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

Abstract

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.

Osteosarcomas are highly malignant bone tumors predominantly seen in childhood and adolescence with an annual incidence rate of about 5.6 per million in the USA. In young patients, it arises most often in the metaphyses of long bones such as the distal femur, and the major cause of death in osteosarcoma is metastasis to the lungs. Although the survival rate has increased 60–70% within the last ten years, drug resistance is still a major concern for the clinical management of osteosarcoma patients. To enhance osteosarcoma therapy, new therapeutic targets must be identified, and therapeutic strategies based on the most effective combinatorial approaches must be developed. Given the dual role of autophagy in cancer cells (either tumor-promoting or tumor-suppressing), molecules that modulate the pathway represent a class of potential anticancer targets. Our recent study demonstrated that the upregulation of high mobility group box 1 (HMGB1) expression during chemotherapy promotes autophagy, and subsequent drug resistance in osteosarcoma (Fig. 1). These findings improve our understanding of both the significance of autophagy in osteosarcoma therapy, and potentially indicate a new role for HMGB1 in cancer.

Figure 1.

HMGB1-mediated autophagy promotes drug resistance in osteosarcoma. Osteosarcoma is a type of bone cancer that usually occurs in childhood and adolescence. Cisplatin, doxorubicin, and methotrexate are commonly used cytotoxic anticancer drugs in the treatment of patients with osteosarcoma. These drugs increase mRNA and protein expression of HMGB1 in osteosarcoma cells by an unknown mechanism. Upregulated HMGB1 competes with BCL-2 to bind BECN1, which increases the formation of the BECN1-PtdIns3KC3 complex and stimulates autophagosome maturation and autophagy. As an upstream signal, activation of the ULK1-mATG13-FIP200 complex is required for the interaction between HMGB1 and BECN1. Knockdown of HMGB1 or inhibition of autophagy increase apoptosis, and reverses drug resistance in osteosarcoma cells in vitro and in vivo. Thus HMGB1-mediated autophagy is a potential therapeutic target for use in osteosarcoma.

Upregulation of HMGB1 in Response to Osteosarcoma Therapy

HMGB1 is a relatively small protein of 215 amino acid residues that is highly conserved across species. Structurally, HMGB1 consists of three different domains: two DNA binding domains (termed A box and B box), and a C-terminal acidic tail. In the nucleus, HMGB1 functions as a DNA chaperone protein and regulates nuclear events such as DNA replication, recombination, and repair. As a stress sensor with redox-sensitive properties, HMGB1 is actively secreted by inflammatory cells or passively released by injured/necrotic cells. In the extracellular space, HMGB1 binds receptors such as toll-like receptors (TLRs) and the receptor for advanced glycation end products (RAGE) to regulate inflammation, immunity and tissue repair. Importantly, HMGB1 has been linked to multiple cancer hallmarks across several tumor types including lymphoma, melanoma, leukemia, breast cancer, cervix, colon, liver, lung, and pancreas. We found that the alkylating agents cisplatin and anthracycline, the antibiotic doxorubicin, and the antimetabolite methotrexate significantly increase protein and mRNA expression of HMGB1 in human p53-deficient osteosarcoma cell lines (e.g., MG-63 and SaOS-2). Moreover, cycloheximide, a protein biosynthesis inhibitor, inhibits chemotherapy-induced HMGB1 protein expression, suggesting that the level of HMGB1 in osteosarcoma cells is regulated by synthesis but not degradation. The upregulation of HMGB1 in the p53 wild-type osteosarcoma cell line U-2 OS after drug treatment is still observed, suggesting that no direct relationship between p53 and HMGB1 expression in these cell lines exists. Aside from p53, the expression of HMGB1 is regulated by other transcription factors such as c-Myc, and Kruppel-like factor (KLF)-4 in various cell types. Thus, it would be interesting to explore whether these transcription factors are required for the upregulation of HMGB1 in response to chemotherapy in osteosarcoma cells.

HMGB1 Regulates Autophagy in Osteosarcoma Therapy

Autophagy is a dynamic process which facilitates the turnover of organelles and proteins, and generates metabolic precursor molecules through the lysosomal-dependent degradation of macromolecules, organelles and other cellular components. The dysregulation of autophagy has been linked to numerous human diseases and has become an important area in cancer research. Many types of cancer cells enhance autophagy following chemotherapy and radiotherapy. We and others have demonstrated that HMGB1 is a critical regulator of nonselective and selective autophagy (e.g., mitochondrial autophagy, or mitophagy). The localization-dependent role of HMGB1 in the regulation of autophagy has been reported in fibroblasts, leukemia, colon and pancreatic cancer cells. For example, nuclear HMGB1 regulates heat shock protein β-1 (HSPB1/HSP27) expression, which influences dynamic intracellular trafficking during autophagy. Cytosolic HMGB1 is a BECN1 (also known as Atg6 in yeast) binding protein which promotes the dissociation of BECN1 from BCL-2 and enhances autophagy. The binding of BCL-2 to BECN1 reduces BECN1's capacity to induce autophagy via interactions with class III phosphatidylinositol 3-kinase (PtdIns3KC3). Reducible HMGB1, but not oxidized exogenous HMGB1, induces autophagy in a RAGE-dependent manner. To explore whether HMGB1 regulates autophagy during osteosarcoma therapy, we assayed autophagy by three widely used methods: western blot analysis of proteolytic processing of endogenous microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, and the expression of SQSTM1/sequestosome 1 (also known as p62); confocal microscopy analysis of LC3 puncta formation by specific antibody stain or RFP-GFP-LC3; and transmission electron microscopy analysis of the ultrastructure of autophagosomes and autolysosomes. We found that suppression of HMGB1 expression by specific shRNA inhibits cisplatin-, doxorubicin-and methotrexate-induced heightened autophagic flux and autophagic vacuole formation. Consistent with previous studies, endogenous HMGB1 forms a complex with BECN1, and knockdown of HMGB1 influences the formation of the BECN1-PtdIns3KC3 complex. However, HMGB1 does not affect the formation of the unc-51-like kinase 1 (ULK1)-mATG13-FAK-family interacting protein of 200 kDa (FIP200) complex, which mediates vesicle nucleation during autophagy. In contrast, knockdown of ULK1 or FIP200 inhibits the interaction between HMGB1 and BECN1, and increases sensitivity to anticancer agent-induced apoptosis. These studies suggest that HMGB1 is a downstream signal from ULK1-mATG13-FIP200 complex formation, and induces autophagy in osteosarcoma cells by interacting with BECN1.

HMGB1-Mediated Autophagy as a Novel Target in Osteosarcoma Therapy

Other studies have demonstrated that HMGB1 modulates the efficacy of other anticancer agents (e.g., cytosine arabinoside, arsenic trioxide, vincristine, melphalan, paclitaxel and gemcitabine) in different tumor models (e.g., leukemia, colon cancer, and pancreatic cancer). We focus on cisplatin, doxorubicin and methotrexate because these drugs are commonly used in osteosarcoma. Suppression of HMGB1 by shRNA decreases autophagy and increases sensitivity to these anticancer agents in vitro in osteosarcoma cells, whereas overexpression of HMGB1 by cDNA transfection increases autophagy and resistance to chemotherapy in vitro. Rapamycin induces autophagy by inhibiting the mammalian target of rapamycin (mTOR). We found that rapamycin pretreatment protects against doxorubicin-induced apoptosis in HMGB1 wild-type cells. However, rapamycin confers less protection in HMGB1 knockdown cells due to diminished autophagic capacity. These findings confirmed that HMGB1 is an important regulator of autophagy-mediated cell survival. Using a xenograft model in which MG-63 cells were transplanted into NOD/SCID mice, we demonstrated that suppression of HMGB1 by shRNA increases sensitivity to doxorubicin, which accompanies decreased autophagy and increased apoptosis. Given that our long-term goal is to improve the outcome of cancer chemotherapy by developing a novel strategy to target HMGB1-mediated resistance in osteosarcoma patients, future directions would include correlating HMGB1 expression levels with chemoresistance and treatment outcomes in human patients diagnosed with osteosarcoma.

Punctum to: Huang J, Ni J, Liu K, Yu Y, Xie M, Kang R, et al. HMGB1 promotes drug resistance in osteosarcoma. Cancer Res. 2011;72:230–238. doi: 10.1158/0008-5472.CAN-11-2001.