The HOXB7 protein renders breast cancer cells
resistant to tamoxifen through activation of the
Kideok Jina,1, Xiangjun Kongb,1, Tariq Shahc, Marie-France Penetc, Flonne Wildesc, Dennis C. Sgroid, Xiao-Jun Mae,
Yi Huanga,2, Anne Kallioniemif, Goran Landbergg, Ivan Biecheh, Xinyan Wua, Peter E. Lobiei, Nancy E. Davidsona,2,
Zaver M. Bhujwallaa,c, Tao Zhub,3, and Saraswati Sukumara,3
aBreast Cancer Program, Department of Oncology, andcRussell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University
School of Medicine, Baltimore, MD 21231-1000;bHefei National Laboratory for Physical Sciences, Hefei, Anhui 230027, People’s Republic of China;
dDepartment of Pathology, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114;eAviaraDx, Carlsbad, CA 92010;fLaboratory of
Cancer Genetics, University of Tampere, FIN-33014, Finland;gBreakthrough Breast Cancer Research Unit, School of Cancer and Enabling Sciences, University of
Manchester, Paterson Institute for Cancer Research, Manchester, M20 4BX, United Kingdom;hInstitut Curie, Hospital René Huguenin, F-92210, St-Cloud,
France; andiCancer Science Institute of Singapore and Department of Pharmacology, National University of Singapore, Singapore 119077
Edited by Kornelia Polyak, Dana-Farber Cancer Institute, Boston, MA, and accepted by the Editorial Board May 25, 2011 (received for review January 27, 2011)
Multiple factors including long-term treatment with tamoxifen are
involved in the development of selective estrogen receptor (ER)
modulator resistance in ERα-positive breast cancer. Many under-
lying molecular events that confer resistance are known but a uni-
fying theme is yet to be revealed. In this report, we provide
evidence that HOXB7 overexpression renders MCF-7 cells resistant
to tamoxifen via cross-talk between receptor tyrosine kinases and
ERα signaling. HOXB7 is an ERα-responsive gene. Extended treat-
ment of MCF-7 cells with tamoxifen resulted in progressively in-
creasing levels of HOXB7 expression, along with EGFR and EGFR
ligands. Up-regulation of EGFR occurs through direct binding of
HOXB7 to the EGFR promoter, enhancing transcriptional activity.
Finally, higher expression levels of HOXB7 in the tumor significantly
correlated with poorer disease-free survival in ERα-positive patients
with breast cancer on adjuvant tamoxifen monotherapy. These
studies suggest that HOXB7 acts as a key regulator, orchestrating
a major group of target molecules in the oncogenic hierarchy. Func-
tional antagonism of HOXB7 could circumvent tamoxifen resistance.
(ERα)-positive breast cancer, the selective estrogen-receptor
modulator (SERM), tamoxifen (TAM) represents a major ad-
juvant treatment in clinical practice. Many of the breast tumors
that initially respond to the tamoxifen therapy eventually de-
velop resistance and recur (1). Among the patients with breast
cancer with acquired resistance, only 20% of patients who pro-
gress on tamoxifen respond to the selective ER down-regulator,
fulvestrant, or to aromatase inhibitors even if ERα expression
is maintained and regulates tumor proliferation (2–4). Thus, far,
studies of tamoxifen resistance have revealed that increased lev-
els of ErbB/HER family members can directly alter the cellular
response to tamoxifen.
Homeobox genes are regulatory genes encoding nuclear pro-
teins that act as transcription factors during normal development
and differentiation (5). One of these, HOXB7, is involved in
a variety of developmental processes, including hematopoietic
differentiation and lymphoid and mammary gland development.
The role of HOX genes in breast cancer development is largely
unexplored. Recently, we identified HOXB7 through microarray
analysis as one of the genes whose expression was significantly
elevated in both the primary cancer and distant metastasis (6).
HOXB7 overexpression promoted cell proliferation in SKBR3
breast cancer cells that acquired the ability to grow as robustly
vascularized xenografts in immunodeficient mice (7). In culture,
HOXB7 transformed mammary epithelial cells, MCF10A, and
promoted epithelial/mesenchymal transition and invasion in a va-
riety of cell lines through activation of the RHO/RAC pathway (6).
Here, we present evidence that HOXB7 overexpression in
ER+breast cancer cells confers TAM resistance through in-
creased expression and signaling of EGFR. By studying ER+,
TAM-resistant cells, and MCF-7 cells over time as they acquired
n postmenopausal women with early-stage estrogen receptor-α
TAM resistance, we show that elevation of HOXB7 expression
might be one of the key steps in the acquisition and maintenance
of SERM resistance in breast cancer. Therefore, HOXB7 could
serve as a critical regulator in the transition of breast cancer cells
to estrogen independence, TAM resistance, and acquisition of an
aggressive phenotype. Further, HOXB7 might be an attractive
target for breast cancer therapeutics.
HOXB7 Expression Promotes Breast Tumorigenesis. Breast cancer
cells, MCF-7, are estrogen dependent for growth in vitro and
in vivo and are susceptible to the cytostatic/cytotoxic effects of
TAM. Stable expression of a HOXB7 expression vector in MCF-
7 cells (three clones: MCF-7-B7 1, 2, and 3) enabled the cells to
proliferate much faster than the vector control cells (MCF-7-
Vec) in monolayer cultures and significantly enhanced colony
formation (Fig. 1 A and B). Magnetic resonance imaging (MRI)
analysis of invasion of cells through the extracellular matrix
revealed that MCF-7-B7 cells, but not MCF-7 cells were highly
invasive in vitro (8) and were significantly hypervascular in vivo
(Fig. 1 C and D) (9) without any significant change in perme-
ability. Consistent with these observations, transplanted to the
athymic nude mice s.c. in the presence of exogenous estrogen
supplementation, MCF-7-B7 cells formed faster growing and
larger tumors compared with the MCF-7-Vec cells (Fig. 1E).
Tumors formed by MCF-7-Vec cells were grossly well defined
and loosely attached to surrounding tissue whereas MCF-7-B7
cells grew as highly invasive tumors firmly attached to sur-
rounding tissues, infiltrating the underlying skeletal muscle and
fat tissue (Fig. S1B). Thus, HOXB7 overexpression promoted
invasive and aggressive growth of MCF-7-B7 cells.
One of the hallmarks of cancer is self-sufficiency in growth
signals (10). A reduced need for estrogen by ER+cells is often
linked to their resistance to TAM treatment (11). HOXB7 over-
expression in MCF-7 cells resulted in a much reduced depen-
dence on nutrients. Although MCF10A-B7 cells proliferated in
low growth factor-supplemented medium and MCF-7-B7 cells
Author contributions: K.J., T.Z., and S.S. designed research; K.J., X.K., T.S., M.-F.P., F.W.,
D.C.S., X.-J.M., Y.H., A.K., G.L., I.B., Z.M.B., and T.Z. performed research; X.W. and P.E.L.
contributed new reagents/analytic tools; K.J., T.Z., and S.S. wrote the paper; and N.E.D.
provided critical input in preparing the manuscript.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. K.P. is a guest editor invited by the Editorial
1K.J. and X.K. contributed equally to this work.
2Present address: University of Pittsburgh Cancer Institute, 5150 Centre Avenue, Suite 500,
Pittsburgh, PA, 15232.
3To whom correspondence may be addressed. E-mail: email@example.com or firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 21, 2012
| vol. 109
| no. 8www.pnas.org/cgi/doi/10.1073/pnas.1018859108
survived (Fig. S1 C and E). In vivo, even in the absence of exog-
enous estrogen supplementation, MCF-7-B7 cells formed rapidly
growing tumors in athymic nude mice (Fig. 1F), whereas MCF-7-
Vec cells did not form palpable tumors. Thus, HOXB7 over-
expression enabled MCF-7 cells to largely circumvent the need
for exogenous estrogen for growth.
HOXB7 in the Acquisition of Anti-Estrogen Resistance. Prolonged
of receptor tyrosine kinases such as EGFR/HER2, which, together
with activation of ER-dependent gene transcription and aberrant
growth/apoptosis, leads to endocrine resistance (12). In addition,
epithelial–mesenchymal transition (EMT) has been observed in
in MCF10A-HOXB7 cells (6). Because of the striking similarity in
phenotype between prolonged endocrine therapy-primed TAM-
resistant models and our HOXB7-overexpressing cells, we ex-
plored the hypothesis that HOXB7 is a mediator of anti-estrogen
resistance. We therefore derived MCF-7 cell lines exposed to ei-
ther vehicle or TAM (0.1 μM or 1 μM) for >12 mo (designated
MCF-7-TMR1 or MCF-7-TMR2, respectively). MCF-7-TMR1
cells exhibited significant resistance to TAM treatment as de-
termined by colony formation assay (Fig. S2D). Long-term TAM
treatment caused elevated expression of EGFR and activated
MAPK and ERα, but no significant change of total ERα. In-
terestingly, we found that HOXB7 was up-regulated in MCF-7-
TMR cells (Fig. 2A and Fig. S2 A and B). In addition, over time
(0, 2, 4, and 6 mo) MCF-7 cells treated with 0.1 μM TAM showed
progressively increasing levels of HOXB7 expression, accompa-
nied by concomitant increase in expression of EGFR (Fig. S2A).
TAM resistance through EGFR expression, we depleted HOXB7
expression with HOXB7 siRNA in MCF-7-TMR cells. Re-
markably, abrogation of HOXB7 expression by siRNA reversed
each of the observed molecular events in the MCF-7-TMR cells
and direct evidence of a role for HOXB7 was sought in an un-
manipulated, TAM-resistant breast cancer cell line, BT474 (Fig.
2B). Here, reduction of endogenous HOXB7 expression using
HOXB7 siRNAs was sufficient to reduce the expression levels of
EGFR and P-MAPK with regained sensitivity to TAM (Fig. 2C).
WhetherHOXB7 overexpression isa molecular feature sharedby
other anti-estrogen resistance models is an interesting question.
Factors contributing to SERM resistance have been previously
studied using at least two well-known model systems: first, an
in vitro, long-term estrogen deprivation (LTED) model, MCF-7-
LTED (14), and second, an in vivo, long-term TAM-treated xe-
nograft model, MCF-7-TAMLT (15). We therefore examined
expression of HOXB7 and EGFR in cell lysates of these two
models along with our MCF-7-TMR model. In line with our
previous observations, HOXB7 and EGFR expression was ele-
vated in both models (Fig. S2C). Abrogation of the EGFR-
dependent pathway is apparently critical for HOXB7 siRNA’s
elicited effect, because an EGFR-specific inhibitor, gefitinib,
dramatically converted TAM to a potent antagonist in MCF-7-
TMR and BT474 cells (Fig. 2D). Thus, HOXB7 might be an
important drug target, whose functional antagonism impinges on
the EGFR pathway important to TAM resistance.
HOXB7 Promotes Tamoxifen Resistance. Receptor tyrosine ki-
nases (RTKs) are major mediators of the signaling network that
transmits extracellular signals into cells and controls cellular
differentiation and proliferation (16). The ErbB/HER receptors
including HER1/EGFR, HER2/Neu, HER3, HER4, and their
cognate ligands are involved in the pathogenesis of different
types of carcinomas including breast cancer (17). Because we
5h 12h 18h 36h 48h
Tumor volume (cm3)
07 111417 21
Tumor volume (cm3)
512 18 36 48
and B) (A) Immunoblot analysis of HOXB7 expression in MCF-7-
Vec and MCF-7-B7 cells (three clones: MCF-7-B7 1, 2, and 3) and
growth curve of MCF-7-Vec and MCF-7-B7 cells grown in
monolayer culture and (B) soft agar colony formation by MCF-
7-Vec and MCF-7-B7 cells. (C) T1-weighted1H MR imaging of
MCF-7-B7 cells, and MCF-7-Vec cells visualizing the degrada-
tion of ECM over a period as indicated. (D) Representative 3D
reconstructed images of vascular volume maps (row 1), per-
meability-surface area product (row 2), and combined vascular
volume and permeability-surface area product (row 3) for
MCF-7 parental (column 1), MCF-7-Vec (column 2), and MCF-7-
B7 (column 3) tumors in mice. (E) Tumor growth curves of MCF-
7-Vec and MCF-7-B7 cells implanted s.c. in athymic mice in the
presence of an exogenous slow release, estrogen implant and
(F) in the absence of exogenous estrogen supplementation.
Effect of HOXB7 expression in breast cancer cells. (A
Jin et al.PNAS
| February 21, 2012
| vol. 109
| no. 8
found that expression of HER1/EGFR was up-regulated in
HOXB7-expressing breast cancer cells, we investigated the
mechanism by which HOXB7 might regulate HER1/EGFR.
Stable overexpression of HOXB7 in MCF-7 cells resulted in
increased expression of EGFR and elevated tyrosine phosphor-
ylation at the kinase domain (Y1068) of EGFR, and, as pre-
viously observed (17), the major downstream effector, p44/42
MAPK, was activated as well (Fig. 3A).
Next, we tested the biological properties of MCF-7-B7 cells. In
culture, TAM treatment of MCF-7-Vec cells led to increased
apoptosis and decreased cell viability, whereas MCF-7-B7 cells
were minimally sensitive (Fig. S3 A and B). The TAM-resistant
property of MCF-7-B7 cells was also verified by the estrogen-
stimulated ERE-luc reporter activity in these cells (Fig. S3C).
Further, MCF-7-B7 cells formed colonies in soft agar in the
presence of tamoxifen at frequencies similar to those of vehicle-
treated cells (Fig. S3D). Notably, unlike vector control cells, MCF-
7-B7 xenografts in immunodeficient mice failed to respond to the
inhibitory effects of TAM (Fig. 3B and Fig. S3 E and F). Thus, by
all these growth criteria, the behavior of the MCF-7-B7 cells was
very similar to that of the MCF-7-TMR cells described above.
Consistent with our predictions of HOXB7–EGFR crosstalk,
abrogation of EGFR activity in MCF-7-B7 cells by the EGFR-
specific inhibitor, gefitinib, significantly resensitized them to
TAM treatment (Fig. 3C). To determine whether the HOXB7–
EGFR crosstalk is rapid and direct, we treated estrogen-deprived
MCF-7-B7 cells with 1 μM TAM for 0–30 min, which led to
elevated phosphorylation of EGFR, activation of p44 MAPK,
and ER phosphorylation at Ser-118 (Fig. S3G). These observa-
tions suggest that under these treatment conditions, nongenomic
ERα action occurred to activate EGFR signaling. This effect was
sustained for longer periods of time: MCF-7-B7 cells grown in
estrogen-deprived conditions exposed to TAM for 24 h also led
to the expression of significantly higher levels of active forms of
EGFR, increased p44/42 MAPK activity, and ERα phosphory-
lation in contrast to control MCF-7-Vec cells (Fig. S3H).
MCF-7-TMR cells are known to use an autocrine signaling
pathway with EGFR ligands through activated EGFR and MAPK
for growth (18–20). Elevated activation of EGFR as a result of
HOXB7 overexpression prompted us to examine the possible
overproduction of known EGFR ligands (i.e., TGF-α, ARG, and
HB-EGF). Indeed, increased mRNA expression of the three
EGFRligands wasobservedin MCF-7-B7cells(Fig.3D),MCF-7-
with increased mRNA levels, a significant increase of TGF-α
and HB-EGF expression was detected at the protein level in
MCF10A-B7 cells (Fig. S3K). The elevated expression of TGF-α
and HB-EGF was significantly abrogated by the pharmacological
inhibition of EGFR activity using the EGFR-kinase inhibitor,
AG1478 and gefitinib. This result suggests the possible existence
of a positive feedback mechanism for the synergistic activation of
the EGFR pathway as a result of HOXB7 expression in MCF10A
cells (Fig. S3 K and L). We also found increased expression of
TGF-α and HB-EGF upon overexpression of exogenous HOXB7
in ER-negative breast cancer cell lines, SKBR3, MDA-MB-231,
and MDA-MB-435. Conversely, the expression level of these
ligands was decreased by depletion of HOXB7 by siRNA (Fig.
S3M). However, it is likely that an alternative pathway in MCF-7-
B7 cells, such as overexpressed HOXB7 acting through ERα,
regulates the overproduction of autocrine/paracrine EGFR li-
gands. To further examine possible cross-talk between EGFR and
ER signaling as a result of HOXB7 overexpression, the ER down-
regulator, fulvestrant (ICI 182780; Faslodex), was used. Fulves-
trant treatment modestly reduced increases in EGFR and p44/42
MAPK activity upon HOXB7 overexpression. Consistent with
reduced EGFR activity, a significant reduction in levels of the
autocrine/paracrine EGFR ligands (ARG and TGF-α) was ob-
served in fulvestrant-treated MCF-7-B7 cells (Fig. S3N). A posi-
tive role for ERα in these effects of HOXB7 was indicated by
partial reduction in levels of ARG and TGF-α upon ERα-specific
siRNA transfection of MCF-7-B7 cells (Fig. 3D). Thus, ER-
dependent pathways might be responsible for elevated levels of
the autocrine/paracrine EGFR ligands in MCF-7-B7 cells, which
may result in elevated EGFR signaling.
To examine whether the interaction between HOXB7 and
EGFR is direct, ChIP assays were performed. A single putative
HOXB7-binding site was identified in the 800-bp EGFR pro-
moter (Fig. 3E and Fig. S3O). To provide supportive evidence,
luciferase reporter constructs containing serial deletions of the
EGFR promoter (21) were cotransfected into MCF-7 and
MCF10A cells, along with the HOXB7 expression plasmids. As
shown in Fig. 3F, both pER6-luc–containing nucleotides −771 to
−16 and pER8-luc–containing nucleotides −481 to −16 were ac-
tivated 2.5- to 3-fold by HOXB7, whereas pER9-luc–containing
nucleotides −342 to −16, −297 to −16, and −150 to −16 were
activated at much lower levels in MCF-7 cells (Fig. 3F). These
results were consistent with the ChIP assay data localizing se-
quences necessary for response of EGFR to HOXB7.
In line with these findings, we also found an up-regulation of
EGFR in HOXB7 overexpressing MCF10A and HBL-100 cells
(Fig. S3P). EGFR and p44/42 MAPK activities were also reduced
by HOXB7 siRNA expression in ER-negative MDA-MB-435
and MDA-MB-468 cells (Fig. S3Q). Moreover, transfection of
HOXB7 siRNA into MCF-7-HOXB7 cells was also able to re-
sensitize them to TAM treatment (Fig. S3R). These results sug-
gest that HOXB7 might be an attractive anticancer target in both
ER+and ER−tumors. Collectively, we have provided data de-
monstratingoverexpression ofreceptors andligands,activation of
downstream effectors, direct binding of HOXB7 to EGFR pro-
moter, and reversal of these effects by siRNA directed against
HOXB7. Thus, multiple lines of evidence strongly support the
between EGFR and ERα signaling that is critical for maintaining
resistance to TAM in MCF-7 cells.
HOXB7 Expression Is Regulated by Estrogen. What might be an un-
derlying mechanism of HOXB7 overexpression in TAM-resistant
breast cancer cells? On the basis of a previous finding in a Swiss
cohort (22), in collaboration with the same investigators (A.K.),
we tested whether HOXB7 overexpression in breast tumors could
be traced to gene amplification by performing FISH on tissue
microarrays of primary breast tumors. Unlike the 10% incidence
observed previously (23), only 2 of 172 Swedish cases and 1 of 108
Finnish cases showed amplification of HOXB7 (Fig. S4A). On the
basis of these data, we concluded that gene amplification is not
Cont B7 Cont B7 : siRNA
- + + -
- + +
+ + -
-+ + --
of Phospho-EGFR (Y1068), EGFR, Phospho-MAPK, MAPK, Phospho-ERα
(S118), ERα, and HOXB7 expression in MCF-7 cells treated long term with
vehicle or 0.1 μM TAM (TMR1) or 1 μM TAM (TMR2). (B) Immunoblot analysis
of EGFR, Phospho-MAPK, MAPK, and HOXB7 expression in MCF-7-TMR cells
and BT474 transfected with either scrambled sequence siRNA or HOXB7-
specific siRNA. (C) Soft agar colony formation in BT474 cells and MCF-7-TMR
cells transfected with either scrambled sequence siRNA or HOXB7-specific
siRNA treated with vehicle or 1 μM TAM. (D) Soft agar colony formation by
BT474 cells and MCF-7-TMR cells treated with 1 μM gefitinib and 1 μM TAM.
HOXB7 promotes acquired TAM resistance. (A) Immunoblot analysis
| www.pnas.org/cgi/doi/10.1073/pnas.1018859108Jin et al.
a major mechanism underlying elevated expression of HOXB7 in
breast cancer, including tamoxifen-resistant cancer.
Couldtamoxifen exposure, actingthrough ERα,bethecause of
mRNAlevels increasedupon treatmentofMCF-7andT47Dcells
with tamoxifen in medium containing 10% FBS (i.e., abundance
of estrogen) (Fig. 4A and Fig. S4 B and E). Also, HOXB7 mRNA
levels increased in estrogen-deprived growth conditions (5%
charcoal-stripped serum) (Fig. S4C). Interestingly, HOXB7 ex-
pression was significantly reduced with estradiol stimulation
under the same conditions (Fig. 4A). The down-regulation of
HOXB7 expression by estradiol is abrogated by tamoxifen (Fig.
S4D). To further test this concept, we overexpressed ERα in ER-
negative cell lines such as MDA-MB-231, MDA-MB-453, and
MCF10A.We observed that HOXB7 expression was dramatically
decreased in the ERα-overexpressing cells (Fig. 4 B and C and
Fig. S4F). These findings support our hypothesis that HOXB7
expression is regulated by estrogen and this action is dependent
on the presence of a functional ERα.
Prognostic Significance of High HOXB7 Expression in Breast Cancer.
On the basis of the ability of overexpressed HOXB7 to lead to
in primary tumors may predict subsequent TAM resistance. We
examined HOXB7 mRNA levels of hormone receptor-positive
primary breast cancers in two different small sets of patients (n =
59 and n = 72) treated with adjuvant TAM monotherapy (24) and
209 patients with no adjuvant therapy after surgery by real-time
PCR. In these discovery sets of tissues, the association between
a higher expression level of HOXB7 and a poorer relapse-free
survivalrate wasstatistically significant(Fig.5AandBandFig.S5
A and B), suggesting that elevated expression levels of HOXB7
were associated with the development of tamoxifen resistance in
patients with breast cancer. To investigate whether there is a
correlation between HOXB7 and EGFR expression in clinical
cohorts, HOXB7 and EGFR mRNA levels were examined by
qRT-PCR in 57 samples from patients with breast cancer and 48
breast cancer cell lines. We also investigated 127 samples from
patients with breast cancer by immunohistochemistry (IHC), us-
ing a breast cancer tissue microarray. Interestingly, a correlation
between HOXB7 and EGFR was observed both at the mRNA
level (r = 0.8772, 0.8290) and under IHC detection (r = 0.6341)
with statistical significance (P < 0.0001) (Fig. 5 C and D and Fig.
S5 C, E, and F). Collectively, we have shown that elevated
HOXB7 expression under various scenarios appears to serve as
a unifying molecular hub directing the development of anti-
Molecular adaptations during acquired TAM resistance use
multiple signaling pathways that are well documented in the lit-
erature. In this paper, evidence has been presented to support the
hypothesis that a common mechanism leading to TAM resistance
is overexpression of HOXB7. We have shown the following: (i)
MCF-7 cells treated over extended periods with TAM in vitro
develop TAM resistance, which is accompanied by a parallel, el-
another two different commonly used anti-estrogen resistance
models. (ii) These molecular events are largely recapitulated in
MCF-7 cells by overexpression of a single gene, HOXB7, leading
to acquired resistance to TAM. (iii) SiRNA-mediated silencing of
HOXB7 significantly reversed many of the malignant traits and
molecular changes in both the native (BT474) and engineered
anti-estrogen resistance models. (iv) Preliminary evidence of po-
0 + 200 -1200 -800 -400
Vec B7 Vec B7
Tumor volume (mm3)
MCF-7-B7;No E2 Pellet
noblot analysis of Phospho-EGFR, EGFR, P-MAPK,
MAPK, and HOXB7 expression levels in MCF-7-Vec and
MCF-7-HOXB7 cells. (B) Tumor growth curve of MCF-7-
HOXB7 cells implanted s.c. in athymic Swiss female
mice and treated with either vehicle or TAM (83.3 μg/
d), in the absence of an exogenous estrogen supple-
ment. (C) Soft agar colony formation by MCF-7-Vec
and MCF-7-B7 cells treated with vehicle, estrogen (10
nM), or TAM (1 μM) and combination with 1 μM
gefitinib as indicated. (D) Semiquantitative RT-PCR
analysis of mRNA expression levels of TGF-α, HB-EGF,
and Amphiregulin in HOXB7-expressing MCF-7 cells
and their vector controls (Left) and Amphiregulin or
TGF-α mRNA expression in MCF-7-Vec and MCF-7-B7
cells treated with either scrambled sequence siRNA or
ERα-specific siRNA (Right). (E) Diagram representing
the HOXB7 binding sites in the EGFR promoter region.
MCF-7 cells were transfected with pcDNA3-Flag-
HOXB7 and vector plasmid, and ChIP was performed
by IP with either anti-Flag M2 antibody or control IgG.
(F) Luciferase activity of deletion/truncation constructs
of the EGFR promoter, with (solid bars) and without
(hatched bars) transfected HOXB7 plasmid, to map the
minimal region necessary for activation by HOXB7.
HOXB7 promotes TAM resistance. (A) Immu-
Jin et al. PNAS
| February 21, 2012
| vol. 109
| no. 8
tential clinical relevance was observed in a small discovery cohort
of ER+patients with breast cancer who received TAM mono-
therapy (24), where a higher level of HOXB7 in cancer spec-
imens correlated significantly with poor relapse-free survival (P =
0.049). Thus, HOXB7, frequently overexpressed in breast cancer,
appears to be a major upstream regulator of events leading to
Mechanistically, HOXB7 acts via simultaneous up-regulation
of receptor tyrosine kinase, EGFR, and EGFR ligands, each of
which was efficiently reversed by HOXB7-specific siRNA. Al-
though HOX genes have been implicated in the regulation of
several pathways involved in embryogenesis and organogenesis,
few target genes have been shown to be under their direct regu-
latory control (5). Here, we show that HOXB7 binds directly to
the EGFR promoter to induce its gene transcription. Moreover,
HOXB7 also up-regulated the expression of multiple EGFR li-
gands, presumably through cross-talk with activated ERα signal-
ing in MCF-7 cells (Fig. 3). Coexistence of an RTK and its ligands
at elevated levels presents a formidable obstacle to successful
breast cancer therapy (2, 25). It is difficult to envision that the
current target-specific (usually one target, one molecule) anti-
cancer therapeutics will efficiently eradicate heterogeneous pop-
ulations of cells, typical of breast cancer. Thus, in a preclinical
blocks multiple signals would be significantly better than single
agents or dual combinations(26). Our data implicated HOXB7as
a unique target acting upstream of important RTKs and cognate
ligands, whose functional antagonism might allow the attack of
multiple therapeutic targets simultaneously in breast cancer.
As suggested here, ERα signaling might render the cells more
dependent on HOXB7-mediated pathways such as EGFR or ERα
itself. It is worthwhile to note that preliminary data from clinical
trials (27) of gefitinib in breast cancer suggested that contrary to
predictions, patients with ER- and high EGFR-expressing tumors
showed a poor response to gefitinib. On the other hand, in two
separate studies, patients with TAM-resistant, ER+breast cancer
86%, respectively) to gefitinib. These observations are in line with
our findings (Fig. 3C). It is likely that TAM-resistant cells use ER-
dependent pathways to produce much more of EGFR-specific
These ligands bind to high-affinity EGFR in ER+cells, rendering
the cells more dependent on the EGFR pathway and therefore
patients with breast cancer treated with gefitinib and anastrozole
had a greater reduction from pretreatment values in the pro-
liferation-related Ki67 labeling index than patients treated with
might have the potential to serve as a marker indicating anti-
There are three questions that remain: First, in tamoxifen re-
sistance, HER2 and HER3 signaling pathways also play important
roles. Because neither AG1478 nor gefitinib have shown a com-
plete response to sensitizing MCF-7-HOXB7 cells to TAM treat-
ment, we should investigate whether other RTK-signaling pathway
are involved in HOXB7-mediated TAMR. Second, what connects
HOXB7 action to ER function? Some hints have surfaced during
our study of HOXB7 × HER2 transgenic mouse mammary tumor
Vehicle 0.1110 100
5% DCC-FBS 5% FBS
+ - + -
analysis of HOXB7 mRNA levels in MCF-7 cells. Cells were incubated in an
estrogen-deprived condition for 72 h [DMEM phenol red-free medium
containing 5% Dextran charcoal-stripped serum (DCC-FBS)]; cells were then
treated with 0.1–100 nM estrogen (E2); and cells were treated with 0.05–
1 μM of TAM in DMEM plus 5% FBS. (Inset) Semiquantitative RT-PCR analysis
of HOXB7 mRNA following 1 uM TAM treatment for 24 h. (B) Real-time
quantitative PCR analysis of HOXB7 mRNA levels in MDA-MB-231 and MDA-
MB-453 cells were transiently transfected with ERα expression plasmid and
vector control for 48 h. ERIN, the ERα overexpressed cell line, was used with
MCF10A as a control. (C) Immunoblot analysis of HOXB7 and ERα expression
levels in transient transfectants of MDA-MB-231-ERα.
ERα regulates HOXB7 expression. (A) Real-time quantitative PCR
P = 0.05
HOXB7 High (>=mean)
HOXB7 Low (<mean)
ER positive (N=59)
0246810 12 1416
P = 0.04
ER positive (N=209)
HOXB7 High (>=mean)
HOXB7 Low (<mean)
Disease free survival
Disease free survival
of estrogen receptor-positive node-negative patients who
received (A) TAM monotherapy (n = 59) and (B) no adjuvant
therapy after surgery (n = 209) were stratified by HOXB7
expression level. (C) Pearson’s correlation between HOXB7
and EGFR mRNA levels in samples from patients with breast
cancer (n = 57). (D) Pearson’s correlation between HOXB7
and EGFR protein levels by IHC in sample TMA from patients
with breast cancer (n = 127).
Kaplan–Meier plots of disease-free survival analysis
| www.pnas.org/cgi/doi/10.1073/pnas.1018859108Jin et al.
cells (32) that deserve attention. These tumors have elevated ex- Download full-text
pression of FoxA1. FoxA1 was reported to interact with ERα to
turn on the transcription of ERα-target genes, which include
directly or indirectly? Because we did not find ERα binding sites in
the HOXB7 promoter regions, on the basis of ERα ChIP-on-ChIP
Thus, in TAM-resistant cells, HOXB7 might orchestrate the co-
ordinated actions of ERα- and EGFR-dependent pathways in
breast tumorigenesis. Further evidence supporting this premise
was elucidated here in acquired TAM resistance models (Fig. 2).
Early effective antagonism of the ER pathway by use of an alter-
ER+patients with higher levels of HOXB7 expression. In sum-
mary, HOXB7 might be a critical regulator on top of the hierarchy
that orchestrates the process of acquisition and maintenance of
anti-estrogen resistance. Thus, HOXB7 might present a unique
drug target to reverse anti-estrogen resistance efficiently.
Materials and Methods
Cell Lines, Cell Culture, and Reagents. pcDNA3 vector or pcDNA3-Flag-HOXB7
was stably transfected into MCF10A cells or MCF-7 cells by use of Effectene
(Qiagen). MCF-7-LTED, the estrogen-hypersensitive MCF-7 subline, was
generated from MCF-7 cells by long-term culture under estrogen-deprived
conditions and thus they are called LTED cells (14) (a kind gift from Richard
Santen). LTED cells are refractory to tamoxifen but sensitive to fulvestrant
(11). MCF-7-TAMLT, long-term tamoxifen-stimulated tumors, were kindly
provided by V. Craig Jordan and were developed by retransplanting grow-
ing estradiol-dependent MCF-7 tumors into new athymic mice and treating
the mice with tamoxifen for >5 y (15). Fulvestrant and Iressa (gefitinib) were
provided by Astrazeneca.
FISH Analysis. HOXB7 copy number levels were determined by FISH on the
Tissue MicroArray (TMA) as described (36). The HOXB7-specific BAC clone
(RP11-361K8) was labeled with SpectrumOrange (Vysis) and SpectrumGreen-
labeled chromosome 17 centromere probe (Vysis) was used as a reference.
The nuclei were counterstained with DAPI. The entire tissue core was
screened, with a minimum of 50 intact nuclei scored for each specimen. A
total of 280 samples were successfully analyzed. Tumor samples containing
athreefoldorhigher increaseinthenumberofHOXB7signals comparedwith
centromere signals were considered to be amplified.
Real-Time PCR of HOXB7 Expression. HOXB7geneexpressionwasquantifiedby
Statistical Analysis. HOXB7 expression levels determined by RT-PCR were di-
chotomized into low and high groups using the median as cutoff. Kaplan–
Meier analysis and log-rank tests were performed to assess the association
twosided,anddifferenceswereconsideredstatisticallysignificantatP < 0.05.
All analyses were performed using SAS (version 9.1) and R (version 2.4.1).
ACKNOWLEDGMENTS. We thank Dr. Ben H. Park for generously providing
the ERIN cell line, Drs. Richard Santen (University of Virginia, Charlottesville,
VA) and V. Craig Jordan (Georgetown University, Washington, DC) for the
LTED and TAMLT cell lysates, and Dr. Cynthia Zahnow and Hexin Chen for
reviewing the manuscript. This work was supported by Specialized Programs
Of Research Excellence (SPORE) in Breast Cancer: National Institutes of
Health Grant P50-CA88843 (to S.S.), Susan G. Komen Postdoctoral Fellow-
ships (to T.Z. and K.J.), the National Key Scientific Program of China
(2010CB912804, 2007CB914801, and 2011CBA01103), and the National Nat-
ural Science Foundation of China (30971492 and 30725015) (to T.Z.).
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| no. 8