Cell-autonomous activation of the PI3-kinasepathway initiates endometrial cancer fromadult uterine epitheliumSanaz Memarzadeha,1, Yang Zongb, Deanna M. Janzena, Andrew S. Goldsteinc, Donghui Chengb, Takeshi Kuritad,Amanda M. Schafenackera, Jiaoti Huange, and Owen N. Witteb,f,g,1

Departments of aObstetrics and Gynecology, ePathology, and gMolecular and Medical Pharmacology, The David Geffen School of Medicine; bThe HowardHughes Medical Institute, cMolecular Biology Institute, and fDepartment of Microbiology, Immunology and Molecular Medicine, University of California, LosAngeles, CA 90095; and dDepartment of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

Contributed by Owen N. Witte, August 24, 2010 (sent for review July 27, 2010)

Epithelial-specific activation of the PI3-kinase pathway is the mostcommon genetic alteration in type I endometrial cancer. In the ma-jority of these tumors, PTEN expression is lost in the epithelium butmaintained in tumor stroma. Currently reported PTEN knockoutmouse models initiate type I endometrial cancer concomitant withloss of PTEN in both uterine epithelium and stroma. Consequently,thebiologic outcomeof selectivelyactivating thePI3-kinasepathwayin the endometrial epithelium remains unknown. To address thisquestion, we established a malleable in vivo endometrial regenera-tion system from dissociated murine uterine epithelium and stroma.Regenerated endometrial glands responded to pharmacologic varia-tions in hormonal milieu similar to the native endometrium. Cell-autonomous activation of the PI3-kinase pathway via biallelic lossof PTEN or activation of AKT in adult uterine epithelia in this modelwas sufficient to initiate endometrial carcinoma. AKT-initiatedtumors were serially transplantable, demonstrating permanent ge-netic changes in uterine epithelia. Immunohistochemistry confirmedloss of PTEN or activation of AKT in regenerated hyperplastic glandsthat were surrounded by wild-type stroma. We demonstrate thatcell-autonomous activation of the PI3-kinase pathway is sufficientfor the initiation of endometrial carcinoma in naive adult uterineepithelia. This in vivo model provides an ideal platform for testingthe response of endometrial carcinoma to targeted therapy againstthis common genetic alteration.

tissue regeneration model | progesterone receptor | uterine cancer

Endometrial cancer, the most common gynecologic cancer inthe United States (1), is a hormonally regulated tumor arising

from epithelial cells lining the uterine cavity. Effective targetedtherapy is unavailable, partly because of lack of knowledge re-garding basic biologic pathways and cellular compartments thatcan initiate this malignancy. Women diagnosed with endometrialcancer are treated in a similar manner irrespective of the hetero-geneity of this disease, with surgery or combinations of radiationand chemotherapy. Although endometrial cancer can be cured inearly stages, side effects associated with the current therapy canhave lifelong debilitating effects on some patients. The majority ofwomen diagnosed with late-stage or metastatic disease die, despiteundergoing radical treatments.Endometrial cancer can be divided into two distinct subtypes

(2). Type I tumors occur in younger patients exposed to high levelsof estrogen unopposed by progesterone. They are typically local-ized with better response to hormonal therapy. Type II tumorsoccur in older patients independent of hormonal status. Thesetumors are more invasive, have a greater propensity for metastaticspread, and are refractory to hormonal treatment. Activation ofdistinct biologic pathways has been reported in type I vs. type IIcancers. Type I tumors are associated with activation of the PI3-kinase pathway, activating mutations of KRAS, mutant β-catenin,and microsatellite instability (reviewed in ref. 3). Type II tumors

are predominantly associated with mutations in p53, amplificationof Her2/neu, and inactivation of p16 (reviewed in ref. 3).One established model for endometrial cancer is the PTEN+/−

mouse, characterized by formation of multiple tumors, includingtype I endometrial carcinoma (4). In this model, one allele ofPTEN is deleted in all tissues (4). In an alternative model, micewith progesterone receptor (PR) promoter-driven Cre werecrossed with a PTENloxP/loxP strain, resulting in endometrial hy-perplasia in prepubertal mice progressing to type I endometrialcarcinoma in adult animals (5). Because PR is expressed in uterineepitheliumand stroma, it is unclear whether the observed tumors inthis model result from genetic alterations in the uterine epithelium,stroma, or both. Human endometrial carcinoma arises pre-dominantly from adult uterine epithelia, unlike transgenic models,where induction of oncogenic signals occur in developing tissue. Intype I endometrial cancers, based on immunohistochemical (IHC)studies, loss of PTEN is predominantly detected in the uterineepithelia and not stromal cells (6). Despite the prevalence of PI3-kinase signaling mutations in type I endometrial cancer, the con-sequence of epithelial-specific activation of this pathway in adultendometrial epithelium remains unknown.To study genetic changes that may have a causative role in ini-

tiating endometrial cancer, we have developed a regeneration andtransformation model from dissociated populations of adultuterine epithelium and cultured neonatal stroma. Previous studiesdemonstrated that recombined uterine tissue fragments implantedsubrenally could regenerate into endometrial-like glands (7, 8).The inability to genetically manipulate tissue fragments limits theutility of this model system. In a separate study, human endome-trial glands were regenerated fromdissociated adult endometrium,but endometrial glands in these grafts were sparse and appearedatrophic, perhaps because of the limited inductive capacity of adultstroma (9). With the inductive capacity of neonatal stroma,regenerated endometrial glands in our model were secretory,functional, and responded to alterations in hormonal milieu.Herein, we report on the outcome of epithelial-specific PI3-kinasepathway activation using a recently established in vivo endometrialregeneration system. Our findings clearly demonstrate that epi-thelial specific loss of PTEN or activation of AKT alone is suffi-cient for initiation of endometrial cancer in adult uterine epithelia.To our knowledge, this in vivomousemodel is unique in accurately

Author contributions: S.M., Y.Z., and O.N.W. designed research; S.M., Y.Z., D.M.J., A.S.G.,D.C., and A.M.S. performed research; T.K. contributed new reagents/analytic tools; S.M.,Y.Z., D.M.J., J.H., and O.N.W. analyzed data; and S.M., D.M.J., and O.N.W. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. E-mail: smemarzadeh@mednet.ucla.edu orowenwitte@mednet.ucla.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012548107/-/DCSupplemental.

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recapitulating the epithelial-specific pattern of PTEN loss seenin human endometrial cancer specimens.

ResultsEndometrial-Like Glands Regenerated from Dissociated Adult Epi-thelium and Neonatal Stroma. We developed an in vivo murineendometrial regeneration model from dissociated uterine stromaland epithelial cells. Endometrial glands from reproductive-agefemale mice were harvested as previously described (7, 8) anddissociated (Fig. 1A). Neonatal uterine stroma was isolated (7, 8),dissociated, and cultured short term (Fig. 1A). Efficient separa-tion of epithelium and stroma was demonstrated by IHC (Fig. S1A and B) and quantitative PCR (Fig. S1C). To differentiallycolor-mark the epithelium, stroma were harvested from WT miceand epithelium was harvested from mice expressing the fluores-cent protein DsRed. Equal numbers of dissociated WT uterinestroma and DsRed epithelium were recombined, placed in a col-lagen plug, and implanted under the kidney capsule of a SCIDmouse for 8 wk (Fig. 1 A and B). Predominance of implants,containing a minimum of 1 × 105 epithelia and 1 × 105 stroma,yielded grafts composed of endometrial-like glands surroundedby normal stroma (Fig. 1B). Fluorescent analysis revealed DsRedmarker in regenerated glands only, demonstrating their originfrom adult epithelial cells (Fig. 1B). To compare the regeneratedtissue to native murine uterus, protein expression patterns ofknown mouse uterine markers were examined by IHC. Similar tomouse uterus, regenerated grafts exhibited cytokeratin 8 (CK8)expression only in epithelial glands and smooth muscle actin(SMA) was present in the outer wall of regenerated grafts (Fig.1C). Estrogen receptor (ER)α predominated in the endometrialepithelium, and PR was distributed equally in both stromal andepithelial compartments (Fig. 1C).

Dissociated Endometrial Glands Were Highly Enriched for EpithelialCells. To verify that isolated adult endometrial cells used in theregeneration assay were enriched for epithelia, we established anin vitro colony-forming assay. Isolated and dissociated endome-trial cells were plated in matrigel coated six-well plates and thenumber and type of colonies were scored after 8 d based onmorphology and expression of epithelial vs. stromal markers.Trop2 is a cell surface marker expressed in uterine epithelium(10) (Fig. 2A). CD90 (Thy-1) is expressed in human endometrialstroma (11) and upon immunostaining, was detected in murinestromal cells adjacent to endometrial glands (Fig. 2A). Isolatedmurine endometrial cells were dissociated, costained for Trop2and CD90, fractionated using FACS (Fig. 2B), and placed in thein vitro colony assay. Lineage depletion was performed to removecontaminating hematopoietic and endothelial cells (Fig S2A).The predominance of isolated endometrial cells was Trop2+/CD90− (91%). These cells expressed high levels of CK8 and E-Cadherin message assessed by quantitative PCR (qPCR) and gen-erated epithelial colonies as determined by morphology and ex-pressionofCK8 (Fig. 2B andC andFig. S2B). Trop2−/CD90+ (7%)cells predominantly yielded stromal colonies expressing SMA andvimentin (Vim) (Fig. 2 B and C and Fig. S2B). All Trop2+/CD90−

cellswereCD49f+andCD24+byFACSand their epithelial-specificexpression pattern was confirmed by immunostaining (Fig. S2C).These results demonstrate that our isolated populations of adult

endometrium used in the regeneration assay were highly enrichedfor endometrial epithelia, and these cells express Trop2, CD49f,and CD24.

Regenerated Endometrial-Like Glands Were Functional and Respondedto Alterations in the Hormonal Milieu. In response tofluctuating levelsof estrogen and progesterone, mouse and human endometria un-dergo cyclic apoptosis/shedding and regrowth during the estrous andmenstrual cycle, respectively (12, 13). Endometrial estrogen- andprogesterone-mediated effects are thought to occur in a paracrine

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Fig. 1. Dissociated populations of uterine epithelium and stroma form en-dometrial-like glands in the in vivo regeneration model. (A) Schematic of theendometrial regeneration system. Dissociated endometrial epithelia har-vested from adult female mice were combined with cultured uterine stromaobtained from neonatal mice. The collagen plug containing both cellularfractions was implanted subrenally into a SCID mouse. (B) Regenerated tissuecontained endometrial-like glands. These glands arose from adult donorepithelium (DsRed transgenic) demonstrated by the expression of fluores-cence in Cy3/RFP channel. (C) The marker expression profile of regeneratedgrafts (Lower) closely resembled the native murine uterus (Upper). Theregenerated endometrial glands expressed CK8 in the endometrial epithe-liumand SMA in the outerwall of the graft. ERαwas predominantly expressedin the epithelium and PRwas detected both in the epithelium and the stroma.(Scale bars, 100 μm.)

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manner from adjacent stroma (14, 15). Alterations in these steroidsinduce changes in the endometriumessential for implantation of theembryo (16), and result in secretion of leukemia inhibitory factor(LIF), essential for blastocyst implantation (17, 18).To assess the optimal condition for regeneration of endometrial

glands, mice harboring grafts were pharmacologically exposed todefined hormonal conditions. To exclude the influence of endog-enous female hormones, recipient mice were oophorectomizedbefore graft implantation. The regeneration of endometrial glandswas poor in the absence of any hormonal supplementation (Fig.S3A). Exposure to unopposed estrogen for 8 wk resulted in re-generation of crowded endometrial glands, histologically consis-tent with simple endometrial hyperplasia, but normal secretoryglands regenerated with the administration of an additional pro-gesterone pulse (Fig. 3A and Fig. S3B). The combination of steady-state estrogen with progesterone pulse was deemed the optimalcondition for in vivo regeneration of the endometrium. With-drawal of progesterone led to breakdown of the endometriumglands similar to the cycling uterus (Fig. 3A and Fig. S3B).To assess if regeneration of endometrial glands was dependent

on stromal inductive effects, dissociated populations of epitheliumand stroma were implanted alone or in varying ratios in the re-generation assay (Fig. 3B and Fig. S4). Implantation of stromalcells resulted in overgrowth of solely stromal tissue, but re-generation of epithelial cells alone produced atrophic endometrialglands lined by flattened cells (Fig. 3B). Combinations of epithe-

lium and stroma generated differentiated secretory endometrialglands, and optimal regeneration was achieved when equal orgreater numbers of stromal cells were used (Fig. 3B and Fig. S4).LIF expression was only detected in regenerated secretory endo-metrial glands and was notably absent in atrophic glandular tissue(Fig. 3C). These results suggest that regeneration of functionalendometrial glands depends on stromal and epithelial interactions.Chronic exposure to unopposed estrogen is a well-known risk

factor for endometrial hyperplasia and cancer (19, 20). In ourmodel, increasing durations of exposure to high-dose estrogen

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Fig. 3. Regenerated endometrial-like glands were functional and respon-ded to hormonal variations. (A) The response of regenerated tissue toalterations in hormonal milieu was similar to normal uterine endometrium.Regenerated glands became hyperplastic in response to unopposed estradiol(E2), secretory with the administration of progesterone pulse (P4), and un-derwent breakdown after the withdrawal of P4. (B) The regeneration ofendometrial glands depends on stromal and epithelial interactions. Combi-nations of epithelium and stroma led to the formation of normal secretoryglands. Implantation of epithelium alone resulted in the formation ofatrophic glands, but glands were absent in the stromal grafts. (C) Similar tothe native uterus, LIF was detected in regenerated grafts composed of epi-thelium and stroma. Importantly, expression of LIF was absent in atrophicglands regenerated from epithelium alone. (D) Prolonged exposure to un-opposed estrogen resulted in the formation of progressive endometrialhyperplasia. Simple endometrial hyperplasia developed within 8 wk. At12 wk, complex hyperplasia was seen, which progressed to atypical complexhyperplasia at 16 wk. (Scale bars, 100 μm.)

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Fig. 2. Isolated populations of endometrium are highly enriched for uterineepithelial cells. (A) Cell surface markers that define endometrial epithelium vs.stroma. Trop2 is a marker that is solely expressed in the endometrial epithe-lium. CD90 marks stromal cells adjacent to the uterine epithelium. (B) Isolatedadult endometria used in the regeneration assay were predominantly Trop2+/CD90− (blue oval, 91% of total isolated cells). Trop2+/CD90− cells were epi-thelial as they gave rise to epithelial colonies in vitro. Trop2−/CD90+ cells (greenoval) were stromal and formed stromal colonies in the 2D in vitro assay. Errorbars represent 1 SD. (C) The epithelial nature of the Trop2+/CD90− colonieswasconfirmedwith the expression of CK8. In contrast, Trop2−/CD90+ colonies wereCK8− and Vim+ or SMA+. (Scale bars, 100 μm.)

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unopposed by progesterone resulted in progressive endometrialpathology, demonstrated by increased gland to stromal ratio, andirregularly contoured glands with cytologic atypia (Fig. 3D andFigs. S3 C and D and S5).These results collectively demonstrate that regenerated endo-

metrial glandsnotonly resemble theendometriummorphologically,but also function in a similar manner to native endometrial tissue.

Cell-Autonomous PI3-Kinase Pathway Activation in Adult NaiveUterine Epithelia Resulted in Type I Endometrial Adenocarcinoma.Common genetic changes in type I endometrial cancer includePI3-kinase pathway activation via loss of PTEN function (21) or

mutations in AKT (22). In human cancer specimens, PTEN is pre-dominantly lost in the epithelium and maintained in the stroma (6).The ability to work with dissociated uterine cells makes our modelan ideal platform for testing consequences of cell-autonomous ge-netic changes in naive adult epithelia.Activating mutations in the pleckstrin homology domain of

AKT oncogene have been detected in endometrial cancers (22).Dissociated populations of WT endometrial epithelium wereinfected with control or myristoylated AKT-expressing lentivirus(23). These cells were combined with WT stroma and placed inthe in vivo regeneration model. Cell-autonomous AKT activationresulted in formation of larger grafts that contained areas ofcomplex endometrial hyperplasia and well-differentiated adeno-carcinoma, as determined histologically and by pankeratin stain-ing (Fig. 4A).Histopathologic criteria used for the diagnosis of type I endo-

metrial carcinoma included uncontrolled proliferation of endome-trial glands, resulting in higher gland/stromal ratio compared withcontrol. Tumor glands were irregular, angulated, back-to-back withminimal intervening stroma, and had an invasive growth pattern.Regenerated abnormal glands in these tumors expressed phos-

phorylated AKT, confirming AKT activation in hyperplastic areas(Fig. 4A). The predominance of abnormal glands expressed ERα(Fig. 4A) but PR expression was variable, with many hyperplasticregions exhibiting decreased levels of epithelial PR (Fig. 4A). Lossof PR is clinically associated with poorer prognosis and resistanceto hormonal therapy (24, 25). These results suggest that cell au-tonomous AKT activation is sufficient to initiate hyperplasia andwell-differentiated carcinoma in naive adult uterine epithelium.To ensure that observed AKT-mediated effects were cell-

autonomous, primary AKT tumors were dissociated, combinedwith WT stroma, and reimplanted as a secondary graft in the re-generation assay. The resulting secondary tumor was phenotypi-cally similar to the primary tumor based on histology and AKTactivation (Fig. 4B and Fig. S6A). These results demonstrate thatactivation of AKT in adult uterine epithelia resulted in permanentcell-autonomous changes that could be serially propagated.A more common genetic alteration in type I endometrial cancer

is PI3-kinase pathway activation via deletion, inactivatingmutation,or promoter hypermethylation of thePTEN tumor-suppressor gene(6, 21, 26, 27).Mutations inPTEN are found in 83%of endometrialcancer specimens (6). IHC analysis revealed undetectable levels ofPTEN in 61% of type I endometrial carcinomas, suggestingbiallelic loss or inactivation of PTEN in these tumors (6). PTENloss not only activates AKT but also leads to the activation ofadditional downstream targets, such as Rac1 and CDC42, in-volved in regulation of cell growth and cell cycle (reviewed in ref.28). To test the consequences of epithelial-specific PTEN loss,endometrial glands were harvested from adult PTENloxP/loxP micewhere PTEN is floxed by loxP sites (29). The dissociated endo-metrial epithelia were infected with FUCRW control or FU-Cre-CRW virus, which expresses Cre-recombinase (Fig. S7), resultingin excision of PTEN. Infected epithelial cells were combined withWT stroma and placed in the in vivo regeneration assay. Epi-thelial-specific PTEN loss in naive adult endometria resulted inthe formation of well-differentiated endometrial cancers (Fig.4C). High levels of ERα were noted in the epithelial tumor cells(Fig. 4C). In contrast to AKT tumors, robust expression of epi-thelial PR was detected in hyperplastic glands (Fig. 4C). Theseabnormal glands had a lower proliferation index compared withAKT tumors (Fig. S6C). Loss of PTEN was observed in tumorepithelia surrounded by WT stroma (Fig. 4C and Fig. S6B). Thecompartment-specific pattern of PTEN loss in these tumorsresembles findings in clinical endometrial cancer specimens.Collectively, these results demonstrate that epithelial-specific

PI3-kinase pathway activation via loss of PTEN or activation ofAKT can initiate type I endometrial carcinoma or hyperplasia innaive adult endometrial cells.

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Fig. 4. Consequences of cell autonomous PI3-kinase pathway activation innaive adult endometrial epithelium. (A) Activation of AKT in endometrialepithelium led to the development of hyperplasia and well-differentiatedadenocarcinoma. Expression of Myr-AKT with a lentiviral vector resulted inthe formation of larger grafts comprised of hyperplastic glands comparedwith controls. Expression of activated AKT was confirmed by immunostaining.Heterogeneous expression of PR was detected in AKT tumors with many areasexhibiting low levels of epithelial PR. (B) AKT initiated tumors result fromcell autonomous genetic changes. Primary AKT tumors were dissociated andretransplanted into a secondary graft with WT stroma. The histology of thesecondary regenerant was consistent with endometrial adenocarcinoma. (C)Loss of PTEN resulted in formation of well-differentiated endometrial ade-nocarcinoma. PTEN was absent only in the epithelial compartment of thesetumors, recapitulating the PTEN expression pattern observed in human en-dometrial adenocarcinoma. (Scale bars, 100 μm.)

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DiscussionWe report an endometrial cancer mouse model that accuratelyrecapitulates the epithelial-specific PTEN loss observed in humanendometrial cancer specimens. Regenerated tumors resultingfrom loss of PTEN in this in vivo model exhibit normal expressionof PTEN in the stroma with loss of PTEN in the epithelium. Cur-rently published PTENmouse models result in concomitant loss ofPTEN in the epithelium and stroma (4, 5). Although the con-sequences of stromal PTEN loss in the endometrium are un-known, loss of PTEN in mammary stromal cells has been shown topromote tumor formation in cooperation with cell-autonomousgenetic changes (30).The ability to accurately recapitulate human endometrial cancer

makes this model system a unique tool for testing therapeutics inan in vivo setting. For example, progesterone is a well-toleratednoncytotoxic drug with 50 to 70% response rates in primary typeI endometrial cancers and 17% response rates in recurrent disease(reviewed in ref. 31). In clinical practice, progesterone therapy isunder-used, as there are no reliable methods for stratification ofendometrial hyperplasia or cancer into those that may respond toprogesterone. Endometrial tumors from PTEN+/− mice were re-fractory to hormonal treatment (32). However, in tumors of thesemice, PTEN was lost in the uterine epithelium and stroma, whichdoes not recapitulate epithelial specific loss of PTEN in humanendometrial cancer specimens. Based on retrospective clinicalreports, it is unclear if loss of PTEN is a predictor of response toprogesterone therapy (33–36). Findings in these clinical studies arewidely variable, with some reporting PTEN loss as a positive pre-dictor of response (34, 35) but others reporting loss of PTEN asa negative predictor for response to progesterone treatment (33,36). These conflicting clinical findings may be because of tumorheterogeneity, resulting from genetic alterations in addition toPTEN loss. The expression of PR in endometrial cancer specimenscorrelates with a positive response to progesterone therapy(25, 37). In our in vivo model, differential expression of epithelialPR was observed in AKT vs. PTEN-initiated tumors. Lower levelsof epithelial PR were detected in AKT tumors compared withPTEN-initiated cancers. To determine predictors of response tohormonal therapy, we plan to use this model to ascertain responseto progesterone treatment in the context of defined geneticchanges in an in vivo setting.The cell of origin for endometrial carcinoma is unknown. Stem

cells are efficient targets of oncogenic transformation in someepithelial cancers (38–40). Stem cells must exist in the uterinelining as estrogen-deprived endometrium undergoes atrophy butmaintains the capacity to regrow with the addition of exogenoushormones. In many hormonally responsive epithelia, stem cellseither lack hormone receptor expression (41) or active signaling(38, 40). Murine uterine epithelial label-retaining cells were foundto be ERα-negative (12), suggesting these progenitors may havequiescent hormone-receptor transcription machinery. Becausetype II tumors are resistant to hormonal therapy and are pre-dominantly ER-/PR-negative, they may arise from endometrialstem cells. In contrast, hormonally responsive type I tumors thatare ER-/PR-positive, may arise from more differentiated endo-metrial progenitors. To date, mouse models have not recapitulatedtype II endometrial cancers. One possible reason for the lack ofanimal models that recapitulate type II tumors could be the in-ability to target endometrial stem cells with oncogenic signals usingthe PR-driven promoter.The ability to genetically manipulate fractionated endometrial

epithelia in our model provides the tools necessary for identifyingcells and differential oncogenic signals that may give rise to type Ivs. type II endometrial cancers. This experimental approach canhelp elucidate genetic pathways that can initiate endometrialcancer in target cells. Results from these studies could lead to

scientifically based clinical trials that can help individualize ther-apeutic interventions for women affected by endometrial cancer.

MethodsExperimental Animals. WT C57BL/6, β-actin DsRed [C57BL/6-Tg(ACTB-DsRed.MST)1Nagy/J], PTENloxPloxP (C;129S4-Ptentm1Hwu/J), and CB17Scid/Scid mousestrains were purchased from The Jackson Laboratory.Mice weremaintained inaccordance with the University of California at Los Angeles, Division Labora-tory of Animal Medicine-approved protocols. All animal experiments wereperformed under University of California at Los Angeles Animal ResearchCommittee approval.

Plasmids and Virus Production. Lentiviral plasmids expressing GFP (FUCGW),myristoylated-AKT and GFP (FU-myrAKT-CGW), and RFP (FUCRW) werepreviously described (23, 42). To construct the cre-expressing vector FU-cre-CRW, cDNA for cre recombinase was cloned into the EcoRI site of the FU-CRW vector. Lentivirus preparation and titering was performed as previouslyreported (23).

Preparation of Endometrial and Stromal Cells. Uterine epithelial and stromalseparation was performed as previously described (7, 8). Uterine epithelialsheets were digested in 0.8 mg/mL collagenase (Invitrogen; 17018–029) inDMEM/10% FBS by incubation at 37 °C with gentle agitation. Dissociated cellpreparations were passed through a 100-μm nylon mesh to remove clumps.Dissociated epithelia were either used directly or virally infected beforeplacement in the regeneration system. Uterine stromal fragments wereobtained from 0- to 2-d-old C57BL/6 pups, as described previously (7, 8). Stro-mal fragments were digested with collagenase, passed through a 100-μm ny-lonmesh, and cultured short term in BFSmedia (DMEM, 5%FBS, 5%Nu-serum,and 5 μg/mL insulin).

Epithelial Infection and Regeneration. Dissociated epithelial cells were lenti-virally infected with centrifugation at a multiplicity of infection of 40. Dis-sociated epithelial and stromal cells were mixed and resuspended in rat-tailcollagen (BD Biosciences; 354236) neutralized according to the manu-facturer’s instructions. Cell and collagen mixtures were dispensed into 15-μLgrafts and incubated for 30 min at 37 °C. Grafts were overlaid with BFSmedia and incubated overnight. The following day, endometrial grafts wereimplanted under the kidney capsule of oophorectomized CB17Scid/Scid mice.Estrogen pellets (90-d time release, 0.72-mg β-estradiol/pellet; InnovativeResearch of America) were implanted subcutaneously unless otherwisenoted. A progesterone pulse (2.5 mg/d) was administered for the final 8 d ofregeneration, unless otherwise indicated. In endometrial hyperplasiaexperiments, two estrogen pellets were implanted per mouse. All surgicalprocedures were performed under the Division of Laboratory Animal Med-icine regulations for the University of California, Los Angeles.

Immunohistochemistry. Tissue was formalin-fixed and paraffin-embedded.Primary antibodies used for IHC are listed in Table S1. Bioiotinylated goatanti-rabbit, rabbit anti-rat, streptavidin-FITC, and streptavidin-HRP werefrom Jackson ImmunoResearch, and biotinylated anti-mouse antibody waspurchased from Vector Laboratories.

FACS and in Vitro Colony Assay. Endometrial cells were suspended in DMEM/10% FBS and stained with antibody for 20 min at 4 °C with shaking. FACS cellsorting was performed using the BD FACS AriaII (BD Biosciences) machine.FACS population analyses were performed on a BD FACSCanto. Antibodiesused for FACS sorting are listed in Table S1. For colony-forming assays, tripli-cate samples were plated at 10,000 cells per well in PrEGM in matrigel-coatedsix-well plates. Colony formation was scored after 8 d. Epithelial and stromalcolony designations were confirmed by immunocytochemical staining.

Quantitative PCR. RNA was extracted using Qiagen RNeasy Mini kit accordingto the manufacturer’s instructions. Reverse transcription was performed witha SuperScript III first-strand synthesis kit (Invitrogen). Quantitative PCR wasperformed using iQ SYBR Green Supermix for Real-Time PCR (Bio-Rad) ona Bio-Rad iCycler and iQ5 2.0 Standard Edition Optical System Software. Datawere analyzed by using the Pfaffl method. Primer sets used are outlined inTable S2.

ACKNOWLEDGMENTS. We thank Ms. Shirley Quan for her contributions inarrangingmice needed for experiments andMs. Barbara Anderson for her helpin preparing this manuscript. S.M. is supported by Building InterdisciplinaryResearch Careers in Women’s Health Grant (National Institutes of Health/Na-

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tional Institute of Child Health and Human Development 5 K12 HD001400),a Prostate Cancer Foundation Young Investigator’s Award, the Stein Oppen-heimer Clinical Translational Seed Grant, the Liz Tilberis Scholars Program fromthe Ovarian Cancer Research Fund, and a Scholars in Translational Medicinegift. J.H. is supported by American Cancer Society RSG-07-092-01-TBE, Depart-ment of Defense Prostate Cancer Research Program PC061456, a DevelopmentResearch Award from the University of California at Los Angeles Prostate Can-

cer Specialized Program in Research Excellence [Primary Investigator (PI):R. Reiter], a Challenge Award from the Prostate Cancer Research Foundation(PI: O.N.W.), and a Creativity Award from the Prostate Cancer Research Founda-tion (PI: M. Rettig). A.S.G. is supported by the Ruth L. Kirschstein National Re-search Service Award GM07185. T.K. is supported by 1R01CA154358-01 throughthe National Cancer Institute. O.N.W. is a Howard Hughes Medical InstituteInvestigator.

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