BECN1 and BIM interactions with MCL-1 determine fludarabine resistance in leukemic B cells

The purine analog fludarabine (Fd) is an essential therapeutic for chronic lymphocytic leukemia (CLL). Innate or acquired resistance to Fd is a significant clinical problem and is largely mediated by increased expression of BCL-2 family members. The antiapoptotic BCL-2 family proteins inhibit both apoptosis and autophagy, therefore, downregulation of antiapoptotic BCL-2 family proteins and enhanced autophagy must coexist in cells dying in response to an apoptosis inducing therapeutic. However, in the drug-resistant cells that have an increased dependence on antiapoptotic proteins, whether autophagy is also inhibited remains unclear. Here, we examined the role of the BCL-2 family in regulating cell death and autophagy in leukemic cell lines and their derivative isogenic Fd-resistant (FdR) cells. MCL-1 degradation following Fd treatment freed the proapoptotic effectors BIM and BECN1, thus leading to cell death-associated autophagy in Fd-sensitive cells. However, in FdR cells, low BIM expression and BECN1 sequestration by MCL-1 prevented cell death. Consistently, in sensitive cells inhibition of apoptosis using siBIM and of both the early-phase autophagy nucleation steps by siBECN1, shATG7 or 3-methyladenine and the late-phase autophagy by shLAMP2, significantly reduced Fd-induced cell death. Paradoxically, FdR cells were addicted to basal autophagy, which was dependent on AMP-activated protein kinase (AMPK) but not BECN1. Moreover, in FdR cells, inhibition of autophagy by shLAMP2, but not siBECN1, enhanced cell death. The BH3-mimetic obatoclax released BIM and BECN1 from MCL-1 in Fd-sensitive and BECN1 from MCL-1 in FdR cells, and was effective at killing both Fd-sensitive and - resistant leukemic cells, including primary CLL cells. Therefore, a differential regulation of autophagy through BECN1 and AMPK signaling in Fd-sensitive and - resistant cells determines the different possible outcomes of autophagy inhibition. These findings suggest effective means to overcome Fd resistance by induction of BIM-dependent apoptosis and activation of BECN1-dependent autophagy.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process

In this study, we report a novel mechanism of action for a cytotoxic derivative of betulinic acid (BA). B10 is a semi-synthetic glycosylated derivative of BA selected for its enhanced cytotoxic activity. Interestingly, although B10 induces apoptosis, caspase-3 downregulation incompletely prevents B10-induced cell death, Bcl-2 overexpression fails to protect cells and DNA fragmentation rates do not reflect cell death rates in contrast to cytoplasmic membrane permeabilization. These results implicate that apoptotic and non-apoptotic cell death coexist upon B10 treatment. Unexpectedly, we found that B10 induces autophagy and also abrogates the autophagic flux. B10 destabilizes lysosomes as shown by Lysotracker Red staining and by cathepsin Z and B release from lysosomes into the cytoplasm. Consistently, the cathepsin inhibitor Ca074Me significantly decreases B10-induced cell death, further supporting the fact that the release of lysosomal enzymes contributes to B10-triggered cell death. Downregulation of ATG7, ATG5 or BECN1 by RNAi significantly decreases caspase-3 activation, lysosomal permeabilization and cell death. Thus, by concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. These findings have important implications for the therapeutic exploitation of BA derivatives, particularly in apoptosis-resistant cancers.     

SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we reported that SPHK1 induced the epithelial-mesenchymal transition (EMT) by accelerating CDH1/E-cadherin lysosomal degradation and facilitating the invasion and metastasis of HepG2 cells. Initially, we found that SPHK1 promoted cell migration and invasion and induced the EMT process through decreasing the expression of CDH1, which is an epithelial marker. Furthermore, SPHK1 accelerated the lysosomal degradation of CDH1 to induce EMT, which depended on TRAF2 (TNF receptor associated factor 2)-mediated macroautophagy/autophagy activation. In addition, the inhibition of autophagy recovered CDH1 expression and reduced cell migration and invasion through delaying the degradation of CDH1 in SPHK1-overexpressing cells. Moreover, the overexpression of SPHK1 produced intracellular sphingosine-1-phosphate (S1P). In response to S1P stimulation, TRAF2 bound to BECN1/Beclin 1 and catalyzed the lysine 63-linked ubiquitination of BECN1 for triggering autophagy. The deletion of the RING domain of TRAF2 inhibited autophagy and the interaction of BECN1 and TRAF2. Our findings define a novel mechanism responsible for the regulation of the EMT via SPHK1-TRAF2-BECN1-CDH1 signal cascades in HCC cells. Our work indicates that the blockage of SPHK1 activity to attenuate autophagy may be a promising strategy for the prevention and treatment of HCC.     

Mechanistic study of TRPM2-Ca2+-CAMK2-BECN1 signaling in oxidative stress-induced autophagy inhibition

Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca²⁺ influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca²⁺ influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca²⁺ influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca²⁺ influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca²⁺-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca²⁺-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X_L bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X(L) bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.     

Berberine attenuates autophagy in adipocytes by targeting BECN1

The lysosomal degradation pathway, autophagy, is essential for the maintenance of cellular homeostasis. Recently, autophagy has been demonstrated to be required in the process of adipocyte conversion. However, its role in mature adipocytes under physiological and pathological conditions remains unclear. Here, we report a major function of BECN1 in the regulation of basal autophagy in mature adipocytes. We also show that berberine, a natural plant alkaloid, inhibits basal autophagy in adipocytes and adipose tissue of mice fed a high-fat diet via downregulation of BECN1 expression. We further demonstrate that berberine has a pronounced effect on the stability of Becn1 mRNA through the Mir30 family. These findings explore the potential of BECN1 as a key molecule and a drug target for regulating autophagy in mature adipocytes.     

Berberine attenuates autophagy in adipocytes by targeting BECN1

The lysosomal degradation pathway, autophagy, is essential for the maintenance of cellular homeostasis. Recently, autophagy has been demonstrated to be required in the process of adipocyte conversion. However, its role in mature adipocytes under physiological and pathological conditions remains unclear. Here, we report a major function of BECN1 in the regulation of basal autophagy in mature adipocytes. We also show that berberine, a natural plant alkaloid, inhibits basal autophagy in adipocytes and adipose tissue of mice fed a high-fat diet via downregulation of BECN1 expression. We further demonstrate that berberine has a pronounced effect on the stability of Becn1 mRNA through the Mir30 family. These findings explore the potential of BECN1 as a key molecule and a drug target for regulating autophagy in mature adipocytes.     

GADD45A inhibits autophagy by regulating the interaction between BECN1 and PIK3C3

GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A₁ treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.     

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.     

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint

Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47^PHOX (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.     

Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis

Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.     

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia

Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.     

Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids

Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.