A role for the TGF?-Par6 polarity pathway in breast
Alicia M. Viloria-Petita, Laurent Davida,1, Jun Yong Jiaa,1, Tuba Erdemira, Anita L. Baneb,c,d, Dushanthi Pinnaduwagee,
Luba Roncaria, Masahiro Narimatsua, Rohit Bosea,f, Jason Moffatf, John W. Wongd,g, Robert S. Kerbelh,i,
Frances P. O’Malleyb,d, Irene L. Andrulisb,c,d,f, and Jeffrey L. Wranaa,f,2
aCenter for Systems Biology, Samuel Lunenfeld Research Institute, Room 1078 Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada M5G 1X5;
bDepartment of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5;cFred A. Litwin Centre for Cancer Genetics,
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5;dDepartment of Laboratory Medicine and Pathobiology,
University of Toronto, Banting Institute, 100 College Street, Room 110, Toronto, ON, Canada M5G 1L5;eProsserman Centre for Health Research, Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, ON, Canada M5G 1X5;fDepartment of Molecular Genetics, 1 King’s College Circle, University of Toronto,
Toronto, ON, Canada M5S 1A8;gDepartment of Anatomic Pathology, Room E4–32, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Toronto, ON,
Canada M4N 3M5;hMolecular and Cellular Biology, Room S-217, Sunnybrook Health Sciences Centre, Toronto, ON, Canada M4N 3M5; andiDepartment of
Medical Biophysics, University of Toronto, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Room 7–411, Toronto, ON, Canada
Communicated by Louis Siminovitch, Mount Sinai Hospital, Toronto, Canada, July 2, 2009 (received for review March 6, 2009)
The role of polarity signaling in cancer metastasis is ill defined.
Using two three-dimensional culture models of mammary epithe-
in breast cancer metastasis. Interference with Par6 signaling
blocked TGF?-dependent loss of polarity in acini-like structures
formed by non-transformed mammary cells grown in three-dimen-
sional structures and suppressed the protrusive morphology of
mesenchymal-like invasive mammary tumor cells without rescuing
E-cadherin expression. Moreover, blockade of Par6 signaling in an
in vivo orthotopic model of metastatic breast cancer induced the
formation of ZO-1-positive epithelium-like structures in the pri-
pathway in tissue microarrays of human breast tumors further
revealed that Par6 activation correlated with markers of the basal
carcinoma subtype in BRCA1-associated tumors. These studies thus
reveal a key role for polarity signaling and the control of morpho-
logic transformation in breast cancer metastasis.
epithelial-to-mesenchymal transition ? cell polarity ? metastasis ?
tumor invasion ? epithelial plasticity
deaths in breast cancer patients (1). Metastasis has been asso-
ciated with epithelial-to-mesenchymal transition (EMT), which
is a complex manifestation of epithelial plasticity, in which
polarized epithelial cells embedded in organized, stratified, or
single cell layers convert into single fibroblastoid cells capable of
locomotion (2). Cellular changes necessary for EMT include
both morphological changes, as well as alterations in gene
expression. While the role of the gene expression program
associated with EMT has been well-described (3), it is unclear
contribute to cancer progression and metastasis in vivo. The
Par6 polarity complex localizes to the tight junction (TJ) and is
an important regulator of the morphological transitions associ-
ated with epithelial cell plasticity (4). The complex is comprised
of three highly conserved proteins, including Par3, Par6, and
aPKC. Par6 is a core component that was initially identified as
one of the six Par (for ‘‘partitioning’’-defective) proteins essen-
tial for asymmetric cell division in the C. elegans zygote, and was
subsequently found to be required for asymmetric division of
neuroblasts and the differentiation of oocytes in Drosophila, as
well as the establishment/maintenance of apical-basal polarity
and polarized migration in both Drosophila and mammalian
by its interaction with Par3 and aPKC, as well as the Crumbs
etastasis, the spread of cancer cells from the primary
tumor site to distant organs, accounts for over 90% of
complex (4). Par6 is regulated directly by TGF? (5) and ErbB-2
receptors (6) to control epithelial cell plasticity and misregula-
tion in expression of polarity proteins, including Scribble and
Par6 itself, have been observed to be associated with breast
cancer progression (7, 8). However, the role of Par6-mediated
signaling in cancer progression has not been well-defined.
Sustained TGF? receptor signaling has been shown to en-
hance metastasis in mouse models of breast cancer (1) and in
advanced human breast cancer, high TGF?1 expression has been
detected at the invasive leading edge of the tumor (9). In
addition, strong associations between tumor levels of TGF?1
and poor prognosis (1, 10), and between a TGF? response gene
signature and lung metastasis (11), have been observed in
patients with breast cancer. Therefore, we used three-
dimensional (3D) in vitro cultures of both normal mammary
gland epithelial cells and metastatic tumor cells, as well as an
orthotopic mouse model of breast cancer to explore the role of
TGF?-polarity signaling in breast cancer progression. We dem-
onstrate that interference with polarity signaling blocks the
morphological changes associated with EMT. Furthermore,
polarity signaling is critical for the distinctive protrusive mor-
phology of metastatic breast tumor cells and blocking it in vivo
suppresses metastasis to the lungs. Moreover, we found that the
Par6 pathway was highly active in a subset of human breast
tumors with basal subtype features, which are generally more
aggressive. These studies thus demonstrate a key role for polarity
signaling in breast cancer metastasis.
The importance of the TGF?-Par6 pathway in breast cancer
progression is unknown. Since Par6 is a key component of the
core pathways that control apical-basal polarity (4, 6) and there
are three Par6 genes (12), RNAi-based approaches were not
feasible. We previously used mutant Par6 S345A to block the
TGF?-Par6 pathway (5). Therefore, to evaluate the role of this
pathway in breast cancer progression under longer term, more
physiologically relevant conditions, we cultured Par6/S345A-
Author contributions: A.M.V.-P., L.D., J.Y.J., T.E., A.L.B., D.P., M.N., R.S.K., F.P.O., I.L.A., and
analytic tools; A.M.V.-P., L.D., J.Y.J., T.E., A.L.B., D.P., J.W.W., F.P.O., I.L.A., and J.L.W.
analyzed data; and A.M.V.-P., D.P., I.L.A., and J.L.W. wrote the paper.
The authors declare no conflict of interest.
1L.D. and J.Y.J. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 18, 2009 ?
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expressing NMuMG cells in 3D cultures using reconstituted
basement membrane (Matrigel) (Fig. 1, Fig. S1, and Movie S1).
After 9 days in culture, 80% of NMuMG structures were hollow,
polarized, and acini-like (Fig. 1 A and B and Fig. S1) and were
characterized by apical ZO-1-, PKC-?-, and F-actin-positive tight
junctions (TJs); basal-lateral ?-catenin- and E-cadherin-positive
adherens junctions (AJs); and basal ?4-integrin (Fig. S1). Par6/
S345A-expressing NMuMG cells were similar to controls (Fig. 1
and Fig. S2). In contrast, 10% or less of the Par6/wt-expressing
cells were polarized (Fig. 1B and Fig. S2B), while the rest were
irregular, generally lacked a lumen, and displayed mislocalized
obtained using EpH4 mouse mammary epithelial cells (Fig. S3),
cell-line specific. When 9-day-old NMuMG structures were
treated with TGF?1 for 2 or 6 days (Fig. 1 B and C and Fig. S2),
we observed loss of polarity that was characterized by the loss of
the lumen and various markers of epithelial polarity that in-
cluded apical-lateral ZO-1 and F-actin, and basal-lateral E-
cadherin (Fig. 1 B and C and Fig. S2, respectively). These effects
were more pronounced in Par6/wt-expressing structures, consis-
tent with their disturbed acinar morphology in the absence of
TGF?1. In contrast, Par6/S345A structures maintained apical
ZO-1, F-actin, lateral E-cadherin, and normal acinar morphol-
ogy. Thus, TGF?-treated NMuMG 3D cultures displayed loss of
polarity, but not acquisition of complete EMT or protrusive
activity. This is likely due to the lack of a transforming oncogene,
since previous studies showed that long-term exposure to TGF?
cooperates with oncogenes to promote complete EMT and
protrusive behavior in 3D culture conditions (3, 13).
TGF?-induced loss of expression and lateral localization of
E-cadherin and ?-catenin is mediated by a Smad-dependent
gene expression program [reviewed in (3)]. To confirm that
interfering with the TGF?-Par6 pathway does not significantly
impact Smad transcriptional signaling, we examined expression
of a panel of 10 TGF? target genes (14) (Table S1) of known
relevance to breast cancer progression. All 10 genes were
regulated similarly by TGF? in Parental, Vector, Par6/wt-, and
Par6/S345A-expressing NMuMG cells (Fig. S4A). Consistent
with this, the anti-proliferative response to TGF?, which is a
well-documented response to Smad signaling (15), was similar in
3D cultures of all three cell types (Fig. S4B). Of note, these
studies also revealed that overexpression of Par6 (wt or the
S345A mutant) induced proliferation, as previously reported for
MCF-10A cells (7). We also examined TGF?-dependent apo-
ptosis in this model, using TUNEL staining. This revealed that
80% of TGF?1-treated vector or Par6/wt-expressing structures
contained apoptotic cells. However, apoptosis was significantly
reduced in Par6/S345A-expressing structures (Fig. S4 C and D),
possibly because their highly polarized phenotype confers resis-
tance to apoptosis (16).
Autocrine TGF? signaling mediates mammary tumor cell
of autocrine TGF? signaling, we used the EMT-6 mouse mam-
mary carcinoma cell line. EMT-6 cells secrete their own (auto-
crine) TGF?, which mediates both their migratory capability ex
vivo, as well as their ability to metastasize to the lungs in vivo
(17). The cells have undergone EMT, as deduced from their
fusiform morphology (19), and they can grow in the mammary
fat pad of syngeneic BALB/c mice (i.e., orthotopically). This
preserves species-specific interactions between secreted factors
and their receptors as well as the host immune response, which
is a key target of TGF? signaling during tumor progression (15).
In Matrigel, we observed that EMT-6 cells formed highly
protrusive structures with a compact spherical core (Fig. 2A and
E). This morphology was suppressed by a neutralizing TGF?1
antibody (Ab) (Fig. 2 A and B), consistent with a key role for
autocrine TGF? in promoting the metastasis of these cells (17).
We also examined the human breast cancer line, MDA-MB-231,
which is subject to autocrine TGF? signaling (Fig. S5A) that
mediates metastasis to lung (20). Like EMT-6, interference with
TGF? suppressed formation of protrusive structures by MDA-
MB-231 cells (Fig. S5B).
To explore regulation of the Par6 pathway, we next generated
an affinity purified rabbit polyclonal phospho-Par6 (pPar6) Ab
to Ser 345P, which is phosphorylated by the TGF? type II
receptor (5). Characterization in NMuMG cells revealed robust
pPar6 levels in anti-Flag immunoprecipitates (IP) from Par6/wt,
but not Par6/S345A-expressing cells (Fig. 2C, left blot). Endog-
enous pPar6 was not detected in NMuMG by IP of total Par6
(Fig. 2C, left blot), but it co-precipitated with TGF?RI and was
stimulated by TGF? treatment (Fig. 2C, right blot). Further, in
of polarized acini-like structures by NMuMG cells. (A) Gross morphology of
9-day-old 3D cultures of NMuMG lines expressing empty vector (Vector), wt,
or S345A Par6. (B) Quantification of acini-like structures. Nine-day-old struc-
tures were treated with TGF? (500 pM; black bars) or without (Control; white
bars) for 2 days (2d) and the percentage of acini-like structures (containing a
lumen) quantified. Most Par6/wt structures lacked a lumen under basal con-
ditions and maintained their abnormal morphology after TGF? treatment. In
sharp contrast, about 60% of Par6/S345A structures remain polarized after
TGF? exposure. (C) The TGF?-Par6 pathway disrupts polarity in NMuMG 3D
structures. TGF? treated or untreated structures as in B were immunostained
for nuclei (DAPI, blue) and polarity markers, followed by confocal microscopy
analysis. Untreated vector and Par6/S345A structures (Top) had well-defined
lumens, with ZO-1 (yellow) and F-actin (red) localized to the apical, TJ region,
and E-cadherin (green) localized to the AJ, basal to ZO-1. Par6/wt structures
had disorganized ZO-1 and F-actin and were lumenless. TGF? treatment
(Bottom) caused ZO-1, F-actin, and E-cadherin mislocalization in both Vector
100 ?m; C, 20 ?m.)
Activation of the TGF?-Par6 pathway interferes with the formation
Viloria-Petit et al.PNAS ?
August 18, 2009 ?
vol. 106 ?
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cells overexpressing Par6/wt, elevated pPar6 was detected bound
to TGF?RI (Fig. 2C, right blot). In EMT-6 cells, pPar6 was
constitutively present, was enhanced by exogenous TGF? treat-
ment and anti-TGF?1 treatment led to down-regulation (Fig.
2D, left blot). As expected, the TGF?RI small molecule antag-
onist, SB431542, had no effect on pPar6, but clearly suppressed
phosphorylation of Smad2, which is a TGF?RI substrate (Fig.
2D, left blot). Analysis of EMT-6 cells expressing Par6/S345A
revealed an absence of Par6 phosphorylation in S345A-
expressing cells (Fig. 2D, right blot).
Next we analyzed EMT6 cells in 3D cultures. Expression of
Par6/S345A had a striking effect on 3D morphology, causing the
usually protrusive structures to become spherical and signifi-
cantly less protrusive (Fig. 2E). In contrast, structures formed by
cells expressing Par6/wt maintained a protrusive phenotype.
Similar observations were made in MDA-MB-231 cells (Fig.
S5C). Furthermore, a significant proportion of the cells that
appeared at the periphery of a small central lumen in EMT-6
Par6/345A 3D structures regained junctional ZO-1 staining
(44% ? 7, n ? 6) compared to cytoplasmic ZO-1 in the controls
(Fig. 3Ai). Analysis of F-actin (Fig. 3Aii) further revealed that
and lamellipodial-like protrusions (Fig. 3Aii, arrows) consistent
with their mesenchymal character. These structures were strik-
ingly absent in Par6/S345A-expressing cells. The general mor-
phology of the Par6/S345A structures, the appearance of junc-
tional ZO-1, and the loss of protrusive behavior (Figs. 2E and
3A) suggest a reversion to a phenotype that displays aspects of
epithelial polarity, albeit not the fully polarized morphology of
non-transformed epithelium (note the low and cytoplasmic
E-cadherin expression in Par6 S345A structures; Fig. 3Ai).
Finally, to investigate pathways downstream of phospho-Par6,
we knocked down Smurf1, an ubiquitin ligase effector of the
phenotype of EMT6 cells via the Par6 pathway. (A and B) EMT6 cells were
grown in Matrigel for 5 days and continuously treated with an anti-TGF?1 Ab
structures are shown in A, while quantification of protrusive structures
formed in control or ?TGF?1-treated cultures is shown in B. (C) Characteriza-
tion of a Par6S345P (pPar6) Ab in NMuMG cells. Lysates from parental cells or
cells expressing Flag-tagged wt or Par6/S345A were subjected to IP with Abs
to Par6 or Flag and immunoblotted for pPar6. A pPar6 band was readily
in NMuMG parental (P) cells after total Par6 IP (Left), but co-precipitated with
TGF? receptor I (TGF?RI), in which case TGF?1 (500 pM, 1.5 h) stimulated Par6
were detected. (D) Analysis of endogenous pPar6 in EMT6 cells. Lysates from
EMT6 cells subjected to irrelevant Ab IP (Ir Ab) or a Par6 IP were then blotted
for pPar6. pPar6 that was present in untreated cells was enhanced by TGF?1
treatment and was reduced by neutralizing TGF? Ab (?TGF?1), but not by 10
?M of the type I kinase inhibitor SB431542 (SB). Lysates were also blotted for
pSmad2, which revealed inhibition of autocrine activation by both the neu-
tralizing Ab and SB431542. In the Right, phosphorylation of Par6 in wt or
Par6/S345A-expressing cells was analyzed by immunoblotting. (E) Bright field
in Matrigel. Quantification of the percent of structures with protrusive mor-
phology (mean ?/? SD from three independent experiments shown at the
bottom of each image; see SI Text for details) shows that Par6/S345A expres-
sion significantly suppresses (P ? 0.005) the percent of protrusive structures
formed by EMT-6 cells. (Scale bar in A, 50 ?m; E, 100 ?m.)
Autocrine TGF? signaling regulates the protrusive, mesenchymal
and confocal microscopy analysis of EMT-6 3D structures. (i) Selected areas of
right with the ZO-1 (yellow), E-cadherin (green), and the merged image (Dapi
merge) stains. Both Vector and Par6/wt structures showed only cytoplasmic
those formed by S345A cells. (ii) F-actin staining showed distinctive filopodial
and lamellipodial-like protrusions (white arrows) in Vector and Par6/wt struc-
tures that were absent in Par6/S345A structures. (B and C) Smurf1 knockdown
empty vector (control), or expression of shRNA to GFP (shGFP) or Smurf1
3D cultures (bright field images, Bottom). Note suppression of protrusive
structures by Smurf1 knockdown that is quantitated in C. (D and E) SB431542
treatment of EMT-6 3D cultures does not interfere with protrusive structures.
EMT6 cells grown in 3D cultures were treated with DMSO or the indicated
concentrations of SB431542 continuously for 11 days. Quantitation of protru-
sive structures is shown in E. (Scale bar in A, 16 ?m; B and D, 100 ?m.)
Par6 phosphorylation mediates morphologic EMT via Smurf1. (A) IF
www.pnas.org?cgi?doi?10.1073?pnas.0906796106 Viloria-Petit et al.
pathway (5) (Fig. 3B). Loss of Smurf1 expression significantly
inhibited the formation of protrusive structures in both EMT-6
(Fig. 3 B and C) and MDA-MB-231 3D cultures (Fig. S5D).
Moreover, treatment with SB431542, which blocks Smad signal-
ing, did not block the protrusive morphology of EMT-6 3D
structures (Fig. 3 D and E), indicating that TGF?’s role in
promoting EMT-6 protrusiveness is not mediated by the Smad
pathway. Taken together, our results demonstrate that the
TGF?-Par6 pathway is activated by autocrine TGF? in trans-
formed cells and promotes morphological EMT and invasive
behavior via the Smurf1 effector.
To analyze metastatic behavior in vivo, we used an orthotopic
mouse model. For this purpose, we surgically implanted EMT-6
cells into the right fourth inguinal mammary fat pad of female
BALB/c mice and allowed tumors to develop for 3–5 weeks.
Animals were then killed and lungs examined for metastases
(Fig. 4A). Although we observed some variability in tumor take,
in neither case did we see significant effects on the growth rate
(Fig. S6 A–C) of tumors derived from any of the lines tested.
However, expression of Par6/S345A in EMT-6 mammary tumors
significantly reduced the incidence and number of macroscopic
lung metastasis (Fig. 4 B–D and another cohort in Fig. S6D). In
contrast, Par6/wt either had no effect or, in a highly expressing
clonal line, increased the number of lung metastases (Fig. 4
B–D). To investigate the mechanism by which Par6/S345A
inhibited the metastatic spreading of EMT-6 tumors, we next
analyzed tumor tissue obtained from 7–14 day old tumors
(average size of 0.2 cm3) using standard IHC and IF. First, using
the S345 pPar6 antibody we determined the status of Par6
phosphorylation in Vector, Par6/wt, and S345A tumors. We
observed that pPar6 immunostaining of either mouse EMT-6
syngeneic tumors (Fig. 5A) or human MDA-MB-231 tumor
xenografts (Fig. S5E), showed a similar granular cytoplasmic
pattern in areas or ‘‘patches’’ of cells that were dispersed
throughout the tumor. We confirmed the specificity of the signal
by preincubating the primary antibody with a S345 phosphopep-
tide, which blocked staining (Fig. 5A). Positive areas of staining
were numerous in EMT-6 Vector (control) tumors and while the
frequency of staining was similar in Par6/wt expressing tumors,
the intensity of staining was increased. It is unclear why pPar6
activation is sporadic in vivo, but this may be due to mechanisms
that restrict TGF? signaling to the complex, or negative regu-
latory pathways, such as phosphatases. In stark contrast, pPar6
staining was virtually absent in tumors expressing Par6/S345A
(Fig. 5A). Thus, Par6/S345A acts as a dominant negative to
suppress Par6 phosphorylation in vivo.
IF analyses of Par6/S345A tumors further revealed morpho-
logical differences. When we used Flag immunostaining on
paraffin sections to specifically identify tumor cells expressing
Flag-tagged Par6, round structures formed by Flag-positive cells
were readily apparent in Par6/S345A-expressing tumors, but not
and ZO-1 in tumor cryosections further showed that these
localized to the membrane (compare vector and Par6/S345A
tumors in Fig. S7B). These structures were not seen in Par6/wt-
expressing tumors. Taken together, these results suggest that
interfering with Par6 signaling suppresses metastasis and pro-
motes a partial rescue of the epithelial phenotype.
To explore the Par6 pathway in human breast cancer we used
a tissue microarray (TMA) from tumors belonging to a cohort
that includes patients with hereditary breast cancer. Our previ-
ous work revealed high TGF? levels in BRCA1-associated
(A) Diagrammatic representation of the orthotopic model used. m.f.p.: mam-
mary fat pad. (B) Relative basal expression of Flag-tagged Par6 in cells im-
planted into the m.f.p. of BALB/c mice as determined by Flag IP followed by
Par6 IB. (C) A significant reduction in the number of macroscopic lung metas-
tases was observed in both S345A#3 and S345A#6 tumor bearing mice when
compared to mice implanted with either Vector control or Par6 wt tumors.
Each bar shade represents an independent experiment. Plotted values corre-
spond to the mean ? SD for n ? 6–10 (mice per group). The wt#6 clone was
lung samples. Metastases appear as white/light yellow spots on the darker
yellow background. The incidence of lung metastasis for experiments (C) is
metastasis as compared to both Vector and Par6/wt tumors (note that similar
results were obtained from another independent experiment shown in Fig.
S6D). (Scale bar, 5 mm.)
Par6/S345A suppresses lung metastasis of EMT-6 mammary tumors.
of pPar6 in EMT-6 tumors. Tissue derived from syngeneic mouse tumor trans-
plants of the indicated cell lines was stained with pPar6 Ab. Negative control
sections were stained in the presence of excess antigen. Note that pPar6
immunoreactivity was present in the cytoplasm and was absent in Par6/S345A
expressing tumors. (Scale bar, 100 ?M.) (B) pPar6 immunostaining in human
breast cancer TMAs. Examples of positive and negative staining, as indicated,
are shown at lower (left images; scale bar, 500 ?m) and at higher magnifica-
tion (right images, scale bar, 50 ?m). pPar6 immunoreactivity was primarily
was considered for pPar6 scoring (see SI Text for details).
Immunostaining of pPar6 in mouse and human tumors. (A) Analysis
Viloria-Petit et al.PNAS ?
August 18, 2009 ?
vol. 106 ?
no. 33 ?
tumors in this cohort (21). Therefore, we analyzed pPar6 levels
in this TMA. Using the Allred method (22), pPar6 positivity
(Score ? 5; Fig. 5B) was detected in 42% (122/289) of the breast
tumors analyzed (Table S2). Since BRCA1-associated tumors are
highly enriched in the basal subtype, which is associated with
EMT and mesenchymal characteristics (23, 24), we further
analyzed pPar6 in the BRCA1 group. We observed that pPar6
positivity was associated with a subgroup of BRCA1-associated
breast tumors that displayed basal features; that is, tumors
expressing basal rather than luminal cytokeratins. Basal tumors
are poorly differentiated invasive carcinomas characterized by,
among other features, the expression of cytokeratin (CK) 5/6, 14
and 17, and vimentin (25). We found that basal CK5- and
CK14-positive tumors in the BRCA1-associated group were
more likely to be pPar6 positive than basal cytokeratin negative
tumors (53.1% vs. 21.1%; P ? 0.039 and 75.0% vs. 28.6%; P ?
0.007, respectively) (Table 1). We also observed that tumors
positive for vimentin were more likely to be positive for pPar6,
although this correlation was of borderline significance (P ?
0.069) (Table 1). Associations between pPar6 and basal markers
seem to be restricted to the BRCA1 group, since an additional
exploratory study did not detect similar associations in the other
showed a clear tendency to reduced overall survival (OS) in
pPar6-positive as compared to pPar6-negative patients (P ?
0.067), particularly after 10 years follow-up (Fig. S8). Taken
together, these results support the notion that Par6 phosphor-
ylation is associated with more invasive, metastatic tumors.
Our studies have revealed an important role for polarity signal-
ing in mediating loss of cellular polarity and morphologic
transformation of mammary cells. Using two 3D culture models
of mammary cells that we characterize in this study: NMuMG
and EMT-6, as well as previously established 3D culture models
tumor progression, we demonstrated that the TGF?-Par6 path-
way promotes protrusiveness in transformed cells (EMT-6 and
MDA-MB-231) that is dependent on the Par6 effector, Smurf1.
These findings correlated with in vivo metastatic potential and
revealed an in vivo role for polarity signaling in regulating
epithelial plasticity within tumors. These studies highlight the
relevance of the TGF?-Par6 pathway to breast cancer invasion
and metastasis in an appropriate in vitro and in vivo tissue-like
In NMuMG normal (immortalized) mouse mammary cells,
activation of the Par6 pathway interferes with the formation of
polarized acinar structures and promotes the formation of
lumenless structures. These results are in contrast to the recently
reported finding that Par6 overexpression in MCF-10A human
mammary cells does not disrupt acinar morphogenesis (7).
Nevertheless, we observed that Par6 overexpression also inter-
feres with acini formation and polarization of 3D structures
formed by EpH4 mouse mammary cells. This suggests that the
effect of Par6 overexpression on 3D morphogenesis may differ
among cell types, but is not specific to NMuMG cells. Since the
effect of Par6 on polarity is a consequence of its ability to
regulate TJ dynamics (5), one possible explanation for this
of Crumbs3 expression, which associates with the Par6 complex
to mediate TJ formation (27). This suggests that our NMuMG
3D model might be more suitable for EMT studies.
Apart from the direct effects on polarity, two other major
cellular outcomes of TGF?-Par6 signaling were unveiled by our
studies of 3D structures, namely its role in TGF?-induced cell
death, and in specifically promoting morphological changes
associated with EMT, independent from the gene expression
reprogramming induced by the Smad pathway. This report,
therefore, describes a regulatory role of the TGF?-Par6 polarity
pathway in mammary cell survival/apoptosis. Since activation of
the Par6 pathway causes loss of polarity, while its blockade by the
Par6/S345A mutant maintains polarized structures, our results
suggest that loss of polarity is a prerequisite for activation of the
TGF?-induced pro-apoptotic cascade. While a detailed molec-
ular understanding of the mechanisms underlying Par6-
dependent regulation of apoptosis is beyond the scope of the
present study, one previous report has linked ?4 integrin-
dependent polarity with resistance to apoptosis in 3D structures
(16), and the Par6 polarity complex (via aPKC) has been shown
to be required for ErbB2/HER-2 dependent survival (28). It will
be interesting to test whether modulation of integrin expression
or signaling by the Par6/pathway also mediates TGF?-induced
The pro-apoptotic and EMT-promoting functions of the
TGF?-Par6 pathway in normal and transformed cells, respec-
tively, are consistent with the similar functions of TGF? itself
during tumor progression (29). However, it is of particular note
that the Par6 polarity pathway predominantly acts on the TJ
complex and is critical for the execution of a program that leads
to the cytoskeletal rearrangements required for the morpholog-
ical events associated with EMT, independent of the canonical
TGF?-Smad pathway and the modulation of AJ. This is a
challenging concept, taking into account that the loss of E-
players in the process of EMT (3). Nevertheless, the importance
of the TJ as a target of the TGF?-Par6 pathway is supported by
our analysis of EMT-6 cells, where blockade of Par6 phosphor-
ylation induced morphological mesenchymal-to-epithelial rever-
Table 1. Association between pPar6 status and basal cytokeratins/Vimentin in BRCA1 tumors
pPar6 Positive (5–8) pPar6 Negative (1–4)
71.4 1025 0.007
64.3 15 270.069
*P-values from Fisher’s Exact Test.
†Positive score (? 4) validated by ALB and FPO (Bane AL, et al. (2007) Am J Surg Pathol 31 (1):121–128).
‡Positive score (? 4) validated by ALB and FPO.
www.pnas.org?cgi?doi?10.1073?pnas.0906796106 Viloria-Petit et al.
sion and rescues junctional/apical ZO1 ex vivo, without an Download full-text
obvious rescue of E-cadherin expression/localization to AJ.
Moreover, in orthotopically implanted EMT-6 mouse mammary
tumors, blockade of the Par6 pathway induced the formation of
tumor cell derived, ZO-1-positive structures; and significantly
reduced the incidence and number of lung metastasis. The good
correlation between our in vitro and in vivo results suggests that
the EMT-6 3D model might be a reliable in vitro alternative to
study the role of TGF? signaling in breast cancer progression,
including testing of signal transduction inhibitors, in an appro-
priate, tissue-like context.
Our finding of a positive association between the activation
status of the Par6 pathway and basal cytokeratins in BRCA1-
associated tumors suggests that this pathway could be implicated
in the aggressive characteristics commonly associated with the
basal subgroup of BRCA1-associated tumors. It is also possible
that TGF? expression and thus activation of the Par6 pathway
may be a molecular event associated with the loss of the BRCA1
gene, which itself favors a ‘‘commitment’’ to the basal subtype.
This hypothesis is further supported by the high TGF? expres-
sion (21) and high incidence of basal carcinoma (30) observed in
BRCA1-associated tumors. Furthermore, loss of BRCA1 has
been associated with a stem cell-like phenotype (31) and TGF?
signaling in mammary tumor cells is associated with both
mesenchymal and stem cell-like properties (32, 33). Detailed
molecular analysis and further multivariate studies with human
tumor samples are necessary to support this hypothesis.
Materials and Methods
Matrigel 3D Cultures and Immunofluorescence. Cells were maintained under
standard culture conditions (see SI Text). Subconfluent monolayers were
trypsinized, washed, resuspended in assay media, and plated as single cell
suspensions on 100% growth factor reduced Matrigel (BD BioSciences) using
the overlay method (28). Assay media contained 2% Matrigel added to
supplemented mammary media (PromoCell) for NMuMG and MDA-MB-231
cells, or to DMEM plus 2% FBS, 0.5 ?g/mL hydrocortisone, 10 ?g/mL insulin,
and standard antibiotics, for EMT-6 cells. Stable cell lines were cultured with
G418. Medium was changed every 3 days. TGF?1 was added after mature
structures were formed. Mouse anti-TGF?1 or the SB431542 inhibitor was
added at the time of plating, and was replenished every 2 days. IF was
performed following a standard methodology (28). All IFs were analyzed
using a confocal microscope provided with a spinning disk camera (Leica
Microsystems). Images were captured and processed using Volocity software
(Improvision Inc.). Final images were slices from Z-stacks unless otherwise
indicated. For methodologies used to quantify acini-like and protrusive 3D
structures see SI Text.
EMT-6 Model of Breast Cancer Metastasis. EMT-6 cells were implanted in the
mammary gland of BALB/c mice following a reported methodology (17).
Tumors were allowed to growth to 1.7 mm3, at which point mice were
euthanized and lungs were harvested for analysis of macrometastases (see SI
Text for details).
pPar6 Determination in Mammary Tumors. pPar6 expression was assessed in
formalin-fixed, paraffin-embedded tissue obtained from mouse or human
mammary tumors using an antibody generated during this study. Immuno-
staining was performed using standard antigen retrieval IHC techniques and
a final pPar6 Ab concentration of 2–10 ?g/mL. Scoring of pPar6 expression in
human TMAs was performed independently by ALB and FPO using the Allred
For a list of reagents and sources, methodological details and statistical
analysis see SI Text.
ACKNOWLEDGMENTS. We thank Shan Man (Sunnybrook Health Sciences
Centre, Toronto, Canada) for advice on orthotopic surgeries, Susie Tjan
(Mount Sinai Hospital, Toronto, Canada) for immunostaining of TMAs, Eliza-
beth Balogun (Department of Molecular and Cellular Biology, University of
Troy Ketela (Department of Molecular Genetics, University of Toronto, Can-
ada) for help with the design of shRNA viruses, and Drs. Etienne Labbe ´ and
Liliana Attisano (Department of Medical Biophysics, University of Toronto,
Breast Cancer Research Alliance Grant 74692 (to J.L.W.); National Cancer
Institute, National Institutes of Health Grant RFA CA-95–011; postdoctoral
research awards from Canadian Institutes of Health Research (A.M.V.-P.) and
Fondation Pour La Recherche Medicale, France (L.D.); and through coopera-
tive agreements with members of the Breast Cancer Family Registry.
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