progesterone inhibits endometrial cancer invasiveness by...

Research Article

Progesterone Inhibits Endometrial Cancer Invasiveness byInhibiting the TGFb Pathway

Amber A. Bokhari1, Laura R. Lee1, Dewayne Raboteau1, Chad A. Hamilton2, George L. Maxwell3,Gustavo C. Rodriguez4, and Viqar Syed1,5

AbstractIncreased expression of TGFb isoforms in human endometrial cancer correlates with decreased

survival and poor prognosis. Progesterone has been shown to exert a chemoprotective effect against

endometrial cancer, and previous animal models have suggested that these effects are accompanied by

changes in TGFb. The goal of this study was to characterize the effect of progesterone on TGFb signaling

pathway components and on TGFb-induced protumorigenic activities in endometrial cancer cell lines.

Progesterone significantly decreased expression of three TGFb isoforms at 72 hours after treatment

except for TGFb2 in HEC-1B and TGFb3 in Ishikawa cells. Progesterone treatment for 120 hours

attenuated expression of the three isoforms in all cell lines. Progesterone exposure for 72 hours reduced

expression of TGFb receptors in HEC-1B cells and all but TGFbR1 in Ishikawa cells. Progesterone

reduced TGFbR3 expression in RL-95 cells at 72 hours, but TGFbR1 and bR2 expression levels were not

affected by progesterone at any time point. SMAD2/3 and pSMAD2/3 were substantially reduced at 72

hours in all cell lines. SMAD4 expression was reduced in RL-95 cells at 24 hours and in HEC-1B and

Ishikawa cells at 72 hours following progesterone treatment. Furthermore, progesterone effectively

inhibited basal and TGFb1-induced cancer cell viability and invasion, which was accompanied by

increased E-cadherin and decreased vimentin expression. An inhibitor of TGFbRI blocked TGFb1-induced effects on cell viability and invasion and attenuated antitumor effects of progesterone. These

results suggest that downregulation of TGFb signaling is a key mechanism underlying progesterone

inhibition of endometrial cancer growth. Cancer Prev Res; 7(10); 1045–55. �2014 AACR.

IntroductionThe TGFb family members are secreted cytokines that

regulate a broad array of cellular responses, includingproliferation, differentiation, migration, and apoptosis(1, 2). The role of TGFb in tumor biology is complex. Ithas tumor-suppressing activity in the early stages of carci-nogenesis, whereas in the later stages of carcinogenesis, itfunctions as a tumor promoter (2, 3). The growth-inhibi-tory function of TGFb is selectively lost in advanced cancersdue to mutational inactivation or dysregulated expressionof various components of the TGFb signaling pathway

resulting in growth, invasion, and metastasis of cancer cells(4).

Human endometrial tumors contain the three TGFb iso-forms in both the epithelial and stromal counterparts of thetumors, and these proteins are responsible for autocrine aswell as paracrine signaling in the microenvironment ofthese tumors. The different TGFb isoforms can sometimesdifferentially activate signaling pathways in cancer cells,leading to isoform-specific effects on cellular phenotype(5, 6).

Overexpression of TGFb1 has been reported in many(pancreas, breast, prostate, ovary, and endometrium)tumors and is associated with metastatic phenotypes andpoor patient outcome (7, 8). TGFb1 signals are largelyknown as tumor promoters of cellular responses such asproliferation, survival, migration, and invasion (4). Com-pared with TGFb2 and TGFb3, high levels of TGFb1 arereported in endometrial cancer cell lines and these findingsare corroborated with abundant TGFb1 immunoreactivityin human endometrial tumors in vivo (8).

TGFb2 is expressed in a variety of cancer cell lines,including glioma, prostate, breast, lung and kidney, andendometrium (5). In endometrial cancer of the three iso-forms, TGFb2 is the least expressed. Increase of TGFb2protein levels was observed in advanced-stage endometrialtumors (9). TGFb2 promotes the survival of tumor cells by

1Department ofObstetrics andGynecology, UniformedServicesUniversityof the Health Sciences, Bethesda, Maryland. 2Division of GynecologicOncology, and Gynecologic Cancer Translational Research Center ofExcellence,Walter ReedNationalMilitaryMedical Center, Bethesda,Mary-land. 3Department of Obstetrics and Gynecology and Women's HealthIntegrated Research Center, Inova Fairfax Hospital, Falls Church, Virginia.4Division of Gynecologic Oncology, North Shore University Health System,University of Chicago, Evanston, Illinois. 5Department of Molecular andCell Biology, Uniformed Services University of the Health Sciences,Bethesda, Maryland.

Corresponding Author: Viqar Syed, Uniformed Services University of theHealth Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. Phone:301-295-3128; Fax: 301-295-6774; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0054

�2014 American Association for Cancer Research.

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activating NF-kB. Suppression of TGFb2 expression bysiRNA attenuates cancer cells growth, indicating that theTGFb2–NF-kB pathway is important for viability of tumorcells (5).

High levels of TGFb3 are detected in the glands inendometrial carcinoma compared with TGFb3 detectedby immunostaining in the glandular epithelial cells of thenormal proliferative and secretory endometrium (6).TGFb3 is shown to increase invasiveness of endometrialcancer (KLE and HEC-1A) cells through two signalingpathways: PI3K-dependent upregulation of X-linkedinhibitor of apoptosis protein and through protein kinaseC–dependent induction of matrix metalloproteinase-9(MMP-9) expression (6). Upon progression toward amore invasive phenotype (from stage I to stage III endo-metrial adenocarcinoma), TGFb3 immunoreactivity grad-ually extends from the epithelial compartment (innormal tissues) to the stroma (in adenocarcinoma), con-sistent with a role for TGFb3 in endometrial cancer cellinvasiveness (6, 8).

TGFb ligands bind to TGFb receptor type 2 (TGFbR2), aconstitutively active transmembrane serine/threoninekinase, and forms an oligomeric complex with TGFb recep-tor type 1 (TGFbR1). TGFbR1 is phosphorylated by theTGFbR2. The activated TGFbR1 initiates intracellular sig-naling through phosphorylation of specific receptor-regu-lated (R)-SMAD proteins (SMAD2 and SMAD3). Activated(R)-SMADs form a complex with SMAD4, and the complextranslocates to the nucleus, where it binds to DNA in asequence-specific manner, and regulates gene transcription(3, 4).

Immunohistochemical analysis has shownhigher expres-sion of TGFbR1, TGFbR2, and TGFbR3 in endometrialtumors compared with normal tissues (10, 11). Deregula-tion of TGFbR3 expression not only results in loss ofantiproliferative activity of TGFb signaling at early stagesof endometrial cancer development but also leads to acqui-sition of cell motility. High TGFbR3 expression plays a rolein mediating cell proliferation and migration in endome-trial tumors (9). TGFbR3 binds all three TGFb isoforms. Thefunction of TGFbR3 is to present ligands to TGFbR2 andTGFbR1, which, upon ligand binding, transphosphorylateto activate the type I TGFbR1 (12, 13).

An important step in the metastatic process is epithelialto mesenchymal transition (EMT), in which adherent andnonmotile epithelial cells acquire motility and invasive-ness (14). Increased expression of TGFb induces EMT byenhancing the expression of zinc-finger transcriptionalfactors Snail and Slug and downregulating expression ofepithelial marker E-cadherin, which is critical in mediat-ing epithelial cell integrity and cell–cell adhesion (15).Subsequently, mesenchymal markers N-cadherin, vimen-tin, and MMP proteins are elevated and thus cell motilityand invasive phenotypes (14, 16). Cancer cells overex-pressing active TGFb have increased metastatic ability(17, 18), and targeting of TGFb signaling prevents metas-tasis in several neoplastic tumors, including breast, pros-tate, and colorectal cancers (19–21).

Progestins are highly effective endometrial cancer–pre-ventive agents. Extensive epidemiologic investigations dem-onstrate that long-term progestins use is associated withsignificant endometrial cancer risk reduction (22–23). Pro-gesterone has also been used as a primary treatment forendometrial carcinoma in premenopausal women, andresponse rates in these women can be as high as 60%,indicating that progesterone is a potent inhibitor of endo-metrial carcinogenesis (24, 25). Recent studies in primateshave demonstrated a potential mechanism underlying thechemopreventive effect of progestins [levonorgestrel andmedroxyprogesterone acetate (MPA)] against ovarian andendometrial cancer. Progestin exposure was associated withincreased apoptosis and a concomitant marked decrease inthe expression of TGFb1 in the ovarian and endometrialglandular epithelium, and a concomitant increase in theexpression of TGFb2 and 3 in the ovarian epithelium andendometrial stroma, respectively (26, 27). This suggests thatthe antitumorigenic effects of levonorgestrel and MPA maybe at least, in part, mediated by altering the expression ofTGFb signaling components.

Despite the importance of TGFb in regulating severalendometrial cellular activities (4, 28), the effect of pro-gesterone on the expression and regulation of TGFbsignaling components in human endometrial cancer hasnot been well characterized. In the present study, weexamined the effect of progesterone on the TGFb/SMADsignaling pathway in endometrial cancer cells andshowed that progesterone inhibits TGFb-induced R-SMAD expression, nuclear translocation, and subsequent-ly attenuates tumor cell growth and invasiveness bydecreasing vimentin and increasing E-cadherin.

Materials and MethodsCell culture and treatment

Human endometrial cancer cell lines HEC-1B and RL-95were obtained from the ATCC and the Ishikawa cell linewasobtained from Sigma. The cell lines were authenticated byDNA short-tandem repeat analysis by the ATCC and Sigma.All three cell lines were initially expanded and cryopre-served within 1 month of receipt. Cells were typically usedfor 3 months, at which time a fresh vial of cryopreservedcells was used. The cells were routinely tested for mycoplas-ma. Ishikawa cellswere grown inDMEM:F12 supplementedwith insulin (Invitrogen), HEC-1B cells were cultured inEagle minimum essential medium (Invitrogen), and RL-95cells were grown in DMEM:F12 medium supplementedwith insulin (0.005 mg/mL). All three media were supple-mented with 10% (v/v) FBS (Invitrogen), 100 U/mL pen-icillin, and 100 U/mL streptomycin. The cells were culturedat 37�C in a humidified atmosphere containing 5% CO2.Forty-eight hours later, the media were replaced withsame media but containing charcoal-stripped FBS. Thecells were treated with progesterone (20 mmol/L, 99.9%pure; Sigma) for 24, 72, and 120 hours and collectedfor protein extraction. The time of treatment and doseof progesterone was based on our previous studies show-ing inhibition of cell growth and apoptosis of cancer

Bokhari et al.

Cancer Prev Res; 7(10) October 2014 Cancer Prevention Research1046




cells (29). For a set of experiments, cells were treatedwith progesterone (20 mmol/L) or TGFb1 (10 ng/mL) inthe presence or absence of TGFbR1 blocker (SD-208,10mmol/L; Santa Cruz Biotechnology) for 72 hours andthe effects on cell viability and invasion were assessed.Progesterone solution was in absolute ethanol, TGFb1solution was in aqueous saline, and TGFbR1 blocker wasin DMSO. The final concentrations of ethanol and DMSOin the media were 0.1%. Untreated control cultures wereexposed to equal concentrations of ethanol, aqueoussaline, or DMSO vehicle alone.

Measurement of TGFb1 concentration in theconditioned mediaTo study the effect of progesterone on the secretion

of TGFb in culture, endometrial cells were treated for72 hours with progesterone or TGFb1 in the absence orpresence of progesterone receptor antagonist Mifepristone(0.1 mmol/L). After culture, cells were removed by centri-fugation and the cell-freemediawere collected and stored at�80�C until use. After thawing at room temperature, themedia were assayed for immunoreactive latent TGFb byenzyme-linked immunosorbent assay (BioLegend) accord-ing to the manufacturer’s specifications with absorbance at450 nm in a plate reader (ELX800). The immunoassay kitused in this experiment offered a dynamic range starting inthe low ng/mL and covered three logarithms. Experimentswere repeated at least three times with different media tominimize the intra- and interassay variation.

Cell viability assayCell viability of endometrial cancer cells treated with

progesterone, TGFb1, or the combination in the presenceor absence of TGFbR1 blocker was evaluated using theCellTiter 96 AQueous One Solution cell viability assay(Promega) according to the instructions of the manufac-turer. CellTiter 96 AQueous One Solution reagent (20 mL)was added into each well of the 96-well assay plate contain-ing the samples in 100 mL of culture medium. Absorbancewas measured at 490 nm using an ELX800 microtiterReader. Relative cell viability was expressed as percentchange of treated cells over vehicle-treated cells.

Cell invasion assayEndometrial cancer cells treated with progesterone,

TGFb1, or the combination in the presence or absence ofTGFbR1 blocker for 72 hours were detached by trypsin andresuspended in serum-free medium. Medium containing10% FBSmediumwas applied to the lower chambers of BDBioCoat Matrigel Invasion Chambers (BD Biosciences) aschemoattractant, and then, cells were seeded on the upperchambers at a density of 2.5� 104 cells per well in 100 mL ofserum-free medium without progesterone or TGFb1. Thechambers were incubated for 16 to 18 hours at 37�C. At theend of incubation, noninvading cells were removed fromthe upper surface of the membrane by scrubbing. The cellson the lower surface of the membrane were fixed for 2minutes in 100% methanol and stained with 1% toluidine

blue in 1% sodium borate for 2 minutes. Excess stain wasremoved by rinsing the inserts with water. Each membranewas removed from the insert and placed on a microscopeslide. The cover slip was placed on the slide and cells werecounted in five randomfields per slide. All slideswere codedto avoid biased counting. The assay was run in triplicates.

Western blot analysisCell lysates were prepared in RIPA buffer [50 mmol/L

Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% deoxycholate,1.0% NP40, and 0.1% SDS] supplemented with a proteaseinhibitor mixture solution (Roche Molecular Biochem-icals). After sonication, cell debris was pelleted by centri-fugation, and protein concentration was determined withthe BCA Protein Assay Reagent (Pierce). Equivalentamounts of proteins were resolved by 10% SDS–PAGE andtransferred to PVDF membranes. The blots were then incu-bated with primary antibodies against TGFb1, TGFb2,TGFb3, TGFbR1, TGFbR2, TGFbR3, pSMAD2/3, SMAD2/3, SMAD4 (Santa Cruz Biotechnology), E-cadherin, vimen-tin (Cell Signaling Technology, Inc.), and b-actin (Sigma-Aldrich). The Enhanced Chemiluminescence DetectionSystem (Pierce), followed by autoradiography, was usedfor protein visualization. Protein bands were quantifiedusing densitometry software (Bio-Rad) and normalizedusing actin as a loading control. To calculate the relativeintensity of eachband, individual bandswere dividedby thecorresponding loading control intensity.

ImmunofluorescenceCells were plated on a glass bottom culture dish (MatTek

Corporation) at a density of 0.3 � 106 cells per dish. Cellswere grown to approximately 50% confluence in chamberslides and then treated with or without TGFbR1 blocker for2 hours before treatment with either vehicle, progesterone,TGFb1, or a combination of progesterone and TGFb1 for a24-hour period. After treatment, cells were washed twicewith PBS and fixed with 4% paraformaldehyde for 15minutes. Cells were then permeabilized with 100% meth-anol at�20�C, and care was taken to prevent desiccation ofthe samples. After complete removal of methanol, thesamples were blocked using buffer prepared with normalgoat serum for 60minutes. SMAD2/3 antibody (Santa CruzBiotechnology) diluted at 1:250 was applied overnight at4�C. After complete removal of primary antibody, thediluted secondary antibody, Alexa Fluor 488 Goat Anti-mouse IgG (Invitrogen; Cat# A-11001), was applied. Sam-ples were incubated for 2 hours at room temperature in thedark. Once excess secondary antibody was removed byrinsing with PBS, 40,6-Diamidino-2-Phenylindole, Dihy-drochloride (DAPI, 1 mg/mL; Sigma) was applied to coverfor 30 seconds in the dark and then rinsed away with PBS.Coverslips were then mounted with Prolong Gold AntifadeReagent (Invitrogen;Cat#P36930). Slideswere imagedonaZeiss Pascal Confocal Microscope (Carl Zeiss MicroscopyGmbH) immediately following preparation. Z stack slicesobtained through confocal microscopy were then summedin ImageJ (NIH).

Progesterone and TGFb Signaling

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Statistical analysisData are presented as themean of triplicate determinants

with SEM. Experiments carried out in triplicate were repeat-ed at least 3 times. Statistically significant differences weredetermined between control and treatment groups usingtwo-way ANOVA followed by the Tukey post hoc test. A Pvalue of less than 0.05 was considered statistically signifi-cant. Western blots were quantified using densitometry. Arepresentative of 3 immunoblots is presented in the figuresalong with average relative density of the bands normalizedto b-actin. Statistically significant differences were deter-mined between control and treatment groups by quantifi-cation of 3 immunoblots, and p values are provided in theresults section.

ResultsProgesterone attenuates TGFb ligands in endometrialcancer cells

Endometrial tumors have been reported to express highlevels of three TGFb isoforms and TGFb receptors in vivo (6).In addition, it has been suggested that TGFb1 plays a majorrole in the initiation of endometrial carcinoma invasion(21). To determine whether progesterone regulates theexpression of TGFbs and their receptors, HEC-1B, Ishikawa,and RL-95 human endometrial carcinoma cell lines wereexposed to progesterone for 24, 72, and 120 hours. Expo-sure of endometrial cells to progesterone for 120 hoursshowed significantly decreased expression of TGFb1, -b2,and -b3 in all cell lines tested. All isoforms showed amarkeddecrease in expression following 72 hours of exposureexcept for TGFb2 and TGFb3 in HEC-1B and Ishikawa cells,respectively. In RL-95 cells, all three TGFb isoforms showeda significant decrease with progesterone in 72 hours ofculture. No alteration of TGFb isoforms was seen in anycell line with progesterone in the first 24 hours of exposure(Fig. 1A).

Progesterone inhibits latent TGFb concentration in theculture medium of cancer cells

To ascertain if progesterone regulates secretion of TGFb,the amount of TGFb secreted into the culture media wasassayed by ELISA. As evident in Fig. 1B, cells exposed toTGFb1 secreted high levels of latent TGFb compared withvehicle-treated cells. TGFb secretion by endometrial cancercells was markedly inhibited by addition of 20 mmol/Lprogesterone for 72 hours compared with vehicle-treatedcells. TGFb1-induced secretion of latent TGFb was attenu-ated by progesterone. No such inhibiting effect wasobserved in the culture media of cells treated with proges-terone receptor antagonist (Mifepristone) and progesteroneor TGFb1 progesterone combination. Mifepristone-treatedcells showed no effect on TGFb. These results clearly suggestthat cancer cells secrete TGFb, and progesterone, at least invitro, inhibits basal and TGFb1-induced secretion. Down-regulation of TGFb was blocked by the antagonist Mifep-ristone providing evidence for the specificity of progester-one action in cancer cells.

TGFb receptors are downregulated by progesterone inendometrial cancer cells

TGFb acts through amembrane-associated receptor com-plex. We evaluated the protein expression of TGFbR1,TGFbR2, and TGFbR3 in endometrial cultures exposed toprogesterone for 24, 72, and 120 hours. In HEC-1B cells,expression of TGFbR1, TGFbR2, and TGFbR3 was signifi-cantly (P < 0.05) downregulated following progesteroneexposure for 72 and 120 hours compared with vehicle-treated cells. Ishikawa cells showed a marked reduction ofTGFbR1 expression at 120 hours of cell culture with pro-gesterone, whereas TGFbR2 and TGFbR3 were inhibited at72 and 24 hours, respectively. Progesterone inhibitedTGFbR3 expression in RL-95 at 72 and 120 hours exposureand had no effect on TGFbR1 and TGFbR2 at any time point(Fig. 2).

SMAD expression is inhibited by progesterone inendometrial cancer cells

In the canonical signaling pathway, SMAD phosphory-lation is an indicator of functional TGFb receptors. Toconfirm that progesterone inhibits TGFb signaling trans-duction, protein levels of downstream SMADs were inves-tigated. We examined levels of total and phosphorylatedSMAD2/3 (p-SMAD2/3) in endometrial cancer cellsexposed to progesterone for 24, 72, and 120 hours. Incu-bation with an antibody recognizing p-SMAD2 and 3 atSerine 423/425 showed that SMAD2/3 is phosphorylated innontreated cancer cells. A significant reduction in SMAD2/3and pSMAD2/3 was apparent 72 and 120 hours afterexposure to progesterone in all cell lines except RL-95,where pSMAD2/3 showed significant inhibition at 24 hoursof treatment. Total and pSMAD2/3 levels were not affectedby progesterone in HEC-1B and Ishikawa cells during thefirst 24 hours of treatment (Fig. 3). These results imply thatprogesterone is a potent TGFb antagonist in endometrialcancer cells. HEC-1B and Ishikawa cells exposed to proges-terone for 72 and 120 hours showed pronounced decreasein SMAD4 expression. RL-95 cells exhibited a markeddecrease in SMAD4 expression after 24 hours of progester-one treatment (Fig. 3).

Furthermore, we investigated the effect of progesteroneon the TGFb1-induced subcellular localization of SMAD2/3in HEC-1B and Ishikawa endometrial cancer cells. In vehi-cle-treated cells, SMAD2/3 was expressed in the cytoplasmand in the nucleus. Progesterone and TGFb1 treatmentdecreased cytoplasmic SMAD2/3 (Fig. 4A and C). To estab-lish that high SMADs localization in cancer cells is activatedvia an autocrine pathway, endometrial cancer cells pre-treated with the TGFb receptor I blocker and then exposedto TGFb1 or a combination of TGFb1 and progesteroneshowed significantly attenuated nuclear localization ofSMAD2/3 (Fig. 4B and D) compared with non-TGFb recep-tor I blocker pretreated cells (Fig. 4A and C). These findingsstrongly suggest that nuclear localization of SMAD2/3,which is an indication of the activated TGFb/SMAD signal-ing pathway, is attributed to TGFbproduced and secreted byendometrial cancer cells.

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Progesterone attenuates TGFb-mediated cell viabilityand invasionTGFb1has a principal role at the initiation of endometrial

carcinoma invasion through the promotion of the EMT thatleads to the acquisition of an invasive phenotype. In addi-tion, although TGFb1 is a potent inhibitor of proliferationof most types of normal epithelia, it has a paradoxicalproliferative effect in carcinomas (30). To explore the effectof progesterone and TGFb1 on cell proliferation and inva-sion, we examined the effect of progesterone, TGFb1, or thecombination in the presence or absence of TGFbR1 blocker(72 hours) on cell viability and cellular invasiveness. Asshown in Fig. 5A, TGFb1 treatment of endometrial cancercells induced a marked increase in cell viability comparedwith cells treated with vehicle. As expected, progesteronealone inhibited cell viability. Interestingly, progesteronemarkedly abrogated TGFb1-induced cell viability in bothcell lines shown. TGFb1 failed to enhance cell viability in thepresence of TGFbR1 blocker. Moreover, progesterone-induced inhibition of cell viability was attenuated byTGFbR1 blocker.The effects of progesterone and TGFb1 on the invasive-

ness of HEC-1B and Ishikawa cell lines were determined byusing a boyden chamber invasion assay. Treatment ofendometrial cancer cells with TGFb1 produced a significantincrease in invasiveness as compared with vehicle-treatedcontrol. In addition, HEC-1B and Ishikawa cells treatedwith progesterone alone, or TGFb1 and progesterone in

combination, underwent significant decreases in invasive-ness when compared with vehicle-treated cells and TGFb1-treated cells, respectively (Fig. 5B). TGFbR1blockermarked-ly reduced the antitumorigenic effect of progesterone on cellinvasiveness. These results demonstrate that progesteroneinhibits both basal and exogenous TGFb1-induced promi-gratory and invasive activity of endometrial cancer cells bysuppressing TGFb signaling.

Progesterone reduces expression of mesenchymalmarkers and inhibits EMT process

The EMT process, which is a crucial step in tumorprogression, can be induced by several cytokines and che-mokines, including TGFb (4). To examine if a progesterone-induced decrease in cell invasiveness is associated with theEMT process, we evaluated the expression of EMT markerproteins (vimentin and E-cadherin) in extracts of HEC-1Band Ishikawa cells treated with vehicle, progesterone,TGFb1, or combination of progesterone and TGFb1 for72 hours. Vimentin was upregulated and E-cadherin wasdownregulated in vehicle-treated cells. The TGFb1-exposedcells showed decreased expression of E-cadherin andenhanced expression of vimentin. Progesterone treatmentcaused a significant decrease in vimentin and an increase inE-cadherin expression in cancer cells. A similar expressionpattern was observed in TGFb1- and progesterone-exposedcells. These results suggest that progesterone inhibits theEMT process in endometrial cancer cells (Fig. 6).

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Figure 1 A, downregulation of TGFbisoforms expression in endometrialcancer cells by progesterone.Endometrial cancer cells (HEC-1B,Ishikawa, and RL-95) treated withprogesterone (PROG, 20 mmol/L)for 24, 72, and 120 hours wereevaluated by Western blot analysisfor expression of TGFb1, TGFb2,and TGFb3. b-Actin was used as aloading control. Data,representative of threeindependent experiments. B,concentration of latent TGFbpresent in the culture media ofendometrial cancer cells treatedfor 72 hours with progesterone(20 mmol/L), TGFb1 (10 ng/mL), orthe combination in the absence orpresence of progesterone receptorantagonist Mifepristone (0.1 mmol/L). The concentrations of TGFbpresent in the conditionedmedia ofcells were measured by ELISA, asdescribed in Materials andMethods. Measurement was madeat least three times per sample andpresented as mean � SD.�, P < 0.05 compared with control.






DiscussionA strong body of clinical evidence has shown that pro-

gestins are potent inhibitors of endometrial carcinogenesis.Routine use of progestin-containing oral contraceptivesmarkedly lowers endometrial cancer risk by as much as30% or more (31, 32). In addition, high-dose progestintherapy has been shown to reverse preexisting PTEN-inac-tivated endometrial latent precursors, as well as endome-trial hyperplasia and even-low grade endometrial cancer insome women (33–35).

TGFb canplaymultiple roles in carcinogenesis, acting as atumor suppressor at early stages and a tumor promoter atlate stages (2, 4). Impairment of the TGFb–SMAD pathwaycan result in loss of restraint of growth, initiation of cellproliferation, and carcinogenesis (36, 37). There is a paucityof information available about the effect of progesterone onTGFb/SMAD signaling in endometrial carcinoma cells. Tofill this data gap, we explored the antitumor effect ofprogesterone on TGFb signaling at three levels: ligands,receptors, and SMADs. High levels of TGFbs are present inendometrial carcinoma cells in vitro and in vivo (38–39).TGFb isoforms have been shown to act on endometrial cell

growth by autocrine or paracrine mechanisms and areimplicated in several processes of tumorigenesis andmetas-tasis (8). In the current study, progesterone inhibitedexpression of TGFb ligands in all of the tested endometrialcancer cell lines. Our study provides clear evidence thatthe TGFb produced and secreted by endometrial cancercells is inhibited by progesterone, and the progesteronereceptor antagonist Mifepristone abolished progesterone-induced inhibition of TGFb secretion from endometrialcancer cells.

TGFb mediates its effects from the surface of cells tothe nucleus through TGFb receptors. Thus, TGFbRs are thegateways of intracellular signaling (4). Therefore, drugsblocking TGFb receptors’ intracellular activity are urgentlyneeded to inhibit TGFb-induced tumor progression. In

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Figure 2. Progesterone inhibited TGFb receptor expression in endometrialcancer cells. Endometrial cancer cells (HEC-1B, Ishikawa, and RL-95)treated with progesterone (PROG, 20 mmol/L) for 24, 72, and 120 hourswere evaluated by Western blot analysis for expression of TGFbR1,TGFbR2, and TGFbR3. b-Actin was used as a loading control. Data,representative of three independent experiments.

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1.01 1.07 1.89 0.05 1.43 0.97

0.25 0.14 1.19 0.02 1.42 0.03

Figure 3. Downregulation of SMADexpression in endometrial cancer cellsby progesterone. Endometrial cancer cells (HEC-1B, Ishikawa, and RL-95) treated with progesterone (PROG, 20 mmol/L) for 24, 72, and 120hours were evaluated by Western blot analysis for expression of p-SMAD2/3, SMAD2/3, andSMAD4. b-Actinwas used as a loading control.Data, representative of three independent experiments.

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the present report, we show that one of the possiblemechanisms by which progesterone inhibits cancer cellsviability and invasiveness is through targeting TGFbRs asevident by a marked decrease in TGFbR1, TGFbR2, andTGFbR3 expression.The TGFbR3 plays a vital role in presenting TGFb to the

signaling receptors, enhancing receptor–ligand interactionsand promoting the cooperation between TGFb type I and IIreceptors. Decreasing the expression of TGFbR3 in endo-metrial cancer cells lines by progesterone may lead todecreased endometrial responsiveness to TGFb, which isa natural modulator of endometrial cell proliferation andultimately inhibition of endometrial cancer cells invasionand metastasis.SMAD proteins are major signaling molecules acting

downstream of TGFb receptors. In normal cells, RSMADs

are predominantly concentrated in the cytoplasm. Uponbinding of TGFb to TGFbR2 and phosphorylation ofTGFbR1, the activated receptor complex phosphorylatesSMAD2/3, which forms a complex with SMAD4. Thiscomplex then translocates into the nucleus (4). We ana-lyzed the localization of SMAD2/3 and its nuclear translo-cation in TGFb1 and progesterone-treated endometrialcancer cells. SMAD2/3 was found in the cytoplasm ofendometrial cancer cells. However, it was found at evenhigher levels in the nucleus, despite the absence of exoge-nous TGFb1. This indicates constitutive activation of theSMAD pathway. This can be explained by the fact thatendometrial cancer cells secrete TGFbs, which activate thesignaling pathway by an autocrine mechanism. As a result,SMADs eventually translocate into the nucleus where theyinteract with other transcription factors to induce or repress

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Figure 4. Localization of SMAD2/3inHEC-1Band Ishikawacells. Cellswere treated with progesterone(PROG, 20 mmol/L), TGFb1(10 ng/mL), or a combination of thetwo for 24 hours with (B and D) orwithout (A and C) TGFbR1 inhibitor(SD-208, 10 mmol/L). The cellimages were aligned into threevertical panels: the first verticalpanel shows nuclear staining withDAPI, the second shows SMAD2/3signals, and the third is the overlayof DAPI and SMAD2/3. Theexperiment was performed threetimes.






transcriptionof anumberof target genes.Ourfindings showthat TGFb1 treatment of the cells caused translocation ofSMAD2/3 from the cytoplasm to the nucleus. Progesteronereduced SMAD2/3 in the cytoplasm. This suggests disrup-tion of SMAD signaling may be in part the mechanism bywhich progesterone affects tumor cell growth. To establishthat SMAD activation in endometrial cancer cells is via anautocrine pathway, cells exposed to TGFbR1 blockershowed lack of SMAD2/3 nuclear localization comparedwith cells where TGFbR1 was not blocked. These findingssubstantiate that endometrial cancer cells secrete TGFb,which activate the signaling pathway by an autocrinemech-anism. The discrepancy between confocal results and time-dependent effect of progesterone on SMAD2/3 expressionin endometrial cancer cells by Western blotting can beexplained by the fact that confocal microscopy is moresensitive than Western blotting. Thus, small decrease ofSMAD2/3 in the cytoplasm was not detected by Western

blotting.However, when cellswere exposed to progesteronefor a longer period of time, a marked decrease in SMAD2/3observed in total cellular extracts can be attributed to anoverall decrease in TGFb/SMAD signaling pathway compo-nents and could be implicated in the observed decrease incell viability and invasion.

Because the findings reported herein suggest a role ofprogesterone in regulation of the TGFb/SMAD signalingpathway, it is conceivable that the response of cells toprogesterone depends on progesterone receptors. In ourrecently published study (40), we have shown reducedexpression of both isoforms (PR-A and PR-B) in cancer(Ishikawa, HEC-1B, and RL95-2) cell lines compared withan immortalized endometrial epithelial (EM-E6/E7-TERT)cell line, and no marked changes were noticed in theexpression of progesterone receptor isoforms in immortal-ized cells and in endometrial cancer cells following proges-terone treatment. Treatment of endometrial cancer cellswith progesterone caused a dose-dependent decrease in cellviability, and progesterone receptor antagonist abrogatedthat effect (29), suggesting the presence of functional pro-gesterone receptors on endometrial cancer cells. Levels ofprogesterone receptor expression vary in cancer cell lines,whichwould explain the differences in the time required forprogesterone to downregulate TGFb, TGFbRs, and SMADsamong cancer cell lines.

Our results showed reduced levels of TGFb secretedinto culture media of cells exposed to progesterone com-pared with vehicle-treated cells. Abrogation of progester-one-induced decrease of TGFb into culture media of cellsexposed to progesterone receptor antagonist is a testamentof the fact that reduction of TGFb by progesterone ismediated through progesterone receptors. Furthermore,treatment of endometrial cancer cells with progesteroneinhibited TGFb-induced SMAD2 translocation in the nucle-us. This effect was not observed when cancer cells werecultured with the progesterone receptor antagonist

160

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Figure5. Progesterone inhibits basal andTGFb1-induced cell viability andinvasion of endometrial cancer cells. HEC-1B and Ishikawa cells werecultured with progesterone, with and without TGFb1 (10 ng/mL) for72 hours in the presence or absence of TGFbR1 (SD-208, 10 mmol/L)blocker, and the effect of treatment on cell viability (A) and invasion (B)was assessed usingMTS and cell invasion assays. Data, mean�SEMofvalues from triplicates. �, P < 0.05 (statistically significant) between thecontrol and the treatment groups. The experiment shown isrepresentative of at least three experiments, all with similar results.

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0.190.112.271.270.280.542.131.47

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Figure 6. Progesterone inhibits EMT by suppressing the expression ofmesenchymal marker. The protein expression of EMT markers wasmeasured by Western blot analysis in endometrial cancer cells treatedwith progesterone in the presence or absence of TGFb1 for 72 hours.Values of densitometric scans were used to quantify the changes of EMTmarkers. The experiment was performed twice.

Bokhari et al.





Mifepristone (results not shown). These findings stronglysuggest that alterations in TGFb signaling pathway compo-nents are specifically mediated by progesterone.Results presentedhereindemonstrated adecrease inbasal

and TGFb1-induced viability, and invasive potential ofendometrial cells exposed to progesterone. TGFbR1 block-ade has shown effects on cellular responses, such as cell-cycle arrest and EMT of epithelial cells in vitro (41, 42). Thefact that TGFbR1 blocker could completely block TGFb1-induced viability and invasion and markedly attenuatedprogesterone-induced suppression of both phenotypes sug-gests that progesterone is exerting its effects via the TGFb/SMAD signaling pathway. A significant decrease in cellviability and invasion seen with progesterone in TGFbR1blocker–treated cells indicates that progesterone uses otherpathways as well to inhibit malignant phenotypes. Theseresults are in agreement with studies demonstrating thatblocking TGFb receptors inhibits TGFb1-induced prolifer-ation and reduces the invasive and metastatic potential ofhuman glioma colon, breast, and pancreatic cancer cells(42–44).In the present study, the inhibitory effect of progester-

one treatment alone on cell viability and invasion ismore potent than blocking TGFbR. In the previous study,we investigated the effects of progesterone on endome-trial cancer cells and identified its targets of action usingmass spectrometry–based proteomics. A total of 278proteins, including histone H1.4 (HIST1H1E), histidinetriad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase(EIF2AK2), and Bcl-2–associated X protein (BAX),showed differential expression with progesterone (40).The dominant inhibitory effect of progesterone on cellviability and invasion can be attributed to upregulation ofseveral tumor suppressor proteins suppressing cancer cellviability and invasive potential independent of the TGFbsignaling pathway.Althoughmany signaling pathways have been implicated

in the regulation of cell migration and invasion, TGFb isknown to be a major modulator of tumor cell migration(4, 45) and invasion, partly due to its promotion of EMT(2). TGFbhas emerged as a promising target for treatment ofcancer metastasis (4). The expression of TGFb1 is increasedin various tumors, including endometrial cancer, and cor-relates with poor patient prognosis (11, 45). TGFb1 triggersEMT by direct phosphorylation of SMAD2/3, nuclear trans-location of active SMAD complexes, and subsequent inter-actions with the general transcription machinery. As aresult, cells become invasive, lose epithelial characteristics,and acquire a mesenchymal phenotype characterized bymigration and invasion (46, 47). This process is associatedwith the reduction of E-cadherin and the increase of N-cadherin and vimentin, allowing cells to become moremotile and invasive (14, 48). Reduced E-cadherin expres-sion in endometrial tumors has been reported (49). Herein,we showed that progesterone decreased the invasiveness ofendometrial cancer cells via upregulation of E-cadherin anddownregulation of vimentin. These findings are supported

by a study that implicates progesterone in inhibiting EMT inendometrial cancer (49).

Our work suggests that endometrial cancer cells have anintact TGFb pathway as they respond to TGFb and prolif-eration is reduced by adding TGFbR blocker. Endometrialcancer cells receive a myriad of signals from the tumormicroenvironment, and the interpretation of these signalsultimately results in the enhancement of cellular responses(e.g., growth, migration, invasion). With respect to TGFb/SMAD signaling, target gene transcription is dictated bynuclear accumulation of the activated SMAD complex andsubsequent interaction with nuclear transcription factorsand cofactors and activation of tumor promoting genes.Hence, suppression of the activated TGFb pathway byprogesterone may be a new avenue for potential therapyin human endometrial tumors.

In summary, these findings suggest that progesteronemarkedly suppresses the TGFb pathway in endometrialcancers. Progesterone suppresses expression of the TGFbligands, thus inhibiting autocrine TGFb-mediated growthoftumor cells. Progesterone also suppresses TGFb down-stream signaling events via suppression of TGFb receptorsand SMADs. These inhibit the metastatic characteristics ofendometrial cancer cells by increasing E-cadherin anddecreasing vimentin expression. These data suggest thatmodification of TGFb signalingmay be amajormechanismunderlying the anticancer effects of progesterone in theendometrium.

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

DisclaimerThe opinions and assertions expressed herein are those of the authors

and should not be construed as reflecting those of the UniformedServices University of the Health Sciences, Department of the Air Force,or the U.S. Department of Defense.

Authors' ContributionsConception and design: C.A. Hamilton, G.L. Maxwell, G.C. Rodriguez,V. SyedDevelopment of methodology: A.A. Bokhari, C.A. Hamilton,G.C. RodriguezAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A.A. Bokhari, L.R. Lee, V. SyedAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A.A. Bokhari, L.R. Lee, G.L. Maxwell,G.C. Rodriguez, V. SyedWriting, review, and/or revision of the manuscript: C.A. Hamilton,G.L. Maxwell, G.C. Rodriguez, V. SyedAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. RaboteauStudy supervision: V. Syed

Grant SupportThis study was funded by the United States Medical Acquisition Activity

Award W81XWH-11-2-0131 (to C.A. Hamilton, G.L. Maxwell, G.C. Rodri-guez, and V. Syed).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 11, 2014; revised July 11, 2014; accepted July 11, 2014;published OnlineFirst July 28, 2014.






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2014;7:1045-1055. Published OnlineFirst July 28, 2014.Cancer Prev Res Amber A. Bokhari, Laura R. Lee, Dewayne Raboteau, et al.

PathwayβInhibiting the TGFProgesterone Inhibits Endometrial Cancer Invasiveness by

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