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Tumor Biology |
Departments of Medicine and Pathology [Y. Z., J. H.], University of Chicago, Chicago, Illinois 60637 [Y. Z., J. H.], and Department of Medicine [F. C. v. L., N. V., G. R. B.], University of California, San Diego, California 92093 [M. B., S. K., R. K. W., L. N., S. S., T. A. B.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% of large human
colonic adenomas and adenocarcinomas (2)
, and mutations in
APC or ß-catenin occur in most colon cancers (1)
.
Mutations in the K-ras gene (5
, 6)
and in
CTNNB1 (7)
can also be detected in colonic
tumors of rats administered the colonic procarcinogen,
1,2-dimethylhydrazine, or its proximate metabolite, AOM. Several lines of evidence from both human (8, 9, 10) and experimental systems (11, 12, 13, 14) have also indicated that epigenetic alterations in a number of important signal transduction elements, including cyclin D1 and p21ras, may lead to the clonal expansion of a variety of malignant cell types. Recent in vitro studies, for example, have found that expression of cyclin D1 is increased by mutations in K-ras or APC/ß-catenin (15, 16, 17) . Cyclin D1 activates cyclin-dependent kinase-4 and cyclin-dependent kinase-6 and, thereby, promotes the G1 to S transition. This growth enhancing regulator is also increased in a subset of AOM-induced tumors (18) .
The K-ras proto-oncogene codes for
p21K-ras, a small monomeric GDP/GTP-binding
(G) protein, which is involved in the regulation of a number of
important normal cellular functions, including proliferation,
differentiation, and apoptosis (19
, 20)
. In its GTP-bound
form, it serves as an active signal transducer, whereas in its
GDP-bound state, p21ras is inactive (20
, 21)
. The conversion of GTP-bound to GDP-bound
p21K-ras is stimulated by GAPs
(20)
. Inactive p21K-ras, in
turn, can be reactivated by replacement of its bound GDP by GTP, via
GNEFs (20
, 21)
. Several receptor tyrosine kinases,
including EGF and c-erbB-2 receptors, have been shown to activate
p21ras by increasing GNEF activity (22
, 23)
. Although GAPs and GNEFs can, thereby, regulate the
activation of wild-type p21K-ras, these two
regulatory proteins do not influence oncogenic p21K-
ras, which is constitutively activated by mutation
(20)
. Because only 50% of colonic carcinomas have
mutant p21K-ras, we hypothesized that
alterations in the activity and/or expression of Ras-GAP and/or GNEFs,
such as Sos, could lead to sustained activation of wild-type
p21K-ras in colonic malignant cells not
possessing K-ras mutations. To date, however, these
possibilities have not been experimentally demonstrated in models of
colonic carcinogenesis in vivo.
Activation of p21K-ras, in turn, might be expected to stimulate a number of its downstream effectors including, for example, the ERK family of MAPK by activating the MEK kinase, Raf-1, thereby phosphorylating and activating MEK (reviewed in Ref. 24 ). This latter dual functioning kinase activates two isoenzymes of MAPK/ERK, pp44ERK-1 and pp42ERK-2. Once activated, these MAPKs translocate to the nuclei of cells, and their phosphorylated substrates, in turn, lead to transactivation of genes involved in the regulation of cellular proliferation, differentiation, and malignant transformation (25) . We, therefore, determined whether the activation status of p21K-ras in AOM-induced tumors was related to alterations in the activities of these MAPKs.
The gene coding for COX-2, an inducible isoform of the enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and other eicosanoids (26) , is another important downstream target of p21ras. COX-2, for example, has been shown to be increased in a number of ras-transformed epithelial cells (27) . Several lines of evidence, moreover, have implicated COX-2, which has been shown to be overexpressed in both human (28) and experimental colonic tumors (29 , 30) , in colonic malignant transformation (31 , 32) . Although no direct evidence has emerged linking aberrant APC/ß-catenin signaling to alterations in COX-2, recent in vitro studies in Rat-1 fibroblasts transfected with an activated Ha-ras oncogene have shown that increases in MAPK activity appear to be required for the induction of COX-2 expression by this proto-oncogene (27) . We, therefore, examined the possible relationship between the activity of p21ras and the expression of COX-2 and the activities of MAPKs in AOM-induced tumors. In addition, this model allowed us to investigate whether MAPK activity and COX-2 and cyclin D1 expression would be altered by ß-catenin mutations.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Protocol.
Weanling male albino Fisher (F344) rats, initially weighing 80–100 g,
were fed a standard rat chow diet (#5001, Purina Mills, Richmond, IN).
Two weeks after being placed on this diet, one-half of the rats
received i.p. injections weekly with AOM (15 mg/kg body weight/week) or
vehicle (saline) for 2 weeks and then maintained on the diet for an
additional 35 weeks. At that time, the animals were sacrificed in the
nonfasted state. Colons were removed, flushed with normal saline,
opened, and examined macroscopically for the presence of tumors. Tumors
were rapidly excised and washed with ice-cold PBS. A small portion from
each tumor was fixed in 10% buffered formalin for microscopic
examination, whereas the remainder was snap frozen in liquid nitrogen
for later analyses. After formalin fixation, tissue specimens were
paraffin embedded, sectioned, and stained with H&E, as described
previously (33)
. All specimens were evaluated by a
pathologist (J. H.), who was unaware of the treatment groups.
Macroscopic lesions were classified as either benign (adenoma) or
malignant (adenocarcinoma). No tumors were present in the group treated
with vehicle. Tissue specimens of colonic mucosa, 1
cm2 in size, were taken from each of these
control rats and processed in the same way as tumor samples.
DNA Isolation, PCR Amplification, and Allele-specific
Oligonucleotide Hybridization.
The snap-frozen samples were mechanically disrupted, and DNA was
extracted using the TRI Reagent. In these samples, a 116-bp sequence of
the K-ras exon 1 gene was amplified by PCR and then ASOH was
carried out as described previously (6)
. To investigate
G
A mutations in the second position of codon 59 or in the first
position of codons 12 or 13, appropriate primers and conditions were
used as described (6)
.
Standard and Enriched Primer-mediated RFLP.
To confirm the ASOH results, PM-RFLP was used to detect
K-ras mutations in codons 12 and 13. Briefly, mismatched 5'
primers created restriction sites for BstN1, or
BglI in wild-type K-ras, which were abolished by
GA mutations in codon 12 or 13, respectively. The primers, PCR
conditions, and restriction digests were as described previously
(5)
. To amplify low abundance codon 12 K-ras
mutant forms, a procedure of enriched PM-RFLP was carried out as
described previously (34)
.
PCR-SSCP Analysis.
Primers for PCR were designed to amplify the consensus sequence for
GSK-3ß phosphorylation in exon 3 of CTNNBI, the gene
coding for ß-catenin, based on the published cDNA sequence of mouse
and used successfully in rat (7)
. PCR and subsequent SSCP
was performed exactly as described previously (7)
.
DNA Sequencing.
K-ras and ß-catenin mutations were also confirmed by
direct sequencing of amplified DNA using automated fluorescent DNA
sequencing.
Measurements of p21ras-bound GTP and GDP.
The GTP and GDP bound to p21ras were measured as
described previously (35)
. Briefly, after
p21ras immunoprecipitation from tumor lysates,
GDP and GTP bound to p21ras were separated and
quantified using coupled reactions and a sensitive luciferin/luciferase
system (35)
.
Western Blotting.
Proteins from control colonocytes and from tumors were extracted in
SDS-containing buffer, quantified (36)
, and subjected to
Western blotting (37)
. Proteins (20 µg) were separated
by SDS-PAGE using a 10% resolving polyacrylamide gel and
electroblotted as described previously (38)
. Blots were
incubated overnight at 4°C with specific primary antibodies, rabbit
polyclonal anti-Sos1,2 (0.2 µg/ml), anti-active MAPKs (1:5000),
anti-Cox-2 (2 µg/ml), anti-cyclin D1 (2 µg/ml), or mouse monoclonal
anti-Ras GAP (0.2 µg/ml), followed by incubation with appropriate
peroxidase-coupled secondary antibodies and subsequent detection by
enhanced chemiluminescence.
IHC.
For EGFR and c-erbB-2 immunostaining, antigens were retrieved by
microwave heating in 0.01 M citrate buffer. Sections were
incubated with primary antibodies, anti-EGFR (1:200), or anti-c-erbB-2
(1:500) and then with Link reagent (LSAB kit), following the
instructions of the manufacturer (DAKO). For ß-catenin, cyclin D1,
and COX-2 antigen retrieval, sections were pretreated with 1% SDS
(ß-catenin), 1% Triton-X-100 (cyclin D1), or left untreated (COX-2).
Sections were incubated overnight at 4°C with 1:50 dilution of
appropriate primary polyclonal antibodies, followed by incubation with
1:200 dilution of biotinylated antirabbit antibodies at room
temperature, which were detected with the ABC complex (Vector) using
diaminobenzidine as substrate. For negative controls, sections were
processed in the absence of primary antibodies.
Statistical Methods.
Data are expressed as mean ± SE. Differences between
groups were compared by Student’s t test or Fisher exact
test, as appropriate. Linear regression was performed by least square
fitting analysis. Values of P < 0.05 were
considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CTNNB1 Mutations in AOM-induced Colonic Tumors.
Among the 84 tumors screened for K-ras mutations, 68 tumors
were also examined for CTNNB1 mutations by SSCP. We
identified 22 mutations (32.4%) in exon 3 of this gene coding for
ß-catenin (Fig. 1
and Table 1
). There were no significant correlations between K-ras and
CTNNB1 mutations (Table 1)
. In agreement with others
(7)
, we also found an increased expression of ß-catenin
and a redistribution of the protein from the membrane to the cytosol
and nucleus in tumors with mutant ß-catenin (Fig. 1
).
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). Interestingly, as shown in Table 2
, we identified 18 tumors with
wild-type K-ras, with p21ras
activation ratios greater than three SEs above that of control
colonocytes (8.0 ± 0.9, n = 14 versus 3.7 ± 0.4, n = 6, P < 0.05, respectively). The
activation ratios of p21ras in these tumors did
not differ significantly compared with those with mutant
K-ras (Table 2)
. Because >50% of all K-ras
mutations occur in codon 12, we also examined these tumors by a highly
sensitive technique of enriched PM-RFLP to detect mutations in this
codon. This assay was negative for each of the tumors classified as
activated but without K-ras mutations (data not shown). We
have operationally defined these tumors as possessing constitutively
activated wild-type p21ras.
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). There was an inverse correlation between GAP expression and
p21ras activation, with decreased GAP expression
in tumors with wild-type but activated p21ras
(Fig. 2B
). In contrast, GAP expression was elevated in
tumors with mutant (GAP-unresponsive)
p21K-ras compared with tumors with
nonactivated wild-type p21K-ras (data
not shown). These findings suggest that a decrease in the expression of
GAP may, at least in part, underlie the activation of wild-type
p21ras in a subset of AOM-induced tumors without
K-ras mutations, whereas the GAP elevation in the
K-ras mutant tumors may possibly reflect a futile attempt to
inhibit their activated ras state. The expression of Sos-1,
however, was not different in tumors with (134 ± 13%;
n = 4) and without (100 ± 17%; n = 4; P > 0.10) constitutively activated p21ras (Fig. 2C
). Sos-2 was not detected in normal or neoplastic tissue
(Fig. 2C
).
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). These three tumors with increased c-erbB-2 receptor expression also
had low GAP expression (see above). Moreover, the remaining single
tumor, in the group with nonmutant ras and activated
p21ras, also had decreased GAP expression. In
contrast, four of four tumors examined with mutant K-ras
were negative for c-erbB-2 staining (Fig. 3
). EGF receptor expression
was not different among the three groups (data not shown). These
findings suggest that c-erbB-2 receptor overexpression may also
account, at least in part, for the increased GTP binding to wild-type
p21ras in the subset of AOM-induced tumors
without K-ras mutations. Because tumors with mutant
K-ras did not show increased c-erbB-2 receptor expression
(Fig. 3
), this suggests that increased receptor expression drives
increased p21ras activation and not the
converse.
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). In tumors with wild-type but activated p21ras,
ERK-1 and ERK-2 were 222 ± 26% and 104 ± 18% above the levels of ERK-1 and ERK-2 from tumors with
normal p21ras activation (n = 4; P < 0.05), respectively. Tumors
with mutant p21ras also had increased ERK-1 and
ERK-2 activation, 156 ± 15% and 223 ± 8% above that of ERK-1 and ERK-2 from tumors with wild-type
nonactivated p21ras (n = 4; P < 0.05), respectively. There
were no differences in the total expression of ERK-1 or ERK-2 among the
three groups (data not shown). As shown in Fig. 4B
, there
were significant correlations between phospho-ERK-1 and phospho-ERK-2
and p21ras activation.
|
). There were no significant differences in COX-2 expression between
tumors with mutant versus wild-type ß-catenin (Fig. 5B
). In contrast, the expression of COX-2 was significantly
greater in tumors with activated p21ras (mutated
or wild-type) compared with tumors with wild-type but nonactivated
p21ras (Fig. 5A
). Interestingly, in
tumors with wild-type but constitutively activated
p21ras, the expression of COX-2 was diffuse and
predominantly cytosolic (Fig. 6
, E–H), as well as present in the surrounding
stroma, whereas in tumors with mutant K-ras, there was
increased nuclear COX-2 expression in the malignant epithelium (Fig. 6
,
I–L). The mechanism for this difference in
distribution is currently unclear and will require further study.
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) and immunohistochemical analysis (Fig. 8
). Tumors with wild-type K-ras but activated
p21ras, however, did not show increased cyclin
D1 expression (Figs. 7
8
). Thus, activation of
p21ras, by mutational versus
nonmutational mechanisms, causes parallel changes in MAPK activation
but differential alterations in cyclin D1 expression and COX-2
localization. Our findings suggest that the signaling cascades involved
in activation of wild-type p21ras may block the
expected increase in cyclin D1 by MAPK that we observed in tumors with
mutant K-ras. Elucidation of the underlying mechanisms for
these differences in cyclin D1 expression in tumors with mutant
versus nonmutant activated p21ras
will, however, require further study.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ratio of GTP:(GTP + GDP) bound to p21ras in the group with wild-type ras, but with activated p21ras, and the group with mutated p21ras, were similar, suggesting that like mutated p21ras, activated wild-type p21ras may also be intimately involved in colonic malignant transformation by altering critical cellular processes (46 , 47) . Because wild-type p21ras was constitutively activated in both adenomas and adenocarcinomas, the constitutive activation of p21ras appears to occur early in colonic malignant transformation. Prior studies have shown that wild-type p21ras was activated in human peripheral nerve sarcomas, although the ras mutational status of these tumors was not assessed (10) . In addition, in several ovarian carcinoma cell lines without mutant ras, wild-type p21ras was found to be activated (39) . The present studies are the first, however, to demonstrate the activation of wild-type p21ras in any animal model of carcinogenesis.
As noted earlier, the activation state of wild-type p21ras is normally regulated by changes in the activity and/or expression of GAP and GNEFs, such as Sos. A decrease in the activity/expression of GAP or an increase in the activity/expression of GNEFs would be expected to activate wild-type p21ras. Indeed, we found that GAP expression was inversely proportional to levels of wild-type p21ras activation, whereas Sos-1 expression was not significantly different between tumors with activated and nonactivated p21ras. Because of the limited quantities of these tumors, the enzymatic activities of GAP and Sos-1 could not be assessed and will be of interest to examine in future experiments. Although the underlying mechanism(s) responsible for the decrease in GAP expression remains to be elucidated, it would appear that alterations in GAP expression, at least in part, are responsible for the activation of wild-type p21ras in this subset of adenocarcinomas.
Alterations in growth factors and/or their receptors, which regulate GAP and GNEF activity, may be involved in activation of wild-type p21ras. For example, tumor-associated increases in growth factors could stimulate their respective tyrosine kinase receptors and, thereby, activate p21ras (41 , 42) . Alternatively, overexpression of these receptors could also lead to p21ras activation (40) . Recent studies in human colon cancer and in several colonic cancer-derived cell lines have demonstrated increased c-erbB-2 receptor expression (48 , 49) . In the present studies, c-erbB-2 receptors were detectable in three of four AOM-induced tumors with activated p21ras compared with 1 of 8 with nonactivated p21ras. Because the overexpression of this receptor has been shown to activate p21ras (40) , alterations in the expression of c-erbB-2 receptors may, at least in part, along with decreases in the expression of GAP, be responsible for the activation of wild-type p21ras.
The present studies also demonstrate that colonic tumors with activated wild-type p21ras, like those with mutated p21ras, have increased activation of ERK-1 and ERK-2, presumably via the activation of Raf-1 and MAPK kinase (46 , 47) . Thus, activated wild-type p21ras, like their mutated counterparts, may be involved in colonic malignant transformation, because activation of these MAPKs may be expected to influence the proliferation and differentiation of these malignant cells (46 , 47) . Recent studies, moreover, have demonstrated that blockade of MAPK activation can inhibit colonic tumor growth in vivo (50) .
In agreement with earlier studies in the AOM model (30) , we found that COX-2 was increased in all of these carcinogen-induced tumors. In agreement with prior in vitro studies (27) , our results also suggest that K-ras mutations, via increases in the activities of MAPKs, may lead to COX-2 overexpression in tumors. Furthermore, our studies indicate that this also occurs in tumors with activated wild-type p21ras. The cytoplasmic localization of COX-2 in the stromal and malignant cells of tumors with wild-type activated p21ras, compared with a nuclear distribution in malignant cells with mutant K-ras, however, underscores important differences in COX-2 expression depending upon the mechanism of p21ras activation. Further studies will be required to understand these differences that may arise from signal transduction pathways driving p21ras activation in the case of wild-type ras, compared with mutant p21ras.
In addition to alterations in ras and its effectors, changes in APC and ß-catenin signaling have been identified in colonic carcinogenesis. A number of downstream targets of APC/ß-catenin have been demonstrated in human colonic adenocarcinoma cells, including cyclin D1 (51 , 52) . We have found that both K-ras and CTNNB1 mutations can drive increased cyclin D1 expression in vivo, in agreement with previous in vitro findings in cultured cell lines (15, 16, 17) . The failure of cyclin D1 to increase in tumors with wild-type but activated p21ras suggests that the signaling involved in this activation may also inhibit the expected increased cyclin D1 expression induced by the downstream ras effector pathway Raf-MEK-MAPK. The mechanisms involved in this inhibition will also require further study.
Although COX-2 expression is increased in most human sporadic colon
cancers, we did not find that ß-catenin mutations altered COX-2
expression in the AOM model. This is in agreement with recent in
vitro studies with colon cancer-derived HT29 cells in which
peroxisome proliferator-activated receptor 
agonists of the
thiazolidinedione class increased ß-catenin expression but failed to
alter COX-2 expression (53)
. Independent of the
ß-catenin status, however, it should be noted that COX-2 expression
was increased in all AOM-induced tumors examined, compared with normal
mucosa.
In summary, the present results demonstrate that a subset of AOM-induced tumors possess activated wild-type p21ras, perhaps as a consequence of changes in the expression of c-erbB-2 receptors and/or GAP. Moreover, like their activated, mutated p21ras counterparts, these activated wild-type small monomeric G proteins appear to play a role in colonic malignant transformation via increases in the activities of the MAPKs which, in turn, may lead to an increase in COX-2 expression. Compared with mutational activation of p21ras, the differential alterations in cyclin D1 expression and COX-2 localization observed in tumors with nonmutational activation of p21ras may involve alternative signaling pathways that activate the gene product of wild-type but not mutant ras. Elucidation of the mechanisms that drive these differences may identify novel new targets for anticancer therapy.
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FOOTNOTES |
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1 These studies were funded in part by the
following grants: USPHS Grant CA36745 (to T. A. B. and M. B.),
DK42086 (to T. A. B., Digestive Diseases Research Core
Center), and CA69532 (to M. B.), as well as by the Samuel
Freedman Research Laboratories for Gastrointestinal Cancer Research.
T. A. B. is the recipient of a Merit Award from the NIH. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Department of Medicine, MC 4076, University of Chicago
Hospitals and Clinics, 5841 South Maryland Avenue, Chicago, IL 60637.
Phone: (773) 702-9898; Fax: (773) 702-2182; E-mail: tbrasitu{at}medicine.bsd.uchicago.edu
4 The abbreviations used are: APC, adenomatous
polyposis coli; AOM, azoxymethane; EGF, epidermal growth factor; EGFR,
EGF receptor; ASOH, allele-specific oligohybridization; PM-RFLP,
primer-mediated RFLP; SSCP, single strand conformational polymorphism;
GAP, GTPase activating protein; GNEF, guanine nucleotide exchange
factor; COX-2, cyclooxygenase-2; MAPK, mitogen-activated protein
kinase; MEK, MAPK kinase; ERK, extracellular signal regulated kinase;
IHC, immunohistochemistry.
Received 1/26/00. Accepted 6/20/00.
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 C Domon-Dell, Q Wang, S Kim, M Kedinger, B M Evers, and J-N Freund Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells Gut, April 1, 2002; 50(4): 525 - 529. [Abstract] [Full Text] [PDF]
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 N. A. C. S. Wong and M. Pignatelli {beta}-Catenin--A Linchpin in Colorectal Carcinogenesis? Am. J. Pathol., February 1, 2002; 160(2): 389 - 401. [Abstract] [Full Text] [PDF]
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T. Shono, P. J. Tofilon, J. M. Bruner, O. Owolabi, and F. F. Lang Cyclooxygenase-2 Expression in Human Gliomas: Prognostic Significance and Molecular Correlations Cancer Res., June 1, 2001; 61(11): 4375 - 4381. [Abstract] [Full Text] [PDF]
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