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Research Article

Divergent Roles of PAX2 in the Etiology andProgression of Ovarian CancerEnsaf M. Al-Hujaily1,2,3,Yong Tang4, De-Sheng Yao5, Euridice Carmona6, Kenneth Garson1,and Barbara C. Vanderhyden1,2

Abstract

PAX2 is an essential transcription factor for development.Aberrant PAX2 expression in adult tissues is associated withcarcinogenesis and experimental evidence shows that PAX2 gen-erally exhibits oncogenic properties. Although PAX2 is notexpressed in normal ovaries, it is highly expressed in low malig-nant potential and low-grade epithelial ovarian tumors, suggest-ing that PAX2 induction in ovarian surface epithelium (OSE)maycontribute to transformation. Herein, we provide evidence thatexpressionof PAX2 innormalmurineOSE cells (mOSE) enhancestheir proliferation and survival and, with loss of p53, inducestumorigenicity. PAX2 expression in murine ovarian cancer cells

enhanced or inhibited tumorigenicity, depending on the modelsystem. In RM cells (mOSE transformed by K-RAS and c-MYC),PAX2 expression inhibited p53 and induced pERK1/2 and COX2,resulting in enhanced angiogenesis and decreased apoptosis oftumors arising from these cells. However, in a murine model ofhigh-grade serous ovarian cancer (STOSE), PAX2 expressionimproved animal survival by reducing proliferation and metas-tasis, which correlated with increasedHtra1 and decreased COX2.Thus, PAX2 may not be a classical oncogene or tumor suppressorbut instead can act in either role by differential regulation ofCOX2 and/or HTRA1. Cancer Prev Res; 8(12); 1163–73. �2015 AACR.

IntroductionEpithelial ovarian carcinoma (EOC) has been divided into two

major subgroups with distinct molecular characteristics and clin-ical behavior (1). Type I tumors include low-grade serous (LGSC),low-grade endometrioid, clear cell, and mucinous carcinomasand are generally associated with mutations in KRAS, BRAF,ERBB2, PIK3CA, and CTNNB1 (2). Patients with type II tumorsencompass 75% of all EOC cases and include high-grade serous(HGSC), endometrioid, and undifferentiated carcinomas (1).They frequently harbor TP53 mutations; 15% have BRCA1 orBRCA2 mutations and 20% have CCNE1 amplification (3).Unfortunately, they are often diagnosed at an advanced stage(2), resulting in poor understanding of the pathogenesis of EOC.

The putative oncogene, PAX2, is highly expressed in lowmalig-nant potential tumors (59%) and LGSC (63%) and ciliatedinclusion cysts, which are thought to be precursor lesions forovarian carcinogenesis (4). PAX2 is not expressed in normal

ovaries (4), suggesting that induction of its expression might bean early step in ovarian tumorigenesis. PAX2 is a member of thepaired-box binding protein family (PAX) of transcription factorsand is expressed in the developing urogenital tract, inner ear, andbrain (5). After development, Pax2 expression is usually attenu-ated (5, 6), except in putative stem cells in breast, kidney, andfemale reproductive tract (4, 6, 7), where it promotes cell survivaland tissue regeneration (8).

Aberrant expression of PAX2, as found in several carcinomas,promotes cell proliferation, survival, and chemoresistance (6, 9).PAX2 enhances tumor progression or chemoresistance in xeno-graft models for endometrial, colon, and renal cancers (10–12).PAX2 was proposed as a potential proto-oncogene, as ectopicexpression of Pax2 led to transformation of rat fibroblast cells(13); however, constitutive expression of PAX2 in transgenicmiceled to cyst formation in kidneys with no evidence of tumorincidence (14).

Although PAX2 expression in ovarian cancers has been exten-sively reported (4, 7, 9, 15), its role in ovarian tumorigenicity isuncertain, due to a limited number of experimental reports andtheir conflicting outcomes. Notably, PAX2 knockdown in someovarian cancer cell lines increased apoptosis and reduced cellularproliferation and/or migration (9, 15). However, overexpressingPAX2 in other cell lines suppressed cell proliferation (15).Although the mechanism behind the divergent roles of PAX2 isstill unknown, comparison of cell lines suggests that PAX2 mightact as a tumor suppressor in serous carcinoma with mutant TP53,whereas it may be oncogenic in cells with wild-type TP53. Todetermine whether induction of PAX2 expression is an earlyevent in ovarian epithelial transformation, we used mouse ovar-ian surface epithelial (mOSE) cell lines with and without p53expression. The role of PAX2 in tumor progression was alsoevaluated in two different ovarian cancer model systems. To ourknowledge, this is the first report that examines both in vitro and

1Centre for Cancer Therapeutics, Ottawa Hospital Research Institute,Ottawa, Canada. 2Department of Cellular and Molecular Medicine,University of Ottawa, Ottawa, Canada. 3King Abdullah InternationalMedical Research Center, Riyadh, Saudi Arabia. 4Department of Urol-ogy, Affiliated Cancer Hospital of Guangxi Medical University, Nan-ning, China. 5Department of Gynecologic Oncology, Affiliated CancerHospital of Guangxi Medical University, Nanning, China. 6Centre derecherche duCentre hospitalier de l'Universit�edeMontr�eal, Institut ducancer de Montr�eal, Montr�eal, Qu�ebec, Canada.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Barbara C. Vanderhyden, Ottawa Hospital ResearchInstitute, 501 Smyth Road, 3rd Floor, Box 926, Ottawa K1H 8L6, Canada. Phone:613-737-7700; Fax: 1-613-247-3524; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-15-0121-T

�2015 American Association for Cancer Research.

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in vivo the consequence of PAX2 induction on the behavior ofnormal ovarian epithelial cells and ovarian cancer cells. Noveltargets of PAX2 that are involved in tumor progression were alsoidentified.

Materials and MethodsCell lines

Two independent isolations of mOSE cells (M0505 andM1102) were obtained from 6-week-old FVB/N female mice asdescribed previously (16). Long-term passage of M0505 cells ledto spontaneous transformation (STOSE; ref. 16). An independentculture of mOSE cells was obtained from Trp53loxP/loxP mice(mOSE-fxp53). Treatment with the recombinant adenovirusAd5CMVCre (AdCre; Vector Development Laboratory) results inthe loss of functional p53 by excision of exons 2–10 (p53D2-10-mOSE), confirmed as described by Clark-Knowles and colleagues(17). Mouse ovarian cancer cells (RM cells) were derived frommOSE cells obtained from Immortomice having temperature-permissive T-Antigen (TAg) expression. Following TAg-inducedimmortalization, cells were maintained at a nonpermissive tem-perature and transduced with retrovirus constructs to achieveexpression of mutant K-Ras (KRASG12D) and Myc (18). All celllines are of murine origin and were validated as syngeneic(STOSE) or confirmed to express RASG12D and MYC (RM cells).All cells were maintained as previously described (16).

InfectionThe murine Pax2 cDNA, corresponding to pax2-b variant, was

cloned from murine oviduct and used to generate a lentivirusexpression vector (WPI-Pax2-IRES-eGFP, hereafter PAX2). Theempty vector, pWPI (Addgene plasmid 12254), was used as acontrol. Lentiviral vectors were prepared by cotransfection ofvector plasmids (pWPI or pWPI-Pax2), packaging plasmidpCMVR8.74 (Addgene plasmid 22036), and the ecotropic enve-lope expression plasmid, pCAG-Eco (Addgene plasmid 35617)into 293T cells. After infecting the cells with either PAX2 or WPI,cells were passaged at least 3 times and sorted for GFP expressionby fluorescence-activated cell sorting (Beckman Coulter, Inc.).Both the WPI and PAX2 integrated proviruses harbor loxP sites inboth 50 and 30 LTRs allowing AdCre-mediated deletion. Deletionof both GFP and Pax2 was achieved by treating PAX2-expressingcells with AdCre and sorting them for GFP negativity (referred toas PAX2þCre). Except for the mOSE-fxp53 cells, infections weredone twice for each cell line, with similar results.

Cell proliferation and survival assaysCells were plated at 2 � 105 cells per well in 12-well plates

(mOSE) or 1� 105 cells perwell in 6-well plates (p53D2-10-mOSE,STOSE, and RM cells). Detached and attached cells were countedusing a Vi-CELL XR Cell Viability Analyzer (Beckman Coulter,Inc.).

For survival analysis, 24 hours after plating, cells were washedwith PBS and refreshed with serum-free, growth factor–freemedia. After 48 hours in this starvation condition, cell viabilitywas measured using a Vi-CELL XR Cell Viability Analyzer.

For chemosensitivity, 1� 104 cells were plated in 96-well platesand treated with cisplatin (12–50 mg/mL) the next day. Viabilitywas assessed after 48 hours by Alamar Blue (Sigma-Aldrich), withabsorbance measured using Fluoroskan Ascent FL (ThermoScientific).

AnimalsAnimal experiments were performed in accordance with the

Canadian Council on Animal Care'sGuidelines for the Care andUseof Animals and the University of Ottawa's Animal Care Commit-tee. Cells (1� 107 in 500mL PBS)were injected into the peritonealcavity of 8-week-old SCID or FVB/N mice (The Jackson Labora-tory). Disease progressionwasmonitored until humane endpoint(>15%weight gain, abdominal distension). Tumors were fixed in10% buffered formalin for 24 hours, paraffin-embedded, andsectioned at 5 mm for histologic analysis or were snap-frozen forRNA or protein analysis.

Gene expression analysisGene expression was determined by quantitative PCR (QPCR),

as previously described (16) using the primer pairs listed inSupplementary Table S1. Microarray analysis (accession numberGSE69238) was performed on M0505 þ WPI, M0505 þ PAX2,and M0505 þ PAX2 þ Cre as described previously (16).

Western blot analysisProteinswere extracted from cell lines and tumors as previously

described (16). Blots were incubated with primary antibodiesovernight (see Supplementary Table S2) and then incubated withthe corresponding secondary antibodies and imaged on aFluorChem E gel documentation system (ProteinSimple).

ImmunohistochemistryProtein expression was visualized with anti-PAX2 (1:1,000,

Abcam), anti-Ki-67 (1:300, Millipore), and anti-CD31 (1:100,Abcam) primary antibodies and Dako Envision system-HRP–labeledpolymer anti-rabbit secondary antibodies (K4003,Dako),with detection using diaminobenzidine as a substrate (0.2%DAB,0.001%H2O2; Sigma-Aldrich). Sectionswere counterstainedwithHarris hematoxylin (Fisher Scientific) and mounted with Per-mount (Fisher Scientific). Images were obtained with the Scan-Scope CS system and ImageScope software (Leica MicrosystemsInc.) and 5 random areas for 3 to 4 tumors per group wereanalyzed for pixel positivity.

Statistical analysesOn the basis of the number of conditions tested, statistical

significancewas determinedby t test, ANOVA(Tukeypost-test), orlog-rank test (Kaplan–Meier), performed by GraphPad Prismsoftware (GraphPad).

ResultsPAX2 enhances proliferation in mOSE cells

Similar to its expression in the human reproductive tract (4, 7),PAX2was expressed in themurine oviduct and uterus, but not theovary (Supplementary Fig. S1). To investigate the biologic con-sequences of PAX2 induction in normal mOSE cells, three inde-pendent isolations of mOSE cells were placed in primary culture(M0505, M1102, and mOSE-fxp53). Forced expression of PAX2proteinswas confirmed by immunoblotting (Fig. 1A; Supplemen-tary Fig. S2A). In all three mOSE cell lines, PAX2 significantlyenhanced cell proliferation (Fig. 1B; Supplementary Fig. S2B)without affecting viability (data not shown). When control cellswere deprived of growth factors, their proliferation was inhibitedbut PAX2 was able to enhance proliferation with and withoutgrowth factors, supporting the notion that PAX2 has this

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characteristic of a potential proto-oncogene (Fig. 1C; Supplemen-tary Fig. S2C).

PAX2 enhances survival in mOSE cellsTo determine the effect of PAX2 on cell survival, cells were

challenged bydeprivation of growth factors, and the percentage ofviable cellswas normalized to viability of cells supplementedwithgrowth factors. Only a limited decrease in cell viability (14%–

18%) was observed in the parental lines (Fig. 1D; SupplementaryFig. S2D). PAX2 expression enhanced survival, albeitmodestly, inM0505 cells, with a similar trend for enhanced survival in PAX2-expressing M1102 and mOSE-fxp53 cells, compared with non-expressing controls (P ¼ 0.0725 and P ¼ 0.0593, respectively;Supplementary Fig. S2D). Exposure to the DNA-damaging agentcisplatin for 48 hours decreased cell viability in a dose-dependentmanner; this decrease was significantly inhibited by PAX2 expres-sion (Fig. 1E; Supplementary Fig. S2E).

PAX2 inhibits p53 induction by cisplatinBecause our results showed that PAX2 enhances resistance to

cisplatin and others have shown that PAX2 has a binding sitein the first exon of Trp53 in murine cells (19), we investigatedthe effect of PAX2 on cisplatin-induced p53. Exposure tocisplatin increased p53 accumulation significantly in two mOSEcell lines (Fig. 2A), and this increase was suppressed in PAX2-expressing cells, reaching only 40% of the levels induced incontrol cells (Fig. 2B). However, PAX2 expression did not changethe levels of Trp53 transcripts (Fig. 2C), indicating that PAX2affects the translation and/or stabilization of p53 but not thetranscription of Trp53.

PAX2 deregulates cancer-associated pathways in mOSE cellsTo better understand the role of PAX2 in mOSE cells, micro-

array analysis was performed on M0505 þ PAX2 and comparedwith M0505 þ WPI and M0505 þ PAX2 þ Cre transcripts.

Figure 1.PAX2 actions in M0505 mOSE cells. A,Western blot analysis confirming thatM0505 transduced with a PAX2expression vector (PAX2) showsincreased PAX2 relative to controls:WPI vector or cre-mediated PAX2deletion (PAX2 þ Cre). B, PAX2increases M0505 proliferation. C and D,PAX2 increases viable cell number and% viability after 48 hours in growthfactor- and serum-free media(starvation). E, PAX2 enhancesM0505 cisplatin resistance. For allexperiments, n ¼ 3; � , P < 0.05;�� , P < 0.01; �� , P < 0.001;�� , P < 0.0001.

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Changes were considered relevant only when a 2-linear-folddifference or more was evident between transcript levels in thePAX2-expressing cells compared with transcripts in bothWPI andPAX2 þ Cre cells. Using these criteria, there were 41 genesupregulated and 58 genes downregulated in PAX2-expressingcells. Ingenuity Pathway Analysis (IPA) showed changes in genesinvolved in cancer and cellular growth, predicting an activation of

cancer-associated functions (hyperplasia and tumor size) and cellproliferation (Fig. 3). Supplementary Table S3 lists the top 10most highly upregulated genes in the PAX2-expressing cells com-pared with the two control cell lines. Many of these genes havebeen reported to be involved in tumor growth and progression,which highlights the potential role of PAX2 in cancer initiationand development (Supplementary Table S4).

Figure 2.PAX2 inhibits p53 induction in mOSE cells. Western blot analysis (A) and densitometric analysis (B) of p53 in M0505 and M1102 cells after cisplatin treatment(12.5 mg/mL; 48 hours), with UV-exposed mouse embryonic fibroblasts as a positive control. C, Trp53 mRNA levels after PAX2 expression. For all experiments,n ¼ 3; � , P < 0.05; �� , P < 0.01.

Figure 3.IPA showing biologic functionsassociated with gene expressionchanges after PAX2 expression.Relationships between genes are color-coded by predicted interaction (seelegend).

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Figure 4.PAX2 actions in RM and STOSE mouse ovarian cancer cells. A, Western blot analysis confirming PAX2 induction in both cell lines. B, proliferation of STOSEand RM cells with PAX2 induction (PAX2) versus controls (parental, WPI, PAX2-Cre). C, viability (left, alamar blue) and cleaved caspase-3 (right, Western blotting)were assessed after 48-hour cisplatin treatment (n ¼ 3; �� , P < 0.01; �� , P < 0.0001). D, PAX2 expression decreased colony formation ability of STOSE butnot RM cells (n ¼ 3; �� , P < 0.0001). E and F, Kaplan–Meier survival curves for mice injected with (E) RM þ WPI or RM þ PAX2 cells or (F) STOSE þ WPI,STOSE þ PAX2, or STOSE þ PAX2 þ Cre cells (�� , P < 0.0001). G, PAX2 expression decreased STOSE tumor weight at endpoint (�� , P < 0.01; � , P < 0.05).






Figure 5.PAX2 inhibited p53 accumulation in RM but not in STOSE tumors. A, PAX2 expression abolished p53 and cleaved caspase-3 (Western blotting) in all RM þ PAX2tumors examined. Positive controls for p53 and cleaved caspase-3 were cisplatin-treated normal and p53-deficient mOSE, respectively. B, Trp53 mRNAlevels in RM and STOSE tumors. C, STOSE tumors did not show p53 accumulation (Western blotting). (Continued on the following page.)

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PAX2 exhibits oncogenic properties in normal mOSE cellsTo determine whether PAX2 induction in mOSE facilitates

oncogenic transformation, SCID mice were injected intraperito-neally with 107 cells ofM0505þWPI,M0505þ PAX2 (6mice pergroup), or M1102 þ PAX2 (5 mice). At 114 days postinjection,animals were sacrificed and none had developed tumors, indi-cating that Pax2 expression is not sufficient for transformation ofovarian epithelial cells.

TP53mutationhasbeen reported in 95%ofHGSC (3), andnullmutations in TP53 have been found in 24% of ovarian cancers(20). To determine whether PAX2 combined with p53 deficiencyis tumorigenic, we generated mOSE cells lacking functional p53(p53D2-10-mOSE; Supplementary Fig. S3A). Although p53D2-10

-mOSE cells are not tumorigenic (21), they show anchorage-independent growth by forming colonies in soft agar and haveenhanced proliferation and survival compared with normalmOSE cells (Supplementary Fig. S3B). PAX2 induction modestlyincreased proliferation and did not alter survival in these p53-deficient cells (Supplementary Fig. S3C), indicating that PAX2enhances these functions, at least partially, in a p53-dependentmanner. To test the effects of PAX2 in p53-null cells, SCID micewere injected with either p53D2-10-mOSE þ WPI or p53D2-10

-mOSE þ PAX2 cells (4 animals per group). Two mice injectedwith PAX2-expressing p53D2-10-mOSE cells developed tumors,whereas there was no evidence of tumor in the p53D2-10-mOSEþWPI group. Animals that did not develop tumors were kept for 6months and examined for tumor incidence. None had developedtumors. Although the number of animals is small, these dataindicate that loss of p53 confers oncogenic potency to PAX2 inmOSE cells.

PAX2 has contradictory effects on ovarian tumor progressionOur results are in accordance with reports showing that PAX2

enhances tumor progression in different cancer models (10, 11).To further determine whether this is the case in ovarian cancers,PAX2 was expressed in two murine ovarian cancer models gen-erated in our laboratory (16, 18). The RMmodelwas derived fromimmortalizedmOSE cells transducedwith retrovirus constructs toachieve ectopic expression of the mutant K-Ras (KRASG12D) andMyc (18). RMcells injected intonudemice formaggressive, poorlydifferentiated tumors with a median survival of 23.3 � 1.7 days.The STOSE model mimics HGSC and arose spontaneously fromlong-term culture of the M0505 cells (16). Expression of Pax2 inboth cell lines (Fig. 4A) resulted in different effects on prolifer-ation, enhancing proliferation in RM, but not STOSE cells (Fig.4B).When the cells were deprived of growth factors, PAX2 had noeffect on their survival (data not shown). However, when cellswere exposed to cisplatin, PAX2 modestly enhanced cisplatin-induced apoptosis in STOSE cells (associated with increasedcleaved caspase-3), but not in RM cells (Fig. 4C). PAX2 alsoreduced the number of colonies produced in soft agar, reachingsignificance only in STOSE cells (Fig. 4D).

To determine the role of PAX2 on ovarian tumor progression,RM þ WPI and RM þ PAX2 cells were injected intraperitoneally

into 20 SCIDmice per group,whereas STOSEþWPI and STOSEþPAX2 were injected intraperitoneally into both immunodeficientand -competent mice (4 SCID and 10 FVB/N per group). Inaddition, STOSE þ PAX2 þ Cre cells were injected intraperitone-ally into 10 FVB/Nmice. Surprisingly, the effect of PAX2 on tumorprogression differed drastically between the two models (Fig. 4Eand F), with decreased survival time in RM-injected animals(median survival, 11 vs. 16 days) but increased survival time inFVB/N (74.5 vs. 27.5 days) and SCID (59 vs. 26.5 days) miceinjected with STOSE cells.

All animals showed widespread tumor dissemination (perito-neum, pancreas, liver, intestine, diaphragm, uterus, and ovaries,with most tumors consolidating with the intestine). Animalsinjected with RM cells were sacrificed because of large tumorburden, whereas STOSE-injected animals reached endpoint dueto large ascites volumes. Although all animals injected withSTOSE cells exhibited extensive ascites, STOSE þ PAX2 tumorshad significantly smaller tumors (Fig. 4G). The data indicate thatPAX2 may affect progression of ovarian cancer positively ornegatively, depending on the tumor context.

PAX2 ablates p53 in RM tumors but not STOSE tumorsTo understand why PAX2 enhanced RM tumor progression

while it inhibited progression of STOSE tumors, we examined theeffect of PAX2 on p53 in both tumor types. PAX2 completelyinhibited p53 accumulation in RM tumors (Fig. 5A), with sup-pression also evident at the mRNA level (Fig. 5B). STOSE cells(with or without PAX2) did not show stable expression of p53(Fig. 5C). RMþPAX2 tumors have reduced apoptosis as shownbythe lack of cleavage of caspase-3 (Fig. 5A), suggesting that PAX2-expressing RM tumor cells were not eliminated by apoptosis. Thismay suggest a mechanism for more rapid tumor progression andshorter survival. Because loss of p53 could result in enhancedproliferation, we examined the proliferative rate of tumors; how-ever, pixel positivity for Ki67 was not different in RM þWPI andRMþ PAX2 tumors (Fig. 5D). In contrast, STOSEþ PAX2 tumorsshowed less positivity than control tumors, indicating a lowerproliferative rate that could explain the smaller tumor size.Proliferation rate was largely restored by deletion of PAX2 in theSTOSE þ PAX2 þ Cre cells (P ¼ 0.0571).

PAX2 targets different pathways in different tumor contextsTo identify targets dysregulated byPAX2 indifferent tumors,we

analyzed the microarray data to search for genes that mightidentify pathways activated by PAX2. The gene most downregu-lated by PAX2 was High-temperature requirement A serine pep-tidase1 (Htra1). Although PAX2 decreased the level of Htra1transcripts in M0505 cells, this effect was only partially reversedwhen PAX2 was deleted (Fig. 5E). Like mOSE cells, Htra1 wasdownregulated in RM cells when PAX2 is expressed. In contrast, inSTOSE, there was no decrease but rather a trend to increased levelsof Htra1 (P ¼ 0.0884; Fig. 5E). HTRA1 has been associated withEGFR activity (22), but RM tumors showed no EGFR expression.Despite the lack of EGFR, PAX2 decreased the levels of PTEN and

(Continued.) D, tumor proliferation was examined by immunohistochemistry for Ki67 (quantified in histograms). E, PAX2 expression reduced Htra1 transcripts inmOSE cells and RM tumors, but not STOSE tumors. F, PAX2 expression reduced PTEN levels in RM tumors (Western blotting). G, immunoblot detection oftotal and phospho-AKT following AKT immunoprecipitation in RM tumors. H, Western blot analysis shows PAX2-associated phosphorylation of ERK1/2 atThr202 and Tyr204 in RM but not STOSE tumors; SCC25 head and neck carcinoma cells were used as a positive control for EGFR. EGF-treated SCC25 cellswere used as positive and (with an EGF inhibitor) negative controls for pERK1/2.






increased phospho-AKT in the RM tumors (Fig. 5F and 5G), anaction for PAX2 that has been reported previously (23). Immu-noblotting showed that PAX2 induced robust dual phosphory-lation of ERK1/2 at Tyr202 and Thy204 only in RMbut not STOSEtumors (Fig. 5H).

IPA also showed the involvement of prostaglandin-endoper-oxide synthase 2 (Ptgs-2 or Cox2), which was downregulated byPAX2 in M0505 mOSE cells in several pathways related to tumorprogression, hyperplasia, and ovarian cancer (Fig. 3; Supplemen-tary Table S4). However, aberrant upregulation of Ptgs2 has beenreported in ovarian tumors and is associated with tumor progres-sion in animal studies (24, 25). COX2 levels are significantlyhigher in RMþ PAX2 tumors (5.7-fold); however, STOSEþ PAX2tumors expressmuch lower levels ofCOX2 (0.14-fold), comparedwith controls (Fig. 6A). Therefore, although both derived fromovarian epithelial cells, PAX2 targets different genes in the twotumor models.

COX2 is associated with angiogenesis in some tumortypes (24). To investigate whether the COX2 induction in RMtumors resulted in changes in tumor microvessel density(MVD), vascular endothelial marker (CD31) was detected byIHC analysis. PAX2-RM tumors have an increased MVD, ascompared with control tumors (Fig. 6B). However, there is nodifference in the MVD among STOSE tumors (with and withoutPAX2). These data indicate that in RM tumors, PAX2 mayenhance angiogenesis, and therefore progression, through theinduction of COX2.

DiscussionThe aim of this study was to determine the role of the

potential oncogene PAX2 in the etiology and progression ofovarian cancer. Because PAX2 is expressed in ovarian cancersbut not in normal OSE, we developed mOSE cells expressingPAX2 to identify whether PAX2 induction is one of the initialevents in transformation. Although PAX2 expression enhancedcell survival and proliferation, it was not sufficient to inducetumorigenicity, unless combined with loss of p53. PAX2 hadopposing actions in two murine ovarian tumor models, accel-erating or inhibiting tumor progression.

Although recent evidence shows that some HGSC originatefrom Mullerian cells (26), OSE cells remain a probable origin ofseveral subtypes of EOCs (reviewed in ref. 27).OSE cells retain thepotential to differentiate into epithelia resembling that of theMullerian system (28, 29) by, for example, induction of HOXgene expression (30). HOX and PAX genes are required forMullerian development and are expressed in a subset of EOCbut not in normal OSE cells (4, 31). PAX2 is expressed in ciliatedinclusion cysts, low-malignant potential, and LGSC (4), suggest-ing that gain of PAX2 expression promotes transformation (asshown in rat fibroblasts; ref. 13). We found that expression ofPAX2 increased mOSE proliferation even when the cells weredeprived of growth factors and increased survival during cisplatintreatment. Proliferation, self-sufficiency in growth signals, andapoptosis avoidance are key factors in tumor formation (32).

Microarray analysis showed that PAX2 disturbs signaling path-ways involved in cancer, reproductive diseases, and cellular pro-liferation. Further experiments in OSE showed that PAX2increased cancer-related characteristics, including inhibition ofp53 accumulation, but was not sufficient to induce transforma-tion. PAX2has a potential binding site on exon1of the Trp53 gene

in mice (19), but our results show that p53 downregulation byPAX2 is a posttranscriptional event.

Previous studies showed that Trp53 mutation is required formOSE transformation by alterations in K-Ras, myc, and/or Akt(21). Because mutations in TP53 have been found in almost all(96%) HGSC (3) and null mutations in 24% of ovarian cancers(20), we expressed PAX2 in p53-deficient mOSE cells. Someanimals injected with p53-null PAX2-expressing cells formedtumors, but p53-null cells did not form tumors, as shown byothers (21). This suggests that loss of p53 predisposes ovarianepithelial cells to transformation by PAX2 induction. Similarly,PAX2 sensitizes the cells to become tumorigenic following Trp53mutation.

In vitro, PAX2 shows oncogenic behavior, as silencing PAX2resulted in decreased tumor volume or enhanced cisplatin-induced tumor regression in xenograft models of human endo-metrioid, colon, and renal carcinoma cells (10–12). However,there is limited and conflicting evidence for its contribution toovarian tumorigenicity. In human ovarian cancer cell lines, PAX2can enhance or inhibit growth (9, 15). Although the mechanismwas not identified in these studies, cancer subtype and/or TP53status may explain these contradictory results.

To understand the role of PAX2 in ovarian tumor progression,we used two different mouse models reflecting LGSC and HGSC.TheRMmodelwas developedby inducingmutant K-RASG12D andMYC in immortalized mOSE cells. Activatingmutations in K-RASor B-RAF are present in two thirds of LGSC (33), and overexpres-sion of c-MYC has been reported in up to 70% of ovarian cancers(34). LGSC cell lines andmurinemodels are limited (35, 36), andthe RM cells were used as a model for LGSC on the basis of theirdependence on K-Ras mutation. Nevertheless, the RM cells formaggressive disease, in contrast to the slower disease progression ofLGSC in women. Unlike STOSE tumors, RM tumors have stablep53 accumulation. This was not due to TAg expression, which isundetectable (Supplementary Fig. S4), but as mutant RAS orincreased levels of c-MYC stimulate p19ARF–MDM2 complexformation, this may sequester MDM2 and stabilize p53 (37, 38).

RM cells expressing PAX2 became more proliferative in cultureand more aggressive in vivo, showing increased angiogenesis anddecreased apoptosis. Molecular analysis showed that PAX2expression eliminated p53 and cleaved caspase-3 expression,decreased PTEN, and increased pAKT and pERK1/2. Therefore,it appears that enhanced angiogenesis and reduced apoptosis inthe RMþ PAX2 tumors account for the accelerated tumor growthand shorter survival.

Both the histotype andmolecular profile for the STOSE ovariancancer model mimic human HGSC. STOSE cells have a highdegree of aneuploidy, increased expression of cyclin D1, andpossibly increased NF-kB activity, but interestingly no mutationsin the Trp53 gene (16). In contrast to the RM model, PAX2improved survival and reduced STOSE tumor mass and dissem-ination in both immunodeficient and -competent mice, suggest-ing that PAX2 is a negative regulator of some aspects of ovariantumorigenicity.

One possible explanation, as shown in Fig. 6C, is the regulationof Htra1 by PAX2 in these tumors. HTRA1 is a putative tumorsuppressor gene that is downregulated inovarian cancers (39) andenhances chemosensitivity by targeting the antiapoptotic proteinXIAP for degradation (40). Downregulation of HTRA1 enhancedperitoneal dissemination in a xenograft study by increasing phos-phorylation of EGFR, AKT, and ERK1/2 (22).We found thatHtra1

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Figure 6.Effect of PAX2 on COX2 and angiogenesis. A, COX2 in ovarian tumors after PAX2 induction (Western blotting). B, MVDwas examined using immunohistochemistryfor CD31. Bottom, CD31-positive pixel selection, quantified in the histograms on the right (� , P < 0.05). Scale bar, 100 mm. C, a proposed model for PAX2actions in normal and cancerous ovarian cells. Dotted lines indicate predicted functional effects.






transcripts were more abundant in STOSE þ PAX2 tumors, butlower in RMþPAX2 tumors, as comparedwith the correspondingcontrols. In STOSE tumors, PAX2 did not alter apoptosis (cleavedcaspase-3) or ERK1/2 phosphorylation, whereas PAX2-expressingRM tumors showed a robust increase in pAKT and pERK1/2, ascompared with controls. This was likely not due to activation ofthe EGFR pathway, as EGFR was undetectable, but it is associatedwith the downregulation of PTEN, a known negative regulator ofthe PI3K pathway (41).

Another relevant PAX2 target with differential expression inRM versus STOSE tumors is PTGS2 (COX2). Aberrant upregula-tion of COX2 has been reported in ovarian tumors and isassociated with chemoresistance (25). The COX2 inhibitor(celecoxib) suppressed growth of ovarian xenografts in mice,reduced angiogenesis, and increased apoptosis (24). In ourstudy, PAX2 increased COX2 in RM tumors but reduced itsexpression in STOSE tumors.

Interestingly, COX2 is induced by TGFb1 (42) andHTRA1mayinhibit TGFb signaling (43). TGFb1 expression in human ovariantumors is associated with poor prognosis (44), and we speculatethat HTRA1 upregulation prevented potential deleterious effectsof TGFb1 in STOSE þ PAX2 tumors. Therefore, PAX2 may conferor inhibit tumor progression at least partially through differentialregulation of COX2 and/or HTRA1, depending on the tumorcontext.

This investigation identifiednovel targets dysregulatedbyPAX2(i.e., COX2 and HTRA1), both of which are involved in cellproliferation (40, 45). Although apoptotic induction wasenhanced by PAX2 in STOSE cells, no apparent apoptosis wasevident in STOSE tumors, where PAX2 promoted proliferation.Interestingly, PAX2 expression reduced STOSE colony formationin soft agar, indicating a higher rate of anoikis. This may beattributed to HTRA1 induction, which has been shown to preventmetastasis by inducing anoikis (22). Thus, HTRA1 inductionmight cause the decreased metastasis seen in PAX2-expressingSTOSE tumors.

This study confirms the differential role of PAX2 in ovariantumor progression reported previously (9, 15) and identifiessomemolecular drivers thatmight be responsible. The differentialrole of PAX2 in ovarian cancer has been suggested previously bySong and colleagues (15). Compelling evidence that PAX2 mightact as a tumor suppressor in well-differentiated epithelia comesfrom studies reporting that PAX2 is completely lost or markedlyreduced in the early stages of tubal and endometrial intraepithe-lial carcinomas (46, 47), often in association with TP53mutationor in the presence of co-existing pelvic serous cancer (48, 49).Interestingly, PAX2 transcription is stimulated by wild-type TP53in kidney cells and this stimulation is inhibited by dominant-negative TP53 mutations (50), a common aberration in HGSC(3). Because HGSC have a low incidence of PAX2 expression (4),one could argue that the forced expression of PAX2 in STOSE cells,which generate HGSC-like tumors, allowed the demonstration ofits tumor-suppressive function. In contrast, expression of K-RAS

(as in RM cells) and PAX2 are both associated with LGSC (4, 33)and induction of PAX2 in this model enhanced tumoraggressiveness.

In summary, PAX2 induces carcinogenic features in OSE cells(enhanced proliferation, survival, and growth factor indepen-dence) and increases their sensitivity to transformation. Inovarian cancer cells, PAX2 enhances or inhibits their aggres-siveness in vivo, depending on the molecular context. In tumorswith K-Ras andMycmutations, PAX2 enhances cell survival andangiogenesis, whereas in tumors resembling HGSC, PAX2decreases proliferation and metastasis. Therefore, in mouseovarian tumors, in agreement with results reported for humanovarian cancer cells (15), PAX2 seems to have contradictoryeffects on progression, depending on the context. The differen-tial effects of PAX2 appear to be mediated by differential reg-ulation of COX2 and/or HTRA1. It remains to be determinedwhether thesemolecular pathways are activated or repressed in amanner dependent on the molecular environment that leads toHGSC versus LGSC.

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

Authors' ContributionsConception and design: E.M. Al-Hujaily, Y. Tang, D.-S. Yao, K. Garson,B.C. VanderhydenDevelopment of methodology: E.M. Al-Hujaily, Y. Tang, D.-S. Yao, K. GarsonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.M. Al-Hujaily, Y. Tang, D.-S. Yao, K. GarsonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.M. Al-Hujaily, Y. Tang, D.-S. Yao, E. Carmona,B.C. VanderhydenWriting, review, and/or revision of the manuscript: E.M. Al-Hujaily, Y. Tang,D.-S. Yao, E. Carmona, K. Garson, B.C. VanderhydenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.C. VanderhydenStudy supervision: B.C. Vanderhyden

AcknowledgmentsThe authors thank Olga Collins and Elizabeth Macdonald for excellent

technical support. They are grateful to Dr. Didier Trono for providing plasmidspPWI and pCMVR8.74 and Dr. Arthur Nienhuis for providing plasmid pCAG-Eco.

Grant SupportThis work was funded by grants from the Marsha Rivkin Center for Ovarian

Cancer Research (B.C. Vanderhyden) and the Canadian Institutes of HealthResearch (MOP-111194; B.C. Vanderhyden), and a scholarship to E.M. Al-Hujaily from the King Abdullah International Medical Research Center.

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 March 24, 2015; revised August 24, 2015; accepted September 8,2015; published OnlineFirst September 15, 2015.

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2015;8:1163-1173. Published OnlineFirst September 15, 2015.Cancer Prev Res Ensaf M. Al-Hujaily, Yong Tang, De-Sheng Yao, et al. Ovarian CancerDivergent Roles of PAX2 in the Etiology and Progression of

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