Endothelial Cell Migration and Vascular Endothelial Growth Factor
Expression Are the Result of Loss of Breast Tissue Polarity
1Paraic A. Kenny,
2and Nancy Boudreau
1Surgical Research Laboratory, Department of Surgery, University of California at San Francisco, San Francisco, California;
Division, Lawrence Berkeley National Laboratory, Berkeley, California; and
and Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York
3Department of Developmental and Molecular Biology,
Recruiting a new blood supply is a rate-limiting step in tumor
progression. In a three-dimensional model of breast carcino-
genesis, disorganized, proliferative transformed breast epi-
thelial cells express significantly higher expression of
angiogenic genes compared with their polarized, growth-
arrested nonmalignant counterparts. Elevated vascular endo-
thelial growth factor (VEGF) secretion by malignant cells
enhanced recruitment of endothelial cells (EC) in heterotypic
cocultures. Significantly, phenotypic reversion of malignant
cells via reexpression of HoxD10, which is lost in malignant
progression, significantly attenuated VEGF expression in a
hypoxia-inducible factor 1A–independent fashion and re-
duced EC migration. This was due primarily to restoring
polarity: forced proliferation of polarized, nonmalignant cells
did not induce VEGF expression and EC recruitment, whereas
disrupting the architecture of growth-arrested, reverted cells
did. These data show that disrupting cytostructure activates
the angiogenic switch even in the absence of proliferation
and/or hypoxia and restoring organization of malignant
clusters reduces VEGF expression and EC activation to levels
found in quiescent nonmalignant epithelium. These data
confirm the importance of tissue architecture and polarity in
malignant progression. [Cancer Res 2009;69(16):6721–9]
It is well established that solid tumors require angiogenesis to
survive (1, 2) and changes in the breast tumor microenvironment
promote formation of new blood vessels (3–5) with high angiogenic
potential linked to poor prognosis (4, 6).
In breast tumors, several key angiogenic factors have been
identified, the most prominent being vascular endothelial growth
factor (VEGF), which acts on adjacent endothelial cells (EC)
through the VEGF receptor 2, initiating growth, migration, and
invasion into adjacent tumor stroma. Recent clinical studies have
shown that function-blocking antibodies against VEGFsignificantly
impair tumor progression (7).
In the earliest stages of malignant breast cancer [i.e., ductal
carcinoma in situ (DCIS)], VEGF may be induced by the increased
metabolic demand and hypoxia (8), as low oxygen tension
stabilizes hypoxia-inducible factor 1a (HIF1a), which binds to,
and activates, transcription of the VEGF promoter (see ref. 9 for
review). Yet, paradoxically, most cells suspend mRNA translation
and protein synthesis when faced with either nutrient depletion or
hypoxia (10, 11) and suggests that additional changes in the breast
tumor microenvironment may facilitate expression of angiogenic
Sustained signaling through a6h4 integrin and elevated
phosphatidylinositol 3-kinase (PI3K) levels also enhance transla-
tion of VEGF mRNA in carcinoma cells (12), and even in the
absence of hypoxia, both PI3K and mitogen-activated protein
kinase (MAPK) signaling are elevated in breast tumors and can
increase the transcription and secretion of VEGF independent of
HIF1a (13, 14). Expression of the transcription factor HoxB7 also
increases VEGF expression in both breast and other epithelial
tumor cells; similarly, h-catenin activation can induce the
expression of angiogenic factors, including VEGF (15, 16). Thus,
a variety of changes in breast tumor cells conspire to activate
expression of angiogenic factors.
Identifying features of the normal breast microenvironment,
which collectively normalize MAPK and PI3K, sequester h-catenin,
and/or attenuate a6h4 expression, may prove to be key in
inhibiting the angiogenic switch. Several studies by our group
have shown that breast tissue architecture plays a fundamental role
in mediating each of these pathways (17–20). Further, despite many
genetic defects, malignant breast epithelial cells, which exhibit a
proliferative, unpolarized morphology when grown in three-
dimensional cultures (21), can be reverted to a polarized, acinar
morphology and growth arrested by agents targeting the MAPK
and PI3K pathways (17–19, 22). We have also shown that restoring
expression of a key morphoregulatory gene, HoxD10, lost in
tumorigenic breast epithelial cells reverts tumorigenic breast cells
to a growth-arrested and organized phenotype (23). Whether the
paralogous HoxA10 gene, which is also lacking in some breast
tumors (24), also stabilizes breast tissue architecture is not known.
Nonetheless, considering the remarkable dominance of the
reverted tumor cell phenotype over the tumor cell genotype, we
hypothesized that proper organization of breast epithelial cells may
suppress expression of angiogenic factors, thus implicating loss of
tissue organization as a key activator of the angiogenic switch in
Materials and Methods
Cell culture. Immortalized human dermal microvascular EC HMEC-1
[a gift from T. Lawley (25), Emory University, Atlanta, GA], the human
breast epithelial cell line MDA-MB-231 (American Type Culture Collection),
and epithelial cell lines HMT-3522 T4-2, S-1, and S1 epidermal growth factor
receptor (EGFR) were grown and maintained in two- and three-dimensional
cultures as previously described (18, 19).
Note: M. Bissell and N. Boudreau contributed equally to this work.
Requests for reprints: Nancy Boudreau, SurgicalResearch Laboratory, Department
of Surgery, University of California at San Francisco, Box 1302, San Francisco, CA 94143.
Phone: 415-206-6951; Fax: 415-206-6997; E-mail: firstname.lastname@example.org.
I2009 American Association for Cancer Research.
Cancer Res 2009; 69: (16). August 15, 2009
Epithelial/endothelial cocultures and migration assay. Cell migra-
tion assays were performed using a modification of procedures previously
described (23, 26). Briefly, 6.5-mm Transwell chambers (8-Am pore; Corning)
were coated with 10 Ag/mL of type I collagen (Cohesion Tech), and 5 ? 104
serum-starved HMEC-1 cells were plated in 300 AL of fibroblast basal
medium (FBM; Lonza) containing 0.5% bovine serum albumin (BSA).
For coculture experiments, T4-2, S1, or MDA-MB-231 cells were cultured
using polymerized laminin-rich extracellular matrix (lrECM; Matrigel, BD
Biosciences) for 72 and 16 h before assays, the medium was changed to
serum-free FBM, and Transwell inserts with EC were added to the upper
chamber. After 4 h at 37jC, ECs on the upper surface were removed and
HMEC-1 cells that migrated onto the bottom of the membrane were stained
with Diff-Quick (VWR Scientific Products) and five fields in each well were
counted by phase-contrast microscopy (magnification, ?20). When
indicated, HMEC-1 cells were preincubated for 30 min with 0.5 Ag/mL of
control IgG or a monoclonal antibody against anti-human VEGF (R&D
Retroviral vectors and transduction. The human 1,100-bp HoxD10
cDNA (Genbank accession no. X59373) was cloned into the EcoRI site of the
pBABE retroviral vector (Clontech). T4-2 and T4-2 Rac1L61 cells were
transduced with control plasmid (pBABE) or pBD10 and selected in
0.5 Ag/mL puromycin (Sigma) as previously described (23).
Reverse transcription/PCR. Cells grown in three-dimensional cultures
were released from lrECM using previously described procedures (18, 23),
and cell pellets were resuspended in RNA lysis buffer and extracted using
the RNeasy Mini isolation kit (Qiagen). One microgram of total RNA was
reverse transcribed using Moloney murine leukemia virus reverse
transcriptase (Qiagen), and one twenty-fifth of this reaction was linearly
amplified for 30 cycles (VEGF and HoxD10) of denaturation (30 s at 95jC),
annealing (30 s at 51jC for VEGF; 58jC for HoxD10), and extension (30 s at
72jC) in a thermal cycler (PTC-200 Peltier thermal cycler, M.J. Research).
The following primers were used: HoxD10, 5¶-CTGTCATGCTCCAGCT-
CAACCC-3¶ (forward) and 5¶-CTAAGAAAACGTGAGGTTGGCGGTC-3¶
(reverse); VEGF, 5¶-CGAAACCATGAACTTTCTGC-3¶ (forward) and 5¶-
CCTCAGTGGGCACACACTCC-3¶ (reverse). Total RNA was normalized
using 18S internal standards at a 1:3 ratio (Ambion).
Quantitative real-time PCR was carried out in triplicate with a 10- to
20-fold dilution of first-strand cDNA using human Taqman probes and
primers purchased as Assays-on-Demand (Applied Biosystems) for
h-glucuronidase (Gus, reference gene control) and VEGF using an ABI
Prism SDS 7000 (Applied Biosystems) according to the manufacturer’s
instructions. Data were analyzed with ABI Prism SDS 7000 companion
VEGF ELISA. For two-dimensional cultures, T4-2 and S1 cells were
plated at a density of 500,000 per well, and for three-dimensional cultures,
cells were plated at 100,000 per well on top of 200 AL of polymerized lrECM
and overlaid with 10% lrECM. Forty-eight hours later, medium was changed
to FBM + 0.5% BSA, and 24 h later, secreted VEGF was assayed in triplicate
by ELISA (DVE00, R&D Systems) according to the manufacturer’s
Angiogenesis profiling arrays. The relative expression of 84 angiogen-
esis-related genes was evaluated using the Human Angiogenesis RT2Profiler
PCR Array system (SuperArray Bioscience Corp.) according to the
manufacturer’s instructions. DNase-treated total RNA was purified from
T4-2 cells treated with either the EGFR-blocking antibody mAb225 or an
IgG control. cDNA was generated by reverse transcription from 1 Ag of total
RNA from each sample using the RT2First Strand kit and then combined
with the RT2qPCR Master Mix and added to lyophilized primer pairs in the
96-well arrays. Thermal cycling was performed in a Bio-Rad iCycler. Relative
gene expression levels were calculated using the DDCtmethod (27) with
normalization to the average expression level of five common genes
(ACTB, B2M, GAPDH, HPRT, and RPL13A).
Microarray analysis. Gene expression analysis was performed on
samples of RNA purified from S1 and T4-2 cells grown in three-dimensional
lrECM cultures in the presence of various signaling inhibitors or vehicle
controls. The Affymetrix High Throughput Array GeneChip system, with
HG-U133A chips mounted on pegs in a 96-well format, was used for the
analysis, as described (28). Data were imported into the Partek Genomics
Suite (Partek, Inc.) and normalized using RMA (29).
Immunoblot analysis. Cells were cultured in three-dimensional lrECM
for 72 h, released, and lysed in 10 mmol/L Tris-HCl (pH 7.4), 1 mol/L
sodium chloride, 1% Triton X-100, 50 mmol/L sodium fluoride, 1 mmol/L
sodium orthovanadate, and protease inhibitor cocktail. Total protein was
determined using the bicinchoninic acid assay (Pierce) and 40 Ag were
electrophoresed on SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and blocked with 5% milk. HoxD10 (E-20) polyclonal antibody
(Santa Cruz Biotechnology, Inc.), a polyclonal phospho-Akt antibody Ser473
(193H12, Cell Signaling), and a mouse monoclonal HIF1a antibody
(NB 100-105, Novus Biologicals) were used and detected with enhanced
chemiluminescence system (Amersham Biosciences). Relative protein
loading was assessed by h-actin (Ab8227, Abcam). Nuclear extracts were
isolated from T4-2 and HoxD10-reverted T4-2 cells, and electrophoretic
mobility shift assays (EMSA) were performed using procedures as
Immunofluorescence. After release from three-dimensional lrECM, cells
were smeared onto slides and fixed in cold 1:1 methanol-acetone as
previously described (23). After blocking with 10% goat serum, cells were
incubated overnight with a 1:100 dilution of antibodies against h4 integrin
(mAb1964, Chemicon) and washed with immunofluorescence buffer
followed by a 1:400 dilution of goat anti-mouse Alexa Fluor 546 IgG
(H+L) (Invitrogen), and nuclei were counterstained with 1:1,000 dilution of
4¶,6-diamidino-2-phenylindole (DAPI; Sigma). Slides were mounted in
Fluoromount G (Southern Biotechnology Associates, Inc.) and images were
collected with a Nikon Eclipse TE300 fluorescence microscope.
Ki-67 proliferation index. Proliferation was assessed by Ki-67
immunostaining using a modification of the previous method (23) with a
1:500 dilution of Ki-67 antibody (VP-K451, Vector Laboratories) overnight at
4jC followed by a 1:400 dilution of goat anti-rabbit Alexa Fluor 546 IgG
(H+L), counterstained with DAPI, and mounted in Fluoromount G.
Proliferation was determined by visually counting at least 300 DAPI-labeled
nuclei and thereafter scoring Ki-67–positive cells as a percentage of a total
Phenotypic reversion of malignant cells restores basal
expression of angiogenic factors. Malignant breast epithelial
cells (T4-2) cultured in three-dimensional lrECM exhibit a
disorganized morphology compared with their growth-arrested,
polarized nonmalignant counterparts (S1). Disrupting either h1
integrin, EGFR, MAPK, or PI3K-mediated signaling restores baso-
lateral polarity and growth arrest and reverts malignant cells to a
phenotype similar to nonmalignant cells and fails to form tumors
in vivo (18, 19, 31, 32). We used microarray analysis to determine
whether malignant T4-2 cells display enhanced expression of
angiogenic factors and whether phenotypic reversion reduces
expression of angiogenic factors. We analyzed RNA samples from
S1, T4-2, and T4-2 cells reverted with an EGFR inhibitor (AG1478;
in duplicate) and T4-2 cells reverted with an EGFR-blocking
antibody (mAb225), a h1-integrin–blocking antibody (AIIB2), or
inhibitors of MAPK/extracellular signal-regulated kinase (PD98059)
and TACE (TAPI-2). Unsupervised hierarchical clustering of all
samples (Fig. 1A) revealed significantly different transcriptional
profiles of S1, T4-2, and reverted T4-2 cells. To identify gene
expression changes associated with polarity, we selected genes
differentially expressed between disorganized T4-2 colonies,
organized nonmalignant S1 colonies, and reverted T4-2 cells (P <
0.001, t test; Fig. 1B). Differentially expressed genes that were
significantly higher in T4-2 compared with nonmalignant or
reverted T4-2 included several of the Gene Ontology angiogenesis
class (GO:0001525; Fig. 1C).
Cancer Res 2009; 69: (16). August 15, 2009
For independent confirmation, we performed an RT2Profiler
PCR array for 84 different angiogenic mediators with RNA
isolated from control (IgG) T4-2 and T4-2 reverted with a
function-blocking antibody against EGFR (mAb225; Fig. 1D).
Expression of VEGF and other angiogenic factors, including
fibroblast growth factor-2, matrix metalloproteinase-9 (MMP-9),
placental growth factor, and VEGF-C (33), was significantly
reduced in reverted cells. Thus, expression of angiogenic genes
may be restrained when cells adopt a polarized tissue
Increased VEGF production by breast tumor cells enhances
EC migration. To assess a functional role for angiogenic factors
linked to tissue polarity, we focused on VEGF. As most soluble
inhibitors that revert tumor cells also inhibit signaling in adjacent
ECs, we transfected T4-2 cells with HoxD10, which attenuates
growth and restores polarity in metastatic breast tumor MDA-MB-
231 cells (23). Although T4-2 cells express low levels of HoxD10
mRNA and protein (Fig. 2A), restoring expression of HoxD10 did
not induce any observable differences in morphology when grown
on conventional polystyrene tissue culture plates (Fig. 2B, top).
Figure 1. Reversion of the malignant phenotype suppresses angiogenic genes. A, unsupervised hierarchical clustering using 22,946 probes for 10 variations
showing distinct transcriptional profiles of S1, T4-2, and T4-2 cells treated with different drugs. B, identification of 294 genes significantly associated (P < 0.001) with
colony organization in S1, T4-2, and T4-2 cells reverted with different agents. Two hundred four were highly expressed in disorganized (T4-2) colonies and 90
were more highly expressed in organized (S1 and T4-2 reverted) colonies. C, profile of seven genes from B belonging to the GO:0001525 class of angiogenic regulators
high in malignant T4-2 and low in polarized S1 and reverted T4-2 cells. The indicated genes correspond to the following Affymetrix probes: VEGF, 210512_at;
VEGFC, 209946_at; CEACAM1, 209498_at; TNFRSF12A, 218368_s_at; ANGPTL4, 221009_s_at; NF1, 211094_s_at; and ELK3, 206127_at. D, relative fold
changes detected in angiogenesis-related genes between control (IgG) or EGFR-blocking antibody (mAb225)–treated T4-2 cells.
Polarity and Breast Tumor Angiogenesis
Cancer Res 2009; 69: (16). August 15, 2009
However, when cultured within a three-dimensional lrECM,
HoxD10-expressing T4-2 cells formed organized, growth-arrested
structures resembling their nonmalignant counterparts, as shown
by phase microscopy and immunofluorescent imaging of h4
integrin in which total levels were reduced (data not shown) and
the remaining redistributed to the basal surface (Fig. 2B, bottom).
In three-dimensional lrECM, both nonmalignant S1 and HoxD10-
reverted T4-2 cells produced significantly less VEGFcompared with
control T4-2 cells (Fig. 2C). In contrast, when cultured on
polystyrene, VEGF mRNA and protein were similar in S1, T4-2,
and HoxD10-expressing T4-2 cells (data not shown).
We developed a modified Boyden invasion assay (Fig. 2D, inset)
to quantify EC migration in response to malignant or nonmalig-
nant cells cultured in lrECM and observed that ECs migrated
significantly more in response to coculture with T4-2 cells than S1
cells (Fig. 2D). Pretreatment of ECs with VEGF-blocking antibodies
reduced EC migration to the basal levels seen in cocultures
with S1 cells (Fig. 2D), implicating VEGF as the primary mediator
of EC migration in EC/T4-2 cocultures. Notably, migration of
ECs cocultured with reverted HoxD10-expressing T4-2 cells was
significantly reduced to levels observed by blocking VEGF or
coculturing with nonmalignant cells (Fig. 2D). No differences in
EC migration were observed when cocultured with S1, T4-2, or
HoxD10-expressing T4-2 cells grown in tissue culture plastic (data
Together, these data suggest that differential expression of VEGF
by nonmalignant and tumorigenic epithelial cells is influenced by
their respective tissue architecture.
Phenotypic reversion of metastatic breast tumor cells also
suppresses VEGF expression and EC migration. We also
compared expression of VEGF in MDA-MB-231 cells, HoxD10-
expressing MDA-MB-231 cells, and MDA-MB-231 expressing the
paralogous HoxA10 (Fig. 3A, top). In contrast to HoxD10, restoring
HoxA10 did not induce organized structures in MDA-MB-231 cells
in three-dimensional lrECM, as evidenced by diffuse immunoflu-
orescence staining of h4 integrin (Fig. 3A, bottom), and growth was
Figure 2. Phenotypic reversion by HoxD10 reduces VEGF and EC migration in three-dimensional cultures. A, top, semiquantitative PCR of HoxD10 mRNA in
control or HoxD10-transfected T4-2 cells; bottom, corresponding Western blot of HoxD10 protein. B, top, phase-contrast photomicrographs of control and
HoxD10-expressing T4-2 cells on tissue culture plastic for 48 h; bottom, phase-contrast photomicrographs, immunofluorescence costaining for h4 integrin (red),
and merged image with DAPI (blue) in control T4-2 or HoxD10-expressing T4-2 cells grown in three-dimensional (3D) lrECM for 72 h. C, real-time PCR of VEGF
mRNA levels in nonmalignant S1 cells, control, or HoxD10-expressing T4-2 cells grown in three-dimensional lrECM for 72 h. **, P < 0.05 (n = 4). Corresponding
ELISA showing relative levels of VEGF protein secreted by S1, T4-2, and HoxD10-reverted T4-2 cells cultured on three-dimensional lrECM for 72 h. **, P < 0.05 (n = 5).
D, inset, schematic of coculture model of HMEC-1 on collagen-coated Transwells with either S1, T4-2, or HoxD10-expressing T4-2 cells cultured in three-dimensional
lrECM in the lower chambers. Plot shows migration of EC after 4 h of coculture with lrECM only (negative control; dots), S1 cells (white), T4-2 cells (black),
HoxD10-expressing T4-2 cells (gray), or pretreatment with neutralizing antibody against VEGF (striped). Columns, mean number of cells that migrated from five
different fields (?20) for each of four independent experiments; bars, SD. **, P < 0.05.
Cancer Res 2009; 69: (16). August 15, 2009
not significantly reduced as determined by Ki-67 labeling (Fig. 3A).
Importantly, levels of VEGF mRNA remained high in nonpolarized
control or HoxA10-expressing cells compared with polarized
HoxD10-expressing cells (Fig. 3B). EC migration during coculture
with MDA-MB-231 could be blocked by antibodies against VEGF or
by reexpression of HoxD10 but not by HoxA10 expression (Fig. 3C).
Thus, only HoxD10, which reverts MDA-MB-231 to a growth-
arrested and basally polarized phenotype, attenuates VEGF
expression and EC migration.
Sustained growth is not responsible for induction of
angiogenic activity. To evaluate whether growth arrest or reestab-
lishment of basolateral polarity was suppressing VEGF, we compared
nonmalignant S1 cells with S1 cells constitutively expressing the
EGFR (S1-EGFR) that maintained basal polarity in lrECM despite a
10-fold increase in proliferation (19). VEGF expression by proliferat-
ing, polarized S1-EGFR cells was not significantly different from
growth-arrested S1cells(Fig.4A). Moreover,therewerenosignificant
differences in EC migration in response to S1 or S1-EGFR cells
(Fig. 4B), indicating that growth arrest was not sufficient to suppress
VEGF expression or EC activation.
Reduced VEGF is not linked to reduced HIF1A expression or
activity. VEGF can be induced by HIF1a, a transcription factor
stabilized by hypoxic tumor microenvironments (34, 35). We
investigated whether reduced VEGF expression in reverted tumor
cells was accompanied by reduced HIF1a. Western blot analysis
shows that relative levels of HIF1a were unchanged in three-
dimensional cultures of control T4-2 or reverted T4-2 cells
(Fig. 4C). We also assessed HIF1a binding to consensus sites in
target DNA using EMSA and observed similar levels of bound
HIF1a in both control and reverted T4-2 cells (Fig. 4C). Thus,
reduced VEGF in reverted, polarized tumor cells is not linked to
changes in HIF1a expression or activity.
Disruption of polarity is sufficient to increase VEGF
expression and EC migration. Given that neither changes in
HIF1a nor continued growth in polarized cells induced VEGF
expression, we exploited our previous findings that proliferation
and polarity are mediated by distinct signaling pathways (22) to
establish whether polarity alone regulated VEGF. A dominant-
active Rac mutant (Rac1L61) was introduced into HoxD10-
expressing T4-2 cells (Fig. 5A), which disrupted acinar polarity, as
indicated by diffuse h4 integrin staining, without significantly
affecting proliferation (Fig. 5B). The loss of polarity was
accompanied by increased production of VEGF to levels found in
nonpolarized control T4-2 cells (Fig. 5C) and significantly increased
EC migration compared with polarized T4-2 cells expressing only
HoxD10-expressing cells (Fig. 5D).
Together, these data emphasize that maintaining tissue structure
is critical for suppressing VEGF expression and subsequent
activation of adjacent EC by malignant cells in the breast tumor
microenvironment (Fig. 6).
Figure 3. HoxD10, but not HoxA10, down-regulates VEGF and EC migration in metastatic MDA-MB-231 cells. A, top, Western blot of HoxA10 in control, HoxD10, or
HoxA10-expressing MDA-MB-231 cells and HeLa cells. Corresponding phase-contrast photomicrographs of control, HoxA10, or HoxD10-expressing MDA-MB-231
cells in three-dimensional lrECM for 72 h and immunofluorescence staining for h4 integrin. Proliferation is indicated by the % Ki-67 index expressed as the mean F SE.
**, P < 0.05. B, real-time PCR of VEGF mRNA in control (black), HoxD10-expressing (gray), and HoxA10-expressing (speckled) MDA-MB-231 cells cultured in
three-dimensional lrECM for 72 h. **, P < 0.05 (n = 4). C, migration of EC after 4 h of coculture with lrECM only (negative control; dots), control MDA-MB-231 (black),
and HoxD10-expressing (gray) and HoxA10-expressing MDA-MB-231 cells (speckled) or EC pretreated with a neutralizing antibody against VEGF and cocultured
with control (dark gray stripes) or HoxA10-expressing MDA-MB-231 cells (light gray stripes), all cultured in three-dimensional lrECM. Columns, mean of migrated
EC in a total of five different fields (?20) for each of three independent experiments; bars, SD. **, P < 0.05.
Polarity and Breast Tumor Angiogenesis
Cancer Res 2009; 69: (16). August 15, 2009
In the present study, we show that production of angiogenic
factors and EC activation is primarily controlled by breast
epithelial cell polarity and tissue architecture. In conventional
(two-dimensional) tissue culture, nonmalignant and malignant
breast epithelial cells do not differentially secrete VEGF or recruit
ECs. However, once provided with three-dimensional lrECM,
nonmalignant cells organize into quiescent, polarized acini,
produce low levels of VEGF, and limit migration of cocultured
ECs. Disorganized, malignant epithelial cells, on the other hand,
produce more VEGF and significantly increase recruitment of ECs,
mimicking the stromal angiogenic response of malignant breast
tumors in vivo. Significantly, phenotypically reverting malignant
epithelial cells to polarized acini via reintroduction of the HoxD10
tumor suppressor or by treatment with various signaling
inhibitors (20) reduces VEGF and migration of cocultured ECs
to levels observed with nonmalignant cells. Hence, differential
expression of VEGF by nonmalignant and malignant breast
epithelial cells is evident only when cells undergo characteristic
morphologic changes in three-dimensional lrECM. Moreover,
reduced VEGF transcription in tumor cells repolarized by HoxD10
is unlikely a direct effect, as HoxD10 did not influence VEGF
production when cells were unable to reorganize into polarized
structures in two-dimensional cultures, although it remains
possible that accompanying morphologic changes in three-
dimensional cultures unmask binding sites within the VEGF
promoter. Still, these findings indicate that activation of the
angiogenic switch is not simply due to genetic changes within the
tumor cells but rather linked to how cells sense their architecture
and interact with their microenvironment.
Importantly, we also show that in tumor cells, which character-
istically exhibit increased growth and metabolic demand compared
with nonmalignant cells, restoring normal tissue architecture is
essential for attenuating their angiogenic potential. Indeed,
a 10-fold increase in proliferation of nonmalignant cells was not
sufficient to explain resistance to chemotherapeutic agents (17)
and is also not sufficient to activate VEGF expression when
growing cells maintain a polarized architecture.
Many studies have established that increased proliferation,
metabolic demand, and hypoxia in tumor cells stabilize HIF1a
Figure 4. Forced proliferation of polarized breast epithelial cells does not induce VEGF expression or EC migration. A, top, phase-contrast photomicrographs of
polarized S1 or S1-EGFR cells cultured in three-dimensional lrECM for 72 h; bottom, corresponding immunofluorescence staining for EGFR (red) and merged image
with DAPI staining (blue). B, left, real-time PCR VEGF mRNA levels in S1 (white) or S1-EGFR cells (horizontal stripes) cultured in three-dimensional lrECM for 72 h,
expressed as percentage of VEGF mRNA levels by malignant T4-2 cells in the same conditions (n = 4). Right, corresponding EC migration over 4-h coculture
with either S1 (white) or S1-EGFR cells (horizontal stripes) in three-dimensional lrECM in the lower chamber. Columns, mean of migrated EC from five different fields
(?20) for each of three independent experiments (n = 4). C, top, Western blot of HIF1a in control T4-2 or HoxD10-expressing T4-2 cells cultured in three-dimensional
lrECM for 72 h. Total protein loading is shown by corresponding levels of h-actin. Bottom, EMSA with nuclear extracts from control or HoxD10-expressing T4-2
cells incubated with a HIF1a consensus oligonucleotide. Top arrow, position of the protein complexes; bottom arrow, free, unbound probe.
Cancer Res 2009; 69: (16). August 15, 2009
and drive VEGFexpression (3, 6, 34–36). Blocking HIF1a expression/
activity reduces VEGF expression, and HIF1a-null mice display
defective angiogenesis and fail to support tumor growth
(8, 35). However, our results show that neither differences in HIF1a
tumor cells and implicate other pathways and/or transcriptional
mediators in regulating the angiogenic switch in this system.
In a mouse model of pancreatic cancer, hypoxia-independent
activation of the angiogenic switch occurs via increased MMP-9
activity, which liberates bioactive VEGF trapped within the tumor
matrix (37). Despite the fact that MMP-9 is highly expressed in
disorganized T4-2 cells and reduced in reverted T4-2,4elevated
secretion of VEGF is reflected by increased VEGF mRNA. Whether
reduced VEGF transcription by repolarized breast tumor cells
increases inhibitory factors (38) and/or attenuates other positive
inducers of VEGF requires further investigation.
Previous studies also showed that PI3K signaling induces VEGF
(13, 39), and our current study suggests that the Rac1 branch of this
pathway directly drives VEGF expression. We reported that PI3K
inhibition phenotypically reverts tumorigenic breast epithelial cells
and is accompanied by down-regulation of both the Akt and Rac1
effector pathways (22). However, although attenuation of Akt
reduces proliferation, it did not restore a polarized phenotype.
Instead, suppression of Rac1 activity was necessary for reestab-
lishing an organized polarized phenotype, and selective reactiva-
tion of Rac1 disrupts polarity without reinitiating growth when Akt
remained blocked (22). Moreover, whereas activation of Rac1 in
Figure 5. Rac1-mediated disruption of polarity restores VEGF expression and EC migration. A, left, levels of HoxD10 in control, HoxD10-expressing, or T4-2 cells
expressing both HoxD10 and dominant-active Rac1; right, Western blot for GTP-loaded Rac 1 (top) in corresponding cells. Bottom right, total protein determined
by reprobing with h-actin. B, top, phase-contrast microscopy of T4-2, HoxD10-expressing T4-2, or T4-2 cells expressing both HoxD10 and Rac1L61 cultured in
three-dimensional lrECM for 72 h; middle, corresponding immunofluorescence staining for h4 integrin (red); bottom, merged images for h4 integrin (red) and nuclear
DAPI (blue). Ki-67 labeling index was assessed after 72 h in three-dimensional lrECM; columns, mean for 10 independent measurements; bars, SE. C, real-time
PCR showing VEGF mRNA in control T4-2 (black), T4-2 cells expressing HoxD10 (gray), and T4-2 cells expressing HoxD10 and Rac1L61 (checkered) after
72 h in three-dimensional lrECM. **, P < 0.05 (n = 4). Corresponding ELISA showing VEGF protein secreted by each cell type. **, P < 0.05 (n = 3). D, relative migration
of EC over 4 h of coculture with lrECM only (negative control; dots), control T4-2 cells (black), HoxD10-expressing T4-2 cells (gray), and T4-2 cells expressing
HoxD10 and Rac1L61 (checkered), cultured for 72 h in three-dimensional lrECM. Columns, mean of five different fields (?20) for each of three independent
experiments. **, P < 0.05.
4A. Beliveau and M.J. Bissell, submitted for publication.
Polarity and Breast Tumor Angiogenesis
Cancer Res 2009; 69: (16). August 15, 2009
nonmalignant breast epithelial cells normally induces apoptosis, in
breast tumor epithelial cells lacking scribble, Rac activation
promotes tumorigenesis and loss of cell polarity (40). In the
present study, we show that reactivation of Rac1 and disruption of
polarity restore VEGF expression and induce EC migration despite
the fact that growth remains suppressed via low phospho-Akt levels
(latter data not shown).
Active Rac1 directly binds and phosphorylates signal
transducer and activator of transcription 3 (STAT3; ref. 41),
which in turn forms SP1/STAT3 complexes that bind the VEGF
promoter (42) and the SP1 site within the ?86 to ?66 region of
the VEGF promoter is linked to HIF1a-independent transcrip-
tion of VEGF (10, 39, 43). Phosphorylated STAT3 can also bind
to HIF1a and prevent its degradation (44). Although we
observed an increase in phospho-STAT3 in Rac1-overexpressing
cells, reversion of malignant cells by HoxD10 did not reduce
phospho-STAT3 (data not shown) and HIF1a protein levels
were similar in both tumorigenic and HoxD10-reverted tumor
cells. Thus, binding of STAT3 to either SP1 or HIF and VEGF
expression may be context dependent as previously suggested
by investigations of pathways impinging on VEGF expression
(39), and our current study also emphasizes the critical role of
tissue polarity in mediating VEGF expression in breast tumor
It is noteworthy that inhibiting EGFRs represses VEGF in both
HIF-dependent and HIF-independent manner, with the latter
attributed to reduced phosphorylation and binding of SP1 to the
VEGF promoter (43). Several EGFR inhibitors, including those used
in this study, not only reduce VEGF expression and inhibit breast
tumor angiogenesis in vivo but also phenotypically revert tumor
cells to a polarized morphology in culture (19, 43, 45, 46). Finally,
mice lacking LKB1, a tumor suppressor that directs cell polarity
and, when mutated, produces epithelial cancers, show markedly
increased VEGF (47, 48).
The present study addresses breast tumor epithelial organization
and VEGF production, but it is important to note that other
components of the tumor microenvironment, including fibroblasts
and macrophages, are also rich sources of angiogenic factors. In
DCIS where newly formed capillaries seem immediately adjacent to
the basal surface of the breast epithelium (5), the basement
membrane is largely intact but most epithelial cells have lost their
characteristic polarized morphology and display attenuated
expression of the HoxD10 tumor suppressor (3, 23). As macrophage
recruitment typically occurs in later stages of malignant progres-
sion accompanied by basement membrane degradation (49), it is
likely that the loss of epithelial cell polarity marks an early and
critical step in activation of the angiogenic switch.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Received 10/23/08; revised 5/7/09; accepted 5/26/09; published OnlineFirst 8/4/09.
Grant support: California Breast Cancer Research Program Award 101B-0157
(N. Boudreau); U.S. Department of Energy, OBER Office of Biological and
Environmental Research (DE-AC02-05CH1123), a Distinguished Fellow Award, and
Low Dose Radiation Program and the Office of Health and Environmental Research,
Health Effects Division (03-76SF00098; M. Bissell); National Cancer Institute awards
R01CA064786, R01CA057621, U54CA126552, and U54CA112970; and U.S. Department
of Defense grants W81XWH0810736 and W81XWH0510338.
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.
Figure 6. Polarity of breast epithelial cells suppresses
VEGF and EC migration. Schematic summarizing the
effect of tissue organization on VEGF expression in
nonmalignant, malignant, or malignant breast epithelial
cells repolarized with HoxD10.
Cancer Res 2009; 69: (16). August 15, 2009
Polarity and Breast Tumor Angiogenesis Download full-text
Cancer Res 2009; 69: (16). August 15, 2009
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