The antibodies to WAVE1 and p62 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies to LC3, actin and tubulin from Sigma Inc. (St. Louis, MO, USA); antibody to mitochondria HSP70 (mHSP70) from Abcam (Cambridge, MA, USA); antibodies to class III phosphoinositide 3-kinase (PI3K-III), p-4EBP1, Beclin1 and Bcl-2 from Cell Signaling Technology (Danvers, MA, USA); 3-methyladenine (3-MA), vincristine (VCR), cytosine arabinoside (Ara-C), adriamycin (ADM), E64D and pepstatin from Sigma.

HL-60 acute promyelocytic leukemia cells, K562 chronic myelogenous leukemia cells, THP-1 acute monocytic leukemia cells, adriamycin-resistant HL-60/ADR cells, A549 human lung cancer cells, human umbilical vein endothelial cells, human lung A549 cancer cells and CNE2 nasopharyngeal carcinoma cells (Xiangya School of Medicine Type Culture Collection, Xiangya, China) were cultured in RPMI-1640 or DMEM medium with 10% heat-inactivated fetal bovine serum (FBS) in 5% CO₂ and 95% ambient air.

WAVE1 small hairpin RNA (shRNA) lentiviral knockdown (GeneCopoeia, Guangzhou, China) or shRNA non-target control (NTC) were packaged with HIV-based packaging mix (GeneCopoeia) for infecting HL-60 cells to establish cells constitutively repressing WAVE1. Stable clones were selected by puromycin. PI3K-III shRNA from Sigma were constructed with FuGENE HD transfection reagent (Roche Applied Science, Stockholm, Sweden) according to the manufacturer's instructions.

After drug dosing, cell viability was evaluated by Cell Counting kit-8 (CCK-8) (Dojindo Molecular Technologies, Tokyo, Japan) according to the manufacturer's instructions.

Total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentration and purity were measured with a spectrophotometer at A260 and A260/280, respectively. RNA was reverse-transcribed into cDNA using a Primescript™ RT reagent kit (Invitrogen) according to the manufacturer's instructions. The sequences of primers used were as follows: for β-actin: forward, 5′-TCCTTCCTGGGCATGGAGTC-3′ and reverse, 5′-GTAACGCAACTAAGTCATAGTC-3′. For WAVE1: forward, 5′-TCTGGGCTACATCCAACTCC-3′ and reverse, 5′-CCTGTTCACGCTGCTCTTCT-3′. β-actin was used as an internal control for evaluating the relative expressions of WAVE1. The conditions of polymerase chain reaction (PCR) for WAVE1 were as follows: denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 sec, 56°C for 30 sec (β-actin, 50°C for 30 sec), 72°C for 30 sec and ultimately by a 5-min elongation at 72°C. The PCR products were analyzed with 1.0% agarose gel electrophoresis, ethidium bromide-stained, photographed and scanned by Band Leader software for grey scale semi-quantitative analysis.

After rinsing with phosphate-buffer solution (PBS), the cells were collected, resuspended in lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) and maintained on ice for 15 min. Cell extracts were cleared by microcentrifugation at 14,000 × g for 30 min at 4°C. Whole cell lysate was separated by 8% (10%/12%) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene difluoride (PVDF) blotting membrane (Beyotime Institute of Biotechnology). After blocking with 5% non-fat dry milk in TBST (50 mM Tris pH 7.5, 100 mM NaCl, 0.15% Tween-20), the membranes were incubated with diluted primary antibodies for 12 h at 4°C and washed thrice with TBST for 10 min. After incubating for 12 h at 4°C with various secondary antibodies, detection was made with enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL, USA) after rinsing thrice with TBST for 10 min. The membranes were exposed to X-ray film and the expressions of targeted proteins quantified by detecting specific bands. In addition, BandScan 5.0 system was used for quantifying and analyzing each specific blotting band (16).

Cells were lysed at 4°C in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% NP-40, 0.5% nadeoxycholate, 0.1% SDS, protease inhibitor cocktail) and cell lysates centrifugated at 12 000 × g for 10 min. The concentrations of proteins in supernatant were determined by bicinchoninic acid (BCA) assay. Prior to immunoprecipitation, the samples containing equal amounts of proteins were pre-cleared with protein A or protein G agarose/sepharose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (4°C, 3 h) and subsequently incubated with various irrelevant IgG or specific antibodies (5 mg/ml) in the presence of protein A or G agarose/sepharose beads for 2 h or overnight at 4°C with gentle vortexing. After incubation, agarose/sepharose beads were rinsed thoroughly with PBS, and the proteins were eluted by boiling in 2X SDS sample buffer prior to SDS-PAGE.

Cellular apoptosis was assessed by FITC Annexin V apoptosis detection kit [Annexin V-FITC, propidium iodide (PI) solution and Annexin V binding buffer]. This assay involved staining cells with Annexin V-FITC (a phospholipid-binding protein binding to disrupted cell membranes) in combination with PI (a vital dye binding to DNA penetrating into apoptotic cells). Flow cytometry (FACS) was performed for determining the percentage of apoptotic cells (Annexin V⁺/PI).

After collection, NB4 cells were fixed in 2.5% glutaraldelhyde for at least 3 h and subsequently treated with 2% paraformaldehyde at room temperature for 60 min, 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h and post-fixation with 1% osmium tetroxide (OsO₄) for 1.5 h. After the second rinsing, the samples were dehydrated with graded acetone and ultimately embedded in Quetol 812. Ultrathin sections were observed under Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan).

Caspase-3 activity was assayed by caspase-3 colorimetric assay kit (Calbiochem, Berlin, Germany) according to the manufacturer's instructions.

The quantitative data were presented as means ± standard deviation. In addition, analysis was performed with specified statistical methods using GraphPad Prism (version 5.04). For calculating P-value, two-tailed parameters with a confidence interval of 95% were used. A P-value <0.05 was considered to indicate a statistically significant result.

A total of 30 patients aged 1–13 years with newly diagnosed AML at our department were enrolled from January 2008 to December 2014. There were 17 males and 13 females (Table I). The median age was 6 years (range, 1–13 years). According to the French-American-British (FAB) classification scheme, the types were M0 (n=1), M1 (n=1), M2 (n=16), M4 (n=7), M5 (n=4) and M7 (n=1). At the time of diagnosis, the median count of white blood cells (WBC) was 14.9×10⁹/l (range, 0.8–102.7×10⁹/l), median hemoglobin level 66.5 g/l (range, 31–241 g/l) and median platelet count 31.2×10⁹/l (range, 2–104.6×10⁹/l). The median relative level of WAVE1 mRNA expression was 0.4635 (range, 0.0–0.781), and 22 (73.3%) achieved a complete remission (CR) with 1 or 2 induction chemotherapies. The survival outcomes revealed that 16/30 patients (48.4%) achieved CR after 6-month chemotherapy, and 10 of them (33.3%) died. Relapsing and refractory leukemias were the commonest cause of mortality (n=8), followed by infection (n=1) and hemorrhage (n=1). All of them received anthracycline and cytarabine-based chemotherapeutic regimens, and 4 patients received hematopoietic stem cell transplantation (HSCT) after induction therapy.

WAVE1 expression patterns were detected by RT-PCR in 30 newly-diagnosed AML patients and 8 normal healthy subjects (Fig. 1A). The relative WAVE1 mRNA levels were summarized in Table I. There was a general trend for a higher incidence of relapsing or refractory leukemia in WAVE1 high-expression patients. Also a poor prognosis was found in patients with leucocytosis at diagnosis (Table II). Moreover, the relative WAVE1 mRNA expression levels were examined for 30 pediatric AML patients at varying clinical status. Higher levels of WAVE1 expression were found in bone marrow mononuclear cells (BMMCs) from patients with primary (n=30) and relapsing (n=6) leukemias (Fig. 1B). By contrast, WAVE1 was not detectable in BMMCs from CR patients (n=12) or normal healthy subjects (n=8) (Fig. 1B). Thus WAVE1 was well-correlated with the clinical status of pediatric AML.

Furthermore, the expression levels of WAVE1 were determined by western blot analysis in 4 leukemia cell lines of HL-60, K562, HL-60/ADR and THP-1, their levels were all upregulated (Fig. 1C). In contrast, the constitutive expression levels of WAVE1 were noticeably lower in non-hematological cancer cell lines, including human lung A549 cancer cells, human umbilical vein endothelial cells and CNE2 nasopharyngeal carcinoma cells. Thus, a different role of WAVE1 is implicated for leukemic tumorigenesis.

A specific shRNA against WAVE1 was transferred into HL-60 for knocking down the expression of WAVE1. The cellular transfection efficiency was determined by visualizing eGFP-positive cells under microscope and confirmed by flow cytometry (Fig. 2A and B). Moreover, RT-PCR and western blot analysis were performed for detecting the expression of WAVE1. Transfection of WAVE1 shRNA resulted in a marked downregulation of WAVE1. In contrast, transfection of blank vector did not interfere with WAVE1 expression at the level of either mRNA or protein (Fig. 2C and D).

As a dynamic process for degrading such cytosolic components as dysfunctional organelles and proteins, autophagy offered a pathway of generating metabolic substrates (28,29). A host of existing cancer therapeutics, including DNA-damaging chemotherapy, radiation therapy and molecular targeted therapies, could induce autophagy in cell culture and animal models (6,30). To investigate whether WAVE1 is a direct activator of autophagy, immunoblot was employed for determining microtubule-associated protein light chain 3 (LC3) and its conversion products (LC3-I to LC3-II) (31). With a depletion of WAVE1 expression, the classical autophagic stimuli, such as starvation (Hank's balanced salt solution, HBSS), decreased the expression of LC3-II versus control groups (Fig. 3A). Similar to HBSS, ADM at a therapeutic dose of 1 μg/ml also decreased LC3-II expression (Fig. 3A), suggesting targeted deletion of WAVE1 inhibited ADM-induced autophagy.

The inhibition of mammalian target of rapamycin (mTOR) signaling pathway has been considered as an important step in the initiation of autophagy (6,29,32), and mTOR activity was further determined through monitoring 4EBP1 (a mTOR substrate) phosphorylation (33). The levels of phosphor-4EBP1 (p-4EBP1) decreased in transfection vector control while a depletion of WAVE1 expression partially rescued p-4EBP1 expression compared with control group (Fig. 3B). An important method for detecting autophagic flux is measuring enhanced degradation of p62 (sequestosome-1), a long-lived scaffolding protein involved in the transport of ubiquitinated protein for proteasomal digestion (34). p62 has LC3 binding domains targeting this protein for incorporation into autophagosome. Thus, it served as a selective substrate of autophagy. Knockdown of WAVE1 decreased the level of LC3-II, yet, the level of p62 increased compared with control group. It suggested that p62 degradation was dependent on WAVE1-induced autophagy (Fig. 3C). Moreover, LC3 accumulation and p62 expression were exaggerated after treatment with lysosomal protease inhibitor E64d and pepstatin A vs. WAVE1 shRNA group (Fig. 3C). Thus, the resulting elevation of LC3-II was not due to a decreased degradation of lipidated LC3, but rather to a higher autophagic flux.

The most reliable and conventional technique for visualizing autophagic vacuolization is transmission electron microscopy capable of revealing the presence of multiple autophagosome-like vacuoles with double-membrane structures (31). Ultrastructural analysis revealed that HL-60 cells exhibited fewer autophagosomes after WAVE1 RNAi treatment vs. control group (Fig. 3D). Altogether, these data demonstrate that WAVE1 is required for initiating autophagy in leukemia cells.

Chemoresistance has become a major obstacle for successful therapeutics of leukemia. A growing body of evidence has suggested that autophagy is an important resistance mechanism for chemotherapy in hematological malignancies (35–37). To characterize the role of WAVE1-mediated autophagy in chemosensitivity of leukemia cells, HL-60 cells were treated with several common chemotherapeutic drugs such as ADM (1 μg/ml), VCR (1 μg/ml) and Ara-C (0.2 μM) (32). After transfection with WAVE1 shRNA or control shRNA, autophagy was suppressed by 3-MA, a PI3K inhibitor. The expression of LC3-II significantly decreased in control group. Similar to using 3-MA, a depletion of WAVE1 expression also decreased LC3-II expression, suggesting that WAVE1 was required for ADM-induced autophagy (Fig. 4A). Moreover, a knockdown of WAVE1 expression in HL-60 cells rendered it significantly more sensitive to ADM, VCR and Ara-C-induced apoptosis associated with high levels of caspase-3 activities (Fig. 4B). Furthermore, a depletion of WAVE1 expression increased early apoptosis with ADM treatment vs. control group (Fig. 4C), supporting a potential prosurvival role for WAVE1-induced autophagy in leukemia cells under chemotherapy.

WAVE1 is expressed abundantly in mitochondria of neuronal cells (37). Similar to neuronal cells, our previous study showed that WAVE1 was localized to mitochondria and cytoplasm in leukemia cells (10). In the present study the expression of WAVE1 was confirmed by western blot analysis in both mitochondria and cytoplasm (Fig. 5A). Its mitochondrial localization provided a basis for its molecular interaction with different mitochondrial proteins. Bcl-2, an integral membrane protein on endoplasmic reticulum (ER) and mitochondria, is an important inhibitor of apoptosis. Yet, its mechanism of localization is not fully understood (38). WAVE1 knockdown decreased the mitochondrial levels of (Mit)-Bcl-2, but increased the cytoplasmic levels of Bcl-2 (Fig. 5B). Thus, a specific role for WAVE1 was evident in the regulation of Bcl-2 intracellular localization in leukemia cells.

An important molecular event in autophagic vesicle nucleation is the disassociation of Bcl-2-Beclin1 complex and the formation of Beclin1-PI3K-III complex (31,40). The disassociation of Bcl-2-Beclin1 complex sustained autophagy (39). In the present study knockdown of WAVE1 blocked the disassociation of Beclin1-Bcl-2 during enhanced autophagy (Fig. 5D). As PI3K-III is required for autophagy initiation (40), the role of PI3K-III was examined in the regulation of WAVE1-mediated autophagy using a target-specific shRNA against PI3K-III expression. Transfection of PI3K-III-shRNA led to a significant decrease in PI3K-III protein and significantly inhibited WAVE1-mediated autophagy (Fig. 5C), suggesting that PI3K-III is required for WAVE1-mediated autophagy. Depletion of WAVE1 expression blocked the interaction between Beclin1 and PI3K-III (Fig. 5D), suggesting that WAVE1 promotes vesicle nucleation. The overall data indicate that WAVE1-mediated autophagy may occur through Beclin1/Bcl-2 and Beclin1/PI3K-III-dependent pathway in HL-60 cells.