Imatinib induces autophagy via upregulating XIAP in GIST882 cells

Accepted Manuscript Imatinib induces autophagy via upregulating XIAP in GIST882 cells Qingqing Xie, Qi Lin, Dezhi Li, Jianming Chen PII:

S0006-291X(17)30977-4

DOI:

10.1016/j.bbrc.2017.05.096

Reference:

YBBRC 37817

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 10 May 2017 Accepted Date: 17 May 2017

Please cite this article as: Q. Xie, Q. Lin, D. Li, J. Chen, Imatinib induces autophagy via upregulating XIAP in GIST882 cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.05.096. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Imatinib induces autophagy via upregulating XIAP in GIST882 cells Qingqing Xie1, Qi Lin1, Dezhi Li1, Jianming Chen2

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1 School of Life Sciences, Xiamen University, Xiamen 361102, Fujian, China

2 Key Laboratory of Marine Genetic Resources, Fujian Collaborative

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Innovation Center for Exploitation and Utilization of Marine Biological

China.

Corresponding authors: Chen:

Third

Institute

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Jianming

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Resources, Third Institute of Oceanography, Xiamen, Fujian 361005,

of

Oceanography,

State

OceanicAdministration, Xiamen 361005, Fujian, China. Tel/Fax:

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+86592-2195337, E-mail: [email protected]

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Abstract Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms originating from the gastrointestinal tract with

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gain of function mutations in receptor tyrosine kinases KIT or platelet-derived growth factor receptor A (PDGFRA). The main effective treatment for GISTs is tyrosine kinase inhibitors, such as imatinib

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mesylate. However, GISTs respond to imatinib treatment eventually

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develop acquired resistance, which is a main obstacle for GISTs therapy. Therefore, it’s urgent to have a better understanding of the mechanisms underlying the imatinib resistance in GISTs to develop novel therapeutic strategies. X-linked inhibitor of apoptosis (XIAP) is the most potent

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apoptosis inhibitor among the inhibitor of apoptosis protein (IAP) family members. Increased cellular expression of XIAP often leads to drug resistance in cancers. Here we report that XIAP is induced upon imatinb

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treatment in GIST882 cells, leading to imatinib-induced autophagy.

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Imatinib-induced autophagy was impaired in XIAP-knockout cells generated by CRISPR/Cas9 system demonstrated by the decreasing of LC3

lipidation.

XIAP

knockout

sensitizes

GIST882

cells

to

imatinib-induced apoptotic cell death, suggesting that XIAP protects GIST882 cells from imatinib-induced cell death by inducing autophagy. Thus, the resistance of the GIST882 cells to imatinib appears to be, in part, due to the increasing of XIAP and subsequent induction of

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autophagy.

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Keywords: XIAP; autophagy; GIST; imatinib

Introduction

Gastrointestinal stromal tumors (GISTs) are the most common

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mesenchymal malignancy that arise from the gastrointestinal tract [1].

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GISTs are characterized by the ligand-independent constitutive activation mutations in receptor tyrosine kinases KIT or platelet-derived growth factor receptor A (PDGFRA) [1,2]. As a result, GISTs markedly respond to treatments with tyrosine kinase inhibitors, such as imatinib mesylate,

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which inhibits KIT and PDGFRA activities, leading to growth arrest of GIST cells [1,3,4]. Currently, imatinib treatment has been shown to be highly effective in impairing the progression of GISTs [1,4,5,6].

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Unfortunately, GISTs treated with imatinib eventually develop acquired

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resistance due to additional intra-allelic KIT mutations that impair imatinib binding [3,5]. Therefore, revealing the molecular mechanisms for imatinib resistance in GISTs is crucial to develop effective therapy. It is well known that imatinib induces cellular autophagy in mammalian cells independent of cell type [3,7]. Increasing evidences indicate that autophagy is induced as a survival pathway and an imatinib resistance mechanism in GIST cells [3,8]. Imatinib induces a reversible quiescence

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state and autophagy in imatinib-sensitive GIST cells, promoting survival of a subset of GIST cells [3]. Impairing autophagy by autophagy inhibitor chloroquine or depletion of autophagy regulators (ATGs) dramatically

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promote GIST cells apoptosis in response to imatinib treatment indicated by the increasing activities of caspase 3/7 both in vivo and in vitro [3].

GIST cells are largely unknown.

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However, the underlying mechanisms of imatinib-induced autophagy in

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X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins (IAP) family which inhibits apoptosis by selectively binding and inhibiting caspases 3, 7 and 9 [9,10,11]. Eight distinct mammalian IAPs, including XIAP, c-IAP1, c-IAP2 and

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ML-IAP/Livin, have been identified. XIAP is the most potent caspase inhibitor of all family members and an attractive target for cancer treatment [12]. A number of studies show that XIAP is overexpressed in

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various human cancers and contributes to chemotherapy resistance

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[12,13,14,15,16,17,18]. Downregulation of XIAP induces apoptosis and enhances chemotherapeutic sensitivity in many kinds of human cancer cells, a fact that can be exploited for cancer therapy [19,20,21]. XIAP and other members of IAPs are found commonly dysregulated in GISTs [13,14,16,18]. Growing evidences indicated that XIAP is involved in autophagy regulation [15,22,23]. However, the role of XIAP in controlling autophagy in cancer cells is quite controversial. It is reported

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that XIAP suppresses starvation-induced autophagy by promoting mdm2 ubiquitination and degradation, leading to p53 stabilization [23]. However, whether XIAP inhibits autophagy in p53-null cells has not been

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illustrated yet. XIAP is also reported to induce autophagy through upregulating the expression of beclin1 [15]. In this study, we are concerned about whether XIAP is involved in imatinib-induced

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autophagy in GIST cells.

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Here we reported that XIAP is induced in response to imatinib treatment in a time and dose dependent manner in GIST882 cells. Interestingly, the upregulated XIAP is positively connected with the increasing of LC3-II, indicating that XIAP is involved in imatinib-induced autophagy in cells.

We

show

that

imatinib-induced

autophagy

in

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GIST882

XIAP-knockout GIST882 cells generated using CRISPR/Cas9 systems is dramatically reduced. We also observed that knockout of XIAP sensitizes

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GIST882 cells to imatinib-induced growth inhibition and apoptosis. It is

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believed that high expression of XIAP in GIST882 cells induces autophagy and protects GIST882 cells from imatinib-induced apoptosis, resulting in decreased cell death, a fact that can be exploited for GISTs therapy.

Materials and methods Cell culture

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GIST882 cells were cultured in RPMI 1640 medium (Gibco), supplemented with 15% fetal bovine serum (FBS) (Hyclone) and 1% penicillin–streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin)

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at 37℃ in 5% CO2. Western blotting

Cells were collected and lysed followed by SDS-PAGE analysis. Lysates

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were separated on 12% SDS-polyacrylamide gels, and separated proteins

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were transferred to PVDF membranes (Millipore). After blocked with 5% skim, the membranes were incubated with primary antibodies and then goat anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase followed by chemiluminescence reaction.

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Antibodies used were as follows: anti-XIAP (ab21278) polyclonal antibody (Abcam), and anti-LC3-Ⅰ/Ⅱ (12741) monoclonal antibody (Cell

signaling

And

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(Sigma-Aldrich);

technology);

anti-β-actin

horseradish

monoclonal

antibody

peroxidade-conjugated

goat

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anti-mouse and anti-rabbit (Thermo Scientific). Cell cycle analysis

GIST882 cells were fixed in ice-cold 70% ethanol overnight. Then the fixed cells were washed with PBS and incubated with 200 µg/ml DNase-free RNase A and 50 µg/ml PI for 30 minutes in the dark, 1×104 cells were counted and DNA content was analyzed by a Flow Cytometer (BD C6).

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Establishment of CRISPR/Cas9-mediated XIAP-knockout GIST882 cell lines

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Two gRNAs targeting XIAP were designed using an online CRISPR Design Tool http://crispr.mit.edu. The sequences of the two gRNAs are as follows:

gRNAs-1:

GCATCAACACTGGCACGAGC;

gRNAs-2:

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ATGACAACTAAAGCACCGCA. The gRNAs were cloned into

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pLentiCRISPRV2 vector (Addgene plasmid #52961) which also expresses Cas9 following Feng Zhang’s protocol. The constructions were then transfected into GIST882 cells with Lipofectamine 3000. 24 hours after transfection, 1µg/ml puromycin was added into GIST882 cells for

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72 hours for selection. Surviving GIST882 cells were collected and passaged. And then T7E1 assay were performed. Genomic DNA of the survival cells was extracted using DNA extracting kit (Tiangen Biotech,

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Beijing, China). PCR was performed to amplify the genomic fragments

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surrounding the sgRNA-targeting sites. Primers are as follows: XIAP-T7E1-F:

GAATGTTTCTTAGCGGTCGTG,

XIAP-T7E1-R:

GCAGGGTTCCTCGGGTAT. Then the purified PCR products were annealed and digested by nuclease T7E1 (Vazyme, Nanjing, China). The colonies which had the specific cleavage bands were then sequenced.

Confocol

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In brief, GIST882 cells were cultured on sterile coverslips and treated with 1 µM imatinib for the indicated times. Then cells were fixed with 4% paraformaldehyde for 15min, permeabilized with 1% triton X-100 for 10

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min, blocked with 5% BSA for 1 hour. Next, cells were incubated with LC3-Ⅰ/Ⅱ antibody at 4℃ overnight followed by fluorescently labeled secondary antibody for 1 hour at 37 ℃. Nuclei were stained with 1

Ubiquitination assay

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confocal microscope (zeiss, 800).

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µg/ml DAPI for 5 min at 37 ℃. Finally, cells were examined under a

GIST882 cells were treated with 1 µM imatinib for the indicated times,

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then cells were lysed with RIPA buffer (0.2% SDS, 0.5% Nonidet P-40, 0.5% sodium deoxycolate, and protease inhibitors). Anti-Ub antibody was to

immunoprecipitated

ubiquitinated

proteins

and

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employed

ubiquitinated-XIAP was detected by Western blot analysis with

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anti-XIAP antibody.

Results

Imatinib induces XIAP in GIST882 cells Gastrointestinal stromal tumor (GIST) is a highly malignant cancer and most patients respond to imatinib eventually acquire imatinib resistance. Given that XIAP plays an important role in apoptosis resistance, we

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sought that XIAP may associate with GIST imatinib resistance. As an E3 ligase, XIAP has been shown to be autoubiquitinated and degraded in response to various apoptosis stimuli, leading to apoptosis. We

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determined to investigate whether XIAP is decreased in GIST cells in response to imatinib treatment. Imatinib-sensitive GIST882 cells were treated with 1µM imatinib and cultured for the indicated times.

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Surprisingly, Fig. 1A shows that XIAP was upregulated in response to

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imatinib treatment at early time points. However, the expression level of XIAP returned to the level of control cells when treated with imatinib for 12 hours. Fig. 1B shows that imatinb also induced XIAP in a dose-dependent manner. To prove that imatinib stabilizes XIAP, we

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treated GIST882 cells with imatinib for 1 hour before adding cycloheximide (CHX) and examined XIAP stability. Indeed, XIAP stability was increased in cells treated with imatinib (Fig 1CD). These

would

stabilize

XIAP.

Given

that

XIAP

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imatinib

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results demonstrate that imatinib induces XIAP. Next, we examined how induces

autoubiquitination and degradation as an E3 ubiquitin ligase, we test the ubiquitination levels of XIAP in GIST882 cells treated with imatinib. As Fig. 1E shows, the ubiquitination of XIAP was dramatically decreased in response to imatinib treatment, leading to XIAP stabilization.

XIAP induces autophagy in GIST882 cells

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It is well known that imatinib treatment induce autophagy, which serves as a survive pathway in GIST cells. To determine whether the induction of XIAP was involved in the induction of autophagy after imatinib monitored the expression levels of XIAP and

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treatment, we

autophagosome formation by detecting the levels of LC3-I and LC3-II the same time. As Fig. 2AB shows, the increasing of XIAP after imatinib

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treatment was accompanied by the increasing ratio of LC3-II/LC3-I. We

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noticed that the punctate LC3-II were decreased at 12 hours when XIAP decreased to the control levels, which is in accordance with an previous study showing that imatinib treatment did not change LC3-II markedly at 8, 16 and 24 hours in GIST882 cells [3]. However, we found that

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autophagy was induced at an early stage after imatinib treatment and subsequently decreased at 12 hours in GIST882 cells. Fig 2CD shows that the induction of XIAP and autophagy was also connected in response to

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imatinib treatments in a dose-dependent manner.

Generation of XIAP-knockout GIST882 cell lines By using CRISPR/Cas9 system, we generated XIAP-knockout GIST882 cell lines to investigate the functions of XIAP in imatinib-induced autophagy. We designed two gRNAs against different sites of the second exon of XIAP (Fig. 3A). E1T7 assay indicated that both gRNAs could target XIAP efficiently in GIST882 cells (Fig. 3B). The target genomic

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regions amplified by PCR for each gRNA were sequenced. Sequencing results confirmed that both gRNAs could lead Cas9 to cleave target sites in GIST882 cells (Fig 3C). Then we analyzed XIAP expression in

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GIST882 cells by Western blot. Fig 3D shows that XIAP-knockout cell lines did not express XIAP, indicating that we successfully generated XIAP-knockout GIST882 cell lines using CRISPR/Cas9 system. However,

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it is difficult to select and culture single clones of XIAP-knockout

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GIST882 cells because GIST882 cells grow slowly in vitro. We generated XIAP-knockout GIST882 cells as a puromycin-selected pool which was successfully transfected with gRNA and Cas9.

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XIAP knockout inhibits autophagy and sensitizes GIST882 cells to imatinib-induced apoptosis

XIAP-knockout cell lines were employed to evaluate the roles of XIAP in

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regulating imatinib-induced autophagy. As shown in Fig 4A, XIAP

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knockout reduced the proliferation rate of GIST882 cells, probably due to XIAP knockout-induced G1 phase cell cycle arrest (Fig 4B). XIAP knockout also sensitized GIST882 cells to imatinib-induced cell growth inhibition (Fig 4A). Next, we test the role of XIAP in imatinib-induced autophagy. As shown in Fig 4C, baseline of LC3-II was observed in XIAP wild-type GIST882 cells, while LC3-II was significantly reduced in XIAP-knockout GIST882 cells. Moreover, LC3II production and the

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number of LC3 puncta were reduced in response to imatinib treatment in XIAP-knockout

GIST882

cells,

indicating

that

imatinib-induced

autophagy in XIAP-knockout GIST882 cells was dramatically decreased

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(Fig. 4CD). We determined whether autophagy inhibition caused by XIAP knockout enhances apoptosis induced by imatinib. Indeed, as shown in Fig 4E, increased caspase3 activation was observed in

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XIAP-knockout cells upon imatinib treatment, suggesting that XIAP

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knockout sensitized GIST882 cells to imatinib-induced apoptosis.

Discussion

Imatinib is a specific tyrosine kinase inhibitor, effectively controlling

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gastrointestinal stromal tumors (GISTs), chronic myeloid leukaemia (CML) and other cancers which possess constitute activation of c-KIT, PDGFRA, bcr-abl, c-abl, Arg (abl-related gene) or c-fms [1,24,25,26].

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However, imatinib-treated cells eventually acquire resistance, resulting in

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ineffective at inducing cell death of cancer cells. Growing evidences indicate that the induction of autophagy, as a key mechanism, contributes to imatinib resistance [3,7]. However, the underlying mechanisms of imatinib-induced autophagy are barely reported. Here we reported that X-linked inhibitor of apoptosis protein (XIAP) is induced upon imatinib treatment and subsequently induces autophagy in GIST882 cells. We show that XIAP is induced upon imatinib treatment in a time and

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dose dependent manner in GIST882 cells. Interestingly, XIAP is induced at an early stagy. The protein levels of XIAP are increased before 6 hours, and decreased at 12 hours. It is possible that XIAP is induced to

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antagonize the toxicity of imatinib. However, caspase 3 is activated at late stage and promotes XIAP degradation by mediating its proteolytic cleavage in a positive feedback loop [27]. The stability of XIAP upon

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imatinib is further demonstrated by the half-life assay, which indicates

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that XIAP in imatinib-treated cells stays stable upon CHX treatment. Given that XIAP is an E3 ubiquitin ligase, we proved that the ubiquitination of XIAP is decreased upon imatinib treatment, leading to increased XIAP stability.

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XIAP is well known as a powerful inhibitor of apoptosis by inhibiting the activity of caspase 3, 7 and 9. Recent studies also indicate that XIAP is involved in the regulation of autophagy. Indeed, we found that the

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induction of XIAP after imatinib treatment is positively connected with

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imatinib-induced autophagy in GIST882 cells. To further test the role of XIAP in autophagy regulation, we generated XIAP-knockout GIST882 cells. We show that the induction of autophagy upon imatinib treatment is impeded in XIAP-knockout GIST882 cells, demonstrated by the decrease of LC3 lipidation. Thus, these results reveal that XIAP induced autophagy in response to imatinib treatment in GIST882 cells. However, XIAP was reported to inhibit starvation-induced autophagy by mediating

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ubiquitination of mdm2 and stabilization of p53. In our work, we employed GIST882 cells to study the role of XIAP in the regulation of autophagy, which was reported to be a p53-null cell line [28]. It is

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possible that XIAP exerts distinct effects on autophagy in p53 wild-type and p53 mutant cells.

It is reported that imatinib treatment completely inhibits AKT activation

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intermediated by KIT in GIST882 cells [29]. However, AKT activation

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plays an important role in imatinib-resistance development in GISTs. AKT/mTOR is highly activated in imatinib secondary resistant GISTs [30]. It is possible that activated AKT phosphorylates and stabilizes XIAP in imatinib-resistant GIST cells, resulting in increased autophagy

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[31].We hypothesize that AKT activation and XIAP stabilization critically mediates survival in the development of imatinib resistance in GISTs. More further studies are needed to test the hypothesis.

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It is critical to conquer the acquired imatinib resistance to control GISTs

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more effectively. We reported that XIAP is induced in GIST882 cells upon imatinib treatment and induces autophagy, which possibly contributes to GIST882 cells imatinib resistance, avoiding GIST882 cells undergoing imatinib-induced cell growth inhibition and apoptosis. Targeting XIAP may be a new strategy to increase the sensitivity of GIST cells to imatinib.

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Acknowledgements This work was supported by the International Science & Technology

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Cooperation Program of China (2015DFA20500).

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Figure legends

Figure 1. Imatinib treatment induces XIAP. (A) GIST882 cells were

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treated with 1 µM imatinib for the indicated times. Cells were harvested and subjected to Western blot analysis with the indicated antibodies. (B) GIST882 cells were treated with various concentrations of imatinib for 3 hours. Cells were collected and the lysates were probed with the indicated

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antibodies. (C) Imatinib prolongs the half-life of XIAP. GIST882 cells were treated with 1 µM imatinib for 1 hour before incubating with 50

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µg/ml CHX for the indicated times. Cells were collected and the cell lysates were blotted with the indicated antibodies. (D) Quantification of

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the XIAP protein levels relative to β-actin from experiments in (C). (E) Imatinib inhibits the ubiquitination of XIAP. GIST882 cells were treated with 1 µM imatinib for the indicated times. Then cells were collected and the cell lysates were immunoprecipitated with anti-ubquitin antibody and immunoblotted with anti-XIAP antibody. Figure 2. Imatinib induces autophagy and XIAP. (A) GIST882 cells were incubated with 1µM imatinib for the indicated times. Then cells were

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lysed and subjected to Western blot analysis with the indicated antibodies. (B) quantification of the experiments shown in (A). (C) GIST882 cells were treated with the indicated concentrations of imatinib for 3 hour

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followed by Western blot analysis with the indicated antibodies. (D) Quantification of XIAP and LC-Ⅱ/LC-Ⅰfrom the experiments in (C).

Figure 3. Generation of XIAP-knockout GIST882 cell lines using the

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CRISPR/Cas9 system. (A) Schematic showing the XIAP-targeting gRNA

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sequences. (B) T7E1 assay confirms the CRISPR/Cas9 system efficiency in GIST882 cells. The XIAP genomic regions were PCR amplified using XIAP-specific primers and then digested by T7 Endonuclease Ⅰ. (C) Both gRNAs work in GIST882 cells. Sequencing results of the genomic

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regions around the gRNAs were shown. (D) XIAP protein levels in the indicated cell lines. Indicated cells were lysed and cell lysates were immunoblotted with anti-XIAP antibody.

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Figure 4. Knockout of XIAP inhibits autophagy and induces apoptosis in

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response to imatinib treatment. (A) Colony-formation assay was performed with 1×103 GIST882 XIAP wild-type and XIAP knockout cells treated with or without 0.1 µM imatinib for 14 days. (B) Indicated cell lines were stained with propidium iodide (PI) followed by flow cytometry analysis. (C) Indicated cell lines were treated with 1µM imatinib for the indicated times. Then cells were lysed and analyzed by Western bolt analysis. (D) The indicated cell lines were treated with 1µM

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imatinib for 6 hours, and then stained with LC3. Cells were visualized by confocal microscopy. (E) Indicated cell lines were treated with 1µM imatinib for 24 hours, cells were harvested and analyzed by Western

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Figure 1

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blotting.

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Figure 2

Figure 3

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Figure 4

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Highlights: Imatinib induces XIAP in GIST882 cells by inhibiting XIAP ubiquitination.

autophagy.

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XIAP induction is positively correlated to imatinib-induced

cells generated by CRISPR/Cas9 system.

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Imatinib-induced autophagy is impeded in XIAP-knockout GIST882

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inhibition and apoptosis.

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XIAP knockout sensitizes GIST882 cells to imatinib-induced growth

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Conflict of interest statement

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The authors declare that they have no conflicts of interest to this work.

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Qingqing Xie, Qi Lin, Dezhi Li, Jianming Chen