecancerous growth. To investigate whether such character-istics could be identified, we isolated Trop2þ/þ:Arf�/� andTrop2�/�:Arf�/� keratinocytes; the Arf deficient back-ground imparts features of cellular immortalization in vitro(30). Loss of Trop2 did not alter cellular proliferation in

standard tissue culture conditions (data not shown), butTrop2�/�:Arf�/� keratinocytes displayed a dramatic prolif-erative advantage in 3-dimensional culture compared withTrop2þ/þ:Arf�/� cells. Measurement of BrdUrd incorpo-ration revealed increased rates of DNA synthesis in Trop2-null cells when grown in 3-dimensional culture, suggesting aproliferative advantage under these more physiologic con-ditions (Fig. 4A–C). We also compared the migratory

Figure 4. Trop2 loss confers a proliferative and migratory advantage in Arf�/� immortalized keratinocytes. A, photomicrographs of Trop2þ/þ:Arf�/� andTrop2�/�:Arf�/� keratinocytes grownonMatrigel for 7 days. 10�magnification. B, graphof colony counts 7days after keratinocyte plating. C, graphof BrdUrdincorporation of keratinocytes grown onMatrigel measured 3 days after plating. �,P¼ 0.025, the Student t test. D, photomicrographs of a scratch assay usingkeratinocytes from Trop2þ/þ:Arf�/� and Trop2�/�:Arf�/�mice showing a completely closed wound in Trop2-null cells by 16 hours. E, immunoblot analysis ofprotein lysates fromTrop2þ/þ:Arf�/�andTrop2�/�:Arf�/� keratinocytes showingdecreasedE-cadherin levels in cells lacking Trop2. F, immunoblot analysis ofTrop2�/�:Arf�/� keratinocytes infected with control (empty vector) or Trop2-expressing lentivirus. Protein levels of Trop2 in Trop2þ/þ:Arf�/� are shown forcomparison.G, graph of BrdUrd incorporation ofTrop2�/�:Arf�/� keratinocytes infectedwith control or Trop2-expressing virus grownonMatrigelmeasured 3days after plating. �, P¼ 0.049, the Student t test. BrdUrd incorporation of Trop2þ/þ:Arf�/� keratinocytes is shown for comparison. For BrdUrd experiments,resultswere normalized to Trop2þ/þ:Arf�/� cells. H, photomicrographs of a scratch assay using keratinocytes from Trop2�/�:Arf�/�mice infectedwith control(EV, empty vector) or Trop2-expressing virus showing retardedmigration in Trop2-expressing cells. Arrows point to scattered cells in the unfilled region of thescratch. Results from each experiment are representative of 3 independent tests.

Wang et al.





potential of Trop2þ/þ:Arf�/� and Trop2�/�:Arf�/� kerati-nocytes and found the latter to possess a significantlyincreased mobility in a 2-dimensional would-healing assay(Fig. 4D). Next, we examined whether Trop2 loss wassufficient to promote features of EMT in immortalizedkeratinocytes. Trop2�/�:Arf�/� did not acquire a mesen-chymal morphology and immunoblot analysis of Twist,Snail, Slug, andfibronectin revealed no detectable alterationsin the levels of these proteins compared with wild-type cells.However, E-cadherin protein levels were found to be sig-nificantly decreased in the absence of Trop2 (Fig. 4E anddata not shown). Finally, we sought to reconstitute Trop2expression inTrop2�/�:Arf�/� cells with a full-length mousecDNA in an effort to reverse the observed phenotypicchanges. After lentiviral transduction of Trop2 and anti-biotic selection, Trop2 protein expression was partiallyrestored relative to levels measured in Trop2þ/þ:Arf�/�

cells (Fig. 4F), and the protein migrated as a doublet,suggesting differential posttranslational modification(s) inthis cellular environment. Importantly, expression ofTrop2 in Trop2�/�:Arf�/� cells was found to be sufficientto reduce (but not normalize) both rates of proliferation in3-dimensional culture as well as migration in a wound-

healing assay relative to those measured in empty vector–transduced cells (Fig. 4G and H). Total E-cadherin levelswere not measurably increased (data not shown), possiblyowing to the need for higher levels of Trop2 proteinexpression to rescue this specific defect in knockout cells.Nevertheless, partial restoration of Trop2 protein levels isable to measurably reduce the proliferative and migratoryadvantages identified in Trop2-null keratinocytes, obser-vations that point to a causal role for Trop2 loss in thedevelopment of these phenotypes.To identify Trop2-dependentmechanisms underlying the

increased susceptibility to tumorigenesis, we examinedwhether Trop2 loss could increase the activity of commononcogenic pathways by measuring the phosphorylationstatus of epidermal growth factor receptor, AKT, extracel-lular signal–regulated kinase (ERK), and Src. This analysisrevealed that keratinocytes derived from Trop2�/�:Arf�/�

mice exhibit an increase in MAPK and Src activity, theformer showing a dramatic increase in the absence ofTrop2 compared with Trop2þ/þ:Arf�/� cells (Fig. 5A). Anincrease in the activity of epidermal growth factor receptorand AKT was not observed (data not shown). In Trop2knockout cells, we also detected an increase in the level of

Figure 5. Activation of MAPKand Src in Trop2�/�:Arf�/�

keratinocytes and tissues.A, immunoblot analysis ofimmortalized Trop2þ/þ:Arf�/� andTrop2�/�:Arf�/� keratinocytesreveals activation of ERK 1/2(Thr202/Tyr204), Src (Tyr416), FAK(Tyr397), and p130Cas (Tyr165) inTrop2-null cells. B, immunoblotanalysis shows reduction in ERKand Src activation in Trop2-expressing Trop2�/�:Arf�/� cellscompared with control cellsharboring an empty vector. Thenumbers indicate densitometricallyquantified protein levels(normalized to total ERK or Src).C, immunohistochemical stainingof p-ERK (Thr202/Tyr204) andp-Src (Tyr416) in papillomas andcarcinomas from tissues arising inthe denotedgenotypes. Arrows andarrowheads point to cells stainingpositive. Sectionswere stainedwithmethyl green as a counterstain. Baris 50 mm.






phosphorylated forms of 2 Src substrates, FAK andp130Cas (Fig. 5A). Importantly and in consonance withthe partial phenotypic rescue achieved by Trop2 expres-sion, we observed reduced levels of active MAPK and Srcin Trop2�/�:Arf�/� cells after infection with Trop2-expressing lentivirus (Fig. 5B). To determine whetherincreased MAPK and Src activity could be identified inthe context of Trop2 deficiency in vivo, we conducted animmunohistochemical analysis of papillomas and tumors.Neither MAPK nor Src hyperactivation was found inpapillomas arising in Trop2þ/þ:Arf�/� mice. In contrast,increased staining of phospho (p)-ERK and phospho (p)-Src was readily detected in papillomas arising in Trop2�/�:Arf�/� mice (n ¼ 5 per group). In addition, extensivestaining was observed in 5 carcinomas examined thatdeveloped in the double knockout animals (Fig. 5C).Interestingly, Src staining was detected primarily in thecytoplasm, a pattern of localization that has previouslybeen associated with poorly differentiated cancer (31).Collectively, these data reveal that Trop2 loss causes anincrease in MAPK and Src activation prior to oncogenictransformation.

Decreased Trop2 levels in squamous cell carcinoma withan EMT gene expression signature and sarcomatoidcarcinomas of the head and neckOur data showing that loss of Trop2 facilitates EMT in

squamous cell tumors in mice prompted us to investigateTrop2 expression in human head and neck cancer. Asubset of head and neck cancers exhibit morphologic and/or molecular features of EMT. In addition to sarcomatoidcarcinomas of the head and neck, which exhibit mesen-chymal markers as well as distinct histopathology sugges-tive of an EMT, a gene expression signature has previouslybeen reported by us that identifies a subtype of tumorswith molecular features of EMT (designated as group 2).Pathologically, these tumors appear as poorly differenti-ated carcinomas (22). To determine whether decreasedTrop2 expression associates with this EMT-like subtype,we queried its expression in our data set that was used todevelop this signature (22). Notably, Trop2 expression wasfound to be decreased in the EMT-like group relative tothe other subtypes and normal tonsillar mucosa. Accord-ingly, reduced Trop2 expression segregates with lowE-cadherin and high vimentin expression in this data set(Fig. 6A). Given the relative decrease in these poorlydifferentiated, EMT-like yet nonsarcomatoid tumors andbased on the results from the knockout animal experi-ments, we suspected that Trop2 expression might becompletely undetectable in the spindle cell componentof sarcomatoid carcinoma of the head and neck. Immu-nohistochemical staining of sarcomatoid carcinoma con-firmed this possibility. Trop2 expression was detected inareas of squamous cell histology in cancers containing puresquamous cell elements and biphasic sarcomatoid carci-nomas. However, in striking contrast, its expression wasabsent in the spindle cell component of sarcomatoidcarcinomas (Fig. 6B and C; Table 1) from various head

and neck sites (n ¼ 5 of each histologic group, P ¼ 0.007,the Fisher exact test). Consistent with their mesenchymalstate, Trop2-negative sarcomatoid carcinomas also lackedE-cadherin expression, whereas tumors containing Trop2-positive cells with squamous histology expressed highlevels (Fig. 6C). Therefore, decreased Trop2 expressionis a feature of squamous cell carcinomas that possessmolecular and histologic characteristics of EMT.

Figure 6. Trop2 expression in head and neck cancers. A, association ofTrop2 expression levels with intrinsic subtypes of head and neck cancersby in silico analysis. Group 2 is the EMT-like subtype consisting of poorlydifferentiated tumors. B, example of loss of Trop2 staining in a biphasichead and neck cancer. Trop2 is absent in the spindle cell component (sp),whereas nests of squamous cancer cells (sq) stain positive. 10�magnification. C, immunohistochemical stainingof Trop2 andE-cadherinshowing concomitant loss in spindle cell components. 20�magnification.

Table 1. Association of negative Trop2 statuswith the spindle cell component of sarcomatoidcarcinomas

Squamous Spindle

Site Trop2status

Site Trop2status

Tongue Positive Tongue NegativeTongue Positive Tongue NegativeGlottic Positive Glottic NegativeGlottic Positive Soft palate NegativeAlveolar ridge Positive Buccal mucous Negative

NOTE: Cancers denoted in the left column contain puresquamous cell histology.

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Discussion

The results presented here were not predicted on thebasis of previous epidemiologic and functional studies ofTrop2, all of which pointed to a gain-of-function role forthis protein in the development of aggressive cancer andtumor susceptibility (2–7, 11, 23, 32–34). As such, weexpected that Trop2 ablation would hinder cancer devel-opment. Instead, through the use of a novel Trop2knockout mouse model generated in our laboratory, weuncovered an unpredicted relationship between Trop2 lossand cancer development. In addition, we were able toconfirm a relationship between Trop2 loss and anincreased progression towards EMT during epithelialtransformation. The epidemiologic analysis presented inthis report is the first description, to our knowledge, of theassociation between Trop2 loss and EMT in any primaryhuman tumor and highlights the clinical relevance ofour findings. Collectively, these observations reveal a farmore complex functional role for this protein in themultistep development of cancer than has been previouslysuspected.It has been reported that germline deletion of EpCAM,

the closest homologue to Trop2, causes early embryoniclethality in mice (26). In addition, Trop2 expression iselevated in stem cell compartments of the prostate (23) andliver (24), and the pattern of expression is dynamic duringdevelopment (25). All of these observations suggested a highlikelihood that Trop2 deletion would result in abnormalitiesin development or adulthood, but these animals are viableand lack overt developmental defects. However, a moredetailed analysis revealed that Trop2 loss promotes skincancer in mice lacking Arf, a tumor suppressor gene com-monly inactivated in many cancers, including those arisingin the head and neck. It is notable that mice lacking this geneare tumor prone and predominantly develop sarcomas andlymphomas rather than skin tumors (30), which do formwhen exposed to DMBA as neonates (35). C57BL/6 mice,the strain used in our experiments, are notoriously resistantto the standard TPA–DMBA-mediated carcinogenesis pro-tocol that we used herein. Given the cancers that develop inTrop2�/�:Arf�/� animals in the C57BL/6 background afterTPA–DMBA exposure and the absence of a detectablecancer phenotype in Trop2�/� mice, our results indicatethat Trop2 participates in multistep tumorigenesis as amodifier in collaboration with other protumorigenic events.In this manner,Trop2 loss resembles the effects of Src, whosecatalytic activity is frequently elevated in cancers. Owing toits weak transforming ability, Src is thought to facilitate otheroncogenic signals rather than function as a dominant onco-gene (36). The identification of Src hyperactivation inkeratinocytes, papillomas and carcinomas derived fromTrop2�/�:Arf�/� mice, and the suppression of this activityby reexpression of Trop2 supports the idea that Trop2-dependent modulation of Src likely contributes to theenhanced tumor susceptibility observed in Trop2�/�:Arf�/� animals. We also found that Trop2 loss causeselevation of MAPK activity, which lies downstream of

several oncogenic pathways. We did not assess the relativecontributions of these 2 pathways to the observed cellularphenotypes but it is notable that crosstalk between theSrc and MAPK pathways is well established. Thesepathways are commonly linked in adhesion signalingmediated by integrin and hyaluronic acid receptors, sug-gesting that Trop2 may be a component of one of suchmodule (37).Strikingly, the squamous cell tumors that arise in

Trop2�/�:Arf�/� mice are primarily sarcomatoid or poorlydifferentiated carcinomas, an observation that is consistentwith the primary human tumor data. The ability of Trop2loss to promote spindle cell histology in primary keratinocytetransformation assays provides additional evidence for a rolefor Trop2 in modulating the epithelial state. Similar to whathas been observed with established inducers of EMT, Trop2loss–induced mesenchymal transdifferentiation must occurin collaboration with other factors and may be cell typespecific (38). This is clear because Trop2 knockout miceare developmentally normal, and the pattern of E-cadherinexpression in 2 tissues that we have examined in situ(skin and breast, data not shown) is similar in wild-typeand knockout animals. In addition, EMT was not observedin immortalized Trop2�/�:Arf�/�–null keratinocytes,although E-cadherin expression is reduced in these cells.The sarcomatoid histology and E-cadherin loss observed intumors arising in Trop2�/�:Arf�/� animals are likely to bemediated at least in part through Src activity, a well estab-lished negative regulator of E-cadherin. Activation of theMAPKpathway has also been implicated in the developmentof aggressive cancer and EMT (15, 39) and may alsocontribute to Trop2 loss–induced EMT. Intriguingly, geneexpression analyses of lung and ovarian cancer cell lines thatexhibit features of EMT (as well as those from the head andneck) show strikingly low Trop2 expression than moreepithelial lines originating from the same tumors. Theseresults point to the possibility that Trop2mediates EMT in abroad range of tissues when considering the data presentedherein (40–42). Further dissection of the relationshipbetween Trop2, Src, and MAPK activation is the goal ofongoing studies.The ability of proteins to exert dual (oncogenic or tumor

suppressor) functions is well established (43, 44). Theobservations that both gain and loss of Trop2 functionprovoke strong phenotypes suggest that this protein pos-sesses potent signaling activities. Given the similarity toEpCAM, which regulates both adhesion (45) and intracel-lular signaling via cleavage products (46), Trop2 may alsopossess a pleiotropic mechanism of action. This possibilitymay reconcile the apparent contradiction that high levels ofTrop2 in oral cancers have been observed and associate witha poor outcome in one study (5). As such, studies in the oralmucosa of Trop2 knockout animals are planned to moreaccurately define the functional role of Trop2 in HNSCC,where EMT is thought to be an important prognosticfactor (47).In summary, while previous studies have highlighted the

potential importance of Trop2 gain of function in the






development of aggressive epithelial cancers, the data pre-sented herein identify for the first time a role for Trop2loss of function in tumorigenesis and mesenchymal trans-differentiation. Ongoing efforts to target Trop2 immu-nologically (10, 48) will need to take into account theeffects of its loss uncovered in this study. Further inves-tigation into Trop2 function through the loss-of-functiongenetics described in this report should provide insightsinto EMT and elucidate novel mechanisms of cancersusceptibility.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Andrea C. Santeford for assistance with image analysis and PeterSiegel, PhD, for helpful comments and suggestions.

Grant Support

L.S. Michel is supported by a Damon Runyon Clinical Investigator Award andSusan G. Komen for the Cure. J.D.Weber is supported byDepartment of Defense Eraof Hope Scholar Award (W81XWH-08-1-0178). C.H. Chung is supported by aDamon-Runyon Clinical Investigator Award (CI-28-05) and 1R01-DE017982.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMay 26, 2011; revised September 8, 2011; accepted September 27, 2011;published OnlineFirst October 4, 2011.

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2011;9:1686-1695. Published OnlineFirst October 4, 2011.Mol Cancer Res Jianbo Wang, Kaihua Zhang, Dorota Grabowska, et al. to Mesenchymal Transition in Squamous Cell Carcinoma

Promotes Carcinogenesis and Features of EpithelialTrop2Loss of

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Loss of Trop2 Promotes Carcinogenesis and Features of ... · preferentially susceptible to transformation (11). These diverse epidemiologic and functional (12) data point to an important