Akt/PKB Activity Is Required for Ha-Ras-mediated Transformation
of Intestinal Epithelial Cells*
Hongmiao
Sheng
,
Jinyi
Shao
, and
Raymond N.
DuBois
§¶
From the Departments of
Medicine and
§ Cell Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt
University Medical Center, Department of Veterans Affairs Medical
Center, Nashville, Tennessee 37232
Received for publication, November 6, 2000, and in revised form, January 16, 2001
 |
ABSTRACT |
Phosphatidylinositol 3-kinase (PI3K)/protein
kinase B (PKB/Akt) is thought to serve as an oncogenic signaling
pathway which can be activated by Ras. The role of PI3K/Akt in
Ras-mediated transformation of intestinal epithelial cells is currently
not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of
PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the
morphological transformation induced by Ha-Ras and resulted in a modest
stimulation of apoptosis. The most pronounced phenotypic alteration
following inhibition of PI3K was induction of G1
phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced
expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27kip. Both
LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the
constitutively active forms of Raf (
Raf-22W or Raf BXB) and Akt
(Akt-myr) resulted in transformation of RIE cells that was not achieved
by transfection with either the Raf mutant construct or Akt-myr alone.
These findings delineate an important role for PI3K/Akt in Ras-mediated
transformation of intestinal epithelial cells.
 |
INTRODUCTION |
The serine/threonine kinase Akt, or protein kinase B
(Akt/PKB)1 is a direct
downstream effector of phosphatidylinositol 3-kinase (PI3K) (1, 2).
Akt/PKB lies in the crossroads of multiple cellular signaling pathways
and acts as a transducer of input initiated by growth factor receptors
that activate PI3K (reviewed in Ref. 3). Akt/PKB regulates gene
transcription by direct phosphorylation of some of the Forkhead
transcription factors such as FKHR, FKHRL, and AFX (4-6) or indirectly
by modifying the cAMP-responsive element-binding protein (7, 8), E2F (9), or nuclear factor-
B (10). Evidence suggests that the PI3K/Akt/PKB pathway promotes growth factor-mediated cell survival and
inhibits apoptosis (11) by modifying the anti-apoptotic and
pro-apoptotic activities in the bcl-2 gene family (12,
13).
Activation of the PI3K/Akt pathway is important for Ras transformation
of mammalian cells and essential in Ras-induced cytoskeletal reorganization (14). The PI3K/Akt signaling pathway plays a critical
role in R-Ras-mediated transformation, adhesion, and cell survival
(15). Additionally, transformation of hematopoietic cells by BCR/ABL
requires activation of the PI3K/Akt signaling pathway (16). Ectopic
expression of active Akt/PKB leads to transformation of NIH 3T3 and
chicken embryo fibroblasts (17, 18). These observations strongly
suggest that the PI3K/Akt pathway is oncogenic and widely involved in
the neoplastic transformation of mammalian cells. Understanding the
role of Akt/PKB in malignant transformation has been greatly enhanced
by recent work on the tumor suppressor gene, PTEN/MMACI. A
large body of evidence demonstrates that PTEN (phosphatase and tensin
homologue deleted on chromosome ten) suppresses tumor formation by
restraining the PI3K/Akt pathway (Refs. 19-22; reviewed in Ref. 23).
PTEN has been shown to dephosphorylate the 3-position of both
phosphatidylinositol-3,4,5-P3 and
phosphatidylinositol-3,4-P2 to reverse the reaction
catalyzed by PI3K, which leads to activation of Akt/PKB (23).
Interestingly, a high incidence of epithelial dysplasia and colonic
carcinoma has been observed in PTEN+/
heterozygous mice
(24, 25), suggesting that Akt activity may be important in colorectal carcinogenesis.
Colorectal cancer is the second leading cause of death related to
cancer in the United States. About 50% of colorectal carcinomas contain Ki-ras mutations (26) and the Ki-ras
oncogene plays a key role during the adenoma to carcinoma sequence of
events involved in the neoplastic transformation of colonic epithelial cells (reviewed in Ref. 27). The activated form of Ras can directly bind to p110, the catalytic subunit of PI3K (28), and ectopic expression of oncogenic Ras in mammalian cells results in the activation of the PI3K pathway. We have established a rat intestinal epithelial cell line (RIE-iHa-Ras) in which oncogenic
RasVal12 can be induced upon addition of
isopropyl-1-thio-
-D-galactopyranoside (IPTG), which
results in morphological transformation (29). In the present study, we
sought to determine the role of the PI3K/Akt in
Ha-RasVal12-mediated transformation of intestinal
epithelial cells to further evaluate the oncogenic potential of this
signaling pathway.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The RIE-iHa-Ras cell line with an inducible
HaRasVal12 cDNA was generated by using LacSwitch
eukaryotic expression system (Stratagene, La Jolla, CA) and described
previously (29). The cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 400 µg/ml G418 (Life Technologies, Inc.), and 150 µg/ml hygromycin B
(Calbiochem, San Diego, CA). The Ha-RasVal12 cDNA is
under the transcriptional control of the Lac operon. IPTG (Life
Technologies, Inc.) at a concentration of 5 mM was used to induce the expression of mutated Ha-Ras. RIE/
Raf-22W cell
was a kind gift of Dr. Robert Coffey (Vanderbilt University Medical
Center) and was described previously (30). PD-153035, PD-98059,
wortmannin, and LY-294002 were purchased from Calbiochem (San Diego, CA).
Northern Analysis--
Total cellular RNA was extracted as
previously described (31). RNA samples were separated on
formaldehyde-agarose gels and blotted onto nitrocellulose membranes.
The blots were hybridized with RNA probes labeled with
[
-32P]UTP using MAXIscriptTM kit (Ambion, Austin,
TX). After hybridization and washes, the blots were subjected to autoradiography.
Immunoblot Analysis and Antibodies--
Immunoblot analysis was
performed as previously described (32). Cells were lysed for 30 min in
radioimmunoprecipitation assay buffer (1× PBS, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin, 1 mM sodium orthovanadate) and then
clarified cell lysates were denatured and fractionated by SDS-PAGE.
After electrophoresis, the proteins were transferred to nitrocellulose
membrane. The filters were then probed with the indicated antibodies
and developed by the enhanced chemiluminescence system (ECL, Amersham
Pharmacia Biotech). The anti-pan Ras antibody was purchased from
Calbiochem. The anti-cyclin D1 antibody was purchased from Upstate
Biotechnology (Lake Placid, NY). Anti-CDK2, CDK4, and p27 antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
anti-phosphorylated Akt antibody was obtained from New England Biolabs
(Beverly, MA), and the anti-active ERK1/2 antibody was from Promega
(Madison, WI).
Flow Cytometry--
RIE-iHa-Ras cells were seeded into 100-mm
plates and treated with 5 mM IPTG or IPTG plus LY-294002 at
indicated concentrations for 48 h. Cells were fixed in 70% EtOH,
digested in 1 ml of 0.1% RNase (Sigma), and stained with propidium
iodide (Sigma). The DNA was analyzed by a flow cytometer, and the cell
cycle profile was expressed as percentage of cells in each cell cycle stage.
Soft Agarose Assay--
1 × 104 cells were
mixed with Sea plaque agarose (Hoeffer, Rockland, ME) at a final
concentration of 0.4% in DMEM, and overlaid onto a 0.8% agarose layer
in 35-mm plates. The plates were incubated for 6-10 days, following
which colonies were photographed. Colony number was manually counted
and is expressed as colony number per microscope field (×40).
Immunofluorescence--
RIE-iHa-Ras cells were grown in 35-mm
tissue culture plates and fixed in methanol/acetone at
20 °C for
10 min. Fixed cells were incubated with 10% normal donkey serum for
1 h and then with anti-FAK antibody (Transduction Laboratories,
Lexington, KY) for 2 h at room temperature. After washing the
cells three times with PBS, they were incubated with Cy3-conjugated
donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) for an
additional 1 h. To carry out rhodamine-phalloidin staining, cells
were fixed in 4% formaldehyde-Triton solution for 10 min and then
incubated with 100 nM fluorescent phalloidin for 30 min.
The stained cells were washed with PBS, mounted, and observed under
fluorescent microscopy with appropriate filters.
Apoptosis--
RIE-iHa-Ras cells were grown to confluence in
60-mm plates. IPTG, PD-98059 (50 µM), or LY-294002 (20 µM) was added to the culture medium. The cells were
incubated for another 48 h, after which floating and attached
cells were collected and lysed in lysis buffer (1% Nonidet P-40 in 20 mM EDTA, 50 mM Tris, pH 7.5). The supernatant
containing fragmented DNA was clarified by centrifugation for 5 min at
1600 × g. The cell lysates were treated with a
solution containing RNase A (5 mg/ml) and proteinase K (2.5 mg/ml). The DNA was then separated on 1.6% agarose gels. For fluorochrome staining, confluent RIE-iHa-Ras cells were treated with 50 µM PD-98059 for 48 h, and the floating cells were
collected, stained with 167 µM Hoechst 33258 (Sigma), and
observed with fluorescent microscopy.
Stable Transfection--
Stable transfections were performed by
using Lipofectin (Life Technologies, Inc.). The myristoylated form of
Akt (Akt-myr) was kindly provided by Dr. Michael White (Southwestern
Medical Center, Dallas, TX) and was inserted into pZeoSV2(+)
(Invitrogen, Carlsbad, CA). The mammalian expression vector (pSR
/Raf
BXB) containing the BXB mutant of c-Raf-1 kinase (provided by Dr.
Michael White) lacking amino acids 26-203. To establish
RIE/
Raf-22W/Akt-myr cell line, RIE/
Raf-22W cells (30) were
transfected with pZeoSV2/Akt-myr plasmid and selected by growth in
media containing zeocin (300 µg/ml). The expression of Akt-myr was
verified by detection of tagged hemagglutinin protein. To establish the
RIE/Akt-myr/Raf BXB cell line, RIE-1 cells were first transfected with
pZeoSV2/Akt-myr and then co-transfected with pSR
/Raf BXB plus
pTK/hyg. Stable clones were selected by growth in media containing
hygromycin (100 µg/ml), and expression was verified by measuring
increased levels of Raf protein. To establish RIE-RasVal12
cells, RIE parental cells were stably transfected with the
pcDNA3/Ha-RasVal12 expression vector and were selected
by growth in neomycin (600 µg/ml).
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RESULTS |
Activation of Akt/PKB by the Induction of
Ha-RasVal12--
Oncogenic Ras is known to activate
Akt/PKB through direct binding and activation of the p110 catalytic
subunit of PI3K (33). In order to elucidate whether expression of
mutated Ha-Ras activates Akt/PKB in intestinal epithelial cells, we
evaluated the effect of inducible expression of Ha-RasVal12
in RIE cells. As shown in Fig.
1A, the expression of
Ha-RasVal12 in RIE-iHa-Ras cells is detectable 4 h
following treatment with IPTG and markedly increased within 24 h.
The levels of phosphorylated extracellular signaling-regulated kinase
(ERK) and Akt were coincidentally induced following induction of Ras
expression. Following Ras induction, RIE cells acquired a transformed
appearance characterized by growth in overlapping clusters and
formation of colonies in soft agarose (Fig. 1B). To block
the Ras-induced activation of Akt/PKB a specific inhibitor for PI3K,
LY-294002 (20 µM) (34) was added to the RIE-iHa-Ras cells
prior to IPTG treatment. The Ras-induced phosphorylation of ERKs was
not affected by the treatment with LY-294002. In contrast, treatment
with LY-294002 almost completely blocked the activation of Akt/PKB
following induction of oncogenic Ha-Ras, but pAkt was maintained at a
basal level (Fig. 1C).

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Fig. 1.
Ha-Ras-mediated transformation and
induction of ERK and Akt. A, Western analysis for Ras,
pERK, and pAkt. RIE-iHa-Ras cells were treated with IPTG and lysed in
radioimmunoprecipitation assay buffer at the indicated time points.
After electrophoresis, the proteins were transferred to nitrocellulose
filters and the filters were blotted with an anti-pan-Ras antibody,
active ERK antibody, or active Akt antibody. The relative expression
levels were determined by autoradiography using the ECL
chemiluminescence system. The results were similar in three independent
experiments. B, morphological transformation of RIE-iHa-Ras
cells. RIE-iHa-Ras cells were grown on 60-mm tissue culture dishes and
treated with vehicle (left panel; original
magnification, ×100) or IPTG (central panel;
original magnification, ×100). For anchorage-independent growth of
RIE-iHa-Ras cells, 1 × 104 cells were mixed with Sea
plaque agarose at a final concentration of 0.4% in DMEM containing
vehicle or IPTG, and overlaid onto a 0.8% agarose layer in 35-mm
plates. Colonies were photographed using an inverted microscope
(right panel; original magnification, ×40).
C, the effect of inhibition of PI3K on the levels of pERK
and pAkt. LY-294002 (20 µM) was added to subconfluent
RIE-iHa-Ras cells 1 h prior to IPTG treatment. Cellular protein
was collected at the indicated time points for Western analysis of pERK
and pAkt. Data shown in this figure represent three independent
experiments.
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Inhibition of Akt/PKB and Apoptosis--
Akt/PKB has been shown to
promote cell survival in a variety of different cell lines (12, 13).
Therefore, we evaluated the role of Akt/PKB in programmed cell death of
Ras-transformed intestinal epithelial cells. As demonstrated in Fig.
2A, RIE-iHa-Ras cells
underwent apoptosis when they reached confluence as indicated by their
DNA banding pattern. In contrast, programmed cell death was almost
completely inhibited following induction of Ha-RasVal12 and
DNA fragmentation was barely detected in IPTG-treated RIE-iHa-Ras cells. Inhibition of mitogen-activated protein kinase/ERK kinase (MEK)
with PD-98059 (50 µM) greatly increased DNA fragmentation despite the induction of oncogenic Ha-Ras. Surprisingly, inhibition of
Akt/PKB activation by LY-294002 only partially blocked the Ras-induced
increase in cell survival. Treatment with PD-98059 resulted in an
increased fraction of apoptotic floating cells (Fig. 2B,
left panel) as determined by fluorochrome
staining (Fig. 2B, right panel).

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Fig. 2.
Inhibition of Akt and apoptosis.
A, DNA fragmentation assay. RIE-iHa-Ras cells were grown to
confluence and subjected to the treatment indicated. PD,
PD-98059 (50 µM); LY, LY-294002 (20 µM). Cells were incubated for 48 h following
treatment, and then floating and attached cells were collected in lysis
buffer and the soluble DNA was isolated. DNA laddering was visualized
on 1.6% agarose gel. B, RIE-iHa-Ras cells were treated with
5 mM IPTG and 50 µM PD-98059 for 48 h.
Adherent cells were photographed (left panel;
original magnification, ×200), and the floating cells were collected,
stained with 167 µM Hoechst 33258 and observed under a
fluorescent microscope (right panel; original
magnification, ×1000). Data shown in this figure are representative of
three independent experiments.
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Akt/PKB and Cell Morphology--
Non-induced RIE-iHa-Ras cells
displayed the same non-transformed morphology as the parental RIE-1
cells, which grew as monolayer cultures with contact inhibition (Fig.
3A, panel
a). Morphological transformation of the RIE-iHa-Ras cells
was observed between 24 and 48 h after IPTG treatment (Fig.
3A, panel b). Inhibition of MEK/ERK
activity by PD-98059 completely blocked the Ras-induced transformation
in RIE-iHa-Ras cells (Fig. 3A, panel
c). Inhibition of Akt/PKB activity resulted in a partial
suppression of Ras-mediated transformation. The spindly appearance of
cells and overlapping growth was still observed in some areas (Fig.
3A, panel d). Fluorescent staining
with rhodamine-phalloidin clearly demonstrated stress fibers in
uninduced RIE-iHa-Ras cells. Induction of Ha-RasVal12
resulted in diffusion of stress fibers (compare Fig. 3B,
panels a and b). Treatment with
LY-294002 restored well organized stress fibers (Fig. 3B,
panel c). Induction of oncogenic Ha-Ras also increased focal adhesion complexes, as determined by immunostaining for
focal adhesion kinase (FAK). Normally, FAK was localized at the ends of
stress fibers (Fig. 3B, panel d), but,
following induction of Ras, the proteins diffusely accumulated in the
cytoplasm (Fig. 3B, panel e).
Treatment with LY-294002 restored the FAK expression to the similar
pattern in uninduced RIE-iHa-Ras cells (Fig. 3B, panel f).

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Fig. 3.
The effect of inhibition of Akt on cell
morphology. A, RIE-iHa-Ras cells were treated with
vehicle (a), 5 mM IPTG (b), 5 mM IPTG plus 50 µM PD 98059 (c),
or 5 mM IPTG plus 20 µM LY 294002 for 48 h and photographed (original magnification, ×100). B,
immunofluorescence. RIE-iHa-Ras cells were treated with vehicle
(a and d), 5 mM IPTG for 24 h
(b and e), or IPTG plus 20 µM LY
294002 for 24 h (c and f). Fixed cells were
directly stained with rhodamine-phalloidin (a-c) or
incubated with anti-FAK antibody (d-f). After washing the
cells with PBS, they were incubated with Cy3-conjugated donkey
anti-mouse IgG for an additional 1 h. The dishes were washed with
PBS, mounted, and observed under fluorescent microscopy with
appropriate filter (original magnification, ×1000). The results were
similar in three independent experiments.
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Akt/PKB and Cell Proliferation--
The rate of cell turnover is
determined by the balance between cell proliferation and cell death.
Akt/PKB activity is known to be involved in the regulation of cell
proliferation (3). In order to elucidate whether Akt/PKB activity was
required for the growth of Ras-transformed RIE cells, their growth
rates following different treatments were determined. As demonstrated
in Fig. 4A, when uninduced
RIE-iHa-Ras cells reached confluence (at day 6), further growth was
inhibited by cell-cell contact. Induction of oncogenic Ras
significantly increased the cell density, and at the end of the
experiment, the number of Ras-induced cells was 2.3-fold greater than
the number of uninduced RIE-iHa-Ras cells. Both PD-98059 (50 µM) and LY-294002 (20 µM) completely blocked Ras-induced growth stimulation. LY-294002 inhibited the growth
of Ras-transformed RIE-iHa-Ras cells in a
concentration-dependent manner and reduced the cell number
by 35%, 55%, and 76% at 5, 10, and 20 µM respectively
(Fig. 4B). Next, we determined the effect of Akt/PKB on cell
cycle regulation using flow cytometry. Induction of
Ha-RasVal12 accelerated the G1/S transition
(Fig. 4C). Addition of LY-294002 led to an accumulation of
Ras-induced RIE-iHa-Ras cells in the G1 phase of the cell
cycle. Interestingly, treatment with PD-98059 barely altered cell cycle
progression in these cells.

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Fig. 4.
Inhibition of Akt and cell growth.
A, growth of RIE-iHa-Ras cells. Cells (2 × 104) were seeded in a 12-well plate and incubated for
24 h prior to the treatment as indicated (CTR,
Me2SO; IPTG, 5 mM IPTG;
LY, 20 µM LY 294002; PD, 50 µM PD 98059). Cell numbers were counted at the indicated
times, and values are means ± S.E. from triplicate wells. All
growth data are representative of three separate experiments.
B, LY 294002 inhibition of cell growth. 2 × 104 RIE-iHa-Ras cells were seeded in 12-well plates and
treated with LY 294002 at the indicated concentrations. Cell numbers
were counted at day 5 after plating, and values are means ± S.E.
from triplicate wells. Cell growth studies were repeated at least three
times. C, cell cycle analysis. RIE-iHa-Ras cells were
treated with 5 mM IPTG or IPTG plus LY 294002 at indicated
concentrations for 48 h. The DNA was analyzed by a flow cytometer,
and the cell cycle profile is presented as percentage of cells in each
stage of the cell cycle. The results were similar in three independent
experiments.
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Growth inhibition that resulted from blocking the activation of
Akt/PKB was also observed when RIE-iHa-Ras cells were grown in an
anchorage-independent manner. Induction of oncogenic Ras led
RIE-iHa-Ras cells to form colonies in soft agarose, and both PD-98059
and LY-294002 reduced the colony number by > 80% (Fig. 5A). To confirm that the
growth-inhibitory effect of LY-294002 was mediated through inhibition
of Akt/PKB activity, a structurally unrelated PI3K inhibitor,
wortmannin, was employed. Both 0.1 and 1 µM wortmannin
reduced the number of Ras-induced RIE-iHa-Ras colonies by >50%.

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Fig. 5.
Colony formation in soft agarose.
A and B, 1 × 104 RIE-iHa-Ras
cells were seeded in 0.4% agarose and treated as indicated in 35-mm
dishes. After 6 days the colonies were photographed using an inverted
microscope, and colony number was presented per microscope field
(original magnification, ×40). Values are means ± S.E. from 27 fields of triplicate plates (9 fields/plate). Data shown in this figure
are representative of three independent experiments.
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To further elucidate the mechanism by which Akt/PKB regulates cell
cycle progression, we analyzed the regulation of cell cycle proteins in
RIE-iHa-Ras cells. Induction of oncogenic Ras increased levels of
cyclin D1 and CDK2, but had less of an effect on expression of CDK4,
cyclin E, and p27kip (Fig.
6A). Treatment with LY-294002
inhibited the expression of cyclin D1 and CDK2 in a
concentration-dependent manner (Fig. 6B), and 20 µM LY-294002 completely blocked the Ras-induced
expression of cyclin D1 and CDK2. LY-294002 also increased the
expression of p27kip. In contrast, treatment
with PD-98059 blocked Ras-induced expression of cyclin D1 and increased
the levels of p27kip but did not inhibit the
Ras-mediated induction of CDK2.

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Fig. 6.
Akt and the expression of cell cycle
proteins. A, expression of cell cycle proteins in
RIE-iHa-Ras cells. RIE-iHa-Ras cells were treated with IPTG and the
levels of cyclin D1, CDK4, cyclin E, CDK2, and
p27kip were analyzed by Western blotting. Data
shown in this figure were repeated in three independent experiments.
B, RIE-iHa-Ras cells were treated with IPTG, IPTG plus
LY-294002, or IPTG plus PD-98059 for 48 h and the levels of cyclin
D1, CDK4, cyclin E, CDK2, p27 were analyzed by Western blotting.
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Akt/PKB and Cell Transformation--
PI3K/Akt/PKB is thought to
represent an oncogenic signaling pathway. In the present study, we
demonstrated that pharmacologic inhibition of Akt/PKB significantly
inhibited the Ras-induced transformation in RIE-iHa-Ras cells. It was
important to determine whether Akt/PKB truly contributes to
Ras-mediated transformation of intestinal epithelial cells. Oldham
et al. (30) demonstrated that ectopic expression of active
Raf (
Raf-22W) was not sufficient to transform RIE cells. We stably
transfected the RIE/
Raf-22W cells with an expression vector
containing myristoylated form Akt (Akt-myr). The vector-transfected
RIE/
Raf-22W cells did not form colonies in soft agarose (Fig.
7A). In contrast, the RIE cells that expressed both
Raf-22W and Akt-myr grew in an
anchorage-independent fashion. None of the clones that were selected
from vector-transfected cells grew in soft agarose, whereas 50% of the
clones isolated from Akt-myr-transfected RIE/
Raf-22W cells formed
colonies in soft agarose. To confirm the finding that ectopic
expression of both active Raf kinase and Akt kinase was sufficient to
cause RIE cell transformation, we first transfected the parental RIE-1 cells with an Akt-myr expression vector and found that this did not
result in a significant phenotypic alteration. Interestingly, introduction of a constitutively active Raf mutant, Raf BXB (35), into
the Akt-myr-transfected RIE cells resulted in morphological transformation of these cells. The morphological appearance of RIE/Akt-myr/Raf BXB cells was similar to
Ha-RasVal12-transformed RIE cells with a spindly appearance
and overlapping growth (Fig. 7B, upper
panels). RIE/Akt-myr/Raf BXB cells grew in soft agarose and
formed colonies that were relatively smaller than the colonies formed
by RIE-RasVal12 cells (Fig. 7B,
bottom panels).

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Fig. 7.
The role of Raf and Akt in the transformation
of RIE cells. A, RIE/ Raf-22W cells were transfected
with a pZeoSV2/Akt-myr plasmid or the empty vector and selected by
growth in medium containing zeocin. 1 × 104 cells
from the pooled clones were seeded in soft agarose. The plates were
incubated for 10 days, and colony numbers were counted. All stable
transfection experiments shown in this figure were repeated at least
twice. B, transformation of RIE/Akt-myr/Raf BXB cells. RIE
parental cells were transfected with pZeoSV2/Akt-myr. Stable clones
were selected and transfected with pSR /Raf BXB. The morphology of
RIE/Akt-myr/Raf BXB cells was compared with the morphology of
Ha-RasVal12 transfected RIE cells (upper
panels; original magnification, ×100). To determine the
anchorage-independent growth of RIE/Akt-myr/Raf BXB cells or
RIE-Ha-RasVal12 cells, they were seeded in 0.4% soft
agarose and were incubated for 10 days (lower
panels; original magnification, ×40). C,
Northern analysis for TGF . Upper panel,
RIE-iHa-Ras cells were treated with 5 mM IPTG or IPTG plus
20 µM LY-294002 for 24 h. The levels of TGF
mRNA were analyzed by Northern blotting. Lower
panel, the expression of TGF mRNA in RIE,
RIE/ Raf-22W (Raf), and RIE/ Raf-22W/Akt-myr
(Raf/Akt) cells. D, inhibition of EGFR in
RIE/ Raf-22W/Akt-myr cells. RIE/ Raf-22W/Akt-myr cells were treated
with Me2SO (control), PD-153035 (1 µM) for
48 h, or PD-98059 (50 µM) for 24 h (original
magnification, ×100).
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Previous reports indicate that an epidermal growth factor receptor
(EGFR)-dependent autocrine growth loop is required for Ras-mediated transformation (36, 37). TGF
levels were increased in
Ras-induced RIE-iHa-Ras cells and treatment with LY-294002 abolished
Ras induction of TGF
(Fig. 7C, upper
panel). The expression of TGF
was elevated in
RIE/
Raf-22W cells and co-expression of
Raf-22W and Akt-myr
further increased the level of TGF
in RIE cells (Fig. 7C,
lower panel). Treatment with an EGFR inhibitor (1 µM of PD-153035) for 48 h almost completely reversed
the transformed appearance of RIE/
Raf-22W/Akt-myr cells (Fig.
7D). PD-153035-treated RIE/
Raf-22W/Akt-myr cells acquired
a cuboidal appearance, and cell-cell contact inhibition was restored in
these cells. As expected, PD-98059 (50 µM) reversed the
transformed morphology of RIE/
Raf-22W/Akt-myr cells by 24 h.
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DISCUSSION |
A previous study by Oldham et al. (36) reports that
Ras(12V/35S) and Ras(12V/37G) mutants that have an impaired ability to
activate PI3K are able to transform RIE cells, suggesting that activation of PI3K is not required for Ras-mediated transformation. Our
data reported here clearly demonstrate that activation of the PI3K/Akt
pathway is involved in RIE cell transformation following induction of
oncogenic Ha-RasVal12. Inhibition of Akt/PKB activity
following treatment with LY-294002 exerted a complex anti-neoplastic
effect on Ras-expressing intestinal epithelial cells, which included
stimulation of apoptosis, reversal of morphological transformation, and
accumulation of cells in the G1 phase of the cell cycle. It
is generally accepted that LY-294002, at low concentrations (5-20
µM), specifically targets PI3K activity (34). The
observation that both LY-294002 and wortmannin (a structurally
unrelated PI3K inhibitor) significantly blocked the
Ha-RasVal12-mediated transformation indicates that PI3K was
the likely target of these compounds in RIE-iHa-Ras cells (reviewed in
Refs. 38 and 39).
The PI3K/Akt pathway is known to be oncogenic in other circumstances.
Ectopic expression of active Akt/PKB leads to transformation of NIH 3T3
and chicken embryo fibroblasts (17, 18). Transformation of
hematopoietic cells by BCR/ABL requires activation of the
PI3K/Akt signaling pathway (16). It has been demonstrated that the
tumor suppressor gene, PTEN, suppresses tumor formation by
restraining the PI3K/Akt pathway (Refs. 19-22; reviewed in Ref. 23).
Several reports indicate that expression of wild type PTEN
that down-regulates Akt/PKB activity results in G1 growth
arrest (19, 20). Interestingly, a high incidence of aberrant
transcripts of the PTEN/MMAC1 gene has been
observed in colorectal neoplasia (40), suggesting that Akt activity
might play some role in colorectal carcinogenesis.
Mounting evidence suggests that Akt/PKB mediates growth factor-induced
cell survival and blocks programmed cell death (11-13). Induction of
oncogenic Ras promoted cell survival and resulted in loss of contact
inhibition. We sought to determine which Ras effector was responsible
for this effect and found that, in Ras-induced RIE-iHa-Ras cells,
inhibition of Akt/PKB activity only partially restored the effect on
programmed cell death, whereas inhibition of ERK activity completely
blocked the Ras-induced increase in cell survival. On the other hand,
Akt/PKB appears to play an extremely important role in cell cycle
progression. Inhibition of Akt/PKB activity led to an accumulation of
RIE-iHa-Ras cells in the G1 phase of the cell cycle. This
effect was not observed in PD-98059-treated RIE-iHa-Ras cells. These
findings suggest that Akt/PKB activity might play a role in overcoming
cell cycle blocks that are necessary to prevent cells from uncontrolled
proliferation and eventual transformation. Progression through the mid
to late G1 phase of the mammalian cell cycle is dependent
upon the cyclin D1-mediated activation of CDK4 or the related CDK6
(41). Previous studies suggest that cyclin D1 is regulated via the Ras
signaling pathway. Activation of ERK1 and ERK2 up-regulates cyclin D1
(42). Several studies suggest that cyclin D1 is also regulated by the
Akt/PKB pathway at both the transcriptional (43) and
post-transcriptional levels (44). Akt/PKB stabilizes cyclin D1 protein
through inhibition of glycogen synthase kinase-3
kinase (45).
We recently reported that induction of Ras activates Akt, inactivates
glycogen synthase kinase-3
, and increases the level of cyclin D1 in
RIE-iHa-Ras cells (31). In the present study, we show that inhibition
of Akt/PKB completely blocked Ras-induced expression of cyclin D1, indicating that both ERK and Akt/PKB are essential for Ras-mediated induction of cyclin D1. Interestingly, forced expression of a stable
mutant of cyclin D1 (T286A) (46) failed to override LY-294002-induced G1 growth arrest in RIE-iHa-Ras cells (data not shown).
Treatment with PD-98059 decreased the levels of cyclin D1 to a similar
extent but did not cause growth arrest at the G1 phase,
suggesting that inhibition of cyclin D1 alone is not sufficient for
LY-294002-induced G1 growth arrest.
G1-S transition also requires the activity of cyclin E/CDK2
which accelerates the phosphorylation of retinoblastoma protein initiated by the cyclin D/CDK complex and prevents its inhibition of
transcription factors (including the E2Fs). In the present study, we
found that induction of oncogenic Ras increased the levels of CDK2,
which was dependent on the activation of Akt/PKB, but not MEK/ERK
activity. p27kip, which represses the activity
of the cyclin E/CDK2 complex, is thought to be directly involved in
restriction point control (47, 48). Recently, Medema et al.
(49) demonstrated that expression of AFX-like forkhead transcription
factors blocks cell cycle progression at the G1 phase
through up-regulation of p27kip. Activation of
Akt/PKB results in phosphorylation of AFX and FKHR-L1 and a reduction
of p27kip expression. In agreement with previous
findings, we observed that inhibition of Akt/PKB resulted in growth
arrest at the G1 phase, which was associated with an
increased expression of p27kip in RIE-iHa-Ras cells.
Oncogenic mutations in ras result in activation of multiple
downstream signaling proteins including Raf/MEK/ERKs (50, 51), Rho
family (52-54), and the PI3K/Akt/PKB pathway (14, 15). Activation of
Raf/MEK/ERKs is known to be sufficient to transform NIH 3T3 fibroblasts
(55, 56) but not sufficient for Ras transformation of RIE epithelial
cells (30). These findings suggest that downstream effectors that are
required for Ras-induced transformation are cell
type-dependent and that additional Raf-independent
pathway(s) are required to complete Ras-mediated morphological and
growth transformation in intestinal epithelial cells. Our results
demonstrate that ectopic expression of activated Akt/PKB did not cause
significant phenotypic alterations in intestinal epithelial cells;
however, expression of both active Raf and active Akt is sufficient to induce transformation of RIE cells. Our data suggest that, in cooperation with Raf, PI3K/Akt signaling promoted the
EGFR-dependent autocrine growth loop that is thought to be
essential for the morphological transformation of RIE cells (36, 37).
Although it has been shown that Raf may be activated by Akt (57), there is no evidence to support direct activation of PI3K/Akt signaling by
the Raf/MEK/ERK pathway, suggesting that they play different roles in
Ras-mediated transformation. Indeed, we observed that the Raf/MEK/ERK
pathway predominantly enhances cell survival and that the PI3K/Akt
pathway predominantly promotes loss of contact inhibition of cell
growth. Since ras mutations are found in a wide variety of
human malignancies and in 50% of colorectal adenocarcinomas (26), our
results are of particular interest in understanding the changes in
molecular signaling pathways that occur during colorectal
carcinogenesis and may have clinical significance by providing
additional therapeutic targets.
 |
FOOTNOTES |
*
This work was supported by the National Institutes of Health
Grants DK-47297, CA-77839 (to R. N. D.), and CA 68485 (to
Vanderbilt-Ingram Cancer Center) and by a Veterans Affairs merit grant.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Medicine/GI, MCN C-2104, Vanderbilt University Medical Center, 1161 21st Ave. S., Nashville, TN 27232-2279. Tel.: 615-322-5200; Fax:
615-343-6229; E-mail: raymond.dubois@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M010093200
 |
ABBREVIATIONS |
The abbreviations used are:
Akt/PKB, protein
kinase B;
RIE, rat intestinal epithelial;
PI3K, phosphatidylinositol
3-kinase;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
EGFR, epidermal growth factor receptor;
DMEM, Dulbecco's modified
Eagle's medium;
PBS, phosphate-buffered saline;
TGF, transforming
growth factor;
CDK, cyclin-dependent kinase.
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