Therapeutic implications of autophagy-mediated cell survival in gastrointestinal stromal tumor after treatment with imatinib mesylate

Article (PDF Available)inAutophagy 6(8):1190-1 · November 2010with5 Reads
DOI: 10.4161/auto.6.8.13430 · Source: PubMed
Autophagy 6:8, 1190-1191; November 16, 2010; © 2010 Landes Bioscience
1190 Autophagy Volume 6 Issue 8
Punctum to: Gupta A, Roy S, Lazar AJ, Wang
WL, McAulie JC, Reynoso D, et al. Autophagy
inhibition and antimalarials promote cell death
in gastrointestinal stroma tumor (GIST). Proc Natl
Acad Sci USA 2010; 107:14333–8; PMID: 20660757;
DOI: 10.1073/pnas.1000248107.
Key words: autophagy, GIST, KIT,
PDGFRA, imatinib mesylate, small-
molecule tyrosine kinase inhibitors,
autophagy inhibitors
Submitted: 08/23/10
Revised: 08/24/10
Accepted: 08/27/10
Previously published online:
DOI: 10.4161/auto.6.8.13430
Correspondence to: Brian P. Rubin and Jayanta
Debnath; Email: and jayanta.
Gastrointestinal stromal tumor (GIST),
the most common sarcoma of the gastro-
intestinal tract, is characterized by ligand-
independent, constitutively activating
mutations in either KIT or platelet-derived
growth factor receptor A (PDGFRA)
receptor tyrosine kinases that drive tumor
cell proliferation. Because of their depen-
dence on these activating mutations,
GISTs are a striking example of “onco-
gene addiction,” which has been exploited
therapeutically using imatinib mesylate
(Gleevec; Novartis Pharmaceuticals), a
small molecule tyrosine kinase inhibi-
tor used as first-line therapy against this
tumor. Imatinib has revolutionized GIST
treatment, with an approximately 80%
response rate. However, GIST patients
generally respond to treatment with qui-
escent/stable disease; complete regres-
sion is only seen in approximately 2% of
patients. Due to clinically stable disease,
patients require lifelong therapy with
imatinib. Moreover, approximately 50%
of such patients develop secondary resis-
tance within two years of the initiation of
imatinib therapy due to the occurrence
of additional intra-allelic KIT mutations
that abrogate imatinib binding. To more
effectively treat GIST patients, it is criti-
cal to elucidate how GIST cells survive
imatinib therapy, in order to develop
therapeutic strategies that augment the
action of KIT inhibition by imatinib.
Accordingly, we have developed tissue cul-
ture models for imatinib-induced quies-
cence in GIST cells. Upon treatment with
imatinib, multiple GIST cell lines demon-
strate KIT-dependent cell cycle and trans-
lational arrest. However, akin to what is
Therapeutic implications of autophagy-mediated cell survival
in gastrointestinal stromal tumor after treatment with imatinib mesylate
Brian P. Rubin
and Jayanta Debnath
Departments of Molecular Genetics and Anatomic Pathology; Cleveland Clinic; Lerner Research Institute; Taussig Cancer Center; Cleveland, OH USA;
Department of Pathology and Diller Family Comprehensive Cancer Center; University of California; San Francisco, CA USA
seen in vivo in GIST patients, apoptosis
is both modest and variable, resulting in
significant clonogenic survival in all cell
lines. Furthermore, cell cycle and transla-
tional arrest are reversible upon removal of
imatinib, further supporting the validity
of these in vitro GIST models.
Since autophagy has been increasingly
implicated as a resistance mechanism dur-
ing cancer chemotherapy, we asked whether
autophagy was induced as a survival path-
way in response to imatinib therapy in
GIST. Treatment with imatinib in vitro
resulted in robust autophagic flux, as evi-
denced by LC3 turnover in the lysosome,
as well as p62/SQSTM1 degradation.
Furthermore, we performed immunohis-
tochemistry for LC3 in formalin-fixed
parafn-embedded GIST samples from
patients that had been treated with ima-
tinib for either 3, 5 or 7 days. Although
the number of samples evaluated was too
small for meaningful statistical analysis,
we were struck by the trend of increasing
numbers of autophagosomes in biopsies
from patients treated for longer times with
imatinib. Based on sequencing analysis
for KIT or PDGFRA mutational status in
these patients, the induction of punctate
LC3 positively correlated with predicted
sensitivity to imatinib. Considerable evi-
dence indicates that autophagy and apop-
tosis are intimately related; inhibition of
autophagy increases apoptosis in both
normal and cancer cells (Fig. 1). We simi-
larly discovered that inhibiting autophagy
using RNAi-mediated depletion of ATGs
leads to increased apoptosis and decreased
clonogenic survival of imatinib-treated
GIST cells. Autophagy 1191
reduced outgrowth of imatinib-sensitive
GIST cells in comparison with cultures
treated with imatinib therapy alone. This
broaches the exciting possibility that com-
bining autophagy inhibition with imatinib
may prevent the development of secondary
resistance in GIST patients treated with
imatinib; further studies are underway to
test this hypothesis.
Overall, our results demonstrate that
autophagy is induced in GIST cells in
response to imatinib, facilitating the
establishment of a quiescent state in
which a subset of GIST cells can sur-
vive indefinitely (Fig. 1). Inhibition of
autophagy synergizes with imatinib to
increase GIST cell death and retard the
outgrowth of residual viable GIST cells in
comparison with imatinib therapy alone.
Important unresolved issues include the
identification of cellular triggers that acti-
vate autophagy in response to imatinib
therapy; the elucidation of these proteins/
pathways may lead to new therapeutic
strategies to specifically inhibit the signals
that stimulate autophagy downstream of
this kinase inhibitor. Moreover, we have
initiated studies to determine whether
or not autophagy plays a role in the sur-
vival of GIST progenitor cells. Finally,
we are exploring whether combination
regimens of imatinib and anti-malarials
can be moved into clinical trials to treat
GIST patients, especially in consideration
that quinacrine and chloroquine are both
well-tolerated, inexpensive drugs with a
long history of use in humans. Ultimately,
these studies may improve clinical out-
come in GIST patients currently undergo-
ing chronic therapy with imatinib.
A grant from the Life Raft Group (to
B.P.R.), National Cancer Institute Grant
R01 CA126792 (to J.D.), a Culpeper
Medical Scholar Award (Partnership For
Cures) (to J.D.), an AACR-Genentech
BioOncology Award (to J.D.) and a
Howard Hughes Medical Institute
Physician-Scientist Early Career Award
(to J.D.) supported this work.
inhibits both baseline and starvation-
induced LC3-II turnover, demonstrating
that quinacrine functions similarly to
other lysosomotropic agents with regard to
its ability to inhibit autophagy at its later
stages. More importantly, both in vitro
and in vivo experiments demonstrated
that quinacrine, when used in combina-
tion with imatinib, results in increased
cell death in comparison to chloroquine,
a more commonly utilized autophagy
inhibitor in diverse therapeutic settings.
Because quinacrine also inhibits NFκB
and other cellular processes, we cannot
exclude that the cytotoxic effects of quina-
crine may be augmented by modulation of
other cellular pathways in addition to its
ability to inhibit autophagy. This possibil-
ity is currently under investigation in our
Our cell-based models also revealed
that imatinib-induced quiescence in GIST
cultures is notable for low levels of tumor
cell proliferation. Theoretically, this slow
proliferation may facilitate the outgrowth
of GIST cells with secondary KIT or
PDGFRA mutations resistant to imatinib.
Upon combining lysosomal inhibitors with
imatinib therapy, we found significantly
Cancer cells exhibiting autophagy
are often sensitive to lysosomal inhibi-
tors that block the terminal stages of
autophagic proteolysis; such agents
include commonly used anti-malarials
such as chloroquine and quinacrine.
We discovered that the pharmacologi-
cal inhibition of autophagy using these
lysosomotropic agents synergized with
imatinib to induce GIST cell death
over a wide range of physiologically rel-
evant drug concentrations. In further
support, RNAi depletion of LAMP-2
corroborated our results obtained with
anti-malarials. Furthermore, to validate
the in vitro results, autophagy inhibitors
were combined with imatinib in vivo to
treat a mouse GIST xenograft model; this
combination regimen results in increased
apoptosis within tumors, as measured by
cleaved caspase-3 immunohistochemis-
try. Notably, in the case of imatinib plus
quinacrine, partial tumor regression was
observed after only 15 days of treatment.
To our knowledge, we are the first to
utilize quinacrine to suppress autophagy
in a preclinical model for cancer therapy.
Our studies corroborated that quinacrine
accumulates in lysosomes and robustly
Figure 1. Interrelationship between apoptosis and autophagy in imatinib-treated GIST. Through
inhibition of the KIT tyrosine kinase, imatinib induces modest levels of apoptosis as well as
autophagy, which facilitates cell survival. (A) Inhibition of autophagy increases apoptosis during
imatinib treatment (B).
    • "Thereby it suppresses survival and proliferation of tumour cells [22,23]. Imatinib improved the prognosis of locally advanced inoperable or metastatic GISTs [24] as well as of stable disease [25] and is currently considered standard therapy for GISTs, in particular those attributed to known genetic defects [20]. It was found that the treatment induces changes in the tumour structure (e.g. "
    [Show abstract] [Hide abstract] ABSTRACT: Clinical studies have indicated that the NV (nanovesicle) concentration in blood samples is a potential indicator of clinical status and can be used to follow the development of the disease. For 32 months, we monitored the effect of imatinib treatment on NV concentrations in blood samples from 12 patients with GIST (gastrointestinal stromal tumour). The NV concentration before the treatment increased with respect to control by a factor of 3.5 on average (range 2.6-9.2). The first week after initiation of the treatment, the NV concentration increased considerably, by a factor of 13 on average (range 5.9-21.2), whereas on average, after 1 month, it decreased to the level of the control and remained at that level for at least 1.5 years. Recent assessment (after 2.5 years) showed a somewhat increased NV concentration, by a factor of 2 on average (range 0.7-3.9). Low NV concentrations in blood samples during the treatment reflect a favourable effect of imatinib in these patients and no remission of the disease was hitherto observed.
    Full-text · Article · Feb 2013
    • "Thus, autophagy appears critical for the establishment of a dormant state in which GIST cells can survive indefinitely [78]. Moreover, these results in GIST broach the exciting idea that autophagy can be more widely exploited to kill or prevent the expansion of quiescent or dormant cancer cells, which are notorious for their resistance to both conventional and targeted therapies [79]. Tumor dormancy is also postulated to be a stress-management mechanism adopted by DTCs to cope with an unfavorable microenvironment by completely withdrawing from the cell cycle [1]. "
    [Show abstract] [Hide abstract] ABSTRACT: The development of metastasis is the major cause of death in cancer patients. In certain instances, this occurs shortly after primary tumor detection and treatment, indicating these lesions were already expanding at the moment of diagnosis or initiated exponential growth shortly after. However, in many types of cancer, patients succumb to metastatic disease years and sometimes decades after being treated for a primary tumor. This has led to the notion that in these patients residual disease may remain in a dormant state. Tumor cell dormancy is a poorly understood phase of cancer progression and only recently have its underlying molecular mechanisms started to be revealed. Important questions that remain to be elucidated include not only which mechanisms prevent residual disease from proliferating but also which mechanisms critically maintain the long-term survival of these disseminated residual cells. Herein, we review recent evidence in support of genetic and epigenetic mechanisms driving dormancy. We also explore how therapy may cause the onset of dormancy in the surviving fraction of cells after treatment and how autophagy may be a mechanism that maintains the residual cells that are viable for prolonged periods.
    Full-text · Article · Jan 2013
    • "Thus, autophagy appears critical for the establishment of a dormant state in which GIST cells can survive indefinitely (Gupta et al., 2010). Moreover, these results in GIST broach the exciting idea that autophagy can be more widely exploited to kill or prevent the expansion of quiescent or dormant cancer cells, which are notorious for their resistance to both conventional and targeted therapies (Rubin and Debnath, 2010). Tumor dormancy is also postulated to be a stress management mechanism adopted by disseminated tumor cells to cope with the unfavorable microenvironment by completely withdrawing from the cell cycle (Aguirre-Ghiso, 2007). "
    [Show abstract] [Hide abstract] ABSTRACT: Autophagy is an evolutionarily conserved lysosomal degradation process that is crucial for adaptation to stress as well as in cellular homeostasis. In cancer, our current understanding has uncovered multifaceted roles for autophagy in tumor initiation and progression. Although genetic evidence corroborates a critical role for autophagy as a tumor suppressor mechanism, autophagy can also promote the survival and fitness of advanced tumors subject to stress, which has important implications during breast cancer progression and metastasis. Here, I discuss the mechanisms and the evidence underlying these diverse roles for autophagy in cancer and speculate on specific circumstances in which autophagy can be most effectively targeted for breast cancer treatment.
    Full-text · Article · Sep 2011
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