ORIGINAL ARTICLE

Gain-of-function prolactin receptor variants are not

associated with breast cancer and multiple
fibroadenoma risk

Zeina Chakhtoura1, Fatima Laki2, Marie Bernadet3,8, Ibtissem Cherifi3,8,

Aurlie Chiche3,8, Natascha Pigat3,8, Sophie Bernichtein3,8, Carine Courtillot1,
Florence Boutillon3,8, Ivan Biche4, Sophie Vacher4, Marie-Laure Tanguy5,
Anne Bissery5, Virginie Grouthier1, Philippe Camparo3, Marc Foretz6,7,8,
Marcio Do Cruzeiro6,7,8, Rmi Pierre6,7,8, Fabienne Rakotozafy9, Jean Tichet9,
Isabelle Tejedor1, Jacques-Emmanuel Guidotti3,8, Brigitte Sigal-Zafrani10,
Vincent Goffin3,8,*,, Philippe Touraine1,3,*,
1
Institut E3M-ICAN, Department of Endocrinology and Reproductive Medicine, HpitauxUniversitaires
Piti Salptrire Charles Foix, Centre des Pathologies Gyncologiques Rares, FIRENDO, Paris, France ;
2
Institut Curie, Department of Oncological Surgery, Paris, France; 3Inserm, U1151, Institut Necker
Enfants Malades, Paris, France ; 4Institut Curie, Department of Genetics, Paris, France; 5Unite de
recherche Clinique, Pitie Salptriere Hospital, Paris, France ; 6Inserm, U1016, Institut Cochin, Paris,
France ; 7CNRS, UMR8104, Paris, France ; 8Universit Paris Descartes, Sorbonne Paris Cit, France ; 9Inter
Rgional pour la Sant (IRSA), LA Riche, France ; 10Institut Curie, Department of Pathology, Paris, France

Context: in a cohort of 95 women with multiple breast fibroadenomas (MFA), we recently iden-
tified patients harboring germline heterozygous variants of the prolactin receptor (PRLR) exhib-
iting constitutive activity (PRLRI146L and PRLRI176V).

Objective: to better delineate the potential role of PRLR gain-of-function variants in benign and
malignant mammary tumorigenesis

Design: observational study and transgenic mouse model analysis

Setting: Department of Endocrinology, Reproductive Disorders and Rare Gynecologic Diseases,

Piti Salptrire, Paris, and Inserm Unit 1151, Paris

Patients or Other Participants: we generated a second MFA cohort (n71) as well as a group of
control subjects (n496) and a cohort of women with breast cancer (BC, n119). We also generated
two transgenic mouse models carrying the coding sequences of human PRLRI146L or PRLRWT.

Intervention: to determine the prevalence of PRLR variants in these three populations, and to
uncover any association of the latter with specific tumor pattern, especially in patients with breast
cancer.

Results: This study did not highlight a higher prevalence of PRLR variants in the MFA group and in
the BC group compared to control subjects. Transgenic mice expressing PRLRI146L exhibited very
mild histological mammary phenotype but tumors were never observed.

Conclusion: PRLRI146L and PRLRI176V variants are not associated with breast cancer or MFA risk.
However, one cannot exclude that low but sustained PRLR signaling may facilitate or contribute
to pathological development driven by oncogenic pathways. Long-term patient follow-up should
help to address this issue.

he role of prolactin (PRL) in breast physiology is well growth factors, PRL plays a role in many steps of breast
T established. In synergy with various hormones and development and is mandatory for terminal tissue differ-

ISSN Print 0021-972X ISSN Online 1945-7197

doi: 10.1210/jc.2016-2372 J Clin Endocrinol Metab press.endocrine.org/journal/jcem 1

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2 gain-of-function PRLR variants in mammary diseases J Clin Endocrinol Metab

entiation during gestation and lactation (1, 2). The impli- I76V, conferred constitutive activity to the receptor vari-
cation of PRL in mammary tumorigenesis is fully admitted ants in various reconstituted cell lines, as reflected by PRL-
in animal models. We and others have shown that systemic independent activation of canonical PRLR signaling cas-
or mammary expression of a PRL transgene induced be- cades (STAT5, ERK1/2) and proliferative/antiapoptotic
nign or malignant mammary tumors (35), while PRL or effects in the lymphoid Ba/F3 cell line (2123). These find-
PRL receptor (PRLR) genetic deficiency delayed onco- ings raised the question whether the expression of gain-
gene-induced mammary tumorigenesis (6, 7). Despite of of-function PRLR variants could participate in the etiol-
this strong experimental evidence, the actual role of PRL ogy of (some) breast pathologies, especially PRLRI146L
in human mammary tumorigenesis remains a matter of that was shown to exhibit the higher basal activity than
debate. Epidemiological studies showed that high-normal PRLRI76V and to be more prevalent in MFA patients than
circulating PRL levels were associated with an increased in control subjects (21).
risk of estrogen receptor (ER) positive breast cancer irre- The aim of the present study was to better delineate the
spective of estrogen levels (8). Accordingly, PRL has been potential role of PRLR gain-of-function variants in mam-
reported to have a mitotic action on luminal (ER) breast mary diseases, using complementary clinical and experi-
cancer cells in vitro (9). Prolactin produced by breast can- mental approaches. First, we generated a second MFA
cer cells was shown to have the same effect in vitro (10), cohort as well as a larger group of control subjects and we
in agreement with the observation that the levels of PRL also included a cohort of women with breast cancer in
expression within breast cancer tissue are correlated to order i) to determine the prevalence of PRLRI146L and
worse patient outcome (11). Otherwise, some authors sug- PRLRI76V variants in these three populations, and ii) to
gested that PRL signaling might be protective for breast uncover any association of the latter with specific tumor
cancer based on the observation that activated signal pattern, especially in patients with breast cancer. Second,
transducer and activator of transcription (STAT) 5, the in order to unambiguously identify any mammary pheno-
major signaling target of the PRLR in mammary epithelial type that may result from PRLRI146L expression, we gen-
cells, was identified as a good prognosis factor in breast erated two transgenic mouse models carrying the human
cancer patients (12). Accordingly, loss of STAT5a expres- (h) PRLRI146L or hPRLRWT coding sequences at the
sion was correlated to breast cancer progression (13). The ROSA26 locus leaving the endogenous mouse (m) PRLR
protective effect of STAT5 was tentatively correlated to gene unaffected.
the prodifferentiation effect of PRL on mammary cells,
which may contribute to prevent metastatic dissemination
(14, 15). Finally, some authors reported associations be- Materials and Methods
tween PRLR and PRL single-nucleotide polymorphisms Patients and study design
(SNPs) and breast cancer risk, but no functional analysis This prospective multicentric study called Prolacsein was car-
was undertaken to support these associations (16 20). In ried out from September 2009 to June 2013. It was approved by
fact, the PRLR gene does not appear to be prone to mu- the local ethical committee, and all patients and controls pro-
tation in any cancer (eg, vided written informed consent before initiation of the study.
Seventy-one patients with multiple breast fibroadenomas
http://cancergenome.broadinstitute.org/index.php).
(MFA group) from 18 to 50 years of age were enrolled in the
We recently described the largest cohort of women with department of Endocrinology and Reproductive Medicine in La
multiple breast fibroadenomas (MFA), a rare benign Piti-Salptrire Hospital, Paris, France. The inclusion criteria
breast disease of unknown etiology (21). In this cohort of included to present at least three fibroadenomas (FA) in one
95 women, MFA was diagnosed at the age of 25.7 9.0 breast, and to be free of any hormonal treatment influencing the
gonadal axis for at least one month before the evaluation. Ex-
years. One third of patients had a family history of breast
clusion criteria were pregnancy and breastfeeding. The evalua-
disease. No hormonal imbalance was observed, except tion consisted in careful recording of personal and familial his-
30.7% of explosive stimulated PRL. Of interest, this study tory, with a particular attention paid to mammary and
led to the identification of patients harboring germline gynecological histories, and in mammary gland examination.
heterozygous variants of the PRLR gene, including one Eventhough a familial history for benign or breast cancer dis-
nucleotide substitution in exon 6 encoding Ile146 to Leu eases was recorded for many MFA patients, in this study re-
cruited patients were all from different families. On the same day,
amino acid change (I146L), and another one in exon 5 patients underwent breast ultrasound and MRI in the depart-
encoding Ile76 to Val amino acid change (I76V). We ment of Radiology in the same hospital.
showed that the I146L substitution, and to a lesser extent One hundred nineteen consecutive women suffering from
Printed in USA Abbreviations:
Copyright 2016 by the Endocrine Society
Received June 13, 2016. Accepted August 26, 2016.

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doi: 10.1210/jc.2016-2372
breast cancer (BC group) older than 55 years were included in the
Department of Oncological Surgery at Institut Curie, Paris,
France. They had to be postmenopausal without hormone re-
placement therapy at diagnosis, and have received no neoadju-
vant chemotherapy. Same anamnesis and clinical data than the
MFA group were collected.
The third group was constituted by 496 control subjects from
18 to 60 years of age who were recruited in the Health Inter-
regional Institute, Tours, France. They did not have personal
history of breast disease, and were submitted to a breast exam-
ination. Exclusion criteria were pregnancy and breastfeeding.
Finally, to assess the prevalence of PRLRI146L in the various
molecular breast cancer subtypes, we took advantage of a larger
retrospective breast cancer cohort established at Institut Curie,
Saint-Cloud - formerly Ren Huguenin Hospital. This group
holds a unique library of tumor mRNA from a series of 442
unilateral invasive primary breast tumors (excised from women
between 1978 and 2008) that was earlier characterized from a
clinical and pathological point (24). This cohort is hereafter re-
ferred to as the BC Huguenin cohort.
Biological samples
Blood samples were taken from the three groups of Prolacsein
study for PRL measurements and germinal PRLR genotyping.
After surgical removal, specimens of breast cancer were fixed
in neutral formalin and embedded in paraffin in order to have
precise histopathological analysis and to perform routine immu-
nohistochemistry (IHC) analyses (ER, progesterone receptor
(PR), human epidermal growth factor receptor 2 (HER2)) in the
department of Pathology at Curie Institute. Some tumor samples
were gathered in order to perform PRLR/STAT5 IHC, and fro-
zen pieces of tumor were used for molecular studies.
Hormone assays
Prolactin was measured at the Biochemistry department in La
Piti-Salptrire hospital. All blood samples were assayed by Im-
mulite 2000 chemiluminescence assay equipment (Diagnostics
Products Corporation, Los Angeles, CA). Normal range went
from 4.8 to 23.3 ng/ml.
DNA sequencing and qRT-PCR
Genomic DNA was extracted from blood cells for the three
groups of Prolacsein study. In addition, for the BC cohort, so-
matic (tumor) DNA could be obtained for 69 patients. For the
Huguenin BC cohort, cDNA was reverse-transcribed from tu-
mor RNA following routine procedures (24). PRLR genotyping
regarding the presence of exon 5 and exon 6 germline SNPs was
performed by differential enzymatic digestion of cognate PCR
amplicons as earlier described (23). Any restriction fragment
length polymorphism (RFLP) was confirmed by Sanger sequenc-
ing in both directions. Typical examples are illustrated in Figure
S1A and B.
Quantitative analysis of tumor hPRLR expression (all iso-
forms) was performed on mRNA from the Prolacsein and Huge-
nin BC cohorts following routine procedures using the primers
reported in Table S1.
Immunohistochemistry and immunofluorescence
Immunohistochemistry and immunofluorescence (IF) proce-
dures have been described in earlier publications (5, 25, 26). For
STAT5 detection in human breast samples, we used an antibody
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press.endocrine.org/journal/jcem 3
widely validated in the lab (Santa Cruz STAT5a/b, sc-845). This
antibody recognizes all forms of STAT5 including nuclear (ac-
tivated) and cytoplasmic (not activated) STAT5. First, a score
between 0 and 3 was determined by a pathologist (P.C.) accord-
ing to staining intensity (Figure S1C). Second, the percentage of
tumor cells stained for STAT5 in the fields was estimated, then
these numbers were multiplied together to obtain a score of total
staining. Based on this methodology, a tumor exhibiting uni-
form, but moderate staining, scored similarly to a tumor dis-
playing foci of intense staining. At least three slides per tumor
sample were analyzed.
The quality (specificity, affinity) of commercial antibodies
directed against the hPRLR is an issue in the field (27, 28). In a
recent systematic comparison of various PRLR commercial an-
tibodies, one (clone 1A2B1, Invitrogen) was demonstrated to be
hPRLR-specific, but its low affinity gave rise to faint signals in a
very limited number of breast tumors (27). Alternatively, the
H-300 antibody (Santa Cruz) was found to give stronger signal
but sometimes at the expense of specificity. In this context, we
used in the present study a rabbit mAb (referred to as D2A3B3)
that was recently generated by Novartis during the development
of the therapeutic anti-hPRLR mAb LFA102 (29). The D2A3B3
mAb was validated internally as specific to the hPRLR using
various reconstituted models expressing or not the hPRLR. Hu-
man PRLR immunostaining in clinical samples was then scored
as described above for STAT5 (Figure S1C).
For mouse studies we used antibodies previously validated in
mouse tissues (25, 26) and directed against phosphorylated
STAT5 (C11C5, Cell signaling), cytokeratin (CK) 5 (clone SP7,
Spring-Eurobio) and CK8 (clone 1E8, Thermofisher).
Development of knock-in mice expressing
hPRLRI146L or hPRLRWT
To generate mice expressing hPRLRI146L or, as a control,
hPRLRWT, we used the homologous recombination strategy to
insert a single copy of the cognate coding sequences at the
ROSA26 locus which was reported to be suitable to avoid in-
sertional effects in transgenic models (30). The two knock-in (KI)
mouse lineages (referred to as KI-hPRLRI146L and KI-
hPRLRWT) were established on C57/black 6 genetic back-
ground. The development of these novel mouse models is de-
scribed in the Results and in Supplemental Information.
Mice were housed in polycarbonate cages in an environment-
controlled room at 22C on a 12-hour dark/light cycle and were
regularly checked for signs of distress during the course of the
study. Female mice of 3, 6 and 12 months of age were sacrificed
by cervical dislocation, then the fourth mammary glands were
harvested and processed for analysis. Mammary whole mounts
were performed as described (5). Alternatively, mammary tissue
was fixed in paraformaldehyde for histological/IHC/IF analyses
following previously published procedures (5, 26). This study
was approved by the Comit dEthique en matire
dExprimentation Animale Paris Descartes - CEEA 34 (autho-
rizations # CEEA34.VG.033.11) and was carried out in strict
accordance with the European Directive 2010/63/UE on the pro-
tection of animals used for scientific purposes.
Statistical analysis
Comparisons of general characteristics of the three groups
(Table 1) were performed with analysis of variance (ANOVA)
for quantitative variable and 2 test for qualitative variables.
4 gain-of-function PRLR variants in mammary diseases J Clin Endocrinol Metab

Table 1. Characteristics of the 442 breast cancers of Ren Huguenin, relative to the mutational status of PRLR
Number of patients (%)
Total population (%) PRLRWT PRLRI146 liter pa
n 420 n 22
Total 442 (100.0) 420 (95) 22 (5)
Age
<50 96 (21.7) 90 (21.4) 6 (27.3) P 0.70
>50 346 (78.3) 330 (78.6) 16 (72.7)
SBR histological gradeb
I 57 (13.1) 54 (13.1) 3 (13.6) P 0.93
II 223 (51.4) 211 (51.2) 12 (54.6)
III 154 (35.5) 147 (35.7) 7 (31.8)
Lymph node statusc
0 117 (26.5) 113 (27) 4 (18.2) P 0.24
13 226 (51.3) 216 (51.5) 10 (45.5)
>3 98 (22.2) 90 (21.5) 8 (36.4)
Macroscopic tumor sizeb
<25 mm 217 (50) 204 (49.5) 13 (59.1) P 0.38
>25 mm 217 (50) 208 (50.5) 9 (40.9)
ER status
Negative 116 (26.2) 109 (26) 7 (31.8) P 0.54
Positive 326 (73.8) 311 (74) 15 (68.2)
PR status
Negative 190 (43) 181 (43.1) 9 (40.9) P 0.84
Positive 252 (57) 239 (56.9) 13 (59.1)
HER2 status
Negative 344 (77.8) 329 (78.3) 15 (68.2) P 0.39
Positive 98 (22.2) 91 (21.7) 7 (31.8)
Molecular subtypes
HR- HER2- 66 (14.9) 63 (15) 3 (13.6) P 0.73
HR- HER2 45 (10.2) 42 (10) 3 (13.6)
HR HER2- 278 (62.9) 266 (63.3) 12 (54.5)
HR HER2 53 (12) 49 (11.7) 4 (18.2)
a: 2Test
b: information available for 434 patients, c: information available for 441 patients
SBR: Scarff-Bloom- Richardson, HR: hormone receptor, ER: estrogen receptor, PR: progesterone receptor (HR means ER and/or PR), HER2:
human epidermal growth factor receptor 2

Prolactin levels were compared with ANOVA. Pairwise com- group than in the two other groups, probably because they
parisons were performed with the Tukey test to account for mul- were younger. The MFA women complained more often
tiple comparisons. The prevalence of PRLR1146L and PRLRI176V
about mastodynia, and so for a longer period, than the BC
were compared with the 2 test. A multivariable logistic regres-
sion was performed to adjust the analysis on ethnicity (Cauca- group and the controls.
sian vs non-Caucasian). Variables introduced in the logistic Focusing on the MFA group, the disease was diagnosed
model were ethnicity, group and a term for interaction of group at 24.4 7.8 years, mostly thanks to self-examination
with ethnicity. All tests were two-sided. The alpha level was set (73.2% of cases). 40.9% of the cohort already went
at 0.05. Analyses were performed using SAS software V9.3 (SAS
through surgery to remove one or more lumps. Patholog-
Institute, Cary, NC).
ical analysis of these 43 tumors identified 39 fibroadeno-
mas (90.7%), three grade 1 phyllod tumors (7.0%), and
one grade 2 phyllod tumor (2.3%). Breast examination
Results
found no lump in 15.7%, one lump per breast in 38.6%,
Clinical description of the three cohorts 2 lumps per breast in 21.4%, 3 lumps per breast in 18.6%,
General characteristics of the MFA group, the BC 4 or more lumps per breast in 5.7% of cases. Breast ul-
group and the control cohort are detailed in Table 1. The trasonography showed 7.4 4.2 FA per woman, with an
MFA group was proportionally enriched in non-Cauca- average size of 12.7 5.4 mm. Breast MRI underlined
sian women compared to the other groups. We observed 8.8 8.1 FA per woman, with an average size of 11.9
a higher rate of familial history of benign breast disease in 4.7 mm. These two technics were not statistically
the MFA group than in the two other groups. Fewer different.
women had pregnancies and miscarriages in the MFA In the BC group, 18.5% of patients reported personal

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doi: 10.1210/jc.2016-2372 press.endocrine.org/journal/jcem 5

history of benign breast disease at a mean age of 59.1 whose tumor tissue matched similar histopathological
19.2 years, mostly identified thanks to mammographic characteristics (Table S2). We first verified that the ex-
screening. Breast cancer was diagnosed at 78.5 6.5 pression levels of PRLR protein were similar for both ge-
years, just before the study inclusion. Characteristics of notypes. As shown in Figure 1A, although the samples
these cancers are depicted in Table 2. displayed staining heterogeneity, the average score was
similar for both genotypes.
Prolactin levels STAT5 is the canonical signaling target downstream of
Average PRL circulating levels were normal in the three the PRLR in the mammary gland. As all STAT proteins,
groups, but higher in the BC group 19.0 31.3 ng/ml, the latent form of STAT5 resides in the cytosol while ac-
compared to the MFA group 12.7 13.0 ng/ml and con-
trols 14.4 9.2 ng/ml (P .01).

The prevalence of PRLR variants is similar in the

three cohorts
We did not highlight difference of PRLRI146L and
PRLRI76V prevalence between the three Prolacsein co-
horts, with 11.3% in the MFA group, 3.5% in the BC
group and 5.9% in controls for PRLRI146L (P .09), and
respectively 5.6%, 5.3% and 5.7% for PRLRI76V (P
.99) (Table 3). Moreover, there was no statistical differ-
ence of PRLR status repartition in each group, whether
patients were Caucasian or non-Caucasian. The same re-
sult (data not shown) was obtained when the analysis in-
volved the 166 MFA samples combining this and our for-
merly published study (21). Strikingly, when all subjects
from the three groups of the Prolacsein study were com-
bined, PRLRI146L appeared to be more frequent in non-
Caucasian women than among Caucasian ones (13.6% vs
5.1%, P .008).
In the BC cohort, the somatic PRLR was also genotyped
when enough tumor material could be made available for
mRNA extraction (n 69). In all cases, somatic and ger-
minal genotypes were strictly identical, indicating that
PRLR genetic variants do not result from mutations oc-
curring during the tumorigenesis process.
In each group, subjects harboring PRLRI146L and
PRLRI76V had the same clinical features and PRL levels
than women with PRLRWT. Radiological data in the MFA
group and breast cancer characteristics in the BC group
were not different between PRLRI146L, PRLRI76V, and
PRLRWT women.

STAT5 signaling does not correlate with PRLR

genotype in breast cancer
Figure 1. PRLRI146L and PRLRWT have undistinguishable expression
Next, we aimed to address whether the gain-of-func- pattern in breast cancer A,B: quantification of PRLR (A) and STAT5 (B)
tion PRLR variants could correlate with increased PRLR immunostaining according to PRLR genotype in 17 selected patients
signaling in vivo. For this analysis, we focused on from the Prolacsein BC group (see Table S2). Representative
immunostaining of the different scores are shown in Figure S1. C,D:
PRLRI146L as the latter was previously shown to exhibit analysis of PRLR distribution among molecular subtypes (C) and overall
higher basal activity than PRLRI76V (21). mRNA expression level (D) in the BC Huguenin cohort according to
To that aim, we obtained fixed tissue samples from the PRLR genotype. In D, the expression level of the PRLR mRNA in BC
samples was normalized to that obtained in 10 samples of healthy
4 patients of the Prolacsein BC cohort harboring the breast tissues, showing 3 fold increased expression in cancer
PRLRI146L variant. We selected 13 PRLRWT BC patients samples, irrespective of the PRLR genotype.

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6 gain-of-function PRLR variants in mammary diseases J Clin Endocrinol Metab

tivated forms translocate into the nucleus (31). Attempts status of the patients. Since mPRL does not activate the
to detect nuclear STAT5 by IHC using two earlier vali- hPRLR (32), hPRLRWT expression in mice was assumed
dated phospho-STAT5 antibodies (26) were unsuccessful to be inert. Accordingly, we assessed in vitro that the co-
(not shown), suggesting low level of STAT5 activation. expression of hPRLRWT and mPRLR did not affect the
We then used antibodies directed against total STAT5. ability of mouse PRL to activate the latter (Figure S3D).
According to the lack of signal obtained using phospho- KI-hPRLRWT mice were used throughout this study to
STAT5 antibodies, very low nuclear staining was obtained reflect any unexpected bias linked to transgenesis, which
using total STAT5 antibodies. As for the PRLR, despite of was not observed as they were virtually undistinguishable
high staining heterogeneity, there was no obvious differ- from nontransgenic mice.
ence of total STAT5 staining between tumors harboring We used the ubiquitous phosphoglycerate kinase
PRLRI146L vs PRLRWT irrespective of their molecular sub- (PGK) promoter to direct transgene expression in order to
type (Figure 1B) which contrasted with the loss of STAT5a mimic the broad distribution of the PRLR gene described
expression linked to breast cancer progression (13). Al- in humans tissues (33). Accordingly, using qRT-PCR and
though any conclusion should remain cautious given the human-specific primers (Table S1), we showed that the
very low number of patients harboring PRLRI146L (n 4), transgene was expressed in several mouse tissues as illus-
this set of IHC analyses suggested that PRLRI146L geno- trated in Figure 2A for hPRLRI146L. Of note, the expres-
type did not correlate with specific pattern of PRLR ex- sion level of the transgene was similar to the levels ob-
pression or STAT5 signaling. served in MCF7 cells, meaning that there was no massive
hPRLR overexpression in mouse tissues, including the
PRLRI146L prevalence does not segregate with a mammary gland. In contrast, mRNA extracted from lit-
particular BC molecular subtypes termates lacking Cre-recombinase expression (ie, before
We then aimed to address whether PRLR variants may excision of LacZ cassette) achieved expression values
correlate with some BC molecular subtypes defined ac- comparable to the MDA-MB-231 cell line in which
cording to the status of hormone receptors (ER, PR) and hPRLR expression is virtually undetectable (Figure 2A).
HER2. To that aim, we took advantage of the large ret- Irrespective of the transgene genotype and animal gen-
rospective BC Huguenin cohort (n 442) in which each der, there was no gross phenotype regarding behavior,
molecular subtype was quantitatively more represented viability, fertility, litter size, pup survival and growth, and
than in the Prolacsein BC cohort (Table 4). The PRLRI146L organ size at sacrifice. We then focused our analyses on the
variant was detected in 22 patients (5%), ie, a similar mammary gland.
percentage compared to the control cohort (n 496). Its
distribution among the various molecular subgroups was Assessment of transgene expression and activity
similar to that of PRLRWT, indicating it did not segregate in the mammary gland
with any particular molecular subtypes (Figure 1C). Of Mammary gland proteins extracted from virgin KI-
note, the PRLRI146L genotype was correlated neither to PRLRI146L, KI-PRLRWT and control mice were analyzed
any molecular or clinical characteristics of the tumors (Ta- by western blot for hPRLR expression (H300 antibody),
ble 4), nor with the level of PRLR (Figure 1D) or Ki-67 (not taking again MCF7 and MDA-MB-231 cell lines as pos-
shown) mRNA expression in the tumor. itive and negative controls, respectively. As shown in Fig-
ure 2B, a band of 90 kDa comigrating with the PRLR
Generation of knock-in mice expressing hPRLRI146L expressed in MCF-7 was observed in the mammary gland
or hPRLRWT extracts of the two KI lineages. A faint band was also
In order to determine the actual impact of PRLRI146L observed in control (non transgenic) mice, likely reflecting
expression on mammary tissue development and homeo- low antibody cross-reactivity with mPRLR.
stasis in vivo, we generated two KI mouse models express- Next, the three genotypes were screened for hPRLR
ing hPRLRI146L or, as a control, hPRLRWT. The trans- expression by IHC using D2A3B3 mAb. The lack of signal
genic vectors, their functional validation in vitro and the in control mouse mammary glands confirmed the absence
complex mouse breeding scheme to generate the animals of D2A3B3 mAb cross-reactivity with mPRLR (Figure
of interest are presented in Supplemental Information 2C,a-c). In contrast, intense immunostaining was ob-
(Figures S2 to S4). In brief, as our transgenesis strategy did served in the mammary glands from KI-hPRLRWT (Figure
not involve the mouse PRLR gene locus, the KI- 2C,d-f) and KI-hPRLRI146L (Figure 2C,g-i), confirming
hPRLRI146L mice that we analyzed expressed both WT immunoblot data (Figure 2B). However, irrespective of
(endogenous mPRLR) and a mutated (transgenic the KI genotype, hPRLR staining displayed both interand
hPRLRI146L) alleles, mimicking the heterozygous PRLR intra-animal heterogeneity suggesting mosaic transgene

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doi: 10.1210/jc.2016-2372 press.endocrine.org/journal/jcem 7

Figure 2. Analysis of transgenic mice expressing hPRLRWT or hPRLRI146L. A. The ubiquitous expression of the transgene was assessed by qRT-
PCR in various tissues from hPRLRI146L littermates harboring (Cre) or not (-Cre) the Cre recombinase gene. Expression levels are normalized to
the values obtained for MDA-MB-231 cells. By removing the LacZ cassette, the Cre recombinase turned on transgene expression in all tissues.
Breast cancer cells lines expressing moderate (MCF7) or undetectable (MDA-MB-231) hPRLR amounts were taken as controls. B. Immunoblot using
H-300 antibody showing the expression of the transgenic PRLR protein in mammary glands from KI-hPRLRWT and KI-hPRLRI146L mice compared
to control mice. The same two breast cancer cell lines as above were taken as controls. C. Immunohistochemical analysis of hPRLR (WT or I146L)
expression in knock-in mice. Symbols: arrows: epithelial expression; arrowheads: adipocyte expression; stars: stromal expression. D.
Immunofluorescence analysis of basal (CK5) and luminal (CK8) cytokeratin expression in mammary glands from 12 month-old KI-hPRLRI146L mice
(panels d-i) compared to age-matched control mice (panels a-c). In KI-hPRLRI146L mice, reduced CK8 expression was observed in some (panels e-f)
but not all (panels h-i) glands.

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8 gain-of-function PRLR variants in mammary diseases J Clin Endocrinol Metab

expression. In a given mammary gland, some acini dis- cascades (STAT5, ERK1/2) and proliferative/antiapop-
played strong epithelial staining (Figure 2C,d-h) while totic effects (2123), and that ii) PRLRI146L (but not
others did not (Figure 2C,i). Stromal (Figure 2C,d) and/or PRLRI176V) was preferentially harbored by MFA patients
adipocyte (Figure 2C,i) staining could also be observed in (4 out of 95) compared to control subjects (0 out of 194,
some glands. P .01). These original findings suggested a possible as-
Finally, we used the earlier validated C11C5 mAb (25) sociation of PRLRI146L with MFA, providing a new track
to determine the status of STAT5 phosphorylation as a to elucidate the etiology of this still poorly understood
reflection of PRLR activity. No nuclear staining could be disease. Thinking more globally, these data also suggested
observed in the mammary gland of any mice, including a possible role of PRLR gain-of-function genetic variants
KI-hPRLRI146L (Figure S5A,a). Intra-peritoneal injection in breast tumorigenesis, which stimulated the analysis of
of hPRL (50 g) in these animals led to clear STAT5 phos- this variant in the breast cancer context.
phorylation in epithelial cells (Figure S5A,b), validating This study did not highlight a higher prevalence of
the experimental IHC procedures. These data suggest that PRLRI146L or PRLRI176V variants in the MFA group or the
the constitutive activity of hPRLRI146L in vivo was below BC group compared to control subjects. Few teams have
the detection threshold. investigated the association between SNPs in PRL and
PRLR genes and breast cancer risk. Canbay et al identified
Expression of hPRLRI146L does not induce PRLRI146L in 2 patients among 38 suffering from breast
mammary overgrowth cancer, vs 0/100 controls (20). Lee and colleagues evalu-
The analysis of mammary whole mount from mice of ated the association between PRL and PRLR tag SNPs and
the three genotypes failed to reveal any evidence for tumor breast cancer risk in a case-control analysis of approxi-
formation up to 12 months of age, including in KI- mately 3500 premenopausal and postmenopausal women
hPRLRI146L mice (Figure S5B). In fact, the only mild and and reported that a noncoding SNP (rs9466314) in the
not fully penetrant (3 out of 7 mice analyzed) phenotype PRL gene was associated with increased breast cancer risk
that could be detected was a trend for a slightly less de- while rs34024951 SNP in PRLR gene was associated with
veloped side branching in KI-hPRLRI146L mice at 12 decreased risk (17). Vaclavicek et al reported that
months of age. Since this phenotype was observed neither rs1341239 and rs12210179 SNPs in PRL promoter were
in younger animals nor in control and KI-hPRLRWT mice, positively associated with familial breast cancer (16). Fi-
it may reflect premature gland atrophy described by others nally, Nyante et al genotyped 8 PRL and 20 PRLR tag
(34). SNPs in 1965 breast cancer cases and 2229 matched con-
At the histological level, there was no obvious abnor- trols in Poland (19). Three PRLR SNPs were associated
malities in KI-hPRLRI146L mammary glands compared to with breast cancer risk: in premenopausal women,
the other genotypes. At best, the IF analysis of CK5 and rs249537, and in postmenopausal women, rs7718468
CK8 expression showed that, in two out of the five KI- and rs13436213. Unfortunately, none of these studies car-
hPRLRI146L mice analyzed for this parameter, some (Fig- ried out functional analysis. Of note, none of them picked
ure 2D, d-f) but not all (Figure 2D, g-i) glands displayed out PRLRI146L.
partial atrophy of the luminal cell layer as reflected by the The group of Lee and colleagues was the only one to
reduced CK8 staining while basal CK5 staining was un- relate some relevant PRL and PRLR SNPs to ethnicity
affected. This mild phenotype was in agreement with the (17). Indeed, the studied population included multiple eth-
ability of KI-hPRLRI146L females to lactate. nic groups, and PRL rs9466314 allele was somewhat com-
mon (5% in controls) among African-Americans only; in
whites, this allele was extremely uncommon (0.25% in
Discussion controls). Of interest, the analysis of 1000 genome (http://
www.1000genomes.org) and ExAC (http://exac.broadin-
To improve our understanding of the role of PRL/PRLR in stitute.org) public databases showed that the allele fre-
human breast tumorigenesis, we performed this prospec- quencies of I146L and I76V PRLR variants (rs72478580
tive multicentric study designed to add new insights on the and rs2228482, respectively) were highly variable among
potential impact of PRLRI146L and PRLRI176V that we ethnic groups. In the present study, PRLRI146L appeared to
previously described as gain-of-function variants. Our be more frequent in non-Caucasian than in Caucasian
prior work showed that i) PRLRI146L and, to a lesser ex- women, subjects of the three groups combined. This ob-
tent, PRLRI176V, exhibited some level of constitutive ac- servation was not made in our earlier reports. Indeed, the
tivity in reconstituted cell systems, as reflected by PRL- four patients harboring PRLRI146L were Caucasians,
independent activation of canonical PRLR signaling while this variant was not identified among the 194 con-

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doi: 10.1210/jc.2016-2372 press.endocrine.org/journal/jcem 9

trols (21, 23). Two hypotheses could explain this discor- this variant that we first reported some years ago (23).
dant data between our two studies: i) the present work Following up this seminal publication, we recently dem-
included much more subjects than our prior studies (686 onstrated that position 146 controlled the intrinsic prop-
vs 289), which could be more representative; ii) non-Cau- erties of the PRLR, including protein folding, PRL-respon-
casians were more represented in the MFA group (25.4%) siveness and ligand-independent activity (22). However,
than in the two other ones (3.4% in the BC group, 11.2% we also showed that several amino acid substitutions en-
in the controls), either because the prevalence of MFA is gineered at position 146 (eg, I146N, I146D, I146R) were
sizeable in non-Caucasian women (they were also 24.2% much more efficient than the natural I146L substitution
of the MFA group in our first project), or because there found in humans to confer constitutive (PRL-indepen-
might be a selection bias in this group. In fact, the main dent) activity to the PRLR (22). Therefore, it is possible
methodological limitation of this study was the recruit- that the basal signaling activity that can be detected when
ment of our population, which was monocentric for each PRLRI146L is ectopically expressed in cell lines in vitro (eg,
group. MFA group was included in the department of Figure S3C) is too low to result in measureable effects in
Endocrinology and Reproductive Medicine in La Piti- vivo. Accordingly, when hPRLRI146L was expressed in the
Salptrire Hospital, which is a referral center for rare mouse mammary gland of transgenic mice, it failed to
breast diseases; BC group was constituted at the Institut trigger STAT5 activation up to detectable levels (Figure
Curie, a cancer center also used to follow-up particular S5A).
cases of breast cancer, familial breast cancer, old patients, Such a threshold effect may not be the only explanation.
etc., One could thus wonder whether MFA and BC groups Indeed, when the very potent gain-of-function variant
had the same characteristics than the overall population PRLRI146D was ectopically expressed in breast cancer
suffering from these breast diseases. Indeed, women from cells, it failed to promote the proliferation of both MCF-7
the BC group were older than 55 years and (ER) and MDA-MB-231 (triple negative) cells; in some
postmenopausal. experimental conditions, it even decreased cell viability
Nyante et al also tried to underline a relationship be- (22). Although this observation contrasted with the many
tween SNPs in PRL and/or PRLR genes, serum PRL levels reports supporting the tumorigenic effect of PRLR signal-
and breast cancer risk, but no difference in the mean serum ing (see references in the Introduction), other groups have
PRL levels for the three SNPs associated with breast cancer recently suggested that the canonical activity of PRL on
risk could be observed (19). Recent epidemiological evi- mammary differentiation may actually protect from,
dence has enhanced the hypothesis that PRL may play a rather than promote, breast cancer progression (14, 15,
role in the etiology of breast cancer. Actually, in the 37). This was nicely supported by the observation that the
Nurses Health Study, a positive association between PRL loss of STAT5 phosphorylation in human breast cancer
concentrations and breast cancer risk was found, espe- specimens correlates with disease progression (12). Of in-
cially among postmenopausal women with ER tumors terest, mild epithelial differentiation defects is the only
(8). The European Prospective Investigation into Cancer mammary phenotype that we could detect in transgenic
and Nutrition (EPIC) recently described an increased risk mice expressing hPRLRI146L, in good agreement with the
of invasive breast cancer among postmenopausal women role of PRLR signaling in mammary gland differentiation.
in the highest quartile of PRL (OR 1.29 (95% CI, 1.05 Since this phenotype was not observed in control mice
1.58), P .09) (35). Moreover, the recent publication of expressing PRLRWT, it also supports the mild functional
a case-control nested EPIC cohort suggested that PRL impact of PRLRI146L expression in vivo. In none of our KI-
might also play a role in the early natural history of breast PRLRI146L mice, however, we could detect any evidence
cancer by increasing the occurrence of in situ tumors. Ac- for tumor formation up to 12 months of age. Although
tually, authors showed that higher PRL levels were asso- PRL transgenic mice have been reported to develop mam-
ciated with a 35% increased risk of in situ breast cancer for mary tumors lately in life (from 11 to 15.5 months de-
each unit of PRL on a continuous log scale (ORlog2 1.35 pending on the models) (3, 4), spontaneous mammary tu-
(95% CI 1.04 1.76), P .03) (36). Together, these ob- morigenesis remains very unlikely in KI-PRLRI146L mice
servations corroborate our data showing normal PRL cir- due to the undetectable level of constitutive PRLR
culating levels in our three groups, but higher in the BC signaling.
group comparing to MFA group and controls, indepen- In conclusion, this prospective multicentric study sug-
dently of PRLR gene status. gests that PRLRI146L and PRLRI176V variants are not as-
In this context, the lack of association between sociated with breast cancer or MFA risk. They were also
PRLRI146L and breast cancer revealed by this study raised not associated with higher PRL levels in these two popu-
some questions regarding the actual gain of function of lations. Hence, clinical evidence is currently lacking to

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10 gain-of-function PRLR variants in mammary diseases J Clin Endocrinol Metab

imply them as major players in the development of breast 3. Wennbo H, Gebre-Medhin M, Gritli-Linde A, Ohlsson C, Isaksson
OG, Tornell J. Activation of the prolactin receptor but not the
pathologies. Although their gain-of-function properties
growth hormone receptor is important for induction of mammary
have been previously ascertained in reconstituted cell sys- tumors in transgenic mice. J Clin Invest. 1997;100:2744 2751.
tems, this was not the case in the ex vivo analysis of mam- 4. Rose-Hellekant TA, Arendt LM, Schroeder MD, Gilchrist K, Sand-
mary tissues of mouse or human origin, suggesting that in gren EP, Schuler LA. Prolactin induces ERalpha-positive and ERal-
pha-negative mammary cancer in transgenic mice. Oncogene. 2003;
the in vivo context, their basal activity beyond the detec- 22:4664 4674.
tion threshold has minimal impact and is insufficient to 5. Manhes C, Kayser C, Bertheau P, Kelder B, Kopchick JJ, Kelly PA,
initiate mammary tumorigenesis. However, one cannot Touraine P, Goffin V. Local over-expression of prolactin in differ-
entiating mouse mammary gland induces functional defects and be-
exclude that low but sustained PRLR signaling may facil- nign lesions, but no carcinoma. J Endocrinol. 2006;190:271285.
itate or contribute to pathological development driven by 6. Vomachka AJ, Pratt SL, Lockefeer JA, Horseman ND. Prolactin
oncogenic pathways. This could be tested using a trans- gene-disruption arrests mammary gland development and retards
T-antigen-induced tumor growth. Oncogene. 2000;19:10771084.
genic approach involving coexpression of a mammary on- 7. Oakes SR, Robertson FG, Kench JG, Gardiner-Garden M, Wand
cogene and PRLRI146L. Long-term patient follow up MP, Green JE, Ormandy CJ. Loss of mammary epithelial prolactin
should also help to address this issue. receptor delays tumor formation by reducing cell proliferation in
low-grade preinvasive lesions. Oncogene. 2007;26:543553.
8. Tworoger SS, Eliassen AH, Zhang X, Qian J, Sluss PM, Rosner BA,
Hankinson SE. A 20-year prospective study of plasma prolactin as
Acknowledgments a risk marker of breast cancer development. Cancer Res. 2013;73:
4810 4819.
The authors thank all the personnel of the animal facility of 9. Biswas R, Vonderhaar BK. Role of serum in the prolactin respon-
siveness of MCF-7 human breast cancer cells in long-term tissue
Necker site for help in the management of mouse colonies and
culture. Cancer Res. 1987;47:3509 3514.
Ghislaine Hamard for precious advice in the generation of trans- 10. Ginsburg E, Vonderhaar BK. Prolactin synthesis and secretion by
genic mice. Terry Cheav and Mostafa Hajji are acknowledged human breast cancer cells. Cancer Res. 1995;55:25912595.
for clinical data management, Monique Leban for measuring 11. Wu ZS, Yang K, Wan Y, Qian PX, Perry JK, Chiesa J, Mertani HC,
PRL and Stphanie Saada for the biochemical management of Zhu T, Lobie PE. Tumor expression of human growth hormone and
human prolactin predict a worse survival outcome in patients with
cancer samples. The authors are grateful for Jason Damiano
mammary or endometrial carcinoma. J Clin Endocrinol Metab.
(formerly at Novartis) for providing the D2A3B3 anti-hPRLR 2011;96:E1619 E1629.
mAb. 12. Peck AR, Witkiewicz AK, Liu C, Stringer GA, Klimowicz AC,
Pequignot E, Freydin B, Tran TH, Yang N, Rosenberg AL, Hooke
Address all correspondence and requests for reprints to: JA, Kovatich AJ, Nevalainen MT, Shriver CD, Hyslop T, Sauter G,

Philippe Touraine, Institut E3M-ICAN, Hpitaux Universita- Rimm DL, Magliocco AM, Rui H. Loss of nuclear localized and
ires Piti Salptrire Charles Foix, Centre des Pathologies Gy- tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical
outcome and increased risk of antiestrogen therapy failure. J Clin
ncologiques Rares, Firendo, 83 Bd de l Hpital, 75 651 Paris
Oncol. 2011;29:2448 2458.
Cedex 13, France ; email : philippe.touraine@psl.aphp.fr; Vin-
13. Peck AR, Witkiewicz AK, Liu C, Klimowicz AC, Stringer GA,
cent Goffin, Inserm U1151 / Institut Necker Enfants Malades, Pequignot E, Freydin B, Yang N, Ertel A, Tran TH, Girondo MA,
Universit Paris Descartes,n Btiment Leriche, 14 Rue Maria Rosenberg AL, Hooke JA, Kovatich AJ, Shriver CD, Rimm DL,
Helena Vieira Da Silva, CS61431, 75 993 Paris cedex 14, France Magliocco AM, Hyslop T, Rui H. Low levels of Stat5a protein in
; email : vincent.goffin@inserm.fr. breast cancer are associated with tumor progression and unfavor-
*
Equal contributions able clinical outcomes. Breast Cancer Res. 2012;14:R130.
Disclosure Summary: the authors declare no conflict of 14. Sultan AS, Xie J, LeBaron MJ, Ealley EL, Nevalainen MT, Rui H.
interest Stat5 promotes homotypic adhesion and inhibits invasive charac-
This work was supported by Funding: This study was sup- teristics of human breast cancer cells. Oncogene. 2005;24:746 760.
15. Nouhi Z, Chughtai N, Hartley S, Cocolakis E, Lebrun JJ, Ali S.
ported by the Dpartement de la Recherche Clinique (DRCD,
Defining the role of prolactin as an invasion suppressor hormone in
grant PAS07008 to P.T.), Novartis (grant 3000782530 to P.T.), breast cancer cells. Cancer Res. 2006;66:1824 1832.
the Fondation de France (grant 2011 00 020 359 to V.G.), the 16. Vaclavicek A, Hemminki K, Bartram CR, Wagner K, Wappen-
Association pour la Recherche sur le Cancer (ARC, grant schmidt B, Meindl A, Schmutzler RK, Klaes R, Untch M, Burwinkel
SFI20101201635 to V.G.) and the Ligue contre le Cancer (grant B, Forsti A. Association of prolactin and its receptor gene regions
RS09/7572 to V.G.). with familial breast cancer. J Clin Endocrinol Metab. 2006;91:
15131519.
17. Lee SA, Haiman CA, Burtt NP, Pooler LC, Cheng I, Kolonel LN,
Pike MC, Altshuler D, Hirschhorn JN, Henderson BE, Stram DO.
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