HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1) is a multifunctional, ubiquitous protein located inside and outside cells that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, immunity, metastasis, metabolism, and death. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance, sustains tumor metabolism requirements and T cell survival, prevents polyglutamine aggregates and excitotoxicity, and protects against endotoxemia, bacterial infection, and ischemia-reperfusion injury in vitro or in vivo. In contrast, HMGB1 may not be required for autophagy in some organs such as the liver and heart. Understanding HMGB1-dependent and -independent autophagy in more detail will provide insight into the integrated stress response and guide HMGB1-based therapeutic intervention.     

The redox protein HMGB1 regulates cell death and survival in cancer treatment

Metabolic and therapeutic stress activates several signal transduction pathways and releases damageassociated molecular pattern molecules (DAMPs) that regulate cell death and cell survival. The prototypical DAMP, high-mobility group box 1 protein (HMGB1) is released with sustained autophagy, late apoptosis and necrosis. Our recent findings reveal that the HMGB1 protein triggers autophagy or apoptosis in cancer cells, depending on its redox status. Reducible HMGB1 binds to the receptor for advanced glycation end products (RAGE), induces Beclin 1-dependent autophagy and promotes pancreatic or colon tumor cell line resistance to chemotherapeutic agents or ionizing radiation. In contrast, oxidized HMGB1 increases the cytotoxicity of these agents and induces apoptosis via the mitochondrial pathway. This suggests a new function for HMGB1 within the tumor microenvironment, regulating cell death and survival and suggests that it plays an important functional role in cross-regulating apoptosis and autophagy.     

MIR34A regulates autophagy and apoptosis by targeting HMGB1 in the retinoblastoma cell

MIR34A (microRNA 34a) is a tumor suppressor gene, but how it regulates chemotherapy response and resistance is not completely understood. Here, we show that the microRNA MIR34A-dependent high mobility group box 1 (HMGB1) downregulation inhibits autophagy and enhances chemotherapy-induced apoptosis in the retinoblastoma cell. HMGB1 is a multifaceted protein with a key role in autophagy, a self-degradative, homeostatic process with a context-specific role in cancer. MIR34A inhibits HMGB1 expression through a direct MIR34A-binding site within the HMGB1 3′ untranslated region. MIR34A inhibition of HMGB1 leads to a decrease in autophagy under starvation conditions or chemotherapy treatment. Inhibition of autophagy promotes oxidative injury and DNA damage and increases subsequent CASP3 activity, CASP3 cleavage, and PARP1 [poly (ADP-ribose) polymerase 1] cleavage, which are important to the apoptotic process. Finally, upregulation of MIR34A, knockdown of HMGB1, or inhibition of autophagy (e.g., knockdown of ATG5 and BECN1) restores chemosensitivity and enhances tumor cell death in the retinoblastoma cell. These data provide new insights into the mechanisms governing the regulation of HMGB1 expression by microRNA and their possible contribution to autophagy and drug resistance.     

HMGB1 Promotes Mitochondrial Dysfunction–Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation

Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP–induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP–induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.     

Autophagic secretion of HMGB1 from cancer-associated fibroblasts promotes metastatic potential of non-small cell lung cancer cells via NFκB signaling

Tumor progression requires the communication between tumor cells and tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major components of stromal cells. CAFs contribute to metastasis process through direct or indirect interaction with tumor cells; however, the underlying mechanism is largely unknown. Here, we reported that autophagy was upregulated in lung cancer-associated CAFs compared to normal fibroblasts (NFs), and autophagy was responsible for the promoting effect of CAFs on non-small cell lung cancer (NSCLC) cell migration and invasion. Inhibition of CAFs autophagy attenuated their regulation on epithelial–mesenchymal transition (EMT) and metastasis-related genes of NSCLC cells. High mobility group box 1 (HMGB1) secreted by CAFs mediated CAFs’ effect on lung cancer cell invasion, demonstrated by using recombinant HMGB1, HMGB1 neutralizing antibody, and HMGB1 inhibitor glycyrrhizin (GA). Importantly, the autophagy blockade of CAFs revealed that HMGB1 release was dependent on autophagy. We also found HMGB1 was responsible, at least in part, for autophagy activation of CAFs, suggesting CAFs remain active through an autocrine HMGB1 loop. Further study demonstrated that HMGB1 facilitated lung cancer cell invasion by activating the NFκB pathway. In a mouse xenograft model, the autophagy specific inhibitor chloroquine abolished the stimulating effect of CAFs on tumor growth. These results elucidated an oncogenic function for secretory autophagy in lung cancer-associated CAFs that promotes metastasis potential, and suggested HMGB1 as a novel therapeutic target.Subject terms: Cancer microenvironment, Non-small-cell lung cancer, Metastasis, Translational research     

Metabolic regulation by HMGB1-mediated autophagy and mitophagy

Autophagy is a dynamic process for degradation of cytosolic components such as dysfunctional organelles and proteins and a means for generating metabolic substrates during periods of starvation. Mitochondrial autophagy ("mitophagy") is a selective form of autophagy, which is important in maintaining mitochondrial homeostasis. High mobility group box 1 (HMGB1) plays important intranuclear, cytosolic and extracellular roles in the regulation of autophagy. Cytoplasmic HMGB1 is a novel Beclin 1-binding protein active in autophagy. Extracellular HMGB1 induces autophagy, and this role is dependent on its redox state and receptor (Receptor for Advanced Glycation End products, RAGE) expression. Nuclear HMGB1 modulates the expression of heat shock protein β-1 (HSPB1/HSP27). As a cytoskeleton regulator, HSPB1 is critical for dynamic intracellular trafficking during autophagy and mitophagy. Loss of either HMGB1 or HSPB1 results in a phenotypically similar deficiency in mitophagy typified by mitochondrial fragmentation with decreased aerobic respiration and adenosine triphosphate (ATP) production. These findings reveal a novel pathway coupling autophagy and cellular energy metabolism.     

Endogenous HMGB1 regulates autophagy

Autophagy clears long-lived proteins and dysfunctional organelles and generates substrates for adenosine triphosphate production during periods of starvation and other types of cellular stress. Here we show that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is a critical regulator of autophagy. Stimuli that enhance reactive oxygen species promote cytosolic translocation of HMGB1 and thereby enhance autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2. Mutation of cysteine 106 (C106), but not the vicinal C23 and C45, of HMGB1 promotes cytosolic localization and sustained autophagy. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin1 and sustaining autophagy. Thus, endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits programmed apoptotic cell death.     

Metabolic regulation by HMGB1-mediated autophagy and mitophagy

Autophagy is a dynamic process for degradation of cytosolic components such as dysfunctional organelles and proteins and a means for generating metabolic substrates during periods of starvation. Mitochondrial autophagy (“mitophagy”) is a selective form of autophagy, which is important in maintaining mitochondrial homeostasis. High mobility group box 1 (HMGB1) plays important intranuclear, cytosolic and extracellular roles in the regulation of autophagy. Cytoplasmic HMGB1 is a novel Beclin 1-binding protein active in autophagy. Extracellular HMGB1 induces autophagy, and this role is dependent on its redox state and receptor (Receptor for Advanced Glycation End products, RAGE) expression. Nuclear HMGB1 modulates the expression of heat shock protein β-1 (HSPB1/HSP27). As a cytoskeleton regulator, HSPB1 is critical for dynamic intracellular trafficking during autophagy and mitophagy. Loss of either HMGB1 or HSPB1 results in a phenotypically similar deficiency in mitophagy typified by mitochondrial fragmentation with decreased aerobic respiration and adenosine triphosphate (ATP) production. These findings reveal a novel pathway coupling autophagy and cellular energy metabolism.     

DAMP-mediated autophagy contributes to drug resistance

Damage-associated molecular pattern molecules (DAMPs) are cellularly derived molecules that can initiate and perpetuate immune responses following trauma, ischemia and other types of tissue damage in the absence of pathogenic infection. High mobility group box 1 (HMGB1) is a prototypical DAMP and is associated with the hallmarks of cancer. Recently we found that HMGB1 release after chemotherapy treatment is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia. Overexpression of HMGB1 by gene transfection rendered leukemia cells resistant to cell death; whereas depletion or inhibition of HMGB1 and autophagy by RNA interference or pharmacological inhibitors increased the sensitivity of leukemia cells to chemotherapeutic drugs. HMGB1 release sustains autophagy as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, degradation of p62 and accumulation of autophagosomes and autolysosomes. Moreover, these data suggest a role for HMGB1 in the regulation of autophagy through the PI3KC3-MEKERK: pathway, supporting the notion that HMGB1-induced autophagy promotes tumor resistance to chemotherapy.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H 2O 2) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular cells

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H₂O₂) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

Poly-ADP-ribosylation of HMGB1 regulates TNFSF10/TRAIL resistance through autophagy

Both apoptosis ("self-killing") and autophagy ("self-eating") are evolutionarily conserved processes, and their crosstalk influences anticancer drug sensitivity and cell death. However, the underlying mechanism remains unclear. Here, we demonstrated that HMGB1 (high mobility group box 1), normally a nuclear protein, is a crucial regulator of TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10)-induced cancer cell death. Activation of PARP1 (poly [ADP-ribose] polymerase 1) was required for TNFSF10-induced ADP-ribosylation of HMGB1 in cancer cells. Moreover, pharmacological inhibition of PARP1 activity or knockdown of PARP1 gene expression significantly inhibited TNFSF10-induced HMGB1 cytoplasmic translocation and subsequent HMGB1-BECN1 complex formation. Furthermore, suppression of the PARP1-HMGB1 pathway diminished autophagy, increased apoptosis, and enhanced the anticancer activity of TNFSF10 in vitro and in a subcutaneous tumor model. These results indicate that PARP1 acts as a prominent upstream regulator of HMGB1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy, which provides new insight into the mechanism of TNFSF10 resistance.     

Poly-ADP-ribosylation of HMGB1 regulates TNFSF10/TRAIL resistance through autophagy

Both apoptosis ("self-killing") and autophagy ("self-eating") are evolutionarily conserved processes, and their crosstalk influences anticancer drug sensitivity and cell death. However, the underlying mechanism remains unclear. Here, we demonstrated that HMGB1 (high mobility group box 1), normally a nuclear protein, is a crucial regulator of TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10)-induced cancer cell death. Activation of PARP1 (poly [ADP-ribose] polymerase 1) was required for TNFSF10-induced ADP-ribosylation of HMGB1 in cancer cells. Moreover, pharmacological inhibition of PARP1 activity or knockdown of PARP1 gene expression significantly inhibited TNFSF10-induced HMGB1 cytoplasmic translocation and subsequent HMGB1-BECN1 complex formation. Furthermore, suppression of the PARP1-HMGB1 pathway diminished autophagy, increased apoptosis, and enhanced the anticancer activity of TNFSF10 in vitro and in a subcutaneous tumor model. These results indicate that PARP1 acts as a prominent upstream regulator of HMGB1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy, which provides new insight into the mechanism of TNFSF10 resistance.     

Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma

Autophagy is a catabolic process critical to maintaining cellular homeostasis and responding to cytotoxic insult. Autophagy is recognized as "programmed cell survival" in contrast to apoptosis or programmed cell death. Upregulation of autophagy has been observed in many types of cancers and has been demonstrated to both promote and inhibit antitumor drug resistance depending to a large extent on the nature and duration of the treatment-induced metabolic stress as well as the tumor type. Cisplatin, doxorubicin and methotrexate are commonly used anticancer drugs in osteosarcoma, the most common form of childhood and adolescent cancer. Our recent study demonstrated that high mobility group box 1 protein (HMGB1)-mediated autophagy is a significant contributor to drug resistance in osteosarcoma cells. Inhibition of both HMGB1 and autophagy increase the drug sensitivity of osteosarcoma cells in vivo and in vitro. Furthermore, we demonstrated that the ULK1-FIP200 complex is required for the interaction between HMGB1 and BECN1, which then promotes BECN1-PtdIns3KC3 complex formation during autophagy. Thus, these findings provide a novel mechanism of osteosarcoma resistance to therapy facilitated by HMGB1-mediated autophagy and provide a new target for the control of drug-resistant osteosarcoma patients.     

Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma

Autophagy is a catabolic process critical to maintaining cellular homeostasis and responding to cytotoxic insult. Autophagy is recognized as “programmed cell survival” in contrast to apoptosis or programmed cell death. Upregulation of autophagy has been observed in many types of cancers and has been demonstrated to both promote and inhibit antitumor drug resistance depending to a large extent on the nature and duration of the treatment-induced metabolic stress as well as the tumor type. Cisplatin, doxorubicin and methotrexate are commonly used anticancer drugs in osteosarcoma, the most common form of childhood and adolescent cancer. Our recent study demonstrated that high mobility group box 1 protein (HMGB1)-mediated autophagy is a significant contributor to drug resistance in osteosarcoma cells. Inhibition of both HMGB1 and autophagy increase the drug sensitivity of osteosarcoma cells in vivo and in vitro. Furthermore, we demonstrated that the ULK1-FIP200 complex is required for the interaction between HMGB1 and BECN1, which then promotes BECN1-PtdIns3KC3 complex formation during autophagy. Thus, these findings provide a novel mechanism of osteosarcoma resistance to therapy facilitated by HMGB1-mediated autophagy and provide a new target for the control of drug-resistant osteosarcoma patients.     

A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin

High-mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac-1-dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1-mediated recruitment was prevented in mice deficient in the beta2-integrin Mac-1 but not in those deficient in LFA-1. As observed by bone marrow chimera experiments, Mac-1-dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of nuclear factor-kappaB in neutrophils required both Mac-1 and RAGE. Together, a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE is described here.     

The IKKα-dependent NF-κB p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.