FEBS Letters 586 (2012) 3104–3110

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Cbl-b-dependent degradation of FLIPL is involved in ATO-induced in leukemic K562 and gastric cancer cells ⇑ Guodong Zhang a, Jing Liu a, Ye Zhang a, Jinglei Qu a, Ling Xu a, Huachuan Zheng b, Yunpeng Liu a, , ⇑ Xiujuan Qu a, a Department of Medical Oncology, The First Hospital of China Medical University, Shenyang 110001, China b Department of Biochemistry and Molecular Biology, College of Basic Medicine, China Medical University, Shenyang 110001, China article info abstract

Article history: Various molecular mechanisms are involved in the efficacy of arsenic trioxide (ATO) against malignant Received 7 June 2012 hematologic and some solid tumors. FLICE-like inhibitory protein (FLIP) is an inhibitor of apoptosis Revised 21 July 2012 mediated by death receptors. In this study, we identified a new link between the down-regulation Accepted 25 July 2012 of cellular FLIP and ATO-induced autophagy. ATO induced the degradation of FLIP in K562 and Available online 1 August 2012 L L MGC803 cells, which was mediated by the –proteasome pathway. Moreover, the casitas B- Edited by Noboru Mizushima lineage lymphoma-b (Cbl-b) was involved in this process, which interacted with FLIPL and promoted proteasomal degradation of FLIPL. Our findings lead to a better understanding of the mechanism of action of ATO, and suggest that a novel signaling pathway is required for ATO-induced autophagy in Keywords: Arsenic trioxide K562 and MGC803 cells. Autophagy

FLIPL Structured summary of protein interactions: Cbl-b FLIP-L physically interacts with CBL-B by anti bait coimmunoprecipitation (View interaction)

Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction only possess anti-apoptotic activity but also serve as an anti- autophagy molecule [14–15]. A previous study suggested that Over the last two decades, arsenic trioxide (ATO) has been used ATO induced apoptosis via down-regulation of FLIP in myelodys- successfully for the treatment of many types of hematologic malig- plastic syndromes and myeloid leukemia cells [16]. However, nancies [1–4]. Recent clinical trials have also demonstrated prom- whether FLIP is involved in ATO-induced autophagy remains ising results with ATO on a variety of solid tumors [5–8]. To date, unclear. various mechanisms of ATO have been investigated [9], such as The casitas B-lineage lymphoma (Cbl) family of E3 ubiquitin li- those involved in the stimulation of differentiation or the induction gases plays a key role in the determination of ubiquitinated sub- of apoptosis. Recently, there is emerging evidence that autophagy strate proteins and in regulating cell function [17–20]. In tumor plays an important role in ATO-induced toxicity [10–12]. However, necrosis factor (TNF)-treated mouse macrophage cells, c-Cbl is in- the exact molecular mechanisms involved in ATO-induced autoph- volved in ubiquitination and degradation of FLIPS [21]. c-Cbl also agy require further investigation. degrades active Src for autophagy in focal adhesion kinase (FAK)- Cellular FLIP (FLICE-like inhibitory protein), which is critical for deficient mouse squamous carcinoma cells [22]. Our prior observa- protection against death receptor-mediated cell apoptosis [13],is tions indicated that ATO could up-regulate the expression of Cbl-b, overexpressed in many tissues, mainly in two major isoforms, FLIP another Cbl family member, in both acute promyelocytic leukemia long (FLIPL) and FLIP short (FLIPS). FLIP has been reported to not and gastric cancer cells [23]. However, the function of Cbl-b up- regulated by ATO is not fully understood.

In the present study, we demonstrated that degradation of FLIPL Abbreviations: ATO, arsenic trioxide; CQ, chlorochine; FLIP , FLICE-like inhibi- L was associated with ATO-induced protective autophagy in hema- tory protein long; FLIPS, FLICE-like inhibitory protein short; Cbl-b, casitas B-lineage lymphoma-b; TNF, tumor necrosis factor; FAK, focal adhesion kinase tologic and solid tumor cells. Most notably, the ubiquitin E3 ligase ⇑ Corresponding authors. Address: Department of Medical Oncology, The First Cbl-b interacted with FLIPL and triggered proteasomal degradation Hospital of China Medical University, No. 155, North Nanjing Street, Heping District, of FLIPL, which facilitated ATO-induced autophagy. These observa- Shenyang 110001, China. Fax: +86 24 83282543. tions suggest that FLIPL is a critical regulator of ATO-induced E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Qu). autophagy in leukemic K562 and gastric cancer cells.

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.067 G. Zhang et al. / FEBS Letters 586 (2012) 3104–3110 3105

2. Materials and methods sequence used was: AAT TCT CCG AAC GTG TCA CGT. FLIPL, FLIPS, Beclin 1, Cbl-b and Ctrl siRNAs were produced from Genechem 2.1. Cell culture Co. (Shanghai, China). Gene silencing effect was verified by Immunoblotting. Human chronic myelogenous leukemic cell line (K562), human T lymphoma cell line (Jurkat), human breast cancer cell line (MCF- 2.8. Statistical analysis 7) and human gastric cancer cell line (MGC803) were obtained from the Type Culture Collection of the Chinese Academy of Sci- All the presented data were confirmed in at least three indepen- ences (Shanghai, China). All cells were cultured in RPMI-1640 med- dent experiments, and are expressed as the mean ± SD. Statistical ium (Gibco) containing 10% heat-inactivated fetal bovine serum comparisons were made by Student’s t test. P < 0.05 was consid- (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml) at ered statistically significant.

37 °C under an atmosphere of 95% air and 5% CO2. 3. Results 2.2. Reagents and antibodies 3.1. ATO affects cell viability, and induces apoptosis and protective autophagy in malignant hematologic and solid tumor cell lines Arsenic trioxide (ATO) and chloroquine (CQ) were purchased from Sigma–Aldrich (St. Louis, MO, USA). PS341 (Bortezomib) was K562, Jurkat, MCF-7 and MGC803 cell lines were treated with purchased from Millenium Pharmaceuticals Inc. (Cambridge, MA, different concentrations of ATO for 24 or 48 h, and viability was USA). Anti-Cbl-b, anti-actin, anti-p62, anti-Beclin 1, anti-PARP and evaluated. The IC values of ATO at 24 h in K562, Jurkat, MCF-7 anti-FLIP antibodies were purchased from Santa Cruz Biotechnol- 50 and MGC803 cells were 4.59 ± 1.53 lM, 5.92 ± 1.39 lM, ogy (USA). Anti-LC3 antibody was purchased from Novus Biological 49.76 ± 8.34 lM, and 59.33 ± 7.90 lM, respectively (Table 1). To (Littleton, CO, USA) and anti-phosphotyrosine 4G10 was purchased investigate whether ATO is a direct activator of autophagic flux, from Upstate Biotechnology (Lake Placid, NY). we detected microtubule-associated protein light chain 3 (LC3) (LC3-I/LC3-II) by immunoblot analysis. As shown in Fig.1A, treat- 2.3. Cell viability assay, flow cytometry and Western blot analysis ment with ATO significantly induced a time-dependent increase in the expression of both LC3-I and LC3-II in K562, Jurkat, MCF-7 These experiments were performed as described previously and MGC803 cells accompanied by PARP cleavages. It has been re- [23]. ported that the sequestosome 1 (p62) protein has LC3 binding do- mains for serving as a selective substrate of autophagy [25]. 2.4. Transmission electron microscopy Consistent with increased autophagic flux following treatment with ATO, there was a time-dependent decrease in the level of Cells were treated and collected by trypsinization, then fixed p62 by immunoblotting (Fig. 1A). Furthermore, ATO-induced with 2.5% phosphate-buffered gluteraldehyde, postfixed in 1% autophagy was confirmed by transmission electron microscopy phosphate-buffered osmium tetroxide. Cells were then embedded, (formation of ) in K562 and MGC803 cells (Supple- sectioned, double stained with uranyl acetate and lead citrate, and mentary Fig. S1). Because autophagy can result in both cell survival analyzed using a JEM-1200EX transmission electron microscope and death, we next determined whether ATO-induced autophagy (JEOL, Japan). was protective or pro-apoptotic. Treatment of K562 cells with either ATO, CQ (an inhibitor of autophagy that stops autophagy at 2.5. Immunoprecipitation assay the late phase), or a combination of ATO and CQ for 24 h revealed that combined treatment with ATO and CQ decreased cell viability, Cells were collected and lysed in an NP40 buffer supplemented and increased apoptotic cell death compared to ATO or CQ treat- with complete protease inhibitor cocktail (Roche). After pre-clear- ment alone. Similar results were also observed in MGC803 cells ing with protein A/G agarose beads for 2 h at 4 °C, whole-cell lysates (Fig. 1B and C). To confirm the effect of autophagy inhibition by were used for immunoprecipitation with the indicated antibodies. the pharmacologic agent CQ on ATO-induced apoptosis, an RNA Generally, 1–4 lg of the commercial antibody or immunoglobu- interference approach was used to suppress the expression of Be- lin-G (Santa Cruz, CA, USA) was added to 1 ml of the cell lysate clin 1, which is required for the initiation of the formation of and incubated at 4 °C for 8–12 h. After adding protein A/G agarose autophagasome [26]. Reduced cell viability in MGC803 cells was beads, incubation was continued for an additional 1 h. The immu- observed with knockdown of Beclin 1 (Fig. 1D). These data indi- noprecipitates were then washed extensively with a lysis buffer cated that blockage of autophagy enhanced the antitumor effect and eluted by boiling them with an SDS loading buffer for 5 min. of ATO in K562 and MGC803 cells.

2.6. Transfections of plasmid constructs 3.2. FLIPL is involved in ATO-induced autophagy

The experiment was performed as described previously [24]. To investigate whether FLIP expression was associated with ATO-induced autophagy, K562, Jurkat, MCF-7 and MGC803 cells 2.7. Small interfering RNA transfections were treated with ATO. Results showed that the levels of FLIPL pro-

Cells were seeded at a density of 3 Â 105 cells/well in 6-well Table 1 plates. After 24 h, cells were transfected with small interfering IC50 values obtained following treatment with ATO in K562, Jurkat, MCF-7 and MGC803 cell lines. Data are means ± SDs. RNA (siRNA) using Lipofectamine 2000 reagent according to the manufacturer’s protocols. The FLIPL siRNA sequence used was: Cells IC50/24 h(lM) IC50/24 h(lM) AAG GAA CAG CTT GGC GCT CAA. The FLIPS siRNA sequence used K562 4.59 ± 1.53 2.82 ± 0.96 was: AAC ATG GAA CTG CCT CTA CTT. The Beclin 1 siRNA sequence Jurkat 5.92 ± 1.39 3.68 ± 1.01 used was: CAG TTT GGC ACA ATC AAT ATT. The Cbl-b siRNA MCF-7 49.76 ± 8.34 20.48 ± 5.66 MGC803 59.33 ± 7.90 26.91 ± 4.43 sequence used was: AAA GTG GTA AGA CTG TGC CAA. The Ctrl 3106 G. Zhang et al. / FEBS Letters 586 (2012) 3104–3110

Fig. 1. ATO induced apoptosis and protective autophagy in malignant hematologic and solid tumor cell lines. (A) K562, Jurkat, MCF-7 and MGC803 cells were incubated with ATO for the indicated times. PARP cleavage and LC3 expression were analyzed by immunoblot. (B) K562 cells and MGC803 cells were treated with either ATO or 20 lM CQ, or a combination of ATO and CQ for 24 h, and cell viability was measured by the trypan blue exclusion assay or MTT assay. Differences between treatment groups were determined using Student’s t test and by calculating subsequent P values. ⁄P < 0.05. (C) Cell apoptosis was determined by flow cytometry. (D) MGC803 cells were transiently transfected with Beclin 1-specific siRNA for 48 h, followed by 20 lM ATO for 24 h, and cell viability was measured by MTT assay. ⁄P < 0.05 vs. cells transfected with control siRNA.

tein were down-regulated in a time-dependent manner in all cell ed FLIPL was evident within 3 h in K562 cells after ATO treatment lines, but the levels of FLIPS in solid tumor cells were not consistent (Fig. 3C left). A similar effect was also obtained in MGC803 cells with leukemia cells (Fig. 2A). We therefore investigated the effect after treatment of ATO within 1 h (Fig. 3C right). These findings of FLIPL on autophagy. To assess the effect of FLIPL on autophagy, suggested that FLIPL was ubiquitinated during ATO-treated K562 FLIPL-targeted siRNA was transfected in MGC803 cells. Suppression and MGC803 cells. of FLIPL induced significant levels of PARP cleavage and LC3-II expression (Fig. 2B), indicating activation of apoptosis and autoph- 3.4. Cbl-b is involved in the ubiquitination of FLIPL agy. Furthermore, specific down-regulation of FLIPL enhanced ATO- induced autophagy in MGC803 cells as indicated by the up-regula- To determine whether the E3 Cbl-b was in- tion of LC3-II (Fig. 2C). The result was further confirmed by trans- volved in ubiquitination of FLIPL, K562 and MGC803 cells were mission electron microscopy (Supplementary Fig. S2). However, treated with ATO, and the level of Cbl-b protein was detected. ATO-induced autophagy in MGC803 cells remained no changes in Immunoblotting showed that the expression of Cbl-b increased down-regulation of FLIPS (Fig. 2D), suggest that FLIPL plays more in a time-dependent manner (Fig. 4A). Furthermore, an interaction important role in the regulation of ATO-induced autophagy. between Cbl-b and FLIPL was found in ATO-treated K562 and MGC803 cells (Fig. 4B). To characterize whether FLIPL was ubiqui- 3.3. Degradation of FLIPL protein by ATO is mediated through the tinated by Cbl-b, previously established MGC803 cells stably trans- ubiquitin–proteasome pathway fected with the shRNA plasmid of Cbl-b [24] were used for further experiments (Fig. 4C). Knockdown of Cbl-b significantly inhibited

To explore whether FLIPL was regulated by the ubiquitin–pro- ATO-dependent ubiquitination of FLIPL (Fig. 4D). These results indi- teasome pathway, the proteasome inhibitor PS341 was used to in- cated that Cbl-b was required for ATO-induced ubiquitination of hibit the function of the proteasomal pathway. In ATO-treated FLIPL in K562 and MGC803 cells. K562 cells, pretreatment with PS341 could effectively prevent

FLIPL degradation (Fig. 3A left). A similar effect was observed in 3.5. Cbl-b is involved in ATO-induced human gastric cancer cell MGC803 cells (Fig. 3A right). Because phosphorylation of tyrosine autophagy residues could serve as a signal for ubiquitination [27], phosphor- ylation of FLIPL was examined in ATO-treated K562 and MGC803 Next, to characterize the effect of Cbl-b on ATO-induced cells. Compared to the control, the results revealed an increase in autophagy, Cbl-b specific shRNA was used to silence Cbl-b expres- phosphorylated FLIPL at 3 h in K562 cells (Fig.3B left) or at 1 h in sion in MGC803 cells. Specific silenced Cbl-b expression induced MGC803 cells (Fig. 3B right). Next, ATO-induced ubiquitination of the down-regulation of LC3-II levels and the up-regulation of FLIP the FLIPL protein was investigated. Consistent with the fact that protein (Fig. 5A). Moreover, shRNA-mediated knockdown of Cbl-b ubiquitination precedes proteasomal degradation, polyubiquitinat- effectively suppressed autophagy induced by ATO or starvation, G. Zhang et al. / FEBS Letters 586 (2012) 3104–3110 3107

Fig. 2. FLIPL is involved in ATO-induced autophagy. (A) K562, Jurkat, MCF-7 and MGC803 cells were incubated with ATO for the indicated times, immunoblot analysis of FLIPL and FLIPS expression in all cell lines. (B) After transiently transfection with FLIPL-specific siRNA for 48 h in MGC803 Cells, the expression of FLIPL, FLIPS, LC3 and PARP cleavage were analyzed by immunoblot. (C) MGC803 Cells were transiently transfected with FLIPL-specific siRNA for 48 h, followed by 20 lM ATO for 12 h. LC3 expression was analyzed by immunoblot. (D) MGC803 Cells were transiently transfected with FLIPS-specific siRNA for 48 h, followed by 20 lM ATO for 12 h. LC3 expression and PARP cleavage were analyzed by immunoblot.

Fig. 3. Degradation of FLIPL protein by ATO is mediated through the ubiquitin–proteasome pathway. (A) K562 and MGC803 cells were without pretreated or pretreated for

12 h with 5 nM PS341, and then incubated with ATO for indicated times, FLIPL was analyzed by immunoblot. (B) K562 and MGC803 cells were incubated with ATO for indicated times, FLIPL was immunoprecipitated and then the phosphorylated tyrosine was analyzed by immunoblot. (C) K562 and MGC803 cells were pretreated for 12 h with

5 nM PS341, and then incubated with ATO, FLIPL was immunoprecipitated and the ubiquitin was analyzed by immunoblot. (Ub) n-FLIPL, polyubiquitinated FLIPL. The non- specific beads was included in immunoprecipitation step as negative control.

as shown by the reduction of LC3-II levels (Fig. 5B and C). Electron tion was reverted by knockdown of FLIPL in Cbl-b silencing microscopy studies also showed a decrease in the number of ATO- MGC803 cells (Fig. 5D). Moreover, siRNA-mediated knockdown of induced autophagic in Cbl-b silencing MGC803 cells when Cbl-b in K562 cells also suppressed ATO-induced autophagy compared to non-silencing control cells (Supplementary Fig. S3). (Fig. 5E). These observations collectively indicated that Cbl-b might Furthermore, the inhibition of autophagy induced by Cbl-b deple- enhance ATO-induced autophagy in K562 and MGC803 cells. 3108 G. Zhang et al. / FEBS Letters 586 (2012) 3104–3110

Fig. 4. Ubiquitination of FLIPL requires Cbl-b. (A) K562 and MGC803 cells were treated with ATO for the indicated times, Cbl-b was analyzed by immunoblot. (B) K562 and

MGC803 cells were treated with ATO for 3 h before immunoprecipitation with anti-FLIPL antibody, Cbl-b was analyzed by immunoblot. (C) Cell viability and Cbl-b expression were analyzed in MGC803 Cells stable transfected with control or Cbl-b-specific shRNA. (D) The Cells were pretreated for 12 h with 5 nM PS341 and then for indicated times with 20 lM ATO, FLIP was immunoprecipitated and the ubiquitin was analyzed by immunoblot.

Fig. 5. Cbl-b is involved in ATO-induced human gastric cancer cell autophagy. (A) Immunoblot analysis of FLIPL and LC3 expression in MGC803 Cells stable transfected with control or Cbl-b-specific shRNA. (B) The cells were treated with 20 lM ATO for 6 h or 12 h, LC3 expression was analyzed by immunoblot. (C) The cells were treated with Hank’s balanced salt solution for 12 h, LC3 expression was analyzed by immunoblot. HBSS, Hank’s balanced salt solution. (D) MGC803 Cells stable transfected with Cbl-b- specific shRNA were transiently transfected with FLIPL-specific siRNA for 48 h, followed by 20 lM ATO for 12 h, LC3 expression was analyzed by immunoblot. (E) K562 Cells were transiently transfected with Cbl-b-specific siRNA for 48 h, followed by 5 lM ATO for 12 h, LC3 expression was analyzed by immunoblot.

4. Discussion troversial [29]. As reported recently, ATO induced not only apoptosis but also autophagy in leukemia cells [11]. It is important to under- Autophagy is a cellular catabolic degradation response to nutrient stand in detail the signaling pathways involved in this process. ATO starvation or metabolic stress. Previous reports have shown that was suggested to induce autophagic cell death in acute myelogenous autophagy is activated by some anti-tumor drugs that induce apopto- leukemia and malignant glioma cells [11,30]. However, in our obser- sis [28]. But, whether autophagy is a death-induced mechanism or a vations, ATO was observed to induce protective autophagy and not protective effort for cellular survival after chemotherapy is still con- autophagic cell death in K562 and MGC803 cells. Therefore, the G. Zhang et al. / FEBS Letters 586 (2012) 3104–3110 3109 opposing effects in response to ATO may be dependent on different References types of malignant cells. 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