Endocrine-Related Cancer (1999) 6 389-404

Prolactin involvement in breast

cancer
B K Vonderhaar
Molecular and Cellular Endocrinology Section, Laboratory of Tumor Immunology and Biology, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland, USA
(Requests for offprints should be addressed to B K Vonderhaar, NCI, NIH, 10 Center Drive, Building 10, Room
5B47, Bethesda, Maryland 20892-1402, USA)

Abstract
Normal development and differentiation of the mammary gland are profoundly influenced by prolactin
(PRL). In rodent mammary cancer PRL plays a well defined role, but its role in human breast cancer
has not been appreciated until recently. It is now clear that breast tissue, both normal and malignant,
is a significant source of extrapituitary PRL. Thus an autocrine/paracrine role of PRL in human breast
cancer may be invoked. Both PRL and PRL receptor mRNA are expressed in the vast majority of
breast cancer biopsies independent of estrogen and progesterone receptor status. An autocrine/
paracrine PRL acting in human breast cancer requires that this hormones action be blocked at the
cellular level, as opposed to suppressing the synthesis and secretion of pituitary PRL. Mutants of PRL
or human growth hormone are being explored which act as selective PRL antagonists. In addition,
tamoxifen has been shown to act locally at the target tissue by binding directly to the PRL receptor
and thus inhibiting PRLs action. These strategies may have clinical relevance in treating PRLresponsive human breast cancer.
Endocrine-Related Cancer (1999) 6 389-404

Breast cancer models and prolactin

Historically, the role of the peptide hormone prolactin
(PRL) in human breast cancer has been an obscure one. It
has been, and still is, difficult to assign a role of this
hormone in either the etiology or progression of the disease. However, it is well established that PRL is intimately
involved in development and differentiation of the normal
gland in mammalian species, and in rodent model systems
it is a key player in the development of mammary cancer.
Much of the literature on human breast cancer and PRL
appears to be contradictory. Thus, it has been difficult to
establish a defined involvement of PRL in human breast
disease. However, it is hard to believe that a hormone so
intimately involved in normal breast growth and
differentiation would have nothing to do with breast
malignancy. This paper examines the basis for a claim that
PRL is a player in human breast cancer and that it is
through an autocrine/paracrine pathway that this hormone
acts.
Rodent mammary cancer
In the normal mammary gland, growth, development and
differentiation is influenced by a variety of growth factors
and hormones; the peptide hormone, prolactin, is consid-

ered a major player (Vonderhaar 1987). Functional differentiation of the gland as measured by induction of milk
protein synthesis both in vivo and in vitro is dependent on
PRL. Extensive growth and development of the alveolar
cells within the lobules require PRL (Vonderhaar 1984,
1987). Progesterone increases the level of PRL receptors
in the developing mammary gland (Nagasawa et al. 1985)
and thus acts synergistically with PRL to induce cell
growth.
Prolactins role in rodent mammary cancer has been
established for some time (Welsch & Nagasawa 1977,
Vonderhaar & Biswas 1987). Multiple pituitary isografts
which secrete large amounts of PRL result in a significant
increase in the incidence of spontaneous mammary tumors
in mice (Muhlbock & Boot 1959). Daily injections of PRL
result in more mice with tumors compared with controls
(Boot et al. 1962). In rats, there is a direct correlation
between serum PRL levels and susceptibility of various rat
strains to induction of mammary tumors by chemical
carcinogens (Boyns et al. 1973). Tumors induced in rats
by either nitrosomethyl urea or 7,12-dimethyl-benz[a]anthracene are dependent on PRL for sustained growth
(Mershon et al. 1995). In rodents there is a direct
correlation between drug-induced hyperprolactinemia and

Endocrine-Related Cancer (1999) 6 389-404

1351-0088/99/006-389 1999 Society for Endocrinology Printed in Great Britain

Online version via http://www.endocrinology.org

Vonderhaar: Prolactin and breast cancer

increased tumor growth, and hypoprolactinemia and retarded tumor growth (Welsch & Gribler 1973, Welsch &
Nagasawa 1977).
Human breast cancer
In contrast to the situation in rodent model systems, the
function of PRL in the etiology and progression of human
breast cancer is not clear. The discovery of extrapituitary
PRL synthesis by breast tissue suggests that a re-examination of the role of PRL in human breast cancer is in
order. For several years significant contradictory evidence in the literature on the role of this hormone in human
disease has clouded the situation. However, there is
significant evidence that PRL may play a role in human
breast disease. In 1984, Holtkamp et al. (1984) reported
that as many as 44% of patients with metastatic breast
disease were hyperprolactinemic during the course of the
disease. Several cases of breast carcinoma in association
with prolactinoma have been reported (Strung et al. 1997).
In a subset of women at risk for familial breast cancer,
basal serum PRL levels were significantly elevated (Love
& Rose 1985, Love et al. 1991). In addition, the circadian
rhythm of PRL secretion from the pituitary differs
between groups at high vs low risk of breast cancer (Haus
et al. 1980), with no seasonal variations (Holdaway et al.
1997). In one study, hyperprolactinemia was found to be
an important indicator of unfavorable prognosis in nodepositive breast cancer patients, both when evaluated singly
and in conjunction with steroid receptor status
(Bhatavdekar et al. 1994). In another study (Patel et al.
1996), hyperprolactinemia and alterations in levels of p53
were associated with aggressiveness of the tumor, early
disease relapse or metastases, and poor overall survival in
patients with node-negative breast cancer. However, in
contrast, a surgery-induced rise in PRL was paradoxically
associated with a longer disease-free survival in operable
breast carcinoma in patients both with or without axillary
node involvement, despite the potential stimulation of
cancer cell growth by the hormone (Lissoni et al. 1995).
In addition, a significant decline in the serum level of
insulin-like growth factor (IGF)-I was associated with
surgery-induced hyperprolactinemia (Barni et al. 1994),
suggesting that the balance of specific hormones and
growth factors may be a key etiological factor.
Prolactin effects in vivo
It has been known for nearly 20 years that more than 70%
of human breast biopsies are positive for PRL receptors
(Codegone et al. 1981, Peyrat et al. 1981, Bonneterre et al.
1982, LHermite-Baleriaux et al. 1984); approximately
80% of breast cancer cells in culture respond to PRLs
mitogenic signal when proper conditions of reduced
serum or serum-free conditions are employed (Das &
Vonderhaar 1997a). PRL itself can be detected in 60-85%

390

of human breast cancer biopsies by immuno-histochemistry (Purnell et al. 1982, Agarwal et al. 1989).
However, while the literature suggests that PRL levels
may influence human breast cancer, there is no clear correlation between circulating PRL levels and the etiology or
prognosis of the disease (Wang et al. 1986, Ingram et al.
1990, Love et al. 1991). Nor is there a clear clinical
response when the secretion of pituitary PRL is impaired.
In earlier studies, treating patients with PRL inhibiting
ergot drugs in order to diminish the levels of circulating
pituitary PRL resulted in no change in disease state over
time (Henson et al. 1972, Pearson & Manni 1978).
However, human growth hormone (hGH), which is also a
lactogen, may have compensated for the diminished PRL
levels. Therefore, another cohort of women with advanced
breast cancer was given a combination therapy of
bromocriptine, to diminish circulating PRL levels, and a
somatostatin analog to block hGH action (Manni et al.
1989). As a result, circulating levels of PRL, detected by
a single assay, were abolished nearly completely in 8 of 9
patients, whereas hGH levels were suppressed in 7 of 9
patients during treatment. However, the patients entering
the study had been pretreated heavily with chemotherapeutic agents, thus making reliable assessment of
overall antitumor effects difficult. Only one patient
experienced disease stabilization. In a subsequent study, 4
of 6 patients with advanced breast cancer who had failed
first and second line endocrine therapies experienced no
evidence of disease progression for periods of up to 6
months when treated for extended periods of time with
bromocriptine and the long-acting, superpotent somatostatin analog octreotide. While 24 h profiles of immunoreactive PRL, growth hormone (GH) and IGF-I in their
serum were greatly reduced by these treatments, GH levels
and diurnal peaks of bioreactive PRL were still apparent,
although much reduced (Anderson et al. 1993).
Prolactin effects in vitro
Investigations in vitro using human breast tumor tissue
and cells show clear responses to PRL in contrast to results
collected in vivo. Among the responses are increased
synthesis of DNA (Salih et al. 1972, Welsch et al. 1976,
Peyrat et al. 1984, Calaf et al. 1986), protein (Burke &
Gaffney 1978) and -lactalbumin (Wilson et al. 1980),
colony formation (Malarkey et al. 1983, Manni et al.
1986) and changes in shape, adhesion, lipid accumulation
(Shiu & Paterson 1984) and estrogen receptor (ER)
content (Shafie & Grantham 1981). Increased growth is
observed when primary breast biopsy samples are grown
in nude mice supplemented with PRL (McManus &
Welsch 1984). Physiological levels of human PRL (hPRL)
and hGH in culture increase the population doubling of
primary breast tumor biopsies (Malarkey et al. 1983). In
addition, we have shown that more than 80% of ER-

Endocrine-Related Cancer (1999) 6 389-404

Table 1 Synthesis of PRL as a function of ER and PRL receptor (PRL-R) status.

Reprinted from Vonderhaar (1999).

Cell line

ER status

PRL-R status

PRL mRNA

ZR 75-1

T47D

MCF-7

T47Dco

MCF 10a

BT20

MDA MB-231

MDA MB-468

MDA MB-453

Hs578t

SK-BR-3

Not done

A1N4

positive, as well as ER-negative, human breast cancer cell

lines express mRNA for PRL receptors, bind PRL
specifically with high affinity, and respond to PRLs
mitogenic signal (Biswas & Vonderhaar 1987, Das &
Vonderhaar 1997a). Both T47D and MCF-7 human breast
cancer cells respond to PRLs growth signal when grown
as solid tumors in nude mice (Vonderhaar & Biswas 1987).
In the past, several reports have shown a lack of growth
stimulation by PRL with these same human breast cancer
cells. However, these studies were performed in the
presence of high levels of fetal bovine serum, a rich source
of lactogenic hormones (Biswas & Vonderhaar 1987).
Only under serum-free conditions or in the presence of
charcoal-stripped serum (CSS) can direct effects of PRL
on growth of human breast cancer cells be achieved (Das
et al. 1994, Lemus-Wilson et al. 1995, Das & Vonderhaar
1997a). We showed that PRL-stimulated growth of MCF7 cells is greater in the presence of 1% CSS compared with
10% CSS or fetal bovine serum (Biswas & Vonderhaar
1987). Growth effects are seen at concentrations as low as
25 ng/ml hPRL; the maximal effect is observed at 100-250
ng/ml. Other lactogens such as hGH, human placental
lactogen and ovine PRL also stimulate growth, but higher
concentrations are required to achieve the same effect.
Bovine PRL had no effect on the growth of these human
breast cancer cells. Thus MCF-7 cells are more sensitive
to the mitogenic effect of hPRL than to other lactogens
or PRL from other species.
The effects of PRL on the growth of breast cancer cells
is affected by the presence of other hormones and growth
factors in the media. Growth of MCF-7 and ZR75.1 cells
induced by hPRL is completely blocked by melatonin, the
primary hormone from the pineal gland (Lemus-Wilson et

al. 1995). When bovine PRL is added simultaneously with

hPRL to the culture media the effect of hPRL on the
growth of MCF-7 cells is completely abolished. As little
as 50-100 ng/ml bovine PRL is able to block the hPRLinduced growth of these cells (Biswas & Vonderhaar 1987,
Das & Vonderhaar 1997a). In contrast, bovine PRL is an
effective mitogen and differentiating agent in normal
mouse mammary cell lines in culture, suggesting that this
hormones action as an antagonist of hPRL may be unique
to human breast cancer cells.

Synthesis and secretion of prolactin

by breast cancer cells
In human breast cancer, the lack of clear clinical data to
correlate with responses to PRL observed in vitro may be
explained, in part, by the fact that breast cancer cells
themselves are a source of extrapituitary PRL (Fig. 1). For
many years, evidence in the literature has hinted at the
possibility of an extrapituitary source of PRL. In one
study, the levels of PRL in serum remained at 30-80% of
the pre-surgical levels for as long as 10 months in breast
cancer patients following total hypophysectomy (Lachelin
et al. 1977). In a similar study by Manni et al. (1979), 128
of 156 breast cancer patients were determined to have a
complete hypophysectomy as shown by the fact that their
serum PRL was undetectable even after stimulation with
chlorpromazine. However, 28 patients (18%) had low to
normal levels of PRL which did not respond to
stimulation. This latter group were catagorized as
incomplete hypophysectomy, but no confirming data
were presented, leaving open the possibility that the PRL
was from another source which would not necessarily be

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Vonderhaar: Prolactin and breast cancer

subject to the same stimulatory mechanisms. In addition,
low levels of circulating, bioactive PRL persisted in
patients given bromocriptine plus a somatostatin analog to
suppress pituitary hormone synthesis and activity
(Anderson et al. 1993). Prolactin is generated by tumors
as well as by a variety of normal tissues. Placenta is the
richest source of extrapituitary PRL (Sinha 1995). The
decidua is responsible for the high concentrations of PRL
in human amniotic fluid. The brain, uterus, dermal fibroblasts and the immune system all produce PRL (Clevenger
et al. 1990, Gellerson et al. 1994, Sinha 1995, Richards &
Hartman 1996). Several laboratories, including our own,
have shown that PRL is produced by both normal and
malignant mammary epithelial cells and thus may be an
autocrine/paracrine factor for this tissue. Steinmetz et al.
(1993) showed by in situ hybridization that PRL gene
transcripts are present in secretory mammary epithelial
cells from pregnant rats. Northern analysis and the reverse
transcriptase-polymerase chain reaction (RT-PCR) have
provided evidence for local synthesis of PRL by
mammary glands from lactating rats (Kurtz et al. 1993),
goats and sheep (LeProvost et al. 1994). More recently, the
presence of PRL mRNA has been demonstrated in human
breast cancer cells lines in our laboratory (Ginsburg &
Vonderhaar 1995) and in some primary human breast
carcinomas (Clevenger et al. 1995a).
In addition, we demonstrated that bioactive PRL is
synthesized by human breast cancer cells in culture and

acts in an autocrine manner to stimulate cell proliferation.

Growth of both T47Dco (ER-negative) and MCF-7 (ERpositive) human breast cancer cell lines was inhibited by
20-90% when cells were treated with monoclonal
antibodies raised against human pituitary PRL (Ginsburg
& Vonderhaar 1995). In addition, antisense RNA directed
against the gene encoding for pituitary PRL significantly
inhibited growth (>50%) of T47Dco cells (Ginsburg et al.
1997). Parallel cultures treated with a randomized
antisense RNA sequence grew at the same rate as
untreated controls. The presence of the mRNA for PRL in
T47Dco and MCF-7 cells was confirmed by RT-PCR,
followed by Southern analysis using pituitary hPRL
cDNA as the probe (Ginsburg & Vonderhaar 1995). In
total, 82% of all breast cancer cell lines tested (Table 1)
contained mRNA for PRL (Ginsburg et al. 1997).
Using 35 S-cysteine to metabolically label the T47Dco
cells, the nature of the protein synthesized was assessed.
Both cell extracts and conditioned media contain a 22 kDa
protein precipitated by anti-hPRL monoclonal antibodies.
Concentrated conditioned media prepared from T47Dco
cells stimulated the growth of PRL-responsive Nb2 rat
lymphoma cells to grow; these cells respond to picogram
quantities of lactogens. The level of biological activity in
the conditioned media is equivalent to 0.7 g/ml (14.5 pg
PRL/cell) pituitary PRL as measured by the Nb2 assay and
is approximately 30% of the amount normally produced
by the rat pituitary cell line, GH3 (Bancroft & Tashjian

Figure 1 The autocrine/paracrine action of PRL in the mammary gland.

392

Endocrine-Related Cancer (1999) 6 389-404

1971, Tanaka et al. 1980). By using a specific RIA for
human pituitary PRL, 0.35 g/ml PRL protein is detected.
When the media are pretreated with an anti-pituitary PRL
antibody, the activity in the conditioned media, like that of
the human pituitary PRL, is abolished (Ginsburg &
Vonderhaar 1995).
More than 80% of all breast cancer cell lines tested
contain mRNA for PRL (Fig. 2). In this sample of cell
lines, we found no correlation of ER status with the presence of PRL receptors or with the ability of the cells to
synthesize PRL (Table 1). PRL gene transcripts were also
found in tumors arising from MCF-7 cells implanted into
nude mice (Ginsburg et al. 1997). Immunologically detectable PRL was present in 60-85% of human breast cancer
biopsies, although the source of PRL is indeterminate
(Purnell et al. 1982, Agarwal et al. 1989). More than 75%
of primary breast cancer surgical samples also contain
mRNA for PRL. In the majority of cases, the amount of
mRNA for PRL and its receptors is significantly elevated
in cancerous vs the adjacent, non-involved tissue from the
same patient (Fields et al. 1993, Ginsburg et al. 1997).
Similar results were reported by Touraine et al. (1998)
who found PRL mRNA in all breast samples tested from
29 patients.
Post translational modifications of PRL
A PRL mRNA variant lacking exon 4 which arises from
alternate splicing has been reported in rat brain (Emanuele
et al. 1992). However, the mRNA for PRL isolated from
late pregnant and lactating sheep and goat mammary
glands differs from pituitary transcripts by only three mutations, two of which are silent (LeProvost et al. 1994).
Our recent data (E Ginsburg & BK Vonderhaar, unpublished observations) suggest at least 90% sequence identity between the PRL mRNA from the pituitary and from
human breast cancer cells. Similar results have been
reported in a variety of human breast cancer cell lines and
neoplastic breast tissue samples (Shaw-Bruha et al. 1997).
Because there is significant evidence that many diverse
activities of PRL are modulated by different post-translational modifications, the key to the role of autocrine PRL
in human breast cancer may lie therein. The biological
activity and immunoreactivity of pituitary PRL are modified by phosphorylation and glycosylation (Markoff et al.
1988, Lewis et al. 1989). Phosphorylated PRL, present in
murine, bovine and avian species (Walker 1994), has less
activity compared with the nonphosphorylated form
(Wicks & Brooks 1995). The phosphorylated form of PRL
is unable to bind to the receptor due to conformational
change in the hormone. The biological activity is restored
by dephosphorylation.
Glycosylation may selectively down-regulate PRL
action in target tissues (Hoffman et al. 1993). Glycosylated PRL, in the mammary casein synthesis and the Nb2

Figure 2 Presence of PRL transcripts in

human breast cancer cell lines and tumor from
nude mice. The PCR product obtained by
using primers designed from the known
sequence of pituitary PRL (Ginsburg &
Vonderhaar 1995) was electrophoresed on a
2% agarose gel and a predicted 612 bp PCR
product for PRL was detected by ethidium
bromide staining. Samples from human breast
cancer cell lines and tumor are marked as
indicated. M=600 bp marker. Reprinted from
Vonderhaar (1999).

proliferation assays, has biological activity similar to or

lower than that of the nonglycosylated form (Sinha et al.
1991, Pellegrini et al. 1992). Receptor binding activity and
immunological cross-reactivity are greatly reduced as a
result of glycosylation. Both glycosylated and nonglycosylated recombinant human PRL can be purified from the

393

Vonderhaar: Prolactin and breast cancer

murine C127 cell expression system. The 23 kDa nonglycosylated form of the PRL is 3-4 times more active in
the Nb2 mitogenesis bioassay compared with the 25 kDa
glycosylated form (Price et al. 1995). The changes in the
ratio of glycosylated and nonglycosylated forms of PRL
may explain physiologically diverse effects of PRL on
target tissues (Young et al. 1990).
The nature of the post-translational alteration of PRL
synthesized by the mammary gland and breast cancer cells
is unknown. Our observations that a panel of monoclonal
antibodies directed against pituitary PRL vary in their
ability to recognize the PRL produced by breast cancer
cells (Ginsburg & Vonderhaar 1995) suggests that there
are marked differences in post-translational modifications
between the product of the pituitary and that of the
mammary gland.
Cleaved prolactin and tumor angiogenesis
The mammary gland cleaves PRL (Wong et al. 1986);
three PRL species (25 kDa, 23 kDa and 14 kDa) are
detected in rat tissue. Following bromocriptine treatment,
the 25 kDa and 14 kDa forms remain, suggesting that they
are the products of the mammary gland and that cleavage
takes place (Lkhider et al. 1996). Explants of normal
mouse mammary tissue cleave PRL to yield two fragments (Baldocchi et al. 1995); the larger fragment of
cleaved PRL, either 14 kDa or 16 kDa, has anti-angiogenic
activity and inhibits the vascular endothelial growth factor
(VEGF)-induced growth of capillary endothelial cells
(Ferrara et al. 1991, DAngelo et al. 1995). VEGF is
essential for initial but not continued in vivo growth of
human breast carcinoma cells (Yoshiji et al. 1997). Explants from a transplantable rat tumor are unable to cleave
PRL (Baldocchi et al. 1995).
Regulation of expression of prolactin
The regulatory mechanisms which control PRL synthesis
and secretion by human breast cancer cells are not known.
In the normal sheep and goat mammary gland, the PRL
gene appears to be transcribed from the same proximal
promoter used by the pituitary (LeProvost et al. 1994).
However, other studies (Shaw-Bruha et al. 1997) suggest
that, in human breast cancer cells, PRL synthesis is regulated by the distal promoter used by decidua and lymphocytes. Our data (E Ginsburg & BK Vonderhaar, unpublished observations) show that one of the most effective
regulators of mammary PRL synthesis is the hor-mone
itself, suggesting an autoregulatory, feedback mechanism.
Such a mechanism may explain the obser-vation that treatment of lactating rats with bromocriptine results in a
decrease in the PRL localization to the endocytic organelles and an increase in localization to the organelles

394

associated with synthesis and exocytosis (Lkhider et al.

1996).

Prolactin receptors
PRL receptors belong to the cytokine hematopoietic
family of receptors (Kelly et al. 1991). The members of
this superfamily are single membrane-spanning receptors
organized into three domains comprising an extracellular
ligand binding domain, a hydrophobic transmembrane domain and an intracellular domain containing a proline-rich
motif. The three different forms of the PRL receptor differ
in their cytoplasmic domain. The long (90 kDa) and short
(40 kDa) forms of the receptor are generated by differential splicing of a single gene and differ only in the length
of the cytoplasmic domain (Kelly et al. 1993). The
intermediate form of the receptor is a deletion mutant of
the long form, lacking 198 amino acids in its cytoplasmic
region. This form of the receptor is found in Nb2 rat
lymphoma cells and is more sensitive to PRL compared
with the other forms (Ali et al. 1992). It may be present in
some human breast cancers (Clevenger et al. 1995a). Both
the long and the intermediate forms of the PRL receptor
transduce a differentiation signal as measured by induction of milk protein gene expression (Lesueur et al. 1991).
All three forms promote mitosis (ONeal & Yu Lee 1994,
Das & Vonderhaar 1995).
Both normal and malignant mammary cells contain
both long and short forms of the receptor. The receptors
also exist as multiple charged forms. Dimerization of the
membrane-associated receptor results from the interaction
of PRL with its receptor (Goffin & Kelly 1997, Sakal et al.
1997). The physiological significance of homodimers vs
heterodimers is unknown.
In human breast cancer, there is a clear correlation of
ER and progesterone receptor (PR) status with a variety of
disease parameters. However, because of the multiple size
and charged forms of the PRL receptor, no similar clear
correlation has been established for this hormone. PRL
receptors have been demonstrated in over 70% of breast
cancer biopsy samples using specific binding assays
(Codegone et al. 1981, Peyrat et al. 1981, Bonneterre et al.
1982, LHermite-Baleriaux et al. 1984). In our study
(Mertani et al. 1998), virtually all breast cancer surgical
samples were positive for PRL receptor mRNA by in situ
hybridization, but the amount varied considerably. Quantitative estimation of receptor mRNA levels was regionally
measured in areas corresponding to tumor cells or adipose
cells in the same section. No correlation emerged according to the histological type of lesion, probably due to the
large individual variation. Receptor immunoreactivity was
detected in some scattered stromal cells but the labeling
intensity was always weaker than for the neoplastic

Endocrine-Related Cancer (1999) 6 389-404

epithelial cells. In addition, the expression of mRNA as
measured by RT-PCR in malignant tissue was always
greater than in adjacent uninvolved tissue. Touraine et al.
(1998) made a similar observation in tissue from 29
patients using quantitative PCR and immunohisto-chemistry. In our study (Mertani et al. 1998) the expression of
PRL receptor occurred regardless of the ER or PR status.
In a similar study, Reynolds et al. (1997) demonstrated by
immunocytochemistry that >95% of breast cancers and
>93% of normal breast tissues expressed the PRL receptor.
They also found no association between the expression of
PRL receptor and ER or PR status. These observations are
in contrast to the report from Ormandy et al. (1997), who
found that the level of PRL receptor expression in breast
cancer cell lines was linearly related to that of the ER and
PR status.
In human breast cancers, the ratio of long and short
forms is unknown. The short form of the human PRL
receptor has not yet been cloned. No message for the
intermediate form of the receptor has been detected in our
samples, in contrast to the report by Clevenger et al.
(1995a).

Prolactin mitogenic signaling

pathways
PRL receptor-associated kinases
Because PRL receptors do not have intrinsic kinase
activity, they recruit several different kinases to transduce
the mitogenic signal. JAK2, a member of the Janus family
of kinases which is associated with the PRL receptor constitutively, is phosphorylated upon PRL binding in rat
lymphoma (David et al. 1994, Rui et al. 1994), mouse
mammary explants (Campbell et al. 1994) and murine
lymphoid BAF-3 cells (Dusanter-Fourt et al. 1994). In
murine lymphoid BAF-3 cells transfected with the long
form of the PRL receptor, JAK1 is associated with the
PRL receptor and is phosphorylated upon ligand binding
(Dusanter-Fourt et al. 1994). The JAK kinase is transphosphorylated and activated as a result of receptor dimerization. Phosphorylation of the JAK proteins may be one of
the earliest cellular events in response to the hormone
which ultimately triggers a chain of events in the PRL
signaling pathway.
The PRL receptor itself is phosphorylated within 1
min of PRL treatment, both in vivo in rabbit mammary
gland and in vitro in Chinese hamster ovary (CHO) cells
transfected with the long form of the PRL receptor cDNA
(Waters et al. 1995). Binding of PRL to its receptors then
induces phosphorylation of cytoplasmic transcription factors, mainly signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5 (DaSilva et al.
1996). STAT-5 is activated for PRLs differentiation signal

(Wakao et al. 1994). Two different STAT-5 proteins have

been isolated from mouse mammary tissue (STAT-5a;
STAT-5b). Both of the transcription factors recognize
GAS sequences. Their expression is concurrent during
mammary gland development, increasing from the virgin
state, reaching a maximum at day 16 of pregnancy and
declining during lactation (Liu et al. 1995). STAT-5a is
essential for full lobuloalveolar development and lactation, as demonstrated by use of knockout mice (Liu et al.
1996). The phosphorylation of STAT proteins has also
been reported in Nb2 cells (David et al. 1994) and in
normal mammary epithelial cells (HC11) (Wakao et al.
1994). We have reported activation of STAT-5 upon PRL
treatment in T47D breast cancer cells (Das & Vonderhaar
1996b). DaSilva et al. (1996) also reported the phosphorylation of STAT-1, STAT-3 and STAT-5 in T47D cells
within 15 min of PRL treatment. Activation of STAT
proteins results in translocation of the transcription factors
to the nucleus and activation of gene transcription (Darnell
et al. 1994). STAT-1, and possibly STAT-3, is constitutively activated in breast cancer tissue (Watson & Miller
1995).
In hepatocytes of lactating rats in which the short form
of the receptor is predominant, PRL induces the association of PRL receptors with pp60 c-src and activation of its
tyrosine kinase activity (Berlanga et al. 1995). PRL stimulation in rat lymphoma Nb2 cells induces the association
and activation of src family protein tyrosine kinase p59
fyn (Clevenger & Medaglia 1994) and also the guanine
nucleotide releasing factor (GNRF)-vav (Clevenger et al.
1995b). In Nb2 cells, a protein tyrosine phosphatase,
PTP1D, which is associated with the PRL receptor-JAK2
complex is essential for PRL signal transduction during
induction of -casein (Ali et al. 1996). Both tyrosine
kinase inhibitors herbimysin A and tyrphos-tin are able to
decrease the expression of a -lactoglobin promoter/chloramphenicol acetyl transferase (CAT) con-struct by over
50% in CHO cells transfected with the rabbit PRL receptor. In addition, orthovanadate, an inhibitor of tyro-sine
phosphatase, is able to substitute for PRL in inducing CAT
responses in these cells (Daniel et al. 1996). This suggests
an intricate role of both kinases and phosphatases during
PRL signal transduction.
The ras-mitogen activated protein kinase
(MAPK) pathway
A variety of growth factors and cytokines mediate
proliferation by activating the ras-MAP kinase pathway of
signal transduction. By measuring the guanine nucleotides
bound to the protein, it is possible to demonstrate activation of ras p21 protein by PRL in a variety of cell systems
(Erwin et al. 1995, Elberg et al. 1996). Raf-1, mitogen
activated protein kinase kinase (MEK) and MAP kinase

395

Vonderhaar: Prolactin and breast cancer

are downstream kinases in the ras-MAPK pathway which
are activated for mitosis induced by PRL. In Nb2 cells,
PRL rapidly phosphorylates c-raf-1 (Clevenger et al.
1994) which is closely associated with the PRL receptor.
We have reported the rapid and transient activation of
these enzymes in both normal and breast cancer cells in
response to 5 min PRL treatment (Das & Vonderhaar
1996a). Tyrosine kinase inhibitors block the MAP kinase
activity and PRL-induced growth in these mammary cells
(Das & Vonderhaar 1996a). Both the long and the short
form of the PRL receptor are able to activate MAP kinase,
with the activity reaching a peak within 5 min of PRL
treatment (Das & Vonderhaar 1995). PRL also activates
MAP kinase in rat liver, in vivo, and in Nb2 cells (Buckley
et al. 1994, Piccoletti et al. 1994). Activation and nuclear
translocation of protein kinase C (PKC) and MAP kinase
have been reported during PRL-induced proliferation of
Nb2 cells (Buckley et al. 1994, Ganguli et al. 1996). A
preliminary report has shown PRL activation of 40S ribosomal protein S6 kinase (Carey & Liberti 1995), an
enzyme downstream from MAP kinase in this signal
cascade in Nb2 cells. The activation of S6 kinase reached
its peak at 1.5-2 h after PRL treatment. Rapamycin, a
specific inhibitor of S6 kinase, inhibited PRL-induced
mitogenesis (Carey & Liberti 1995).
The PKC pathway
The PKC pathway of signal transduction may be involved
in PRL-induced cell proliferation (Kiley et al. 1996). The
phosphoinositide cycle is activated during PRL signal
transduction, which then activates PKC. The phosphorylation and activation of phosphatidylinositol-3 (PI3)kinase for signal transduction of PRL in Nb2 cells have
been reported (Alsakkaf et al. 1996). Endogenously added
phospholipase C, an enzyme that hydrolyzes L--phosphoinositol 4,5-diphosphate (PIP2) to D-myo-inositol
triphosphate (IP3) and diacylglycerol, elicited PRL-like
effects on ornithine decarboxylase activity and RNA synthesis in pregnant mouse mammary gland explants in
culture (Rillema et al. 1983). In NOG-8 mammary cells,
PRL activates and translocates PKC to the membranes
within 5-10 min (Banerjee & Vonderhaar 1992). In addition, activation of PKC by PRL occurs in splenocytes,
liver, hypothalamus and Nb2 cells (Buckley et al. 1988,
Rillema et al. 1989). Waters and Rillema (1989) showed
translocation of PKC upon PRL treatment in explants
from mouse mammary glands. In addition activation of
nuclear PKC by PRL has been reported in Nb2 cells
(Buckley et al. 1988, Fan & Rillema 1993, Ganguli et al.
1996).
The SHC-Grb pathway
The JAK-SHC pathway is activated in 3T3-F442A cells
through the GH receptor which is a member of the same
396

receptor family as the PRL receptor (VanderKuur et al.

1995). The association of JAK2 with SHC occurs in a
rapid and transient manner in Nb2 cells upon 15 min of
PRL treatment (Erwin et al. 1995). In breast cells, both
normal and malignant, we have shown that PRL can
phosphorylate SHC proteins within 1 min of hormone
treatment followed by association with the Grb2-son of
sevenless (Sos) complex in these cells. Also, JAK2 is
rapidly phosphorylated and associated with the SHC
protein upon PRL binding to its receptors (Das &
Vonderhaar 1996b). Thus for PRL signal transduction
during the induction of the mitogenic response in human
breast cancer cells, both the JAK-STAT and the SHC-rasMAP kinase pathways are operative. Possible cross talk
between the MAP kinase pathway and the JAK-STAT
pathway has been suggested (David et al. 1995).
Early response genes
Cell growth results from modulation of early response
genes and late response genes in the signaling pathways of
a mitogen. Expression of a variety of early genes,
including myc (Zabala & Garcia-Ruiz 1989), is induced
by PRL in a number of systems. After 30 min PRL
treatment in hepatocytes, there is a rapid induction of the
expression of the proto-oncogenes c-fos, c-jun and c-src,
even in the presence of cycloheximide (Berlanga et al.
1995). This suggests that PRL stimulates the expression of
genes with AP-1 or serum response elements sequences in
their promoter regions. An early response gene, Pim-1,
which is a proto-oncogene encoding a conserved cytosolic
serine/threonine protein kinase, is stimulated by PRL
during mitogenesis of Nb2 and hematopoietic cells
(Buckley et al. 1995). Peak expression occurs at 2-4 h of
PRL treatment and is not affected by cycloheximide.
Interferon regulatory factor-1 (IRF-1), another early
activation gene, is induced over 20-fold in Nb2 cells by
PRL. This gene is induced twice by PRL in a single cell
cycle. First, it is induced during G1 at 30-60 min and then
again during early S phase at 10-12 h of hormone
treatment (Stevens et al. 1995). The second peak of IRF-1
mRNA expression in early S phase is dependent on the
continuous presence of PRL throughout G1, and is
correlated tightly with DNA synthesis and subsequent cell
proliferation. The GAS site in the IRF-1 promoter is
thought to act as a PRL responsive element that responds
to the mitogenic signal of PRL in T cells. Its activation in
breast cells has not been established.
Late response genes
Several cyclins, important regulators of cell cycle progression (Musgrove et al. 1996), are amplified in malignant
cells compared with their normal counterpart. Cyclin D1
is among the most commonly overexpressed oncogenes in
breast cancer. Cyclin D1 knockout mice are devoid of

Endocrine-Related Cancer (1999) 6 389-404

PRL-dependent lobuloalveolar structures in the mature
gland (Sicinski et al. 1995). In addition, following PRL
stimulation of quiescent Nb2 cells, the cyclin D2 mRNA
level increases in mid G1 phase and decreases sharply
before S phase. The cyclin D3 level increases in late G1/
early S phase and gradually decreases during S phase
(Hosokawa et al. 1994). Further elucidation of the
activation of specific late response genes should help
clarify the distinguishing events in PRL-induced mitogenesis vs differentiation.

Inhibition of prolactin action

Because the mammary gland synthesizes its own PRL and
the majority of human breast tumors contain PRL receptors, clinical approaches to controlling this disease in
PRL-responsive tumors need to incorporate antagonists of
PRL acting directly at the receptor level. Included among
these are mutants of hGH and hPRL, some of which have
been shown to have anti-prolactin activity in vitro under
defined growth conditions (Fuh et al. 1992, 1993, Fan &
Rillema 1993, Dattani et al. 1995, Fuh & Wells 1995,

Figure 3 Inhibition of ligand-induced receptor dimerization. (A) Sequential hormone

binding first through binding site 1, forming an inactive complex. The hormone then
binds to a second receptor through site 2, which leads to dimerization and formation
of an active complex. (B) Site 2 mutants prevent formation of active dimers. (C)
Inhibitors, such as TAM, interfere with the ability of the hormone to bind to the receptor.
Reprinted from Vonderhaar (1999).

397

Vonderhaar: Prolactin and breast cancer

Mode et al. 1996). These mutants usually involve that
portion of the hormone identified as site 2 (Fig. 3) where
amino acids with small side chain residues are replaced
with amino acids carrying large side chains. Steric hindrance prevents binding of the mutant to the second
receptor in the dimer and hence they act as hormone
antagonists (Goffin & Kelly 1997).
The anti-lactogen binding site (ALBS)
In addition, tamoxifen (TAM), the first line of therapy in
pre- and post-menopausal, ER-positive breast cancer
patients, is also an anti-prolactin or anti-lactogen
(Vonderhaar & Banerjee 1991). In some reports, TAM has
been shown to be effective in a small number of ERnegative breast cancer patients (Hawkins et al. 1980, Furr
& Jordan 1984, Nolvadex Adjuvant Trial Organization
1985). Work in vitro has also suggested that TAM can
work through other ER-independent mechanisms. Frequently, estrogens can reverse TAM inhibition of cell
proliferation in vitro when the anti-estrogen is used at low
concentrations. However, the growth rates cannot be
restored by estradiol in the presence of higher concentrations of the anti-estrogen (Sutherland et al. 1987).
Growth of several ER-negative human breast cancer cell
lines is inhibited by micromolar concentrations of TAM
(Green et al. 1981, Chouvet et al. 1988, Das et al. 1994,
Perry et al. 1995). Several other cellular proteins besides
the ER may be directly affected by TAM (Vonderhaar &
Banerjee 1991). Pollak et al. (1990) have suggested that,
during TAM therapy, there is a decrease in circulating

IGF-I which could account for the ER-independent response in some patients. Others have suggested that direct
interaction of TAM with PKC may be responsible
(OBrian et al. 1985). We have shown that the antilactogenic activity of TAM results from interaction with
the ALBS (Das et al. 1993) which is located on the PRL
receptor.
The ALBS is a member of the family of high affinity
membrane-associated binding sites called anti-estrogen
binding sites or AEBS (Sutherland & Foo 1979,
Sutherland et al. 1980) which have been identified in a
variety of tissues (Gulino & Pasqualini 1982, Mehta et al.
1984). A TAM resistant clone of MCF-7 cells, RTx6,
differs from the parent MCF-7 cells in having no AEBS,
but is identical to the parent cells with respect to the ER
content and affinity (Faye et al. 1987). These cells could
not be inhibited by TAM, whereas MCF-7 cells were.
Anti-estrogens, acting through the ALBS, inhibit the
growth of PRL-responsive cells, even in the absence of ER
(Das et al. 1994, Das & Vonderhaar 1997a). The ALBS,
located on cellular membranes, binds TAM and related
non-steroidal anti-estrogens with high affinity, but does
not bind estrogens (Fig. 4) (Biswas & Vonderhaar 1991,
Vonderhaar & Banerjee 1991). It is through the ALBS that
TAM inhibits PRL-induced growth of ER-negative, Nb2
rat lymphoma cells (Biswas & Vonderhaar 1989); the
effects are not reversed by estradiol. In addition, binding
of PRL to particulate and solubilized microsomal membranes isolated from normal mammary glands of lactating
mice is inhibited by direct addition of 10-10 M or greater

Figure 4 Interactions at the anti-lactogen binding site (ALBS) on the PRL receptor.
E2, estradiol. Reprinted from Vonderhaar (1999).

398

Endocrine-Related Cancer (1999) 6 389-404

concentrations of TAM to the binding assays (Biswas &

Vonderhaar 1991); estradiol did not have this effect. Maximal inhibition of PRL binding by TAM is observed in the
light microsomes that contain the plasma membranes.
Characterization of the relationship of the ALBS to the
PRL receptor (Das et al. 1993), showed that TAM decreases the number of binding sites without changing the
receptors affinity for PRL. TAM acts by inhibiting the
binding of PRL to its receptor (Fig. 3), rather than promoting dissociation of the hormone-receptor complex. The
PRL receptor and the ALBS co-purify on either PRLSepharose or TAM-Sepharose affinity columns. Both are
recognized by the anti-PRL receptor monoclonal antibody
B6.2 (Das et al. 1993). ER-negative T47Dco as well as
ER-positive T47D cells bind lactogenic hormones specifically and this binding is inhibited by 30-50% when 10-11
M TAM is added directly to the whole cell binding reaction (Das et al. 1994). At 10-9 M, TAM inhibits PRL
binding by 70-90% with complete inhibition at 10-7 M
TAM. Subsequently, PRL-induced growth for both cell
lines is inhibited. In the ER-negative mouse mammary
epithelial cell line, NOG-8, TAM inhibits PRL binding
and at concentrations as low as 10-9 M rapidly inhibits
PRL signal transduction through raf-1, MEK and MAP
kinase. This, in turn, leads to inhibition of PRL-induced
growth of these PRL-responsive mammary cells (Das &
Vonderhaar 1997b). Thus, we conclude that the ALBS is
on the PRL receptor, and that TAM and other related, nonsteroidal, triphenylethylene anti-estrogens may inhibit
growth of ER-negative human breast cancer cells in vitro
through this mechanism.

Conclusion
The ability of PRL to stimulate growth of human breast
cancer cells in culture, coupled with the presence of active
receptors for PRL on the majority of breast carcinomas,
suggests that this peptide hormone is an active player in
this disease. Understanding its role is complicated by the
fact that human breast cancer cells synthesize and secrete
significant amounts of biologically active PRL. These data
suggest that clinically useful reagents should be sought
which act at the level of the target tissue. The drug tamoxifen, in addition to its action as an anti-estrogen, is also an
anti-lactogen and hence may be a useful tool for understanding the potential mechanism of action of PRL in
tumors which are PRL receptor-positive even if they are
ER-negative.

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