Oncogenic Kit signaling and therapeutic intervention
in a mouse model of gastrointestinal stromal tumor
Ferdinand Rossi*, Imke Ehlers*, Valter Agosti*†, Nicholas D. Socci‡, Agnes Viale§, Gunhild Sommer*, Yasemin Yozgat*,
Katia Manova*¶, Cristina R. Antonescu*?, and Peter Besmer*,**††‡‡
Departments of *Developmental Biology and§Molecular Biology,‡Computational Biology Center, and¶Molecular Cytology Facility, Sloan–Kettering
Institute, New York, NY 10021; and?Department of Pathology, Memorial Sloan–Kettering Cancer Center, **Gerstner Sloan–Kettering Graduate School of
Biomedical Sciences, and††Cornell University Weill Graduate School of Medical Sciences, New York, NY 10021
Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved June 20, 2006 (received for review December 30, 2005)
Kit receptor-activating mutations are critical in the pathogenesis of
gastrointestinal stromal tumors (GIST). We investigated mecha-
nisms of oncogenic Kit signaling and the consequences of thera-
peutic intervention in a mouse model of human GIST. Treatment of
GIST mice with imatinib decreased cell proliferation and increased
apoptosis in the tumor. Analysis of tumor tissue from imatinib-
treated mice showed diminished phosphatidylinositol 3-kinase
(PI3-kinase) and mammalian target of rapamycin (mTOR) signaling
suggesting that oncogenic Kit signaling critically contributes to the
translational response in GIST. Treatment with RAD001 (everoli-
mus), an mTOR inhibitor, diminished the translational response
and cell proliferation in tumor lesions, pointing to mTOR inhibition
as a therapeutic approach for imatinib-resistant GIST. Analysis of
RNA expression profiles in GIST lesions with and without imatinib
treatment showed changes in expression of IFN-inducible genes
and cell cycle regulators. These results convincingly show that
KitV558?/?mice represent a unique faithful mouse model of human
familial GIST, and they demonstrate the utility of these mice for
preclinical investigations and to elucidate oncogenic signaling
mechanisms by using genetic approaches and targeted pharmaco-
imatinib ? Kit receptor tyrosine kinase ? signal transduction
as well as in hematopoietic cell populations, gametogenesis, and
organism (1–3). The finding of Kit receptor-activating mutations in
human tumors, including gastrointestinal stromal tumor (GIST),
seminomas, and mastocytosis, as well as some acute myelogenous
common mesenchymal tumor of the gastrointestinal tract. GISTs
express Kit and are thought to derive from a Kit?or KitlowICC
progenitor or ICC. The vast majority of GISTs contain Kit recep-
found predominantly in the juxtamembrane domain of the Kit
receptor, but mutations in the extracellular and kinase domains of
Kit have been described as well (6, 7). Imatinib mesylate (Gleevec,
STI571), an inhibitor of the Kit, PDGFR, and BCR-ABL tyrosine
kinases, is used to treat patients with GIST and chronic myeloge-
in a majority of patients with metastatic or recurrent disease. The
clinical response correlates with a decrease in tumor cellularity and
myxoid degeneration of the tumor. Although the clinical response
to imatinib is quite well described, the molecular response of Kit
inhibition by imatinib in GIST is poorly understood. Imatinib is
most effective in GISTs with Kit-activating mutations in the jux-
tamembrane domain, some kinase domain mutations, or extracel-
lular domain mutations. But Kit mutations that destabilize the
inactive form of the kinase are resistant to inhibition by imatinib.
the development of drug resistance, and in some cases, resistance
he Kit receptor tyrosine kinase has a critical role in the normal
9). Therefore, the development of new strategies for the treatment
of GIST is highly relevant.
Several cases of human familial GIST syndrome with associated
interstitial cells of Cajal hyperplasia, hyperpigmentation, and?or
urticaria pigmentosa with germ-line Kit mutations have been
reported (10, 11). Based on these findings, we produced a mouse
The KitV558?/?mutation is located in the juxtamembrane domain
of Kit (exon 11), a negative regulatory region of the receptor where
the majority of the somatic GIST mutations in patients occur.
Patchy hyperplasia of Kit-positive cells is observed within the
myenteric plexus of the GI tract, and neoplastic lesions indistin-
guishable from human GIST are found with complete penetrance
a unique opportunity to investigate the development of GIST and
the consequences of therapeutic intervention.
The Kit receptor has a role in distinct cellular responses, includ-
ing cell proliferation, survival, adhesion, chemotaxis, and secretory
responses, as well as the desensitization of the activated receptor in
various cell types in vitro and in vivo. The downstream signaling
cascades that are known to be activated by Kit include the Ras?
MAP kinase, Rac?Rho-JNK, phosphatidylinositol 3-kinase (PI3-
kinase)?AKT?PDK1?FOXO, and src family kinase (SFK)?STAT
signaling networks. Cell-type-specific responses depend in part on
the cellular context and the presence of signaling components, and
therefore signaling cascades that may be activated by Kit vary in
different cell types. Consequently, disruption of Kit-specific signal-
ing pathways by knock-in mutations produces cell-specific effects,
e.g., disruption of Kit-induced PI3-kinase signaling was shown to
impair male fertility, whereas melanogenesis and hematopoiesis
age-dependent lymphopoietic defects (14). We have used imatinib
to investigate oncogenic Kit signaling in mouse GIST in vivo. We
lesions. Analysis of gene expression profiles in placebo- and ima-
tinib-treated mice revealed roles for cell cycle regulators and
IFN-inducible genes in GIST. Biochemical analysis of tumor tissue
from imatinib-treated mice showed diminished PI3-kinase and
for the translational response in oncogenic Kit signaling in GIST.
To investigate the role of the translational response in GIST, mice
were treated with the mTOR inhibitor RAD001 (everolimus).
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: GIST, gastrointestinal stromal tumor; PI3-kinase, phosphatidylinositol
3-kinase; ABC, ATP-binding cassette; mTOR, mammalian target of rapamycin.
†Present address: Department of Experimental and Clinical Medicine, University Magna
Graecia, University Campus, Germaneto, 88100 Catanzaro, Italy.
‡‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
August 22, 2006 ?
vol. 103 ?
no. 34 ?
Effects of Imatinib Treatment in the GIST Mouse Model.Todetermine
whether GISTs in KitV558?/?mice respond to imatinib treatment,
heterozygous KitV558?/?mice were treated with 45 mg?kg imatinib
twice per day. After 7 days of treatment, a decrease in cellularity
and an increase in myxoid stroma were observed microscopically in
H&E-stained tumor sections. Whereas in 8 of 10 treated mice, the
degree of response varied from a mild response with patchy
acellular areas comprising 10–50% of the lesion, to a moderate
stroma and necrosis, in two mice, the response was minimal,?10%
were observed for shorter time treatments, i.e., 6, 12, and 24 h (Fig.
1 A–D). These results showed that after 7 days of treatment, the
murine GIST lesions responded to imatinib treatment, and that
some variability was observed between mutant mice.
To confirm that imatinib inhibited Kit receptor activation and
autophosphorylation in the murine GIST lesions, tumor protein
extracts were prepared from mice treated with placebo or imatinib.
To assess the range of individual variability in the molecular
response to the drug after short treatment periods, tumor protein
We had previously shown that Kit is constitutively activated and
autophosphorylated in tumor lesions of untreated mice using the
of treatment and was maintained at 6 and 24 h of treatment (Fig.
3 and results not shown). Total Kit protein levels were not affected
by the treatment. Similarly, immunohistochemical analysis of
GISTs showed similar Kit staining patterns independent of treat-
ment (Fig. 6 A and B, which is published as supporting information
on the PNAS web site). These results indicated that imatinib
treatment did not affect Kit receptor expression and?or turnover
but only inhibited its kinase activity.
We then investigated the effect of imatinib treatment on GIST
cell proliferation and apoptosis. GISTs from placebo-treated mice
the tumors were viable and proliferating (Fig. 7A, which is pub-
index was quite low, ?4%. Treated GISTs showed a reduction of
Ki67-positive cells after a 6-h treatment time, and virtually no
Ki67-positive staining was observed after 24 h or 7 days of imatinib
treatment (Figs. 2A and 7 B–D), indicating an almost complete
arrest of cell proliferation in tumor lesions as early as 24 h of
treatment. We next determined whether there was an increase in
apoptosis in the tumor by measuring cleaved caspase 3 levels by
immunohistochemistry. GISTs from placebo-treated mice showed
no obvious cleaved caspase 3 staining. However, positive staining
pronounced effect was observed after 7 days of treatment (Figs. 2B
and Fig. 7 E–H). Taken together, these results show that GIST
lesions in KitV558?/?mice are sensitive to imatinib treatment.
Phosphorylation of Downstream Targets of Oncogenic Kit Signaling in
GIST. Because imatinib treatment abolishes Kit signaling in GIST
lesions, a comparison of known signaling cascades that might be
activated by Kit receptor signaling in tumor lysates before and after
drug treatment should provide insights into the mechanisms of
Kit-mediated responses in GIST. Our immunohistochemical anal-
ysis in imatinib-treated KitV558?/?mice indicated that drug treat-
ment induced both apoptosis and arrest of cell cycle progression in
murine GISTs. To establish which signaling pathways are activated
in GIST in vivo, we assessed the phosphorylation state of down-
stream substrates of the oncogenic Kit receptor. Kit is thought to
mediate proliferation and survival by the PI3-kinase- and mitogen-
activated protein kinase (ERK1 and -2) pathways and by signaling
through STAT transcription factors (15). We prepared tumor
lysates from placebo and 6-h-treated mice and evaluated protein
activation by Western blotting with phospho-specific antibodies.
treatment. H&E-stained tumor sections of placebo and imatinib-treated mice
for the indicated time intervals (?20).
Histological response of GIST lesions in KitV558?/?mice to imatinib
Table 1. Individual response of GIST lesions in KitV558???mice
to 7-day imatinib treatment
(n ? 10)
?? ???? ???
8.50 ? 1.29
9.25 ? 2.21
15.75 ? 2.5
13.75 ? 1.70
4.20 ? 1.30
4.40 ? 2.50
23.4 ? 1.51
3.83 ? 1.47
4.20 ? 3.70
17.4 ? 3.5
Histologic response based on microscopic findings of necrosis, increased
stromal fibrosis, and myxoid changes, scored as: minimal or no (?10% re-
(?90% response). Cleaved caspase 3-positive cells were counted in 20 fields,
histological response and number of cleaved caspase 3-stained cells. (?) and
(???) indicate no obvious staining or strong staining, respectively, for Ki67
(see Figs. 2 and 7) and P-S6 protein (see Fig. 11, which is published as support-
ing information on the PNAS web site).
ptosis in tumor lesions of imatinib-treated KitV558?/?mice. Quantification of
proliferating (A) and apoptotic (B) cells (percent of control) in tumor sections
significantly at P ? 0.05 (see Materials and Methods).
Imatinib treatment decreases cell proliferation and increases apo-
www.pnas.org?cgi?doi?10.1073?pnas.0511076103Rossi et al.
Protein extracts from individual tumors of several mice were
analyzed to assess potential variability. The ras?MAP kinase path-
way is activated by many receptor tyrosine kinases, including Kit.
Although ERK1?2 phosphorylation was observed in tumor lysates
from untreated mice, there was no effect of imatinib treatment on
ERK1?2 activation (Fig. 3). Thus ERK signaling is not critical for
cell proliferation and survival response in tumor cells.
The PI3-kinase?Akt pathway, a key pathway in the control of
apoptosis and the translational response, is known to play a role in
malignancies (for reviews, see refs. 16 and 17). In tumor extracts
from untreated control animals, PDK1 and Akt, as well as the
downstream components GSK3? and mTOR, and ribosomal pro-
tein S6 were all strongly phosphorylated with minimal variability in
all placebo-treated samples (Fig. 3). Phosphorylation of 4EBP1, a
mice, PDK1 and GSK3? phosphorylation was still visible but
reduced compared to placebo controls (compare lanes 1–4 to 5–8;
Fig. 3). Interestingly, Akt and its downstream targets (mTOR, the
ribosomal protein S6, and 4EBP1) showed the strongest reduction
of phosphorylation upon imatinib treatment. The Ser?Thr kinase
among others, ribosomal protein S6 and 4EBP-1, components of
the protein synthesis machinery. Phosphorylation of 4EBP-1 re-
of mTOR, ribosomal protein S6, and 4EBP1 upon imatinib treat-
ment indicates a strong down-regulation of the translational re-
sponse upon imatinib treatment in GIST.
in tumor extracts from imatinib- and placebo-treated mice. STAT1
reliably assess its expression or phosphorylation state. However,
STAT3 and -5 were found to be expressed and consistently phos-
phorylated in placebo-treated GIST, and their phosphorylation
appeared to be diminished upon treatment with imatinib. Interest-
ingly, the decrease of phosphorylation of the smaller STAT3?
isoform was more pronounced than that of the ? isoform (Fig. 3).
The reasons for this difference are not clear. Therefore, STAT5
response of GIST tumor cells.
Gene Expression Profile of Kit Oncogenic Signaling in GIST.Tofurther
elucidate the mechanisms underlying the response of murine GIST
to imatinib, we compared gene expression profiles in RNA isolated
from GISTs from placebo and imatinib-treated animals. Total
RNA was isolated from the GISTs of five mice for each group:
placebo and 6- and 24-h imatinib-treated and -labeled cRNA were
synthesized and hybridized to an Affymetrix MOE430A murine
expression array platform.
A one-way ANOVA was performed to determine which genes
changed significantly between the three treatment groups, 0, 6, and
24 h. The genes found to be significantly different [68 and 76 probe
identifications (IDs)] are listed in Table 2, which is published as
into 11 categories, including cell signaling, transcription, metabo-
lism, and IFN response as the most represented categories. To
determine which genes were differentially expressed after 6 or 24 h
of imatinib treatment, a t test analysis between the placebo and the
6- or 24-h groups was performed. One hundred twenty-four (138
expressed after 6 and 24 h of treatment, respectively (Tables 3 and
Genes belonging to the IFN response group were consistently
down-regulated upon treatment with imatinib in all three types of
used to validate that IFN?-induced GTPase (Igtp) expression was
reduced after 6 and 24 h of treatment by 2.3 and 3 times,
respectively, consistent with the array data (Table 5, which is
published as supporting information on the PNAS web site).
1 (Ifi1) expression was reduced in treated GIST (Fig. 8, which is
published as supporting information on the PNAS web site).
Another observation from the ANOVA and t test analysis was
that cyclins D1, D2, and?or D3 were down-regulated upon drug
treatment. The down-regulation of cyclins D2 and D3 was con-
firmed by real-time and semiquantitative PCR, respectively (Table
5 and Fig. 8), and the results were consistent with the array data.
Inversely, the cyclin-dependent kinase inhibitor p18 (Cdkn2c) was
up-regulated almost 2-fold, a result confirmed by semiquantitative
PCR (Fig. 8). Interestingly, down-regulation of the eukaryotic
translation initiation factor 1A (Eif1a) was observed only after 24 h
(Table 5). Taken together, the analysis of RNA expression profiles
showed that IFN-responsive genes are expressed in GIST, and that
their expression is diminished upon Kit inhibition. Furthermore,
cyclin D proteins were also down-regulated by imatinib treatment,
consistent with the cell cycle arrest observed in GIST lesions of
imatinib-treated KitV558?/?mice (Fig. 2A).
The ATP-binding cassette (ABC) transporters are a family of
At present, 10 ABC transporters have been implicated in the
development of drug resistance (see refs. 18 and 19 for review).
resistance protein BCRP?ABCG2 (20), and overexpression of the
MDR1?ABCB1 gene decreased imatinib uptake and conferred
imatinib resistance in cancer cell lines (21–23). Because ABC
transporters are regulated at the level of transcription (19), we
sought to determine whether RNA expression of any of these
transporters was modified upon imatinib treatment in GIST. We
analyzed the expression level of 10 transporters implicated in
resistance to chemotherapeutics (19). Although none of these
transporters were identified in our previous ANOVA and t test
analysis, we reexamined the effect of imatinib treatment with the
less-stringent t test analysis (Tables 6 and 7, which are published as
supporting information on the PNAS web site). Only ABCC5 was
found to have a reasonably significant P value of 0.0095 after 6 h of
protein lysates from four placebo (lanes 1–4) and four 6-h imatinib-treated
mice (lanes 5–8) were fractionated by SDS?PAGE and subjected to Western
blotting to assess protein activation using the following phospho-specific
antibodies: P-Akt T308, P-mTOR S2448, P-S6 S235?236, P-4EBP1 S65, P-GSK3?
S9, P-PDK1 S241, P-STAT5 Y694, P-STAT3 Y705, P-ERK Y202?204, Kit, and
P-Y719-Kit. Protein extracts were prepared from tumors of several mice to
assess individual variability in response to the drug.
Imatinib down-regulates the translational response in GIST. Tumor
Rossi et al. PNAS ?
August 22, 2006 ?
vol. 103 ?
no. 34 ?
treatment, but MDR1?ABCB1 and BCRP?ABCG2 were also found
to be up-regulated, with P values of 0.0016 and 0.0040, respectively,
after 24 h of treatment. These results were validated by real-time
PCR (Table 5) and, whereas the three genes were found to be
up-regulated after 6 and 24 h of imatinib treatment, the high
standard deviation indicated variability between tumors for the
regulation of these genes. These results may suggest that imatinib
treatment can induce an up-regulation of ABC transporters in
GIST, and this could contribute to the development of imatinib
Human GISTs have been shown to have rather distinct gene
expression profiles. To further validate whether the GIST mouse
model replicates the human disease, we compared the expression
profile of murine GIST with the human GIST signature derived
from the comparison of 181 different sarcomas (24). The human
GIST signature represents a list of 295 weighted genes and among
them, 173 are present on the MOE430A chip and thus could be
compared with the mouse GIST expression profile. Importantly,
144 genes from the human GIST signature were found to be
expressed in the mouse GIST, and 29 were absent (Table 8, which
is published as supporting information on the PNAS web site). The
top discriminators of the human signature, including KIT,
GIST. This indicates a substantial degree of similarity between
human and mouse GIST expression profiles. Interestingly, none of
the human signature genes found in the mouse GIST expression
profiles are affected by imatinib treatment.
Akt signaling by mTOR may be involved in oncogenic Kit signaling
in GIST. To determine whether inhibition of mTOR is sufficient to
reproduce the effects of imatinib on murine GIST, we treated
KitV558?/?mice with the mTOR inhibitor RAD001. Mice were
treated by gavage with 5 mg?kg RAD001 daily. Tumor extracts
were prepared after 24 h and characterized by Western blot
analysis. We observed that ribosomal protein S6 phosphorylation
was completely abolished after treatment, indicating that the dose
administered was sufficient to block mTOR signaling in GIST (Fig.
mTOR signaling, including Y719 phosphorylation of Kit and
phosphorylation of Akt, were not affected by RAD001 treatment;
only a variable slight increase in Akt phosphorylation was observed
(Fig. 4 and results not shown). These results indicate rapid and
effective inhibition of mTOR signaling in GIST by RAD001.
Despite the effect of RAD001 blocking mTOR activity,
KitV558?/?mice treated with 5 mg?kg RAD001 for up to 4 weeks
did not show a histologic response in tumor sections prepared from
treated mice, and no significant decrease in tumor cellularity or
increase in myxoid stroma was observed compared to control mice
(Fig. 5 A–D). Similarly, RAD001 treatment did not produce
apoptosis, as demonstrated by the absence of cleaved caspase 3
5 I–L and Fig. 9C, which is published as supporting information on
the PNAS web site). Treatment of mice with a higher dose of 10
mg?kg RAD001 still produced no effect on histology or apoptosis
in a group of five treated animals (Fig. 9A and results not shown),
but near-complete arrest of cell cycle progression was evident after
only 7 days of treatment (Figs. 9 B and C). Of note is that the onset
of cell cycle arrest upon treatment with RAD001 is delayed
compared to cell cycle arrest induced by imatinib.
Combination Treatment of GIST with Imatinib and RAD001. The
the mouse GIST model. They also showed that signaling through
mTOR is strongly affected by imatinib treatment. To determine
whether imatinib treatment synergized with RAD001 in GIST,
mice were treated with 45 mg?kg imatinib and 10 mg?kg RAD001
treatment with a decrease in cellularity and increase in myxoid
PNAS web site). However, the response observed in the eight mice
treated with both imatinib and RAD001 varied from mild to
moderate and was similar to that obtained by imatinib treatment
similar to that in imatinib treatment alone (not shown). We
conclude that the two drugs do not synergize in mouse GIST.
The protein tyrosine kinase inhibitors imatinib, erlotinib, and
gefitinib, inhibitors of the Kit, PDGFR, Bcr-Abl, and EGFR
tyrosine kinases are being used successfully to treat patients with
GIST, chronic myelogenous leukemia, and lung adenocarcinomas,
Tumor extracts from four placebo and four RAD001-treated mice were frac-
tionated by SDS?PAGE and subjected to immunoblot analysis with antibodies
S235?236 and GAPDH (B).
RAD001 treatment inhibits mTOR signaling in GIST of KitV558?/?mice.
mice. (A–D) H&E staining of tumor sections of placebo and RAD001-treated
mice (5 mg?kg). (E–L) Immunostaining of tumor sections of placebo and
Ki67 (I–L). The quantification of proliferating cells is presented in Fig. 9.
www.pnas.org?cgi?doi?10.1073?pnas.0511076103Rossi et al.
respectively. However, the development of drug resistance is a
limiting factor in targeted single-agent therapy. Resistance often
involves the acquisition of second-site receptor tyrosine kinase
mutations, which interfere with tyrosine kinase inhibition. A de-
tailed understanding of the signaling pathways involved in the
development and maintenance of GIST may help to identify
effector molecules that could be targeted with other specific
inhibitors and to uncover combinational therapies to more effec-
to investigate Kit-mediated oncogenic signaling pathways and eval-
uate the consequences of imatinib inhibition on posttranscriptional
modifications and gene expression in vivo. In our GIST mouse
model, the juxtamembrane domain KitV558?mutation found in a
by using a knock-in strategy. The KitV558?/?mice develop GIST
with complete penetrance and indistinguishably from the human
model is similar to the histologic response in human GIST patients,
with replacement of cellular areas by myxoid stroma and focal
necrosis. These features provided a rationale to investigate the
consequences of imatinib treatment in the GIST mice.
Normal Kit ligand-induced Kit receptor signaling is known to
activate several signaling molecules and cascades in vitro, including
the PI3-kinase signaling network, the Ras?-MAP kinase cascade,
Src kinase family signaling, SHP1?2 signaling, and the E3 ubiquitin
ligase c-cbl. The characterization of posttranslational modifications
of signaling molecules suspected to have a role in Kit signaling in
GIST indicates strong activation of the PI3-kinase signaling cas-
cades, including the translational response and down-regulation of
the activating modifications upon drug treatment. Furthermore,
phosphorylation of the STAT transcription factors, STAT3 and
STAT5, was inhibited by imatinib as well. Surprisingly, the Ras-
MAP kinase pathway, although activated in GIST samples, was not
affected by imatinib treatment. Because all tumor samples showed
histologic and biochemical evidence of response to imatinib, the
lack of an effect on ERK1?2 activation suggests this pathway is
insufficient for oncogenic Kit signaling. However, it is possible that
were inhibited in imatinib-treated mice. The availability of such
inhibitors will help to clarify the role of MAP kinase signaling in
The analysis of gene expression profiles revealed that the
genes affected by imatinib fall into several categories. First,
imatinib treatment affects the expression of cell cycle regulators.
The cyclins E and D and their associated kinases are positive
regulators of cell division. The cyclin D family members (D1, D2,
and D3) are expressed in various combinations in different cell
types. They bind and activate the cyclin-dependent kinases Cdk4
and –6, which lead to the phosphorylation and inactivation of
pRb and subsequent transcription of E2Fs-dependent genes
required for S phase entry. Two families of inhibitors restrain
cyclin?cdk activity: the ink4 and Cip?Kip families (for reviews,
see refs. 25–27). Our gene expression profiling experiments
revealed that the three members of the cyclin D family were
down-regulated upon imatinib treatment, whereas the inhibitor
p18ink4cwas up-regulated. This is in agreement with a decrease
in cell proliferation, and it underlines the importance of cyclin
D in oncogenic Kit signaling in GIST. Cyclin D expression is
induced by growth factors and mitogenic signals to mediate
progression of the cell cycle, and their overexpression is ob-
served in several cancer types (25). In agreement with our
upon imatinib treatment, STAT5 has been shown to bind and
activate the cyclin D1 promoter (28). Small molecule inhibitors
of cyclin?cdk activity have been developed recently as possible
patients resistant to imatinib therapy could benefit from these
Both Imatinib and RAD001 similarly down-regulate the trans-
lational response in GIST, as demonstrated by the greatly
diminished ribosomal protein S6 phosphorylation, and both
drugs induced cell cycle arrest. But, in contrast to imatinib,
RAD001 treatment did not induce apoptosis. This difference
could be explained by the fact that mTOR forms two different
complexes, the raptor–mTOR and the rictor–mTOR complex.
The raptor–mTOR complex has been shown to directly phos-
phorylate the hydrophobic motif site of S6K1 and to regulate cell
growth (31) by ribosomal protein S6 phosphorylation. This
complex is sensitive to rapamycin and RAD001 inhibition. In
contrast, rapamycin does not associate with the rictor–mTOR
complex (32), which has been shown to be a kinase for Akt in
Drosophila and human cells (33) and therefore may play an
important role in Akt activation also in GIST. One can hypoth-
which is consistent with a decrease in cell proliferation observed
in mice treated for 7 days but leaves the rictor–mTOR complex
free to activate Akt.
Whereas mTOR inhibition by RAD001 induced cell cycle
arrest, no concomitant histological or apoptotic response was
observed in tumor lesions. In contrast, imatinib inhibited cell
cycle progression and induced an increase in apoptosis as well as
a histological response in GIST. This indicates that mTOR
inhibition induces cell cycle arrest but, to achieve better thera-
peutic efficacy, other components of oncogenic Kit signaling
have to be targeted. These components may lie upstream of
mTOR in the PI-3 kinase or STAT pathways. In addition to
activating the translational response, the PI3-kinase effector Akt
also phosphorylates and inhibits the proapoptotic BAD protein
and the forkhead transcription factor, which may account for the
lack of induction of apoptosis.
Rapamycin has been reported to have a weak antitumor
activity in vivo. In mouse models of lymphoma and chronic
myelogenous leukemia, rapamycin alone did not improve sur-
vival of transplanted mice significantly. However, when rapa-
mycin was used in combination with cytotoxic agents or with
imatinib, synergistic antitumor activity was observed (34, 35).
Our results show no benefit of using the rapamycin derivative
RAD001 in combination with imatinib to improve the histolog-
ical response in GIST, indicating that RAD001 does not improve
the therapeutic efficacy of imatinib inhibition in GIST carrying
the KitV558?mutation. Targeting of other pathways in addition
to mTOR may improve the efficacy of GIST treatment. High
mitotic index values are associated with malignant behavior of
human GISTs and poor prognosis. By virtue of abolishing tumor
cell proliferation, RAD001 may be of useful in the treatment of
patients with imatinib-resistant GIST. Our previous description
and the current study convincingly show that KitV558?/?mice
represent a unique faithful mouse model of human familial
GIST. The current study demonstrates the utility of these mice
for preclinical investigations and for elucidating oncogenic sig-
naling mechanisms by using genetic approaches and targeted
Materials and Methods
Mice. Heterozygous KitV558?/?mice were described in ref. 12. The
KitV558?/?mice used in these experiments were backcrossed with
C57BL?6J for seven to nine generations. Mice selected for treat-
ment had no apparent signs of disease and were 3–5 months old.
Drug Treatment of Heterozygous KitV558?/?Mice.Imatinib(Gleevec,
STI571) was kindly provided by Novartis. Imatinib was dissolved
in water as a 10-mM solution and stored at ?20°C. Heterozygous
KitV558?/?mice were treated by i.p. injection with 45 mg?kg
Rossi et al.PNAS ?
August 22, 2006 ?
vol. 103 ?
no. 34 ?
for 24 h, 7 days, and 3 weeks. RAD001 (everolimus) [40-O-(2-
hydroxyethyl)-rapamycin] was also provided by Novartis as a
20-mg?g emulsion. An emulsion placebo was also provided. The
emulsion was diluted in 5% sucrose before administration by
4-week treatment. After the indicated treatment times, mice
were killed 6 h after the last administration of the drug, and the
tumors were quickly harvested and fixed in freshly prepared 4%
paraformaldehyde for histology and immunohistochemistry or
snap-frozen in liquid nitrogen for protein analyses. Five to 10
mice per group of treatment were used.
Microarray Expression Analysis. Total RNA was prepared from
GIST by the TRIzol method (Invitrogen). Biotin-labeled c-RNA
prepared from 5 ?g of total RNA was fragmented and hybridized
files were quantitated with Affymetrix’ MAS 5 software. Initially,
the lists of genes were filtered to remove those with ?25% present
done to find genes whose expression value differed among the
groups. Then, to find genes differentially expressed at a given time
point, a standard t test was used. To correct for multiple testing, the
False Discovery Rate method was used.
Real-Time PCR. Two micrograms of total RNA was reverse-
transcribed at 42°C for 30 min using the iScript cDNA Synthesis kit
(Bio-Rad). Forty nanograms of resultant cDNA was used in a
quantitative PCR by using an iCycler (Bio-Rad) and predesigned
TaqMan gene expression assays (Supporting Text, which is pub-
lished as supporting information on the PNAS web site). Triplicate
cycle threshold values were averaged, and amounts of target were
interpolated from the standard curves and normalized to hypoxan-
Statistical Analysis. The mitotic index (percentage of proliferating
Ki67-positive cells by using MetaMorph software in 20 pictures of
10 placebo-treated tumors. To determine cell proliferation and
apoptosis in tumors, Ki67 and cleaved caspase 3-positive cells were
counted under a microscope on 20 fields of at least five different
tumors for each time point. Student’s t test assuming unequal
variances between the two samples was used to determine the
significance of differences of proliferating and apoptotic cells
between the placebo and imatinib or RAD001-treated GISTs.
Groups were judged to differ significantly at P ? 0.05.
Histological and Immunohistochemical Analyses. For microscopic
with H&E. Immunohistochemistry antibodies were purchased
from Cell Signaling Technology (no. 9661) for cleaved caspase 3,
Novacastra (no. NCL-Ki67P) for Ki67, Oncogene (no. PC34) for
Kit, and Cell Signaling Technology (no. 2211) for phospho-S6
Immunoprecipitation and Western Blotting. Western blotting and
(12). Anti-p85 rabbit polyclonal antibody was purchased from
Upstate Biotechnology, and anti-GAPDH were purchased from
GeneTex. All other antibodies were rabbit polyclonals obtained
from Cell Signaling Technology.
We thank Sandra Gonzales, Craig Farrell, and Ahmed Fadl of the
Molecular Cytology Facility for help with histological analyses and Drs.
Yuhong She and Ju Haiying of the Antitumor Assessment Facility for
technical assistance with drug administration. We thank Drs. Elisabeth
Buchdunger and Heidi Lane of the Novartis Institutes for BioMedical
Research and Oncology, Basel, respectively, for providing STI571 and
RAD001. We also thank Drs. Eva Besmer and Joe Scandura and Robert
supported by the National Cancer Institute and National Institutes of
Health Grants CA 102774 and HL?DK55748 (to P.B.).
1. Huizinga, J. D., Thuneberg, L., Kluppel, M., Malysz, J., Mikkelsen, H. B. &
Bernstein, A. (1995) Nature 373, 347–349.
2. Maeda, H., Yamagata, A., Nishikawa, S., Yoshinaga, K., Kobayashi, S. & Nishi,
K. (1992) Development (Cambridge, U.K.) 116, 369–375.
3. Torihashi, S., Ward, S. M., Nishikawa, S., Nishi, K., Kobayashi, S. & Sanders,
K. M. (1995) Cell Tissue Res. 280, 97–111.
4. Antonescu, C. R., Sommer, G., Sarran, L., Tschernyavsky, S. J., Riedel, E.,
Woodruff, J. M., Robson, M., Maki, R., Brennan, M. F., Ladanyi, M., et al.
(2003) Clin. Cancer Res. 9, 3329–3337.
5. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S.,
Kawano, K., Hanada, M., Kurata, A., Takeda, M., et al. (1998) Science 279,
6. Lasota, J., Wozniak, A., Sarlomo-Rikala, M., Rys, J., Kordek, R., Nassar, A.,
Sobin, L. H. & Miettinen, M. (2000) Am. J. Pathol. 157, 1091–1095.
7. Rubin, B. P., Singer, S., Tsao, C., Duensing, A., Lux, M. L., Ruiz, R., Hibbard,
M. K., Chen, C. J., Xiao, S., Tuveson, D. A., et al. (2001) Cancer Res. 61,
8. Antonescu, C. R., Besmer, P., Guo, T., Arkun, K., Hom, G., Koryotowski, B.,
Res. 11, 4182–4190.
9. Debiec-Rychter, M., Cools, J., Dumez, H., Sciot, R., Stul, M., Mentens, N.,
Vranckx, H., Wasag, B., Prenen, H., Roesel, J., et al. (2005) Gastroenterology
10. Nishida, T., Hirota, S., Taniguchi, M., Hashimoto, K., Isozaki, K., Nakamura,
H., Kanakura, Y., Tanaka, T., Takabayashi, A., Matsuda, H., et al. (1998) Nat.
Genet. 19, 323–324.
11. Robson, M. E., Glogowski, E., Sommer, G., Antonescu, C. R., Nafa, K., Maki,
R. G., Ellis, N., Besmer, P., Brennan, M. & Offit, K. (2004) Clin. Cancer Res.
12. Sommer, G., Agosti, V., Ehlers, I., Rossi, F., Corbacioglu, S., Farkas, J., Moore,
M., Manova, K., Antonescu, C. R. & Besmer, P. (2003) Proc. Natl. Acad. Sci.
USA 100, 6706–6711.
13. Kissel, H., Timokhina, I., Hardy, M. P., Rothschild, G., Tajima, Y., Soares, V.,
Angeles, M., Whitlow, S. R., Manova, K. & Besmer, P. (2000) EMBO J. 19,
14. Agosti, V., Corbacioglu, S., Ehlers, I., Waskow, C., Sommer, G., Berrozpe, G.,
Kissel, H., Tucker, C. M., Manova, K., Moore, M. A., et al. (2004) J. Exp. Med.
15. Timokhina, I., Kissel, H., Stella, G. & Besmer, P. (1998) EMBO J. 17, 6250–6262.
16. Luo, J., Manning, B. D. & Cantley, L. C. (2003) Cancer Cell 4, 257–262.
17. Wendel, H. G. & Lowe, S. W. (2004) Cell Cycle 3, 847–849.
18. Borst, P. & Elferink, R. O. (2002) Annu. Rev. Biochem. 71, 537–592.
19. Scotto, K. W. (2003) Oncogene 22, 7496–7511.
20. Burger, H., van Tol, H., Boersma, A. W., Brok, M., Wiemer, E. A., Stoter, G.
& Nooter, K. (2004) Blood 104, 2940–2942.
21. Hamada, A., Miyano, H., Watanabe, H. & Saito, H. (2003) J. Pharmacol. Exp.
Ther. 307, 824–828.
22. Mahon, F. X., Belloc, F., Lagarde, V., Chollet, C., Moreau-Gaudry, F.,
Reiffers, J., Goldman, J. M. & Melo, J. V. (2003) Blood 101, 2368–2373.
23. Thomas, J., Wang, L., Clark, R. E. & Pirmohamed, M. (2004) Blood 104, 3739–3745.
24. Baird, K., Davis, S., Antonescu, C. R., Harper, U. L., Walker, R. L., Chen, Y.,
Glatfelter, A. A., Duray, P. H. & Meltzer, P. S. (2005) Cancer Res. 65, 9226–9235.
25. Sherr, C. J. & Roberts, J. M. (1999) Genes Dev. 13, 1501–1512.
26. Sherr, C. J. & Roberts, J. M. (2004) Genes Dev. 18, 2699–2711.
27. Weinberg, R. A. (1995) Cell 81, 323–330.
28. Bromberg, J. F. (2001) BioEssays 23, 161–169.
29. Malumbres, M. & Barbacid, M. (2001) Nat. Rev. Cancer 1, 222–231.
30. Swanton, C. (2004) Lancet Oncol. 5, 27–36.
31. Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H. & Sabatini, D. M.
(1998) Proc. Natl. Acad. Sci. USA 95, 1432–1437.
32. Sarbassov, D. D., Ali, S. M., Kim, D. H., Guertin, D. A., Latek, R. R.,
Erdjument-Bromage, H., Tempst, P. & Sabatini, D. M. (2004) Curr. Biol. 14,
33. Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. (2005) Science
34. Mohi, M. G., Boulton, C., Gu, T. L., Sternberg, D. W., Neuberg, D., Griffin,
J. D., Gilliland, D. G. & Neel, B. G. (2004) Proc. Natl. Acad. Sci. USA 101,
35. Wendel, H. G., De Stanchina, E., Fridman, J. S., Malina, A., Ray, S., Kogan, S.,
Cordon-Cardo, C., Pelletier, J. & Lowe, S. W. (2004) Nature 428,
www.pnas.org?cgi?doi?10.1073?pnas.0511076103Rossi et al.