Download Involvement of the PI 3-kinase signaling pathway

Carcinogenesis vol.25 no.2 pp.241±248, 2004
DOI: 10.1093/carcin/bgg195
Involvement of the PI 3-kinase signaling pathway in progression of colon
adenocarcinoma
Kianoush Khaleghpour1, Yang Li1, Denis Banville1,
Zhenbao Yu1 and Shi-Hsiang Shen1,2,3
1
Mammalian Cell Genetics, Biotechnology Research Institute, National
Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec
H4P 2R2, Canada and 2Department of Medicine, McGill University,
Montreal, Quebec H3A 1B1, Canada
3
To whom correspondence should be addressed at Mammalian Cell Genetics,
Biotechnology Research Institute, National Research Council of Canada,
6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada. Tel: ‡1 514
496 6318; Fax: ‡1 514 496 6319; Email: [email protected]
The phosphoinositide 3-kinase (PI 3-kinase) signaling
pathway has been shown to play a pivotal role in intracellular signal transduction pathways involved in cell growth,
cellular transformation and tumorigenesis. Analysis of several colon adenocarcinoma cell lines indicates that the PI
3-kinase signaling pathway is up-regulated in colon cancers. In particular, the protein levels and phosphorylation
status of Akt and p70 S6 kinase are up-regulated in colon
adenocarcinoma cell lines. More significantly, we have
demonstrated for the first time that the phosphorylation
of FKHR, a downstream target of Akt, is increased in these
cell lines. Intriguingly, phosphorylation of three components of the PI 3-kinase signaling pathway, namely Akt,
p70 S6 kinase and FKHR, are in direct correlation with the
degree of tumorigenic potential of the colon cell lines
tested. No differences in the protein levels of the two subunits of PI 3-kinase, p85 and p110a, and PTEN were noted.
Real-time quantitative PCR indicated an increase in levels
of Akt message only, and not of the other signaling pathway
components. Inhibition of the PI 3-kinase with wortmannin
decreased the anchorage-independent growth of colon cells
in a soft agar assay. Hence, the components of the PI
3-kinase signaling pathway could serve as potential candidates for drug development in treatment of colon cancer.
Introduction
Protein kinases participate at multiple levels along signal
transduction pathways. These signaling cascades are involved
in diverse biological responses, ranging from cell cycle control
to differentiation to cell proliferation, as well as determination
of organ size and body growth (1±3). A vast body of literature
has documented the involvement of the phosphoinositide
3-kinase (PI 3-kinase) signaling pathway in such functions
(3±17). The involvement of PI 3-kinase signaling pathway
effectors in various forms of cancers has been documented.
PIK3CA elevation was noted in ovarian cancers (18), while
deletion of the catalytic subunit of PI(3)Kg in mice resulted
in colorectal carcinomas (19). Increased expression and
Abbreviations: DTT, dithiothreitol; FBS, fetal bovine serum; FKHR,
Forkhead transcription factor; PMSF, phenylmethylsulfonyl fluoride; PI
3-kinase, phosphoinositide 3-kinase.
Carcinogenesis vol.25 no.2 # Oxford University Press; all rights reserved
activation of Akt was noted in primary human gastric
adenocarcinomas, pancreatic ductal adenocarcinomas, colon
carcinomas, glioblastomas and thyroid carcinomas, as well as
prostate, breast, cervical, ovarian and small cell lung cancers
(7,20±31). Similarly, elevation of p70 S6 kinase was noted in
small cell lung cancer cells, pancreatic cancer cells and liver
tumors (24,32±34).
PI 3-kinase, a heterodimeric protein composed of a catalytic
subunit (p110a) and a regulatory subunit (p85), is implicated
in suppression of apoptosis (6,9,10,35). Upon stimulation of
cells with a wide range of extracellular stimuli, PI 3-kinase
phosphorylates the D-3 position of phosphoinositides to form
the second messengers phosphatidylinositol-3,4-bisphosphate
and phosphatidylinositol-3,4,5-trisphosphate. The survival
mechanism initiated by these lipid products is exerted via
downstream components of this kinase, Akt, mTOR and p70
S6 kinase (36). An intracellular inhibitor of PI 3-kinase activity is PTEN, a lipid phosphatase acting on the phosphatidylinositides generated by PI 3-kinase, thereby functioning as
a tumor suppressor (37±39).
Akt regulates a number of critical cellular pathways, including those leading to cellular proliferation and inhibition of
apoptosis (6,9±11,40,41). The survival signals of Akt upon
its phosphorylation and activation are mediated in part via
identified downstream targets of this kinase. Phosphorylation
of BAD (42,43), Caspase 9 (44) and Forkhead transcription
factor (FKHR) (4,45±47) by Akt suppresses the pro-apoptotic
function of these proteins. FKHR, together with AFX and
FKHRL1, belongs to a small subset of the Forkhead family
of transcription factors (48). Five phosphorylation residues
have been identified in FKHR (Thr24, Ser256, Ser319,
Ser322 and Ser325) (46,47,49). Phosphorylation of FKHR
results in its inactivation and nuclear exclusion (4,47,50±53).
Thus, Akt also promotes cell survival by negative regulation of
the transcriptional activity of AFX, FKHR and FKHRL1,
through their phosphorylation.
Another downstream target of Akt is mTOR. Akt phosphorylates mTOR on Ser2448 (54±56). mTOR has numerous regulatory functions, including activation of p70 S6 kinase, as a
means of translational modulation. p70 S6 kinase contains
several phosphorylation sites (57) which are targeted by various upstream kinases, including mTOR and PDK (58±62).
Phosphorylation and activation of p70 S6 kinase results in
phosphorylation of the 40S ribosomal protein S6, which in
turn stimulates translation of 50 TOP mRNAs, messages
encoding for ribosomal proteins and other components of the
translational machinery (63±65).
This study analyses involvement of the PI 3-kinase signaling
pathway effector molecules in progression of colon adenocarcinoma. Colorectal cancer constitutes the third leading cause
of cancer-related death in North America (Cancer Facts &
Figures 2002, American Cancer Society; Canadian Cancer
Statistics 2000, Canadian Cancer Society). We examined
total protein level and phosphorylation status of various
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K.Khaleghpour et al.
members of the PI 3-kinase pathway in an array of colon
adenocarcinoma cell lines which were previously shown to
demonstrate varying degrees of tumorigenic potential (66).
We show that protein levels and phosphorylation levels of
Akt and p70 S6 kinase are elevated in colorectal carcinoma
cell lines, and this elevation positively correlates with the
increased tumorigenic potential of colon adenocarcinoma cell
lines. More significantly, we found that increased levels of
FKHR and its phosphorylation at Ser265 correlate directly
with the degree of transformation potential of the cell lines
tested. Inhibition of the PI 3-kinase signaling pathway with
wortmannin resulted in a reduction in colony size in a soft agar
assay, indicating that inhibition of this pathway plays an
important role in oncogenicity, at least in colon cell lines.
Materials and methods
Cell lines
Human colon adenocarcinoma cell lines: SW1417, CaCo2, Colo201, Colo205,
SW1222, SW403, Lovo, LS174T and LS180 were grown in a-MEM medium
supplemented with 10% fetal bovine serum (FBS). Human kidney 293A cells
were grown in DMEM supplemented with 10% FBS.
Western blotting
To test the specificity of the phospho-specific antibodies, 293A cells were
starved in DMEM medium without FBS for 16 h. For serum induction, cells
were stimulated with medium containing 10% FBS for 30 min. To test rapamycin and wortmannin sensitivity, cells were incubated with rapamycin (20 ng/ml)
or wortmannin (100 nM) for 30 min and then stimulated with medium containing 10% FBS for 30 min. Cells were rinsed twice with ice-cold phosphatebuffered saline and incubated with the appropriate lysis buffer. For analysis of PI
3-kinase and PTEN, the lysis buffer contained 10 mM Tris±HCl, pH 7.5, 1%
Triton X-100, 50 mM KCl, 1 mM dithiothreitol (DTT), 2 mM MgCl2 and
0.2 mM phenylmethylsulfonyl fluoride (PMSF). For analysis of Akt, the lysis
buffer contained 20 mM Tris±HCl, pH 7.5, 140 mM NaCl, 10% glycerol, 1%
NP40, 2 mg/ml aprotonin, 2 mg/ml leupeptin, 1 mM PMSF, 20 mM NaF, 1 mM
Na4 P2 O7 and 1 mM Na3 VO4 . For analysis of p70 S6 kinase, the lysis buffer
contained 50 mM sodium phosphate, pH 7.2, 2 mM EGTA, 25 mM NaF,
25 mM b-glycerophosphate, 0.5% Triton X-100, 100 mM Na3 VO4 , 1 mM
PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM benzamidine and 2 mM
DTT. Lysates were clarified by centrifugation at 14 000 r.p.m. for 10 min and
total protein concentration was determined in triplicate using the Bio-Rad
Protein Assay (Bio-Rad) and quantified against a standard curve of bovine
serum albumin protein concentrations (Pierce). For each experiment, equal
amounts of total protein, as indicated in the figure legend, were electrophoresed
on 6±15% SDS±PAGE gels and electroblotted onto nitrocellulose membranes
(Millipore). Filters were then blocked in Tris-buffered saline containing 0.2%
Tween 20 (TBST) and 5% (w/v) dry milk at 25 C for 2 h. Membranes were
incubated overnight at 4 C with one of the following antibodies at the indicated
dilutions: mouse monoclonal anti-actin (ICN Biomedicals) at 1:500, anti-PI
3-kinase p85 (N-SH3 clone AB6) (Upstate Biotechnology) at 1:1000, anti-PI
3-kinase p110a (BD Biosciences) at 1:150, rabbit polyclonal anti-Akt, antiPTEN, anti-p70 S6 kinase, anti-phospho-specific Akt (Ser473) (Cell Signal
Technology) at 1:1000 and anti-phospho-specific p70 S6 kinase (Thr389) (Cell
Signal Technology) at 1:300. Membranes were washed with TBST three times
and incubated as follows: for rabbit polyclonal antibodies, membranes were
incubated with donkey anti-rabbit horseradish peroxidase-conjugated IgG (Cell
Signal Technology) at 1:2000; for mouse monoclonal antibodies membranes
were incubated with goat anti-mouse horseradish peroxidase-conjugated IgG
(Bio-Rad) at 1:5000 for 1 h. Membranes were washed with TBST three times
and signals were detected using an enhanced chemiluminescence kit (LumiGLO
reagent, Cell Signal Technology) after exposure to X-ray film (Kodak).
Soft agar assay
Six-well tissue culture plates were covered with 4 ml of 0.5% Bacto Agar
(Difco) growth medium containing 20% FBS in the presence or absence of
100 nM wortmannin (Calbiochem). Cells (20 000) were plated in duplicate in a
3 ml suspension of 0.35% agar medium. After 24 h, medium containing 20%
FBS was added to each well and refreshed twice weekly. For wortmannin
treatment of cells the drug was added to the solid underlay and overlay agar
medium, as well as the liquid top layer. Cells were photographed after 2 weeks.
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Quantitative PCR
Total cellular RNA was extracted with Trizol (Gibco BRL) according to the
manufacturer's instructions. Any potential traces of genomic DNA were
removed by treatment with DNA-Free DNaseTM according to the manufacturer's instructions (Ambion). A two-step RT±PCR was carried out. The first
strand cDNA synthesis was generated using the SuperscriptTM First-Strand
Synthesis System for RT±PCR (Invitrogen) using an oligo(dT)12ÿ18 primer
according to the manufacturer's instructions. The second strand cDNA was
synthesized using gene-specific primers with the Light Cycler-FastStart DNA
Master SYBER Green 1 kit (Roche), according to the manufacturer's instructions, on a Light Cycler machine (Roche).
PI 3-kinase activity assay
Cells were washed with ice-cold buffer A (137 mM NaCl, 20 mM Tris±HCl,
pH 7.4, 1 mM CaCl2 , 1 mM MgCl2 ) once and lysed with buffer A containing
1% NP-40 and proteinase inhibitor mixture (tablets from Roche Diagnostics).
The whole cell lysates containing 2.8 mg of total protein were subjected to
immunoprecipitation with 5 mg of anti-PI 3-kinase antibody (Upstate Biotechnology). After 1 h incubation at 4 C, 50 ml of a 50% slurry of protein
A±Sepharose was added and the samples were incubated with rocking for another
hour at 4 C. The beads were washed three times with buffer A containing 1%
NP-40, three times with 0.1 M Tris±HCl, pH 7.4 and 5 mM LiCl and twice with
TNE buffer (10 mM Tris±HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA) and
suspended in 50 ml of TNE. Ten microliters of phosphatidylinositol (2 mg/ml)
(Avanti Polar Lipids Inc.), 10 ml of 100 mM MgCl2 and 5 ml of [g-32 P]ATP
(0.5 mM ATP containing 30 mCi of [g-32 P]ATP) were added to the immunoprecipitants and the samples were incubated with agitation for 10 min at 37 C.
The phosphatidylinositol solution was prepared by suspending phosphatidylinositol in 10 mM Tris±HCl, pH 7.4, 1 mM EGTA buffer and sonicating at 4 C
for 10 min with 20 30 s pulses. The reaction was stopped by adding 20 ml of
6 N HCl and the lipid was extracted by adding 160 ml of chloroform:methanol
(1:1). The lipid in the lower phase was loaded on a silicon TLC plate (Fisher
Scientific) and the plate was developed by chromatography in chloroform:
methanol:H2 O:NH4 OH (60:47:11.3:2). The radiolabeled lipid was detected by
exposure of the TLC plate to X-ray film (Kodak).
Northern blot
Total RNAs from the individual cell lines were extracted by the Trizol method
(Life Technologies Inc.) and loaded onto agarose gels at 20 mg/lane. The
separated RNA was transferred to nylon membrane and the blot used for
hybridization. PTEN cDNA containing the full coding region (1.2 kb) and
human b-actin cDNA were labeled with 32 P using Ready-To-GoTM DNA
Labelling Beads from Amersham Biosciences according to the manufacturer's
instructions. Blots were prehybridized in ULTRAhybTM solution from Ambion
Inc. for 30 min. Hybridization was performed in the same solution containing
106 c.p.m./ml 32 P-labeled cDNA fragment at 42 C overnight. Then the blots
were washed twice with 2 SSC, 0.1% SDS for 10 min and twice with 0.1
SSC, 0.1% SDS for 15 min at 42 C and exposed to X-ray films for 24 h.
Results and discussion
Akt
We initially set out to observe the protein expression level of
Akt (PKB) in a panel of colon adenocarcinoma cell lines with
varying degrees of transformation, as assayed previously for
their tumorigenic potential in nude mice (66). We performed
western blot analysis on nine colon adenocarcinoma cell lines.
In cell lines displaying low tumorigenic potential, such as
SW1417, CaCo2, Colo201 and Colo205, the level of Akt protein was low (Figure 1A, lanes 1±4) in comparison with cell
lines with high tumorigenic potential, such as SW1222, SW403,
Lovo, LS174T and LS180 (lanes 5±9). Since the activity of Akt
is regulated by its phosphorylation at residues Ser473 and
Thr308 (67,68), we examined its phosphorylation state using
phospho-specific antibodies directed against these two sites. To
demonstrate the specificity of the anti-phospho antibodies for
phosphorylated Akt, serum-deprived 293A cells were stimulated with serum to induce phosphorylation. The antibodies
failed to interact with Akt from serum-deprived or wortmannintreated cells, but they bound to Akt from serum-fed or rapamycin-treated cells (Figure 1B and C, compare lanes 10 and 13
PI 3-kinase signaling and colon adenocarcinoma
Fig. 1. Up-regulation of Akt content and phosphorylation in colon adenocarcinoma cell lines. Colon adenocarcinoma cell lines were grown in a-MEM containing
10% FBS (lanes 1±9). Increasing tumorigenic potential of colon adenocarcinoma cells is indicated from left to right with triangles. 293A cells were grown in
DMEM containing 0.5% FBS for 16 h (lanes 10±13), after which cells were either mock treated (lane 10), incubated for 30 min in the presence of 20 ng/ml
rapamycin (lane 12) or 10 nM wortmannin (lane 13) prior to stimulation with 10% FBS for 1 h, or stimulated with 10% FBS alone (lane 11). Total cell extract
(50 mg) was electrophoresed on 12% SDS±PAGE gels and electroblotted onto a 0.45 mm pore size nitrocellulose membrane. One membrane was probed with a
rabbit polyclonal anti-Akt antibody which recognizes Akt irrespective of its phosphorylation state (A), while the others were probed with a rabbit polyclonal
anti-phosphopeptide antibody which is specific for phospho-S473 in Akt (B) and phospho-T308 (C). A representative blot was probed with a mouse monoclonal
anti-b-actin antibody (D).
with 11 and 12). Phosphorylation of Akt at Ser473 and Thr308
was increased in colon cell lines with high tumorigenic
potency (Figure 1B and C, lanes 5±9) in comparison with cell
lines with low tumorigenic potency (Figure 1B and C, lanes 1±4).
Thus, with the exception of Colo201 and Colo205, both total
Akt and phosphorylated Akt are correlated with increasing
tumorigenic potential of the cell lines. To determine whether
the increase in Akt protein was at the level of translation or
transcription, we performed real-time quantitative PCR analysis
of Akt. Our results indicated that Akt message was increased in
the various cell lines, within a 2- to 4-fold amplification range
in comparison to the level of Akt mRNA in SW1417 cells, the
most non-tumorigenic colorectal adenocarcinoma cell line in
our panel (Table 1). However increases in Akt message did not
correlate with the tumorigenic potential of the available colon
cell lines. As a control, we examined the levels of the PRL-2
gene, which is not directly involved in the PI 3-kinase signaling
pathway. Levels of PRL-2 mRNA were similar in our entire
panel of colon adenocarcinoma cell lines (Table I).
During preparation of this manuscript, Itoh et al. (23) and
Roy et al. (27) reported similar findings upon examining the
role of Akt in colon carcinomas. Itoh et al. examined Akt
phosphorylation at Ser473 in two colon cell lines and 65
human colorectal carcinomas; they noted high expression of
phosphorylated Akt in 46% of the tumors. Roy et al. demonstrated that Akt overexpression is an early event during
Table I. Quantitative RT±PCR of PI 3-kinase signaling genes
Cell lines
Akt
p70S6k
FKHR
PRL-2
SW 1417
CaCo2
Colo201
Colo205
SW1222
SW403
Lovo
LS174T
LS180
HT29
1
1.4
2.5
2.2
4.2
0.9
2.3
1.1
1
1.2
1
1.1
0.9
0.5
0.8
0.6
1.6
1
0.8
1
1
3.5
0.6
1.1
18.4
0.5
1.2
1.3
0.7
0.9
1
1
1
1
1
1
1
1
0.9
1
Total cellular RNA (2.5 mg) was processed for RT±PCR as described in
Materials and methods. Levels of Akt, p70 S6 kinase, FKHR and PRL-2
message were normalized to those of two housekeeping genes, cyclophilin
and 28S rRNA. Values reported are fold amplification of various messages
in comparison to the most non-transformed cell line, SW1417.
sporadic colon carcinogenesis, as Akt was detected in 57% of
the sporadic carcinomas (21 out of 37 tumors) and adenomas
(17 out of 30) examined, with phosphorylation of Akt at Ser473
detected only in neoplastic rather than normal epithelium. Here
we have noted transcriptional, translational and posttranslational modification increases in Akt in colon adenocarcinoma cells. However, only increases in the latter two correlate
well with the increased tumorigenic potential of the colon
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K.Khaleghpour et al.
adenocarcinoma cells, indicating that activation of Akt
could play an essential role in the progression of colon
carcinomas.
FKHR
Since the level and activity of Akt increased with the tumorigenic potential of the colon adenocarcinoma cell lines, we proceeded to examine the protein expression pattern of FKHR, a
downstream target of Akt (4,48±50,52,53,69,70). Western blot
analysis indicated a gradual increase in the expression of total
FKHR and phosphorylation of this protein at Ser256, which
correlated well with increasing tumorigenicity of the colon cell
lines with the exception of a relatively low phosphorylation level
in Colo201 and Colo205, a result similar to Akt (Figure 2A and B,
lanes 1±9). Although the phosphorylation of FKHR at Thr24
was increased in three of the high tumorigenic colon cell lines
(Figure 2C, compare lanes 1±6 with 7±9), we did not observe a
gradual increase in phosphorylation of this site accompanying
the progressive tumorigenic potential of the colon cell lines. The
specificity of thephospho-specific antibodies was demonstrated as
they did not detect FKHR from serum-deprived or wortmannintreated cells, but rather from serum-stimulated and rapamycintreated cells (Figure 2B and C, compare lanes 10 and 13 with 11
and 12). In contrast, antibodies directed against FKHR recognized the protein in 293A cells irrespective of its phosphorylation state (Figure 2A, lanes 10±13). A representative blot was
immunoblotted with b-actin to demonstrate equal protein content of the samples (Figure 2D). Real-time quantitative PCR
analysis indicated that the levels of FKHR message were essentially the same across the colon cell lines, with the exception
of CaCo2 and SW1222, in which 3- and 18-fold elevations in
FKHR mRNA, respectively, were noted in comparison with the
levels observed in SW1417 cells (Table 1). High level expression of mRNA in these two cell lines may be due to their
intrinsic, tumor-unrelated characteristics. Therefore, only the
translational and post-translational modification increases in
FKHR are correlated with the tumorigenic potential of the cells.
It is worth noting that despite elevations in FKHR levels
observed in colon adenocarcinoma cells, we suspect that the
majority of FKHR in these cells is inactivated due to enhanced
phosphorylation. Phosphorylation of FKHR results in its
exclusion from the nucleus and decreases its transcriptional
activity on targeted cell cycle regulating genes (4,47,50,53,64).
A number of studies have aimed at understanding the role of the
various FKHR phosphorylation sites. Phosphorylation of Ser256
has been shown to inhibit transactivation, by inhibiting nuclear
import through suppression of a nuclear localization signal
(51,72,73). Phosphorylation of Thr24 has been suggested to
induce interaction of FKHR with 14-3-3 proteins (4,73). This
may sequester FKHR isoforms in the cytosol, contributing to the
growth factor-induced nuclear exit of these transcription factors.
Thus differences in phosphorylation pattern of FKHR at the two
sites examined in colon cell lines may provide clues as to the
mechanism of PI 3-kinase involvement in progression of colon
carcinomas.
PI 3-kinase and PTEN
Since the activity of Akt and its downstream effector FKHR
correlated well with the increased transformed state of the colon
adenocarcinoma cell lines, we wished to determine whether upregulation of the PI 3-kinase signaling pathway also occurred for
the upstream regulator of Akt, PI 3-kinase (41,74). To this end,
we examined protein levels of the regulatory and catalytic subunits, p85 and p110a, respectively, of PI 3-kinase. Western blot
analysis indicated that the levels of p110a and p85 are similar
across the colon cell lines (Figure 3A and C). The only exception
was that p85 was not detected in cell line SW1417 (Figure 3C,
lane 1). This observation was reproducible and did not appear to
be due to degradation of proteins during protein extraction or
sample preparation. Again, both blots were probed with an anti
b-actin antibody to demonstrate equal loading of total cellular
proteins (Figure 3B and D). We next determined if PI 3-kinase
activity is involved in the tumorigenic potential of the colon cell
lines. To determine PI 3-kinase activity, we immunoprecipitated
PI 3-kinase with anti-P85 antibody and carried out in vitro PI
3-kinase activity assay. As shown in Figure 3E, PI 3-kinase activity
appeared to display some correlations with the increased transformed state of the colon adenocarcinoma cell lines as PI
3-kinase was hardly detected in SW1417 cells but was greatly
activated in cell line LS180, a cell line with the highest tumorigenic potential (Figure 3E). Undetectable activity of PI 3-kinase
in SW1417 cells is consistent with lack of expression of the P85
subunit in this cell line. It is not known why p85 could not be
detected by the anti-p85 antibody in the SW1417 cell line.
Fig. 2. Elevation of FKHR and its phosphorylation in colon cells. Total cell extracts (50 mg) were processed for western blotting as described in Materials and
methods, using 10% SDS±PAGE. One membrane was probed with a rabbit polyclonal anti-FKHR antibody which recognizes FKHR irrespective of its
phosphorylation state (A), while the others were probed with a rabbit polyclonal anti-phosphopeptide antibody which is specific for phospho-S256 (B) and
phospho-T24 (C) in FKHR. A representative blot was probed with a mouse monoclonal anti-b-actin antibody, to normalize for protein loading.
244
PI 3-kinase signaling and colon adenocarcinoma
Fig. 4. PTEN expression levels are unaltered in most of the tested colon cell
lines except for SW1222. (A) Colon adenocarcinoma cell extracts (50 mg)
were subjected to western blotting with a rabbit polyclonal anti-PTEN
antibody. (B) The full-length coding region of PTEN was used for northern
blot analysis. Each lane of the blot contains 20 mg of total RNA from the
cell lines as indicated.
Fig. 3. Comparable PI 3-kinase levels and activity in colon cells. (A±D)
Total cell extracts (50 mg) were processed for western blotting, using 10%
SDS±PAGE, and probed with mouse monoclonal anti-PI 3-kinase (p110a)
(A) and anti-PI 3-kinase (p85) (C) antibodies. Blots were probed with a
mouse monoclonal anti-b-actin antibody (B and D). (E) PI 3-kinase was
immunoprecipitated with anti-P85 antibody from the cell lines as indicated
and subjected to kinase activity assay as described in Materials and
methods.
northern blot (Figure 4B) in SW1222 cells. As controls, it was
readily detectable in other cell lines (Figure 4B and data not
shown). These results suggest that the lack of expression of
PTEN in cell line SW1222 is very likely caused by gene deletion
or promoter silencing, but not by nonsense or mismatch mutation. We also extracted genomic DNA and tried to amplify the
PTEN gene using three pairs of primers. We failed to amplify
PTEN genomic DNA from cell line SW1222, supporting the
notion that the PTEN gene is probably deleted in this cell line.
PTEN is a lipid phosphatase acting on PI 3-kinase. We also
checked the levels of PTEN, to determine whether alterations
in its levels may play a role in up-regulation of the activity of
downstream targets of PI 3-kinase. Western blot analysis indicated that the PTEN expression profile was not altered amongst
colon adenocarcinoma cell lines (Figure 4A), with the exception
that the antibody did not detect observable amounts of the
protein in SW1222 cells (lane 5). This observation was reproducible and did not appear to result from degradation of proteins
during protein extraction or sample preparation. Taniyama et al.
(75) also reported that PTEN protein expression is maintained in
sporadic colorectal tumors. Although our examination of PTEN
protein levels did not evaluate loss of function due to genetic
mutations, a phenomenon normally associated with cancer formation, it is possible that PTEN may be genetically modified in
colon adenocarcinoma cells and rendered inactive. The molecular mechanism of lack of PTEN expression in SW1222 cells was
further investigated by RT±PCR and northern blot analysis. This
mRNA could not be detected by RT±PCR (data not shown) or
Soft agar
The soft agar assay is often used to test for contact inhibition or
non-adherent growth of transformed cells. Since our data indicated that the PI 3-kinase signaling pathway is up-regulated in
colon cell lines, we were interested to see whether the transformation potential of the cells would be reduced by addition
of wortmannin, an inhibitor of this pathway, in a semi-solid
agarose medium. Cell lines with low transformation potential,
SW1417, CaCo2 and Colo201, formed few and small foci in
the agar medium suspension (Figure 5, left panels). In contrast,
the Lovo cell line, which has high transformation potential,
displayed abundant and large colonies (Figure 5, left panels).
Addition of wortmannin to colon adenocarcinoma cells did not
result in a reduction in colony efficiency (data not shown), but
rather resulted in a decrease in colony size (Figure 5, right
panels). These findings further indicate that the PI 3-kinase
signaling pathway plays a role in progression of colon carcinoma and that control of the activity of the pathway may have
potential in the treatment of colon carcinomas.
245
K.Khaleghpour et al.
Fig. 5. Wortmannin reduces colony size formation in transformed cells. Cells (20 000) were plated in duplicate in semi-solid agar medium as described
in Materials and methods in the presence or absence of wortmannin. Cells were photographed and counted for colony growth after 14 days. The colony
size is expressed as average diameter of at least six representative colonies.
A
B
C
Fig. 6. p70 S6 kinase elevation in colon cells. Total cell extracts (50 mg) were processed for western blotting using 8% SDS±PAGE, as described in Materials
and methods. One membrane was probed with a rabbit polyclonal anti-p70 S6 kinase antibody which recognizes p70 S6 kinase irrespective of its phosphorylation
state (A), while the other was probed with a rabbit polyclonal anti-phosphopeptide antibody which is specific for phospho-T389 in p70 S6 kinase (B).
A representative blot was probed with a mouse monoclonal anti-actin antibody, to normalize for protein loading (C).
P70 S6 kinase
Another component of the PI 3-kinase signaling pathway is
p70 S6 kinase. Western blot analysis for p70 S6 kinase indicated that its protein level was also up-regulated in colon cell
lines, and the gradual increase in the protein content correlated
well with the increased tumorigenic potential of the colon cell
lines (Figure 6A, lanes 1±8). Since the activity of p70 S6
kinase is also regulated by its phosphorylation (57,62,76±78), we
examined its phosphorylation state at Thr389 using a phosphospecific antibody directed against this site (Figure 6B). The
phosphorylation of this residue is rapamycin sensitive and
plays an important role in the regulation of p70 S6 kinase
activity (79). To demonstrate the specificity of the phosphospecific antibody for phosphorylated p70 S6 kinase, serumdeprived 293A cells were stimulated with serum to induce
phosphorylation. The antibodies failed to interact with p70
246
S6 kinase from serum-deprived, wortmannin- and rapamycintreated cells, but it bound to p70 S6 kinase from serum-fed
cells (Figure 6B, compare lanes 9, 11 and 12 with lane 10). The
increase in p70 S6 kinase phosphorylation at Thr389 correlated well with the tumorigenic potential of the colon adenocarcinoma cell lines (Figure 6B, lanes 1±8). Interestingly,
while total and phosphorylated Akt and FKHR were relatively
low in both Colo201 and Colo205 cells, the levels of total
and phosphorylated p70 S6 were elevated in Colo205 but not
Colo201 cells, indicating that there is additional complexity
in the expression of these downstream components in the
pathway in these two cell lines. Quantitative PCR analysis
did not reveal any increase in p70 S6 kinase message across
the colon cell lines (Table 1), indicating that only translational
and post-translational modification increases of the protein
correlate with increasing tumorigenic potential of the cells.
PI 3-kinase signaling and colon adenocarcinoma
To our knowledge, this is the first report on elevated p70 S6
kinase level and activity in colon adenocarcinoma cells.
Acknowledgements
We thank Christiane Cantin, Normand Jolicoeur and Yves Fortin for technical
assistance, Andre Migneault for assistance in preparation of the figures and
Dr Hui-Fen Zhao for valuable discussions. Colon adenocarcinoma cell lines
were a generous gift from Dr Clifford Stranners (McGill University). This
work was in part supported by a Cancer Genomics Grant from the NRC GHI to
S.H.S. K.K. is the recipient of a Visiting Fellowship from the Natural Science
and Engineering Council Canada.
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Received November 30, 2002; revised August 21, 2003;
accepted October 8, 2003