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Constitutive Activation of Mitogen-activated
Renal Cell Carcinoma1
Protein (MAP) Kinases in Human
Hiroya Oka, Yuji Chatani, Rika Hoshino, Osamu Ogawa, Yoshiyuki Kakehi, Toshiro Terachi, Yusaku Okada,
Masashi Kawaichi, Michiaki Kohno, and Osamu Yoshida2
Di'fitirlmt'HI (if Urologv. Fdcldtv of Medicine. K\'<Ho University, Kiiwahimi-cho 54, Sukyo-kit. Kyoto 606 ¡H.O., O. O., Y. K., T. T.. Y. O.. O. YJ: Department of ßiology. Gißt
Pharmaceutical
Universitv.
M¡Ã-uhom-hi\Ã-ushi.
Gijit 502 ¡Y.C., K. H., M. Kt>.¡;und Division (ff Gene Function in Animais. Nitrii Insiliate of Science und Technology, Tukayamuclw
M16-5. Iktima.
Nani '630-01
¡M.KuJ. Ja/Hin
variety of transcription factors such as p62'c/,
ABSTRACT
Mitogen-activated
protein kinases (MAPKs) play a pivotal role in the
mitogenic signal transduction pathway and are essential components of
the MAPK cascade, which includes MEK (also known as MAP kinase
kinasc), Kaf-1, and Kas. In this study, we examined whether constitutive
activation of the MAPK cascade was associated with the carcinogenesis of
human renal cell carcinomas in a series of 25 tumors and in corresponding
normal kidneys. Constitutive activation of MAPKs in tumor tissue, as
determined by the appearance of phosphorylated forms, was found in 12
cases (48%), and this activation was confirmed by a direct in vitro kinase
assay of immunoprecipitate using myelin basic protein as the substrate.
The phosphorylation of Ml K and of Kaf-1, as monitored by a mobility
shift in SDS-PAGE, which is reportedly associated with the activation of
these kinases, occurred in 9 of 18 cases (50%) and in 6 of 11 cases (55%)
respectively. The activation of MAPKs was correlated with MEK activa
tion (P = 0.0045) and with Raf-1 activation (P = 0.067). Furthermore,
overexpression of MEK was found in 13 of 25 cases (52% ) by Western blot
analysis, and this overexpression was associated significantly with MAPK
activation (P = 0.034). No mutations were noted in H-,K-, or N-ras genes
by PCK direct sequencing in any of the 25 tumor samples. Of the patients
studied, 8 of 18 (44%) stage pT, patients and four of six (67%) stage pT,
patients showed MAPK activation. The single stage pT, patient did not
evidence MAPK activation. Furthermore, one of seven (14%) grade 1
patients, 9 of 13 (69%) grade 2 patients, and two of five (40%) grade 3
patients showed MAPK activation (grade 1 versus grades 2 and 3,
/•
= 0.046). Our results suggest that constitutive activation of MAPKs may
be associated with the carcinogenesis
of human RCCs.
INTRODUCTION
c-Fos, and c-Myc (5,
6). Therefore, MAPKs are thought to integrate a variety of mitogenic
signals, and the constitutive activation of this cascade could be asso
ciated with carcinogenesis. In this respect, constitutively active mu
tant of MEK has recently been shown to transform NIH3T3 cells (19).
Moreover, it has been reported that expression of MAPK niRNA was
more elevated in mammary adenocarcinoma cell lines with higher
metastatic potential (20). Few studies, however, have examined the
possible association of MAPK cascade disorders with the carcinogen
esis of human neoplasms.
RCC is the most common malignant tumor of the adult kidney (21 ).
Several investigators have demonstrated a high frequency of enhanced
expression of epidermal growth factor receptor gene (c-erbB-l) in
RCCs, although amplification of this gene has not been detected (22).
One study has demonstrated a shift in the c-raf-1 locus in RCCs from
the terminal portion of 3p to the breakpoint region (23). Interleukin-6
is considered to be an autocrine growth factor for RCCs (24), and ras
gene mutations have been demonstrated in RCCs, as well as in other
tumors (25). Therefore, disorders of such factors, which are involved
in the MAPK cascade, may be related to the carcinogenesis of RCCs.
In this study, we have examined whether constitutive activation of
the MAPK cascade was associated with the carcinogenesis of human
tumors. We found that constitutive activation of MAPKs occurred in
a majority of RCCs. In addition, the activation of MAPKs was
correlated not only with increased phosphorylation of both MEK and
Rat-1, but also with the overexpression of MEK. Furthermore. MAPK
activation showed a significant correlation with the histológica! grade
of RCCs.
The 41- and 43-kilodalton MAPKs' (ERK2 and ERK1, respective
ly), which have been originally identified as molecules activated by
growth factor-stimulation of fibroblusts, are key kinases in the intracellular signal transduction pathways (1-6). MAPKs are rapidly phos
phorylated and activated in response to many of the growth factors,
hormones, and neurotransmitters that influence cell proliferation and
differentiation. Moreover, MAPK activation has also been shown to
be induced by nongrowth factor-mediated signals, such as ionizing
radiation, hydrogen peroxide, and UV light (7). The activation of
MAPKs is a result of the sequential activation of a dual specificity
kinase, known as MEK or MAPKK (8-10). The MAPK pathway also
involves both Ras and Raf-1 proto-oncogene products (11-15). MEK
and Raf-1, themselves, are activated by phosphorylation (5, 16-18).
MAPKs send signals into the cell nucleus by phosphorylating
a
Received 5/1/95; accepted 7/19/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants-in-aid from the Ministry of Education.
Science, and Culture of Japan.
: To whom requests for reprints should be addressed.
' The abbreviations used are: MAPK. mitogen-activated protein kinase; ERK. extra
cellular signal-regulated kinase; MEK. MAP kinase or ERK kinase; MAPKK. MAPK
kinase. RCC. renal cell carcinoma; MBP. myelin basic protein; T:N ratio, MAPK activity
or MEK expression in tumor tissue:MAPK activity or MEK expression in corresponding
normal kidney tissue ratio.
MATERIALS
AND METHODS
Tissues. Twenty-five primary tumors and one metastatic s.c. tumor, as well
as normal kidney tissues, were surgically obtained from 25 patients with
RCCs. Tissues samples were snap frozen in liquid nitrogen and stored at
—
80°Cuntil the experiments were performed.
Immunoblot Analysis. Tissues were homogenized on ice in buffer A
containing 25 mM Tris-HCl (pH 7.4). 25 mM NaCI, 1 mM sodium orthovanadate, 10 mM NaF. 10 mM sodium PP¡,25 mM /)-nitrophenylphosphate.
25 mM
ß-glycerophosphate, 10 nM okadaic acid. 0.5 mM EGTA. 1 mM /)-amidinophenyl methanesulfonyl fluoride hydrochloride. 0.2 mM sodium molybdatc, and
1% aprotinin and centrifuged at 10,000 x g for 30 min (26, 27). The protein
concentration was determined with the aid of a Bio-Rad Protein Assay Kit
(Bio-Rad Laboratories, Richmond. CA). The lysates containing 40 pig of
protein were subjected to 10% or 7.5% SDS-PAGE and transferred electrophoretically onto a Immobilon-P membrane (Millipore Corporation, Bedford,
MA). Blots were blocked in 2% BSA or 5% nonfat dried milk in 20 mM
Tris-HCl (pH 7.6). 137 mM NaCI. and 0.05% Tween 20 and then probed with
anti-MAPK antibody (raised against the peptide RIEVEQALAHPYLEQYYDPSDEP based on residues 299-321 of ERK.2: 3, 27). anti-phosphotyrosine
antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), anti-MEKl
antibody (Transduction Laboratories, Lexington. KY), or anti-Raf-1 antibody
(C-12; Santa Cruz Biotechnology. Inc., Santa Cruz, CA). Immunoreactive
bands were detected using enhanced chemiluminescence reagents (Amcrsham.
Buckinghamshire, UK) (15, 17, 27, 28). In the MEK activation assay, 12%
polyacrylamide separating gels containing 6 M urea were used to improve the
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CONSTITUTIVE
ACTIVATION
OF MAI'K CASCADE
resolution of phosphorylaled and nonphosphorylatcd forms of MEK (17). The
signal intensily of the autoradiograms was quantified by using a Bio-Profil 1-D
densitometer (Vilber Lourmat. Marne-La-Vallee, France). MAPK phosphoryl-
The amplified DNA fragments were purified by using Microcon (Amicon. Inc..
Beverly. MA) and sequenced directly. Sequencing reactions were performed
using a Takara Taq Cycle Sequencing Kit (Takara. Kyoto. Japan) with the
l:P-cnd-labeled primers used in the PCRs. The products were subjected to
clcctrophoresis on denaturing W't polyacrylamide gels.
Statistical Analysis. Fisher's exact probability test was used for all 2 X 2
alion was arbitrary defined as the intensity of the pp4l (phosphorylated active
form of p4rrk'; sec Fig. 1) band/the intensily of the p41 band. MAPK was
considered lo be constitulively active when the phosphorylation of the tumor
was >2-fold the value measured in the matched normal kidney. MHK and
Raf-l were considered to be active when the phosphorvlated hands were
tables. The Wilcoxon test was used for comparison between m vitro MAPK
activity in 25 tumor tissue samples and thai in 25 normal kidney tissue
samples. Linear correlation of MAPK phosphorylation, as assayed by Western
blotting, and MAPK activity, as assayed by the IH vitro kinase assay, were
determined by calculating Pearson's correlation coefficient. Data were ana
detected in tumor tissue, because, in no case, were the phosphorvlated forms
of these protein detected in normal tissue. Overexpression of MEK protein was
considered to have occurred when the signal of MEK protein in the tumor
tissue was >2-fold the value measured in the respective normal kidney tissue.
/;; Vitro MAPK Assay. Lysates containing 4(1 p.e. of protein were immunoprccipitated with anti-MAPK antiserum in the presence of 0.1''Ã- SDS (2(i.
lyzed with the Slat View statistical software package (Ver. 4.0; Abacus
Concepts. Inc.. Berkely, CA). P values less than 0.05 were considered to be
statistically significant.
27), and the immune complexes were captured with protein A-Sepharose.
Immunoprecipitates were washed three times in buffer A and three times in
kinase buffer [50 mM Tris-HCl (pH8.0). 25 mM MgCU I HIM EDTA. 1 nisi
RESULTS
DTT, 0.5 mM EGTA. and 10% glycerol]. Kinase activity was assayed by
incubating the Sepharosc beads with 30 /¿Iof reaction buffer [kinase buffer
with 20 JIM ATP. 1 /iCi [7-12P]ATP, and 0.5 mg/ml of MBP (Sigma Chemical
Co., St. Louis. MO)]. After W) min at 30°C,the raction was terminated by
Activation of MAPKs in RCCs. The appearance of the active
forms of both ERK2 (p41) and ERK1 (p43), which showed reduced
mobility in SDS-PAGE as a result of phosphorylation of specific
threoninc and tyrosine residues (4 —
6, 26), was noted in several of the
adding 10 /¿Iof a slop solution (0.6% HC1, \% BSA. and 1 mM ATP). Thirty
ju.1of the reaction mixture were spotted onto 1.5 X 1.5-cm squares of phos-
human RCC cases (Fig. \A ). The active forms of both ERK2 and
ERK1 contained phosphotyrosine when analyzed by Western blotting
using anti-phosphotyrosinc antibodies (data not shown). ERK1, de
tected by the anti-MAPK antiserum used, was much less abundant
than was ERK2 (3, 15, 28). Therefore, the activation of MAPKs was
determined by quantifying the increase in the amount of the more
slowly migrating form of ERK2 (p41) in tumor tissue compared with
that in corresponding normal kidney tissue, as described in "Materials
and Methods." Activation of MAPKs in renal tumors (T:N ratio >2)
phocellulose paper (PS1: Whatman International Ltd.. Maidstone. UK) and
washed in 1X0 HIMphosphoric acid (12-14). The radioactivity on the filter was
measured by a liquid scintillation counter (Packard Instrument Company.
Meridian. CT). The MAPK activity was defined as radioactivity incorporated
into MBP under these conditions.
DNA Preparation
and Direct Sequencing. High-molecular
weight
genomic DNA was prepared from the tissue samples by the phenol/chloroform
method after protein K digestion. The regions centering on codons 12, 13. and
61 of the H-. K-. and N-ni.v genes were amplified selectively using PC'R (21)).
3
T~N
IN HUMAN RCC
1
T~N
Fig. 1. A. Western hlot aiuilysis of seven repre
sentative RCCs and mulched normal kidneys. Numhfr\ above Ihe lanes show case numbers; 7'. Iunior
TN
.-
tissue; N. corresponding normal tissue; M. mclastatie s.c. lesion; /W/. inactive ERK2; [>[>JI. phos
phorvlated (active) ERK2;/>-/.(. inactive ERK1 ; and
Pl>43.phosphorylalcd (active) ERKI. Cases 3. 1.5.
S, 11. and ft show the more slowly migrating (phos
phorylalcd) forms of bolh ERK2 and ERKI in
tumor tissues. However, longer exposure was nec
essary to visuuli/e the phosphorylated form of
F.RKI clearly, because KRK1 delected by Ihe antiMAPK antiserum used was much less abundant
than was ERK2. C'ase 17 is an example of the
11
8
TNTN
..
17
TMN
ft*
negative case. H. in viint MAPK assay of the same
matched carcinoma ('/') and normal kidney (¿V)
tissues shown in A. MAPK activity was determined
as described in "Materials and Methods." Him,
radioactivity incorporated into MBP in duplicale
samples. Similar results were obtained in Iwo to
tour independent experiments. C. expression of
MliK protein as detected by Western hlol analysis.
The same blots that were shown in /\ were reprohed
with anti-MEK-anlibodics. Cases 3. I, 5. S. and 11
with MAI'K activation also show MEK overexpression. Case 6 shows MAPK activation hut not
MIÃŒKoverexpression.
Cuse 17 shows neither
MAPK activation nor MEK overexpression.
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CONSTITUTIVE
ACTIVATION
OF MAPK CASCADE
was detected in 12 of the 25 cases analyzed (48%). The degree of
activation varied from tumor to tumor, with a 2- to 18-fold variation
when the signal was compared with that in normal kidney tissue (Fig.
2 and Table 1). Seven representative cases are shown in Fig. IA. Cases
3, 1, 5, 8, 11, and 6 showed varying degrees of MAPK phosphorylation, whereas MAPK activation was not detected in case 17.
The activation of MAPKs was confirmed by a subsequent in vitro
kinase assay using MBP as the substrate. The MAPK activity in 25
tumor tissue samples (average, 1178 cpm ±288 SE) was significantly
greater than that in normal kidney tissues (average, 423 cpm ±71 SE;
P = 0.0188). Increased MAPK activity in renal tumors (T:N ratio >2)
8
30O
•a
t 20-
was found in 12 of 25 cases (48%; Fig. 2 and Table 1), and the degree
of increased activity was correlated closely with increases in the
quantity of the more slowly migrating form of ERK2 (r = 0.742:
P < 0.0001). Seven representative cases are shown in Fig. Ifi.
Activation and Overexpression of MEK Protein in RCCs. To
detect the activation of MEK, we examined the phosphorylation of
MEK as monitored by a mobility shift on 12% polyacrylamide sep
arating gels containing 6 M urea (17). Phosphorylated bands, which
were shifted up, were found in 9 tumor tissue samples of the 18 cases
(50%) examined (Table 1). Four representative cases are shown in
Fig. 3A. Nine of 12 cases positive for MAPK-activation also showed
MEK activation, whereas none of the 6 cases negative for MAPKactivation showed MEK activation (Table 2). Therefore, the activation
of MEK was correlated significantly
with MAPK-activation
(P = 0.0045).
I
10-
f
Western blot
analysis
IN HUMAN RCC
In vitro
kinase assay
Fig. 2. MAPK .i. in uu in in 25 RCCs. Activation of MAPK was determined both by the
appearance of the more slowly migrating form of ERK2 in Western blots and by the
ability of anti-MAPK antibody-immunoprecipitates
to phosphorylate MBP, as described
in "Materials and Methods." A T:N ratio >2 was considered to represent constitutive
activation.
To determine the expression level of MEK protein in RCCs, the
same membranes that were probed with anti-MAPK antibodies were
reprobed with anti-MEK antibodies. Although the amount of MAPK
protein was not significantly different between tumor and normal
kidney tissues, MEK protein expression varied considerably from
tumor to tumor when compared with that in matched normal
kidney tissue (Fig. 1, A and C). Quantitation of relative band
intensities showed that elevated expression of MEK in carcinomas
(T:N ratio > 2) was detected in 13 of the 25 cases (52%; Table 1),
Table 1 Activation of the MAPK cascade in 25 RCCs
activation''StagepT3aNXMOpT2NXMIpT2NOM()pT3aNOMODT2NOMOpT2NXMlpT2NOMOpT2NOMOpT2NXMODT2NOMXpT3aNXMOpT3aNOMlpT2NOMOp
mutation'-———---------------Case12345678910111213141516171819202122232425TypeClearClearClearCleaCleaCleaCleaCleaCleaClearMixedClearClearCleaCleaCleaCleaSpindleCleaCleaGranularGranularClear(iranularClearTumor"Grade32322
In vitro
Overexpression32.9
assay
activation+•f+++•f+•f---+NDN
activationND''ND+ND++ND+NDND-+-NDNDNDN
+1.77.6
+9.4
+32.4
+7.23.2
+4.5
+8.13.0
+3.1
+1.5
+1.03.70.540.803.50.72
DND-----ND-NDNDNDRaf-1
+1.81.0
+0.171.6
+0.660.170.18
+MEK
" Determined according to the criteria of the Japanese Pathological Society (30).
^N
ratio.
' Codons 12, 13. and 61 of H-, K-, and N-r«.vgenes were analyzed.
'' ND, not determined.
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C'ONSTITUTIVE
ACTIVATION
OK MAI'K CASCAI)!-: IN HUMAN RC'C
patients (grade 1 was significantly different from grades 2 and 3,
P = 0.046; grade 1 was significantly different from grade 2,
P = 0.029). Furthermore, MAPK activation was not found in the 1
stage pT, patient, but was found in 8 of the 18 (44%) stage pT3
patients and in four of the six (67%) stage pT, patients. This differ
ence, however, was not statistically significant. No association was
observed between MAPK activation and the age of the patient, the
presence of lymph node/distant métastases,or histológica! classifica
tion. Activation of MAPK, MEK .and Raf-l and mutation of'ras gene
8
3T3T N
MEK-P-O
MEK
T N
T N
T N
^
in 25 RCCs together with their clinical/histopathological
tics are summarized in Table 1.
characteris
DISCUSSION
B
17
TN
TN
TN
In this study, we demonstrated a high frequency of MAPK activa
tion in human RCCs. This activation in tumor tissues was considered
to be constitutive, because MAPK activation in response to a wide
variety of extracellular stimuli reaches the maximal level within 5 min
and subsides rapidly within 30-60 min (2). To confirm that this
TN
Raf-1-P Raf-1-
Fig. 3. Activation of MEK und Ruf-1 ¡nfour representative matehed RCCs and in
normal kidney (issues. M<w/?rry above the lanes represent case numbers and correspond
with those in Fig. 1; ruf 3T3, v-ra/'-transformed NIH 3T3 as positive control: 7". tumor
tissue; N, corresponding normal tissue. A. activation of MEK. The 12% polyacrylamide
separating gels containing o M urea were used to improve the resolution of closely
migrating forms of MF.K. as described in "Materials and Methods." The activation of
MEK was detected by the presence of several forms with reduced mobility (MEK-P). In
v-r(i/Ltransformcd NIH3T3 cells. MEK activation is observed with several more slowly
migrating forms. Cases 3. X. and 6 show different patterns of MEK activation only in the
tumor tissues, whereas case 17 is not activated, ß.activation of Raf-l. The activation of
Raf-1 was detected by the presence of its more slowly migrating forms as a result of
phosphorylation (Ktij-l-l1) in Western blot analyses. Cases 3, 8, and 6 show Raf-l
activation in the tumor tissues only, whereas case 17 is not activated.
and the degree of overexpression
was in the 2-10-fold range.
Furthermore, MEK overexpression
was correlated significantly
with MAPK activation (P = 0.034; Table 3). Representative cases
are shown in Fig. 1C. Cases 3, 1, 5, 8, and 11 show both MEK
overexpression and MAPK activation. Case 6 shows MAPK acti
vation but not MEK overexpression.
Activation ofRaf-1 in RCCs. Raf-l activation is accompanied by
increased serine/threonine phosphorylation in the protein and by a
characteristic decrease in its mobility in SDS-PAGE (18). To examine
the possible participation of Raf-l kinase in the constitutive activation
of MAPKs in RCCs, we studied the activation of Raf-l in 11 cases by
monitoring Raf-l mobility shifts in SDS-PAGE. The activation of
Raf-l was found in 6 of the 11 tumors (55%) examined (Table 1).
Four representative cases are shown in Fig. 3A. Five of 6 cases
positive for MAPK-activation were also characterized by Raf-l acti
vation, whereas one case was not (Table 2). Therefore, although Raf-l
activation seemed to be associated with MAPK activation, this rela
tionship was not statistically significant (P = 0.067).
Ras Gene Mutations in RCCs. To examine whether ras gene
mutations were associated with the constitutive activation of MAPKs,
the ras genes of 25 tumors were examined for point mutations. No
mutations were detected at codons 12, 13, and 61 of the H-, K-. and
N-ra.s genes by PC'R direct sequencing (Table 1).
Relationship between MAPK Activation and the Clinical and
Histopathological Characteristics of RCCs. MAPK activation was
correlated significantly with the histológica! grade of the tumors
(Table 4). MAPK activation was found in the tumors of one of seven
(14%) grade 1,9 of 13 (69%) grade 2, and two of five (40%) grade 3
MAPK activation was not the consequence of increased cell prolifer
ation in the malignant state, the MAPK activity of freshly isolated
normal kidney cells in primary culture and of human renal cancer cell
lines was measured in both exponentially growing and in densityarrested quiescent states. No significant difference in MAPK activity
was observed between cells in the growing phase and those in the
quiescent state (data not shown). This observation suggests that
MAPK activation may be a cause rather than a consequence of RCC
malignancy. In clinical and pathological analyses. MAPK activation
was more frequent in high-grade RC'C's than in low-grade RCCs
(Table 4). Carcinogenesis is a complex, multistep process involving
various genetic and epigenetic events within both proto-oncogenes
and tumor suppressor genes. In RCCs, oncogenes such as rax, raf-\,
c-erbE-\, and mvc, which are involved in the MAPK cascade, have
been demonstrated to participate in carcinogenesis (21-23, 25).
MAPKs are thought to act in the integration of a multiplicity of
mitogenic signals. Therefore, the malignant potential of tumors may
be increased by convergence of multiple oncogenic stimuli on
MAPKs, concurrent with increased MAPK activity.
MAPK activation is a result of the sequential activation of a series
of protein kinases, including MEK and Raf-l (8-10. 13-15). There
fore, we examined the participation of MEK/Ral-1 activation in the
constitutive activation of MAPKs in RCCs. None of the 6 cases
lacking MAPK activation showed MEK activation. This observation
supports the hypothesis that MEK activation is necessary for MAPK
activation. Of the 12 cases showing MAPK activation. 9 cases (75%)
also showed MEK activation. Three cases did not show MEK activa
tion, although MAPK was activated (Table 2). There are at least three
possible explanations for the latter observation: (a) MAPK kinases
other than MEK may exist. Segar et al. (9) demonstrated that the
amount of MEK transcript detected in human tissues by Northern blot
analysis was not always related to the level of MEK activity and
suggested the possible existence of another type of MEK. Williams
and Roberts (18) also suggested that ra.v-dependent MAPK kinases.
distinct from MEK, may exist. In support of these observations,
MEK2, which has an amino acid sequence 80% homologous to the
human and murine MEK1, has been identified (10). As a result, MEK
protein, like the MAPK family of proteins, may comprise a family of
enzymes regulated by a variety of stimuli and in different cell types (5,
10); (/>) disorders of phosphatase(s) that negatively regulate MAPK
activity may exist. Certain phosphatases. such as 3CHI34 (31) and
PACI (32), have been shown to dephosphorylate and inactivate
MAPKs. Sun et al. (31). for example, have reported that a catalytically inactive mutant of 3CH134 increased MAPK phosphorylation;
41X5
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CONSTITUTIVE
ACTIVATION
OF MAPK CASCADE
Table 2 Correlation between MAPK activation anil MEK/Raf-l activation in RCCs
MAPK
activationPositive
(20)6(55)"
NegativeTotaln12
(%)"9(75)
0(0)9(50)n6
618MEKActivation
We examined the existence of point mutations in the three ras
genes in an attempt to detect the origin of the constitutive MAPK
cascade activation. Mutations in codons 12, 13, and 61 of any one of
the three ras genes H-, K-, and N-ra.s will convert these genes into
active oncogenes, and activated Ras has been shown to trigger the
activation of the MAPK cascade (11, 12, 28). In other experiments,
we found that MAPKs were constitutively active in the T24/EJ human
bladder cancer cell line, in which H-ra.v is known to be mutated
(codon 12, Gly to Val).4 However, no ras gene mutations were
(%)*5(83)
1
511Raf-1Activation
P = 0.0045.
* P = 0.067.
detected in the 25 tumors examined. These results are in good agree
ment with the observation of Nanus et al. (25), which suggested that
the overall incidence of ras gene mutations in human RCCs was only
approximately 2%. Although the precise cause of the constitutive
activation of the MAPK cascade is unclear in the majority of human
RCCs analyzed in this study, it is likely caused by some dis
order upstream of Ras [e.g., receptor tyrosine kinases, GRB2, son of
sevenless (SOS), or even G-proteins; Ref. 38], which remains to be
Table 3 Correlation bem-een MAPK activation and MEK overexpressitm in 25 RCCs"
MEK overcxpression
MAPK
activationPositiveNegativeTotalH12
1325Positive9
413Negative3
IN HUMAN RCC
912
' P = 0.034.
elucidated in future work.
In conclusion, constitutive activation of MAPKs, MEK, and Raf-1
Table 4 Correlation between tumor grade/stage and MAPK activation in 25 RCCs
was detected in a relatively high number of human renal cancers, and
MAPK
good correlation between the activation of these kinases was ob
Histopathological
classification"Grade123StagepTIpT2PT3Totaln7135118625Activation
(%)'1(14)9(69)2
served. Moreover, MAPK activation was detected more frequently in
high-grade RCCs than in low-grade RCCs. Therefore, it is suggested
that constitutive activation of the MAPK cascade may play an important
role in the carcinogenesis of RCCs, and that greater activation of MAPK
(40)0(0)8(44)4(67)12(48)
could be associated with increased malignant potential in tumors.
ACKNOWLEDGMENTS
We thank M. Hashimoto and F. Higuti for their technical assistance. H. O.
is grateful to Dr. C. Oka (Nara Institute of Science and Technology) for
continuous support during this study.
" Determined according to the criteria of the Japanese Pathological Society (30).
* Grade 1 versus grade 2 (P = 0.029); grade 1 versus grades 2-3 (P = 0.046).
(c) an oncogenic form of MEK that is constitutivcly active in the
absence of phosphorylation may exist. Mansour et al. (19) have
demonstrated that a constitutively active mutant of MEK transformed
NIH3T3 cells. Although oncogenic forms of MEK have not as yet
been found in vivo, it is worthwhile to examine whether constitutively
active forms of MEK and MAPKs themselves are involved as oncogenes in human RCCs.
Of the six tumor cases that showed MAPK activation, five were
characterized by Raf-1 activation. One carcinoma showed MAPK
activation without Raf-1 activation (Table 2). In this case, MAPK
might be activated through the ra/-independent pathway. Indeed,
Raf-1 is not the exclusive activator of MEK. Mos (33), MEKK (34),
and ras p21-dependent ERK-kinase stimulator (REKS; Ref. 35) have
all been recently shown to be MEK activators. Furthermore, MAPK
has been shown to phosphorylate both Raf-1 (36) and MEK (37), and
a cyclic relationship that generates amplification or inhibitory feed
back loops has been suggested among Raf-1, MEK, and MAPK (5, 6).
The cases in which discrepancies in Raf-1. MEK, and MAPK activa
tion were observed could be explained in the context of this compli
cated network.
Another interesting observation was that the expression of MEK
protein was increased in approximately one-half of the RCCs ana
lyzed by Western blotting. Furthermore, this overexpression was
correlated significantly with the activation of MAPK. Although there
have been no reports indicating that MAPK activation is regulated by
the amount of MEK protein in vivo, Zheng and Guan (10) showed that
MAPK activation was correlated linearly with increasing concentra
tions of MEK2 protein in vitro. Although the significance of overex
pression and its mechanistic basis require additional investigation, it is
intriguing to speculate that abnormal regulation of MEK expression
may be involved in the carcinogenesis of some RCCs.
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Constitutive Activation of Mitogen-activated Protein (MAP)
Kinases in Human Renal Cell Carcinoma
Hiroya Oka, Yuji Chatani, Rika Hoshino, et al.
Cancer Res 1995;55:4182-4187.
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