From the Institute of Molecular and Cell Biology and
Department of Medicine, National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore and ¶ CNRS
Unité Mixte Recherche 5578, Physiologies Energetiques
Cellulaires et Moléculaires, Université Claude Bernard,
Lyon 1, France
Received for publication, November 26, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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Increased mammary epithelial expression of the
human growth hormone (hGH) gene is associated with the acquisition of
pathological proliferation. We report here that autocrine hGH
production by human mammary carcinoma cells increased the expression
and transcriptional activity of the homeobox domain containing protein
HOXA1. Forced expression of HOXA1 in human mammary carcinoma cells
resulted in increased total cell number primarily by the promotion of
cell survival mediated by the transcriptional up-regulation of Bcl-2. HOXA1 also abrogated the apoptotic response of mammary carcinoma cells
to doxorubicin. Forced expression of HOXA1 in mammary carcinoma cells,
in a Bcl-2-dependent manner, resulted in dramatic
enhancement of anchorage-independent proliferation and colony formation
in soft agar. Finally, forced expression of HOXA1 was sufficient to
result in the oncogenic transformation of immortalized human mammary
epithelial cells with aggressive in vivo tumor formation. Herein, we have therefore provided a molecular mechanism by which autocrine hGH stimulation of human mammary epithelial cells may result
in oncogenic transformation.
Signal transduction pathways that regulate mammary epithelial cell
proliferation, differentiation, apoptosis, and transformation have been
delineated and utilized as therapeutic targets for the treatment of
mammary gland neoplasia (1, 2). Examples of such include selective
estrogen modulators (3) and herceptin (4) targeting estrogen receptor
and HER-2 pathways, respectively. However, many carcinomas of
the mammary gland neither express the estrogen receptor (5) nor
overexpress HER-2 (6) and are, therefore, not responsive to these
specific therapeutic strategies. Clinical data for the treatment of
cancer has clearly demonstrated the superiority of combinatorial
therapy over a single-agent approach (7). Delineation,
characterization. and development of therapeutic regimes targeting
novel signaling pathways involved in oncogenic transformation of
mammary epithelial cells are therefore required.
Growth hormone (GH)1 is
obligatory for normal pubertal mammary gland development (8).
Specifically, GH acts on both the mammary stromal and epithelial
components to result in ductal elongation and the differentiation of
ductal epithelia into terminal end buds (9, 10). Expression of the hGH
transgene in mice results in precocious development of the mammary
gland (11, 12) and the development of neoplasia (12). Conversely,
spontaneous or experimentally engineered functional deficiency of GH
(13-16) results in severely impaired mammary gland development and
virtual resistance to the spontaneous development of hyperplastic
alveolar nodules (13) and chemically induced mammary carcinogenesis
(14, 16). Similarly, in a primate model, hGH administration results in
marked hyperplasia of the mammary gland with an increased epithelial proliferation index (17). Thus, the somatotropic axis represents one
potential and unutilized approach for novel therapeutic approaches to
the treatment of mammary epithelial neoplasia.
The hGH gene is also expressed in epithelial cells of the normal human
mammary gland (18). Increased epithelial expression of the hGH gene is
associated with the acquisition of pathological proliferation, and the
highest level of hGH gene expression is observed in metastatic mammary
carcinoma cells (18). hGH receptor gene expression per mammary
epithelial cell remains constant throughout the process of neoplastic
progression (19), and therefore, alterations in the local concentration
of ligand are likely to be pivotal in determining the effects of hGH on
mammary epithelial cell behavior. We have recently generated a model
system to study the role of autocrine-produced hGH in mammary carcinoma
by stable transfection of either the hGH gene or a
translation-deficient hGH gene into mammary carcinoma cells (20). The
autocrine production of hGH by mammary carcinoma cells results in a
hyperproliferative state with marked synergism between trophic agents
such as insulin-like growth factor 1 (20). The increase in mammary
carcinoma cell number as a consequence of autocrine production of hGH
is a result of both increased mitogenesis and decreased apoptosis (21). Thus, autocrine production of hGH by mammary carcinoma cells may direct
mammary carcinoma cell behavior to impact on the final clinical
prognosis, and therefore, systematic analysis of the relevant
mechanistic features by which it exerts its cellular effects is required.
One major mechanism by which GH affects cellular and somatic function
is by regulating the level of specific mRNA species (22). We have
previously utilized cDNA microarray analyses to identify genes
regulated by autocrine production of hGH in human mammary carcinoma
cells (MCF-7) (23). One gene demonstrated to be up-regulated by
autocrine hGH in MCF-7 cells was the homeobox containing transcription
factor HOXA1 (23). Homeobox-containing genes are a family of genes
encoding transcription factors that possess pivotal roles in
development (24). For example, HOXA1 has been demonstrated to be
required for vertebrate hindbrain segmentation (25). Several reports
also suggest the involvement of homeobox-containing genes in the
control of cell proliferation and, when dysregulated, in oncogenesis
(26, 27). Moreover, alterations of HOX gene expression have been
detected in a variety of human tumors (26-28), including those of the
mammary gland (29, 30). Accordingly, HOXA1 has been detected in
carcinoma of the mammary gland in the mouse but not in normal mammary
tissue (31), and HOXA1 expression has also been detected in neoplastic
lesions of the human mammary gland (32).
Herein we provide a molecular mechanism by which hGH stimulation of
human mammary epithelial cells results in oncogenic transformation. hGH-regulated HOXA1 increases mammary epithelial cell number primarily by prevention of apoptotic cell death in a Bcl-2-dependent
manner, with resultant anchorage-independent growth and tumor formation in vivo. Functional antagonism of hGH, and the molecular
pathways it utilizes, will therefore constitute novel adjunct
therapeutic approaches to the treatment of mammary gland neoplasia.
Cell Culture--
The MCF-7 cell line and MCF-10A cell line were
obtained from the ATCC. MCF-7 cells and derivatives (see below) were
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. MCF-10A cells and derivatives
(see below) were cultured in Dulbecco's modified Eagle's medium/F-12
medium (Invitrogen, Carlsbad, CA) supplemented with 5% horse serum
(Invitrogen) plus 2 mM glutamine, 100 µg/ml streptomycin,
100 IU/ml penicillin, 0.25 µg/ml ampicillin B, 100 ng/ml cholera
toxin, 20 ng/ml epidermal growth factor (Upstate Biotechnology, Lake
Placid, NY), 0.5 µg/ml hydrocortisone (Calbiochem, La Jolla, CA), and
10 µg/ml insulin. Fetal bovine serum was purchased from HyClone
Laboratories (Logan, UT), and all other cell culture materials were
obtained from Sigma Chemical Co. (St. Louis, MO).
The MCF-7 cell line was stably transfected with an expression plasmid
containing the wild-type hGH gene (pMT-hGH) (33) under the control of
the metallothionein 1a promoter (designated MCF7-hGH) (20). For control
purposes the ATG start site in pMT-hGH was disabled via a mutation to
TTG generated by standard techniques (pMT-MUT) (33), and MCF-7 cells
stably transfected with this plasmid were designated MCF7-MUT. A
detailed description of the characterization of these cell lines has
been published previously (20). MCF-7 and MCF-10A cell lines were
stably transfected with an HOXA1 expression plasmid (pSG5-HOXA1) and
pcDNA3 empty vector in a ratio of 5:1 by use of Effectene
transfection reagent obtained from Qiagen (Hilden, Germany) according
to the manufacturer's instruction. pSG5-HOXA1 is the generous gift of
Dr. Vincenzo Zappavigna (Milano, Italy). Positive transfectants were
selected in 800 µg/ml G418 (Calbiochem) in the appropriate culture
medium for the respective cell lines. Individual colonies were selected
to determine the HOXA1 expression level. Cell lines were established as
MCF7-HOXA1 and MCF10A-HOXA1, respectively, by pooling five individual
colonies. Likewise, MCF-7 and MCF-10A cells, stably transfected with
vector, were also established (MCF7-VECTOR and MCF10A-VECTOR) as control.
Reverse Transcriptase-PCR--
Extraction of total RNA and the
RT-PCR assay were performed as described previously (23). To compare
the PCR products semi-quantitatively, 15-40 cycles of PCR (annealing
temperature, 55 °C) were performed to determine the linearity of the
PCR amplification, and the amplified
The sequences of the oligonucleotide primers used for RT-PCR are as
follows: HOXA1 (sense), 5'-GGGAAAGTTGGAGAGTACGGC-3';
HOXA1 (antisense), 5'-CCTCAGTGGGAGGTAGTCAG-3';
Luciferase Reporter Assay for EphAZ-r42B and Bcl-2 Promoter
Constructs--
Cells were cultured to 40-60% confluence in six-well
plates. Transient transfection was performed by use of Effectene as
described (23). Briefly, 0.2 µg of the respective luciferase
constructs (and other expression constructs as appropriate) were
transfected per well in serum-free RPMI medium for 12 h before the
medium was changed to fresh serum-free RPMI with or without 50 nM hGH and 10% FBS. After a further 24 h, cells were
washed with PBS, and luciferase assays were performed as described
previously (34). Results were normalized to the level of
Western Blot Analysis--
Cells were treated and harvested as
described. Whole cell lysates and crude nuclear extracts were prepared
according to the protocol described (23). Proteins were resolved by
SDS-polyacrylamide (12%) gel electrophoresis, transferred to a
nitrocellulose membrane, and blotted with the antibodies as indicated.
The anti- 5'-Bromo-2'-deoxyuridine Incorporation Assay--
Mitogenesis
was directly assayed by measuring the incorporation of BrdUrd as
described (21). For incorporation of BrdUrd, subconfluent cells were
pulse-labeled with 20 mM BrdUrd for 30 min, washed twice
with PBS, and fixed in cold 70% ethanol for 30 min. BrdUrd detection
was performed by use of the BrdUrd staining kit from Zymed
Laboratories Inc. (San Francisco, CA) according to the
manufacturer's instructions. A total population of over three times
300 cells was analyzed in several arbitrarily chosen microscopic fields
to determine the BrdUrd labeling index (percentage of cells
synthesizing DNA).
Determination of Anchorage-dependent and -independent
Growth of Transfected Cells--
Cells (5 × 104)
were seeded into six-well plates in monolayers in serum-free media or
in serum-free media supplemented with either 50 nM hGH or
10% FBS. After 48 h, cells were trypsinized with 0.5% trypsin,
and the cell number was determined using a hematocytometer. Cells
(5 × 104) in suspension culture were grown in 30-mm
plastic bacteriological dishes (Sterilin, Teddington, United Kingdom).
On the days indicated, cells were harvested and counted as for
monolayers. For soft agar colony formation assay, MCF-7 cell and its
derived cell lines were cultured in six-well plates first covered with
an agar layer (RPMI 1640 with 0.5% agar and 10% FBS). The middle
layer contained 5 × 103 cells in RPMI 1640 with
0.35% agar and 10% FBS with or without 50 nM hGH or Bcl-2
inhibitor. Medium was added as the top layer to prevent drying of the
agarose gels. The MCF10A-VECTOR and MCF10A-HOXA1 cell soft agar
colony formation assay were processed the same as above, except that
the medium was changed to Dulbecco's modified Eagle's medium/F-12
containing appropriate components as described, and 104
cells were seeded to the middle layer of the soft agar. The plates were
incubated for 9 days (for MCF-7 cells) or 14 days (for MCF-10A cells),
after which the cultures were inspected and photographed.
Measurement of Apoptosis--
Apoptotic cell death was measured
by fluorescent microscopic analysis of cell DNA staining patterns with
Hoechst 33258 from Sigma Chemical Co. (St Louis, MO) (35). Cells were
trypsinized with 0.5% trypsin and washed twice with serum-free medium.
The cells were then seeded to glass cover slips in six-well plates and
incubated in serum-free medium with or without 50 nM hGH, 10% FBS, doxorubicin, Bcl-2 inhibitor (Calbiochem), or
Bcl-2 antisense oligonucleotide (Calbiochem) as indicated.
After a culture period of 24 h, the cells were fixed in 4%
paraformaldehyde in PBS (pH 7.4) and stained with the karyophilic dye
Hoechst 33258 (20 µg/ml) for 10 min at room temperature. Following
washing with PBS, nuclear morphology was examined under a UV-visible
fluorescence microscope (Zeiss Axioplan). Apoptotic cells were
distinguished from viable cells by their nuclear morphology
characterized by nuclear condensation and fragmentation as well as the
higher intensity of the blue fluorescence of the nuclei. For
statistical analysis, three times 300 cells were counted in eight
random microscopic fields at ×400 magnification.
Use of Bcl-2 Antisense Oligonucleotide to Deplete Cellular
Bcl-2--
Cells were cultured for 24 h in serum-free medium and
then transfected with 800 nM oligonucleotide (Calbiochem)
for 8 h in DOTAP with a final concentration of 10 µl/ml
as previously described (33): Bcl-2 (sense),
5'-TACCGCGTGCGACCCTCT-3'; Bcl-2 (antisense), 5'-TCTCCCAGCGTGCGCCAT-3'.
Xenograft Analyses--
Severe combined immunodeficient (SCID)
4-week-old mice were purchased from the animal holding facility of the
National University of Singapore and acclimated for 7-10 days. For
subcutaneous implantation, 5 × 106 control
MCF10A-VECTOR or MCF10A-HOXA1 cells were suspended in 100 µl of PBS
or Matrigel and injected into the first mammary (axillary) fat pad of
SCID mice, which simultaneously received a 60-day release pellet
containing 0.72 mg of E2 (Innovative Research of America,
Toledo, OH). At necropsy, primary tumors and all organs were evaluated
macroscopically for the presence of tumors. Tissue samples of the
primary tumor and organs were fixed in 4% paraformaldehyde in PBS (pH
7.4) and embedded in paraffin. Tissue sections of the tumors were
stained with H&E to assess morphology. Total RNA extraction and RT-PCR
were performed by use of fresh lung and liver tissue to detect possible
metastasis of the tumors.
Statistics--
All experiments were repeated at least three to
five times. All numerical data are expressed as mean ± S.E., and
the data were analyzed using Instat 3.0 from GraphPad Software Inc.
Autocrine hGH Stimulation of Mammary Carcinoma Cells Increases
HOXA1 mRNA, Protein, and Transcriptional Activity--
By use
of cDNA microarray analysis we had previously identified
HOXA1 as an autocrine hGH-regulated gene in human mammary carcinoma cells (23). To verify the autocrine hGH-dependent up-regulation of HOXA1 mRNA observed by cDNA array
screening, we first examined the level of HOXA1 mRNA in
MCF7-MUT and MCF7-hGH cells by semi-quantitative RT-PCR. The validation
that the conditions of RT-PCR used here yielded semi-quantitative
estimates of mRNA is described under "Experimental Procedures"
and has been utilized previously by us (23, 36). One amplified fragment
of the predicted size (359 bp) appropriate for HOXA1
mRNA was detected in MCF7-MUT and MCF7-hGH cells (Fig.
1A). Autocrine production of
hGH by MCF7-hGH cells resulted in an increased level of
HOXA1 mRNA in comparison to MCF7-MUT cells. Exogenous
hGH and FBS also increased HOXA1 mRNA in MCF7-MUT cells
and potentiated the increased level of HOXA1 mRNA
observed in MCF7-hGH cells due to autocrine production of hGH. The
level of
HOXA1 cDNA transfected into MCF-7 cells results in the appearance
of two distinct protein species of 33 and 35 kDa (for example see Fig.
2B) as expected (37). To
determine if the autocrine hGH-stimulated increase in HOXA1
mRNA also resulted in increased HOXA1 protein, we examined the
level of HOXA1 protein in nuclear extracts collected from MCF7-MUT and
MCF7-hGH cells by Western blot analysis. Both the 33- and the 35-kDa
forms of HOXA1 were detected in nuclear extracts from MCF7-MUT and
MCF7-hGH cells (Fig. 1B) with increased HOXA1 protein
observed in MCF7-hGH cells. FBS also increased the level of HOXA1 in
MCF7-MUT cells and potentiated the level of HOXA1 in MCF7-hGH cells
(Fig. 1B). Exogenous hGH did not stimulate an increase in
HOXA1 protein in MCF7-MUT cells and only minimally enhanced the level
in MCF7-hGH cells.
To determine if the autocrine hGH-stimulated increase in HOXA1 protein
also resulted in increased HOXA1-mediated transcription, we examined
luciferase reporter activity from the EphA2-r42B enhancer, which contains four HOX-PBX binding sites (38). Autocrine production of
hGH by MCF7-hGH cells resulted in increased luciferase activity from
the EphA2-r42B enhancer compared with MCF7-MUT cells (Fig. 1C) indicative of increased HOXA1 transcriptional activity.
Transient transfection of HOXA1 cDNA increased HOXA1
transcriptional activity in MCF7-MUT cells and dramatically enhanced
the already greater HOXA1 transcriptional response in MCF7-hGH cells.
Transient transfection of MCF7-MUT cells with PBX1 cDNA was without
effect, whereas transient transfection of cDNA for the HOX binding
partner PBX1 dramatically increased HOXA1-mediated transcription in
MCF7-hGH cells. Transient transfection of MCF7-MUT cells with both
HOXA1 and PBX1 cDNAs did, however, result in
a robust transcriptional response through the EphA2-r42B
enhancer. Transient transfection of MCF7-hGH cells with both
PBX1 and HOXA1 cDNAs synergistically
increased HOXA1-mediated transcription above the enhancement observed
with either HOXA1 or PBX1 alone (Fig. 1C). Thus autocrine
hGH production by mammary carcinoma cells results in increased
HOXA1-mediated transcriptional activity.
Forced Expression of HOXA1 in Human Mammary Carcinoma Cells Results
in Increased Total Cell Number--
To determine the functional
consequences of the autocrine hGH-stimulated increased HOXA1 expression
in mammary carcinoma cells we generated a mammary carcinoma cell line
(MCF-7) with increased expression of HOXA1 by stable transfection of
HOXA1 cDNA (termed MCF7-HOXA1). Forced expression of
HOXA1 mRNA in MCF7-HOXA1 cells compared with vector
transfected (MCF7-VECTOR) cells was verified by RT-PCR analysis (Fig.
2A), and MCF7-HOXA1 also demonstrated higher levels of HOXA1
protein compared with MCF7-VECTOR cells (Fig. 2B). Analysis
of HOXA1 transcriptional activity by use of the EphA2-r42B
enhancer demonstrated increased HOXA1-mediated transcriptional activity
in MCF7-HOXA1 cells compared with MCF7-VECTOR cells under serum-free
conditions or when stimulated with exogenous hGH or FBS (Fig.
2C). Transient transfection of cDNA for the HOXA1 binding partner PBX1 in MCF7-VECTOR cells did not alter the level of
HOXA1-mediated transcriptional activity under serum-free conditions or
exogenous hGH stimulation and only slightly enhanced HOXA1-mediated transcriptional activity of MCF7-VECTOR cells in serum. In contrast, transient transfection of PBX1 cDNA in MCF7-HOXA1 cells
synergistically amplified under all conditions, the increased
HOXA1-mediated transcription as a result of forced expression of HOXA1
(Fig. 2C).
We therefore examined the effect of forced expression of HOXA1 in
mammary carcinoma cells on total cell number. MCF7-VECTOR and
MCF7-HOXA1 cells were plated in identical number under serum-free conditions and with either 50 nM exogenous hGH or 10% FBS,
and the cell number was determined after 48 h. Exogenous hGH
stimulated an increase in MCF7-VECTOR cell number as did FBS (although
to a greater extent). MCF7-HOXA1 cells cultured in serum-free media increased in number dramatically more than MCF7-VECTOR cells (Fig. 3A). Exogenous hGH and FBS
also increased cell number in MCF7-HOXA1 above that observed in
serum-free conditions but similar to the percentage increases observed
with hGH and FBS stimulation of MCF7-VECTOR cells. In any case, forced
expression of HOXA1 in mammary carcinoma cells resulted in a
significant increase in cell number. Increased cell number is achieved
by either increased proliferation or decreased apoptotic cell death,
and we therefore proceeded to determine the relative contribution of
these processes to the observed increase in cell number as a
consequence of forced expression of HOXA1.
The D family of cyclins is pivotal to initiate progression through the
G1 phase of the cell cycle (39). CyclinD1 is the predominant member of this family expressed in mammary gland (40), and
we have previously demonstrated its requirement for autocrine hGH-stimulated mammary carcinoma cell cycle progression (36). We
therefore examined the level of cyclin D1 protein in MCF7-VECTOR compared with MCF7-HOXA1 cells. The level of cyclin D1 protein was
increased in MCF7-HOXA1 compared with MCF7-VECTOR cells (Fig. 3D) under serum-free conditions. Both exogenous hGH and FBS
increased cyclinD1 protein in MCF7-VECTOR cells and accentuated the
increase in cyclinD1 observed upon forced expression of HOXA1 in
MCF7-HOXA1 cells (Fig. 3D). We consequently also examined
the expression of the cdk inhibitor p21waf1/cip1 in
MCF7-VECTOR and MCF7-HOXA1 cells. MCF7-HOXA1 cells exhibited a marked
decrease in p21waf1/cip1 protein in comparison to MCF7-VECTOR
cells (Fig. 3D). We next examined the protein level of the
cyclin-dependent kinase inhibitor p27Kip1. Decreased expression of p27Kip1 is
associated with G1/S phase transition (41). The level of p27Kip1 was not altered between MCF7-VECTOR and MCF7-HOXA1
cells (Fig. 3D) under the conditions studied. Equal loading
of the cell extracts was verified by reprobing the stripped membrane
for
We therefore examined the effect of forced expression of HOXA1 on a
variety of proteins involved in the apoptotic process. The level of
Bax, Bak, Bcl-xL, Fas, and FADD did not differ between MCF7-VECTOR and
MCF7-HOXA1 cells under serum-free conditions nor with stimulation by
either exogenous hGH or FBS (Fig. 3E). The level of p53 did
not differ between MCF7-VECTOR and MCF7-HOXA1 cells in serum-free media
nor by stimulation with exogenous hGH but was dramatically reduced in
both cell lines in the presence of FBS. Interestingly, however, the
level of Bcl-2 was dramatically increased in MCF7-HOXA1 cells compared
with MCF7-VECTOR cells in serum-free media, and the level of Bcl-2 was
even further increased in MCF7-HOXA1 cells in the presence of FBS.
Apoptotic cell death was also dramatically reduced in MCF7-HOXA1
cells compared with MCF7-VECTOR cells in serum-free conditions (Fig.
3C). Exogenous hGH slightly reduced apoptotic cell death
in both cell lines. FBS functioned as a powerful survival stimulus for
both cell lines, although apoptosis was still less in MCF7-HOXA1
cells compared with MCF7-VECTOR cells (Fig. 3C).
HOXA1 Expression in Human Mammary Carcinoma Cells Prevents
Apoptotic Cell Death in a Bcl-2-dependent
Manner--
Forced expression of HOXA1 in mammary carcinoma cells
resulted in decreased apoptotic cell death associated with a specific increase in Bcl-2 protein. To determine if HOXA1 regulated
Bcl-2 at the transcriptional level, we first examined the
effect of forced expression of HOXA1 in mammary carcinoma cells on the
level of Bcl-2 mRNA. Increased Bcl-2 mRNA
was observed in MCF7-HOXA1 in comparison to MCF7-VECTOR cells in
serum-free media (Fig. 4A). FBS stimulation of MCF7-VECTOR cells also increased Bcl-2
mRNA in comparison to the serum-free state, and FBS enhanced the
level of Bcl-2 mRNA in MCF7-HOXA1 cells. Examination of
Bcl-2 P1 promoter activity demonstrated that the effect of
forced expression of HOXA1 on Bcl-2 mRNA expression was
mediated at the transcriptional level. Thus, Bcl-2 P1
promoter activity was significantly higher in MCF7-HOXA1 cells in
comparison to MCF7-VECTOR cells in serum-free conditions (Fig.
4B). Exogenous hGH was without effect on Bcl-2 P1
promoter activity, but FBS stimulated Bcl-2 gene
transcription in MCF7-VECTOR cells and dramatically enhanced
Bcl-2 P1 promoter activity in MCF7-HOXA1 cells. Thus HOXA1
regulates Bcl-2 at the transcriptional level.
To determine if Bcl-2 was responsible for the dramatic decrease in
apoptosis as a consequence of forced expression of HOXA1 we prevented
expression of Bcl-2 in MCF7-VECTOR and MCF7-HOXA1 cells by use of
antisense oligonucleotides (42) as observed in Fig. 4C.
Transfection of antisense oligonucleotides to Bcl-2 prevented the HOXA1-stimulated expression of Bcl-2 in
MCF7-HOXA1 cells, whereas sense control oligonucleotides did not
significantly affect the level of Bcl-2 (Fig. 4C).
Transfection of Bcl-2 antisense oligonucleotides in
MCF7-HOXA1 increased apoptotic cell death above that in control or
sense oligonucleotide-transfected MCF7-VECTOR cells (Fig.
4D), and Bcl-2 antisense also increased apoptotic cell death in MCF7-VECTOR cells. To further verify the Bcl-2 dependence of the survival effect of forced expression of HOXA1 in mammary carcinoma cells, we utilized a novel Bcl-2 inhibitor that antagonizes Bcl-2 function by blocking the binding of the Bak BH3 peptide to Bcl-2
in vitro (43). Bcl-2 expression was not altered by cellular
treatment with the Bcl-2 inhibitor (Fig. 4E). Use of the
Bcl-2 inhibitor abrogated the protection from apoptotic cell death as a
consequence of forced expression of HOXA1 (Fig. 4F) similar
to the effect observed with Bcl-2 antisense
oligonucleotides. The anti-apoptotic effects of forced expression of
HOXA1 in mammary carcinoma cells are therefore mediated by increased
Bcl-2 gene transcription.
HOXA1 Expression in Mammary Carcinoma Cells Protects against
Doxorubicin-induced Apoptosis--
As forced expression of HOXA1 in
mammary carcinoma cells offered dramatic protection from apoptosis, we
reasoned that increased HOXA1 expression would also result in decreased
sensitivity to cell death as a result of exposure to anti-neoplastic
agents. We therefore examined the effect of forced expression of HOXA1 on the apoptotic response of mammary carcinoma cells to doxorubicin. Doxorubicin decreased Bcl-2 promoter activity in MCF7-VECTOR
cells and decreased the HOXA1-enhanced promoter activity in MCF7-HOXA1 cells, although Bcl-2 promoter activity in MCF7-HOXA1 cells
treated with doxorubicin remained higher than in vehicle-treated
MCF7-VECTOR cells (Fig. 5A).
The level of Bcl-2 mRNA as determined by RT-PCR demonstrated concordance with Bcl-2 P1 promoter activity
(Fig. 5B). Furthermore, Western blot analysis for Bcl-2
demonstrated an equivalent decrease in Bcl-2 protein upon treatment of
MCF7-VECTOR cells and relative maintenance of Bcl-2 protein in
doxorubicin-treated MCF7-HOXA1 cells. Bcl-xL protein was also slightly
decreased after doxorubicin treatment but to equivalent levels in both
cell lines. The level of Bak and Bax did not differ between the two
cell lines, and no effect of doxorubicin was observed. A dramatic
increase in p53 in both MCF7-VECTOR and MCF7-HOXA1 demonstrated the
efficacy of doxorubicin treatment (Fig. 5C). Doxorubicin
dramatically increased apoptosis in MCF7-VECTOR cells, and in
comparison MCF7-HOXA1 exhibited marked resistance to the apoptotic
effects of doxorubicin (Fig. 5D). Therefore, increased
expression of HOXA1 in human mammary carcinoma cells produces a
chemoresistant phenotype.
Forced Expression of HOXA1 in Human Mammary Carcinoma Cells
Results in Increased Anchorage-independent Growth in a
Bcl-2-dependent Manner--
Anchorage-independent growth
is one pivotal characteristic of malignant transformation (2). To
determine if forced expression of HOXA1 would alter the oncogenicity of
mammary carcinoma cells, we examined the anchorage-independent growth
of MCF7-VECTOR and MCF7-HOXA1 in both soft agar and suspension culture.
MCF7-VECTOR cells formed colonies in soft agar as expected, and forced
expression of HOXA1 in MCF7-HOXA1 cells dramatically enhanced colony
formation in soft agar (Fig.
6A). The size of the
individual colonies formed by MCF7-HOXA1 was also significantly larger
than the small cell aggregates observed with MCF7-VECTOR cells (Fig.
6D). MCF7-HOXA1 cells also increased in number significantly
greater than MCF7-VECTOR cells in suspension culture and after 10 days
were approximately tripled in number compared with MCF7-VECTOR cells
(Fig. 6B). To determine if Bcl-2 expression was required for
the observed enhancement of anchorage-independent growth as a result of
forced expression of HOXA1, we examined soft agar colony formation of
MCF7-VECTOR and MCF7-HOXA1 cells in the presence of the Bcl-2
inhibitor. As is observed in Fig. 6C, use of the Bcl-2
inhibitor completely prevented HOXA1 enhancement of soft agar colony
formation by mammary carcinoma cells (Fig. 6D). Thus, forced
expression of HOXA1 enhances oncogenicity of human mammary carcinoma
cells.
Forced Expression of HOXA1 Results in Oncogenic Transformation of
Immortalized Human Mammary Epithelial Cells--
To determine if
forced expression of HOXA1 would result in oncogenic transformation of
human mammary epithelial cells, we utilized the immortalized human
mammary epithelial cell line MCF-10A (44). When grown attached to a
plastic substrate these cells display a typical epithelial morphology,
do not form colonies in soft agar, and are not capable of growth in
immunocompromised mice (44). To examine the potential oncogenic
capacity of HOXA1, we therefore stably transfected HOXA1
cDNA in MCF-10A cells (MCF10A-HOXA1). These cells expressed higher
levels of both HOXA1 mRNA (Fig.
7A) and protein (Fig.
7B) compared with vector-transfected controls (MCF10A-VECTOR). Forced expression of HOXA1 in MCF10A-HOXA1 cells also
resulted in enhanced HOXA1 transcriptional activity as evidenced by
reporter activity from the EphA2-r42B enhancer (Fig.
7C). Transient transfection of the cDNA for HOXA1
binding partner PBX1 further dramatically enhanced HOXA1
transcriptional activity in MCF10A-HOXA1 cells as expected (Fig.
7C).
We therefore proceeded to examine the growth characteristics of both
MCF10A-VECTOR and MCF10A-HOXA1 cells. MCF10A-HOXA1 total cell number
was increased significantly greater under all examined conditions
(serum free, hGH, and FBS) than MCF10A-VECTOR cell number (Fig.
7D). Comparison of nuclear BrdUrd incorporation between the
two cell lines under serum-free conditions, 50 nM exogenous hGH, or FBS demonstrated that forced expression of HOXA1 significantly increased cell cycle progression of mammary carcinoma cells (Fig. 7E). Apoptotic cell death was also dramatically reduced in
MCF10A-HOXA1 cells compared with MCF10A-VECTOR cells in serum-free
conditions (Fig. 7F) as previously described for forced
expression of HOXA1 in MCF-7 cells. Exogenous hGH slightly reduced
apoptotic cell death in both MCF10A-VECTOR and MCF10A-HOXA1 cells but
proportionate to the serum-free condition for each cell. FBS functioned
as a powerful survival stimulus for both cell lines, although apoptosis was still less in MCF10A-HOXA1 cells compared with MCF10A-VECTOR cells
(Fig. 7F).
MCF10A-HOXA1 cells formed large numerous colonies in soft agar, whereas
MCF10A-VECTOR cells were largely ineffective in colonization of soft
agar (Fig. 8, A and
B). Thus forced expression of HOXA1 conferred tumorigenic
potential upon human mammary epithelial cells. We also verified this
result with a second independently generated pool of HOXA1
cDNA-transfected cell clones (data not shown). MCF10A-HOXA1
colonization of soft agar was also Bcl-2-dependent as it
was entirely prevented by the Bcl-2 inhibitor described above (Fig.
8A). We also observed that forced expression of HOXA1 in
mouse NIH-3T3 cells resulted in their oncogenic transformation as
indicated by dramatic colony formation in soft agar in comparison to an
effective lack of colony formation by vector transfected NIH-3T3 cells
(data not shown).
In vitro analyses of oncogenic transformation are not always
concordant with tumorigenic potential in vivo. We therefore
implanted both MCF10A-VECTOR and MCF10A-HOXA1 cells into the first
mammary (axillary) fat pad of female severe combined immunodeficient
(SCID) mice with use of either PBS or Matrigel as vehicle. MCF10A-HOXA1 cells formed large palpable and visible tumors in all mice injected, whereas MCF10A-VECTOR cells did not (Fig. 8, C and
D). The average volume of the tumors formed by MCF10A-HOXA1
cells was related to the injection vehicle with Matrigel resulting in
larger tumor volume (Table I). Necropsy
revealed that the tumors were attached to the underlying
axillary muscle and surrounded by a vascular fibrous capsule.
Histologically the neoplastic cells were locally invasive and
associated with fibrous connective tissue (Fig. 8, E and
F). The cells exhibited moderate cytoplasmic and nuclear pleomorphism and formed a solid mass often with areas of central necrosis. Necropsy of SCID mice injected with MCF10A-VECTOR cells failed to identify any growth. Macroscopic and histological examination of lung and liver of SCID mice injected with MCF10A-HOXA1 cells failed
to identify metastatic extension of the implanted MCF10A-HOXA1 cells.
Mammary carcinoma cells derived from the in vivo growth of
MCF10A-HOXA1 cells expressed HOXA1 at the same level as the injected
cell indicative of phenotypic retention (data not shown).
We have demonstrated here that forced expression of HOXA1 in
immortalized human mammary epithelial cells (MCF-10A) results in
oncogenic transformation and the development of a rapidly growing carcinoma in vivo. This is remarkable, given that forced
expression of other oncogenes (e.g. Ras, Her2/neu, and TC21)
are insufficient to convey tumorigenic potential on MCF-10A cells (45,
46). Furthermore, cyclin D1 (overexpressed in over 50% of human
mammary carcinomas (47)), although able to support
anchorage-independent growth of MCF-10A cells, does not result in tumor
formation in vivo (48). However, one gene (EphA2) directly
regulated by HOXA1 has also been reported to transform MCF-10A cells
with consequent in vivo tumor formation (49). Indeed, here
we utilized the HOX binding region of the EphA2
promoter (EphA2-r42B) to measure HOXA1 transactivation, and
therefore, EphA2 may also constitute a pivotal component of the
oncogenic program of HOXA1. The aberrant expression of
homeobox-containing genes has been reported in a variety of neoplasias
(for review see Ref. 27). However, it is debated whether
homeobox-containing genes should be classified as oncogene/tumor suppressor genes per se or only as "tumor modulators"
that tip the balance of tumor progression (27, 28). Herein, we provide the first direct evidence that a homeobox-containing protein is a
bona fide mammary gland oncogene, the enhanced expression of which is sufficient for tumorigenesis of human mammary epithelial cells.
Primary rodent cells are easily transformed by two concomitantly
introduced oncogenes (50, 51). Only recently have human cells been
transformed by a combination of the genomic version of the SV40 Large T
antigen, the hTERT gene that encodes the telomerase catalytic subunit, and an oncogenic allele of the H-Ras gene, H-RasV12
(52). hTERT allows mammary epithelial cells to bypass both
senescence (M1) and crisis (M2) with resultant
immortalization of cells. Immortalization of cells is not sufficient to
create an oncogenically transformed cell (52, 53), and we have driven the oncogenic process here in immortalized human mammary epithelial cells by introduction of only the HOXA1 cDNA. Other
members of the homeobox gene family have also been reported
to be oncogenic (54, 55). Such examples include HOXB7 (56) and Cdx1
(57). Interestingly, certain homeobox family members have also been demonstrated to result in cellular immortalization (58). This raises
the intriguing possibility that HOXA1 may both immortalize human
mammary epithelial cells and result in their oncogenic transformation (shown here). Indeed, in preliminary
experiments2 we have
demonstrated that forced expression of HOXA1 does indeed increase
transcription of the hTERT gene with a resultant
increase in telomerase activity in primary human mammary epithelial
cells. HOXA1 may therefore both immortalize and oncogenically transform human mammary epithelial and potentially constitute a "complete oncogene" for human mammary epithelial cells. This exciting
possibility is currently under active investigation in our laboratory.
It is likely that autocrine production of hGH also transcriptionally regulates other homeodomain-containing proteins, and we are currently characterizing the role of two other homeobox family members in the
effect of autocrine hGH on mammary carcinoma cell function.
We have observed here that HOXA1 gene expression and
transcriptional activity is regulated by hGH. Since we demonstrated
that forced expression of HOXA1 resulted in oncogenic transformation of
human mammary epithelial cells, it raises the possibility that hGH
itself is oncogenic. However, exogenous hGH does not support the
anchorage-independent growth of immortalized human mammary epithelial
cells3 despite the
requirement of endocrine GH for optimal growth of mammary tumor
xenografts (59). This is concordant with the fact that, although
exogenous hGH increased HOXA1 mRNA, it did not result in an
increase in HOXA1 protein nor transcriptional activity. Interestingly,
however, and consistent with our previous studies (18),
autocrine-produced hGH does indeed confer immortalized mammary
epithelial cells with the capacity for anchorage-independent growth and
tumor formation in
vivo.4 Autocrine hGH may
therefore utilize HOXA1 to execute its oncogenic program, and this
possibility is currently under investigation. The observation that
exogenous hGH regulates HOXA1 mRNA but not protein nor
transcriptional activity is suggestive that autocrine hGH production in
human mammary epithelial cells also regulates HOXA1 at the
translational or post-translational level. Such potential regulation of
HOXA1 other than at the transcriptional level may be requisite for its
transforming activity.
By use of a functional Bcl-2 inhibitor, we have demonstrated that
HOXA1-dependent transformation is dependent on the
interaction between Bcl-2 and Bak BH3 peptide (43). It is unlikely that this pathway constitutes the sole mechanism by which HOXA1 confers oncogenic transformation of human mammary epithelial cells and further
pathways presumably will be delineated by microarray and promoter
mining experiments in progress. It may be that Bcl-2 is simply required
for human mammary epithelial cell survival, and functional inhibition
of Bcl-2 would consequently drive the cell to apoptosis. Indeed, high
concentrations of the Bcl-2 inhibitor prevented soft agar colony
formation by both MCF7-VECTOR and MCF7-HOXA1 cells (data not shown).
However, titration of the Bcl-2 inhibitor allowed selective inhibition
of the enhanced colony formation as a consequence of forced expression
of HOXA1 with no alteration in colony formation by MCF7-VECTOR cells.
Thus, it is apparent that Bcl-2 is required specifically for HOXA1
enhancement of mammary carcinoma cell colony formation in soft agar.
Chemotherapeutic agents such as doxorubicin result in the induction of
p53 to execute an apoptotic program (60, 61). Bcl-2 functions to
prevent mitochondrial cytochrome c release and subsequent cell death (62, 63), and therefore, increased Bcl-2 expression could be
expected to result in chemoresistance (64). Indeed, Bcl-2 has been
observed to be overexpressed in a large percentage of human neoplasias
(64), and Bcl-2 expression is associated with resistance to
chemotherapy in many cancers, including mammary carcinoma (65, 67). As
could be expected, forced expression of HOXA1 resulted in resistance to
doxorubicin-induced cell death as a consequence of increased Bcl-2
expression. Inhibition of HOXA1 transactivation may therefore
constitute a novel therapeutic target to abrogate resistance to
chemotherapeutic agents. However, Bcl-2 is not the only mechanism
utilized by hGH to prevent apoptotic cell death of mammary carcinoma
cells, and simple antagonism of hGH signal transduction may represent a
more efficacious approach to enhance the response of mammary carcinoma
cells to chemotherapy. We have previously described that autocrine hGH
production promotes mammary carcinoma cell survival by at least two
other mechanisms (23, 36). First, autocrine hGH increases
transcription of the CHOP gene, and increased p38
mitogen-activated protein kinase-dependent CHOP-mediated
transcription results in mammary carcinoma cell survival (23).
Autocrine hGH production in mammary carcinoma cells also conversely
results in transcriptional repression of the p53-regulated placental
transforming growth factor- In conclusion, we have demonstrated that autocrine hGH regulates the
expression and activity of HOXA1 in human mammary carcinoma cells. We
observed that forced expression of HOXA1 protected human mammary
carcinoma cells from apoptotic cell death in a
Bcl-2-dependent manner. We also identified HOXA1 as a
powerful oncogene for human mammary epithelial cells. Further
functional characterization of HOXA1 and other hGH-regulated homeobox
genes in the human mammary epithelial cell will be instrumental in
understanding the development and formation of
hormone-dependent neoplasia in the mammary gland.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin cDNA served as an
internal control for cDNA quantity and quality. All RNA samples
were treated with DNase I to avoid genomic DNA contamination.
-actin (sense), 5'-ATGATATCGCCGCGCTCG-3';
-actin (antisense), 5'-CGCTCGGTGAGGATCTTCA-3';
Bcl-2 (sense), 5'-TGCACCTGACGCCCTTCAC-3'; Bcl-2
(antisense), 5'-TAGCTGATTCGACGTTTTGCCTGA-3'.
-galactosidase activity and protein concentration in the samples.
EphA2-r42B-LUC was a generous gift of Dr. Jin Chen
(Nashville, TN), and the PBX1 expression vector was a generous gift of
Dr. Vincenzo Zappavigna (Milano, Italy). The Bcl-2 P1
promoter reporter plasmid was a kind gift of Dr. John Kurland (Houston, TX).
-actin, anti-HOXA1, and anti-cyclin D1 antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA); the
anti-p27Kip1 and anti-p21waf/cip antibodies were
from Transduction Laboratories (Lexington, KY); and anti-Fas,
anti-Fadd, anti-p53, anti-Bax, anti-Bak, anti-Bcl-2, and anti-Bcl-xL
were obtained from Oncogene (San Diego, CA). Immunolabeling was
visualized by ECL detection kit from Amersham Biosciences (Little
Chalfont, UK) according to the manufacturer's instruction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin mRNA did not differ between the two cell lines under the different treatment conditions and was used as a
control for RNA quality (Fig. 1A).
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Fig. 1.
Effect of autocrine hGH on HOXA1 mRNA,
protein, and transcriptional activity in mammary carcinoma cells.
MCF7-MUT and MCF7-hGH cells were cultured in serum-free media or in
serum-free media supplemented with either 50 nM hGH or 10%
FBS. Experiments were performed as described under "Experimental
Procedures." The level of HOXA1 mRNA was determined by
RT-PCR (A), HOXA1 protein by Western blot analysis
(B), and HOXA1 transcriptional activity by reporter assay
(C) as indicated. -Actin was used as loading control
where appropriate. *, p < 0.01.
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Fig. 2.
Demonstration of functional overexpression of
HOXA1 protein upon stable transfection of MCF-7 cells with HOXA1
cDNA. MCF-7 cells were stably transfected with either
the empty vector (MCF7-VECTOR) or the vector containing
HOXA1 cDNA (MCF7-HOXA1). The level of
HOXA1 mRNA was determined by RT-PCR (A) and
HOXA1 protein by Western blot analysis (B) under serum-free
conditions. -Actin was used as loading control. HOXA1
transcriptional activity (in the presence and absence of its binding
partner PBX1) was determined by reporter assay (C) as
indicated. *, p < 0.01.
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Fig. 3.
Effect of forced expression of HOXA1 in
mammary carcinoma cells on cell number, cell cycle progression, and
apoptosis. MCF7-VECTOR and MCF7-HOXA1 cells were cultured in
serum-free media or in serum-free media supplemented with either 50 nM hGH or 10% FBS as indicated. Total cell number
(A), cell cycle progression (BrdUrd incorporation)
(B), and apoptotic cell death (C) were determined
in both cell lines under the indicated conditions as detailed under
"Experimental Procedures." Western blot analysis was utilized to
determine the level of proteins involved in cell cycle progression
(cyclinD1, p21waf1/cip1, and p27Kip1)
(D) and apoptosis (Fas, Fadd, p53, Bax, Bak, Bcl-xL, and
Bcl-2) (E) as indicated. -Actin was used as loading
control. Results represent means ± S.D. of triplicate
determinations. *, p < 0.01; **, p < 0.05.
-actin (Fig. 3D). To determine the effects of forced
expression of HOXA1 on cell cycle progression in MCF7-VECTOR and
MCF7-HOXA1 cells, we examined the nuclear incorporation of
5'-bromo-2'-deoxyuridine in these cells. Although MCF7-HOXA1 cells
exhibited a significantly higher percentage of nuclear BrdUrd
incorporation compared with MCF7-VECTOR cells (Fig. 3B) the
differences were small and could not account for the observed
differences in total cell number between MCF7-VECTOR and MCF7-HOXA1 cells.
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Fig. 4.
HOXA1 expression in human mammary carcinoma
cells prevents apoptotic cell death in a Bcl-2-dependent
manner. Bcl-2 mRNA levels in MCF7-VECTOR and
MCF7-HOXA1 cells were determined by RT-PCR (A), and
Bcl-2 promoter activity was determined by reporter assay
using the Bcl-2 P1 promoter (B) under the conditions
indicated (serum-free, 50 nM hGH, and 10% FBS). Bcl-2
antisense oligonucleotide (800 nM) depleted cellular Bcl-2
(C), whereas a pharmacological inhibitor of Bcl-2 (10 µM) did not (E). Bcl-2 levels in C
and E were determined by Western blot analysis, and
-actin was used as loading control. Both Bcl-2 antisense
oligonucleotide (D) and Bcl-2 inhibitor (F)
abrogated protection from apoptotic cell death as a consequence of
forced expression of HOXA1 as indicated. All experiments were performed
as described under "Experimental Procedures." *, p < 0.01.
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Fig. 5.
Forced expression of HOXA1 in mammary
carcinoma cells protects against doxorubicin-induced apoptosis.
Forced expression of HOXA1 in mammary carcinoma cells (MCF7-HOXA1)
maintains Bcl-2 promoter activity (A) and
Bcl-2 mRNA levels (B) above that observed in
vector-transfected control cells upon treatment of both cell lines with
1 µg/ml doxorubicin. Western blot analysis (C)
demonstrates dramatic doxorubicin-induced expression of p53 in both
MCF7-VECTOR and MCF7-HOXA1 cells and maintenance of Bcl-2 expression in
MCF7-HOXA1 cells compared with MCF7-VECTOR cells. -Actin was used as
loading control. Doxorubicin-induced apoptotic cell death
(D) was abrogated by forced expression of HOXA1 in mammary
carcinoma cells as indicated. All experiments were preformed as
described under "Experimental Procedures." *, p < 0.01.
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Fig. 6.
Forced expression of HOXA1 enhances
anchorage-independent growth of mammary carcinoma cells in a
Bcl-2-dependent manner. Soft agar colony formation by
MCF7-VECTOR and MCF7-HOXA1 cells (A) and
anchorage-independent growth of MCF7-VECTOR and MCF7-HOXA1 in
bacteriological dishes (B) were performed as described under
"Experimental Procedures." Bcl-2 inhibitor was used at a
concentration of 1 µM to demonstrate Bcl-2 dependence of
HOXA1-stimulated enhancement of soft agar colony formation as indicated
(C and D). The results are given as means ± S.D. of triplicate determinations. *, p < 0.001.
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Fig. 7.
Functional expression of HOXA1 in
immortalized mammary epithelial cells (MCF-10A) results in increased
cell growth. Forced expression of HOXA1 in immortalized mammary
epithelial cells (RT-PCR to demonstrate HOXA1 mRNA
(A) and Western blot (B) to demonstrate HOXA1
protein) results in enhanced HOXA1-mediated transcription
(C). HOXA1 transcriptional activity (in the presence and
absence of its binding partner PBX1) was determined by reporter assay
(C) as indicated. Total cell number (D), cell
cycle progression (BrdUrd incorporation) (E), and apoptotic
cell death (F) were determined in both MCF10A-VECTOR and
MCF10A-HOXA1 cell lines under the indicated conditions as described
under "Experimental Procedures." *, p < 0.01.
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Fig. 8.
Forced expression of HOXA1 in immortalized
mammary epithelial cells results in oncogenic transformation and tumor
formation in vivo. Forced expression of
HOXA1 in immortalized mammary epithelial cells permits soft agar colony
formation (A and B). Assays were performed as
described under "Experimental Procedures." Bcl-2 inhibitor (0.5 µg/ml) was utilized to demonstrate Bcl-2 dependence of oncogenic
transformation (A). MCF10A-HOXA1 cells implanted into the
first mammary (axillary) fat pad of female severe combined
immunodeficient mice formed a large visible tumor mass
(C and D). Histological appearance of the tumor
visualized with hematoxylin and eosin (E), and local
invasion of MCF10A-HOXA1 tumor cells into surrounding skeletal muscle
(F). *, p < 0.01.
HOXA1 overexpression induces in vivo tumor formation
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene with subsequent decreases in its
protein product, Smad-mediated transcription, and its cellular effects,
which include cell cycle arrest and apoptosis (36). Several avenues
exist for antagonism of the somatotropic axis. Non-dimerizing hGH
receptor antagonists have been used clinically to treat acromegaly (68)
and constitute one potential therapeutic approach. Indeed, we have
utilized this antagonist in vitro to demonstrate that the
effects of autocrine hGH on mammary carcinoma cell proliferation (both
mitogenesis and apoptosis) are mediated via the hGH receptor (21).
GH-releasing hormone antagonists have also been utilized to decrease
mammary GH production and, consequently, decrease the growth of mouse mammary carcinoma xenografts (69). It is therefore conceivable that
antagonism of hGH, utilized as adjuvant therapy, will increase the
efficacy of conventional chemotherapeutic regimes currently utilized to
treat mammary carcinoma.
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FOOTNOTES |
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* This work was supported by grants from the Agency for Science, Technology and Research (A*STAR) of Singapore (to P. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Tel.:
65-6874-7847; Fax: 65-6779-1117; E-mail: mcbpel@imcb.nus.edu.sg.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M212050200
2 Y. Chen, T. Zhu, X. Zhang, and P. E. Lobie, unpublished data.
3 T. Zhu and P. E. Lobie, unpublished data.
4 T. Zhu, H. C. Mertani, X. Zhang, K.-O. Lee, and P. E. Lobie, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: GH, growth hormone; hGH, human growth hormone; RT, reverse transcriptase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; SCID, severe combined immunodeficient mice; DOTAP, N-[1-(2,3-dioleoyl(oxy)propyl]-N,N,N-triethyl ammonium methyl sulfate.
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