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Original Research

Loss of progesterone receptor links to high proliferationand increases from primary to metastatic endometrialcancer lesions

Ingvild Løberg Tangen a,b,⇑, Henrica M.J. Werner a,b, Anna Berg a,b, Mari K. Halle a,b,

Kanthida Kusonmano

a,b,c

, Jone Trovik

a,b

, Erling A. Hoivik

a,b

, Gordon B. Mills

d

,Camilla Krakstad a,b, Helga B. Salvesen a,b

a Centre for Cancer Biomarkers, Department of Clinical Science, University of Bergen, Norwayb Department of Gynecology and Obstetrics, Haukeland University Hospital, Norwayc Computational Biology Unit, University of Bergen, Bergen, Norwayd Department of Systems Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA

Received 2 June 2014; received in revised form 12 August 2014; accepted 10 September 2014Available online 30 September 2014

KEYWORDSEndometrial cancerProgesterone receptorSurvivalCDK inhibitors

Abstract Objective: In endometrial cancer loss of progesterone receptor (PR, gene namePGR) is associated with aggressive disease and altered response to hormonal treatment. Theaim of this study was to investigate changes in PR expression level with disease progression,and explore whether differences in gene expression according to PR status can be linked toprocesses involved in cancer development elucidating new therapeutic opportunities.Methods: 686 primary endometrial cancers and 171 metastatic lesions were investigated forPR expression in relation to clinical and histopathological data. Protein levels were investi-gated by immunohistochemistry and reverse phase protein array, and mRNA levels byDNA oligonucleotide microarray.Results: PR protein level was significantly associated with PGR mRNA expression (P < 0.001)and patient survival (P < 0.001). Loss of PR increased with disease progression, with 23% of the primary tumours and 76% of metastases demonstrating PR loss. Using a cell cycle progres-sion signature score, PR loss was associated with increased proliferation for both oestrogen

receptor (ER) positive and negative tumours. Through a Connectivity Map search, CDKinhibitors and other drugs with anti-proliferative effects were suggested in particular for treat-ment of patients with loss of PR.

http://dx.doi.org/10.1016/j.ejca.2014.09.003

0959-8049/ 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Clinical Science, Section for Gynecology and Obstetrics, University of Bergen, Jonas Lies Vei 72, 5020Bergen, Norway. Tel.: +47 55 97 42 00; fax: +47 55 97 49 68.

European Journal of Cancer (2014) 50, 3003 – 3010

A v a i l a b l e a t w w w . s c i e n ce d i r e c t . c o m

ScienceDirect

j o u r n a l h o m e p a g e : w w w . e j c a n c e r . c o m

http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://www.sciencedirect.com/http://www.sciencedirect.com/http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://dx.doi.org/10.1016/j.ejca.2014.09.003http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.ejca.2014.09.003&domain=pdf


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Conclusion: Loss of PR in endometrial cancer is associated with increased proliferation, poorsurvival, and increases from primary to metastatic lesions. Based on expression profiles, CDKinhibitors may have activity in PR negative tumours, supporting further testing in clinical tri-als for patients with systemic endometrial cancer dependent on PR status.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

When endometrial cancer is identified and treated atan early stage there is subsequent good prognosis. How-ever, for patients with systemic disease either recurrent ormetastatic at presentation, the prognosis is poor,unchanged over the last several decades, and with limitedtreatment options [1]. Although endometrial cancer is themost common gynaecological malignancy in developedcountries and the incidence is increasing [2], the progressin development of treatment for advanced or recurrentdisease has been slow. Effective new targeted therapies,

combined with robust biomarkers to identify patient sub-groups that will benefit most from emerging as well asavailable treatments will improve patient care.

Progesterone is important for regulation of normalreproductive function, and is involved in controllingchanges in the uterus and ovaries during the menstrualcycle. The effect of progesterone is mediated throughprogesterone receptor (PR), and PR is expressed in avariety of human tissues, including the uterus, mam-mary gland and ovary [3].

In breast cancer progesterone plays a role in control-ling tumour promotion [4], whilst in the endometriumand the ovaries it has a suppressive effect on tumourdevelopment [5,6]. Although the effect of progesteronediffers depending on the target tissue, the PR expressionprofile has demonstrated a prognostic value in uterine,breast and ovarian malignancies, and loss of PR is asso-ciated with worse outcome [7–10].

In the endometrium oestrogen induces proliferationwhilst progesterone suppresses the oestrogen mediatedsignals and has a differentiating effect [6]. Oestrogendependent endometrial cancers are thought to arise fromunopposed oestrogen exposure, not balanced by the dif-ferentiating effect of progesterone [11]. Currently, bothdrugs antagonising oestrogen effects, and progesterone

analogues are used in endometrial cancer treatment. Inadvanced or recurrent disease, treatment with progester-one has shown modest response rates [12]. However, inpremenopausal woman with well differentiated endome-trial cancer the response rates are reported to be higher,allowing fertility preserving treatment [13,14]. Althoughresponse to progesterone therapy is reported to bedependent on progesterone receptor (PR) status[12,15], and PR is reported to be a prognostic markerin endometrial cancer [9,10,16], evaluation of PR expres-sion is not routinely performed in endometrial cancer toguide treatment decisions.

The aim of this study was to investigate changes inprogesterone receptor expression during disease pro-gression, and to explore if genes differentially expressedaccording to PR status, can be linked to biological pro-cesses involved in cancer development. The identifiedgenetic alterations were explored to search for newpotential drug candidates.

2. Materials and methods

2.1. Patient series

A population based patient series was prospectivelycollected from 2001 to 2013, and includes 686 primarytumours from patients diagnosed with endometrial can-cer in Hordaland County (Norway). Patients were surgi-cally staged according to the International Federation of Gynecology and Obstetrics (FIGO) 2009 criteria. Clinicaldata were collected as described earlier [17]. Biopsies frommetastatic tissue were available from 76 patients (in total171 lesions). When available, fresh frozen tissue was col-lected in parallel with formalin fixed paraffin embedded(FFPE) tissue and used for mRNA and protein extrac-tion. Tissue microarrays (TMA) were generated from

FFPE tissue as previously described [18]. The indepen-dent endometrial cancer patient series with data for Ki-67 and PR has previously been described and published[19,20]. All parts of the study have been approved accord-ing to Norwegian legislation, including the NorwegianData Inspectorate, Norwegian Social Sciences Data Ser-vices and the Western Regional Committee for Medicaland Health Research Ethics, (NSD15501; REK 052.01).Participants gave written informed consent.

2.2. Protein detection

TMAs were dewaxed in xylene, and rehydrated in eth-anol before microwave antigen retrieval and stained forPR expression using M3569 (Dako). The staining wasevaluated as previously described [19]. Staining index 0was considered PR negative, and 1–9 PR positive. Kappavalue was calculated to be 0.82 for PR in two groups.Oestrogen receptor (ER) was stained and scored as pre-viously described [21,22]. When evaluating multiple met-astatic lesions from the same patient, PR was defined aslost if any of the metastatic lesions demonstrated loss.Reverse phase protein array (RPPA) was performed on358 primary tumours as previously described [23,24].

3004 I.L. Tangen et al. / European Journal of Cancer 50 (2014) 3003–3010


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Briefly, fresh frozen patient samples were homogenisedin lysis buffer before proteins were denatured usingSDS and serial diluted in lysis buffer. Samples wereprinted on nitrocellulose coated slides before stainingfor PR (ab32085 Abcam) and proliferation cell nuclearantigen (PCNA) (ab29 Abcam) expression. Relative pro-tein levels were determined by fitting each dilution curvewith a logistic model (‘Supercurve Fitting’ http://bioin-formatics.mdanderson.org/OOMPA).

2.3. Gene expression analyses

RNA was extracted from fresh frozen tissue from 18hyperplasias, 174 primary tumours and 42 metastasesusing the RNeasy Mini Kit (Qiagen). Samples were hybri-dised to Agilent Whole Human Genome Microarrays 44k(Cat. No. G4112F), scanned and normalised as previ-ously described [21]. Differentially expressed genes wereidentified by Significance Analysis of Microarray(SAM). A False Discovery Rate (FDR)of


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in cell proliferation. We continued to explore the PRrelated genes, excluding genes found in both the PRand the ER list, in GSEA. Gene sets associated with cellcycle regulation were significantly enriched in PR nega-tive compared to positive tumours (SupplementaryTable 3).

Cuzick et al. have published a cell cycle progression(CCP) gene signature [25]. In our dataset this CCP scoreincreased with disease progression (SupplementaryFig. 2A), and high CCP score was also reflected in signif-icantly worse survival (Supplementary Fig. 2B). Weinvestigated if hormone receptor status also reflectedchanges in CCP score. Both in the whole population(Fig. 2A) and in subgroups according to ERa status(Fig.2B) high CCP score was significantlyassociated with

loss of PR. In contrast, we found no association betweenERa status and CCP score within the PR positive or PRnegative subgroups (Fig. 2B). PCNA levels, assessed byRPPA, were also found to be significantly associated withPR status within subgroups of both ERa positive andnegative cases, contrasting the lack of associationbetween ERa and PCNA levels within subgroups of PRpositive or PR negative cases (Fig. 2C). We also reexam-ined an additional independent endometrial cancer seriespreviously published with data available for PR statusand the alternative proliferation marker Ki-67 [19,20]confirming the pattern for a significant increase in

proliferation related to PR loss in ER positive tumours(Supplementary Fig. 3).

To explore if transcriptional alterations related to PRloss in the tumours could suggest new targets for treat-ment, Connectivity Map [26] was queried for drug signa-tures negatively correlated with the gene expressionprofile of PR negative tumours. Amongst the top scoringdrugs were two targeting the phosphoinositide 3-kinase(PI3K) signalling pathway (Sirolimus and LY294002).The observation that mammalian target of rapamycin(mTOR) inhibitors are active in endometrial cancer [27]supports the utility of the Connectivity Map analysis.Interestingly, several of the top ranked compounds areknown to be anti-proliferative, including cyclin-dependent kinase 2 (CDK2) inhibitors (Table 2 and

Supplementary Table 4) suggesting a potential newtreatment opportunity in systemic endometrial cancer.

4. Discussion

In this study we investigate PR protein level withrelated transcriptional alterations in a large and uniquecollection of extensively annotated primary and meta-static endometrial cancer cases. We find, as already welldocumented from earlier studies [9,10,16], that loss of PR is associated with markers for aggressive diseaseand predicts poor survival. In addition, we find a

Table 1Clinico-pathological variables related to progesterone receptor (PR) status (protein level evaluated byIHC).

Variable PR positive n (%) PR negative n (%) P -value

Age 0.010


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dramatic change in proportion of patients with PR lossin metastatic compared to primary endometrial cancerlesions. Importantly, the effect of treatment withsynthetic progesterone is reported to be dependent onPR status [12,15], yet assessment of receptor status isnot routinely performed before treatment initiation. Ina review of randomised clinical trials investigating theeffect of hormonal therapy in advanced or recurrentendometrial cancer, information regarding hormonereceptor status was not included in patient stratification

in five of six trials [28], suggesting that hormonaltherapy may have a yet not fully explored potential bothin the primary setting, and for systemic disease.

In the primary lesions we observe loss of PR in 23% of patients, and many tumours lose PR expression from pri-mary to their metastatic counterpart, demonstrated by76% PR loss for metastases. It is interesting to note thatthe proportion of the poor-prognosis non-endometrioidpatients with intact expression of PR in the primary set-ting is relatively high (36%) and it is tempting to speculatethat this patient group could benefit from hormonal treat-ment if biomarker status was implemented. Likewise, the

response rate for hormonal treatment is currentlyreported to be low in advanced disease [28] where, at pres-ent, often the endometrioid subtype is urging the clinicianto consider hormonal therapy. Determination of hor-mone receptor status in recurrent disease after re-biopsyfrom the metastatic lesion would give a valid presentationof PR as target in the systemic disease setting in endome-trial cancers and would better tailor the treatment topatients with suspected benefits. This suggests that theeffect of PR targeted therapy if restricted to patients with

PR positivity in tumours may be higher, warrantingdevelopment of trials to test this hypothesis. The findingthat expression of PR changes during endometrial cancerprogression is in line with a recent study in breast cancerindicating that levels of ER, PR and HER-2 are unstablethroughout tumour progression [29]. Investigation of biopsies from metastatic lesions is also recommended inthis study as it may have important implications for ther-apeutic strategy.

In this study we have investigated total progesteronereceptor expression in endometrial tissue. There arehowever two principal isoforms of progesterone

Length follow up (months)

60360

D i s e a s e

s p e c i f i c s u r v i v a l

1.0

0.8

0.6

0.4

0.2

0 P


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receptor, PRA and PRB, and these are known to playdifferent roles in cell physiology [6,30]. PRB is knownto be a stronger transcriptional activator compared to

PRA [31,32], whilst PRA has an inhibitory effect on ste-roid hormone receptors, including ER [33,34]. There-fore, in future studies, investigating the expressionlevel of the two different PR isoforms during cancer pro-gression, and their association with clinical outcome willbe of interest to better understand the response to hor-mone treatment in endometrial cancer.

Abnormal proliferation and cell cycle dysregulationare common in all cancer types. We show that PR lossis associated with increased proliferation, measured byPCNA and Ki-67. PCNA has been reported as a validmarker for proliferation and has prognostic value[35,36]. Ki-67 has however been suggested to be morespecific [37]. The clinical utility of Ki-67 versus PCNAas proliferation marker in endometrial cancer is notyet established. However, we identify a similar patternof significant increase in proliferation estimated by bothPCNA and Ki-67. This may support that use of bothproliferation markers detects the increase in prolifera-tion related to PR loss in endometrial cancer. Progester-

one is known to inhibit proliferation through opposingthe proliferative effect of oestrogen in the normal endo-metrium [38], through inhibition of ER gene expression,enhanced degradation of ER and possibly by opposingER-mediated gene regulatory events (reviewed in [39]).Furthermore, progesterone induces differentiation of endometrial cells rendering them less sensitive to theeffect of ER as well as other growth factors [40]. How-ever, progesterone has also been shown to inhibit cellgrowth in ER negative tumours suggesting that para-crine interactions may be important. Furthermore, Daiet al. showed that in an ER negative endometrial cancer

cell line, progesterone limited cell growth through induc-tion of the cyclin-dependent kinase inhibitors p21 andp27 [41]. Due to its involvement in processes that inhibittumour development and progression, progesterone hasbeen referred to as the ultimate endometrial tumour sup-pressor [6], and losing PR can be compared to losing thebrake in processes inhibited by progesterone. Our resultsclearly support such anti-proliferative effect of PR,underscoring its clinical relevance.

The primary treatment for patients with endometrialcancer is surgery, and for the intermediate to high-riskpatients, adjuvant treatment with radiation and/or che-motherapy is widely used, although with an uncertain

survival benefit [42]. The response to conventional sys-temic treatment for patients with advanced or recurrentdisease is limited. As patients with retained expressionof hormone receptors benefit most from progesteronetreatment [12,15], this appears to be reasonable if PRstatus is confirmed in metastatic lesion(s). For tumourswith PR loss, our data suggest that drugs inhibitingproliferation such as CDK inhibitors and in particularCDK2 inhibitors may be particularly relevant forfuture analysis in clinical trials. Development of drugsthat inhibit CDKs has been an area of research for

PR negativePR positive

13

12

11

10

9

8

7

PR+

C e l l c y c l e p r o g r e s s i o n s i g

n a t u r e s c o r e

13

12

11

10

9

8

7

PR- PR+ PR-

P=0.053 P=0.015

P=0.31

P=0.98

C e l l c y c l e p r o g r e s s i o n s i g n a t u r e s c o r e

P C N A

( R P P A )

1.5

1.0

0.5

0.0

-0.5

-1.0

P=0.45

P=0.83

P=0.001 P=0.023

PR+ PR- PR-PR+ER positive ER negative

ER positive ER negative

P=0.001

A

B

C

Fig. 2. Loss of progesterone receptor (PR) is significantly associatedwith markers for high proliferation demonstrated by the correlationwith high level of the mRNA cell cycle progression score, both in thewhole population (A) and in subgroups of ERa positive and ERanegative samples (B). High proliferation documented by assessingprotein level for the proliferation cell nuclear antigen (PCNA) in caseswith PR loss is validated, both within ERa positive and ERa negativelesions (C).



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many years. The first generation of CDK inhibitorsdemonstrated only modest effect [43]. One of these,flavopiridol, which is a weak pan CDK inhibitor, wastested as second line treatment in a phase II study onpatients with recurrent or persistent endometrial can-cer, however in this population flavopiridol as a singleagent had minimal effect [44]. New small moleculeCDK inhibitors such as the dual CDK4/6 inhibitor pal-

bociclib show more encouraging results with prolonga-tion of progression free survival in combination withletrozole in breast cancer patients [45]. Whether PRnegative endometrial cancer patients will benefit fromselective CDK1/2 or CDK4/6 inhibitors warrants bothpreclinical and clinical evaluations. Our results suggestCDK inhibitors as potential therapy in particular forPR negative endometrial cancer patients, supportingdifferent clinical trial strategies being tested for PRpositive and PR negative endometrial carcinomapatients.

Financial support

This study was supported by Helse Vest, the Univer-sity of Bergen, the Norwegian Cancer Society (HaraldAndersen Legat), the Research Council of Norwayand Bergen Medisinske Forskningsstiftelse.

Conflict of interest statement

None declared.

Acknowledgements

We thank Ellen Valen, Britt Edvardsen, KadriMadissoo and Bendik Nordanger for technical assis-tance. G.B.M. was supported by P50 CA098258. RPPAanalysis was supported by MD Anderson Cancer CenterSupport Grant (CCSG) P30 CA016672 from theNational Cancer Institute.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at http://dx.doi.org/10.1016/j.ejca.2014.09.003.

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