Regulation of HMGB1 release by autophagy

The characteristics of tumor cell killing by an anti-cancer agent can determine the long-term effectiveness of the treatment. For example, if dying tumor cells release the immune modulator HMGB1 after treatment with anti-cancer drugs, they can activate a tumor-specific immune response that boosts the effectiveness of the initial treatment. Recent work from our group examined the mechanism of action of a targeted toxin called DT-EGF that selectively kills Epidermal Growth Factor Receptor-expressing tumor cells. We found that DT-EGF kills glioblastoma cells by a caspase-independent mechanism that involves high levels of autophagy, which inhibits cell death by blocking apoptosis. In contrast, DT-EGF kills epithelial tumor cells by caspase-dependent apoptosis and in these cells autophagy is not induced. These differences allowed us to discover that the different death mechanisms were associated with differences in the release of HMGB1 and that autophagy induction is required and sufficient to cause release of HMGB1 from the dying cells. These data identify a new function for autophagy during cell death and open up the possibility of manipulating autophagy during cancer treatment as a way to influence the immunogenicity of dying tumor cells.     

Immunogenic cell death modalities and their impact on cancer treatment

It is still enigmatic under which circumstances cellular demise induces an immune response or rather remains immunologically silent. Moreover, the question remains open under which circumstances apoptotic, autophagic or necrotic cells are immunogenic or tolerogenic. Although apoptosis appears to be morphologically homogenous, recent evidence suggests that the pre-apoptotic surface-exposure of calreticulin may dictate the immune response to tumor cells that succumb to anticancer treatments. Moreover, the release of high-mobility group box 1 (HMGB1) during late apoptosis and secondary necrosis contributes to efficient antigen presentation and cytotoxic T-cell activation because HMGB1 can bind to Toll like receptor 4 on dendritic cells, thereby stimulating optimal antigen processing. Cell death accompanied by autophagy also may facilitate cross priming events. Apoptosis, necrosis and autophagy are closely intertwined processes. Often, cells manifest autophagy before they undergo apoptosis or necrosis, and apoptosis is generally followed by secondary necrosis. Whereas apoptosis and necrosis irreversibly lead to cell death, autophagy can clear cells from stress factors and thus facilitate cellular survival. We surmise that the response to cellular stress like chemotherapy or ionizing irradiation, dictates the immunological response to dying cells and that this immune response in turn determines the clinical outcome of anticancer therapies. The purpose of this review is to summarize recent insights into the immunogenicity of dying tumor cells as a function of the cell death modality.     

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     

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.     

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.     

Blocking the interleukin 2 (IL2)-induced systemic autophagic syndrome promotes profound antitumor effects and limits toxicity

Cancer is the leading cause of death in the United States in those dying under the age of 85. Although cancer is increasingly controlled as a chronic disease, true cures of patients with metastatic epithelial malignancies have rarely been obtained with currently available systemic therapies. For example, administration of high-dose recombinant interleukin 2 (IL2), enhancing cytolytic immune cell proliferation and delivery, promotes complete antitumor responses in < 10% of treated individuals. Means to reduce the toxicity, attributed to a cytokine storm and an associated “systemic autophagic syndrome” as well as enhance efficacy and increase the potential set of malignancies in which it is applied (currently patients with renal cancer and melanoma) would be of great interest. IL2 promotes both T-cell and NK cell induction of immune cell-mediated autophagy (iC-MA) in tumor targets. We have demonstrated that HMGB1 is detected at high levels in the serum of IL2-treated mice with translocation to the cytoplasm from the nucleus in the liver, consistent with HMGB1’s release in response to stress, and ability to sustain autophagy. Limiting autophagy in mice with coadministration of chloroquine (CQ) diminishes serum levels of HMGB1, cytokines (IFNG and IL6 but not IL18), and autophagic flux, attenuating weight gain, enhancing DC, T-cell and NK cell numbers, and promoting long-term tumor control in a murine hepatic metastases model. Autophagy (programmed cell survival) is a metabolic process associated with promotion of late cancer growth. In tumor cell lines, CQ treatment limits ATP production through inhibition of oxidative phosphorylation and promotion of apoptosis. CQ increases autophagic vacuoles and LC3-II levels in tumor cells, associated with increased annexin V⁺/PI^- cells, cleaved-PARP, cleaved-CASP3, and cytochrome c release from mitochondria. These observations, limiting toxicity and prolonging antitumor effects, with a combination of IL2 and autophagy inhibition in murine models are now being tested by the Cytokine Working Group in patients with advanced renal cell carcinoma.     

HMGB1 is a bone-active cytokine

High mobility group box 1 (HMGB1) is a chromatin protein that acts as an immunomodulatory cytokine upon active release from myeloid cells. HMGB1 is also an alarmin, an endogenous molecule released by dying cells that acts to initiate tissue repair. We have previously reported that osteoclasts and osteoblasts release HMGB1 and release by the latter is regulated by parathyroid hormone (PTH), an agent of bone remodeling. A recent study suggests that HMGB1 acts as a chemotactic agent to osteoclasts and osteoblasts during endochondral ossification. To explore the potential impact of HMGB1 in the bone microenvironment and its mechanism of release by osseous cells, we characterized the effects of recombinant protein (rHMGB1) on multiple murine bone cell preparations that together exhibit the various cell phenotypes present in bone. We also inquired whether apoptotic bone cells release HMGB1. rHMGB1 enhanced the RANKL/OPG steady state mRNA ratio and dramatically augmented the release of tumor necrosis factor-alpha (TNFalpha) and interleukin-6 (IL6) in osteoblastogenic bone marrow stromal cell (BMSC) cultures but not in the calvarial-derived MC3T3-E1 cells. Interestingly, rHMGB1 promoted GSK-3beta phosphorylation in MC3T3-E1 cells but not in BMSCs. Apoptotic bone cells released HMGB1, including MLO-Y4 osteocyte-like cells. MLO-Y4 release of HMGB1 was coincident with caspase-3 cleavage. Furthermore, the anti-apoptotic action of PTH on MC3T3-E1 cells correlated with the observed decrease in HMGB1 release. Our data suggest that apoptotic bone cells release HMGB1, that within the marrow HMGB1 is a bone resorption signal, and that intramembraneous and endochondral osteoblasts exhibit differential responses to this cytokine.     

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.     

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.     

Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy with TLR4 agonists

Immunogenic cell death induced by anticancer chemotherapy is characterized by a series of molecular hallmarks that include the exodus of high-mobility group box 1 protein (HMGB1) from dying cells. HMGB1 is a nuclear nonhistone chromatin-binding protein. It is secreted at the late stages of cellular demise and engages Toll-like receptor4 (TLR4) on dendritic cells (DCs) to accelerate the processing of phagocytic cargo in the DC and to facilitate antigen presentation by DC to T cells. The absence of HMGB1 expression by dying tumor cells exposed to anthracyclines or oxaliplatin compromises DC-dependent T-cell priming by tumor-associated antigens. Here, we show that transplantable tumors exhibiting weak expression of nuclear HMGB1 respond to chemotherapy more effectively if the treatment is combined with the local or systemic administration of a highly purified and physiochemically defined and standardized lipopolysaccharide solution, which acts as a high-potency and exclusive TLR4 agonist, called Dendrophilin (DEN). The synergistic antitumor effects mediated by the combination of chemotherapy and immunotherapy relied upon the presence of the MyD88 (myeloid differentiation primary response gene) adapter of TLR4 (but not that of the TIR-domain-containing adapter-inducing interferon-β adapter), in line with the well-characterized action of DEN on the MyD88 signaling pathway. DEN and anthracyclines synergized to induce intratumoral accumulation of interferon-γ-producing CD4⁺ and CD8⁺ T lymphocytes. Moreover, DEN could restore the immunogenicity of dying tumor cells from which HMGB1 had been depleted by RNA interference. These findings underscore the potential clinical utility of combination regimens involving immunogenic chemotherapy and certain TLR4 agonists in advanced HMGB1-deficient cancers.     

Chromatin and cell death

HMGB1, a very mobile chromatin protein, leaks out from necrotic cells and signals to neighbouring cells that tissue damage has occurred. At least one receptor for extracellular HMGB1 exists, and signals to different cells to divide, migrate, activate inflammation or start an immune response. Remarkably, apoptotic chromatin binds HMGB1 irreversibly, thereby ensuring that it will not diffuse away to activate responses from neighbouring cells. Thus, dying cells use their own chromatin to signal how they have died. We argue that the nuclear events in apoptosis serve to control the molecular signals that dying cells send out.     

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.     

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.     

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.     

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.     

Cigarette smoke‐induced HMGB1 translocation and release contribute to migration and NF‐κB activation through inducing autophagy in lung macrophages

High‐mobility group box 1 (HMGB1) shows pro‐inflammatory activity in various inflammatory diseases and has been found up‐regulated in chronic obstructive pulmonary disease (COPD). Lung macrophages play an important role in airway inflammation and lung destruction in COPD, yet whether HMGB1 is involved in cigarette smoke (CS)‐induced lung macrophage dysfunction is unknown. We sought to evaluate the intracellular localization and release of HMGB1 in lung macrophages from COPD patients and CS‐exposed mice, and to investigate the role of HMGB1 in regulating autophagy in CS extract (CSE)‐treated lung macrophages (MH‐S cells). Our results showed that HMGB1 was highly expressed in lung tissues and sera of COPD patients and CS‐exposed mice, along with predominantly cytoplasmic exporting from nuclei in lung macrophages. In vitro experiments revealed that CSE promoted the expression, nucleocytoplasmic translocation and release of HMGB1 partly via the nicotinic acetylcholine receptor (nAChR). Blockade of HMGB1 with chicken anti‐HMGB1 polyclonal antibody (anti‐HMGB1) or glycyrrhizin (Gly) attenuated the increase of LC3B‐II and Beclin1, migration and p65 phosphorylation, suggesting the involvement of HMGB1 in autophagy, migration and NF‐κB activation of lung macrophages. Hydroxychloroquine (CQ), an autophagy inhibitor, enhanced the increase of LC3B‐II but not Beclin1 in CSE or rHMGB1‐treated MH‐S cells, and inhibition of autophagy by CQ and 3‐methyladenine (3‐MA) abrogated the migration and p65 phosphorylation of CSE‐treated cells. These results indicate that CS‐induced HMGB1 translocation and release contribute to migration and NF‐κB activation through inducing autophagy in lung macrophages, providing novel evidence for HMGB1 as a potential target of intervention in COPD.     

Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.     

Strange attractors: DAMPs and autophagy link tumor cell death and immunity

Resistance to ‘apoptotic'' cell death is one of the major hallmarks of cancer, contributing to tumor development and therapeutic resistance. Damage-associated molecular patterns (DAMPs) are molecules released or exposed by dead, dying, injured, or stressed non-apoptotic cells, with multiple roles in inflammation and immunity. Release of DAMPs not only contributes to tumor growth and progression but also mediates skewing of antitumor immunity during so-called immunogenic tumor cell death (ICD). Autophagy is a lysosome-mediated homeostatic degradation process in which cells digest their own effete organelles and macromolecules to meet bioenergetic needs and enable protein synthesis. For tumor cells, autophagy is a double-edged sword. Autophagy, in balance with apoptosis, can function as a tumor suppressor; autophagy deficiency, associated with alterations in apoptosis, initiates tumorigenesis in many settings. In contrast, autophagy-related stress tolerance generally promotes cell survival, which enables tumor growth and promotes therapeutic resistance. Most anticancer therapies promote DAMP release and enhance autophagy. Autophagy not only regulates DAMP release and degradation, but also is triggered and regulated by DAMPs. This interplay between autophagy and DAMPs, serving as ‘strange attractors'' in the dynamic system that emerges in cancer, regulates the effectiveness of antitumor treatment. This interplay also shapes the immune response to dying cells upon ICD, culling the least fit tumor cells and promoting survival of others. Thus, DAMPs and autophagy are suitable emergent targets for cancer therapy, considering their more nuanced role in tumor progression.