Vps34 complex

Vps34 complex

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Journal of Pharmacological Sciences xxx (2017) 1e6

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The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex Siyu Wang a, e, Jie Li a, e, Yanyun Du a, Yujia Xu a, Yali Wang a, Zubin Zhang a, Zhuan Xu a, b, Yuanying Zeng a, c, Xinliang Mao a, d, *, Biyin Cao a, ** a

Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, China Department of Neurology, The First Affiliated Hospital of Soochow University, Suzhou, China c Department of Oncology, Suzhou Municipal Hospital East Campus, Suzhou, China d Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 March 2017 Received in revised form 15 June 2017 Accepted 19 June 2017 Available online xxx

S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissociated the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two critical targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment. © 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Keywords: S14161 PI3K Beclin 1 Vps34 Autophagy

1. Introduction The small molecule chemical compound S14161, or 8-ethoxy-2(4-fluorophenyl)-3-nitro-2H-chromene, is an inhibitor of pan-Class I phosphatidylinositol 3-kinases (PI3Ks) and displays potent antileukemia and anti-myeloma activities.1 PI3Ks are a class of membranous lipid kinases that catalyze the production of PI(3,4,5)P3, a

* Corresponding author. Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren Ai Road, Room E2213, Suzhou Industrial Park, 215123, Suzhou, China. Fax: þ86 (512) 65882152. ** Corresponding author. Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren Ai Road, Room E2213, Suzhou Industrial Park, 215123, Suzhou, China. Fax: þ86 (512) 65882152. E-mail addresses: [email protected] (X. Mao), [email protected] (B. Cao). Peer review under responsibility of Japanese Pharmacological Society. e These authors contributed equally to the study.

key second messenger, and regulate various cellular functions. Based on the structure and function, PI3Ks can be grouped into 3 classes, of which the Class I family is composed of both regulatory and catalytic subunits (PI3Ka, b, g and d) and promotes cell survival2; while the Class III family contains only single member, PI3KC3 or Vps34.3 Vps34 is a major player in autophagy, a conserved survival manner of cells under stress, such as nutrient depletion.4 Vps34 complexes with Beclin 1 (BENC) and PIK3R4/ p150 to provide a PI(3)P-enriched domain for autophagosome formation. These core components bind different partners, including pro-survival protein Bcl-2, which inhibits autophagy by preventing Beclin 1 from binding to Vps34.5 Thus, it is possible that the PI3K/AKT/mTOR pathway and the Vps34 complexes coordinate autophagy initiation but lack evidence. It is well known that inhibition of the Class I PI3Ks is a great strategy for cancer therapy and a specific inhibitor of PI3Kd has been approved for the treatment of several subgroups of leukemia.2 S14161 induced apoptosis in myeloma and leukemia cells by inhibiting the activities of PI3Ka, b,

http://dx.doi.org/10.1016/j.jphs.2017.07.001 1347-8613/© 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Wang S, et al., The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex, Journal of Pharmacological Sciences (2017), http://dx.doi.org/10.1016/j.jphs.2017.07.001

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S. Wang et al. / Journal of Pharmacological Sciences xxx (2017) 1e6

d, and g isoforms.1 Because the PI3K signaling pathway is a critical regulator of the autophagy flux, we wondered whether S14161 also inhibited the autophagy in addition to its induction of apoptosis. Whether S14161 modulates the crosstalk between Class I PI3Ks and Vps34 thus inducing autophagy is not known. The present study showed that S14161 also induces autophagy by inhibiting the PI3K/ AKT/mTOR signaling pathway and by promoting the formation of the Vps34/Beclin 1 complex. 2. Materials and methods 2.1. Cell culture MM cell lines (RPMI-8226, OPM2, KMS11) and leukemia cell line (K562) were purchased from American Type Culture Collection or provided by Dr. Aaron Schimmer, the University of Toronto, Canada. MM and leukemia cells were maintained in Iscove's modified Dulbecco medium and RPMI-1640 medium (Invitrogen, CA, USA), respectively, in an incubator humidified with 95% air and 5% CO2 at 37  C. All the media were supplemented with 10% fetal bovine serum (HyClone, MA, USA), penicillin (100 units/ml) and streptomycin (100 mg/ml). 2.2. Chemicals and antibodies S14161 was purchased from Maybridge Chemical Co., Ltd, Tintagel, UK. Bafilomycin A1 (BafA1) was purchased from Abcam Biochemicals (Cambridge, MA, USA); 3-methyladenine (3-MA) was obtained from SigmaeAldrich (St. Louis, MO, USA); Wortmannin and idelalisib were purchased from Selleck Chemicals (Houston, TX, USA). The antibody against LC3 was purchased from Abcam. Antibodies against Beclin 1, Bcl-2, mTOR, p-mTOR (Ser2448), 4E-BP1, p4E-BP1 (Ser65), p62 were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against PI3KC3/Vps34, p70S6K and p-p70S6K (Thr389) were purchased from Abgent (Suzhou, China). The Antibody against GAPDH was purchased from Sigma. Horseradish peroxidase-conjugate secondary antibodies, DAPI (40 ,6-diamidino-2-phenylindole), Cy3-labeled goat anti-rabbit IgG were purchased from Beyotime Institute of Biotechnology (Nantong, China). Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IRDye 680 goat antimouse and IRDye 800CW goat anti-rabbit antibodies were from Odyssey (San Ramon, CA, USA).

proteins were subjected to fractionation by SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) and probed with indicated antibodies. 2.5. Immunofluorescence OPM2 and K562 (5  104 cells) were spread on glass slides by centrifugation at 500 rpm (400g) for 5 min using a cytospin system (Thermo Fisher, MA, USA). Cells were fixed in cold 4% paraformaldehyde for 10 min. After fixation, cells were washed in phosphate buffer solution (PBS) and then blocked for 1 h with 1% bovine serum albumin containing 0.1% Triton X-100 at room temperature. Samples were incubated overnight with anti-LC3 antibody at 4  C, followed by staining with Cy3-labeled goat anti-rabbit IgG and DAPI (5 mg/ml) as described previously.9 All samples were analyzed on a Nikon confocal microscopy (EZ-C1 FreeViewer, Nikon). 2.6. Cycloheximide chase assay Cycloheximide (CHX) was purchased from SigmaeAldrich (USA). RPMI-8226 was pretreated with CHX, an inhibitor of protein de novo synthesis, followed by S14161 or DMSO treatment for 2e10 h and Western blotting to measure the protein level of p62. 2.7. Co-immunoprecipitation (Co-IP) The process was described previously.9 Briefly, cells were collected and washed with ice-cold PBS after treatment, then in lysis buffer for 20 min on ice, followed by clarification with highspeed centrifugation at 4  C for 30 min. Supernatants were incubated overnight at 4  C with specific primary antibodies as needed followed by incubation with 40 ml of a 50% slurry of protein A/G agarose beads with gentle rotation at 4  C for 2 h. Agarose beads were collected and washed 5 times with lysis buffer, followed by re-suspension in 20 ml of 2  SDS loading buffer. Samples were

2.3. Cell viability assay by MTT The MTT assay was performed as described previously.6 Briefly, to evaluate cell viability, each well containing cells for analysis was added 10 mL of MTT dye 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (SigmaeAldrich), cells were further incubated for 2e4 h until purple precipitates were visible. To evaluate cell viability, the precipitates were solubilized with 20 mL of 10% SDS (in 0.01 M HCl). The plates were then subjected to absorbance reading at 570 nm, the reference wavelength was 650 nm. 2.4. Western blot Whole cell lysates were prepared from myeloma and leukemia cells as described previously.7,8 Briefly, cells were washed with Trisbuffered solution (TBS) and re-suspended in a cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, b-glycerophosphate, EDTA, Na3VO4 and leupeptin. Protein concentrations were determined by the BCA assay kit (Thermo Fisher, MA, USA). Equal amount (30 mg) of

Fig. 1. S14161 inhibits the activation of the mTOR/p70S6K/4E-BP1 signaling pathway. A, S14161 inhibited the activation of mTOR and p70S6K in a time-dependent manner. RPMI-8226 cells were treated by S14161 (S1) at 20 mM for 1, 2, 6, or 12 h, followed by the analysis of the expression of p-mTOR and p-p70S6K. B, S14161 inhibited the activation of 4E-BP1 in a concentration-dependent manner. OPM2 and RPMI-8226 cells were treated with S14161 at increasing concentrations for 10 h. Expression of p-4E-BP1 and 4E-BP1 was measured by Western blot. GAPDH was used as a loading control.

Please cite this article in press as: Wang S, et al., The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex, Journal of Pharmacological Sciences (2017), http://dx.doi.org/10.1016/j.jphs.2017.07.001

S. Wang et al. / Journal of Pharmacological Sciences xxx (2017) 1e6

boiled before being subjected to fractionation and Western blot analysis. 2.8. Statistical analysis Results were expressed as mean ± SD. Two-group comparisons were performed using Student's t-test. Values of p < 0.05 were defined as statistically significant. 3. Results 3.1. S14161 inhibits the activation of mTOR signaling pathway S14161 was demonstrated to be a pan-Class I PI3K inhibitor.1 Since mTOR is a key downstream regulator of the PI3K signaling pathway and the central regulator of autophagy progress, we wondered whether S14161 inhibited the mTOR signaling. RPMI8226 cells were treated with S14161 in a time course at 20 mM. Western blot demonstrated that both mTOR and p70S6K

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expression and phosphorylation were inhibited by S14161 in a time-dependent manner (Fig. 1A and Supplemental Fig. 1). To view whether S14161 could inhibit cell growth, RPMI-8226 and other three cell lines (OPM2, KMS11 and K562) were incubated at 20 mM of S1416 for 0e10 h. As shown in Supplemental Fig. 2 and S14161 inhibited the proliferation of these cell lines in a time-dependent manner, which was consistent with our previous study.1 In the process of cell proliferation and survival, mTOR regulates protein synthesis and energy supply. Therefore, we next evaluated two best-characterized substrates of mTOR, the ribosomal protein S6 kinase (p70S6K) and the eukaryotic initiation factor 4E binding protein (4E-BP1).10 Consistent with the inhibition of mTOR, the activation of p70S6K (Fig. 1A) and 4E-BP1 in both OPM2 and RPMI8226 cells were also suppressed by S14161 (Fig. 1B). 4E-BP1 is the major protein in control protein translation.11 There are three isoforms in 4E-BP1, namely a, b, and g. a has the greatest mobility and represents the least-phosphorylated form; g is least mobile and represents the most highly phosphorylated form.12 Upon the treatment with S14161, the level of g form was markedly decreased,

Fig. 2. S14161 induces LC3-II expression in myeloma and leukemia cells. AeB, S14161 induced LC3-II expression in a concentration-dependent manner. RPMI-8226 cells were treated with S14161 (0, 10, 20 mM) or Idelalisib (0, 100, 200 mM), or Wortmannin (0, 1, 2 mM) for 24 h, LC3 expression were detected by Western blot. C, S14161 induced LC3-II expression in various cancer cells. MM cells (OPM2, RPMI-8226, KMS11) and leukemia cells (K562) were treated with 20 mM S14161 or DMSO for 10 h. After incubation, cells were processed for analysis of LC3 and GAPDH by Western blot. D, S14161 induced LC3-II expression in a time-dependent manner. KMS11 and K562 cells were treated with 10 mM S14161 at 0, 12, 24 or 36 h, and then LC3 was detected by Western blot. E, S14161 induced LC3-II expression analyzed by a confocal microscope. K562 and OPM2 cells were starved or treated with S14161 (20 mM) alone or combination with 3-MA (0.2 mM) or BafA1 (20 nM) for 10 h. Then, cells were fixed and stained with antibodies against LC3 (red color) and DAPI (blue color) before being applied to analyze on a confocal microscope. Autophagosome formation was defined by the accumulation of LC3, arrows indicate autophagosomes. The expression ratio of LC3II over GAPDH was analyzed by densitometry.

Please cite this article in press as: Wang S, et al., The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex, Journal of Pharmacological Sciences (2017), http://dx.doi.org/10.1016/j.jphs.2017.07.001

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in contrast, a form was raised (Fig. 1B). Therefore, these data indicated that S14161 suppressed the PI3K/AKT/mTOR signaling pathway and suppresses protein synthesis. 3.2. S14161 induces autophagy in myeloma and leukemia cells Because mTOR is a critical switch of the autophagy flux, we wondered whether S14161 triggered autophagy. To this end, we measured the expression of LC3-II, a typical biomarker of autophagy. As shown in Fig. 2A and B, S14161 induced the expression of LC3-II in all examined cell lines. To verify this finding, LC3-II expression was measured in cells treated with idelalisib, the firstin-class inhibitor of PI3Ks,13 or Wortmannin, one of the typical and moste PI3K inhibitors. As shown in Fig. 2A and 2B, idelalisib and Wortmannin also induced LC3-II expression, which was consistent with the previous report.13 Therefore, these results demonstrated that PI3K inhibitors induced autophagy in MM cells. To further examine autophagy induced by S14161 in other hematological malignant cells, RPMI-8226, KMS11, OPM2, and K562 were treated with S14161 followed by LC3 analysis. As expected, S14161 induced the expression of LC3-II (Fig. 2C) and these effects were time-dependent (Fig. 2D). To visualize the formation of LC3 in autophagosomes, an immunofluorescent assay was applied. OPM2 and K562 cells were treated by S14161 for 10 h before being stained with a LC3 specific antibody. Immunofluorescent analysis revealed that starvation as a positive control induced the appearance of LC-3 punctate dots (Fig. 2E). Similar to the starvation treatment, S14161 induced the LC3 punctate dots (Fig. 2E). Notably, these dots were reduced by 3-MA, an inhibitor of autophagy initiation, but increased by BafA1, a selective inhibitor of lysosomes. Therefore, these results suggested that suppression of the Class I PI3Ks by S14161 induced autophagy in MM and leukemia cells. 3.3. S14161 triggers the autophagic flux Autophagy is a process with multiple stages. After maturation, autophagosomes fuse with lysosomes to form autolysosomes where the cargos are degraded by the lysosomal enzymes.14 To examine the autophagosome delivery and degradation, we measured the protein level of p62/SQSTM1, a hallmark of autophagic flux.15,16 RPMI-8226, KMS11, OPM2, and K562 cells were treated with S14161 and the expression of p62 was measured afterwards by Western blot. As shown in Fig. 3A, p62 protein was markedly decreased in all cell lines examined. The decrease of p62 was concentration-dependent (Fig. 3B). Consistent with this finding on p62, LC3-II was also increased in a similar manner (Fig. 3B). Notably, cell growth was also suppressed in this concentration range (Supplemental Fig. 2). Since autophagy is completed in lysosomes, we next applied BafA1, a vacuolar Hþ-ATPase inhibitor of lysosomes,17,18 to demonstrated the autophagic flux affected by S14161. As shown in Fig. 3C, LC3-II was induced and p62 was decreased by S14161, but these changes were abolished by BafA1. Interestingly, when protein synthesis de novo was halted by cycloheximide, S14161 markedly promoted the degradation of p62 (Fig. 3D), which further suggested that S14161 modulated p62 stability probably via the autophagy-lysosomal pathway. 3.4. Beclin 1 is induced by S14161 Beclin 1 is one of the key players frequently elevated in autophagy. To investigate the effects of S14161 on Beclin 1 expression, MM and leukemia cell lines treated with S14161 were subjected to Western blot analysis. As shown in Fig. 4A and B, Beclin 1 was induced by S14161 in all cells examined and in a dose- and timedependent manner. Notably, when cells were treated with S14161

Fig. 3. S14161 mediates p62 degradation. A, S14161 decreased p62 protein. OPM2, RPMI-8226, KMS11 and K562 cells were treated with 20 mM S14161 or DMSO for 10 h. After incubation, cells were processed for analysis of p62 and GAPDH by Western blot. B, S14161 decreased p62 expression in a concentration-dependent manner. RPMI-8226 and KMS11 cells were treated with S14161 at indicated concentrations for 10 h, followed by analysis for p62 and LC3. C, BafA1 prevented p62 degradation induced by S14161. RPMI-8226 and KMS11 cells were treated with S14161, BafA1 (20 nM) alone or co-treated with S14161 and BafA1 (20 nM) for 10 h. After incubation, cells were harvested and cell lysates were measured the expression of p62 and LC3 by Western blot. GAPDH was used as a loading control. D, S14161 accelerated p62 degradation. RPMI8226 was pretreated with cycloheximide (CHX) followed by S14161 (20 mM) treatment for 2e10 h and Western blot to measure the protein level of p62.

in the presence of 3-MA, the elevated Beclin 1 was blocked, along with decreased LC3-II (Fig. 4C). This result was consistent with the finding in the immunofluorescence assay (Fig. 2E), in which the LC3 punctate dots were decreased by 3-MA.

3.5. S14161 induces autophagy by increasing the formation of Beclin 1/Vps34 complex The above study suggested that Beclin 1 is critical for S14161induced autophagy. In normal status, the anti-apoptotic protein Bcl-2 interacts with and restricts Beclin 1 function therefore preventing autophagy.19 To view the effects of S14161 on the expression of Bcl-2, RPMI-8226 and KMS11 cells were treated by S14161, followed by Western blot. The assay showed that S14161 decreased Bcl-2 and increased Beclin 1 in a concentration dependent manner (Fig. 5A). Next, we evaluated the association of Beclin 1 and Bcl-2 in the treatment of S14161. Endogenous Bcl-2 and Beclin 1 was reciprocally co-immunoprecipitated by a Beclin 1 and a Bcl-2 antibody, respectively, in RPMI-8226 and KMS11 cells. The Co-IP assay showed that the Beclin 1 protein was dramatically decreased by S14161 in the Beclin 1/Bcl-2 complex (Fig. 5B). Consistently, when cell lysates were immunoprecipitated by a Beclin 1 antibody in the same amount of input proteins, Bcl-2 protein was found to be decreased in the Beclin 1 complex

Please cite this article in press as: Wang S, et al., The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex, Journal of Pharmacological Sciences (2017), http://dx.doi.org/10.1016/j.jphs.2017.07.001

S. Wang et al. / Journal of Pharmacological Sciences xxx (2017) 1e6

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(Fig. 5C). Thus, the results suggested that the induction of autophagy by PI3K inhibition was highly associated with the dissociation of Beclin 1/Bcl-2 complex. It is well known that Beclin 1 complexes with Vps34 in mediating autophagy by promoting initial nucleation and assembly of the primary autophagosome membrane.14 To evaluate the effects of S14161 on the Beclin 1/Vps34 complex, we next measured Vps34 and its association with Beclin 1. As shown in the whole cell lysates, the Vps34 protein was not affected by S14161 (Fig. 5B and C). However, there were more Vps34 in the Beclin 1 Co-IP complex, while in the Bcl-2 precipitation assay, Vps34 was decreased in contrast to Beclin 1 (Fig. 5B and C). Therefore, these results suggested that S14161 promoted the formation of the Beclin 1/Vps34 complex but dissociated the Beclin 1/Bcl-2 complex. 4. Discussion

Fig. 4. S14161 induces the expression of Beclin 1. A, S14161 upregulates Beclin 1 in various cancer cells. OPM2, RPMI-8226, KMS11 and K562 cells were treated with 20 mM S14161 (S1) or DMSO for 10 h. B, S14161 upregulates Beclin 1 in a timedependent manner. KMS11 and K562 cells were treated with 10 mM S14161 for 0, 12, 24 and 36 h. After incubation, cells were processed for analysis of Beclin 1 and GAPDH by Western blot. C, 3-MA abolished Beclin 1 expression induced by S14161. RPMI-8226 and KMS11 cells were starved (st) or treated with S14161 (20 mM) alone or with 3-MA (0.2 mM) for 10 h, followed by Beclin 1, LC3 expression analysis using specific antibody.

Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their degradation within lysosomes.20,21 Vps34 (PI3KC3) plays a key role in positively modulating the process of autophagy, especially in the formation of phagophore,3 in contrast, the Class I PI3K superfamily mainly regulates cell proliferation and survival. The present study demonstrated that S14161 as a pan-Class I PI3K inhibitor triggers autophagy by modulating the cross-talk between PI3K and Vps34. Vps34 is a unique PI3K that uses phosphatidylinositol (PI) as the substrate to generate PI(3)P, which is essential for phagophore elongation and recruitment of other Atg proteins to the phagophore.4 In contrast, Class I PI3Ks are extensively involved in cell growth, proliferation, differentiation, motility, survival and intracellular trafficking by the PI3K/AKT/mTOR signaling pathway.2 As

Fig. 5. S14161 interferes with the formation of the Beclin 1/Bcl-2 complex but enhances the interaction between Beclin 1 and Vps34. A, S14161 downregulated Bcl-2 and increased Beclin 1. RPMI-8226 and KMS11 cells were treated with increasing concentrations of S14161 for 24 h. Bcl-2 and Beclin 1 were detected by Western blot. B, S14161 decreased the interaction between Bcl-2 and Vps34/Beclin 1. RPMI-8226 and KMS11 cells were treated with 20 mM S14161 or vehicle (DMSO) for 10 h, the lysates were coimmunoprecipitated (IP) using a Bcl-2 specific antibody. The immunoprecipitates (pull down) and the whole cell lysates (input) were analyzed by Western blot for Beclin 1, Vps34 or Bcl-2 using specific antibodies. C, S14161 increased the interaction between Beclin 1 and Vps34. RPMI-8226 and KMS11 cells were treated with 20 mM S14161 or vehicle (DMSO) for 10 h, the lysates were co-immunoprecipitated using a Beclin 1 specific antibody. The pull down and the input were analyzed for Bcl-2, Vps34 or Beclin 1 using specific antibodies. GAPDH was used as an internal loading control.

Please cite this article in press as: Wang S, et al., The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex, Journal of Pharmacological Sciences (2017), http://dx.doi.org/10.1016/j.jphs.2017.07.001

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shown in the present study, S14161 triggers autophagy by inducing Beclin 1 expression (Fig. 4) and promoting the formation of Vps34/ Beclin 1 complex (Fig. 5). Beclin 1 is at the central stage of phagophore formation or autophagy initiation.22 In normal status, Beclin 1 interacts with Bcl-2 therefore its activity is restricted by Bcl-2.5 However, S14161 can downregulate Bcl-2 and increase Beclin 1 (Figs. 4 and 5). Moreover, S14161 dissociates the complex of Beclin 1/Bcl-2 but increases the association of Vps34/Beclin 1 complex (Fig. 5), suggesting that Class I PI3Ks can switch the interacting partners of Beclin 1, therefore modulating autophagy process. Autophagy is a conserved survival manner in response to nutrient depletion and cellular stress. mTOR, as the main sensor of energy supply, is a major player in autophagy. In growth-promoting conditions, mTOR inhibits autophagy through its inhibitory effects on Atg1 kinase activity.14 In the situation of starvation, mTOR activity is lifted thus activating the autophagy signaling. mTOR is also a major protein in the PI3K/AKT signaling pathway. When Class I PI3Ks are activated by growth factors, mTOR and its downstream substrates p70S6K and 4E-BP1 are subsequently activated thus modulating protein synthesis.23 Inhibition of Class I PI3Ks by S14161 activates autophagy probably via this pathway because S14161 inhibits activation of mTOR, p70S6K and 4E-BP1 (Fig. 1). Protein synthesis is an energy consumptive process requiring amino acids and energy. It is estimated that the proportion of cellular energy used in protein synthesis is as high as 30e40% of total ATP and GTP.24 It is reported that knockdown of p70S6K by siRNA leads to enhanced starvation-induced autophagy.25 Therefore, inhibition of protein synthesis by S14161 probably contributes to autophagy. In conclusion, the present study demonstrated that S14161 as a Class I PI3K inhibitor modulates cross-talks between PI3K/mTOR and Vps34 in autophagy. S14161 induces both autophagy and apoptosis in hematological malignant cells. This study also suggests that inhibition of the Class I PI3Ks triggers pro-death autophagy by inhibiting protein synthesis and promoting the formation of the Vps34/Beclin 1 complex. Authorship contributions X.M. and B.C. designed the study; S.W., Y.X., J.L., Y.D., Y.W., Z.Z., Z.X., Y.Z., and B.C. conducted experiments; X.M. and B.C. analyzed data; X.M. and B.C. wrote the manuscript. Disclosure of conflicts of interest None of the authors has a conflict of interest with this work. Acknowledgements This work was partly supported by Natural Science Foundation of Jiangsu Province (BE2014630 and BK20160347), by Jiangsu Key Laboratory for Translational Research and Therapeutics of NeuroPsycho-Diseases (BK2013003), by Suzhou Key Medical Center (SZZX201506) and by Suzhou Key Laboratory for Pediatric Leukemia (SZS201615).

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jphs.2017.07.001.

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Please cite this article in press as: Wang S, et al., The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex, Journal of Pharmacological Sciences (2017), http://dx.doi.org/10.1016/j.jphs.2017.07.001