ARTICLE

Progesterone Receptor Isoform Ratio A Breast Cancer

Prognostic and Predictive Factor for Antiprogestin

Responsiveness

Paola A Rojas Marıa May Gonzalo R Sequeira Andres EliaMichelle Alvarez Paula Martınez Pedro Gonzalez Stephen HewittXiaping He Charles M Perou Alfredo Molinolo Luz GibbonsMartin C Abba Hugo Gass Claudia LanariAffiliations of authors Laboratory of Hormonal Carcinogenesis Instituto de Biologıa y Medicina Experimental (IBYME) Consejo Nacional de Investigaciones Cientıficasy Tecnicas (CONICET) Buenos Aires Argentina (PAR MM GS AE MA CL) Hospital de Agudos Magdalena V de Martınez General Pacheco Buenos Aires Argentina(PM PG HG) Center for Cancer Research National Cancer Institute Bethesda MD (SH) Department of Genetics Lineberger Comprehensive Cancer Center Universityof North Carolina Chapel Hill NC (XH CMP) Moores Cancer Center University of California San Diego CA (AM) Instituto de Efectividad Clınica y Sanitaria BuenosAires Argentina (LG) CINIBAndashCONICET Escuela de Ciencias Medicas UNLP La Plata Argentina (MCA)

Authors contributed equally to this workCorrespondence to Claudia Lanari PhD Vuelta de Obligado 2490 CP 1428 CABA Buenos Aires Argentina (e-mail address lanariclaudiagmailcom)

Abstract

Background Compelling evidence shows that progestins regulate breast cancer growth Using preclinical models wedemonstrated that antiprogestins are inhibitory when the level of progesterone receptor isoform A (PR-A) is higher than thatof isoform B (PR-B) and that they might stimulate growth when PR-B is predominant The aims of this study were to investi-gate ex vivo responses to mifepristone (MFP) in breast carcinomas with different PR isoform ratios and to examine their clini-cal and molecular characteristicsMethods We performed human breast cancer tissue culture assays (nfrac1436) to evaluate the effect of MFP on cell proliferationPR isoform expression was determined by immunoblotting (nfrac14282) Tumors were categorized as PRA-H (PR-APR-B 12) orPRB-H (PR-APR-B 083) RNA was extracted for Ribo-Zero-Seq sequencing to evaluate differentially expressedgenes Subtypes and risk scores were predicted using the PAM50 gene set the data analyzed using The Cancer Genome AtlasRNA-seq gene analysis and other publicly available gene expression data Tissue microarrays were performed using paraffin-embedded tissues (PRA-H nfrac1453 PRB-H nfrac1424) and protein expression analyzed by immunohistochemistry All statisticaltests were two-sidedResults One hundred sixteen out of 222 (523) PRthorn tumors were PRA-H and 64 (288) PRB-H Cell proliferation was in-hibited by MFP in 19 of 19 tissue cultures from PRA-H tumors A total of 139 transcripts related to proliferative pathways weredifferentially expressed in nine PRA-H and seven PRB-H tumors PRB-H and PRA-H tumors were either luminal B or A pheno-types respectively (P frac14 03) PRB-H cases were associated with shorter relapse-free survival (hazard ratio [HR] frac14 270 95confidence interval [CI] frac14 171 to 620 P frac14 02) and distant metastasisndashfree survival (HRfrac14417 95 CIfrac14218 to 797 Plt 001)PRB-H tumors showed increased tumor size (Plt 001) Ki-67 levels (Plt 001) human epidermal growth factor receptor 2expression (Pfrac14 04) high grades (Pfrac14 03) and decreased total PR (Pfrac14 004) compared with PRA-H tumors MUC-2 (Plt 001) andKRT6A (Pfrac14 02) were also overexpressed in PRB-H tumorsConclusion The PRAPRB ratio is a prognostic and predictive factor for antiprogestin responsiveness in breast cancer

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Received July 27 2016 Revised October 14 2016 Accepted December 1 2016

copy The Author 2017 Published by Oxford University Press All rights reserved For Permissions please e-mail journalspermissionsoupcom

1 of 9

JNCI J Natl Cancer Inst (2017) 109(7) djw317

doi 101093jncidjw317First published online March 9 2017Article

The progesterone receptor (PR) is a member of the steroid nu-clear receptor family There is increasing clinical (12) and ex-perimental evidence (3ndash5) suggesting its relevance in regulatingbreast cancer development and growth However PR levels arestill only used as a surrogate marker for estrogen receptor alpha(ERa) activity (67)

Two PR isoforms are transcribed from a single gene (8) andeach isoform possesses unique tissue-specific functions (9) PR-A(94 kDa) is entirely contained within the larger PR-B (115 kDa) pro-tein making them difficult to discriminate by immunohistochem-istry (IHC) or polymerase chain reaction (PCR) Few breast cancerstudies have evaluated the PR isoforms (10ndash15) Among those thathave the consensus is that PR-A is the prevailing isoform al-though contradictory data exist concerning its prognostic valueCurrently immunoblotting (IB) is the most reproducible method ofdiscriminating between the isoforms to obtain the PR-APR-B ratio

Using medroxyprogesterone acetate (MPA)ndashinduced murinecarcinomas and human breast cancer xenografts engineered toexpress different PR isoform ratios we demonstrated that onlytumors with PR-A levels higher than PR-B levels regress withantiprogestins (16ndash18) Moreover constitutive resistant tumorswere resensitized to antiprogestin treatment when they re-expressed PR-A after treatment with demethylating agents andHDAC inhibitors (1819)

Antiprogestins have induced different responses in clinicaltrials (reviewed in [20ndash22]) We hypothesized that antiproges-tins together with conventional antiestrogens offer a validtherapeutic approach for tumors with high PR-A levels The aimof this study was to investigate the responses of breast cancerswith different PR isoform ratios to MFP treatment and to evalu-ate their molecular portraits and clinical features

Methods

Patient Tumor Specimens

Tissue samples were obtained from 395 patients undergoingbreast cancer surgery from 2007 to 2015 The study was approvedby the institutional review boards of the Hospital RivadaviaBuenos Aires (n frac14 20) Hospital Magdalena V de MartınezGeneral Pacheco Buenos Aires (n frac14 375) and IBYME-CONICETInformed consent was obtained from all patients The sampleswere flash-frozen andor kept in tissue culture medium Patientswere excluded when inconsistent data between IB and IHC wereobtained or if the quality of an IB was not acceptable

Primary Cultures and Organotypic Tissue Cultures

Fresh tissues were processed within four hours of resectionPrimary cultures were performed as previously described(Supplementary Methods available online) (23)

Tissue slices (100mm thick) were obtained using a McIlwainTissue Chopper (Ted Pella Inc Redding CA) and incubated inDMEMF-12 without phenol red (Sigma-Aldrich St Louis MO)with 10 fetal calf serum (FCS Life Technologies CarlsbadCA) with or without MFP (Sigma-Aldrich) After 48 hours tis-sues were harvested formalin-fixed and paraffin-embedded(FFPE) (24) A pathologist (MM) examined morphological integ-rity and the presence of viable cancer cells by hematoxylin andeosin (HampE) staining If adequate Ki-67 was evaluated by IHC(ab15580 Abcam Cambridge MA) as previously described (25)The percentage of positive cells was calculated in a minimumof five fragments per treatment The average number of cells

per tumor was 1039 (median frac14 869 range frac14 197ndash3134 cells) Aninhibitorystimulatory effect was registered if a statistically sig-nificant difference was recorded (Plt 05)

Immunohistochemical Studies and Tissue Arrays

ER and PR expression were extracted from the clinical historiesHuman epidermal growth factor receptor 2 (HER2) expressionwas evaluated by Gabriela Acosta-Haab (Roche LaboratoriesBuenos Aires Argentina) Four tissue microarrays (TMAs) onewith 64 samples and the other three with 24 samples each wereconstructed using samples segregated by PR isoform contentAnti-MUC-2 (sc-15334 1200) anti-FGF-10 (sc-7917 1100) andanti-glucocorticoid receptor (GR sc-1004 1150) antibodies wereobtained from Santa Cruz Biotech (Santa Cruz CA) anti-CRISP-3(14847-1-AP 1250) from Proteintech Group Inc (Rosemont IL)anti-KRT6A (RB-169p 1500) from Covance Inc (Princeton NJ)and anti-androgen receptor (AR 5153P 1250) from Cell Signaling(Danvers MA)

Frozen Samples

Frozen samples were pulverized and separated in TRIzol reagent(Life Technologies) for RNA extraction and in DNA digestion buf-fer and Pierce buffer (Thermo Scientific Waltham MA) for IB

Immunoblotting

T47D breast cancer cells (ATCC Manassas VA) were cultured asdescribed (26) and cytosol and nuclear proteins extractedProteins were measured using a Lowry protein assay (27) ali-quots of standard preparations were stored at 70 C

For IB 100 mg of extracted protein was electrophoresed on an8 polyacrylamide gel (16) T47D standards were included aspositive controls Anti-PR (PR1294 Dako Agilent TechnologiesSanta Clara CA or H190 Santa Cruz) both at 1500 and anti-ERK (sc-94 Santa Cruz 11000) antibodies were used Band in-tensities were densitometrically measured using Image J(National Institutes of Health Bethesda MD) ERK was evalu-ated to control sample quality

Criteria for Patient Classification According to PRIsoform Ratio

We established a priori that a nuclear PR-APR-B ratio of 12 orgreater is representative of patients with high PR-A (PRA-H)whereas a PR-BPR-A ratio of 12 or greater is representative of pa-tients with high PR-B (PRB-H) Patients with either ratio falling be-tween 12 and 083 were considered equimolar This cutoff valuewas selected based on the 20 variability observed when quanti-fying PR-A and PR-B using IBs performed with the same samplesat different times Usually nuclear and cytosol fractions gave co-incident values In borderline cases in which the nuclear valuewas higher than 11 or lower than 09 and the cytosolic value wasclearly PRA-H or PRB-H the cytosolic value was considered

RNA Isolation and RNA-Seq Data Analysis

RNA extracted from 22 samples was processed at the Universityof North Carolina Sixteen samples proved suitable for RNA se-quencing analysis (Ribo-Zero-Seq) two samples were excludedas outliers RNA was isolated and purified as described in the

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2 of 9 | JNCI J Natl Cancer Inst 2017 Vol 109 No 7

Supplementary Methods (available online) Heatmap visualiza-tion of differentially expressed transcripts and PAM50 geneswas achieved using MultiExperiment Viewer software (MeV v49) (28) Intrinsic subtype classification of breast cancer sampleswas performed using the 50-gene (PAM50) predictor bioclassifierR script (29) and functional enrichment analyses using theClueGo Cytoscapersquos plug-in (httpwwwcytoscapeorg) andInnateDB resource (httpwwwinnatedbcom) based on a listof deregulated transcripts between PRA-H and PRB-H tumors(FDR lt 005 log2 FC gt 61) To further explore the prognosticvalue of the PR isoforms in invasive breast carcinomas we eval-uated information from two publicly available gene expressiondata sets from the UCSC Xena resource (httpsxenaucscedu)We analyzed 586 luminal-like and ERthornPRthorn primary invasivebreast carcinomas derived from the The Cancer Genome AtlasndashBreast Invasive Carcinoma project (30) and 355 from a studyreported by Yau et al (31) Tumor classification in PRA-H andPRB-H cases was performed based on low or high expression lev-els of PR-associated signatures (eg PRB-H tumors were definedas having high expression of PRB-H genes and low of PRA-Hgenes) using the StepMiner one-step algorithm (32) implementedin R PRA-H and PRB-H cases were then compared

Statistical Analysis

Absolute and relative frequencies of the main patient character-istics were calculated To compare the effect of MFP on individ-ual primarytissue cultures the Mann Whitney test was usedThe chi-square test was used to compare the proportion of PRA-H and PRB-H samples inhibited by MFP A total of 19 patients pergroup was predicted to detect statistically significant differencesbetween both groups (80 vs 40 type I error 5 type II error10 httpsselect-statisticscoukcalculatorssample-size-calculator-two-proportions) The Wilcoxon test was used to evaluatepossible associations between ER AR and GR expression andMFP responsiveness To compare clinicopathological parametersamong the two groups the chi-square test and Fisherrsquos exacttest were used for categorical variables The Studentrsquos t test wasused to compare the mean age between the two groups TheCochran-Armitage trend test was used to evaluate possible

trends in histologic grade PR status and ER status Log-ranktests were used to analyze Kaplan-Meier curves using theSurvival R package A P value of less than 05 was considered sta-tistically significant and all statistical tests were two-sided

Results

Patient Distribution According to PR Isoform Ratio

In total 282 samples were included patient and tumor featuresare shown in Table 1 The median PR-APR-B ratio across allPRthorn samples was 12 (range frac14 01ndash202 250 percentile 0825750 percentile 2 95 confidence interval [CI] frac14 148 to 194)Of the 222 PRthorn breast cancers 116 were PRA-H predominant(523) 64 were PRB-H predominant (288 ) and 42 were equi-molar (Figure 1A) Figure 1B illustrates the frequency distribu-tion of the PR-APR-B ratio

Effect of MFP in Cultures Of Breast Cancer SamplesClassified According Their PR-APR-B Ratio

From 70 primary culture attempts successful subcultures wereachieved in only nine cases (13 efficiency) In four out of fiveof the PRA-H samples MFP decreased the number of cells(Pfrac14 03) (Supplementary Figure 1 available online) different

Table 1 Clinicopathological parameters of patients

Characteristics No ()

Age (nfrac14 247) ylt40 13 (53)40ndash50 50 (182)51ndash60 57 (231)gt60 127 (514)

Tumor size cm (nfrac14 202)lt2 37 (183)2ndash5 137 (678)gt5 28 (139)

Histologic grade (nfrac14247)I 47 (190)II 71 (287)III 129 (522)

PR-positive (nfrac14 282) 222 (787)ERa-positive (nfrac14 234) 197 (842)HER2-positive (nfrac14 222) 40 (180)Triple-negative (nfrac14 239) 17 (71)

ERa frac14 estrogen receptor alpha HER2 frac14 human epidermal growth factor receptor 2

PRfrac14 progesterone receptor

A

ln PR-APR-B

0

2

4

6

8

10

12

No

of t

umor

s0-1-2 1 2 3

2

4

6

8

10

12

PRB-H

PRA-HEquimolar

B

PRA-H 523PR-APR-B ge12

Equimolar 189PR-APR-B lt12 and gt 083

PRB-H 288PR-APR-B le083

n = 282

PR-A

PR-B

PR-A

PR-B

PR-A

PR-B

PR (ndash) 213

PR (+) 787

Figure 1 Classification of breast tumors according to their progesterone receptor

isoform A (PR-A)progesterone receptor isoform B (PR-B) ratios A) Left Diagram

showing the percentage of PRthorn tumors Right The PR isoform ratio was evalu-

ated densitometrically measuring the band intensity of each isoform in immu-

noblots PRthorn tumors were classified into three categories according to the PR-A

PR-B ratio PRA-H (523) equimolar (183) and PRB-H (288) A representative

immunoblot of each category is shown B) Frequency diagram showing the dis-

tribution of the PR-APR-B ratio for all evaluated PRthorn tumors PR frac14 progesterone

receptor PR-A frac14 PR isoform A PR-B frac14 PR isoform B PRA-H frac14 tumors with higher

levels of PR-A than PR-B PRB-H frac14 tumors with higher levels of PR-B than PR-A

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P A Rojas et al | 3 of 9

responses were observed in non-PRA-H samples Consideringthe low efficiency of successful subculture we explored a noveltissue culture method (described in the ldquoMethodsrdquo section)Using this methodology 36 of 112 cases (321) were successfulincreasing the efficiency previously obtained with primary cul-tures In vitro studies were performed by researchers blinded tothe IB results Statistically significant inhibition of Ki-67 expres-sion was observed after 48 hours of MFP treatment in 19 of 19 ofthe PRA-H samples and three of 10 of the PRB-H samples (Figure2A) MFP stimulated Ki-67 expression in two of 10 of the PRB-Hsamples (Figure 2 A and B) In total one of four of the equimolarand one of three of the PR-negative samples were inhibited byMFP (data not shown) Figure 2C shows two representative exam-ples of Ki-67 staining Overall the tissue cultures maintained themorphological features of their parental tumors (SupplementaryFigure 2 available online) Supplementary Table 1 (available on-line) shows the individual characteristics of the tumors thatwere included as well as their ERa PR AR and GR statusCollectively the results from these in vitro studies demonstratethat PRA-H tumors were consistently inhibited by MFP whereasPRB-H equimolar or PR-negative tumors were only occasionallyinhibited Most importantly MFP may stimulate cell proliferationin some PRB-H samples ERa (Pfrac14 93) AR (Pfrac14 18) and GR (Pfrac14 82)status were not associated with MFP responsiveness

Transcriptome Analysis of PRB-H and PRA-H Samples

RNA-seq analysis of nine PRA-H and seven PRB-H tumors re-vealed 139 genes that were differentially expressed (FDR lt 005Log2 FC gt 1) 84 were upregulated in the PRB- H tumors (anddownregulated in PRA-H) while 55 were upregulated in the

PRA-H tumors (Figure 3A Supplementary Table 2 available on-line) Pathway enrichment analysis of the deregulated tran-scripts revealed that they were related to specific bioprocessesassociated with the cell proliferation signature of breast cancercells including the Aurora B (Plt 001) and FOXM1 (Pfrac14 004) sig-naling pathways M phase (Plt 001) and other cell cycle (Plt 001) processes (Figure 3B)

Using the PAM50 gene set to analyze gene expression we ob-served that the genes overexpressed in the PRB-H tumors were 1)highly concentrated within the group of genes that characterize theluminal B subtype and 2) proliferation-related genes (Figure 3C)Based on this expression pattern these tumors could be classifiedas high risk In contrast the PRA-H tumors were associated withthe luminal A subtype which indicates they were of a lower risk

Further analysis of the deregulated transcripts discriminat-ing the PRA-H tumors from the PRB-H tumors among two inde-pendent luminal-likeER- and PR-positive breast cancer datasets suggested that PRB-H patients have a worse prognosis andare more likely to have luminal B carcinomas whereas PRA-Hpatients are more likely to have the luminal A intrinsic subtype(Pfrac14 03) (Figure 4 A and C) Survival analysis revealed that PRB-H cases are associated with shorter relapse-free survival (haz-ard ratio [HR] frac14 270 95 CIfrac14 171 to 620 P frac1402) (Figure 4B) anddistant metastasisndashfree survival (HRfrac14 417 95 CIfrac14 218 to 797Plt 001) (Figure 4D) compared with their PRA-H counterparts

KRT6A and MUC-2 Expression in PRA-H and PRB-HSamples

Four of the deregulated genes were selected for protein expres-sion evaluation by IHC in TMA performed using both PRA-H and

C o n tro l M F P0

5

1 0

1 5

2 0

K

i-6

7+

to

tal

ce

lls

C o n tro l M F P0

1 0

2 0

3 0

4 0

5 0

K

i-6

7+

to

tal

ce

lls

P R A -H tu m o rs

K

i-6

7+

to

tal

ce

lls

Co n tr

o l

MF

P

0

1 0

2 0

3 0

4 0

5 0

P R B -H tu m o rs

K

i-6

7+

to

tal

ce

lls

Co n tr

o l

MF

P

0

1 0

2 0

3 0

4 0

5 0

Control MFP Control MFP

Ki-6

7

Hamp

E

Ki-6

7

HampE

MFP treatment

Decreasedcell proliferationcasestotal ()

Increasedcell proliferationcasestotal ()

Ki-67 indexcontroltreated

Mean plusmnSD

PRA-HPRB-H

1919 (100)a

310 (30)b019 (0)

210 (20)209 plusmn 065c

137 plusmn 068d

A

B

C

P = 03 P = 01

Figure 2 Effects of mifepristone (MFP) in tissue cultures of breast cancer samples classified according their progesterone receptor isoform A (PR-A)progesterone recep-

tor isoform B (PR-B) ratio A) Proliferative responses (Ki-67 staining) of primary breast cancer tissues cultured ex vivo with or without 10 nM MFP for 48 hours The per-

centage of cells with nuclear Ki-67 staining with respect to all tumor cells was calculated in at least five different sections per experimental group The mean values

were plotted and connected with a line A statistically significant decrease in Ki-67 staining was observed in each of the 19 cultures tested (Plt 001 two-sided paired t

test) whereas variable responses were obtained in 10 PRB-H cases (Pfrac14 54) B) Table illustrating the number of tumors that were inhibited or stimulated by MFP accord-

ing to the PR-APR-B ratio a vs b Plt 001 two-sided Fisherrsquos exact test c vs d Pfrac14 01 two-sided Studentrsquos t test C) Two representative cases of PRA-H (left) and PRB-H

(right) tissue cultures are shown Top Hematoxylin and eosin images of paraffin-embedded tissue cultures and Ki-67 immunohistochemistry showing nuclear stain-

ing bar frac14 50 lm Bottom Quantification of Ki-67thorn cellsall tumor cells in five different explants of the examples shown in (B) P values were calculated using the two-

sided Mann Whitney test HampE frac14 hematoxylin and eosin MFPfrac14mifepristone PR frac14 progesterone receptor PR-A frac14 PR isoform A PR-B frac14 PR isoform B PRA-H frac14 tumors

with higher levels of PR-A than PR-B PRB-H frac14 tumors with higher levels of PR-B than PR-A

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4 of 9 | JNCI J Natl Cancer Inst 2017 Vol 109 No 7

PRB-H samples Transcription levels of KRT6A (Pfrac14 02) andMUC-2 (Plt 001) upregulated in PRB-H were corroborated at theprotein level by IHC (Figure 5 A and B) No statistically signifi-cant differences were found regarding CRISP-3 (upregulated inPRB-H) or FGF-10 (upregulated in PRA-H) expression IntenseKRT6A expression was observed in isolated cells mainly in thePRA-H tumors (Figure 5A) a more uniform cytoplasmic stainingprevailed in the PRB-H tumors

Clinical Features of PRA-H and PRB-H Patients

No statistically significant differences were found between thePRA-H and PRB-H patients in average age although the latterhad a mean age that was three years younger than the formerCompared with the PRA-H patients at the time of surgery thePRB-H patients had larger tumors (Plt 001) with worse histologi-cal grade (Pfrac14 03) and lower levels of total PR (Pfrac14 004) No differ-ences or trends were observed regarding ERa status number ofpositive lymph nodes or distant metastases Positive HER2 ex-pression (Pfrac14 04) and high Ki-67 expression (Plt 001) prevailedin the PRB-H tumors (Table 2) Both are biomarkers of worseprognosis

Discussion

In this study we showed that PRA-H breast cancers can be in-hibited by MFP ex vivo and that the PR-APR-B ratio serves as aprognostic factor with PRB-H patients having a worse progno-sis Our study emphasizes the relevance of determining the PRisoform ratio before starting antiprogestin treatments Of thetwo clinical trials currently evaluating antiprogestins for PRthornbreast cancermdashtrial NCT02052128 is assessing onapristone andtrial NCT01800422 is assessing telapristone acetatemdashneither isconsidering the PR isoform ratio as a possible inclusion crite-rion On the basis of the data reported herein we have recentlystarted a trial evaluating the use of neo-adjuvant MFP treatmentfor patients with PRA-H breast cancer (NCT02651844 MIPRA)

The prognostic value of PR isoforms in breast cancer remainscontroversial and this has been extensively discussed in recentreviews (3334) Isoform expression has been evaluated by IB infew other studies (101113) Bamberger et al (11) and Hopp et al(13) suggested that high PR-A levels potentially correlate withworse prognosis or recurrence after tamoxifen treatment Ourstudy which focused on the PR-APR-B ratio rather than on ab-solute PR isoform values indicates that patients with lower PR-A levels than PR-B levels may have a worse prognosis Similar

CDH3PHGDHFGFR4FOXC1EGFRSFRP1PGRMIASLC39A6KRT5KRT14KRT17NAT1MMP11CXXC5BLVRAERBB2MYCBIRC5UBE2CMYBL2RRM2UBE2TNUF2EXO1CEP55PTTG1MELKGRB7MAPTBCL2NDC80CDC6ACTR3BBAG1TMEM45BTYMSORC6MLPHESR1FOXA1CCNE1GPR160MDM2CENPFMKI67ANLNCCNB1KIF2CCDC20

AR

G1

57

AR

G2

45

AR

G1

26

AR

G2

06

AR

G9

2

AR

G1

2

AR

G1

0

AR

G5

6

AR

G1

89

Subtypes

PAM

50 g

ene

mod

el

Down Up

PRA-H tumors PRB-H tumors

Risk

Log2(PostFC PRA-HPRB-H)-10 -5 N 0 5 10

10

08

06

04

02

0

PPD

E

CutoffFDRlt005Log2FCgt1

Genes upmodulated in PRA-H tumors

Genes upmodulated in PRB-H tumors

55 genes84 genes

LumA LumBM-Risk H-Risk

A

B

C

Statistical

significance

threshold

Aurora B signalingCell cycleM phase

SLC-mediated transportFOXM1 network

-Log10(P)15 25 35

Figure 3 Transcriptome analysis of invasive breast carcinomas according to progesterone receptor (PR) isoforms A) Volcano-like plot of differentially expressed genes

among the tumors with higher levels of isoform A of progesterone receptor (PR-A) than isoform B (PR-B PRA-H) and tumors with higher levels of PR-B than PR-A (PRB-

H) breast carcinomas (false discovery rate [FDR] lt 005 fold-change gt 2) The x-axis represents the posterior fold-changes in the expression levels measured in PRA-H

vs PRB-H tumors and the y-axis represents the posterior probability of differential expression (posterior probabilities of being differentially expressed gt 095 FDR lt

005) B) Functional enrichment analysis of the differentially expressed genes between the PRA-H and PRB-H breast carcinomas C) Prediction of breast cancer intrinsic

subtypes using the PAM50 gene model based on RNA-seq profiles Tumors were denominated as high risk (H-Risk) or moderate risk (M-Risk) based on the intrinsic sub-

type assigned and on their proliferative score (Risk of recurrence P value) Proliferation-related genes are highlighted in red All statistical tests were two-sided FDR frac14false discovery rate Log2FC frac14 logarithm2 fold-change LumA frac14 luminal A LumB frac14 luminal B M phase frac14mitotic phase PPDE frac14 posterior probabilities of being differen-

tially expressed PR frac14 progesterone receptor PR-A frac14 PR isoform A PR-B frac14 PR isoform B PRA-H frac14 tumors with higher levels of PR-A than PR-B PRB-H frac14 tumors with

higher levels of PR-B than PR-A SLC-mediated transport frac14 solute-carrier gene-mediated transport

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P A Rojas et al | 5 of 9

results associating PR-A silencing due to promoter methylationwith worse prognosis have been communicated by Pathirajaet al (14) Whereas the previously mentioned studies used cyto-solic or total extracts to perform IB ours is the only study thatused nuclear extracts to define the PR-APR-B isoform ratioMoreover the conditions we used to define a tumor as PRA-H orPRB-H are considerably more stringent than those used in previ-ous studies (1113)

Immunofluorescence (IF) IHC andor PCR have been usedto define the PR-APR-B ratio in two breast cancer studies(1215) Different antibodies have been reported to specificallyrecognize PR-A in FFPE tissues (1535) however the measure-ment of a ratio of intensities using different antibodies withdifferent affinities may not be as accurate as the use of IBSimilarly in PCR studies PR-A values are obtained by subtract-ing values obtained using primers that recognize both PR iso-forms and those recognizing only PR-B (1236ndash39) A correlationbetween the protein and mRNA expression of PR isoformshas not yet been proven using large numbers of tumors Mote

et al (15) used IF to quantity PR isoforms and showed that ex-ogenous hormone replacement therapy results in higher levelsof PR-B relative to PR-A and identified a correlation betweenthe PR-APR-B ratio and recurrence in patients treated withtamoxifen but not in those treated with the combination oftamoxifenaromatase inhibitors Thus they proposed thatmeasurements of PR isoforms may help identify the most ap-propriate ER-targeted therapy

In vitro studies using cells genetically modified to expressdifferent levels of PR-A andor PR-B have associated PR-A witha gene expression signature that indicates aggressiveness(4041) However when the modified cells were transplantedinto immunosuppressed mice they did not behave as expectedInterestingly T47D-YA tumors expressing PR-A showed aslower growth rate than T47D-YB tumors expressing PR-B andonly the former were statistically significantly inhibited by ta-moxifen (42) or MFP (18) These results are concordant with ourcurrent data and also with data obtained in preclinical murinehuman and canine models (17ndash194344)

Luminal A Luminal B

PRA-HPRB-H

Up

in P

RA-H

Up

in P

RB-H

HIST2H3CATP1A3C2CD4CHIST2H2AA3MYBL2BIRC5KIF18BSRRM3C19orf24TROAPWDR62PRR11CCNB2UBE2SBCAT2ESPL1RIMS1NCAPHPAFAH1B3LLGL2KIF4AHIST2H2ACLENG1ZGPATPIF1KIF14ARHGAP11B

Time mo

Rela

pse-

free

sur

viva

l

P = 02

PRA-HPRB-H

n = 258

Dis

tant

met

asta

sis-

free

sur

viva

l

Time mo

PRA-HPRB-H

P lt 001 n = 190

Luminal A Luminal B

PRA-HPRB-H

Up

in P

RA-H

Up

in P

RB-H

NKX6-1CIDEAPRSS3IVLSCNN1GPRSS21ATP1A3MYBL2BIRC5TROAPWDR62CCNB2BCAT2ESPL1RIMS1PAFAH1B3LLGL2KIF4AZGPATKIF14

A

C

100

075

050

025

00 50 100 150

100

075

050

025

00 50 100 150 200 250

Number at risk by timeGroup

Number at risk by timeGroup

B

D

Figure 4 Comparative analysis of tumors with higher levels of progesterone receptor isoform A (PR-A) than progesterone receptor isoform B (PR-B PRA-H) and tumors

with higher levels of PR-B than PR-A (PRB-H) gene expression signatures and the prognostic value of predicted PR isoforms across two independent breast cancer data

sets A) Heatmap of PRA-H- and PRB-H-associated gene expression signatures among 586 luminal-like and PRthorn primary invasive breast carcinomas obtained from the

The Cancer Genome Atlas (TCGA)ndashBreast Invasive Carcinoma project Black lines at the bottom indicate the predicted PR isoforms B) Relapse-free survival curves of

the predicted PRA-H and PRB-H cases among the TCGA data set C) Heatmap of PRA-H- and PRB-H-associated gene expression signatures among 355 luminal-like pri-

mary invasive breast carcinomas obtained from a data set reported by Yau et al in 2010 Black lines at the bottom indicate the predicted PR isoforms D) Distant metas-

tasis-free survival curves of the predicted PRA-H and PRB-H cases among the same data set P values were calculated using two-sided log-rank test PR frac14 progesterone

receptor PR-A frac14 PR isoform A PR-B frac14 PR isoform B PRA-H frac14 tumors with higher levels of PR-A than PR-B PRB-H frac14 tumors with higher levels of PR-B than PR-A

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This is the first study to perform ex vivo evaluations of theeffect of antiprogestins in human breast cancers categorizedaccording to the prevailing PR isoforms expressed Initially weadapted a tissue culture method used in the field of neurobiol-ogy (45) however a similar method was recently reported forbreast cancer samples (46) The effect of MFP on PRA-H tumorswas consistent over all 19 samples studied which implied thatour decision to stop the analysis was warranted even thoughwe only achieved success with 10 PRB-H tumors Even if thenine remaining samples would have been inhibited by MFP thedifference would have still reached the predicted statistical sig-nificance A stimulatory effect of MFP was detected in two PRB-H samples Interestingly the second sample was a relapse fromthe first reinforcing the reliability of the tissue culture methodutilized Low concentrations of MFP were used to reduce thedrugrsquos anti-glucocorticoid (47) and anti-androgenic effects (48)Although no association was found between ERa GR and AR ex-pression and MFP responsiveness in the PRA-H tumors a largernumber of PRB-H equimolar or PR-negative samples must beevaluated to determine whether GR and AR contribute to the in-hibitory effect of MFP observed in selected cases

Molecular and clinical features were concordant and tumorswith higher levels of PR-A than PR-B were associated with bio-markers of better prognosis and a luminal A phenotype Onelimitation of our study is that we included all patients whounderwent surgical tumor resection regardless of tumor stageFurther validation with independent and larger cohorts of pa-tients with different tumor stages is warranted When we di-vided our cohort of luminal patients into phenotype A or Baccording to the Ki-67 expression there was a statistically sig-nificant association between the luminal A phenotype and thePRA-H status (not shown) However this association was notstrong enough to exclude all PRB-H tumors Data-mining resultssupported these findings These results emphasize the need toidentify alternative biomarkers that may help categorize PRA-Hand PRB-H tumors for cases in which IB cannot be performed

According to RNA sequencing data MUC-2 and KRT6A werehighly expressed in PRB-H tumors and these results were con-firmed by IHC MUC-2 is exclusively expressed in breast cancerand not in normal cells (49) KRT6A is highly expressed in basaltumors (50) and in progenitor mammary cells (51) Studies arenow being performed to develop an IHC algorithm that includes

A K R T 6 A

0

1 0 0

2 0 0

3 0 0IH

C s

co

re

M U C -2

0

1 0 0

2 0 0

3 0 0

IHC

sc

ore

B

PRA-H PRB-H

P = 02

P lt 001

Figure 5 KRT6A and MUC-2 expression in progesterone receptor isoform A (PR-A) than progesterone receptor isoform B (PR-B PRA-H) and tumors with higher levels of

PR-B than PR-A (PRB-H) samples Protein expression levels for two out of four genes selected from a list of 139 deregulated genes identified in RNA-seq studies Protein

expression was studied by immunohistochemistry (IHC) in TMAs constructed using PRA-H (nfrac14 53) and PRB-H (nfrac1424) samples A score was calculated to represent the

percentage of stained cells and the intensity of the staining (0ndash3) A) KRT6A was upregulated in the transcriptome analysis of PRB-H samples IHC studies confirmed a

corresponding increase in protein expression (Pfrac14 02) Images illustrate the heterogeneous cytoplasmic staining observed (left) Occasionally single cells were inten-

sively stained (right) B) MUC-2 was also upregulated in the transcriptome analysis of PRB-H samples and IHC studies confirmed a corresponding increase in MUC-2

protein expression (Plt 001) Images illustrate the typical MUC-2 intense staining pattern observed in a mucinous area (left) as well as the representative cytoplasmic

staining observed (right) Scale bar frac14 50 mm Error bars represent SD P values were calculated using two-sided Mann Whitney test IHC frac14 immunohistochemistry PR frac14progesterone receptor PR-A frac14 PR isoform A PR-B frac14 PR isoform B PRA-H frac14 tumors with higher levels of PR-A than PR-B PRB-H tumors with higher levels of PR-B

than PR-A

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P A Rojas et al | 7 of 9

assessments of Ki-67 HER2 MUC-2 and KRT6A expression aswell as other factors such as tumor size differentiation grademolecular subtype and total PR to define a PRA-H or PRB-H phe-notype without the need of IB

Preclinical data suggest that combined targeting of ER andPR improves the effectiveness of single endocrine treatments(52) If this is the case adjuvancy seems the best scenario forcombined therapies using antiprogestins It has recently beendemonstrated that progestins may induce ER rewiring thusinhibiting estrogen-induced cell proliferation (53) Standard en-docrine therapies involve the use of tamoxifen aromatase in-hibitors or fulvestrant resulting in the better positioning ofantiprogestins than progestins for the treatment of PRA-H pa-tients Ongoing studies are expected to provide data supportingif progestins are better options for tumors with low PRAPRBratios

In summary in this study we provided strong evidence thatantiprogestins should be a therapeutic option for luminal breastcancers with higher levels of PR-A than PR-B

Funding

This work was supported by Agencia Nacional de Promocionde Ciencia y Tecnologıa (ANAPCYT PICT2007932 PICT 20121091) CONICET PIP 2013-16 Fundacion RoemmersFundacion Gador Fundacion Baron and Fundacion FioriniFundacion Sales supported the study until March 2014 GSand MM are CONICET fellows and PR MCA and CL aremembers of the Research Career AE and MA are fellows ofInstituto Nacional de Cancer (INC) CMP was supported byfunds from the National Cancer Institute (NCI) BreastSpecialized Programs of Research Excellence (SPORE) pro-gram (P50-CA58223-09A1) and by the Breast Cancer ResearchFoundation A Molinolo is supported at 25 effort from theCancer Center Support Grants as the Director ofBiorepository and Tissue Technology Shared Resource (3 P30CA023100 [Lippman Scott] May 1 2014 to April 30 2019)

Notes

The study funders had no role in the design of the study thecollection analysis or interpretation of the data the writing ofthe manuscript or the decision to submit the manuscript forpublication

We wish to thank Dr Jose Belizan from the Institute forClinical Effectiveness and Health Policy (IECS) Buenos Aires forhis help with data analysis We also thank Dr Marcos Liguorifor his comments Dr Ana Bagnati from the Hospital Rivadaviafor providing 20 samples in 2007 Dr Alejandro de Nicola andDr Florencia Labombarda for generously sharing their McIlwainTissue Chopper and Dr Gabriela Acosta Haab and RocheLaboratories for measuring human epidermal growth factor re-ceptor 2

References1 Rossouw JE Anderson GL Prentice RL et al Risks and benefits of estrogen

plus progestin in healthy postmenopausal women Principal results from theWomenrsquos Health Initiative randomized controlled trial JAMA 2002288(3)321ndash333

2 Beral V Million Women Study Collaborators Breast cancer and hormone-replacement therapy in the Million Women Study Lancet 2003362(9382)419ndash427

3 Aupperlee MD Smith KT Kariagina A Haslam SZ Progesterone receptor iso-forms A and B Temporal and spatial differences in expression during murinemammary gland development Endocrinology 2005146(8)3577ndash3588

4 Lange CA Sartorius CA Abdel-Hafiz H Spillman MA Horwitz KB JacobsenBM Progesterone receptor action Translating studies in breast cancer mod-els to clinical insights Adv Exp Med Biol 200863094ndash111

5 Lanari C Lamb CA Fabris VT et al The MPA mouse breast cancer modelEvidence for a role of progesterone receptors in breast cancer Endocr RelatCancer 200916(2)333ndash350

6 Nardulli AM Greene GL OrsquoMalley BW Katzenellenbogen BS Regulation ofprogesterone receptor messenger ribonucleic acid and protein levels in MCF-7 cells by estradiol Analysis of estrogenrsquos effect on progesterone receptorsynthesis and degradation Endocrinology 1988122(3)935ndash944

7 Horwitz KB McGuire WL Estrogen control of progesterone receptor in hu-man breast cancer Correlation with nuclear processing of estrogen receptorJ Biol Chem 1978253(7)2223ndash2228

8 Giangrande PH McDonnell DP The A and B isoforms of the human proges-terone receptor Two functionally different transcription factors encoded bya single gene Recent Prog Horm Res 199954291ndash313

9 Mulac-Jericevic B Lydon JP DeMayo FJ Conneely OM Defective mammarygland morphogenesis in mice lacking the progesterone receptor B isoformProc Natl Acad Sci U S A 2003100(17)9744ndash9749

10 Graham JD Yeates C Balleine RL et al Characterization of progesterone re-ceptor A and B expression in human breast cancer Cancer Res 199555(21)5063ndash5068

11 Bamberger AM Milde-Langosch K Schulte HM Loning T Progesterone receptorisoforms PR-B and PR-A in breast cancer Correlations with clinicopathologic tu-mor parameters and expression of AP-1 factors Horm Res 200054(1)32ndash37

Table 2 Clinicopathological parameters of patients by isoform PRstatus

Clinopathologicalparameter PRA-H No () PRB-H No () P

PRthorn patients 116180 (644) 64180 (356) lt001Age mean 6 SD y 63 6141 60 6 129 21daggerTumor size cm lt001Dagger

Nonpalpable 199 (10) 055 (00)lt2 4899 (485) 955 (164)2ndash5 4299 (424) 3455 (618)gt5 899 (81) 1255 (218)

Positive nodes 4992 (533) 1937 (514) 84Positive metastases 466 (61) 138 (26) 65DaggerHistologic type 51

IC NST 91112 (813) 4758 (810)ILC 14112 (125) 558 (86)SS 7112 (63) 658 (103)

Histologic grade 01sectI 988 (102) 146 (22)II 3588 (397) 1246 (260)III 4488 (500) 3346 (717)

PR status 004sect0 0108 (00) 052 (00)1ndash25 22108 (204) 1852 (346)26ndash50 11108 (102) 1352 (250)51ndash75 22108 (204) 452 (77)76ndash100 53108 (491) 1752 (327)

ERa status 23sect0 1107 (09) 152 (19)1ndash25 12107 (112) 752 (135)26ndash50 4107 (37) 452 (77)51ndash75 19107 (178) 1152 (212)76ndash100 71107 (664) 2952 (558)HER2-positive 6102 (59) 850 (160) 04Ki-67 status (lt14) 4358 (741) 723 (304) lt001

P value was calculated using a two-sided chi-square test ERa frac14 estrogen recep-

tor alpha IC NST frac14 invasive carcinoma of no special type ILC frac14 invasive lobular

carcinoma PR frac14 progesterone receptor PRA-H frac14 tumors with higher levels of

isoform A of progesterone receptor (PR-A) than isoform B (PR-B) PRB-H frac14 tumors

with higher levels of PR-B than PR-A SS frac14 special subtype

daggerP value was calculated using a two-sided Studentrsquos t test

DaggerP value was calculated using a two-sided Fisher exact test

sectP value was calculated using a two-sided Cochran-Armitage trend test

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8 of 9 | JNCI J Natl Cancer Inst 2017 Vol 109 No 7

12 Ariga N Suzuki T Moriya T et al Progesterone receptor A and B isoforms inthe human breast and its disorders Jpn J Cancer Res 200192(3)302ndash308

13 Hopp TA Weiss HL Hilsenbeck SG et al Breast cancer patients with proges-terone receptor PR-A-rich tumors have poorer disease-free survival ratesClin Cancer Res 200410(8)2751ndash2760

14 Pathiraja TN Shetty PB Jelinek J et al Progesterone receptor isoform-specific promoter methylation Association of PRA promoter methylationwith worse outcome in breast cancer patients Clin Cancer Res 201117(12)4177ndash4186

15 Mote PA Gompel A Howe C et al Progesterone receptor A predominance is adiscriminator of benefit from endocrine therapy in the ATAC trial BreastCancer Res Treat 2015151(2)309ndash318

16 Helguero LA Viegas M Asaithamby A Shyamala G Lanari C Molinolo AAProgesterone receptor expression in medroxyprogesterone acetate-inducedmurine mammary carcinomas and response to endocrine treatment BreastCancer Res Treat 200379(3)379ndash390

17 Wargon V Helguero LA Bolado J et al Reversal of antiprogestin resistanceand progesterone receptor isoform ratio in acquired resistant mammary car-cinomas Breast Cancer Res Treat 2009116(3)449ndash460

18 Wargon V Riggio M Giulianelli S et al Progestin and antiprogestin respon-siveness in breast cancer is driven by the PRAPRB ratio via AIB1 or SMRT re-cruitment to the CCND1 and MYC promoters Int J Cancer 2015136(11)2680ndash2692

19 Wargon V Fernandez SV Goin M Giulianelli S Russo J Lanari CHypermethylation of the progesterone receptor A in constitutiveantiprogestin-resistant mouse mammary carcinomas Breast Cancer Res Treat2011126(2)319ndash332

20 Klijn JG Setyono-Han B Foekens JA Progesterone antagonists and progester-one receptor modulators in the treatment of breast cancer Steroids 200065(10ndash11)825ndash830

21 Lanari C Wargon V Rojas P Molinolo AA Antiprogestins in breast cancertreatment Are we ready Endocr Relat Cancer 201219(3)R35ndashR50

22 Goyeneche AA Telleria CM Antiprogestins in gynecological diseasesReproduction 2015149(1)R15ndashR33

23 Vazquez SM Mladovan A Garbovesky C Baldi A Luthy IA Three novelhormone-responsive cell lines derived from primary human breast carcino-mas Functional characterization J Cell Physiol 2004199(3)460ndash469

24 Vaira V Fedele G Pyne S et al Preclinical model of organotypic culture forpharmacodynamic profiling of human tumors Proc Natl Acad Sci U S A 2010107(18)8352ndash8356

25 Vanzulli S Efeyan A Benavides F et al p21 p27 and p53 in estrogen andantiprogestin-induced tumor regression of experimental mouse mammaryductal carcinomas Carcinog 200223(5)749ndash757

26 Cerliani JP Guillardoy T Giulianelli S et al Interaction between FGFR-2STAT5 and progesterone receptors in breast cancer Cancer Res 201171(10)3720ndash3731

27 Lowry OH Rosebrough NJ Farr AL Protein measurements with the Folin phe-nol reagent J Biol Chem 1951193265ndash275

28 Saeed AI Sharov V White J et al TM4 A free open-source system for micro-array data management and analysis Biotechniques 200334(2)374ndash378

29 Parker JS Mullins M Cheang MC et al Supervised risk predictor of breastcancer based on intrinsic subtypes J Clin Oncol 200927(8)1160ndash1167

30 Cancer Genome Atlas Network Comprehensive molecular portraits of hu-man breast tumours Nature 2012490(7418)61ndash70

31 Yau C Esserman L Moore DH Waldman F Sninsky J Benz CC A multigenepredictor of metastatic outcome in early stage hormone receptor-negativeand triple-negative breast cancer Breast Cancer Res 201012(5)R85

32 Sahoo D Dill DL Tibshirani R Plevritis SK Extracting binary signals from mi-croarray time-course data Nucleic Acids Res 200735(11)3705ndash3372

33 Diep CH Daniel AR Mauro LJ Knutson TP Lange CA Progesterone action inbreast uterine and ovarian cancers J Mol Endocrinol 201554R31ndashR53

34 Abdel-Hafiz HA Horwitz KB Role of epigenetic modifications in luminalbreast cancer Epigenomics 20157(5)847ndash862

35 Mote PA Johnston JF Manninen T Tuohimaa P Clarke CL Detection of pro-gesterone receptor forms A and B by immunohistochemical analysis J ClinPathol 200154(8)624ndash630

36 Kumar NS Richer J Owen G Litman E Horwitz KB Leslie KK Selective down-regulation of progesterone receptor isoform B in poorly differentiated humanendometrial cancer cells Implications for unopposed estrogen action CancerRes 199858(9)1860ndash1865

37 Attia GR Zeitoun K Edwards D Johns A Carr BR Bulun SE Progesterone re-ceptor isoform A but not B is expressed in endometriosis J Clin EndocrinolMetab 200085(8)2897ndash2902

38 Turgeon JL Waring DW Differential expression and regulation of progester-one receptor isoforms in rat and mouse pituitary cells and LbetaT2 gonado-tropes J Endocrinol 2006190(3)837ndash846

39 Ke W Chen C Luo H et al Histone deacetylase 1 regulates the expression ofprogesterone receptor A during human parturition by occupying the proges-terone receptor A promoter Reprodsci 201623(7)955ndash964

40 Khan JA Bellance C Guiochon-Mantel A Lombes M Loosfelt H Differentialregulation of breast cancer-associated genes by progesterone receptor iso-forms PRA and PRB in a new bi-inducible breast cancer cell line PLOS ONE20127(9)e45993

41 Mote PA Graham JD Clarke CL Progesterone receptor isoforms in normaland malignant breast Ernst Schering Found Symp Proc 20071(1)77ndash107

42 Sartorius CA Shen T Horwitz KB Progesterone receptors A and B differen-tially affect the growth of estrogen-dependent human breast tumor xeno-grafts Breast Cancer Res Treat 200379(3)287ndash299

43 Esber N Le Billan F Resche-Rigon M Loosfelt H Lombes M Chabbert-BuffetN Ulipristal acetate inhibits progesterone receptor isoform A-Mediated hu-man breast cancer proliferation and BCl2-L1 expression PLOS ONE 201510(10)e0140795

44 Guil-Luna S Stenvang J Brunner N et al Progesterone receptor isoform Amay regulate the effects of neoadjuvant aglepristone in canine mammarycarcinoma BMC Vet Res 201410296

45 Labombarda F Ghoumari AM Liere P De Nicola AF Schumacher MGuennoun R Neuroprotection by steroids after neurotrauma in organotypicspinal cord cultures A key role for progesterone receptors and steroidal mod-ulators of GABA(A) receptors Neuropharmacology 20137146ndash55

46 Faversani A Vaira V Moro GP et al Survivin family proteins as novel molec-ular determinants of doxorubicin resistance in organotypic human breast tu-mors Breast Cancer Res 201416(3)R55

47 Gaillard RC Riondel A Muller AF Herrmann W Baulieu EE RU 486 A steroidwith antiglucocorticosteroid activity that only disinhibits the humanpituitary-adrenal system at a specific time of day Proc Natl Acad Sci U S A198481(12)3879ndash3882

48 Song LN Coghlan M Gelmann EP Antiandrogen effects of mifepristone oncoactivator and corepressor interactions with the androgen receptor MolEndocrinol 200418(1)70ndash85

49 Mukhopadhyay P Chakraborty S Ponnusamy MP Lakshmanan I Jain MBatra SK Mucins in the pathogenesis of breast cancer Implications in diag-nosis prognosis and therapy Biochim Biophys Acta 20111815(2)224ndash240

50 Perou CM Soslashrlie T Eisen MB et al Molecular portraits of human breast tu-mours Nature 2000406(6797)747ndash752

51 Bu W Chen J Morrison GD et al Keratin 6a marks mammary bipotential pro-genitor cells that can give rise to a unique tumor model resembling humannormal-like breast cancer Oncogene 201130(43)4399ndash4409

52 Singhal H Greene ME Tarulli G et al Genomic agonism and phenotypic an-tagonism between estrogen and progesterone receptors in breast cancer SciAdv 20162(6)e1501924

53 Mohammed H Russell IA Stark R et al Progesterone receptor modulates ERaaction in breast cancer Nature 2015523(7560)313ndash317

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• djw317-TF1
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