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Molecular and Cellular Biology, March 2001, p. 2203-2212, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.2203-2212.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Activation of Akt (Protein Kinase B) in Mammary
Epithelium Provides a Critical Cell Survival Signal Required for
Tumor Progression
John
Hutchinson,1
Jing
Jin,2
Robert D.
Cardiff,3
Jim R.
Woodgett,2 and
William
J.
Muller1,*
MOBIX, McMaster University,
Hamilton,1 and Ontario Cancer Institute,
Toronto,2 Ontario, Canada, and
University of California at Davis, Davis, California3
Received 18 September 2000/Returned for modification 30 October
2000/Accepted 13 December 2000
 |
ABSTRACT |
Activation of Akt by the phosphatidylinositol 3'-OH kinase
(PI3K) results in the inhibition of proapoptotic signals and the promotion of survival signals (L. P. Kane et al., Curr. Biol. 9:601-604, 1999; G. J. Kops et al., Nature 398:630-634, 1999). Evidence supporting the importance of the PI3K/Akt signaling pathway in
tumorigenesis stems from experiments with transgenic mice bearing polyomavirus middle T antigen under the control of the mouse mammary tumor virus long terminal repeat promoter. Mammary epithelium-specific expression of polyomavirus middle T antigen results in the rapid development of multifocal metastatic mammary tumors, whereas transgenic mice expressing a mutant middle T antigen decoupled from the
phosphatidylinositol 3'-OH kinase (MTY315/322F) develop extensive
mammary gland hyperplasias that are highly apoptotic. To directly
assess the role of Akt in mammary epithelial development and
tumorigenesis, we generated transgenic mice expressing constitutively
active Akt (HAPKB308D473D or Akt-DD). Although expression of Akt-DD
interferes with normal mammary gland involution, tumors were not
observed in these strains. However, coexpression of Akt-DD with
MTY315/322F resulted in a dramatic acceleration of mammary
tumorigenesis correlated with reduced apoptotic cell death.
Furthermore, coexpression of Akt-DD with MTY315/322F resulted in
phosphorylation of the FKHR forkhead transcription factor and
translational upregulation of cyclin D1 levels. Importantly, we
did not observe an associated restoration of wild-type metastasis
levels in the bitransgenic strain. Taken together these
observations indicate that activation of Akt can contribute
to tumor progression by providing an important cell survival
signal but does not promote metastatic progression.
| |
INTRODUCTION |
The growth and development of the
mammary gland is regulated by a complex set of factors including
hormones, cell-substratum interactions, and growth factors and their
associated receptors. Activation of growth factor receptors leads to
the recruitment of a number of cytoplasmic signaling molecules,
including the phosphatidylinositol 3'-OH kinase (PI3K). Recruitment of
the PI3K to the cell membrane by these activated growth factors or
docking molecules then results in the activation of a number of
molecules. PI3K-dependent generation of phosphatidylinositol 3'
phosphate provides docking sites for several Pleckstrin homology (PH)
domain-harboring molecules including Akt (also known as protein kinase
B [PKB]) as well as its upstream kinases, PDK1 and the proposed PDK2
(2, 16). These latter enzymes phosphorylate Akt at
threonine 308 and serine 473, respectively, causing full Akt activation
(1, 2). Activation of Akt subsequently results in the
inhibition of proapoptotic signals from such proteins as BAD
(9), caspase 9 (4), and the forkhead
transcription factor family (3, 22, 34) and the promotion
of survival signals from such proteins as NF-
B (20).
Although evidence suggests roles for PI3K and Akt in normal mammary
development (15) and tumorigenesis (5, 30, 31,
35), the role of these signaling molecules in these processes
remains to be elucidated.
Evidence supporting the importance of the PI3K/Akt signaling pathway in
tumorigenesis stems from experiments with transgenic mice bearing
polyomavirus (PyV) middle T antigen (mT) under the control of the mouse
mammary tumor virus long terminal repeat promoter (MMTV-LTR). The
MMTV-LTR is transcriptionally active throughout mammary development,
and its transcriptional activity increases during pregnancy
(26). Mammary epithelium-specific expression of PyV mT
results in the rapid development of multifocal metastatic mammary
tumors (18) due to its ability to associate with and
activate the Src family kinases, PI3K, and the Shc adapter protein
(6, 7, 14). In contrast to the rapid tumor progression observed in transgenic mice carrying the PyV mT oncogene (MT634), transgenic mice expressing a mutant mT decoupled from the PI3K pathway
(MMTV/MTY315/322F) develop extensive mammary gland hyperplasias that
are highly apoptotic (35). Focal mammary tumors do
eventually arise in these strains and are further correlated with
upregulation of the ErbB-2 and ErbB-3 growth factor receptors
(35). In addition, these tumors show defects in metastatic
progression (35).
The defects in tumor progression in the mutant mT strain suggested that
Akt may play important roles in tumorigenesis by inhibiting apoptosis
and/or promoting metastasis. In this report we show that activation of
Akt alone can interfere with the apoptotic process of mammary gland
involution and promote tumor progression by providing an important cell
survival signal but does not promote metastasis. The dramatic
acceleration of tumor progression in these strains was further
correlated with the phosphorylation of FKHR, a member of the forkhead
class of transcription factors, and induction of cyclin D1. Together
these observations suggest that activation of Akt provides
complementary cell survival signals that are required for mammary tumorigenesis.
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MATERIALS AND METHODS |
Generation and identification of transgenics.
The cDNA
encoding HAPKB308D473D was subcloned into pMMTV-SV40Pa (p206)
(18). This construct was prepared and injected as previously described (35). Transgenic progeny were
identified by Southern analysis using the
EcoRI-BamHI (3900 to 4775) fragment of p206
(18) as a probe. Akt-MTY315/322F bitransgenics were generated by crossing MMTV/MTY315/322F males to MMTV/Akt7 females and
were subsequently identified by identical Southern analysis.
Histology and apoptosis assays.
Lower left mammary fat pad
tissues were fixed in 4% paraformaldehyde, blocked in paraffin,
sectioned at 5 µm, stained with hematoxylin and eosin, and examined.
Whole-mount preparations were prepared from the lower right mammary fat
pad as previously described (35). In situ apoptosis assays
were performed with the Apoptag Peroxidase In Situ Apoptosis Detection
Kit (Intergen) as described previously (35).
RNA isolation and analysis.
RNA was isolated from mammary
glands and analyzed by RNase protection using simian virus 40 (SV40)
polyadenylation-specific (SPA) and PGK-1 ribonucleotide protection
probes as previously described (35).
Protein extraction and analysis.
Tissue from various organs
was flash frozen in liquid nitrogen and stored at 
80°C or
immediately lysed. Protein lysates were prepared as previously
described (35). All immunoblots and immunoprecipitations were carried out as previously described (35) with the
following exceptions. Antihemagglutinin (anti-HA) immunoblot analyses
of mammary tissue from the FVB/n, Akt7, MTY315/322F, and Akt7 × MTY315/322F strains were performed on 250 µg of total protein lysate.
Lysates were precleared in protein G-Sepharose and subjected to anti-HA immunoblot analysis with HA-11 monoclonal antibody (Babco) (1:1,000). PyV mT was immunoprecipitated from 2 mg of total protein lysate with 2 µg of mouse monoclonal Pab762 (courtesy of S. Dilworth) and subjected
to anti-mT immunoblot analysis with rat monoclonal Pab701 (1:1,000).
Anticytokeratin immunoblot analysis was carried out on 250 µg of
total protein lysate using Troma-1 rat monoclonal antibody from ascites
(1:50). Cyclin D1 analysis was carried out on 50 µg of total protein
using the anti-cyclin D1 72-13G monoclonal antibody from Santa Cruz.
FKHR analysis was carried out on 50 µg of total protein using the
anti-FKHR N-18 polyclonal antibody from Santa Cruz and the
anti-phospho-FKHR (Ser256) antibody from New England Biolabs.
Akt immunoblotting was carried out on total lysate using the anti-Akt
antibody from New England Biolabs. Akt kinase activity assays were
carried out on immunoprecipitates from total lysate using the anti-PH
domain PKB antibody from UBI and the cross-tide peptide as substrate.
Glycogen synthase kinase 3 (GSK-3) analysis was carried out on total
lysate using anti-GSK-3 antibodies from New England Biolabs.
| |
RESULTS AND DISCUSSION |
To further assess the importance of the PI3K/Akt signaling pathway
in PyV mT-induced tumorigenesis and metastasis, we derived transgenic
mice that express a constitutively active version of Akt (HAPKB308D473D
or Akt-DD), which mimics the active phosphorylated state of the protein
(1), in the mammary gland (Fig.
1a). To distinguish between the
transgene-derived and endogenous Akt protein, an HA epitope tag was
placed in frame at the amino terminus of the activated Akt protein
(Fig. 1a). Initially, 11 activated MMTV/Akt founder lines were derived.
Nine of these lines passed the transgene to their offspring, and a
screen for expression of the activated Akt transgene revealed
expression in the mammary gland in three of these lines (Table
1). The tissue specificity of transgene protein product expression of two of these lines (MMTV/Akt7 and MMTV/Akt10) was determined, and the higher expresser (MMTV/Akt7) was
chosen for further study (Table 2). To
confirm that activated Akt protein product was expressed in the mammary
epithelium of transgenic mice, multiple mammary tissue extracts from
the Akt7 line were subjected to anti-HA immunoblot analysis. The
results revealed that virgin mammary glands from these strains were
expressing significant levels of the transgene-derived Akt protein
(Fig. 1b).
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FIG. 1.
Activated Akt transgene expression. (a) Structure of the
MMTV/Akt transgene. The Bluescript vector backbone is represented by a
thin line on either side of the expression cassette, with the white
region corresponding to the MMTV-LTR derived from plasmid pAp, the
black portion corresponding to the hemagglutinin tag, the dark grey
region corresponding to the Akt (HAPKBT308D/S473D) cDNA with aspartate
substitutions at amino acid positions 308 and 473, and the mid-grey
region corresponding to the transcriptional processing sequences
derived from the SV40 early transcription unit. The transcription start
site is indicated by an arrow. (b) Immunoblot analysis of expression of
HAPKB and PyV mT in bitransgenic Akt7 × MTY315/322F strains. Note that
Akt7 × MTY315/322F tumor samples coexpress both Akt and PyV mT
proteins. The numbers above each lane indicate individual mouse
identification numbers.
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To ascertain whether elevated expression of activated Akt could
interfere with normal mammary gland development, whole-mount analyses
of both virgin and involuting mammary glands were conducted. Virgin
female glands from MMTV/Akt strains were histologically and
morphologically identical to FVB/n female controls (Fig. 2a and
b). Consistent with these observations,
female virgin Akt transgenic mice have yet to develop mammary tumors
after a year of observation. This observation is further supported by
the observation that multiparous Akt transgenic females, which would
have undergone multiple periods of high transgene expression, have also
failed to exhibit tumors. Given the importance of apoptotic cell death in mammary gland involution, we next examined whether mammary gland
involution was adversely affected in the activated Akt strain. To
explore this possibility, mammary glands from the wild-type and
activated Akt strains were examined at 1, 3, and 7 days
postparturition. In contrast to wild-type control animals, which
exhibited extensive involution at 1 and 3 days postparturition (Fig.
3a, c, e, and g), the Akt7 animals
displayed a dramatic defect in mammary gland involution (Fig. 3b, d, f,
and h). However, the Akt7 mammary glands eventually underwent full
involution at 7 days postparturition (data not shown), likely due to a
drop in the hormonally responsive MMTV-driven transgene expression in
the activated Akt strain.
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FIG. 2.
Coexpression of Akt and mutant PyV mT oncogene results
in the induction of multifocal mammary tumors. These digital images
illustrate the histological patterns observed in the Akt7 (a and b),
MTY315/322F (c and d), and Akt7 × MTY315/322F (e and f) bigenic
mice. Note that the whole-mount preparations (a, c, and e) demonstrate
that the Akt strains have a relatively normal mammary tree (a) compared
to the cystic hyperplasias seen in the MTY315/322F strains at the same
age (c) (8 weeks) (scale bar = 1 mm). In contrast, the bigenic
mammary gland does not fill the fat pad (e) and is a solid mass at this
age (f). The histological patterns seen at high magnification (scale
bar = 0.01 mm) demonstrate that the Akt7 strain has a normal
epithelium (b), while the MTY315/322F strain has a cystic hyperplasia
of the ducts and glands without significant atypia (d). In contrast,
the Akt7 × MTY315/322F cross has acinar or lobular hyperplasia
with low-grade atypia at 8 weeks (f). Normal mammary gland morphologies
for the FVB strain can be viewed at the following website:
http://ccm.ucdavis.edu/tgmouse/wmtable.htm.
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FIG. 3.
Mammary epithelial expression of Akt results in defect
in mammary gland involution. Digital images of the involution patterns
in wild-type (a, c, e, and g) and Akt7 (b, d, f, and h) mammary glands.
The images compare whole-mount preparations (a, e, b, and f) of the
mammary gland (scale bar = 1 mm) with the histological pattern (c,
d, g, and h) (scale bar = 0.1 mm) on days 1 (a to d) and 3 (e to
h) of involution. Note the delayed involution in the Akt7 mouse mammary
gland (b to h).
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To assess whether the observed delay in mammary gland involution was
due to a defect in the induction of apoptotic cell death, terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) analyses were conducted on involuting mammary epithelium
derived from FVB/n and Akt7 strains (Fig.
4). The results revealed that mammary
glands derived from the involuting FVB/n glands exhibited elevated
levels of apoptotic cell death relative to mammary epithelium of the
Akt7 strain (compare Fig. 4a and b). Taken together, these observations
argue that activation of Akt can interfere with normal mammary gland
involution by attenuating apoptotic death in the involuting mammary
gland.
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FIG. 4.
Mammary epithelial expression of Akt results in
decreased mammary gland apoptosis during involution. (a and b) TUNEL
analysis of involuting mammary glands from FVB/n (a) and Akt7 (b) at 3 days postparturition. Arrows indicate representative apoptotic cells.
(c) Mammary apoptotic indices of FVB/n and Akt7 at 3 days
postparturition. Values shown represent the percentage of total cells
stained positive for apoptosis by TUNEL assay in age-matched singly
parous female mice at 15 weeks of age.
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Although these data suggest that the Akt-DD mutant can interfere with
apoptotic cell death during mammary gland involution, its role in
mammary tumorigenesis is unclear. To explore whether active Akt
expression could complement the defect in tumorigenesis exhibited by
transgenic mice expressing the mutant PyV mT decoupled from the
PI3K/Akt signaling pathway, bitransgenics expressing both the Akt
transgene and the mutant mT transgene (MTY315/322F) were derived (Fig.
1b) and monitored for tumor formation by physical palpation. The
results of these analyses revealed that bitransgenic mice developed
multifocal mammary tumors with 100% penetrance with an average latency
of 46 days (Fig. 5a). In contrast,
physical palpation of two independent cohorts of female mice carrying
the mutant PyV mT transgene alone revealed a significant delay in the
onset of tumor formation with average latencies of 123 and 119 days,
respectively (Fig. 5a). In addition, these tumors were focal in nature,
arising next to hyperplastic mammary epithelium. Consistent with these
kinetic analyses, whole-mount analyses of virgin mammary glands of
bitransgenic mice revealed a dramatic difference in the extent of tumor
growth (compare Fig. 2e and f to c and d). In contrast to the diffuse
cystic hyperplasias exhibited by the mutant PyV mT strains, female
transgenic mice coexpressing the mutant PyV mT and activated Akt
transgenes exhibited polyclonal differentiated carcinomas. In agreement
with these analyses, these lesions could be subcutaneously transplanted
into syngeneic recipients. To confirm that bitransgenics expressing MTY315/322F and activated Akt exhibited elevated Akt kinase activity, we examined the total Akt kinase activity against a peptide substrate in virgin FVB/n, MTY315/322F, and bitransgenic mammary glands. These
studies revealed an approximately fivefold increase in the total Akt
kinase activity in the bitransgenic mammary glands as compared to those
of MTY315/322F transgenics (Fig. 6a). The
minimal increases in endogenous Akt phosphorylation (Fig. 6b) would
suggest that the majority of the Akt kinase activity is derived from
the activated mutant. However, these results do not completely preclude a mechanism whereby endogenous Akt is in some way activated via the
combination of Akt-DD and MTY315/322F and contributes to tumor formation.
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FIG. 5.
Mammary tumor kinetics and apoptotic indices in
transgenic strains. (a) Mammary tumor kinetics of MTY315/322F and
Akt7 × MTY315/322F strains. Two different kinetics curves are
shown for the MTY315/322F strain, from the original published data
(MTY315/322F-1) and confirmatory data us (MTY315/322F-2), to account
for possible differences in palpation technique between researchers.
The age indicated is that at which a mammary tumor is first palpable in
each transgenic strain. The number of animals analyzed for each strain
(n) and the median age at which tumors were palpable are
also shown. (b) Mammary apoptotic indices of FVB/n, MT634 (wild-type
mT), Akt7, MTY315/322F, and Akt7 × MTY315/322F strains. Values shown
represent the percentage of total cells stained positive for apoptosis
by TUNEL assay in virgin female mice at 10 to 12 weeks of age.
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FIG. 6.
Akt kinase activity in transgenic strains. (a) Total Akt
kinase activity analysis in 8- to 10-week-old virgin females from FVB/n
(lane 1), MTY135/322F (lanes 2 and 3), and bitransgenic Akt7 × MTY315/322F (lanes 4 and 5) strains. Assays were conducted using the
cross-tide peptide as an Akt kinase substrate. Kinase activities were
quantified by phosphorimager analysis and are represented here both
graphically and numerically. (b) Immunoblot analysis of expression of
HA-Akt-DD, phospho-S473-Akt, and Akt in 8- to 10-week-old virgin
females from FVB/n (lane 1), MTY135/322F (lanes 2 and 3), and
bitransgenic Akt7 × MTY315/322F (lanes 4 and 5) strains. All tissues
were derived from 8- to 10-week-old virgin mammary glands. The arrows
indicate the migration of transgenic HA-Akt-DD (upper panel),
phospho-S473-Akt (middle panel), and total Akt (bottom panel). The
numbers above each lane indicate individual mouse identification
numbers.
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As the mammary epithelial hyperplasias associated with the mutant PyV
mT strains exhibit elevated levels of apoptotic cell death, we measured
the degree of apoptotic cell death in mammary glands derived from the
mutant PyV mT or bitransgenic mice. The results revealed that mammary
epithelial expression of activated Akt resulted in a dramatic
repression of the high rates of apoptotic cell death in PyV mT mutant
tissue decoupled from the PI3K (Fig. 5b). Taken together, these
observations argue that the dramatic acceleration of mammary
tumorigenesis exhibited by these strains is due to the ability of
activated Akt to suppress the elevated apoptotic cell death displayed
by mutant PyV mT mammary epithelium.
Although the active, transgenic Akt is able to complement the mutant
PyV mT strains for the induction of mammary tumors, only 20% of the
tumor-bearing mice have developed lung metastases more than 8 weeks
after the initial palpation of the mammary tumor (n = 10) at tumor loads comparable to those observed in mice expressing wild-type mT at similar time points. The penetrance of the metastatic phenotype is comparable to the 30% metastasis levels exhibited by the
parental mutant PyV strains. In contrast, 100% of mice expressing
wild-type mT show multiple lung metastases at comparable time points
and tumor loads (18). These observations argue that while
expression of active Akt can complement the defect in mammary tumor
progression, it is unable to rescue the defect in metastatic progression.
To further explore the molecular basis for the observed cooperative
interaction between activated Akt and the mutant PyV mT oncogene,
we assessed the status of some of the known targets of Akt,
including BAD (9), I--B (20), and the FKHR
forkhead transcription factor (34).
No significant differences in either BAD-Ser136 phosphorylation or
I--B levels were observed between the various transgenic strains
(data not shown). Caspase 9, another Akt substrate (4), was not examined, as the Akt phosphorylation site found in human caspase 9 is absent in mouse caspase 9 (17). However,
analysis of protein lysates from mammary tissues of 8-week-old virgin
FVB/n, Akt7, mutant PyV mT, and bigenic mice subjected to immunoblot analyses with phospho-specific antisera to serine 256 of FKHR (Fig.
7a) revealed that the mammary tissue
samples derived from the bitransgenic animals expressed elevated levels
of phosphorylated FKHR protein relative to the other tissue samples
(second panel). The differences in the phosphorylation status of FKHR
proteins were not due to levels of FKHR protein, since most of the
tissues expressed comparable levels of FKHR protein (upper panel). In addition, the differences in the phosphorylation status could not be
due to variation in epithelial content, since these samples expressed
comparable levels of cytokeratin 8 (lower panel). Consistent with these
observations, we have demonstrated an identical pattern of FKHR
phosphorylation in a second independent cohort of samples (data not
shown). To further explore this observation we examined the status of
p27 (Kip1), as forkhead transcription factors have been shown to target
expression of the cell cycle regulator p27 (13, 23, 24).
In particular, adenoviral expression of a constitutively active version
of FKHR in the human renal cancer cell line 786-O cells induces
expression of p27 (24). However examination of p27 levels
by Western blotting revealed no apparent decreases in p27 levels in the
bitransgenic animals as compared to MTY315/322F and FVB/n controls
(Fig. 7b, lower panel). This apparent discrepancy may be due to the
different nature of the tissues and signals involved in these
experiments.
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FIG. 7.
Coexpression of activated Akt and MTY315/322F results in
FKHR phosphorylation at serine 256 and increased cyclin D1 levels but
does not affect GSK-3 phosphorylation or p27 levels. (a) Immunoblot
analysis of expression of FKHR, phospho-FKHR (Ser256), cyclin D1, and
cytokeratin in 8- to 10-week-old virgin females from FVB/n (lane 1),
Akt7 (lanes 2 and 3), MTY135/322F (lanes 4 and 5), and bitransgenic
Akt7 × MTY315/322F (lanes 6 and 7) strains. All tissues were
derived from 8- to 10-week virgin mammary glands. The arrows indicate
the migration of FKHR (upper panel), phospho-FKHR (Ser256) (second
panel), cyclin D1 (third panel), and cytokeratin proteins (lower
panel). The numbers above each lane indicate individual mouse
identification numbers. (b) Immunoblot analysis of expression of
phospho-S21-GSK-3 , phospho-S9-GSK-3 , GSK-3/, and p27 in 8- to 10-week-old virgin females from FVB/n (lane 1), MTY135/322F (lanes 2 and 3), and bitransgenic Akt7 × MTY315/322F (lanes 4 and 5)
strains. All tissues were derived from 8- to 10-week virgin mammary
glands. The arrows indicate the migration of phospho-S21-GSK-3 and
phospho-S9-GSK-3 (upper panel), GSK-3/ (middle panel), and p27
(lower panel). The numbers above each lane indicate individual mouse
identification numbers.
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Nevertheless, another potential target for the PI3K/Akt kinase axis is
the cell cycle machinery. Indeed, it has previously been demonstrated
that suppression of the PI3K signaling pathway by expression of the
PTEN tumor suppressor results in downregulation of cyclin D1 expression
and cell cycle arrest (32, 36). To determine whether the
levels of cyclin D1 could be influenced by Akt activation, the
identical set of mammary tissues were subjected to immunoblot analyses
with cyclin D1-specific antibodies. The results of these analyses
revealed that the bitransgenic tissues coexpressing both Akt-DD and the
mutant PyV mT oncogene exhibited dramatically elevated levels of cyclin
D1 (Fig. 7a, third panel). The differences in cyclin D1 protein were
not due to increased levels of cyclin D1 transcripts, since these
samples expressed comparable levels of cyclin D1 transcript (data not
shown). Taken together these observations suggest that activation of
FKHR and cyclin D1 proteins are involved in promoting tumor progression in these strains.
The studies outlined above provide compelling evidence that expression
of activated Akt is involved in promoting tumor progression by
providing a critical cell survival pathway. Consistent with this
contention, mammary epithelial expression of Akt can result in profound
delays in mammary gland involution, a process involving extensive
apoptotic cell death. Moreover, coexpression of activated Akt can
suppress the elevated rates of apoptotic cell death that are observed
in mammary epithelial hyperplasias induced by the mutant PyV mT
decoupled from the PI3K signaling pathway. However, because mammary
epithelial expression of activated Akt does not result in the induction
of mammary tumors itself, tumorigenesis requires the constitutive
activation of other signaling pathways that are recruited by the mutant
PyV mT oncogene, including the Src family kinases and Shc/Grb2/Ras
pathway. Consistent with this view, we have observed that efficient
phosphorylation of the FKHR protein requires the concerted activation
of both Akt and the mutant PyV mT oncogene (Fig. 7). A similar
requirement for coactivation of Akt and mutant PyV mT was also noted
for the induction of cyclin D1. In this regard, it has recently been
reported that the cooperation of Ras and Akt are required for the
efficient transformation of primary glial cells (19). A
potential mechanism for the increased levels of cyclin D1 was suggested
by the ability of Akt and mitogen-activated protein kinases to
phosphorylate and inhibit GSK-3 (8, 33), which has been
shown to target cyclin D1 for proteasomal degradation (12). However, analysis of GSK-3 phosphorylation showed no
significant increases in the bitransgenic strain as compared to FVB/n
and MTY315/322F controls, once differences in GSK-3 levels were
accounted for (Fig. 7b, upper and middle panels). Even so, these
results suggest that the concerted activation of both cell survival and proliferative signaling pathways may be a common requirement for oncogenic transformation of primary cells.
Although our studies suggest that activated Akt can cooperate with
these signaling pathways to efficiently induce mammary tumorigenesis, the observed low rates of metastasis suggest the involvement of other Akt-independent signals downstream of middle T in the potent metastatic phenotype exhibited by wild-type PyV mT.
While these signals are in all likelihood PI3K dependent, we cannot
exclude the possibility that signaling molecules other than PI3K may
bind to and be activated via the 315 and 322 phosphorylated tyrosine
residues. However, PI3K activation does modulate the activity of
members of the Rho family of GTP-binding proteins (21, 25, 27,
29) and the integrin-linked kinase (11). This
modulation is highly relevant, as the roles of these sets of signaling
molecules in cell migration and adhesion implicates them in metastatic
progression (10, 28). Further exploration of these
PI3K-dependent pathways will provide important insight into the
molecular basis of the metastatic phenotype.
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ACKNOWLEDGMENTS |
We thank Dinsdale Gooden for oligonucleotide synthesis and Brian
Allore for automated DNA sequence analysis (MOBIX Central Facility,
McMaster University). We are grateful to S. Dilworth for generously
providing the PAb701 and PAb762 anti-PyV mT antibodies. We are also
grateful to Monica Graham and Judy Walls for technical support.
This work was supported by grants awarded to W.J.M. by the United
States Army Medical Research's Breast Cancer Research Program and the
Canadian Breast Cancer Research Initiative and by NCIC and MRC grants
awarded to J.R.W. W.J.M. is a recipient of a Medical Research Council
of Canada Scientist award, J.R.W. is a recipient of an MRC Senior
Scientist award, and J.N.H. is supported by a scholarship from the
United States Army Medical Research's Breast Cancer Research Program.
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FOOTNOTES |
*
Corresponding author. Mailing address: MOBIX, McMaster
University, 1280 Main St. W., Hamilton, Ontario L8S 4K1, Canada. Phone: (905) 525-9140, ext. 27306. Fax: (905) 521-2955. E-mail:
mullerw{at}mcmaster.ca.
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Molecular and Cellular Biology, March 2001, p. 2203-2212, Vol. 21, No. 6
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.6.2203-2212.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
