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Molecular and Cellular Biology, January 2001, p. 575-594, Vol. 21, No. 2
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.2.575-594.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Architectural Transcription Factor HMGI(Y) Promotes
Tumor Progression and Mesenchymal Transition of Human
Epithelial Cells
Raymond
Reeves,1,*
Dale D.
Edberg,1 and
Ying
Li2
Departments of
Genetics2 and
Biochemistry,1 School of Molecular
Biosciences, Washington State University, Pullman, Washington
99164-4660
Received 14 July 2000/Returned for modification 18 September
2000/Accepted 24 October 2000
 |
ABSTRACT |
Numerous studies have demonstrated that overexpression or aberrant
expression of the HMGI(Y) family of architectural transcription factors
is frequently associated with both neoplastic transformation of cells
and metastatic tumor progression. Little is known, however, about the
molecular roles played by the HMGI(Y) proteins in these events. Here we
report that human breast epithelial cells harboring tetracycline-regulated HMGI(Y) transgenes acquire the ability to form
both primary and metastatic tumors in nude mice only when the
transgenes are actively expressed. Unexpectedly, the HMG-Y, rather than
the HMG-I, isoform of these proteins is the most effective elicitor of
both neoplastic transformation and metastatic progression in vivo.
Furthermore, expression of either antisense or dominant-negative HMGI(Y) constructs inhibits both the rate of proliferation of tumor
cells and their ability to grow anchorage independently in soft agar.
Array analysis of transcription profiles demonstrates that the HMG-I
and HMG-Y isoform proteins each modulate the expression of distinctive
constellations of genes known to be involved in signal transduction,
cell proliferation, tumor initiation, invasion, migration, induction of
angiogenesis, and colonization. Immunohistochemical analyses of
tumors formed in nude mice indicate that many have undergone an
epithelial-mesenchymal transition in vivo. Together, these findings
demonstrate that overexpression of the HMGI(Y) proteins, more
specifically, the HMG-Y isoform protein, is causally associated with
both neoplastic transformation and metastatic progression and suggest
that induction of integrins and their signaling pathways may play
significant molecular roles in these biological events.
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INTRODUCTION |
The mammalian HMG-I, HMG-Y, and HMGI-C proteins
are members of the HMGI(Y) family of nonhistone chromatin
proteins that have been implicated in both positive and negative
regulation of gene transcription in vivo (reviewed in references
14 and 96). The human HMG-I (11.5-kDa) and HMG-Y
(10.4-kDa) proteins are produced by translation of alternatively
spliced mRNAs coded for by the Hmgiy gene at chromosomal
locus 6p21 (39), while the related, but distinct, HMGI-C
protein (12 kDa) is coded for by the Hmgi-c gene at
chromosomal locus 12q15 (15). Members of the HMGI(Y) family are often referred to as architectural transcription factors because of their ability to regulate gene activity through the recognition and alteration of the structure of DNA and chromatin substrates (14, 100). The HMGI(Y) proteins not only bind
with high specificity to the narrow minor groove of A · T-rich,
random sequence B-form DNA (97, 107) but also have the
ability to bind with high affinity to bent or distorted DNA structures
such as synthetic four-way junction DNAs (55, 56),
supercoiled plasmid substrates (87), and nucleosome core
particles (98, 100). The ability of the HMGI(Y) proteins
to bind to a variety of DNA substrates is a consequence of the high
degree of intrinsic flexibility of the proteins, particularly within
their A · T hook DNA-binding motifs (97) whose
structure in a cocomplex with a synthetic substrate has recently been
determined (61). In addition to recognizing structure
rather than sequence, the HMGI(Y) proteins also have the ability to
bend, straighten, unwind, and supercoil DNA substrates (14, 33,
75, 87). It is this unusual combination of substrate-binding
characteristics, combined with the ability to specifically interact
with other transcription factors, that allows the HMGI(Y) proteins to
function as gene-regulatory factors in vivo by participating in the
formation of stereospecific multiprotein enhanceosome complexes
(66, 122). Among the known transcription factors that
interact with HMGI(Y) in vitro or in vivo are NF-
B, ATF-2/c-Jun,
Elf-1, Oct-2, Oct-6, SRF, NF-Y, PU-1, RAR, Sp1, NFAT, and others
(14, 19, 24, 57, 58, 66, 86, 121). Their many potential
protein partners provide the HMGI(Y) proteins with considerable
flexibility in regulating the transcriptional activity of a large
number of different genes in vivo (14), among which some,
such as the intercellular adhesion molecule E-selectin
(77), often exhibit aberrant expression patterns in human
malignancies (6) whereas others, such as tumor necrosis
factor beta (35) and beta interferon (IFN-
) (83), are associated with angiogenesis, a necessary
component of solid-tumor formation.
The level of expression of HMGI(Y) mRNAs and proteins is low
(68, 69, 82) or undetectable (40, 44) in most
differentiated or nonproliferating normal cells but is rapidly induced
in response to growth-stimulatory factors such as serum (67,
69), transforming growth factor 
(TGF-) (95),
epidermal growth factor (EGF) (59), platelet-derived
growth factor, and fibroblast growth factor (FGF) (74), as
well as phorbol esters (22, 39), beta 1 interferon
(IFN-1), and endotoxin (91). These observations, combined with the fact that deletion of the Hmgi-c gene
results in the diminutive pygmy phenotype in mice (7),
implicate the HMGI(Y) proteins in the control of cell proliferation.
Consistent with this suggestion, in neoplastically transformed cells,
as well as in embryonic cells that have not yet undergone overt
differentiation, the constitutive level of HMGI(Y) gene products is
often exceptionally high, with increasing concentrations being
correlated with increasing degrees of malignancy or metastatic
potential (14, 40, 44, 116). This correlation is so
consistent and widespread in tumors that it has been suggested that
elevated concentrations of HMGI(Y) proteins are diagnostic markers
of both neoplastic transformation (43, 44) and increased
metastatic potential (13, 117). For example, HMGI(Y)
overexpression has been suggested to be a diagnostic indicator for
human prostate tumors (118), thyroid neoplasia
(18), uterine cervix cancers (4), and
colorectal cancers (1), among others (reviewed in
reference 116).
Of particular interest for the present study is the fact that increased
expression of HMGI(Y) proteins has been correlated with the increased
metastatic potential of both mouse (94, 95) and human
(59, 79) mammary epithelial cancers. Importantly, HMGI(Y)
overexpression has also recently been detected in primary human breast
cancers by a combination of serial analysis of gene expression and
array technologies (85, 92). Overexpression of full-length
HMGI(Y) proteins induces anchorage-independent growth of both mouse
breast epithelial cells (94) and Rat-1 fibroblast cells
(132) in soft agarose, as does overexpression of either
truncated or chimeric forms of the HMGI-C protein in murine fibroblasts
(37). Furthermore, antisense-mediated suppression of
HMGI(Y) protein synthesis suppresses retrovirus-induced neoplastic transformation of rat thryoid cells (8), as well as
inducing apoptotic death in anaplastic human thyroid carcinoma cell
lines but not in normal thyroid cells (102). Of additional
interest is the fact that chromosomal rearrangements in which the
A · T hook DNA-binding domains of the HMGI(Y) proteins are fused to ectopic peptide sequences to form chimeric oncogenic proteins are among
the most common lesions in human cancer, being observed in a high
percentage of benign mesenchymal neoplasias, including lipomas,
leiomyomas, fibroadenomas, aggressive myxomas, pulmonary hamartomas,
and endometrial polyps (reviewed in references 54 and
116). These previous studies, when considered together, present a compelling case for involvement of the HMGI(Y) proteins, and their
A · T hook DNA-binding motifs, in both neoplastic transformation and metastatic tumor progression. Nevertheless, they did not directly and unambiguously demonstrate a causal in vivo relationship between the
HMGI(Y) proteins and cancer in intact organisms. These previous studies also did not elucidate the molecular events underlying the
biological consequences that overexpression or aberrant expression of
HMGI(Y) proteins might have on processes such as oncogenic transformation, increased tumor metastatic potential, and aggressive neoplastic malignancy.
In the present study, we demonstrated that nontumorigenic human breast
epithelial cells containing either a tetracycline-regulated HMG-I or
HMG-Y transgene acquire the abilities to both grow anchorage independently in soft agar and form metastatic tumors when injected into nude mice only when the HMGI(Y) proteins are overexpressed. While overexpression of either the HMG-Y or HMG-I protein alone readily
induced neoplastic transformation, as evidenced by the acquired ability
of cells to grow in soft agar, somewhat surprisingly, only the HMG-Y
isoform elicited efficient tumor formation and metastasis in vivo when
expressing cells were injected into nude mice. Furthermore, expression
of either antisense or dominant-negative HMGI(Y) constructs in
tumor cells inhibited both their rate of proliferation and their
ability to grow anchorage independently in soft agar. Analysis of the
transcription profiles of cells employing cDNA arrays demonstrates
that overexpression of HMGI(Y) proteins modulates the expression of
a complex set of genes that are known to be involved in cell signaling,
proliferation, tumor initiation, invasion, migration, induction of
angiogenesis, and colonization. Importantly, immunohistochemical
analyses of tumors formed in nude mice by cells overexpressing
HMGI(Y) suggest that they have undergone an epithelial-mesenchymal
transition (EMT) in vivo, an event consistent with the results obtained
from gene transcription array analyses. These data represent direct
experimental support for the hypothesis that overexpression of the
HMGI(Y) proteins is causally associated with neoplastic
transformation, metastatic progression, and mesenchymal transition of
human breast epithelial cells in the context of an intact living organism.
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MATERIALS AND METHODS |
Cell culture and selection of transgenic clones.
The human
breast epithelial cell line MCF-7 (catalog no. HTB-22) was obtained
from the American Type Culture Collection (ATCC; Manassas, Va.) and was
grown in Dulbecoco's modified Eagle's medium (DMEM) supplemented with
2 mM L-glutamine, 10 mM HEPES, 10% fetal calf serum (FCS),
penicillin G sodium at 100 U/ml, and streptomycin sulfate at 100 µg/ml and maintained as recommended by the supplier. MCF-7/PKC
cells (a generous gift of D. K. Ways, East Carolina University
School of Medicine [128] were grown in the same medium as MCF-7 cells but supplemented with G418 at 100 µg/ml. The human epithelial cell lines Hs578Bst (ATCC catalog no. HTB-125) and Hs578T
(ATCC catalog no. HTB-126), as well as the human HeLa cervical carcinoma cell line (ATCC catalog no. CCL-2), were also obtained from
the ATCC and maintained as recommended by the supplier. The tetracycline-regulated M/tet, M/tet/Vec, M/tet/HA-I, and M/tet/HA-Y human breast epithelial cells were obtained as follows. The parental M/tet (MCF-7/Tet-off) cell line was purchased from Clontech, Palo Alto,
Calif. (catalog no. C30071, lot no. 7080502). The M/Tet cell line was
derived by stable transfection of MCF-7 cells with a
tetracycline-controlled transgene coding for the tetracycline transactivator protein and exhibits normal slow-growth characteristics, is contact inhibited, and does not form tumors when injected into nude
mice. The cell lines designated M/tet/HA-I or M/tet/HA-Y (e.g.,
M/tet/HA-IC7, M/tet/HA-YC21, etc.) are clonal derivatives of M/tet
cells that have been stably transfected with a plasmid vector
(Clontech) containing the tetracycline response element (pTRE) driving
the expression of either a hemagglutinin (HA)-tagged HMG-I or HA-tagged
HMG-Y cDNA transgene (69). The control M/tet/Vect cells are clonal derivatives of M/tet cells that have been stably transfected with the empty pTRE vector without any insert. M/tet cells
were cultured in DMEM containing 10% FCS (tetracycline free), 2 mM
L-glutamine, G418 at 100 µg/ml (for maintenance of
selection of the gene for the tetracycline transactivator protein),
penicillin G sodium at 100 U/ml, and streptomycin sulfate at 100 µg/ml. Selection and maintenance of the M/tet/Vect, M/tet/HA-I, and
M/tet/HA-Y cell clones were done with the same medium used for the
M/tet cells, except that it contained either hygromycin at 100 µg/ml for M/tet/Vect and M/tet/HA-I cells or zeocin at 50 µg/ml for M/tet/HA-Y cells. In addition, the M/tet/HA-I and M/tet/HA-Y clones were routinely maintained in tetracycline at 2 to 10 µg/ml (depending on the individual clone) to keep expression of the HA-I and HA-Y transgenes turned off.
Construction and use of HMGI(Y) plasmid expression
vectors.
Wild-type HMG-I and wild-type HMG-Y plasmid expression
vectors were created by subcloning the PCR products of the coding
regions of human HMG-I and HMG-Y cDNAs (69) into the
pcDNA3.1/Zeo+ plasmid expression vector (Invitrogen) between the
HindIII and BamHI sites of the polylinker.
The construction and use of both the antisense expression vector
pRcCMVIGMH (57, 99) and the dominant-negative expression vector HMG-I (cytomegalovirus-HMGImII,mIII)
(58) have been described previously. HA-tagged HMG-I and
HA-tagged HMG-Y expression vectors were created by fusing a synthetic
DNA oligonucleotide encoding the peptide MYPYDVPDYASL from the
influenza virus HA protein (i.e., HA tag) in frame to the N-terminal
end of the HMG-I and HMG-Y cDNAs by standard PCR protocols
(3). The PCR fragments containing either HA-tagged HMG-I
or HA-tagged HMG-Y were then subcloned into the pTRE expression vector,
and the sequences of the resulting expression constructs were confirmed by automated DNA sequencing prior to use.
Cell transfection and clone selection.
Plasmid expression
vectors were introduced into cells by employing either the DMRIE-C, the
Lipofectamine, or the Lipofectamine Plus transfection reagent following
the manufacturer's (Gibco BRL, Gaithersburg, Md.) instructions.
Transfection efficiencies in transient-transfection experiments using
antisense and dominant-negative HMGI(Y) expression vector
constructs were routinely monitored by cotransfection of a
cytomegalovirus-based plasmid vector expressing green fluorescent
protein (GFP) at a DNA ratio of 1:1. The total viable cells and viable
GFP-expressing cells were counted using a UV microscope. The percentage
of GFP-expressing cells was then obtained and served as an estimate of
the transfection efficiency. In many experiments, transfection
efficiencies were also monitored by cotransfection of cells with a
luciferase reporter plasmid and determination of luminescence activity
in cell lysates using a luciferase assay kit (catalog no. E4030;
Promega, Madison, Wis.) and a Top Count Luminometer (Packard Instruments).
Cell proliferation assays.
Cells were trypsinized and
suspended in DMEM with 10% FCS and counted using either a Coulter
Counter (Coulter Corp., Hialeah, Fla.) or a hemacytometer. Cell
viability was assessed using trypan blue exclusion (to obtain the
percentage of viable cells). For growth curve determinations, 2 × 104 or 4 × 104 viable cells were
initially seeded in 2 ml of medium per 35-mm diameter dish. In all
experiment, cells in four or more separate culture dishes were counted
on each day of a time course study in order to ensure statistical
accuracy of the growth curves.
Acid extraction and Western blot analysis of total cellular
HMGI(Y) proteins.
Acid extraction of HMG proteins from cells
with 5% perchloric acid was performed as previously described
(99). The acid-soluble proteins, which include not only
HMGI(Y) but the other HMG proteins and histone H1 as well, were
electrophoretically separated on either a 4 to 20% gradient or a
sodium dodecyl sulfate (SDS)-15 or 18% polyacrylamide gel
electrophoresis (PAGE) minigel and then transferred to an Immobilon-P
membrane (Millipore) by electrotransfer overnight. Immunodetection of
the HMG-I (Y) proteins on the membrane was performed using a rabbit
polyclonal anti-HMG-I antibody (MR-19) whose production and use have
been described previously (99) and an
enhanced-chemiluminescence detection procedure using the SuperSignal
substrate supplied by Pierce Chemical Co., Rockford, Ill.
Immunoprecipitation and Western blot assays of HA-tagged
proteins.
After washing with phosphate-buffered saline, cells were
directly lysed in their culture dishes (about 5 × 106
cells/dish) by addition of 1 ml of radioimmunoprecipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 00.5% sodium deoxycholate, 0.1% SDS, phenylmethylsulfonyl fluoride and aprotinin [Sigma catalog no. A6279] at 30 µg/ml each, and sodium
orthovanadate at 10 µl/ml). The lysate was passed through a 21-gauge
needle to shear the DNA and incubated on ice for 60 min. Particulates were removed from the lysate by centrifugtion at 15,000 × g for 20 min at 4°C, and the supernatant containing the total
soluble cellular proteins was collected. Approximately 50 µl of
anti-HA tag mouse monoclonal antibody (12CA5; a generous gift of
J. J. Chen [16]) was added to 100 µl of lysate,
and the mixture was incubated on ice for 1 h. Protein A-agarose
beads (50 µl; Sigma) were then added, and the mixture was incubated
overnight at 4°C while rotating. The beads were collected by
centrifugation, and the bound proteins were removed by washing the
beads three times with radioimmunoprecipitation assay buffer. An equal
volume of SDS-PAGE sample buffer was added to the eluate, and the
mixture was boiled for 2 min. The proteins in the eluate were separated by electrophoresis on an SDS-15% PAGE minigel and then transferred to
a nitrocellulose membrane using a semidry electrophoretic transfer cell
(Trans-Blot SD; Bio-Rad). The enhanced-chemiluminescence detection
procedure described above was then used to identify the membrane-bound,
HA-tagged proteins.
Soft agar anchorage-independent growth assays.
Seven
milliliters of molten 0.6% Difco agar (44°C) in DMEM with 10% FCS
and the desired antibiotic (such as G418, tetracycline, hygromycin, or
zeocin, etc.) was poured into 60-mm diameter petri dishes and allowed
to solidify for 30 min. The desired numbers of cells and the desired
antibiotics were then layered over the hardened agar in 1.5 ml of
molten (44°C) 0.33% low melting temperature Difco agar containing
DMEM and 10% FCS, and the dishes were allowed to sit for 10 min before
transfer to an incubator. Cultures were incubated at 37°C in 5%
CO2 with over 90% humidity for about 30 days. The colonies
were stained using a 0.25-mg/ml solution of [2-(p-iodophenyl)-3-
(p-nitropheyl)-5-phenyl
tetrazolium chloride (Sigma) for 6 to 24 h at 37°C in a 5%
CO2 incubator. Colonies containing more than 50 cells were
visually counted.
Analysis of gene expression profiles using cDNA arrays and
quantitative RT-PCR.
The commercial Atlas Human Cancer cDNA
Expression Array kit (catalog no. 7742-1) supplied by Clontech was used
to establish gene expression array profiles. The isolation of total
cellular RNA, the preparation of cDNA probes, and the hybridization
of the cDNA array membranes followed the protocols supplied by the manufacturer (Clontech manual PT3140-1, version PR89832). Following hybridization, the membranes were exposed to a PhosphorImager screen, the screen was developed, and the resulting hybridization images were analyzed and quantitatively assessed using ImageQuant software (Molecular Dynamics, Inc.).
Standard protocols for quantitative reverse transcriptase PCR (RT-PCR;
63) were used to evaluate the levels of specific gene transcripts in
cells. Total cellular RNA was isolated using Trizol reagent (Life
Technologies, GIBCO; catalog no. 15596) following the instructions of
the manufacturer. A mixture of 1 µg of RNA and 20 µM primer
poly(T25) oligonucleotides (Collaborative Research) was
reverse transcribed with 10 U of Moloney murine leukemia virus reverse
transcriptase (Promega Corp.) in a PCR thermal cycler at 42°C for 60 min to produce cDNA, and the mixture was then heat denatured and
cooled. Diluted samples of this cDNA were then mixed with two
different PCR primer pairs, one that is specific for the gene
transcript of interest and a second that is specific for a
constitutively synthesized message that acts as an internal reference
standard (e.g., hypoxanthine-guanine phosphoribosyltransferase [HPRT]) for the amplification reaction. The mixture was then PCR amplified in a thermal cycler for 25 cycles of 95°C for 1 min, 60°C
for 1 min, and 72°C for 1 min. The resulting RT-PCR products were
separated by either electrophoresis on a native agarose gel or
non-denaturing PAGE and detected and quantified by PhosphorImager analysis.
Nude mouse tumor formation assays.
Female BALB/c
(nu/nu) mice 4 to 6 weeks old were purchased from
The Jackson Laboratory, Bar Harbor, Maine. For tumor formation assays,
approximately 5 × 106 M/tet/Vect cells,
HA-HMG-I-expressing cells, or HA-HMG-Y-expressing cells in serum-free
medium were injected into either the mammary fat pad or the
subcutaneous space under the skin of the nude mice. The mice were
maintained in a germfree facility, and tumor incidence and size were
observed at regular intervals and documented.
Histology and immunohistochemistry.
Tumor nodules were fixed
in 10% formalin and embedded in paraffin, and 4- to 6-µm sections
were mounted onto slides and stained with hematoxylin and eosin by the
histochemistry core facility of the Center for Reproductive Biology,
Washington State University. Immunostaining of tissue sections that had
been preserved in Histochoice fixative (Sigma) followed standard
protocols. Antigen retrieval for vimentin detection was done by
microwave heating in citrate buffer. Before adding horseradish
peroxidase-conjugated reagents, endogenous peroxidase activity was
blocked by a methanol-H2O2 solution. For
immunolocalization studies, tissue sections were reacted with a primary
antibody (either a monoclonal mouse antibody or a polyclonal rabbit
antibody), washed, and then reacted with either biotinylated goat
anti-mouse immunoglobulin G (IgG; Vector Laboratories; 1:200) or
biotinylated goat anti-rabbit IgG (Bio-Rad; 1:1,000). Biotinylated
antibodies were visualized by reaction with horseradish
peroxidase-streptavidin reagent (Vector Laboratories) and
diaminobenzidine staining, followed by counter staining of nuclei with
Meyer's hematoxylin. The antibodies and dilutions used for tumor
immunostaining were anti-HA tag mouse monoclonal antibody 12CA5 (1:50
dilution), anti-pan-cytokeratin (C11) mouse monoclonal antibody (1:50
dilution; Santa Cruz Biotechnology), and anti-vimentin (V9) mouse
monoclonal antibody (1:50 dilution; Santa Cruz Biotechnology). Both the
mouse monoclonal anti-human procollagen alpha type I antibody M-38
(1:50 dilution; developed by J. A. McDonald) and the mouse
monoclonal anti-procollagen alpha type III antibody SP1.D8 (1:50
dilution; developed by H. Furthmayr) were obtained from the
Developmental Studies Hybridoma Bank, a contract facility supported by
the National Institute of Child Health and Human Development and
maintained by the Department of Biological Sciences, University of
Iowa, Iowa City. Rabbit polyclonal antibody 8691 (1:40 dilution), a
general anti-human alpha 1 (XI) collagen antibody, and rabbit
polyclonal antibody 1703 (1:40 dilution), specific for the v2 and vlav2
isoforms of human alpha 1 (XI) collagen associated with mesenchymal and
not cartilaginous tissues, were both generous gifts from Julia Oxford, Oregon Health Sciences University.
| |
RESULTS |
Endogenous HMGI(Y) protein levels correlate with the
tumorigenic potential of human mammary epithelial cells.
In
preliminary experiments, a number of different normal and malignant
human breast epithelial cell types were examined for both the levels of
endogenous HMGI(Y) proteins and the ability to respond to
artificial manipulation of these levels by the introduction of
antisense or dominant-negative HMGI(Y) expression vectors. Among those tested were two matched pairs of human mammary
epithelial cell lines (lines Hs578Bst and Hs578T and lines MCF-7 and
MCF-7/PKC). Each pair was originally derived from the same parent,
but the individually lines exhibit markedly different tumorigenic
phenotypes. The aneuploid Hs578T cell line originated from a human
mammary epithelial carcinoma (46) and grows aggressively
in soft agar, exhibits invasive properties in in vitro matrigel
outgrowth assays, and causes tumors that extensively metastasize in
immunodeficient nude mice (111). In contrast, the diploid
Hs578Bst cell line is a nontumorigenic myoepithelial cell line
established from normal breast tissue of the same patient
(46) but, unlike Hs578T, does not grow in soft agar, is
not invasive in matrigel assays, and does not cause tumors in nude
mice. The human MCF-7 breast epithelial cell line is estrogen receptor
positive and retains many of the biochemical and phenotypic
characteristics of normal mammary epithelial cells (76).
It has only a very limited ability to grow in soft agar
(128), is noninvasive in matrigel assays, and does not
form tumors in nude mice (111, 128). On the other hand,
the MCF-7/PKC cell line is a derivative of MCF-7 cells that has been
stably transformed with a transgenic cDNA that codes for the
transforming oncogene protein kinase C (PKC). This PKC-over
expressing cell line readily growns in soft agar in vitro, is invasive
in matrigel assays, and readily produces aggressive tumors that
metastasize when injected into nude mice (128).
Figure
1A shows a Western blot in which
the endogenous HMGI(Y) proteins (which migrate together under these
gel conditions)
present in MCF-7 (lane 3), MCF-7/PKC
(lane 2),
Hs578Bst (lane
5), and Hs578T (lane 4) cells were detected by specific
anti-HMGI(Y)
polyclonal antibody MR-19 (
99). Also
shown as a positive control
on this blot are the amounts of endogenous
HMGI(Y) proteins found
in the malignant HeLa cervical
adenocarcinoma cell line (lane
1), which is known to contain high
levels of these proteins (
81).
Figure
1B shows a Commassie
blue-stained gel of the H1 proteins
present in the same protein samples
used for the immunoblot in
panel A that served as an internal standard
for total protein
loading on the gels. From Fig.
1, it is evident that
the amount
of endogenous HMGI(Y) proteins present in these mammary
epithelial
cell lines closely correlates with their degree of in vivo
tumorigenicity,
a finding consistent with many previous studies
(
14,
116).
Specifically, the nontumorigenic Hs578 and
MCF-7 cell lines contain
low or nondetectable levels of endogenous
HMGI(Y) proteins whereas
these proteins are exceedingly abundant in
the highly malignant
Hs578T, MCF-7/PKC
, and HeLa cell lines.
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FIG. 1.
Expression of endogenous HMGI(Y) proteins correlates
with tumor phenotype. Equal amounts of total cellular protein (~5
µg) extracted from cells with different tumorigenic potentials were
separated by electrophoresis on a 4 to 20% gradient
polyacrylamide gel, and the proteins were transferred onto a
nitrocellulose membrane. (A) Western blot obtained with rabbit
MR19 polyclonal antibody against the HMGI(Y) proteins. Under the
electrophoretic conditions employed, the HMG-I and HMG-Y isoforms of
the proteins comigrate as a single band. Lanes: 1, HeLa cells; 2, MCF-7/PKC cells; 3, MCF-7 cells; 4, Hs578T cells; 5, Hs578Bst cells.
(B) Histone H1 proteins on the same membrane stained with Coomassie
brilliant blue. The H1 histones served as an internal standard for
equal protein loading on the gel.
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Antisense HMGI(Y) expression inhibits cell proliferation and
anchorage-independent growth.
To further explore the role of
HMGI(Y) in tumorigenesis, transfection experiments were performed
in order to modulate the amounts of these proteins in various
epithelial cell lines in order to determine whether there is a
mechanistic connection between the endogenous level of HMGI(Y)
proteins and cell proliferation rates or other phenotypic
characteristics. Previous studies have demonstrated that the endogenous
levels of HMGI(Y) proteins can be dramatically reduced in cells by
the introduction of antisense expression vectors (8, 102)
and that both antisense and dominant-negative expression vectors can
significantly inhibit the in vivo transcription of genes regulated by
the HMGI(Y) proteins (57, 58, 121). Figure
2 shows the results of experiments in
which either the antisense pRcCMVIGMH (57) vector or, as a
control, the empty parental pRcCMV vector without any insert was
individually transfected into MCF-7, Hs578T, and HeLa cells. The
transfectants were selected with G418 for a week, and pools of the
G418-resistant cells from each cell line were then subcultured and
their relative growth rates were determined by cell counting. As is
evident from Fig. 2, expression of antisense HMGI(Y) inhibits the
growth of MCF-7 cells (panel A) and Hs578T cells (panel B) but not HeLa
cells (panel C). Control experiments with cotransfected reporter genes as internal reference standards demonstrated that this difference in
the degree of growth inhibition in the different cell lines as a result
of antisense HMGI(Y) expression is not due to the differences in
transfection efficiency between the different cell lines (data not
shown). The question therefore arises of why growth of the MCF-7 and
Hs578T cells is significantly inhibited by antisense HMGI(Y)
expression but growth of HeLa cells is not. A likely answer lies in the
relative levels of endogenous HMGI(Y) proteins found in these
different cell lines (Fig. 1). As shown in Fig. 2D, when the levels of
HMGI(Y) proteins in the transfected cells were determined by
Western blot analysis, it was seen that the antisense vector reduced
the amount of proteins remaining in the MCF-7 and Hs578T cells (lanes 3 and 5, respectively) to a far greater extent than it did in the HeLa
cells (lane 7). Thus, it is reasonable to propose that the reason why
the growth rate of HeLa cells is unaffected by the antisense expression
vector is that it is unable to reduce the very high concentrations of
endogenous HMGI(Y) proteins found in HeLa cells to a level that
would slow cell proliferation. Nevertheless, these transfection results
clearly indicate that antisense HMGI(Y) expression impacts the
growth rate of cells with low-to-moderate levels of endogenous
HMGI(Y) proteins. Additional transfection experiments also
demonstrated that antisense expression inhibited the
anchorage-independent growth of some lines of mammary epithelial tumor
cells in soft agar (data not shown). Taken together, the results from
these two independent lines of antisense experiments indicate that
reduction of endogenous HMG-I(Y) levels in cells can lead to a
decrease in their proliferation rates and a reduction of the ability of
some tumor cells to grow anchorage independently.
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FIG. 2.
Expression of antisense HMG-inhibits tumor cell
proliferation. MCF-7 (A), Hs578T (B), and HeLa (C) cells were
transfected with 6 µg of either the empty pRcCMV expression vector
( ) or the pRcCMV-IGMH (antisense HMG-I) vector ( ). The
transfectants were selected with G418 at 150 µg/ml in culture medium
for 7 days, and then the viable cells were subjected to a cell growth
rate assay. Data were collected from three independent experiments with
four dishes for each time point per experiment. (D) Western blots of
HMGI(Y) proteins in MCF-7 cells (lanes 2 and 3), Hs578T cells
(lanes 4 and 5), and HeLa cells (lanes 6 and 7) transfected with either
the empty control vector (lanes 2, 4, and 6) or the antisense HMG-I
vector (lanes 3, 5, and 7). Approximately equal amounts of total
acid-soluble proteins were loaded into all of the lanes, and the
Western blots were probed with a rabbit polyclonal antibody (MR19)
against the HMGI(Y) proteins. On these gels, the HMG-I and HMG-Y
proteins comigrated as a single band.
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Overexpression of exogenous HMGI(Y) proteins promotes
anchorage-independent growth in soft agar.
A series of preliminary
transfection experiments demonstrated that transfection of MCF-7 cells
with a wild-type HMG-I or HMG-Y vector whose transcriptional expression
was driven by a highly efficient constitutive promoter resulted in a
marked increase in the total amount of HMGI(Y) proteins in the
recipient cells and also resulted in a significant increase in their
ability to grow anchorage independently in soft agar (data not shown).
There is, however, an intrinsic problem that interferes with an
unambiguous interpretation of these and other, similar studies
(37, 94, 132). Namely, owing to the fact that HMGI(Y)
proteins can influence the transcriptional activity of the host cell's
endogenous Hmgiy gene promoter (unpublished observations),
it is difficult, if not impossible, to distinguish between the amount
of HMGI(Y) proteins in the transfected cells contributed by
expression of the introduced transgene and the amount of these proteins
contributed by expression of the endogenous host cell gene.
To circumvent the problem of protein identification, plasmid expression
vectors were constructed that produced either HMG-I
or HMG-Y proteins
containing a nine-amino-acid peptide derived
from the HA protein of
influenza virus fused to their N-terminal
ends. Furthermore, these
transgenes were under the transcriptional
control of a
tetracycline-regulated promoter element (
30) and
were
transfected into a genetically engineered MCF-7 cell line
that
synthesizes the Tet regulatory protein, whose activity was
controlled
by the presence or absence of tetracycline in the cell
culture medium.
Specifically, in the Tet-off expression system
employed, genetically
engineered MCF-7 cells that were stably
transfected with either the
HA-tagged HMG-I or HMG-Y gene produced
large amounts of the
corresponding HA-tagged protein in the absence
of tetracycline in the
culture medium. In contrast, when tetracycline
was added to the cell
culture medium, production of the HA-tagged
transgenic protein was shut
off. Thus, by using this tetracycline-regulated
gene transcription
system, the expression of either the HA-HMG-I
or HA-HMG-Y transgenic
protein in MCF-7 cells could be reversibly
controlled at will and the
resulting levels of protein expression
could be quantitatively
assessed. A major advantage of using HA-tagged
proteins is that the
exogenous HMG-I(Y) proteins can be easily
distinguished from the
endogenous proteins in transgenic cells
by means of a monoclonal
antibody (12CA5) directed specifically
against the HA tag peptide that
was used for immunoprecipitation
of HA-tagged proteins during screening
for clones expressing high
levels of exogenous HMGI(Y) proteins
(
30). As shown in Fig.
3, by
using immunoprecipitation and Western blotting analysis,
several
independently derived transgenic MCF-7 clones that express
high levels
of either HA-HMG-I or HA-HMG-Y protein were identified.
In these
experiments, the HA-HMGI(Y) proteins were first immunoprecipitated
from the transgenic clones by using the 12CA5 antibody and then
the
HMG-I and HMG-Y proteins in the immunoprecipitates were identified
by
Western blotting using the MR-18 antibody directed against
wild-type
HMGI(Y) proteins. As is evident in Fig.
3, all of the
clonal MCF-7
cell lines containing the HA-tagged HMG-I (i.e.,
HA-I-Cs, HA-I-C7,
and HA-I-C14) or HMG-Y gene produced high concentrations
of the
respective HA-tagged transgenic protein when the gene was
transcriptionally on (i.e., in the absence of tetracycline). In
contrast, expression of either the HA-HMG-I or HA-HMG-Y transgenic
protein could be completely inhibited (i.e., turned off) in these
transgenic cells by the addition of 2 to 10 µg of tetracycline
(depending on the clone) to the cell culture medium, with the
exception
of clone of HA-I-C7, which required higher levels of
the drug to be
completely repressed (data not shown). Importantly,
control Western
blot experiments in which total acid-soluble proteins
isolated from the
on cells were run under electrophoretic conditions
that separated the
HMG-I from the HMG-Y isoform protein also demonstrated
high-level
expression of the HA-tagged transgenic proteins in
the on transfected
cells when an anti-HMGI(Y) antibody was used
as a probe (data not
shown).
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FIG. 3.
Identification of HA-HMG-I- and HA-HMG-Y-expressing
clones using immunoprecipitation and Western blots. Total cellular
proteins were isolated from 5 × 106 cells from
individual transgenic MCF7 clones treated without (ON) or with (OFF)
tetracycline at 10 µg/ml, and the extracts were immunoprecipitated
using the 12CA5 monoclonal antibody directed against the HA tag peptide
of the transgenic proteins. The precipitated proteins were separated by
SDS-15% PAGE and then transferred onto a nitrocellulose membrane.
Western blotting of the membrane was performed using the MR19 antibody
against the HMGI(Y) proteins. Under these electrophoresis
conditions, the HMG-I and HMG-Y isoform proteins comigate on the gel.
Individual transgenic clones expressing either the HMG-I (HA-I-Cs,
HA-I-7C, and HA-IC14) or the HMG-Y (HA-Y-C10, HA-Y-C19, and
HA-Y-C21) isoform protein are indicated. The +/ designation
indicates that tetracycline was added (+) to expressing ON cells ()
several days prior to protein extraction in order to inhibit transgene
expression.
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To test the effects of expression of the exogenous HMG-I or HMG-Y
protein, MCF-7 clones producing these transgenic proteins
were tested
in the soft agar assay for the ability to exhibit
anchorage-independent
growth. Figure
4 shows a typical example
of cells containing the exogenous HMG-I or HMG-Y-encoding gene
growing
in soft agar with (expression off) or without (expression
on)
tetracycline treatment. As summarized in Table
1, the HMG-Y-expressing
clones HA-Y-C10,
HA-Y-C19, and HA-Y-C21 produced, depending on
the clone, 13- to
39-fold more colonies in soft agar when the
gene is on than when it is
off. Similarly, HMG-I-expressing clones
HA-I-C14 and HA-I-Cs formed
about 10- to 20-fold more colonies.
We therefore conclude from these
experiments that overexpression
of either the HMG-I or HMG-Y isoform
protein in MCF-7 cells induces
reproducible changes in the phenotype of
these cells, including
an ability to exhibit anchorage-independent
growth in soft agar.
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FIG. 4.
Anchorage-independent growth of MCF-7/HA-I and
MCF-7/HA-Y clones. Assays for growth in soft agar of MCF-7/HA-Y cells
(A and B) and MCF-7/HA-I cells (C and D) were performed as described in
Materials and Methods. The agar medium in the plates shown in panels A
and C contained tetracycline at 10 µg/ml (i.e., transgene expression
was off), whereas those in panels B and D were without tetracycline
(i.e., transgene expression was on).
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TABLE 1.
Overexpression of HA-HMG-I(Y) proteins in MCF-7
cells leads to anchorage-independent growth in soft agar
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Array and RT-PCR analyses of gene transcription profiles in cells
overexpressing HMG-I and HMG-Y.
To identify the tumor-promoting
genes that are altered by HMG-I and HMG-Y overexpression in vivo,
Atlas Human Cancer cDNA Expression Arrays (Clontech Inc.) were used
to generate tumor gene expression profiles. The array was a carefully
selected collection of cDNA fragments arranged on nylon membranes
for rapid assessment of the differential gene expression of 588 tumor-associated genes. These well-characterized genes broadly
represented many crucial cellular signaling pathways and other complex
biological functions that have been associated at the molecular level
with changes underlying tumor progression and metastasis.
Using these arrays, gene transcription expression profiles were
compared in two clonal transgenic cell lines, HA-Y-C21 and
HA-I-C7,
which, as shown in Fig.
3, express high levels of HA-tagged
HMG-Y and
HA-tagged HMG-I proteins, respectively, when on. For
each of these
experiments, total RNA was isolated from both control
(i.e., off) cells
not expressing transgenic proteins and from
cells expressing high
levels of HA-tagged protein (i.e., on) and
reverse transcribed into
cDNA with
32P labeling. The two cDNA probe
populations were then separately
hybridized with two identical Atlas
Array nylon membranes on which
the 588 genes were spotted in duplicate.
The membranes were then
developed by exposure to a phosphorimaging
screen, and the expression
profiles of the two RNA populations were
compared side by side,
as shown in the representative result for
HA-HMG-Y-OFF and HA-HMG-Y-ON
profiles in Fig.
5. Similar hybridization profiles were
generated
for HA-HMG-I-OFF and HA-HMG-I-ON clones (data not shown).
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FIG. 5.
Array analysis of gene expression in transgenic cells
over- expressing HA-HMG-Y. Total RNA was isolated from control off
M/tet/Vect cells not expressing HMG-Y and on HA-Y-C21 cells expressing
high levels of HA-HMG-Y protein and reverse transcribed into cDNA
with 32P labeling. The two cDNA probes were separately
hybridized with identical Atlas Array nylon membranes (Clontech) on
which 588 genes associated with human cancer were spotted in duplicate.
The membranes were then developed by exposure to a phosphorimaging
screen, and the profiles of the two RNA populations were quantitatively
compared. (A) One small section of the large array membrane hybridized
with a probe from control off cells. (B) A corresponding region of a
duplicate membrane hybridized with a probe from HA-HMG-Y on cells. The
small diagonal arrows indicate corresponding positions on the two
membrnaes where significant differences in spot intensities can be
observed.
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The intensity of each of the hybridizing spots on the membrane was
quantified by PhosphorImager analysis using six housekeeping
genes
placed on each array membrane in order to normalize the
hybridization
signals. These housekeeping genes (which generated
nearly
equal-intensity hybridization signals for all of the samples
being
compared) were those for HPRT, ubiquitin, ribosomal protein
S9, 23-kDa
highly basic protein, beta-actin, and phospholipase
A2. In addition,
the hybridization signals of cDNAs surrounding
a specific target
cDNA were used both as references for normalization
and for
confirmation of relative
intensities.
The criterion we chose for identifying the most important
differentially regulated genes is that their expression had to be
altered fourfold or more compared to other populations of mRNAs.
A
4-fold difference as a standard is quiet stringent considering
that
many other studies using gene array analyses have considered
1.4- to
3-fold differences in expression to be biologically significant
(
2,
34,
64,
92). Furthermore, this degree of difference
in
transcriptional expression can be easily detected and independently
confirmed by quantitative RT-PCR techniques (see below). Table
2 lists some of the
proteins encoded by genes that are induced
or repressed in the
HA-Y-C21 cell line that is overexpressing
the transgenic HMG-Y
protein. Of the 588 genes in the array, 33
(5.6%) showed a 10-fold or
greater difference in their level of
expression and 52 (8.8%) were
differentially expressed in the
range of 9- to 4-fold. Thus, a total of
85 genes (~14%) had significantly
altered levels of expression
whereas 503 genes (~86%) exhibited
changes in expression of less
than fourfold. Of the 85 genes with
significantly altered expression,
72 (~12%) were upregulated and
13 (~2%) were downregulated,
resulting in an up-to-down ratio
of 6, indicating that significantly
more genes are positively
regulated by overexpression of the transgenic
HMG-Y protein. Similar
analyses of gene expression profiles in cells
overexpressing the
HMG-I isoform protein were performed yielding
generally comparable
results (data not shown).
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TABLE 2.
Proteins encoded by genes in transgenic human MCF-7
breast epithelial cells whose transcriptional expression is
modulated by overexpression of HMG-Y protein as determined by
cDNA array analyses
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To independently verify the reliability of the cDNA array
hybridization data, quantitative RT-PCR analyses were also performed
on
selected genes for three independently derived HMG-Y-expressing
clones
and two independently derived HMG-I-expressing clones.
As shown in Fig.
6, among the representative genes
analyzed by
RT-PCR were those coding for integrin-
1 (panel A),
matrix metalloprotease-13
(MMP13) (panel B), Notch-4 (panel C), and
Jagged-1 (panel D).
In each RT-PCR, amplification of HPRT transcripts
served as an
internal control. Quantitative assessment of these and
other RT-PCR
results indicated that the differential expression
patterns and
the relative expression levels of these genes are similar
to those
observed with the cDNA arrays, confirming the
reliability of the
array expression profile data. Interestedly, and
perhaps significantly,
these detection methods confirm that HMG-Y
and HMG-I upregulate
integrin-
1 and MMP13 to about the same extent,
but only HMG-Y
upregulates Notch-4 and Jagged-1. This is the first
experimental
data demonstrating differential regulation of gene
expression
by the HMG-Y and HMG-I isoform proteins. Northern blot
analysis
of representative mRNAs isolated from on and off cell
clones likewise
confirmed both the array and RT-PCR results
(unpublished data).
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FIG. 6.
RT-PCR analysis of gene expression profiles regulated by
HMG-I and HMG-Y indicates that the two protein isoforms do not have
identical functions in vivo. The DNA-free RNAs from two
HMG-Y-expressing clones (M8AC10 and M8AC19), two HMG-I-expressing
clones (M7CC7 and M7CCs), and control M/tet/Vect cells were subjected
to semiquantitative RT-PCRs as described in Materials and Methods. The
PCR products were electrophoretically separated by SDS-15%
nondenaturing PAGE. The results of this analysis demonstrate that while
both HMG-I and HMG-Y upregulate the expression of integrin 1 and
MMP13, HMG-Y, but not HMG-I, upregulates Notch-4 and Jagged-1. (A)
Co-RT-PCR of integrin 1 and HPRT messages from different transgenic
cell clones. Lanes: 1, M/Tet/Vec; 2, M8AC10; 3, M8AC19; 4, M8AC21; 5, M7CC7; 6, M7CCs. (B) Co-RT-PCR of MMP13 and HPRT messages from
different transgenic cell clones. Lanes: 1, M7CCs; 2, M7CC7; 3, M8AC21;
4, M8AC19; 5, M8AC10; 6, M/Tet/Vec. (C) Co-RT-PCR of Notch-4 and HPRT
messages from different transgenic cell clones. Lanes: 1, M/Tet/Vec; 2, M8AC10; 3, M8AC19; 4, M8AC21; 5, M7CC7; 6, M7CCs. (D) Co-RT-PCR of
Jagged-1 and HPRT messages from different transgenic cell clones.
Lanes: 1, M/Tet/Vec; 2, M8AC10; 3, M8AC19; 4, M8AC21; 5, M7CC7; 6, M7CCs.
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Genes regulated by HMG-Y overexpression.
The 588 cDNAs
present on the membrane array represent 13 different categories of
genes that have been reported to play key roles in different biological
processes of tumor progression and metastasis. These categories include
the following: A, cell cycle and growth regulators (79 genes); B,
intermediate filament markers (19 genes); C, apoptosis regulators (69 genes); D, oncogenes and tumor suppressors (29 genes); E, DNA damage
response, repair, and recombination genes (33 genes); F, cell fate and
development regulators (23 genes); G, receptors (42 genes); H, cell
adhesion, motility, and invasion genes (82 genes); I, angiogenesis
regulators (16 genes); J, invasion regulators (39 genes); K, Rho family
small GTPases and their regulators (21 genes); L, cell-cell
interaction genes (38 genes); M, growth factors and cytokines (98 genes).
The 85 genes whose transcriptional expression was significantly altered
in response to overexpression of HMG-Y protein were
distributed in all
13 categories. However, as shown in Fig.
7,
the patterns of alteration of gene
expression were found to be
quite different within the various
individual categories. For
example, in category H (i.e., cell adhesion,
motility, and invasion
genes) and category J (i.e., invasion
regulators), expression
of ~24% (20 of 82) and ~21% (8 of 39) of
the genes, respectively,
were significantly altered and all were
upregulated. In marked
contrast, in category E (i.e., DNA damage
response, repair, and
recombination genes), alteration of the
expression of only ~9%
(3 of 33) of the genes was observed and all
of these were downregulated.
It may be argued that the grouping and
classification of the cDNA
clones of the Atlas Array (as suggested
by the manufacturer) are
in some ways artificial and/or arbitrary;
nevertheless, the observed
expression pattern variations provide strong
evidence that overexpression
of the HMGI(Y) proteins differentially
regulates, either directly
or indirectly, the transcriptional activity
of various categories
of genes in vivo.
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FIG. 7.
Diversity of genes that are regulated by overexpressed
HMG-Y proteins. Transcription profiles determined by cDNA array
analyses indicate that overexpression of HMG-Y protein either up- or
downregulates a broad range of genes thought to be involved with
various aspects of neoplastic transformation, tumor progression, and
metastasis in human cells. The graph depicts the percentages of genes
in various groups or categories of cancer-related genes present on the
cDNA array membrane. Conservatively, we consider only those genes
whose expression is either up- or downregulated by a factor of 4 or
more to be the most important ones modulated in vivo by HMG-Y
overexpression. At the bottom is a list of the various categories of
genes represented on the cDNA array membrane. For example, in group
G, HMG-Y upregulates ~12% and downregulates ~10% of the receptor
genes present on the membrane.
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Overexpression of HMG-Y promotes tumor formation and metastasis in
nude mice.
The tumorigenicity and metastatic potential of MCF-7
cells overexpressing tetracycline-regulated exogenous HMG-I and HMG-Y were evaluated by injection of cells expressing these HA-tagged proteins from a number of different clones into immunodeficient BALB/c
(nu/nu) nude mice. When 5 × 106
cells were injected into either the mammary fat pad or the subcutaneous space, the HMG-Y-expressing clones (HA-Y-C19 and HA-Y-C21) formed tumors. No mice injected with HA-I-C7 clones expressing the HMG-I protein or control clones expressing only the empty M/tet parental vector developed tumors within a 3-month observation period, as summarized in Table 3. When inoculated
subcutaneously into the backs of mice, HA-Y-C19 cells formed solid
primary tumor nodules with local invasion of the surrounding tissues
(data not shown). Morphological examination indicated that these tumors
were well circumscribed and bordered by what appeared to be a network
of thin, fibrous tissue. Histological examination of the
HA-HMG-Y-induced primary tumors revealed that they were composed of
solid masses of tightly packed carcinoma cells with a highly necrotic
center. No metastases of cells to other areas of the body were observed when cells were injected subcutaneously (unpublished observations).
In marked contrast, when HA-HMG-Y-expressing cells were inoculated
directly into mammary fat pats, not only were primary tumors
formed at
the site of injection but numerous secondary tumors
were found to have
metastasized into most of the mesenchymal tissues
of the abdominal
cavity, as well as into the connective tissues
surrounding and
supporting various organs (Fig.
8A to C).
The
sites of metastasis included the mesentery supporting the large
and
small intestines and the colon, the greater omentum, the mesentery
lining of the diaphragm, and the fibrous capsule around the kidneys.
Histological examination revealed that these secondary metastatic
tumors were often collagen-rich carcinomas that showed a complex,
pleiomorphic cytology with different regions of the tumors exhibiting
markedly different morphological characteristics (Fig.
9). Some
areas of the
metastatic carcinomas exhibited a compact, more-or-less
epithelial
cell-like, cobblestone morphology (Fig.
9A and B),
whereas other areas
were more loosely organized and often surrounded
by, or infiltrated
with, collagen fibers (Fig.
9C). In some cases,
the metastatic tumors
were not confined to the mesentery and fibrous
connective tissues but
exhibited considerable anaplasia with invasion
of underlying organ
stroma structures, such as the striated muscle
tissue of the diaphragm
(Fig.
9B). Importantly, the presence of
blood vessels within or near
masses of tumor tissue was commonly
observed (e.g., Fig.
9A and B),
suggesting that the tumor cells
had induced angiogenesis and/or that
they had extravasated from
nearby blood vessel during their migration
and metastasis.
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FIG. 8.
MCF-7 cells overexpressing HMG-Y protein form tumors in
nude mice. Metastatic tumors developed in the mesentery membranes and
in the lining of the peritoneal cavity when the mammary fat pads of
nude mice were injected with approximately 5 × 106
cells (in this case, clone HA-Y-21) expressing the HA-HMG-Y protein.
Many of the metastatic tumor nodules were seen in mesenteric membranes
within and lining the peritoneal cavity (A), as well as in the greater
omentum and other mesenteries supporting the small intestines (B) and
in the mesenteries supporting and lining the colon (C).
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FIG. 9.
Histological analysis of sections of metastatic tumors
formed in nude mice overexpressing HA-HMG-Y protein. (A to F)
Hematoxylin-and-eosin staining. (G to L) Immunohistochemical analysis
with specific antibodies and counterstaining with Meyer's hematoxylin.
(A) Paraffin section of a secondary metastatic carcinoma with a
cobblestone-like epithelial morphology (stained blue) formed by
HA-Y-C21 cells located near a blood capillary shown in the upper part
of the picture. Magnification, ×100. (B) Section of a secondary
metastatic carcinoma formed by clone HA-Y-C21 cells showing that the
tumor (blue) has invaded the striated muscle of the diaphragm (pink
with striations) in close proximity to blood capillaries filled with
red cells. Magnification, 100×. (C) Region of a metastatic tumor
showing the coexistence of collage-rich and non-collagen-rich areas
within the tumor mass. In this section, an island of cobblestone-like
carcinoma cells containing little extracellular collagen (lower left)
is surrounded by areas of more elongated, spindle-like cells with a
dense extracellular collagen matrix (stained pink). Magnification,
40×. (D to F) Regions of carcinosarcoma-like cells found in three
independently derived HA-Y-C21-induced tumors. Magnification, 60×.
(G) Carcinoma region reacted with a control nonimmune mouse IgG
antibody. Magnification, 100×. (H) Carcinoma region reacted with mouse
monoclonal IgG (12CA5) specifically directed against the HA tag
peptide. Magnification, 100×. Note the prominent nuclear staining
(arrows), although cytoplasmic staining is also evident. (I)
Carcinosarcoma-like region reacted with anti-HA tag mouse monoclonal
IgG. Magnification, 40×. (J) Carcinosarcoma-like region reacted with
mouse monoclonal IgG (M-38) directed against human fibroblast
procollagen type I (which has no cross-reactivity with mouse
collagens). Magnification, 40×. (K) Disorganized region of tumor
reacted with a polyclonal rabbit antibody (1703) directed against the
v2 and vlav2 isoforms of human alpha 1 (XI) collagen associated with
mesenchymal, but not cartilagenous, tissues. Magnification, ×60. (L)
Mixed carcinoma and carcinosarcoma-like regions reacted with mouse
monoclonal IgG (V9) directed against human vimentin (no reaction with
vimentin of mouse origin or other intermediate filaments.
Magnification, ×40.
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Another commonly observed feature in many of the HMG-Y-induced
metastatic tumors was the presence of areas of less organized,
more
elongated cells with variant morphologies that contained
appreciable
concentrations of extracellular collagen. For example,
as shown in Fig.
9C, islands of epithelioid carcinoma cells with
little apparent
collagen were often observed adjacent to nearby
areas of more elongated
and spindle-shaped tumor cells surrounded
by a dense, collagen-rich
matrix. Nevertheless, as illustrated
in Fig.
9D to F, perhaps the most
interesting and potentially
significant feature observed in the
metastatic tumors was the
presence of localized areas of less
differentiated cells that
seemed to possess an unorganized,
carcinosarcoma-like morphology
(
38). These results suggest
that some of the metastatic tumor
cells have undergone a
phenotypic change from their original epithelial
cell-like
phenotype to a more mesenchymal cell-like phenotype
as a
consequence of overexpression of the HMG-Y protein. Such
phenotypic
changes require extensive alterations in gene expression
patterns and
are called EMTs (
51). The phenomenon of EMT is
a common
feature of both embryonic development (
123) and advanced
epithelial tumors where epithelial cells have dedifferentiated
to a
more fibroblast-like state and regained the ability to invade,
migrate,
and/or proliferate in an uncontrolled fashion (
9).
The EMT
of epithelial tumors is characterized by a number of phenotypic
changes, including loss of cell-to-cell interactions; the acquisition
of a more fibroblast-like cellular morphology, the downregulation
of
epithelial cell products such as cytokeratins, the upregulation
of
mesenchymal cell marker proteins such as vimentin, a marked
increase in
the production of cytoplasmic procollagen and extracellular
collagen
fibers, and the acquisition of a motility machinery that
allows cells
to interact in three dimensions with the extracellular
matrix (ECM)
(
9,
10).
HMG-Y overexpression induces EMT in tumor cells.
Immunohistochemical staining of tumor sections was performed using a
number of monoclonal and polyclonal antibodies directed against
specific proteins to confirm that the tumors formed in nude mice had,
indeed, originated from the transgenic MCF-7 cells and to further
investigate the possibility that these cells had undergone an EMT. The
antibodies used for these studies were selected on the basis of the
gene products indicated by cDNA array analysis to be produced in
the transgenic cells (Table 2) and on the basis of the histological
appearance of the tumors themselves (Fig. 9A to F). For example,
reaction of tumor sections with a monoclonal antibody directed against
the HA tag peptide show that the transgenic human HMG-Y protein was
localized primarily in the nuclei (but with detectable cytoplasmic
staining) of cells in both regions of carcinoma morphology (Fig. 9H)
and regions of carcinosarcoma morphology (Fig. 9I). These results
unambiguously demonstrate that the tumors originated from the injected
human MCF-7 cells overexpressing the HMG-Y protein, a fact also
confirmed by the ability to reisolate tetracycline-regulated transgenic
cell lines from the tumors by growth in selective medium (data not shown).
The list of genes differentially regulated by HMG-Y shown in Table
2
indicates that a number of types of collagen proteins,
especially
pro-alpha I-type collagens, are dramatically upregulated
in the
HMG-Y-overexpressing transgenic cells. This finding is
confirmed by the
immunoreactive staining of the cytoplasm of tumor
cells with a peptide
monoclonal antibody that is specific for
human fibroblast pro-alpha
I-type collagen and does not cross-react
with mouse proteins (Fig.
9J).
Likewise, a polyclonal rabbit antibody
directed against the isoforms of
human alpha I (XI) collagen that
are associated with mesenchymal and
noncartilaginous tissues stains
the extensive extracellular collagen
accumulations found in tumor
tissues, indicating that the tumor cells
are synthesizing and
secreting these proteins (Fig.
9K). These findings
are significant
because excess collagen production is a characteristic
feature
of EMT. High levels of type I and type III procollagen proteins
are also observed in the most aggressive human breast cancers,
with the
level of collagen expression being positively correlated
with the
degree of malignancy of the tumor (
12,
71). The cDNA
array analyses also indicated that expression of the gene coding
for
the classical mesenchymal cell marker protein vimentin, a
diagnostic
protein for EMT, is significantly upregulated (~5.6-fold)
in
transgenic cells. This expression is confirmed by the reaction
of tumor
tissue with a monoclonal antibody that is specific for
human vimentin
and does not cross-react with other human intermediate
filament
proteins, with mouse vimentin, or with other murine proteins
(Fig.
9L).
Finally, a pancytokeratin monoclonal antibody against
human proteins
that does not cross-react with mouse cytokeratins
also stains the
cytoplasm of tumor cells, consistent with the
array analyses (Table
2)
that indicated that cytokeratins 10
and 12 are upregulated in the
transgenic cells (data not shown).
Although cytokeratins are usually
thought of as epithelial cell
differentiation markers, the most
aggressive human breast cancers
that have undergone EMT coexpress both
vimentin and cytokeratins
(
28,
109), as do some malignant
mouse mammary epithelial tumors
that have undergone in EMT
(
113). Together, these immunostaining
results not only
demonstrate the transgenic human cell origin
of the tumors formed in
nude mice but are also entirely consistent
with previous results
indicating that some of the HMG-Y-overexpressing
cells having undergone
an
EMT.
| |
DISCUSSION |
HMGI(Y) proteins are causal agents in tumor progression.
As recounted in the introduction, published reports of human clinical
studies, as well as numerous studies of animal and cell culture model
systems, have firmly established that aberrant expression or
overexpression of members of the HMGI(Y) gene family positively correlates with neoplastic transformation and metastatic progression of
a wide variety of tumors. Nevertheless, the results reported here
represent the first direct demonstration that overexpression of the
HMGI(Y) proteins is causally involved in modulating the expression
of genes involved in all stages of tumor progression from
transformation to anchorage-independent growth in vitro, to the
formation of primary and metastatic tumors in nude mice, and the
induction of in EMT in vivo. The current data unambiguously confirm the
long-held suspicion the Hmgiy is a bone fide proto-oncogene and also provide insights into probable molecular events underlying its
in vivo mode of action.
The results of gene array analyses revealed that induced overexpression
of HMGI(Y) proteins in human mammary epithelial cells
modulated,
either upward or downward, the transcriptional expression
of
constellations of genes associated with regulation of cell
signaling,
cell proliferation, cell migration, tissue invasion,
induction of
angiogenesis, and metastatic colonization (Table
2; Fig.
7). Whether
the HMGI(Y) proteins function directly as
transcription factors by
regulating the promoters of these genes
or whether their effect is
indirect and is mediated through other
intermediary genes or proteins
is unknown. Nevertheless, it is
quite remarkable how closely the
cDNA expression array profiles
induced by HMGI(Y)
overexpression correspond to the phenotypic
patterns of the metastatic
tumors formed by transgenic cells injected
into nude mice. Examination
of the pathology in host mice clearly
showed that the HMG-Y
transgene-containing cells injected into
the mammary fat pad had
metastasized to many distant sites in
the mesenteries lining the body
cavity and various organs and
had also invaded muscle tissues, such as
the diaphragm (Fig.
8 A to C). These biological results could only be
achieved if many
of the tumor progression gene products detected by the
cDNA array
analyses were indeed functional in vivo, a conclusion
also supported
by the histochemical and immunolocalization results
shown in Fig.
9.
In vivo, the HMG-I and HMG-Y isoform proteins are not functionally
equivalent.
One of the most unexpected findings in this study was
that transgenic cells overexpressing the HMG-Y isoform protein were much more effective in inducing both primary and metastatic tumors when
injected into nude mice than were transgenic cells overexpressing the
HMG-I isoform protein (Table 3). In less than 2 months following injection, over half (four of seven) of the nude mice injected with
cells overexpressing the HMG-Y protein had developed tumors whereas
during this same time period none of the five mice injected with cells
overexpressing HMG-I developed tumors. Only much later (after about 4 months) did one of the mice injected with cells overexpressing the
HMG-I protein develop small primary tumors (data not shown). Thus,
HMG-Y is far more efficient in promoting tumor progression in vivo
than HMG-I even though these isoform proteins differ only by an
internal deletion of 11 amino acids in the former. These results were
initially puzzling, since expression of both isoform proteins
efficiently induced anchorage-independent growth in soft agar (Fig. 4;
Table 1). Nevertheless, they were consistent with an earlier report
demonstating that the tumor promoter 12-O-tetradecanoylphorbol acetate preferentially induces HMG-Y
protein expression in transformation-sensitive, but not in
transformation-resistant, mouse JB6 epithelial cells in culture (22). The cumulative data therefore strongly imply that,
in addition to sharing many common biological functions, the HMG-I and
HMG-Y proteins also exhibit important differences in their in vivo
functions. This view is further supported by the finding that in vivo,
the HMG-Y protein contains many more secondary biochemical modifications than the HMG-I isoform and that these modifications differentially affect the substrate-binding properties of the two
proteins (5). A potential difference in function between the two isoforms is likewise supported by the results of the cDNA expression profiles and RT-PCR experiments reported earlier (Fig. 5 and
6; Table 2). The data from these experiments demonstrate that the HMG-I
and HMG-Y proteins do, indeed, regulate the transcriptional expression
of a large constellation of common genes but also indicate that, in
addition, each protein can regulate its own independent set of
genes. For example, both proteins upregulate the expression of genes
such as those which encode the p38 mitogen-activated protein kinase
(MAPK), collagen type XVIII, integrin 1, MMP13, and TIMP
(tissue inhibitor of MMP3), but on the other hand, only HMG-Y
upregulates genes such as those for Notch-4, Jagged-1, and Wnt-10B
(Fig. 6) and only HMG-I downregulates the genes for Wnt-10B and Wnt-8B
(data not shown). It is reasonable to suspect that the constellation of
genes regulated by both the HMG-I and HMG-Y proteins is responsible for
the anchorage-independent growth in soft agar of cells overexpressing
these proteins. On the other hand, it is likely that some combination
of the individual genes that are either up-or downregulated by the
HMG-Y or HMG-I isoform protein is responsible for the much greater in
vivo tumorigenic and metastatic potential of the HMG-Y protein. It
should be stressed that the cDNA array analyses reported here
sampled the transcriptional activity of only a very limited number of
cellular genes. To fully address the issue of the extent to which the
HMG-I and HMG-Y isoform proteins have distinct biological functions in
tumor progression, a much more comprehensive transcriptional expression
analysis employing high-density oligonucleotide array technology is necessary.
Genes modulated by HMGI(Y) overexpression.
The list of
genes differentially regulated by HMG-Y overexpression in Table 2
demonstrates a broad range of functional activity of the HMG-Y
architectural transcription factor. During mouse development, very high
expression of HMGI(Y) proteins has been detected in all embryonic
tissues up to 8.5 days of gestation, a period during which the most
critical events of organogenesis start to take place, and its
expression continues at lower levels to the end of gestation (17,
78). Based on these patterns of embryonic expression, the
suggestion has been made that the HMGI(Y) proteins are not only
involved with cell proliferation but also are likely to be involved
with the establishment of various cell types during organogenesis. This
suggestion is supported by the present data, which demonstrate that
overexpression of HMG-Y significantly upregulates genes known to be
involved in development, such as those for Frizzled 5 (161-fold),
Wnt-13 (14.6-fold), Wnt-10B (4.1-fold), as well as Jagged-1 and its
receptor, Notch-4 (7.5- and 4.1-fold, respectively). HMG-Y also
upregulates a number of genes involved in both cell cycle regulation
and signal transduction. These include the genes for CLK-1 (14.6-fold),
cdc25A (98.6-fold), cdc25B (9.1-fold), cyclin C (5.1-fold), JNK2
(7.4-fold), and p38 MAPK (5.7-fold) and others. Each of these genes has
been demonstrated not only to be involved in either the control of cell
cycle or signaling processes but also to participate in neoplastic
transformation and/or tumor progression in various systems. For
example, the genes coding for the protein phosphatase enzymes cdc25A
and cdc25B are well known as G1/S transition and
G2/M transition regulators, respectively. In addition, they
are all proto-oncogenes whose overexpression has been implicated in
many types of tumors, including breast cancers (60, 135).
HMG-Y modulates expression of EMT genes.
Although studies
analyzing the molecular characteristics of both mesenchymal and
epithelial cells are still at an early stage, a group of genes has
already been identified that is both characteristic of these two
different cell types and influential in EMT (reviewed in reference
9). Analysis of the cDNA profile of epithelial cells
overexpressing HMG-Y protein indicates that transcriptional expression
of several EMT marker genes is modulated in HMG-Y overexpressing cells
(Table 2). Among the group of upregulated genes are type I, III, and IV
collagens, vimentin, certain cytokeratins, a number of matrix
metalloproteases, basic FGF receptor 1, FGF7, and SF (scatter
factor-hepatocyte growth factor) protein. Amoung the group of
downregulated genes are those coding for the EGF receptor and TGF-
receptor III, as well as those coding for both the Met and FGFR2b proteins.
As noted previously, excess collagen production is a characteristic
feature of EMT and clinical studies have correlated high
levels of
procollagen proteins with the most aggressive human
breast cancers and
those with the worst prognosis (
12,
71).
The cells of many
of these highly malignant breast tumors also
coexpress elevated levels
of both vimentin and cytokeratins (
29,
108,
113).
Extracellular and membrane-bound MMPs are a family of enzymes involved
in much of the degradation of ECM molecules that occurs
in
developmental and pathological processes, including tumor invasion
and
metastasis and EMTs (
84,
113). Overexpression of HMG-Y
in
human epithelial cells induces the expression of MMP16 (a potent
activator of gelatinase MMP2) 13.5-fold, that of MMP13 (collagenase
3)
4.7-fold, that of MMP8 (collagenase 2 or neutrophil collagenase)
4.3-fold, and that of MDC9 (a membrane-anchored
metalloprotease-disintegrin)
5.8-fold. Investigations have implicated
the expression of all
of these MMPs in the in vivo spread of tumor
cells. For example,
increased expression of MMP13 has been observed in
many different
human cancers, including breast carcinoma
(
53). Interestingly,
in these breast carcinomas, MMP13 is
expressed by tumor stromal
cells or fibroblast-like tumor cells (also
referred to as mesenchymal
cells) (
53). In addition to
upregulating metalloproteases, overexpression
of HMG-Y also increases
the metalloprotease inhibitor TIMP-3 and
plaminogen
activator inhibitors PAI-1 and PAI-3 4.6-, 5.4-, and
74.2-fold,
respectively. Overexpression of both TIMP-3 (
125)
and
PAI-1 (
101) has been detected in breast carcinomas, and
high
levels of PAI-1 are associated with a higher incidence of lymph
node involvement in breast cancer and poor response to tamoxifen
therapy (
65). The role of MMP-inhibitory proteins such as
TIMP-3
and PAI in tumor invasion and metastasis is unresolved and
controversial,
but many investigator now lean toward the view that
there is a
delicate interplay between these inhibitory proteins and the
MMPs
that regulate the invasiveness of tumor cells by controlling
cellular
adhesion to, and release from, the ECM (
27).
SF and its specific receptor, c-Met, have been implicated in EMTs
during both development (
110) and tumor invasion and
metastasis
(
9). SF is a potent pleiotrophic cytokine with
multiple biological
effects on various epithelial cells, including
morphogenesis,
proliferation, migration, and differentiation. It
promotes development,
regeneration, and reconstitution of normal
organ architecture.
The normal physiological roles played by SF
and c-Met have been
established in embryological studies. In the
majority of tissues,
SF is expressed exclusively on mesenchymal cells
and c-Met, a
receptor tyrosine kinase, is expressed on epithelial cells
(
110).
The exchange of signals between the mesenchymal and
epithelial
cells has long been recognized as a major driving force in
embryogenesis,
and SF and c-Met constitute one such major paracrine
signaling
system (
114). In the development of epithelium
lined organs,
for example, SF-expressing mesenchymal cells play an
essential
role in inducing epithelial cells to proliferate, migrate,
and
differentiate in a coordinated fashion. The c-Met receptor on
the
surface of the epithelial cells transduces the SF signal to
induce
motogenic, mitogenic, and morphogenetic and differentiation
activities.
The effects of SF and c-Met on the EMT and the generation
of motile
cells in normal developmental processes appear to be
indistinguishable
from the generation of motile carcinoma cells
during tumor metastasis
in vivo. Clinical studies have shown that
the level of expression of SF
by breast carcinoma cells correlates
with the state of tumor
progression (
134). Interestingly, overexpression
of HMG-Y
in human breast epithelial cells upregulates SF 14.5-fold
and
downregulates c-Met 4.9-fold (Table
2). This strongly suggests
that, in
some cases, overexpression of HMG-Y promotes an EMT by
coordinately
upregulating the expression of mesenchymal cells
genes and
downregulating the expression of epithelial cell
genes.
FGF7, which is produced by fibroblastic cells, and its receptor,
FGFR2b, which is found on the surface of epithelial cells,
represent
another example of a paracrine system that actively
mediates
communication between mesenchymal and epithelial cells
during embryonic
development (
130). In this case, too, overexpression
of
HMG-Y results in 4.1-fold upregulation of FGF7 and 29.3-fold
down-regulate of FGFR2b in transgenic epithelial cells, again
reinforcing the idea that overexpression induces EMT by coordinated
up-and downregulation of
genes.
HMGI(Y) upregulates integrins and their signaling
pathways.
Integrins are a large family of transmembrane
heterodimeric proteins that are the principal receptors on
mammalian cells for the binding of ECM proteins. They are also the
primary mediators of cell-cell interactions. At least 20 different
integrin heterodimers, comprised of 14 types of subunits and 8 types of subunits, are known (62). Many of the
different and subunits come in alternatively spliced forms
which are expressed in a tissue-specific manner with different cell
types producing multiple, but distinctive, sets of heterodimers.
Although expressed on many different cell types, integrins are
expressed predominantly in the mesenchyme during embryonic development
(36).
Integrins functions as transmembrane linkers (or integrators) and
mediate the interactions between the cytoskeleton, cytoplasmic
kinases,
and the ECM. Owing to this property, integrins enable
the inside of
cells and the ECM to communicate with each other
across the plasma
membrane in a bidirectional fashion (
20,
41).
That is, the
extracellular binding activity of integrins is regulated
from the
inside of the cell (inside-out signaling) while binding
of the ECM
elicits signals that are transmitted into the cell
(outside-in
signaling). As one of the key signaling transducers,
integrins
influence cell shape, movement, proliferation, development,
differentiation, and death (
62). These cellular effects
involve
changes in the expression of many genes, including those coding
for integrins themselves. Transcription factors are often the
final
executors of signals emanating from integrins and mediate
the changes
observed in gene expression
patterns.
Overexpression of HMG-Y significantly upregulates the expression of
several integrin genes, including those coding for the
1,
3,
8,
1,
6,
9, and
E proteins, as well as those coding
for
certain of the cellular components of integrin signaling pathways,
such
as integrin-linked kinase (ILK), protein tyrosine kinase
2 (also known
as cell adhesion kinase
), c-Jun N-terminal kinase
2, p38 MAPK, and
RhoC, a member of the Rho family of small GTP-hydrolyzing
proteins
(Table
2; Fig.
10). Since the cDNA
array used in the
present expression studies included only a restricted
subset of
the many genes known to be involved in integrin signaling,
the
data set is incomplete. Nevertheless, even with the limited
amount
of information available, it is clear that overexpression of
HMG-Y
upregulates the expression of many integrins and their signaling
pathways and these, in turn, are likely to be causally involved
in
tumor progression.
View larger version (24K):
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|
FIG. 10.
Diagram of integrins and their signaling pathways that
are upregulated by overexpression of HMGI(Y) proteins in human
mammary epithelial cells (see text for details). PyK2, protein tyrosine
kinase 2; JNK2, c-Jun N-terminal kinase 2; FAK, focal adhesion kinase;
ERK, extracellular response kinase; Rho/Rac G., Exc. Fac., Rho/Rac G
exchange factor.
|
|
Of particular interest is the fact that HMG-Y overexpression
upregulates transcription of the integrin
1 gene. Integrins
containing the
1 subunit are normally activated through binding
to
components of the ECM. The cytoplasmic domain of the
1 subunit
is
thought to interact directly with components of the actin cytoskeleton,
such as
actinin and talin, thereby forming focal adhesion plaques
(
49) in the cytoplasm at points of contact between
integrins
and the ECM. The essential nature of the integrin
1
protein in
linking the ECM and the actin cytoskeleton was shown by gene
knockout
experiments which demonstrated that
1 integrin deficiency
in
mice results in inner-cell mass failure and peri-implantation
lethality (
112). Elevated levels of
1 integrins are
correlated
with high proliferation potential and also serve as markers
for
stem cells (
70,
90,
120). Overexpression of
1
integrin has
been detected in many types of cancers and associated with
metastasis.
In human breast cancer, the total
1 integrin level is
significantly
higher in malignant epithelial cells than in
nontumorigenic cells
(
129). By using a function-blocking
antibody that inhibits
1
integrin, malignant breast epithelial cells
have been demonstrated
to undergo a striking morphological and
functional normalization;
moreover, these reverted cells became growth
arrested and were
found to exhibit reduced tumorigenicity in nude mice
(
129). In
ovarian carcinomas, aggregation of
1
integrins mediates tumor
invasion and metastasis by stimulation of the
expression of MMP2
and MT1 MMP (
31). The notion of
1
integrins promoting metastasis
has been supported by other studies as
well. Targeted disruption
of the
1 integrin gene in T-cell lymphomas
greatly reduces their
metastatic capacity (
115).
Reciprocally, re-expression of
1
integrin in
1-deficient
epithelial cells led to a malignant,
cell-scattering phenotype that
requires activation of the Rho
family of GTPases (
45).
Most interestingly, small-cell lung
cancer cells are apparently able to
utilize
1 integrin to resist
chemotherapeutic agents
(
103). In small-cell lung cancer tumors,
1
integrin-mediated adhesion of cells to ECM proteins around
the tumor
enhances tumorigenicity and activates protein tyrosine
kinase activity
that inhibits the activation of caspase-3 and
apoptosis that is
normally induced by chemotherapeutic agents
(
103).
The
9 protein is a recently identified integrin subunit that forms
only one integrin heterodimer, the
9
1 integrin (
90).
Expression of
9
1 has been detected on various types of cells,
including human carcinomas (
90,
120).
9
1 promotes
cell proliferation
and migration when it interacts with tenascin, one
of the components
of the ECM (
136), the gene for which is
also upregulated (4.2-fold)
by HMG-Y overexpression (Table
2). The
cellular effects of
9
1
are mediated by phosphorylation and
activation of the protein
tyrosine kinase focal adhesion kinase (FAK)
and the mitogen-activated
kinase Erk2 (
136). Recent
studies have shown that
9
1 expressed
on neutrophil cells
interacts with VCAM-1, which is expressed
on activated endothelial
cells, and mediates neutrophil passage
through the endothelial cell
layer (
105,
120). The implication
of these studies in
terms of cancer progression is striking. Overexpression
of
9
1 in
metastatic cells may well lead to tumor cells gaining
extravasation
capabilities and thus allow them to colonize various
sites at the end
state of the metastatic process. As with
9
1,
overexpression of
6
1 can also contribute to cell proliferation
and migration in
mammary and prostate carcinomas (
23,
80).
Overexpression
of HMG-Y upregulates the integrin
1 subunit 10.7-fold
and the
integrin subunits
9,
6, and
1, 5.2-, 60.6-, and 10.7-fold,
respectively (Table
2).
As diagrammed in Fig.
10, these results support a model in which HMG-Y
promotes tumor cell proliferation and metastasis, at
least in part,
through control of the level of expression of integrins
and their
signaling pathways. The following findings support such
a model.
Engagement of integrin
1-containing heterodimers activates
ILK, a
focal adhesion plaque-associated serine/threonine protein
kinase that
is emerging as a key regulatory protein that functions
at one of the
early convergence points of integrin-and growth
factor-stimulated
signaling (
25,
50). The active form of ILK
phosphorylates
and activates PKB (also called Akt), a potent antiapoptosis
enzyme that
enable many types of tumors to evade programmed cell
death (
26,
32). ILK also directly phosphorylates glycogen
synthase kinase
3, glycogen synthesis kinase thereby inhibiting
3 activity and leading
to a dramatic relocalization of
-catenin
to the cell nucleus
(
124). In the nucleus,
-catenin pairs with
lymphoid
enhancer factor 1 to form a complex that acts as an active
transcription factor and plays an essential role in tumor progression
(reviewed in reference
60). Interestingly, overexpression
of
ILK in epithelial cells leads to a complete EMT that is accompanied
by a loss of cell-cell adhesions and an increase in vimentin expression
(
88,
133). In addition, overexpression of ILK also leads
to
anchorage-independent cell growth, as determined by growth in
soft
agar, suppression of apoptosis, increased invasion of the
ECM, and
tumorigenicity in nude mice (
50,
93).
Heterodimers containing integrin
1 subunits are also know to be
involved with activation of protein tyrosine kinase 2, the
p38 MAPK,
the focal adhesion kinase, and the Shc signaling pathways
(reviewed in
references
20,
41, and
127). Overexpression
of integrins
and the activation of one or more of the resulting
signaling pathways
are often associated with both tumor progression
and increased
metastatic potential of cancer cells (
11,
72,
73,
106,
119).
Integrins also regulate cell spreading, migration, and scattering
through activation of the Rho family of proteins, GTPases
that interact with multiple downstream effectors to control the
actin
cytoskeleton (
47,
126). Recent work by Clark et al.
(
21)
has also demonstrated that overexpression of the
wild-type RhoC
protein alone induces a poorly metastatic human melanoma
cell
line to become highly metastatic when injected into nude mice
whereas a dominant-negative Rho inhibits metastasis. Analysis
of the
phenotype of cells expressing dominant-negative Rho or
RhoC indicated
that the RhoC GTPase is important in invasion by
tumor cells
(
21). Importantly, as shown in Table
2, overexpression
of
HMG-Y induces the upregulation of a number of Rho family proteins,
including RhoC (3.9-fold), RhoG (3.7-fold) RhoGDI
(3.5-fold),
and
Rho-Rac G exchange factor (3.8-fold), a finding consistent
with the
proposed model the involvement of the HMGI(Y) proteins
in the
promotion of tumor metastasis (Fig.
10).
Implications for human cancer.
There are more than 100 distinct types of cancer in humans, and subtypes of tumors can be found
in each specific organ. Available evidence suggests that cancer cells
have defects in the regulatory mechanisms that govern normal cell
proliferation and homeostasis. The complexity and variety of tumor
types raise the question of how many distinct regulatory mechanisms
within a normal cell must be disrupted in order for it to become
cancerous. The current view is that tumorigenesis is a multstep process
(i.e., the multihit theory) and that each step reflects a genetic
alteration that drives the progressive transformation of normal human
cells into highly malignant derivatives. However, all types of cancers
have common hallmarks, i.e., have acquired several common capabilities (48). Therefore, the simplicity elucidated from the
complexity provokes another, even more important question i.e. whether
there is an essential mechanism that is "hard wired" throughout all types of cancer? The cumulative results of numerous correlative studies
have shown that HMGI(Y) genes are overexpressed in such diverse
human epithelial tumor types as malignant prostate, breast, thyroid,
and colon cancers, as well as aberrantly expressed in benign
mesenchymal tumors such as a leiomyomas, adenomas, hamartomas, and
lipomas. Recent studies employing gene expression array analyses of
transcription profiles have also confirmed the overexpression of
HMGI(Y) in biopsies of many human breast tumors (85, 92, 104). An even more interesting finding is that these array
analyses also show that many of the genes listed in Table 2 are
likewise overexpressed in surgical breast tumor biopsies, with the
expression of several (including Notch-4, cyclin A, Rho GDL, and
others) closely paralleling the expression of HMGI(Y) in the tumors
(92) (e.g., see the primary data tables at the website
http://genome-www.stanford.edu/molecularportraits). Combined with the
presented results, the cumulative data therefore suggest that
HMGI(Y) may play the role of a master gene, perhaps one of several,
whose overexpression impacts multiple steps involved in breast cancer
neoplasia. If so, a second critical question arises: which initial
events turn on the overexpression of the Hmgiy gene?
The promoter regions of both the human (
39; M. L. Pedulla, N. Treff, L. Beckenbauer, L. M. Resar, and R. Reeves,
submitted
for publication:) and mouse (
132)
Hmgiy genes contain binding
sites for the transcription
factors AP-1 and c-Myc. Phorbol esters,
tumor-promoting substances that
activate AP-1 transcription factors,
have been demonstrated to
stimulate transcription of both the
human (
89) and mouse
(
22) genes. In addition, Resar and her
colleagues also
recently demonstrated that the c-Myc protein,
along with its partner,
Max, regulates transcription of the mouse
Hmgiy gene in vivo
and that overexpression of either c-Myc or
HMGI(Y) proteins leads
to anchorage-independent growth of Rat-1
cells in soft agarose
(
132). These findings, coupled with the
fact that
mutations in the adenomatous polyposis coli tumor suppressor
gene leads
to overexpression of c-Myc and uncontrolled cell growth
(
52), logically lead to a number of plausible multistep
mechanisms
that might explain the widespread involvement of HMGI(Y)
in neoplastic
transformation and metastatic progression. In one
experimentally
testable scenario, the initial tumor lesion is
envisioned as a
mutation in a tumor suppressor gene (e.g., adenomatous
polyposis
coli) which allows the aberrant expression or overexpression
of
one or more proto-oncogenes (e.g., c-
myc) coding for
transcription
factors that can induce the promoter of the
Hmgiy gene. These
initial lesions might result in neoplastic
transformation of the
cells but not necessarily lead to permanent
activation of the
Hmgiy gene. Subsequent exposure of these
mutation-containing cells
to substances that promoter tumor progression
(e.g., phorbol esters
that activate AP-1 transcription factors) could
then induce constitutive
overexpression of the
Hmgiy gene
(c.f. reference
22), setting
in motion a cascade of events
leading to tumor progression and
metastasis. Regardless of the actual
sequence of molecular events
involved, however, the present work
demonstrates that the HMGI(Y)
proteins are attractive targets for
antitumor drug development
and also for other, novel forms of cancer
treatment.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant RO1-GM46352 (to R.R.) and
U.S. Army Breast Cancer Research Grant DAMD17-96-16249 (to Y.L.).
We sincerely thank M. Nissen and other members of both the Reeves
laboratory and that of M. Griswold (Washington State University) for
helpful discussions and technical assistance during the course of this
work. Our thanks also go to Charles W. Leathers (Washington State
University College of Veterinary Medicine) for assistance with
histological analyses, Adrea Cupp (Center for Reproductive Biology,
Washington State University) for advice on immunohistochemical staining, and Eric Nilsson (also of the Center for Reproductive Biology, Washington State University) for assistance with tumor dissections.
| |
ADDENDUM |
It has recently been reported (131) that Rat-1a
fibroblast cells overexpressing either the HMG-I, HMG-Y, or HMGI-C
protein form tumors and distant metastases when injected into nude mice.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, School of Molecular Biosciences, Washington State
University, Pullman, WA 99164-4660. Phone: (509) 335-1948. Fax: (509)
335-9688. E-mail: reevesr{at}mail.wsu.edu.
| |
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Molecular and Cellular Biology, January 2001, p. 575-594, Vol. 21, No. 2
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.2.575-594.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
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