Download Defining the Role of Prolactin as an Invasion

Research Article
Defining the Role of Prolactin as an Invasion Suppressor
Hormone in Breast Cancer Cells
1,2
1,2
1,2
1
Zaynab Nouhi, Naila Chughtai, Strachan Hartley, Eftihia Cocolakis,
1
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Jean-Jacques Lebrun, and Suhad Ali
1
Hormones and Cancer Research Unit, and 2The Division of Hematology, Department of Medicine,
McGill University, Montreal, Quebec, Canada
Abstract
Prolactin hormone (PRL) is well characterized as a terminal
differentiation factor for mammary epithelial cells and as
an autocrine growth/survival factor in breast cancer cells.
However, this function of PRL may not fully signify its role in
breast tumorigenesis. Cancer is a complex multistep progressive disease resulting not only from defects in cell growth but
also in cell differentiation. Indeed, dedifferentiation of tumor
cells is now recognized as a crucial event in invasion and
metastasis. PRL plays a critical role in inducing/maintaining
differentiation of mammary epithelial cells, suggesting that
PRL signaling could serve to inhibit tumor progression. We
show here that in breast cancer cells, PRL and Janus-activated
kinase 2, a major kinase involved in PRL signaling, play a
critical role in regulating epithelial-mesenchymal transformation (EMT), an essential process associated with tumor
metastasis. Activation of the PRL receptor (PRLR), achieved
by restoring PRL/JAK2 signaling in mesenchymal-like breast
cancer cells, MDA-MB-231, suppressed their mesenchymal
properties and reduced their invasive behavior. While blocking PRL autocrine function in epithelial-like breast cancer
cells, T47D, using pharmacologic and genetic approaches
induced mesenchymal-like phenotypic changes and enhanced
their invasive propensity. Moreover, our results indicate that
blocking PRL signaling led to activation of mitogen-activated
protein kinase (extracellular signal-regulated kinase 1/2) and
transforming growth factor-B/Smad signaling pathways, two
major prometastatic pathways. Furthermore, our results
indicate that following PRL/JAK2 inhibition, ERK1/2 activation precedes and is required for Smad2 activation and
EMT induction in breast cancer cells. Together, these results
highlight PRL as a critical regulator of epithelial plasticity and
implicate PRL as an invasion suppressor hormone in breast
cancer. (Cancer Res 2006; 66(3): 1824-32)
Introduction
Prolactin has been described as the most versatile hormone of
the anterior pituitary gland displaying biological activities related
to reproduction, behavior, and immunomodulation (1). Prolactin
hormone (PRL) is best known for its effect in mammary gland
development. It is required for lobuloalveolar formation of the
Note: Z. Nouhi and N. Chughtai contributed equally to this work.
S. Ali and J-J. Lebrun are research scientists of the National Cancer Institute of
Canada.
Requests for reprints: Suhad Ali, Royal Victoria Hospital, H5-81, 687 Pine
Avenue West, Montreal, Quebec, Canada H3A 1A1. Phone: 514-934-1934 ex. 34863;
Fax: 514-982-0893; E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-2292
Cancer Res 2006; 66: (3). February 1, 2006
mammary ducts during pregnancy, terminal differentiation of the
mammary epithelial cells, and for synthesis of milk components
during lactation (2). PRL binding to its receptor, the PRL receptor
(PRLR), a member of the class I cytokine receptor family, induces
receptor dimerization and activation of cytoplasmic tyrosine
kinases (3). Knock-out mice of PRL, its receptor, and components
of the Janus-activated kinase-2 (JAK2)/signal transducer and
activator of transcription-5a (STAT5a), the main signaling cascade
downstream of the PRLR, confirmed the critical role of PRL
in mammary gland alveolar growth/differentiation as these mice
show limited alveolar growth and are unable to lactate (4–7).
Whereas PRL is a potent mammary epithelial differentiation factor,
in breast carcinomas, autocrine/paracrine PRL is now recognized
to contribute to tumor cell viability (8–10). Both the hormone
and its receptor are expressed in mammary tumors and breast
cancer cell lines, thereby creating autocrine/paracrine modes of
action for PRL (11–13). Interfering with PRL autocrine/paracrine
loop in breast cancer cells with the use of PRL (14–16) and PRLR
antagonists (17) led to inhibition in cell growth and induction
of apoptosis. Furthermore, in transgenic mouse model systems,
autocrine PRL led to mammary tumor formation (18). In addition,
PRL was shown to play a permissive role in oncogene-induced
breast tumors (19). Together, these results indicate a pro-oncogenic
effect of PRL in breast cancer. However, this function of PRL may
not fully explain its role in breast carcinogenesis, particularly in
processes related to tumor progression leading to metastasis.
Cancer is now recognized as a multistep disease resulting from
abnormalities in processes related to cell growth and cell
differentiation (20). Indeed, epithelial plasticity, a fundamental
property of epithelial cells originally described during embryogenesis, also contributes to tumor metastasis for many epithelial
tumors including breast cancer (21, 22). During progression of
epithelial tumors, cells undergo a complex dedifferentiation
process designated as epithelial-mesenchymal transformation
(EMT), leading to loss of epithelial polarity and acquisition of
mesenchymal phenotype, ultimately resulting in an increase in
the migratory/invasion potential of tumor cells. The hallmark of
EMT is down-regulation of adherence junction proteins, such as
E-cadherin, and gain of various mesenchymal proteins, such as
vimentin, leading to the emergence of a fibroblastoid-invasive
phenotype. The signaling pathways regulating the motile-invasive
phenotype of epithelial tumors are not fully characterized.
However, recent studies have clearly implicated robust activation
of Ras/mitogen-activated protein kinase [MAPK; extracellular
signal-regulated kinase 1/2 (ERK1/2)] pathway and the transforming growth factor-h (TGF-h)/Smad pathway in inducing EMT
in vitro and metastasis in vivo (23–26).
Here, we show that PRL, through activation of its downstream
kinase JAK2, plays a critical role in regulating the morphogenic
program of breast cancer cells, leading to suppression of their
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Prolactin Negatively Regulates Prometastatic Pathways
invasion capacity. Furthermore, we show that PRL regulates these
epithelial plasticity variables by negatively regulating prometastatic
pathways, the MAPK (ERK1/2) and the Smad pathways. Our results
point to a dual activity of PRL in breast carcinogenesis as a prooncogenic and as a tumor progression suppressor factor.
Materials and Methods
Plasmid constructs, antibodies, and reagents. Expression plasmid
encoding the rat long-form PRLR NH2-terminal FLAG-tagged was a
gift from Dr. Dominic Devost (McGill University, Montreal, Canada).
Expression plasmid encoding kinase-inactive form of JAK2 was obtained
from Dr. Dwayne Barber (Ontario Cancer Institute, Toronto, Canada).
Reporter construct 3TP-Luc was a gift from Dr. Joan Massague (Memorial
Sloan-Kettering Cancer Center, NY). Polyclonal antibodies (pAb) against
phospho-(ERK1/2)-T202/Y204 and ERK1/2 (New England Biolabs,
Pickering, Ontario, Canada), transcription factor IID (TBP; SI-1; Santa Cruz
Biotechnology, Santa Cruz, CA), PRL and JAK2 (Upstate Biotechnology,
Charlottesville, VA), phospho-Smad2 (kindly provided by Dr. Aris Moustakas,
Ludwig Institute for Cancer Research, Sweden) were used. Monoclonal
antibodies (mAb) against phospho-STAT5Y-694 (Intermedico, Markham,
Ontario, Canada), vimentin, E-cadherin, STAT5 (BD Transduction Laboratories, Ontario, Canada), FLAG-M2, and h-tubulin (Sigma-Aldrich,
Ontario, Canada) were used. Goat anti-mouse horseradish peroxidase
(HRP) and goat anti-rabbit HRP were from Santa Cruz Biotechnology. Goat
anti-mouse Rhodamine Red X was from Jackson ImmunoResearch Laboratories (West Grove, PA). Ovine prolactin (oPRL) was from Sigma-Aldrich.
Human prolactin (hPRL) was a gift from Dr. Vincent Goffin [Institut
National de la Sante et de la Recherche Medicale (INSERM) Unit 344, Paris,
France]. Human TGF-h1 was obtained from PeproTech, Inc. (Rocky Hill, NJ).
Phosphorothioated oligodeoxynucleotides were from Alpha DNA (Quebec,
Canada). PD98059 was from Cell Signaling Technology, and AG490 was from
Calbiochem (CA). Transwell filter discs (8 Am) for migration assay by
Corning were from Fisher Scientific (Nepean, Ontario, Canada). LipofectAMINE 2000 reagent and Hoechst stain 33258 were from Invitrogen (Ontario,
Canada). Enhanced chemiluminescence Hyperfilm and Protein A-Sepharose
beads were from Amersham Biosciences/GE Healthcare (Quebec, Canada).
Cell culture and transfections. Human breast cancer cell lines, MDA
MB-231, T47D, and MCF7 and Chinese hamster ovary (CHO) cells were
grown in DMEM containing 10% fetal bovine serum (FBS) and starved in
DMEM phenol red–free media. Phase-contrast images were taken with a
Zeiss Axiovision 135 microscope (Carl Zeiss Canada, Ltd., Toronto, Ontario,
Canada) with a 10 objective. MDA-MB-231, T47D, and CHO cells were
transfected using LipofectAMINE 2000 following manufacturer’s instructions. T47D cells were also transfected by electroporation.
Antisense oligodeoxynucleotides treatments. Antisense phosphorothioated oligodeoxynucleotides (ASO) to hPRL mRNA 5V-TGGCGATCCTTTGATGTTCAT-3V and control phosphorothioated oligodeoxynucleotides
5V-GATGAATTACGTAACTGGCCCGCCC-3V were used. T47D cells were
treated with 200 Amol/L of ASO to hPRL or control oligodeoxynucleotides
in starvation media.
Cell lysis, immunoprecipitations, and Western blotting. For wholecell lysates and immunoprecipitations, cells were lysed in radioimmunoprecipitation assay lysis buffer as described previously (27). For nuclear
extracts, cells were lysed in a hypotonic buffer, and nuclear pellets were
lysed with a high salt buffer as described previously (27). Immunoprecipitations were done for 3 hours at 4jC using a pAb to JAK2 and protein
A-Sepharose beads. Proteins were run on SDS-PAGE and transferred
to nitrocellulose membrane for Western blotting analysis using the appropriate antibodies.
Immunostaining experiments. Cells were plated on coverslips precoated with poly-L-Lysine in a 24-well plate. Following different treatments,
cells were fixed in 2% PFA/PBS, blocked in 5% bovine serum albumin (BSA)/
PBS, and incubated with various antibodies in 5% BSA/PBS. Goat antimouse Rhodamine Red X was used as secondary antibody. Hoechst stain
was used to stain the nucleus. Coverslips were observed under confocal
microscope (Zeiss LSM-510 META laser scanning microscope, Carl Zeiss,
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Jena, Germany) using a 60 oil immersion lens. Photographs were analyzed
using LSM software.
Invasion assay. For Boyden chamber invasion assay, cells were plated
in a 24-transwell plates coated with Matrigel (100 Ag/cm2; VWR, Montreal,
Quebec, Canada). Following 24 hours of treatment (as described for the
different experiments), migrated cells were counted using five fields of
triplicates for each experimental point using Zeiss Axiovision 135
microscope (Carl Ziess Canada, Ltd., Toronto, Ontario, Canada) with a
10 objective and Northern Eclipse program version 6.0 (Empix Imaging,
Mississauga, Ontario, Canada).
Luciferase assay. Luciferase activity was measured as described
previously (ref. 28; EG& G Berthold Microplate Luminometer LB96V) in
relative light units.
Gelatin zymography assay. Gelatinase activity was done as described
previously (29). Briefly, MDA MB-231 cells were transfected with LipofectAMINE 2000 for 5 hours at DNA/reagent of 1:3 in serum-free media. Cells
were recovered in 5% FBS DMEM for an O/N period. The following day,
cells were stimulated with 2 Ag/mL of oPRL in 2%FBS DMEM for an O/N
period. Conditioned medium (500 AL) was collected and mixed with
nonreducing 2 loading buffer. An 8% SDS-PAGE gel was prepared as
usual with a final concentration of gelatin at 1 mg/mL. Samples were
run using regular running buffer. The gel was renatured in zymogram
renaturation buffer for 1 hour at room temperature and developed in
zymogram assay buffer O/N at room temperature. The following day, the
gel was stained with 0.5%w/v Coomassie blue R-250 for 1 hour at room
temperature. Gel was detsained until white bands appeared against a dark
blue background.
Results and Discussion
Based on estrogen receptor expression, cellular phenotype (i.e.,
epithelial as opposed to mesenchymal markers expression),
and invasion capacity breast cancer cell lines can be divided into
epithelial-like cells of low invasion capacity, such as T47D and
MCF-7, or mesenchymal-like cells exhibiting high invasion capacity,
such as MDA-MB-231 (30). Due to the well-known effects of PRL on
induction and maintenance of a differentiated epithelial phenotype
in mammary cells, we investigated whether PRL may play a role in
regulating the epithelial phenotype and hence the invasion capacity
of breast cancer cells. Recently, the MDA-MB-231 cells were shown
to lack expression of the PRLR due to gene promoter hypermethylation (31). Using this cell system, we considered whether or
not restoring PRLR signaling would alter the mesenchymal invasive
phenotype of these cells. We restored PRLR signaling by transiently
co-overexpressing the PRLR and JAK2, as not only these cells fail
to express the PRLR but also JAK2 expression level is weaker
compared with the epithelial-like breast cancer cells T47D and
MCF-7 (data not shown). As shown in Fig. 1A, whereas MDA-MB231 cells did not respond to PRL, a clear ligand-dependent increase
in JAK2 phosphorylation was observed in cells overexpressing the
PRLR and JAK2, as detected by Western blotting using a specific
antibody to phospho-JAK2 (Y1007/1008) detecting activation of
JAK2. In addition, to examine the level of PRLR phosphorylation
after restoring PRLR/JAK2 expression in these cells, PRLR was
immunoprecipitated, and immunocomplexes were blotted with
a mAb to phosphotyrosine. As expected, two phosphorylated
proteins were detected corresponding to the PRLR and JAK2 in
the MDA/PRLR/JAK2 cells compared with the MDA/pcDNA3 cells
(Fig. 1B). Collectively, these results indicate that overexpressing
the PRLR and JAK2 in MDA-MB-231 cells is sufficient to restore
PRLR/JAK2 activation.
To next investigate whether reestablishing PRLR/JAK2 activation would alter the mesenchymal phenotype of the aggressive
breast cancer MDA-MB-231 cells, we examined the effects of
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Figure 1. Restoring PRLR/JAK2 signaling
suppresses the mesenchymal phenotype
and the invasion potential of MDA-MB-231
cells. A, MDA-MB-231 cells were
transiently cotransfected with plasmids
encoding a FLAG-tagged PRLR long form
(10 Ag) and JAK2 (10 Ag) or with vector
alone (20 Ag). Cells were then incubated in
DMEM (2% FBS) for an O/N period and
stimulated or not with oPRL (2 Ag/mL)
for 10 minutes. Cell lysates were
immunodetected using a pAb to
phospho-JAK2. Membrane was reprobed
with a pAb to JAK2 And then with a mAb to
h-tubulin. B, similar transfections and cell
treatments as in (A ). Cell lysates were
immunoprecipitated using the mAb to
FLAG epitope and immunoblotted with a
mAb to phosphotyrosine. Membrane was
reprobed with a pAb to JAK2 (middle ) and
then with a mAb to FLAG epitope (bottom ).
C, similar transfections as in (A). Cells
were stimulated or not with oPRL (2 Ag/mL)
in DMEM (2% FBS) for 24 hours.
Immunodetection was done on cell lysates
using a mAb to vimentin. The membrane
was reprobed with a mAb to h-tubulin
(bottom ). D, similar transfections as in (A ).
The following day, cells were stimulated
with oPRL (2 Ag/mL) in DMEM (2% FBS)
for 24 hours. Equal volumes of conditioned
media were processed for zymography.
E, MDA-MB-231 cells were cotransfected
as in (A) and incubated in growth media for
24 hours. The following day, cells were
trypsinized, and equal number of cells was
plated for 24 hours in the presence of oPRL
(2 Ag/mL) for invasion assay. Columns,
mean of three different experiments.
Microscopic images of a representative
field of invaded cells (left).
restoring PRLR/JAK2 signaling on the expression level of the
mesenchymal marker vimentin. MDA-MB-231 cells overexpressing
PRLR/JAK2 or not were incubated in 2% serum and stimulated
or not with PRL for 24 hours. Cell lysates were subsequently
subjected to Western blot analysis using a mAb to vimentin. As
shown in Fig. 1C, expression levels of vimentin were significantly
decreased in cells overexpressing the PRLR and JAK2, and this
effect was further marked upon PRL stimulation of the cells.
Another hallmark of invasive breast cancer cells is reflected by an
increase in expression and activity of matrix metalloproteinases
(MMP) that contribute to their invasion potential (32). Therefore,
we next examined whether restoring PRLR/JAK2 expression in
MDA-MB-231 cells would modulate their MMP activity using
gelatinase assay (33). As can be seen in Fig. 1D, wild-type MDAMB-231 cells (transfected with vector only) exhibited a major
gelatinase activity at 72 kDa. However, in cells overexpressing
the PRLR/JAK2, a marked reduction in this activity was observed.
We then determined whether these PRLR-induced phenotypic
changes were associated with changes in the invasion capacity of
MDA-MB-231 cells, using Matrigel invasion assays. As illustrated
in Fig. 1E, crystal violet staining of the cells revealed that
restoring PRLR/JAK2 in these cells dramatically altered their
invasion capacity. After counting cells that migrated through the
Matrigel, our results indicated that in fact restoring PRLR/JAK2
signaling reduced by 50% the invasion capacity of the MDA-MB231 cells (Fig. 1E, right). Altogether, these results indicate that
restoring PRL signaling strongly suppresses the mesenchymal/
invasive phenotype of aggressive breast cancer cells, highlighting
Cancer Res 2006; 66: (3). February 1, 2006
PRLR/JAK2 pathway as a negative regulator of EMT process and
cell invasion.
Contrary to MDA-MB-231, T47D human breast cancer cells still
maintain epithelial-like features and are considered to be of weak
invasive phenotype (30). As well, these cells are known to express
both PRL and its receptor, thereby creating an autocrine/paracrine
growth and survival loop for PRL (8). Using this cell system,
we investigated whether blocking the PRL loop would lead to
phenotypic changes typical of EMT process and increase the
invasive capacity of these cells. To block JAK2 kinase, we used
the pharmacologic inhibitor AG490 (34). As shown in Fig. 2A,
treatment of T47D cells with AG490 for 24 hours at a concentration (50 Amol/L), sufficient to significantly inhibit PRL-induced
STAT5 phosphorylation (Fig. 2A, bottom), led to changes in cell
morphology, from epithelial/cuboidal growing as tight colonies to
mesenchymal/fibroblastic phenotype growing as dispersed colonies. Furthermore, this was found to be associated with the loss
of membrane E-cadherin (Fig. 2B) and increased expression of
the mesenchymal marker vimentin (Fig. 2C), also suggestive of an
EMT. Invasion assays using Matrigel-covered transwell plates
proved that these phenotypic changes were accompanied with an
augmented invasive capacity of T47D cells when treated with JAK2
inhibitor compared with control cells (190% of control; Fig. 2D).
Thus, our data indicate that blocking JAK2 kinase activity lead to
epithelial-mesenchymal phenotypic changes and an increase in the
invasive capacity of T47D cells.
To further confirm that blocking JAK2 leads to EMT, we
overexpressed a kinase-inactive form of JAK2 (JAK2KI), in which
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Prolactin Negatively Regulates Prometastatic Pathways
the predicted type VIII phosphotransferase motif in the COOHterminal JH1 kinase domain of JAK2 has been mutated (35). As
seen in Fig. 2D, expression of the epithelial marker E-cadherin was
markedly reduced in T47D cells overexpressing JAK2KI compared
with control cells. Similar results were obtained in the other
nonaggressive human breast cancer cell line MCF7 upon overexpression of the kinase-inactive form of JAK2 (data not shown).
Together, these results indicate that blocking JAK2 kinase activity
in breast cancer cells leads to induction of the EMT process
and cell invasion. Although JAK2 is a key kinase in mediating
PRL signals in mammary cells, it can also be activated by growth
factors other than PRL (36). Thus, we next determined whether
PRL hormone itself could regulate EMT in a manner similar
to JAK2. As shown in Fig. 2E, down-regulation of autocrine PRL
levels in these cells clearly led to a significant loss in E-cadherin
expression, further strengthening our previous results and
confirming the role of PRL itself in this process. Although the
functionality of the RRL autocrine loop previously established
(8–10), suggests that PRL signaling pathways, such as JAK2 and
STAT5a, to be constitutively active; however, under our experimental condition set-up of serum starvation in combination with
a possible limitation in detection, we do not observe constitutive
activation of this pathway, as measured by phosphorylation of
STAT5a, and indeed, STAT5a phosphorylation occurs upon exogenous PRL stimulation as shown in Fig. 2A. Altogether, these
results indicate that inhibition of autocrine PRL loop in epitheliallike breast cancer cells leads to EMT and enhances their invasion
potential.
The MAPK (ERK1/2) pathway is a key pathway implicated in
epithelial plasticity (EMT), leading to breast cancer progression/
metastasis (21, 37, 38). As our results indicated that blocking
JAK2 kinase also leads to EMT and cell invasion, we next investigated whether these effects could be mediated through activation of the MAPK (ERK1/2) pathway. Interestingly, blocking
JAK2 kinase with AG490 in T47D cells (Fig. 3A, top) or in MCF-7
cells (Fig. 3A, bottom) led to a marked increase in the basal
Figure 2. Blocking PRLR/JAK2 leads to
EMT in T47D cells. A, top, phase-contrast
microscopic images of T47D cells treated
with DMSO or AG490 (50 Amol/L) for
24 hours. Bottom, T47D cells were treated
with DMSO or AG490 (50 Amol/L) in
starvation media for 24 hours before
stimulation with hPRL (100 ng) for 10
minutes. Cells were then lysed, and total
cell lysates were analyzed using a mAb to
phospho-STAT5 Y694. The membrane
was reprobed with a mAb to STAT5.
B, top, immunostaining using a mAb to
E-cadherin (red). Bottom, immunostaining
with a mAb to vimentin (red ) of T47D cells
treated with either AG490 (50 Amol/L)
or DMSO for 24 hours. C, equal number of
T47D cells was plated on Matrigel-coated
24-transwell plates and treated with
AG490 (50 Amol/L) or DMSO for 24 hours,
and invaded cells were quantified.
Columns, mean of three separate
experiments. Microscopic images of a field
of invaded cells (left ). D, T47D cells
were transiently transfected with empty
vector or expression plasmid encoding for
JAK2KI. Cell lysates were immunoblotted
with a mAb to E-cadherin. The membrane
was reprobed with a mAb to h-tubulin
(middle ). Lysates from the same
transfection were immunoprecipitated
using a pAb to JAK2 and immunodetected
with the same antibody (bottom ). E, T47D
cells were treated with ASO to hPRL or
control oligodeoxynucleotide for the
indicated time points. Cell lysates were
immunodetected using a mAb to
E-cadherin (top ) or a pAb to PRL (middle ).
Equal loading was monitored using a
pAb to ERK1/2 (bottom ).
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activation of MAPK (Fig. 3A, compare DMSO-treated with AG490treated samples). In addition, our results indicate that whereas
PRL is able to stimulate MAPK (ERK1/2) activation as has been
previously described (39, 40), however, PRL-induced MAPK
activation was greatly amplified following inhibition of JAK2
(Fig. 3A). Moreover, as shown in Fig. 3B, in contrast to PRL,
AG490 treatment of T47D cells led to ERK1/2 activation similar
to what was observed in cells treated with epidermal growth
factor, a known mitogen for breast cancer cells. Furthermore,
activation of ERK1/2 was also seen following 5 to 10 minutes of
treatment of T47D cells with AG490 (Fig. 3C). Together, these
results indicate that in human breast cancer cells JAK2 exerts
a negative regulatory effect on the MAPK pathway and that
JAK2 may limit PRL-dependent activation of the MAPK (ERK1/2)
cascade.
We next confirmed these inhibitory effects of JAK2 on MAPK
activation using the kinase-deficient form of JAK2 (JAK2KI).
Overexpression of JAK2KI in T47D cells or in MCF-7 cells led to
a significant increase in basal activation/phosphorylation of
ERK1/2 pathway (Fig. 3B). These results indicate that blocking
JAK2 kinase in breast cancer cells leads to activation of the MAPK
pathway. To specifically address the role of PRL in this process,
we next blocked the autocrine/paracrine function of PRL in T47D
cells using ASO to hPRL gene. As can be seen in Fig. 3C, blocking
PRL expression in T47D cells led to a marked increase in MAPK
(ERK1/2) pathway, after 48 and 72 hours of treatment compared
with control cells incubated with scrambled oligodeoxynucleotides.
Together, these results indicate that blocking PRL expression or
PRLR signaling by inhibiting JAK2 kinase activity results in a robust
and prolonged activation of MAPK (ERK1/2) pathway in breast
cancer cells, suggesting that the PRL/JAK2 pathway serves to
mediate negative regulatory signals to MAPK and consequently cell
invasion.
Another critical signaling pathway (TGF-h/Smad) has also
recently been shown to contribute to EMT and metastasis of
mammary tumors (as described in Introduction). In various
epithelial cells, including mammary cells, cooperation between
Ras-MAPK (ERK1/2) and TGF-h pathways was found to induce
EMT (23). As blocking PRL/JAK2 induces ERK1/2 pathway and
EMT, therefore, we next examined whether blocking PRL
signaling in addition to activation of the ERK1/2 pathway would
also activate the TGF-h pathway. As shown in Fig. 4A and B,
treatment of T47D cells with AG490 for different time points
or overexpressing kinase-inactive form of JAK2 led to Smad2
activation as measured by Western blotting using a specific
phospho-Smad2 antibody recognizing the two COOH-terminal
serine residues phosphorylated by activated type I receptors of
the TGF-h superfamily. Moreover, blocking JAK2 activity in MCF7 breast cancer cells also led to Smad2 activation (Fig. 4C).
To further confirm the activation of Smad proteins, we examined
the effects of blocking JAK2 function on the transcriptional
activity of Smad2. For this, CHO cells were transfected with TGFh/Smad-responsive reporter construct 3TP-luc and were treated
or not with AG490 for different time points. As shown in Fig. 4D,
AG490 treatment of the cells led to a marked increase in
luciferase activity. Similarly, as shown in Fig. 4E, blocking
Figure 3. Blocking PRL autocrine loop induces ERK1/2 activation in breast cancer cells. A, T47D (top ) or MCF7 (bottom ) cells were treated with DMSO or AG490
(50 Amol/L) for 24 hours before stimulation with hPRL (100 ng/mL) for the indicated time points. B, T47D cells were treated with DMSO, hPRL (100 ng/mL),
AG490 (50 Amol/L), AG490 (50 Amol/L) and hPRL (100 ng/mL), or epidermal growth factor (EGF ; 10 ng/mL) for 24 hours. C, T47D cells were treated with AG490
(50 Amol/L) for the indicated time points. D, T47D or MCF-7 cells were transiently transfected with empty vector or expression plasmid encoding JAK2KI. E, T47D
cells were treated with ASO to hPRL or control oligodeoxynucleotide for 48 and 72 hours. A-E, cell lysates were immunodetected using a phosphospecific pAb to
ERK1/2 (top ). The membranes were reprobed with a pAb to ERK1/2 (bottom ). E, immunoblot of PRL was done using a pAb to PRL (middle ).
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Figure 4. Blocking JAK2 leads to activation of Smad2. A, T47D cells were treated with DMSO or AG490 (50 Amol/L) for the indicated time points. Nuclear extracts
were immunodetected using a pAb to phospho-Smad2 (top ). The membrane was reprobed with a pAb to TBP (bottom ). B, T47D cells were transfected by
electroporation with vector alone (10 Ag) or expression plasmid encoding JAK2KI (10 Ag). Nuclear extracts were immunodetected using a pAb to phospho-Smad2 (top ).
The membrane was reprobed with a pAb to TBP (middle ). Cell lysates of a parallel transfection were prepared for immunoprecipitation using a pAb to JAK2 and
immunodetected using the same antibody (bottom ). C, MCF-7 cells were either transfected with vector (2 Ag) or JAK2KI (2 Ag) using LipofectAMINE 2000 or cells were
treated with AG490 (50 Amol/L) for 8 hours or TGF-h1 (0.5 nmol/L) for 15 minutes. Cell lysates were immunodetected using a pAb to phospho-Smad2. D, CHO
cells were transfected with the reporter gene 3TP-Luc (1.5 Ag). The following day, cells were treated with DMSO or AG490 (50 Amol/L) for the indicated time points, and
luciferase assays were done. Columns, mean normalized luciferase light units of three experiments. E, left, CHO cells were cotransfected with 3TP-Luc construct
(1.0 Ag), an expression plasmid for h-galactosidase (0.25 Ag), and kinase-inactive JAK2 (JAK2KI) or pcDNA3 as indicated and 24 hours later luciferase assays were
done. Columns, mean of normalized luciferase light units of seven experiments (P < 0.001, ANOVA statistical analysis). Right, MCF-7 cells were cotransfected
with 3TP-Luc construct (2 Ag), an expression plasmid for h-galactosidase (0.5 Ag), and kinase inactive JAK2 (JAK2KI) or pcDNA3 as indicated, and 48 hours later,
luciferase assays were done. Columns, mean of normalized luciferase light units of four experiments (P < 0.001, ANOVA statistical analysis).
JAK2 activity by overexpressing kinase-inactive mutant form of
JAK2, JAK2KI, in both CHO and MCF-7 cells also led to a
significant increase in 3TP-luc reporter activity. Together, these
results indicate that blocking JAK2 leads to increased Smad
phosphorylation and Smad-mediated transcriptional activity.
Thus, inhibiting PRL/JAK2 signaling induces activation of two
well-characterized prometastatic pathways ERK1/2 and TGF-h/
Smad and promotes high-invasion capacity of human breast
cancer cells.
The mechanism by which Ras pathway cooperates with TGF-h/
Smad to induce EMT is still not fully characterized. It has been
shown previously that in epithelial cells transformed with Raf
autocrine production of TGF-h leads to Raf-induced EMT (41). In
addition, it was shown in squamous carcinoma cells of murine
keratinocytes that activation of Ras led to Smad2 nuclear
accumulation, and this effect was required for EMT and metastasis
(42). Furthermore, RON receptor tyrosine kinase activation was
recently shown to induce Smad2 activation through ERK1/2dependent mechanism leading to EMT in epithelial cells (43),
suggesting that MAPK (ERK1/2) and TGF-h/Smad pathways may
contribute to EMT and cell invasion in a sequential and temporal
manner. Therefore, we next investigated whether ERK1/2 pathway
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is required for Smad2 activation and EMT induction following
inactivation of PRL/JAK2 cascade in breast cancer cells. For this,
we blocked ERK1/2 activation using PD98059, a chemical inhibitor of MAP/ERK kinase-1 (MEK-1) kinase. As shown in Fig. 5A,
AG490-induced ERK1/2 activation was blocked in the presence of
PD98059. Interestingly, PD98059 also inhibited AG490-induced
Smad2 activation (Fig. 5B). In addition, AG490-induced epithelial
to mesenchymal morphology changes and loss of E-cadherin
expression was reversed in the presence of PD98059 (Fig. 5C and D)
in T47D cells.
The use of the kinase-inactive form of JAK2 confirmed these
results. As shown in Fig. 5E, JAK2KI-induced phosphorylation
of Smad2 was markedly blocked in the presence of the MEK-1
inhibitor. In addition, loss of E-cadherin expression in cells
overexpressing JAK2KI was reversed in the presence of PD98059
(Fig. 5F). Collectively, these results indicate that induction of EMT
in response to a loss of PRL/JAK2 signaling is a mutistep process
and that following inhibition of PRL/JAK2, activation of Smad2,
and induction of EMT are mediated through an ERK1/2-dependent
mechanism.
PRL has long been known as an important factor in regulating
mammary epithelial cell growth and differentiation. Furthermore,
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Cancer Research
it is now accepted that PRL functions as an autocrine factor
promoting the growth/survival of breast cancer cells. Our studies
here describe for the first time a novel function of the hormone
PRL and its downstream signaling kinase JAK2 as critical regulators
of EMT and invasion of breast cancer cells by negatively regulating
biochemical pathways involved in tumor invasion and metastasis.
Consistent with our study, STAT5a, a downstream mediator of
PRL in mammary cells, was recently shown to display invasion
suppressive activity in breast cancer (44). Therefore, in light of the
current study, the hormone PRL and its main downstream effector
molecules JAK2 and STAT5a are critical regulators of human breast
cancer cell invasion and suggest that PRL may suppress invasion
through multiple mechanisms.
We now propose a new model for the role of PRL in breast
carcinogenesis. PRL is required as a growth and survival factor
in normal mammary epithelial cells and in breast cancer but
functions as an invasion/metastasis suppressor hormone by
blocking the prometastatic MAPK (ERK1/2) and TGF-h/Smad
pathways (Fig. 6).
Tumor metastasis significantly contributes to death in cancer
patients. Although precise criteria have been defined to classify
oncogenes/proto-oncogenes and tumor suppressor genes, no
such variables exist to characterize metastasis suppressor genes/
pathways. Recently, based on the identification of several metastasis suppressor genes, such as NM23 and KAI-1, certain criteria
for metastasis suppressor genes/pathways have been proposed
(45, 46). Interestingly, PRL fulfills these variables. It is proposed
that metastasis suppressor genes are involved in regulating/
maintaining differentiation of target cells, and indeed, PRL is
known as a differentiation factor in mammary epithelial cells. In
Figure 5. Blocking JAK2 leads to activation of ERK1/2 resulting in activation of Smad2 and EMT. T47D cells were treated with DMSO, AG490 (50 Amol/L), PD98059
(10 Amol/L), or AG490 and PD98059 for an overnight period. A, cell lysates were immunoblotted with a pAb to phospho-ERK1/2 (top ), and the membrane was
reprobed with a pAb to ERK1/2 (bottom ). B, nuclear extracts were immunoblotted using a pAb to phospho-Smad2 (top ), and the membrane was reprobed with a pAb to
TBP (bottom ). C, phase-contrast microscopic images. D, fluorescent images of immunostained cells with a mAb to E-cadherin. E, T47D cells were transiently
transfected with empty vector (10 Ag) or expression plasmid encoding JAK2KI (10 Ag). Twenty-four hours following transfections, cells were treated or not with PD98059
overnight. Nuclear fractions were immunodetected using a pAb to phospho-Smad2. The membrane was reprobed with a pAb to TBP (middle). Parallel
transfections were carried out, and cells were used for immunoprecipitation using a pAb to JAK2 and immunoblotted using the same antibody (bottom ). F, T47D
cells were transiently transfected with vector alone (3 Ag) or expression plasmid encoding JAK2KI (3 Ag). The day after, cells were either treated or not with PD98059 for
an overnight period. The membrane was immunodetected using a mAb to E-cadherin (top ). The membrane was reprobed with a mAb to h-tubulin (bottom ).
Cancer Res 2006; 66: (3). February 1, 2006
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Prolactin Negatively Regulates Prometastatic Pathways
Figure 6. Dual role of prolactin in mammary tumorigenesis. Tumor progression to a metastatic phenotype occurs through complex processes results not only
from uncontrolled cell growth but also from dedifferentiation. In this model, we put forth the concept that PRL possesses a dual role in breast carcinogenesis as a growth
and survival factor (pro-oncogenic) but also as an invasion/metastasis suppressor hormone by influencing the MAPK (ERK1/2) and TGF-h/Smad pathways.
addition, it is proposed that the expression level of metastasis
suppressor genes is reduced/lost in aggressive cancers compared
with nonaggressive cancers. Similarly, loss of PRLR expression was
shown in aggressive breast cancer cells (31). Finally, functionally,
these metastasis suppressor genes affect pathways that are known
to be prometastatic. Both NM23 and KAI-1 metastasis-inhibitory
activity was related to their ability to negatively regulate MAPK
(ERK1/2) pathway (47, 48). Likewise, our data presented here
clearly show the negative role of PRL and JAK2 kinase in MAPK
and Smad activation in breast cancer cells. Thus, PRL/JAK2
signaling cascade can now be included in the growing list of
metastasis suppressor pathways.
Furthermore, based on our results, it is evident that PRL/JAK2
limits ERK1/2 activation in breast cancer cells. We can envisage
that this PRL/JAK2 activity would categorize PRL as a negative
regulator of potentially many oncogenic signals that operate
through the Ras-ERK1/2 cascade. Therefore, it is of importance to
define the mechanisms by which PRL negatively regulates the
MAPK pathway. Indeed, multiple kinases, phosphatases, and
scaffold proteins regulate ERK1/2 activation (49, 50). In addition
to GTP hydrolysis by GTPase-activating protein (Ras/GAP) to
convert Ras from GTP-bound active form to GDP-bound inactive
form and ERK1/2 inactivation by MAPK phosphatases (MKP-1 and
MKP-3; ref. 51), there exist several other negative regulators of
the Ras/MAPK pathway. For example, the protein Ras-interacting
protein-1 (RIN-1) competes with Raf for binding to Ras thereby
blocking MAPK (ERK1/2) activation (52), the protein Raf kinase
inhibitor protein (RIKP) competitively disrupts the interaction
between Raf and MEK kinases leading to suppression in MAPK
activation (53) and the protein impedes mitogenic signal
propagation (IMP; ref. 54) blocks the functional assembly of MEK
and Raf kinases through kinase suppressor of Ras (KSR). Other
studies have shown that adaptor proteins, such as sprouty (spry),
sprouty-like protein spred (55, 56), and members of the protein
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family of the Ras/GAP-associated p62 downstream of tyrosine
kinase (Dok; refs. 57, 58) also inhibit the MAPK pathway. One
mechanism by which JAK2 was previously shown to inhibit
ERK2 activation downstream of the angiotensin II hormone is by
modulating the expression levels of the MAPK phosphatase MKP-1
(59). The mechanism by which PRL negatively regulates the MAPK
pathway in mammary and breast cancer cells is unknown and is
currently under investigation.
Moreover, our results also highlight an important role for JAK2
kinase in negative regulation of EMT and cell invasion. Chromosomal translocations and, recently, activating mutations involving
JAK2 have been described and were found to cause myeloproliferative diseases (60). In addition, activation of JAK2 has been
implicated in solid tumors in addition to breast, such as ovarian
and prostate (61). Therefore, efforts are being devoted to target
JAK2 for cancer treatment (62). In light of our study, it will be
important to determine whether JAK2-induced signaling would
also modulate epithelial plasticity and invasion in these types of
tumors.
In summary, our results here define a novel mechanism
controlling invasiveness/metastasis of breast cancer cells through
PRL/JAK2 pathway that could be exploited for therapeutic
manipulation.
Acknowledgments
Received 6/29/2005; revised 10/31/2005; accepted 11/15/2005.
Grant support: Canadian Institutes of Health Research grants MOP-13681 (S. Ali)
and MOP-53141 (J-J. Lebrun), FRSQ studentship award (E. Cocolakis), and Canadian
Cancer Society (S. Ali and J-J. Lebrun).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Dominic Devost; Dwayne Barber, Vincent Goffin, Joan Massague,
and Aris Moustakas for providing us with various reagents used in this study and Eric
Lizotte for technical help with confocal microscopy (Hormones and Cancer Research
Unit core facility).
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Cancer Research
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Defining the Role of Prolactin as an Invasion Suppressor
Hormone in Breast Cancer Cells
Zaynab Nouhi, Naila Chughtai, Strachan Hartley, et al.
Cancer Res 2006;66:1824-1832.
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