An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial … · investigated the expression, regulation,...

Molecular and Cellular Pathobiology

An EGFR-ERK-SOX9 Signaling Cascade Links UrothelialDevelopment and Regeneration to Cancer

Shizhang Ling1, Xiaofei Chang3, Luciana Schultz5, Thomas K. Lee1, Alcides Chaux1, Luigi Marchionni2,George J. Netto1, David Sidransky3, and David M. Berman1,2,4

AbstractLike many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better under-

standing of this association could inform improved strategies for preventing and treating this disease. Weinvestigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobilitygroup box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were highduring periods of prenatal urothelial development and diminished with maturation after birth. In adulturothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure tothe carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extra-cellular signal–regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resultedfrom activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramaticallyinduced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressedby EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion.Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n ¼ 84)compared with noninvasive cancers (n ¼ 56) or benign adjacent urothelium (n ¼ 49). These results identify anovel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasiblethrough a variety of pharmacologic approaches and may have clinical utility. Cancer Res; 71(11); 3812–21.�2011AACR.

Introduction

Cancer growth and spread requires coordinated cell migra-tion, proliferation, and stromal remodeling. Similar programsoperate in both organogenesis and injury repair (1). Repeatedinjury repair vastly increases the risk of epithelial cancers(carcinomas), particularly bladder cancer (2, 3). The processof injury repair recapitulates aspects of normal organogen-esis (4, 5), with transient reactivation of certain genes that areactive in embryonic organogenesis and quiescent in maturetissues. Chronic injury, however, may lead to sustained activa-tion of these genes, and such perseverative signals may lead, inturn, to carcinogenesis (1).

In investigating the molecular links between injury andcancer, transcription factors are appealing targets because

they have distinctive and dynamic expression profiles and canthemselves coordinate complex genetic programs. Theseproperties are illustrated by SRY-related high-mobility group(HMG) box (Sox) 9 (SOX9). SOX9 belongs to group E (SOX8,SOX9, and SOX10) of the SOX transcription factor family (6)defined by a commonHMG box domain originally identified inSRY, the sex-determining gene on the Y chromosome. SOX9has roles in epithelial invasion, migration, and proliferation asshown in developing prostate (7–9), and similar roles inprostate cancer (8). In chondrocyte development, Sox9 is amaster chondrogenic factor whose expression is induced byreceptor tyrosine kinase (RTK) signaling (10). Sox9 inductionby RTKs requires activation of mitogen-activated proteinkinase [p44/42 mitogen-activated protein kinase (MAPK) orErk1/2; ref. 10]. In this study, we investigate RTK induction ofSox9 in urothelial development, regeneration, and cancer.

Here we identify Sox9 as a molecular link between urothelialinjury and urothelial cancer. Sox9 expression coincides withurothelial proliferation during bladder organogenesis, is quies-cent in adult urothelium, and is reactivated during acutebladder injury. Sox9 induction occurs through ligand-stimu-lated activation of epidermal growth factor receptor (EGFR)and subsequent MAPK pathway activation. In contrast tobenign bladder, urothelial carcinomas (UroCa) show constitu-tive SOX9 induction through autonomous EGFR activation,and SOX9was significantly upregulated in invasive carcinomas.SOX9 knockdown significantly impaired UroCa cell migrationand invasion, suggesting its role in UroCa pathogenesis. These

Authors' Affiliations: Departments of 1Pathology, 2Oncology, 3Otolar-yngology, and 4Urology, The Johns Hopkins University School of Medi-cine, Baltimore, Maryland; and 5Pathology, Instituto de AnatomiaPatol�ogica de Paraciba, Paraciba, Brazil

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

Corresponding Author: David M. Berman, The Johns Hopkins UniversitySchool of Medicine, 1550 Orleans St, Room 545 Baltimore, MD 21231.Phone: 443-287-0878; Fax: 410-502-5742; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-3072

�2011 American Association for Cancer Research.

CancerResearch

Cancer Res; 71(11) June 1, 20113812

Research. on October 23, 2020. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 21, 2011; DOI: 10.1158/0008-5472.CAN-10-3072

http://cancerres.aacrjournals.org/

data identify a novel link between urothelial development,regeneration, and cancer.

Materials and Methods

Cell lines and cell culturesBFTC905 (German Collection of Microorganisms and Cell

Cultures) was cultured in Dulbecco's modified Eagle's medium(DMEM; Gibco, Invitrogen) with 10% FBS (Sigma) and 1%penicillin/streptomycin (Invitrogen). Human SCaBER bladdercancer cells [American Type Culture Collection (ATTC)] werecultured in DMEM with 10% FBS. UROtsa (11) cells providedby S. H. Garrett (University of North Dakota, Grand Forks, ND),were cultured in DMEM with 2 g/L glucose and 5% FBS. Themouse UroCa line MB49 (12) was provided by Dr. Yi Luo(University of Iowa, Iowa City, IA) and cultured in RPMI1640with 10% FBS. Cell line identity was assured by use within6 months of receipt from ATCC or short tandem repeatconfirmation by using reference material provided by thecontributor (UROtsa cells) or by an online database main-tained by the Deutsche Sammlung von Mikroorganismen undZellkulturen repository.

Compounds and reagentsErlotinib was purchased from Johns Hopkins Hospital

Pharmacy. All other chemicals were purchased from Sigma,unless otherwise indicated. EGF and Matrigel were purchasedfrom BD Pharmingen and Collagen I from Invitrogen.

Antibodies and immunoblottingAntibodies against EGFR, phospho-EGFR (Tyr1068), Akt,

phospho-Akt (Ser473), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204), and anti-phospho-STAT3were purchased from Cell Signaling Technology, Inc. Immu-noblotting was carried out as previously described (13). Anti-bodies against human EGFR, b-actin (#A5316), and GAPDH(#sc-32233) were purchased fromDako, Sigma, and Santa CruzBiotechnology, respectively. Anti-Sox9 antibody (#AB5535)was purchased from Chemicon. Cells were cultured inserum-free medium overnight (16 hours), pretreated withinhibitors for 3 hours, and then with EGF or heparin-binding(HB)-EGF for 24 hours.

Animals and mouse bladder injury modelC56BL6 mice (age, 6–8 weeks) were obtained from The

Jackson Laboratory. The protocol was approved by the AnimalCare and Use Committee of the Johns Hopkins University.Mice were randomly selected for a single 0.2 mL intraperito-neal (i.p.) injection of 250 mg/kg body weight of cyclopho-sphamide (CPA) or PBS (control). This dose is similar to thatused in humans receiving high dose CPA (14).

Explant cultureBladder strips were laid flat on tissue culture inserts (Milli-

cell-CM 0.4 mmol/L, 30 mm; Millipore) floated in DMEM/F-12(1:1) serum-free media with 1% ITS (10 mg/mL insulin, 5.5 mg/mL transferrin, 5 ng/mL sodium selenite; Sigma; refs. 15, 16).Inhibitors of EGFR (erlotinib) or MEK/ERK kinase [(MEK1/2)

U0126] were added and tissues were cultured for 1 day beforeprocessing for histology.

RNA extraction, reverse transcription, and real-timePCR

RNA was extracted by using Trizol (Invitrogen) followed byRNAeasy mini kit cleanup (Qiagen). RNA was reverse tran-scribed with Superscript III (Invitrogen). Primer sequences areshown in Supplementary Table S1. iTaq SYBR green Supermixwith Rox dye (Bio-Rad) was used for real-time PCR andreactions were carried out in triplicate. Quantification oftarget transcripts was calculated relative to hypoxanthinephosphoribosyltransferase (Hprt1) by using the DDCt methodwith values from injured (CPA exposed) bladders normalizedto values from uninjured (PBS) controls. Data were expressedas mean � SEM and the Student t test was used to comparethe difference in means between control and CPA-treatedsamples.

ImmunohistochemistryImmunohistochemistry (IHC) was carried out as previously

described (13). Briefly, tissue sections were serially incubatedin PBS/3% H2O2 (10 minutes at 22�C), PBS (rinse), 10% goatserum (block; 30 minutes at 22�C) rabbit anti-SOX9 antibody(1 hour at 22�C), PBS (rinse), goat anti-rabbit biotinylatedsecondary antibody (DAKO; 30 minutes at 22�C), PBS (rinse),streptavidin-HRP (DAKO; 30 minutes at 22�C), and PBS(rinse). Staining was visualized with 3,30-diaminobenzidinetetrahydrochloride (Zymed).

Growth factor and inhibitor treatmentCells cultured in fresh complete or serum-free media over-

night (16 hours) were treated with inhibitors of one of thefollowing targets: phosphoinositide 3-kinase (LY294002),MEK1/2 (U0126), p38 MAPK (PD 169316), EGFR (erlotinib),or vehicle control (DMSO) in fresh serum-free medium for 2hours, then EGF (10 ng/mL), or HB-EGF (10 or 50 ng/mL) foran additional 24 hours.

SOX9 knockdownLentiviral vector-based SOX9 and control microRNA-

adapted short hairpin small interfering RNA (shRNA mir)constructs were obtained from Open Biosystems. shRNAswere transfected into human BFTC905 UroCa cells by usingArrest-In reagent (Open Biosystems), followed by puromycin(10 mg/mL) selection for 4 weeks to generate stable clones.Clonal colonies isolated were validated by Western blot forSOX9.

Proliferation assay, scratch wound healing, andinvasion assay

Proliferation assays were carried out in 96-well platesaccording to manufacturer's instruction (ATCC) by usingMTT colorimetric assay kit. Plates were read by using aSpectraMax plate reader (Molecular Devices Corp.) at570 nm with a reference wavelength of 650 nm.

Wounds in 6-well plates were produced with a modifiedP1000 pipette tip and monitored daily. Wound area was

Sox9 Induction in Urothelial Injury and Cancer

www.aacrjournals.org Cancer Res; 71(11) June 1, 2011 3813




measured by using the measurement function in the AnalysisTab of Adobe Photoshop CS4 Extended and then exported asan Excel file for statistical analysis.

In vitro invasion assays were done with the use of 24-welltranswell Boyden chambers (17). Polycarbonate membraneinserts (Costar) were precoated with a mixture of growthfactor–reduced Matrigel (Invitrogen) and DMEM (1:1 ratio) orof Collagen I (Invitrogen) and DMEM (1:1 ratio). Bottomchambers were filled with DMEM containing 10% FBS asa chemoattractant. A total of 5�104 cells were seeded onthe top chamber and incubated for 24 hours. Invasion wasquantified as described (13). Aliquots of the same culture werealso plated in 24-well plate for MTT assay on the same day.

Tissue microarrays, IHC staining, and scoringConstruction and composition of the 2 tissue microarrays

(TMA) used in this study have been previously described (18,19). Cases were included on the basis of available tissue andfollow-up data. Control samples and cancer samples weredeemed informative when they contained morphologicallyrecognizable benign urothelium or cancer cells, respectively.The noninvasive cohort (18) included biopsies of benignurothelium, paired with corresponding low- (n ¼ 30) andhigh- (n ¼ 30) grade noninvasive papillary carcinomas eval-uated at our institution between 1971 and 1995. Of these, 28low-grade and 28 high-grade carcinomas were deemed infor-mative. In addition, 14 cases had paired benign controlsavailable for analysis. The invasive cohort (19) comprised132 cystectomies carried out in our institution between 1994and 2002. Of these, 84 invasive and 15 adjacent CIS lesions weredeemed informative. Four-micron TMA sections were stainedfor SOX9 by IHC as above. Pathologic stages for informativeinvasive UroCa cases were pT1 (n¼ 4), pT2 (n¼ 28), pT3 (n¼40), and pT4 (n ¼ 12). Intensity of SOX9 nuclear staining was

evaluated and assigned an incremental 0, 1þ, 2þ, 3þ score.Distribution of staining was categorized as absent, focal (1%–25% of cells), multifocal (25%–75%), or diffuse (>75%). Tointegrate intensity and distribution of staining, an H-scorefor SOX9 score was calculated by multiplying the intensityscore and the distribution score as previously described (19). H-scores were compared by using the 1-way ANOVA test withthe Bonferroni's post hoc pairwise comparison test. A 2-tailedP < 0.05 was required for statistical significance. Data wereanalyzed by using PASW Statistics version 18.0 (IBM Inc.).Upregulation (Fig. 6A) was calculated separately for each TMAas the ratio of H-score in cancer to H-score in benign.

Results

Induction of Sox9 during urothelial organogenesisThe rudiment of the mouse urinary bladder forms at

embryonic day 14 (e14). Urothelium proliferates and differ-entiates until the perinatal period (e17-birth; ref. 20), givingrise to the mature relatively quiescent trilaminar state con-sisting of basal stem cells, intermediate transit amplifyingcells, and fully differentiated superficial/umbrella cells (21, 22).In rapidly growing epithelium (e14), Sox9 protein inductionwas detected by IHC staining in the basal and intermediatelayers, but not in the superficial layer, with occasional stainingseen in stromal cells (Fig. 1A). Sox 9 staining diminished nearterm (e18, Fig. 1B), was barely detectable by postnatal day 2(P2; Fig. 1C), and was undetectable by P7 (Fig. 1D). SOX9 wasalso quiescent in human urothelium from organ donorsranging from age 4 to 40 (data not shown).

Urothelial injury reactivates Sox9 expressionThe chemotherapeutic agent CPA induces urothelial

injury and can cause bladder cancer in humans. In modeling

e14

SU

S U

SU

S

U

××100

A B C D

××400

e18 P2 P7

Figure 1. Elevated Sox9 levels inembryonic urothelium diminishwith maturation. IHC stains with aSox9 specific antibody show (A)intense nuclear staining in basaland intermediate cells of mousebladder mucosa at e14. B, by e18,Sox9 staining is reduced andmainly seen in the intermediatelayer. Rare superficial andintermediate cells express Sox9 atP2 (C) and Sox9 is undetectable atP7 (D). S, stroma; U, urothelium.The scale bar ¼ 50 mm.

Ling et al.

Cancer Res; 71(11) June 1, 2011 Cancer Research3814




the onset of such injury in mice, we found that Sox9 levelsrose and fell in parallel with the urothelial repair program.Urothelial necrosis, sloughing, and regeneration beganshortly after CPA administration, with peak urothelial pro-liferation at 36 hours and return to baseline by 7 days, wheninjury had healed (refs. 23, 24; Supplementary Fig. S1). Sox9was undetectable by IHC in adult controls. Within 20 hoursof CPA injection, Sox9 protein was readily detected in all 3urothelial cell layers. By 40 hours, we observed Sox9 expres-sion only in the basal cell layer, and by 7 days, the proteinwas undetectable (Fig. 2A). Thus, induction of Sox9 is atightly regulated event with temporal kinetics that trackclosely with urothelial injury repair.

EGFR pathway induction in urothelial injury repairGiven its known roles in injury repair (25, 26) and pro-

posed roles in the urothelium (26, 27), EGFR and its familymembers are likely candidates to induce Sox9. To separatelyexamine epithelial and stromal responses to injury, werapidly separated the 2 compartments for analysis at thetime of harvest after mice had been injected with PBS or CPAfor 20 or 40 hours. By reverse transcriptase (RT)-PCR ana-lysis, Egfr, Her2, and Her3 were readily detected at equiva-lent levels in urothelium (mucosa) and in the muscularbladder wall (stroma), whereas Her4 was nearly undetect-able in mucosa (Fig. 3A). Injury caused little change inreceptor transcripts (Fig. 3A). In contrast, levels of Egfrligands were markedly induced (up to 42-fold, P < 0.001)in urothelial mucosa. Amphiregulin (Areg), HB EGF-likegrowth factor, epiregulin, and epigen were the most highlyinduced ligands, with peak induction within 20 hours ofinjury (Fig. 3B; P < 0.001). Because urothelial basal cells

express EGFRs (27, 28), the induction of EGFR ligands inurothelial cells by injury suggests an autocrine/paracrinemechanism through which injury could lead to Sox9induction.

EGFR induces Sox9 through ERK1/2 signalingWe confirmed that Egfr induces Sox9 expression through

Erk1/2 signaling by using cultured strips of mouse bladder(explant culture), with the cut tissue edges simulatingurothelial injury (29). Unlike intact bladder, urothelialSox9 expression was readily detected (Fig. 3C), but thisinduction was completely blocked by culture with erlotinib(Fig. 3C), confirming Egfr-dependent activation of the path-way. Sox9 induction was also blocked by a MEK1/2 inhibitor,U0126 (Fig. 3C), indicating that canonical Egfr signalingthrough the Erk1/2 pathway is required for induction ofSox9 by injury.

We confirmed and further investigated this pattern ofSOX9 regulation in benign immortalized human UROtsacells. This line phenocopies the basal cells from which it wasderived, as evidenced by the expressions of EGFR and p63(Supplementary Fig. S2; ref. 30). Under normal cultureconditions, SOX9 protein was undetectable (Fig. 4A; Sup-plementary Fig. S2B and C). Proteolysis through the ubi-quitin-proteasome pathway likely contributes to such lowSOX9 levels, because treatment with the proteosomeblocker MG132 increased the levels of SOX9 protein (Sup-plementary Fig. S4A). SOX9 induction also resulted fromculture with EGFR ligands, including EGF, which is physio-logically enriched in urine (27), and HB-EGF, which isphysiologically synthesized by urothelial cells (31) andupregulated in urothelial cells following injury (Fig. 3A).

Figure 2. Sox9 expression israpidly but transiently induced inurothelial injury/repair. Sox9 wasundetectable in the bladders ofmice s.c. injected with PBS(control). Intense nuclear Sox9staining was detected 20 and40 hours after CPA injection, butnot at 7 days when repair wascomplete (SupplementaryFig. S1). Sox9 staining wastrilaminar at 20 hours andpredominantly basal at the40-hour time point. The scalebar is 50 mm.

××100

Time line

××400

U

S

Control 20 h 40 h 7 d






When mediated by EGF or HB-EGF, SOX9 induction wasdetectable within 6 hours of ligand exposure (Supplemen-tary Fig. S4B), consistent with the kinetics seen with CPAexposure in vivo (data not shown). Induction in this caselikely resulted from enhanced de novo synthesis of theSOX9 protein rather than enhanced protein stabilitybecause the protein translation inhibitor cycloheximideblocked SOX9 induction (Supplementary Fig. S4B). Consis-tent with results from organ culture (Fig. 3C), SOX9 induc-tion in cell lines was completely blocked by ERK1/2 or EGFRinhibition (Fig. 4A).Thus, the urothelial EGFR-ERK1/2-SOX9axis seems to operate similarly across mouse and humanspecies and can do so in the absence of stromal–epithelialinteractions.

Injurious chemicals present in urine, such as NaCl andurea (32), or released from injured cells, such as H2O2 (33)and ATP (34), are known to activate EGFR. In UROtsa cells,treatment with H2O2, NaCl, or ATP-g-S (a nonhydrolyzableform of ATP) induced coordinate EGFR phosphorylationand elevated levels of SOX9 (Fig. 4B; SupplementaryFig. S5A). Interestingly, treatment with urea affectedneither EGFR phosphorylation (Supplementary Fig. S5B),nor SOX9 levels (data not shown), suggesting that distinctpathways may mediate responses to different injuries.Thus, a variety of physiologically relevant injuries induceSOX9 in urothelial cells, and such induction may involveactivation of EGFR.

Autocrine EGFR signaling maintains constitutive SOX9elevation in carcinomas

In contrast to undetectable levels of SOX9 found in benignurothelial cells, SOX9 protein levels were high in 8 of 9 UroCacell lines tested (Supplementary Fig. S2B). As shown in humanBFTC905 (Fig. 4A; Supplementary Fig. S6B), J82 (Supplemen-tary Fig. S6A) and murine MB49 UroCa cells (SupplementaryFig. S6B), and confirmed in human SCaBER squamous bladdercarcinoma cells (Supplementary Fig. S6A), treatment with theEGFR inhibitor or with the MEK1/2 inhibitor effectively sup-pressed SOX9 to undetectable levels. In contrast, effectivepharmacologic inhibition (Supplementary Fig. S4) of Akt,p38 MAPK, STAT3, c-MET, insulin-like growth factor-1R, orplatelet-derived growth factor did not significantly alter SOX9expression (Fig. 4; Supplementary Fig. S6A and B). SOX9expression in urothelial cancer cells was further increasedby treatment of EGF or HB-EGF, but not when cells werepretreated with erlotinib or U0126 (Fig. 4A; SupplementaryFig. S6A). In addition to UroCa, we found evidence for an activeEGFR/ERK1/2/SOX9 signaling axis in a variety of other humancarcinomas, including those arising in lung, prostate, orophar-yngeal mucosa, and skin (Supplementary Fig. S7A). These dataindicate that constitutive activation of SOX9 expressionthrough EGFR and ERK1/2 is a common feature of carcinomas.This constitutive activation is also present in vivo as shown inthe corresponding xenografts of UroCa and some other carci-nomas (Supplementary Figs. S2D and 7B).

3Egfr and Her receptors

Vehicle

EGFR blockade(erlotinib)

MEK1/2 blockadeU0126

2

Fol

d ch

ange

/con

trol

Fol

d ch

ange

/con

trol

1

0

50

Mucosa

Egfr ligands

Stroma

Mucosa Stroma

40

30

20

10

0

*

*

* *

*

** *

*

20 h 40 h

20 h 40 h

Egf

rA

reg

Hb-

egf

Epg

nE

reg

Btc

Egf

Tgf

aA

reg

Hb-

egf

Epg

nE

reg

Btc

Egf

Tgf

a

Her

3

Her

2

Her

4

Egf

r

Her

3

Her

2

Her

4

A

B

C Figure 3. Upregulation of Sox9 inurothelial injury by Egfr familyreceptors and ligands. A, real-timeRT-PCR shows the levels ofmRNA transcripts encoding Egfr,Her2, Her3, and Her4 receptors inthe bladder wall (stroma, right) andurothelial mucosa (left), comparedwith controls. The inductions werenot significant, except for Her4,which showed decreasedexpression in CPA-injured mouseurothelial mucosa. *, P < 0.05. B,transcripts encoding Egfr familyligands Areg, Hb-egf, Epigen(Epgn), and Epiregulin (Ereg), weremarkedly induced in urothelialmucosa (left) by CPA injury, butnot in bladder wall (stroma, right).The quantitative data shown in Aand B were averaged from 5bladders in each condition (PBScontrol and CPA injury). Values areexpressed as mean � SEM.*, P < 0.001. C, photomicrographs(bar ¼ 50 mm) show intensenuclear IHC staining for Sox9 insurgically injured bladder explantscultured in control media (vehicle).Twenty four-hour culture witherlotinib or U0126 resulted inundetectable Sox9 levels.Representative images from 6independent experiments (n ¼ 6).

Ling et al.





Constitutive SOX9 expression may result fromautocrine/paracrine signaling by HB-EGF, a phenomenon pre-viously observed to promote growth in urothelial cell cultures(31). Heparin binds EGFR ligands, HB-EGF, and AREG with highaffinity. Heparin reduced SOX9 expression (SupplementaryFig. S6C), suggesting that UroCa cells produce EGFR ligands,HB-EGF, and AREG, to sustain constitutive SOX9 expression.

Requirement for SOX9 in cell migration and invasion,but not proliferationUrothelial injury repair likely requires coordinated prolifera-

tion anddifferentiation of basal cells as theymigrate to cover thewound and reconstitute the urothelial barrier. To addresspotential roles of SOX9 in this process, weused stable expressionof 2 different shRNAs to generate humanBFTC905UroCa clonesdeficient in SOX9 (Supplementary Fig. 8A & B). Comparingclones with low (clone 16), intermediate (clone 5), and high(control) levels of SOX9 (Fig. 5A; Supplementary Fig. S8B),effective reduction of SOX9 elicited no significant change ingrowth rate (Fig. 5B).However, inproportion to the effectivenessof SOX9 reduction, SOX9-deficient BFTC905 cells showedmark-edly impaired abilities to migrate and cover a wound scrapedacross a culture dish (Fig. 5C). Consistent with a migrationdefect, SOX9-deficient BFTC905 cells displayed decreased abil-ities to invade Matrigel- and collagen-coated membranes intranswell (Boyden) chamber assays (Fig. 5D; SupplementaryFig. S9). This phenotype was confirmed by using separateshRNAs targeting 2 distinct regions of the SOX9 transcript,indicating that the migration effect is an "on-target" effect. Byusing transient SOX9 knockdown, this effect was further con-firmed in another human UroCa cell line, UM-UC-3 (Supple-mentary Fig. S10). T24 UroCa cells, in contrast, were unaffected,indicating that they use substitute or redundant pathways formigration. Although not universal, these results confirm a rolefor SOX9 in UroCa invasion and migration.

Sox9 is reexpressed in UroCaExpression analysis in primary human samples suggests a

general role for SOX9 in UroCa, particularly in invasive can-cers. UroCa arises through 2 divergent pathways (reviewed inref. 35). The majority originates in an indolent form withpapillary formations that extend into the bladder lumen. Aminority originates as flat or invasive forms, metastasizesearly, and is often lethal. In analysis of SOX9 IHC by H-score(intensity� proportion of stained cells), cancers scored higherthan benign urothelia (P ¼ 0.0001). Compared with noninva-sive papillary cancers, SOX9 induction was more dramatic inmore aggressive flat/invasive cancers (P < 0.03). SOX9 induc-tion was 7-fold for CIS and 14-fold for invasive cancers(Fig. 6A). Induction was observed frequently. Compared withbenign urothelium, SOX9 staining was elevated in 75% of CIScases and 89% of invasive cases (data not shown). However,SOX9 staining was as high in early stage (pathologic stage pT1)invasive carcinomas as in advanced (pT3 or greater) cases(data not shown). Thus, SOX9 induction seems to be a generalproperty of UroCa, particularly of the more aggressive flat/invasive pathway, consistent with the notion that the proteinis induced early in the course of urothelial carcinogenesis.

Discussion

In embryonic urothelium and urothelium undergoing injuryrepair, Sox9 is expressed in basal and intermediate cells, butnot in terminally differentiated superficial cells. This expres-sion pattern suggests that the transcription factor may antag-onize urothelial differentiation, a role consonant with thatseen in preadipocytes (36), chondrocytes (37), pyloric sphinc-ter epithelial cells (38), and early differentiation of prostatebud epithelia (7).

Sox9 expression is limited to the invasive front of epithelialbuds in developing prostate (7–9) and lung (39) as the buds

Figure 4. Regulation of SOX9 bygrowth factors and injury. A, SOX9was induced in UROtsa andBFTC905 cells by treatment ofEGF or HB-EGF. Induction wasblocked by small moleculeinhibitors of ERK1/2 (U0126) orEGFR (erlotinib), but not PI3Kkinase (LY294002) or p38 MAPK(PD169316). B, immunoblotsshowing induction of EGFRphosphorylation and SOX9protein levels in UROtsa cellsfollowing treatment with hydrogenperoxide (15 minutes or 24 hours)or sodium chloride (15 minutes or24 hours).

EGF

A

B

HB-EGFSOX9

SOX9

β-Actin

β-Actin

β-Actinβ-Actin

SOX9

10100 250

SOX9

SOX9

β-Actin

β-Actin

– – + ––– 10 50 10 50 10 50+ + + + + + +Con

trol (

CM)

Con

trol

Con

trol

Con

trolEG

F

EG

F

Contro

l (CM

)

DMSO

Erlotin

ib

Contro

l (SFM

)

DMSO

Contro

l (DM

SO)

Contro

l (DM

SO)

LY29

4002

U0126

U0126

PD1693

16

Erlotin

ib

UROtsa

BFTC905

H2O2 (μmol/L)

H2O2 (μmol/L)10 100 250 500 7501000 10 50

NaCl (mmol/L)

p-EGFR

EGFR

100 250 500

UROtsa

BFTC905






enter surrounding mesenchyme. Tissue-specific knockout inthe prostate prevents bud outgrowth, likely through defectivegrowth, migration, or both. shRNA-mediated knockdown ofSOX9 expression in UroCa cells can result in a migrationdefect without affecting tumor cell growth. Although morestudies would be needed to distinguish roles in embryonicgrowth versus injury repair, it may be that the proliferativerole of SOX9 operates mainly in primitive embryonic cellswhereas the migratory role is common to both organogenesisand injury repair.

In normal undamaged bladder, an overlying urine–bloodbarrier formed by superficial cells (20, 40) is hypothesized toprotect the EGFR-enriched basal cell layer (27, 28, 41) fromcontact with urinary EGFR ligands (especially EGF). Inaddition to the possibility that basal urothelial cells mightrespond to urinary ligands, urothelium and adjacent smoothmuscle can produce EGFR ligands in response to injury,including TGFa (42), HB-EGF, and Epiregulin (43). It has notbeen previously shown that injury to urothelial tissue acti-vates EGFR signaling, and the sequelae of such activationhave not been previously determined. Here we provide newevidence that HB-EGF and AREG are significantly inducedby injury in urothelial tissue (Fig. 3A), supporting an auto-crine mode of action in activating EGFR. Of particularinterest, HB-EGF has been found to be an autocrine growthfactor for human urothelial cells (31). This finding, coupledwith the induction of this ligand by urothelial tissue injury(Fig. 3) and evidence that HB-EGF induces SOX9 in UroCacells (Supplementary Fig. S6E and F), suggest a mechanismlinking migration induced by injury to that operating incancer.

We have discovered a role for SOX9 in cancer cellmigration and invasion, indicating that SOX9 might med-iate EGFR-induced cancer spread. We further provideevidence for this hypothesis by showing that SOX9 hassignificantly higher expression in the flat/invasive path-way of UroCa compared with noninvasive tumors orbenign urothelium (Fig. 6A). The role of SOX9 in cellmigration is also consistent with the notion that urothe-lial cells are very mobile during injury repair and need tomigrate to the superficial layer and to differentiate toheal. In UroCa cells, aberrant expression of EGFR recep-tors and ligands that lead to constitutive induction ofSOX9 also support the invasive migratory phenotype ofthese cells. This notion is further supported by our recentfinding that EGFR ligands, HB-EGF, and NRG2, werehighly expressed in a highly tumorigenic basal cell com-partment in UroCa (44) that is also enriched for EGFR(Supplementary Fig. S2A) and SOX9 expression (Supple-mentary Fig. S2D).

Our findings have implications for bladder injury repairand carcinogenesis. Cancer is long been thought as a chronicwound that does not heal (45). The ultimate source of cellsfor repairing injured tissue is stem/progenitors cells. Fortissues with a slow turnover like urothelium, stem/progeni-tor cells remain quiescent. However, when injury occurs,cells can rapidly migrate (a process known as epithelialrestitution), proliferate, differentiate, and remodel to healthe wound.

A common trait of both cancer and repair is the activa-tion of signaling pathways best known for their roles inembryonic growth and patterning. We (1, 46) hypothesize

Con

trol

SOX9

clo

ne 5

SOX9

clo

ne 1

6

shRNA

Control shRNA Sox9 shRNA #16

SOX9

ββ-Actin

A

C

B D

0 h

72 h

70Control shRNA

SOX9 shRNAClone #16SOX9 shRNA

ControlshRNA

SOX9 shRNAClone #16

ControlshRNA Control shRNA

SOX9 shRNA S16

Matrigel

SOX9 shRNAClone #16

2.00

1.50

1.00

OD

570

0.50

0.00Day 1 Day 2 Day 3 Day 4

Clone #5

60

50

40

Per

cent

age

of w

ounf

clo

sure

Num

ber

of c

ells

/vie

wfie

d (2

00×)

30

20

10

00 24

** *

** *

* *

* *

*

Hours after scratch wounding

48 72

300

200

100

0Matrigel

Figure 5. SOX9 is required forUroCa migration and invasion.A, immunoblot showing SOX9protein levels in independentBFTC905 UroCa clonesengineered to stably expresscontrol shRNA or shRNAs thattarget SOX9. Note that eachimmunoblot was done on thesame membrane as theSupplementary Fig. S8B withunrelated lanes removed andrepresented as gaps. B, MTTassay showing no effect of SOX9knockdown on cell growth.C, control clones covered woundsscratched more rapidly than SOX9shRNA clones 5 or 16. D,representative photomicrographs(top and middle panels) showingcells that invaded through theMatrigel layered onto a filter with8-mm pores (modified Boydenchamber assay). The quantitativedata shown are averaged across 3independent experiments. Valuesare expressed as mean � SEM.*, P < 0.05; **, P < 0.01.

Ling et al.





that chronic injury acts on tissue stem cells/progenitors topermanently activate survival, proliferation, and migration,all of which are prominent features of cancer. As part ofthis process, we propose that urothelial cells become cellautonomous for EGFR activation during urothelial injury,and that such activation induces SOX9 expression to sup-port epithelial migration and wound repair. In chronicinjury, unknown genetic or epigenetic mechanisms couldlock this signaling circuit in the active state, contributingto malignant transformation of urothelial cells. Futurestudies will be needed to expand this pathway upstreamto identify mediators of sustained EGFR ligand expressionand downstream to discover the molecular links betweenSOX9 and the migration machinery.In the bladder, epidemiologic and experimental evi-

dence has linked human and animal bladder cancers,both squamous and urothelial (reviewed in ref. 47) tochronic injury through chronic parasitic infection (48),or environmental exposure to arsenic (49, 50) or inorganiccadmium (50). Recent evidence indicates that arseniccan also induce EGFR ligands and activate urothelialEGFR and Erk1/2 (26). In these situations, aberrant acti-vation of EGFR, as well as SOX9 is anticipated based onour data.

Effective treatment of any cancer will likely require combi-nations of targeted therapies that overcome resistancemechanisms and redundant signaling circuits. A better under-standing of inducers and effectors of this newly recognizedEGFR-ERK1/2-SOX9 pathway has the potential to aid in thiseffort.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Dr. Yi Luo (University of Iowa) and Dr. Scott H. Garrett(University of North Dakota) for MB49 and UROtsa cell lines, respectively, and toPaula Hurley and Will Brandt for critical reading of the manuscript.

Grant Support

This study is funded by NIH R01 grant (project number: 5R01DK072000-05).The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 23, 2010; revised February 18, 2011; accepted March 7, 2011;published OnlineFirst April 21, 2011.

A B

Benign

Cancer

SOX9

SOX9

Repair

Migration

SOX9ERK1/2

MEK1/2

RAF

RAS

EGFR

Nucleus

ROSHyperosmolarity

INJURY

U0126

Erlotinib

EGFR Ligands(HB-EGF, EGF, etc)

20O

vere

xpre

ssio

n(F

old) 15

1050

Gradestage

Low

**

*

Papillary Flat InvasiveHigh

Figure 6. SOX9 is overexpressed in human bladder cancer tissues. A, IHC SOX9 staining showed undetectable or low levels in benign urothelium(n ¼ 49; representative section in A) staining (both distribution and intensity) was slightly elevated in noninvasive carcinomas (n ¼ 56; staining not shown),more widespread and intense in the majority of high-grade flat carcinoma in situ (Flat) lesions (n ¼ 15; staining not shown) and invasive human UroCas(n ¼ 84; A, middle panel). Overall, mean SOX9 scores for cancer exceeded those for benign urothelium (*, P ¼ 0.0001), and mean scores for invasivecancers exceeded those for noninvasive cancers († P < 0.03). Compared with benign urothelium, SOX9 scores were slightly elevated in low-gradepapillary noninvasive cancers (not significant) but became increasingly elevated in high-grade and invasive tumors (*, P < 0.05). B, model of EGF ligand-receptor-ERK1/2-SOX9 induction by injury. Note that the system turns off in response to completion of injury repair. With chronic injury, the systemmay remain activated and facilitate cancer formation.






References1. Beachy PA, Karhadkar SS, Berman DM. Mending and malignancy.

Nature 2004;431:402.2. Kundu JK, Surh YJ. Inflammation: gearing the journey to cancer.

Mutat Res 2008;659:15–30.3. Michaud DS. Chronic inflammation and bladder cancer. Urol Oncol

2007;25:260–8.4. Martin P, Parkhurst SM. Parallels between tissue repair and embryo

morphogenesis. J Embryol Exp Morphol 2004;131:3021–34.5. Ingber DE, Levin M. What lies at the interface of regenerative medicine

anddevelopmental biology?JEmbryol ExpMorphol 2007;134:2541–7.6. Lefebvre V, Dumitriu B, Penzo-Mendez A, Han Y, Pallavi B. Control

of cell fate and differentiation by Sry-related high-mobility-groupbox (Sox) transcription factors. Int J Biochem Cell Biol 2007;39:2195–214.

7. Thomsen MK, Francis JC, Swain A. The role of Sox9 in prostatedevelopment. Differentiation 2008;76:728–35.

8. Wang H, Leav I, Ibaragi S, Wegner M, Hu GF, Lu ML, et al. SOX9 isexpressed in human fetal prostate epithelium and enhances prostatecancer invasion. Cancer Res 2008;68:1625–30.

9. Schaeffer EM, Marchionni L, Huang Z, Simons B, Blackman A, Yu W,Parmigiani G, Berman DM. Androgen-induced programs for prostateepithelial growth and invasion arise in embryogenesis and are reacti-vated in cancer. Oncogene 2008;27:7180–91.

10. Murakami S, Kan M, McKeehan WL, de Crombrugghe B. Up-regula-tion of the chondrogenic Sox9 gene by fibroblast growth factors ismediated by the mitogen-activated protein kinase pathway. Proc NatlAcad Sci U S A 2000;97:1113–8.

11. Rossi MR, Masters JRW, Park S, Todd JH, Garrett SH, Sens MA, et al.The immortalized UROtsa cell line as a potential cell culture model ofhuman urothelium. Environ Health Perspect 2001;109:801–8.

12. Summerhayes IC, Franks LM. Effects of donor age on neoplastictransformation of adult mouse bladder epithelium in vitro. J NatlCancer Inst 1979;62:1017–23.

13. Kleeberger W, Bova GS, Nielsen ME, Herawi M, Chuang AY, EpsteinJI, et al. Roles for the stem cell associated intermediate filament nestinin prostate cancer migration and metastasis. Cancer Res 2007;67:9199–206.

14. Klein J, Rey P, Dansey R, Karanes C, Abella E, Cassells L, Hamm C,et al. Cyclophosphamide and paclitaxel as initial or salvage regimenfor the mobilization of peripheral blood progenitor cells. Bone MarrowTransplant 1999;24:959–63.

15. Berman DM, Desai N, Wang X, Karhadkar SS, Reynon M, Abate-ShenC, et al. Roles for Hedgehog signaling in androgen production andprostate ductal morphogenesis. Dev Biol 2004;267:387–98.

16. Grishina IB, Kim SY, Ferrara C, Makarenkova HP, Walden PD. BMP7inhibits branching morphogenesis in the prostate gland and interfereswith Notch signaling. Dev Biol 2005;288:334–47.

17. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA,Kozlowski JM, et al. A rapid in vitro assay for quantitating the invasivepotential of tumor cells. Cancer Res 1987;47:3239–45.

18. Cheung WL, Albadine R, Chan T, Sharma R, Netto GJ. Phosphory-lated H2AX in noninvasive low grade urothelial carcinoma of thebladder: correlation with tumor recurrence. J Urol 2009;181:1387–92.

19. Schultz L, Albadine R, Hicks J, Jadallah S, DeMarzo AM, Chen YB,et al. Expression status and prognostic significance of mammaliantarget of rapamycin pathway members in urothelial carcinoma ofurinary bladder after cystectomy. Cancer 2010;116:5517–26.

20. Jezernik K, Pipan N. Blood-urine barrier formation in mouse urinarybladder development. Anat Rec 1993;235:533–8.

21. Lewis SA. Everything you wanted to know about the bladder epithe-lium but were afraid to ask. Am J Physiol Renal Physiol 2000;278:F867–74.

22. Brandt W, Matsui W, Rosenberg J, He X, Ling S, Schaeffer E, et al.Urothelial carcinoma: stem cells on the edge. Cancer Metastasis Rev2009;28:291–304.

23. Farsund T. Cell kinetics of mouse urinary bladder epithelium. II.Changes in proliferation and nuclear DNA content during necrosisregeneration, and hyperplasia caused by a single dose of cyclopho-sphamide. Virchows Arch B Cell Pathol 1976;21:279–98.

24. Anton E. Delayed toxicity of cyclophosphamide on the bladder ofDBA/2 and C57BL/6 female mouse. Int J Exp Pathol 2002;83:47–53.

25. Wang Z, Chen JK, Wang SW, Moeckel G, Harris RC. Importance offunctional EGF receptors in recovery from acute nephrotoxic injury. JAm Soc Nephrol 2003;14:3147–54.

26. Eblin KE, Bredfeldt TG, Buffington S, Gandolfi AJ. Mitogenic signaltransduction caused by monomethylarsonous acid in human bladdercells: role in arsenic-induced carcinogenesis. Toxicol Sci 2007;95:321–30.

27. Messing EM, Hanson P, Ulrich P, Erturk E. Epidermal growth factor–interactions with normal and malignant urothelium: in vivo and in situstudies. J Urol 1987;138:1329–35.

28. Chow NH, Liu HS, Yang HB, Chan SH, Su IJ. Expression patterns oferbB receptor family in normal urothelium and transitional cell carci-noma. An immunohistochemical study. Virchows Arch 1997;430:461–6.

29. Sun TT. Altered phenotype of cultured urothelial and other stratifiedepithelial cells: implications for wound healing. Am J Physiol RenalPhysiol 2006;291:F9–21.

30. Eblin KE, Bredfeldt TG, Gandolfi AJ. Immortalized human urothelialcells as a model of arsenic-induced bladder cancer. Toxicology2008;248:67–76.

31. Freeman MR, Yoo JJ, Raab G, Soker S, Adam RM, Schneck FX, et al.Heparin-binding EGF-like growth factor is an autocrine growth factorfor human urothelial cells and is synthesized by epithelial and smoothmuscle cells in the human bladder. J Clin Invest 1997;99:1028–36.

32. Lezama R, Diaz-Tellez A, Ramos-Mandujano G, Oropeza L, Pasantes-Morales H. Epidermal growth factor receptor is a common element inthe signaling pathways activated by cell volume changes in isosmotic,hyposmotic or hyperosmotic conditions. Neurochem Res 2005;30:1589–97.

33. Goldkorn T, Balaban N, Matsukuma K, Chea V, Gould R, Last J, et al.EGF-receptor phosphorylation and signaling are targeted by H2O2redox stress. Am J Respir Cell Mol Biol 1998;19:786–98.

34. Yin J, Xu K, Zhang J, Kumar A, Yu FS. Wound-induced ATP releaseand EGF receptor activation in epithelial cells. J Cell Sci 2007;120:815–25.

35. Wu XR. Urothelial tumorigenesis: a tale of divergent pathways. NatRev Cancer 2005;5:713–25.

36. Wang Y, Sul HS. Pref-1 regulates mesenchymal cell commitment anddifferentiation through Sox9. Cell Metab 2009;9:287–302.

37. de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S,Huang W. Transcriptional mechanisms of chondrocyte differentiation.Matrix Biol 2000;9:389–94.

38. Moniot B, Biau S, Faure S, Nielsen CM, Berta P, Roberts DJ, et al.SOX9 specifies the pyloric sphincter epithelium through mesenchy-mal-epithelial signals. J Embryol Exp Morphol 2004;131:3795–804.

39. Okubo T, Knoepfler PS, Eisenman RN, Hogan BLM. Nmyc plays anessential role during lung development as a dosage-sensitive regu-lator of progenitor cell proliferation and differentiation. J Embryol ExpMorphol 2005;132:1363–74.

40. Lavelle J, Meyers S, Ramage R, Bastacky S, Doty D, Apodaca G, et al.Bladder permeability barrier: recovery from selective injury of surfaceepithelial cells. Am J Physiol Renal Physiol 2002;283:F242–53.

41. Messing EM. Clinical implications of the expression of epidermalgrowth factor receptors in human transitional cell carcinoma. CancerRes 1990;50:2530–7.

42. Baskin LS, Sutherland RS, Thomson AA, Nguyen HT, Morgan DM,Hayward SW, et al. Growth factors in bladder wound healing. J Urol1997;157:2388–95.

43. Mysorekar IU, Mulvey MA, Hultgren SJ, Gordon JI. Molecular regula-tion of urothelial renewal and host defenses during infection withuropathogenic Escherichia coli. J Biol Chem 2002;277:7412–9.

44. He X, Marchionni L, Hansel DE, Yu W, Sood A, Yang J, et al.Differentiation of a highly tumorigenic basal cell compartment inurothelial carcinoma. Stem Cells 2009;27:1487–95.

45. Dvorak HF. Tumors: wounds that do not heal. Similarities betweentumor stroma generation and wound healing. N Engl J Med1986;315:1650–9.

Ling et al.





46. Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cellrenewal in carcinogenesis. Nature 2004;432:324–31.

47. BurinGJ,GibbHJ,Hill RN.Humanbladder cancer: evidence for a potent-ial irritation-induced mechanism. Food Chem Toxicol 1995;33:785–95.

48. Gelfand M,Weinberg RW, Castle WM. Relation between carcinoma ofthe bladder and infestation with Schistosoma haematobium. Lancet1967;1:1249–51.

49. Smith AH, Goycolea M, Haque R, Biggs ML. Marked increasein bladder and lung cancer mortality in a region of northernchile due to arsenic in drinking water. Am J Epidemiol 1998;147:660–9.

50. Sens DA, Park S, Gurel V, Sens MA, Garrett SH, Somji S. Inorganiccadmium- and arsenite-induced malignant transformation of humanbladder urothelial cells. Toxicol Sci 2004;79:56–63.






2011;71:3812-3821. Published OnlineFirst April 21, 2011.Cancer Res Shizhang Ling, Xiaofei Chang, Luciana Schultz, et al. Development and Regeneration to CancerAn EGFR-ERK-SOX9 Signaling Cascade Links Urothelial

Updated version

10.1158/0008-5472.CAN-10-3072doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2011/04/21/0008-5472.CAN-10-3072.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/71/11/3812.full#ref-list-1

This article cites 50 articles, 8 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/71/11/3812.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/71/11/3812To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-10-3072

http://cancerres.aacrjournals.org/content/suppl/2011/04/21/0008-5472.CAN-10-3072.DC1

http://cancerres.aacrjournals.org/content/71/11/3812.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/11/3812.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/71/11/3812


An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial … · investigated the expression, regulation,...

Documents

Transcript of An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial … · investigated the expression, regulation,...