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PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation, which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as ATG1, ATG5 and PI3KC3, and by autophagy inhibitors (e.g., 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with the ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.
Acute promyelocytic leukemia (APL) is a common variant of acute myeloid leukemia (AML) accounting for 10–15% of all cases, and is characterized by molecular features that render it amenable to combined molecular targeting therapy.1 In APL, all-trans retinoic acid (ATRA) induces degradation of the fusion protein encoded by the PML-RARα oncogene [a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα)], differentiation of leukemic cells and clinical remission.2–5 At the molecular level, ATRA binds PML-RARα and triggers its degradation. The degradation of PML-RARα involves activities of both the proteasome and caspase pathways, although neither is sufficient for complete degradation,2–5 suggesting that other mechanisms are involved in PML-RARα degradation. Thus, a thorough understanding of the mechanism by which PML-RARα is degraded will identify novel targets for APL treatment.
Autophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester organelles and proteins for delivery to the lysosome or vacuole where the cargo is degraded and recycled.6–9 A number of clinically available cancer therapeutics, including DNA-damaging chemotherapy, radiation therapy and molecular targeted therapies have been found to induce autophagy in cell culture and animal models.10–12 Autophagy presently is viewed as a “doubled-edged sword” whereby downregulation of this process promotes tumorigenesis and upregulation in an established tumor promotes cell survival under stress.10 In recent years, several cell differentiation and cell death-inducing agents were shown to activate autophagy in acute myeloid leukemia (AML) cell lines.13 Since the ubiquitin-proteasome system and autophagic machinery share common components and exert overlapping functions,14 we reasoned that autophagy may play a role in the degradation of PML-RARα during cell differentiation. And indeed, recent research has shown that autophagy contributes to therapy-induced degradation of the PML-RARα,15 but the mechanism and significance are still poorly defined.
In this study we demonstrate that autophagy is important for myeloid cell differentiation by degrading PML-RARα. We demonstrate that inhibition of autophagy by shRNA that targets essential autophagy genes and pharmacological inhibition of autophagy blocked ATRA-induced PML-RARα degradation and subsequently myeloid cell differentiation. Futhermore, induction of autophagy enhanced ATRA-induced cell differentiation. Notably, we identified an interaction between PMLRARα and p62/SQSTM1, a long-lived scaffolding protein that is a substrate of autophagy and is involved in the transport of ubiquitinated proteins destined for proteasomal digestion.16,17 Thus, autophagy is essential for PML-RARα degradation and cell differentiation, suggesting a selective strategy to eradicate leukemic cells.
We first examined whether ATRA at therapeutic doses (1 µM) induced autophagy in human myeloid cells.3 Since the microtubule-associated protein 1 light chain 3 (LC3) specifically associates with autophagosomes,9 the numbers of endogenous LC3-positive vesicles reflecting autophagosomes was monitored in the human ATRA-responsive myeloid cells HL-60 (AML M2) and NB4 (AML M3).18 Indeed, LC3 puncta were found in these cells after treatment with ATRA for 24–72 h (Fig. 1A). A similar effect was observed when human primary monocytes (PBMC) were treated with ATRA (Fig. 1A). Additionally, the number of LC3 vesicles was decreased with the addition of an autophagy inhibitor, 3-methyladenine (3-MA), which blocks the activity of phosphatidylinositol 3-kinase (PtdIns3K) and the formation of autophagosomes (Fig. 1A).9
During autophagy, LC3 is processed post-translationally into soluble LC3-I, and subsequently converted to membrane-bound LC3-II, which correlates with the number of autophagosomes.19 ATRA treatment triggered induction of LC3-II (Fig. 1B). The addition of the lysosomal protease inhibitors pepstatin and E64D led to a further increase in the amount of LC3-II (Fig. 1B), consistent with increased autophagic flux.9
The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway is thought to be the important step in the initiation of autopahgy.6–8 Similar to rapamycin, ATRA inhibited phosphorylation of mTOR substrates e.g., 4EBP1 (Fig. 1C), suggesting that ATRA decreased mTOR activity. Moreover, we substantiated our evidence for ATRA-induced autophagy by electron microscopy, the most convincing and standard method to detect autophagy,9 which revealed the presence of multiple autophagosome-like vacuoles with double-membrane structures in ATRA-treated HL-60 cells (Fig. 1D). Thus, ATRA is sufficient to induce autophagy in human myeloid cells.
Autophagy-related (ATG) gene products are the main proteins involved in the process of autophagy and can be categorized into four functional groups: (1) the Atg1 (ULK1) protein kinase complex regulating the induction of autophagy, (2) the class III PtdIns 3-kinase (PI3KC3) lipid kinase complex controlling vesicle nucleation, (3) the Atg12-Atg5 and Atg8-phosphatidylethanolamine conjugation pathways for vesicle expansion and completion and (4) the Atg protein retrieval system.6–8,20 The expression of Atg1 and PI3KC3, but not Atg5, was increased after ATRA treatment for 48 h by western blot analysis (Fig. 2A).
To explore the potential role for ATG proteins in the regulation of cell death of leukemia cells, target-specific shRNA against ATG1, ATG5 and PI3KC3 was transfected into HL-60 and NB4 cells. Transfection of the target-specific shRNA against these ATG genes led to a significant decrease in these proteins in HL-60 and NB4 cells (Fig. 2B). Depletion of ATG1, ATG5 and PI3KC3 expression in these cells inhibited ATRA-induced autophagy (Fig. 2C and D) and rendered them significantly more sensitive to ATRA-induced apoptotic cell death (Fig. 2E), indicating that autophagy genes are involved in sensitivity to chemotherapeutic agents. Indeed, the cytoprotective role of autophagy following chemotherapy has been confirmed by other investigators.21,22
ATRA is a widely used differentiating agent in the clinic. To determine the impact of autophagy on myeloid differentiation, HL-60 and NB4 cells were incubated with an autophagy inhibitor (e.g., 3-MA) or an autophagy inducer (e.g., rapamycin) in the presence or absence of ATRA, followed by flow cytometric analysis of the myeloid differentiation marker CD11b. Co-treatment with rapamycin and ATRA for 48–72 h resulted in marked induction of CD11b expression and a higher level of maturation, relative to that seen with ATRA or rapamycin alone (Fig. 3A and B). In contrast, 3-MA inhibited ATRA-induced CD11b expression and NB4 cell differentiation as judged by morphological observation (Fig. 3A and B). These data suggested that autophagy is involved in myeloid cell differentiation.
To further characterize the role of autophagy in myeloid cell differentiation after ATRA treatment, we used an RNA interference approach. Knockdown of ATG1, ATG5 or PI3KC3 reduced both the ATRA-induced expression of CD11b and functional differentiation determined by the nitroblue tetrazolium (NBT) reduction assay and morphological assay (Fig. 3C–E). Moreover, knockdown of ATG5 impaired the capacity of other differentiating agents [e.g., phorbol 12-myristate 13-acetate (PMA), arsenic trioxide (As2O3) and vitamin D3] to induce differentiation of HL-60 cells (Fig. 3F). Together, these findings suggest a role for autophagy in differentiation of myeloid leukemia cells.
The PML-RARα oncoprotein is a direct molecular target of ATRA in human myeloid cells and mediates differentiation.3,23 PML-RARα is catabolized in response to ATRA either in a proteasome- or caspase-dependent manner.2,3,5 To determine the mechanism by which autophagy alters myeloid cell differentiation, we examined the expression of RARα when degradation by the proteasome, caspases or autophagy was blocked. Consistent with a previous study,3 ATRA induced degradation of PML-RARα in NB4 cells (Fig. 4A). Protease inhibitor cocktails, caspase inhibitors (z-VAD) or autophagy inhibitors (e.g., 3-MA) all significantly (but not completely) blocked ATRA-induced degradation of PML-RARα at 24 h (Fig. 4A). In contrast, induction of autophagy with rapamycin promoted ATRA-induced degradation of PML-RARα (Fig. 4A). Consistent with this finding, knockdown of ATG5 decreased degradation of PML-RARα (Fig. 4B). The colocalization between PML-RARa and LC3 (autophagy marker)/LAMP-2 (lysosomal marker) was increased after ATRA treatment (Fig. 4C). In contrast, inhibition of autophagy by knockdown of ATG5 decreased colocalization of these markers (Fig. 4C). These data suggested that PML-RARα degradation is not only mediated by the previously documented proteasome and caspase pathways,2,3,5 but also through autophagy.
Protein degradation by autophagy is an important mechanism to mitigate the accumulation of polyubiquitinated protein aggregates. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy.16,17 Previous studies have demonstrated that PML-RARα is a polyubiquitinated protein.5 To explore whether p62 binds to PML-RARα, we performed co-immunoprecipitation (Co-IP) analysis using p62 and RARα antibodies. We found that under basal conditions endogenous p62 and RARα Co-IP with each other in HL-60 and NB4 cells and this interaction significantly increased after ATRA treatment (Fig. 5A). Consistently, the colocalization between PML-RARa and p62 were increased after ATRA treatment (Fig. 5B), confirming an interaction between p62 and PML-RARα.
Knockdown of p62 reduced the ATRA-induced expression of the myeloid differentiation marker CD11b, functional differentiation as determined by NBT reduction assay and higher level of maturation based on morphological assay (Fig. 5C and D). Notably, knockdown of p62 impaired the degradation of PML-RARα during cell differentiation (Fig. 5E). The protein synthesis inhibitor cycloheximide causes a drastic reduction in autophagy-induced protein degradation.24 Indeed, cycloheximide inhibited ATRA-induced p62 and PML-RARα degradation (Fig. 5F). Previous studies have demonstrated that overexpression of p62 inhibits the clearance of ubiquitinated proteins destined for proteasomal degradation,25 and the degradation of PML-RARα partially involves activities of the proteasome.2–5 Consistently, overexpression of p62 prevented the degradation of PML-RARα and cell differentiation after ATRA treatment (Fig. 5G and H). Furthermore, knockdown of p62 decreased ATRA-induced colocalization between PML-RARα and the lysosomal marker LAMP-2 (Fig. 5I), suggesting that the p62 adaptor protein delivers PML-RARα proteins to the lysosome where they are subsequently degraded. In contrast, overexpression of p62 increased the delivery of PML-RARα proteins to the lysosome (Fig. 5I) where PML-RARα degradation was inhibited due to the inhibition of proteasomal degradation by p62 overexpression.25 These studies suggest that appropriate levels of p62 contribute to sustained PML-RARα levels during cell differentiation by the coordination of proteolytic crosstalk between the proteasomal and autophagic pathways (Fig. 6).
This study demonstrates a relationship between ATRA-induced degradation of PML-RARα fusion protein and autophagy activation, which may contribute to myeloid cell differentiation. In addition, the present study establishes human p62 as a direct mediator of ATRA-induced degradation of RARα and provides mechanistic insight into the role of p62 in regulating cell differentiation.
Autophagy is an evolutionarily conserved lysosomal self-digestion process essential for cellular homeostasis and survival. As an adaptive response, it protects organisms against a wide range of pathologies including cancer, infection, neurodegeneration, heart disease and aging.6–8 Cell differentiation is often associated with decreased cell growth, indicating an altered rate of macromolecule synthesis and degradation. A recent study demonstrated that autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K562 cell line.26 Knockdown of the autophagy genes LC3 and beclin 1 by specific siRNAs impairs phorbol ester PMA and p38 inhibitor SB202190-mediated megakaryocytic differentiation.26 Similarly, Beclin 1 is required for Vitamin D3-induced autophagy and differentiation in HL-60 cells.13 In this study, we demonstrated that autophagy mediated the effects of ATRA on myeloid cell differentiation, as: (1) Inhibition of autophagy by shRNAs that target essential autophagy genes such as ATG1, ATG5 or PI3KC3 decreased ATRA-induced differentiation in HL-60 and NB4 cells; (2) an autophagy inhibitor (3-MA) inhibited ATRA-induced myeloid cell differentiation whereas an autophagy inducer (rapamycin) promoted myeloid cell differentiation. Moreover, autophagy also is required for differentiation of nonhematologic cells and tissues, such as adipose mass and neuroblastoma.27,28 Together, these findings indicate that an appropriate level of autophagic activity is important for cell differentiation.
Furthermore, we demonstrate that autophagy promoted myeloid cell differentiation activities potentially via controlling degradation of PML-RARα. A hallmark of the ATRA response in APL is PML-RARα degradation, which subsequently promotes cell differentiation.2–5 Previous studies indicate that the proteasome and caspase pathways are involved in the degradation of PML-RARα, although neither is sufficient for complete degradation.2–5 The current data show that ATRA not only directly targets PML-RARα fusions to the proteasomal and caspase-mediated pathways, but also triggers an autophagy-dependent degradation of PML-RARα. Knockdown or pharmacological inhibition of autophagy inhibited PML-RARα degradation, whereas induction of autophagy using rapamycin promoted ATRA-induced degradation of PML-RARα. Rapamycin, a lipophilic, macrolide antibiotic, induces autophagy by inactivating mTOR.29 Others have demonstrated that the activity of mTOR regulates differentiation of memory CD8 T-cell,30 regulatory T cells31 and megakaryocytes32 in vivo and in vitro. Moreover, inactivation of mTOR by RAD001 potentiates the ability of MS-275, a synthetic benzamide histone deacetylase inhibitor, to induce differentiation of HL60 and NB4 cells.33 Recent study consistently finds that both ATRA and arsenic trioxide (ATO) induce autophagy via the mTOR pathway in APL cells and that autophagic degradation contributes therapy-induced proteolysis of PML-RARα.15 These results suggest that mTOR signaling is important for autophagy and myeloid cell differentiation.
Our results also demonstrated that autophagy promoted PML-RARα degradation through an interaction between p62 and RARα. p62 is an adaptor protein that binds ubiquitin and regulates signaling cascades through ubiquitination;34 it may regulate the activation of NFκB in response to upstream signals.34 More recently, a critical role for p62 in macroautophagic removal of intracellular protein aggregates has also been proposed.16 Studies involving the cellular depletion of p62 have indicated a critical role for its association with LC3 and aggregate proteins to facilitate correct formation of the autophagosome.16,35 p62 bodies are found both as membrane-free protein aggregates (sequestosomes) and as membrane-confined autophagosomal and lysosomal structures in the cytosol or nucleus.35,36 Although most of the PML-RARα fusion protein is localized to the nucleus, significant levels of PML-RARα are also found in the cytoplasm.37 Moreover, a recent study demonstrated that the localization of the large phosphoinositide-binding protein ALFY to nuclear PML bodies is dependent on p62 in HeLa cells.38 In the current study, p62-mediated degradation of PML-RARα links the ubiquitin-proteasome and autophagy-lysosome pathways, the two main routes utilized by cells to degrade intracellular proteins.
Collectively, the current study describes a direct link between autophagy and ATRA-induced degradation of PML-RARα, both of which target myeloid cell differentiation.26 Based upon the work reported here and previous evidence,13,26 we propose a more nuanced conceptual model incorporating autophagic processes and myeloid cell differentiation (Fig. 6). In this model, ATRA inhibits the mTOR pathway and activates the Atg1-PI3KC3-Atg5-dependent autophagy pathway, which promotes autophagosome formation. Furthermore, the interaction between p62 and PML-RARα regulates degradation of PML-RARα. Inhibition of p62 impaired the degradation of PML-RARα during cell differentiation. In contrast, inhibition of autophagy results in an accumulation of p62,35 which, through a negative feedback mechanism, inhibits the activity of the proteasome as well as degradation of PML-RARα.25 Thus, autophagy plays an important role in regulating degradation of the PML-RARα oncoprotein and myeloid cell differentiation by p62.
The antibody to LC3 was obtained from MBL International (M152-3) or NOVUS (NB100-2220). The antibodies to Atg5 (2630), PI3KC3 (3811), p-4EBP1 (2855) and GFP (2956) were obtained from Cell Signaling Technology. The antibody to Atg1 was from Novus (NBP1-03502). The antibody to CD11b was from BD Biosciences (557701). The antibody to LAMP-2 was from Novus (NB110-11470). The antibody to p62 was obtained from Santa Cruz Biotechnology (Sc-28359) or Novus (NBP1-03489). The antibodies to RARα (sc-551) and PML (Sc-5621) were obtained from Santa Cruz Biotechnology. Other anticancer agents and inhibitors were from Sigma.
HL-60 and NB4 cells were grown in RPMI-1640 with 10% FBS. NB4 is the only genuine promyelocytic leukemia cell line, whereas HL-60 represents a discrete stage of differentiation between the late myeloblasts and the promyelocyte.18 These ATRA-responsive cell lines provide unique in vitro model systems for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic myelomonocytic cells.18
Whole-cell lysates were resolved on a denaturing 10% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride membranes via semidry transfer. After blocking the membrane at room temperature for 3 h, the membrane was incubated overnight at 4°C with various primary antibodies. After incubation with peroxidase-conjugated secondary antibodies for 1 h at 25°C, the signals were visualized using enhanced chemiluminescence.39,40
Cells were lysed at 4°C in ice-cold RIPA lysis buffer (Cell Signaling Technology, 9806), and cell lysates were cleared by brief centrifugation (12,000 g, 10 min). Concentrations of proteins in the supernatant fraction were determined by BCA assay. Prior to immunoprecipitation, samples containing equal amounts of proteins were pre-cleared with protein A or protein G agarose/sepharose (4°C, 3 h) and subsequently incubated with various irrelevant IgG or specific antibodies (5 µg/mL) in the presence of protein A or G agarose/sepharose beads for 2 h or overnight at 4°C with gentle shaking.39,41,42 Following incubation, agarose/sepharose beads were washed extensively with PBS, and proteins eluted by boiling in 2x SDS sample buffer before SDS-PAGE electrophoresis.
Transfection with human Atg1-shRNA (SHCLN-NM_003565), Atg5-shRNA (SHCLN-NM_004849), PI3KC3-shRNA (SHCLN-NM_002647), p62-shRNA (SHCLN-NM_003900) and control shRNA (SHC001) from Sigma or pEGFPC1 control and pEGFPC1-p62 plasmids (a gift from Dr. Eileen White, The Cancer Institute of New Jersey, USA), were performed using the FuGENE® HD Transfection Reagent (Roche Applied Science, 04709705001) according to the manufacturer's instructions.42,43
Cells were fixed in 4% formaldehyde for 30 min at room temperature prior to cell permeabilization with 0.1% Triton X-100 (4°C, 10 min). Cells were saturated with PBS containing 2% BSA for 1 h at room temperature and processed for immunofluorescence with anti-LC3 antibody followed by Alexa Fluor 488-conjugated IgG (Invitrogen, A11034) and Hoechst 33258 (Invitrogen, H3569). Between all incubation steps, cells were washed three times for 3 min with PBS containing 0.2% BSA. Fluorescence signals were analyzed using an Olympus Fluoview 1000 confocal microscope (Olympus Corp.). The average LC3 puncta per cell from at least 200 cells was determined using Image-Pro Plus 5.1 software (Media Cybernetics).44–46
Cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4), followed by 1% OsO4. After dehydration, thin sections were stained with uranyl acetate and lead citrate for observation under a JEM 1011CX electron microscope (JEOL). Images were acquired digitally from a randomly selected pool of 20 fields under each condition. The quantification of autophagosomes was performed as previously described in reference 43, 44 and 47.
Differentiation of HL-60 or NB40 cells was assessed by NBT reduction assay as described previously in reference 48 or by measuring the surface CD11b antigen expression by flow cytometry analysis or morphological examination (Wright-Giemsa staining).
Data are expressed as means ± SD of three independent experiments. Significance of differences between groups was determined by two-tailed Student's t test or ANOVA LSD test. A p-value <0.05 was considered significant.
This work was supported by grants from The National Natural Sciences Foundation of China (30571982, 30772353, 30973234 to L.C., 30500485 to D.T.), and Doctoral Program of Higher Education of China (20070533042 to L.C.), and a grant from University of Pittsburgh (D.T.).