The functional relationship and cross-regulation between autophagy and apoptosis is complex. Here we show that high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anti-cancer agents enhanced autophagy and apoptosis as well as HMGB1 release. HMGB1 release may be a pro-survival signal for residual cells following various cytotoxic cancer treatments. Diminished HMGB1 by shRNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led to predominantly apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGE) but not Toll-like receptor 4 (TLR4), induces Beclin1-dependent autophagy, and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA-intercalators (oxaliplatin or adriamycin). Oxidized HMGB1 conversely increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release as well as its redox state thus link autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.

Activation of the induced receptor for advanced glycation endproducts (RAGE) leads to initiation of NF-κB and MAP kinase signaling pathways resulting in propagation and perpetuation of inflammation. RAGE knock out animals are less susceptible to acute inflammation and carcinogen induced tumor development. We have reported that most forms of tumor cell death result in release of the RAGE ligand, HMGB1. We now report a novel role for RAGE in the tumor cell response to stress. Targeted knockdown of RAGE in the tumor cell, leads to increased apoptosis, diminished autophagy and decreased tumor cell survival . In contrast, overexpression of RAGE is associated with enhanced autophagy, diminished apoptosis and greater tumor cell viability. RAGE limits apoptosis through a p53 dependent mitochondrial pathway. Moreover, RAGE-sustained autophagy is associated with decreased phosphorylation of mTOR and increased Beclin-1/VPS34 autophagosome formation. These findings demonstrate that the inflammatory receptor RAGE plays a heretofore unrecognized role in the tumor cell response to stress. Furthermore, these studies establish a direct link between inflammatory mediators in the tumor microenvironment and resistance to programmed cell death. Our data suggest that targeted inhibition of RAGE or its ligands may serve as novel targets to enhance current cancer therapies.

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1–RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression.

Neutrophil extracellular traps (NETs) are formed when neutrophils expel their DNA, histones and intracellular proteins into the extracellular space or circulation. NET formation is dependent on autophagy and is mediated by citrullination of histones to allow for unwinding and subsequent expulsion of DNA. NETs play an important role in the pathogenesis of several sterile inflammatory diseases, including malignancy, therefore we investigated the role of NETs in the setting of pancreatic ductal adenocarcinoma (PDA). Neutrophils isolated from two distinct animal models of PDA had an increased propensity to form NETs following stimulation with platelet activating factor (PAF). Serum DNA, a marker of circulating NET formation, was elevated in tumor bearing animals as well as in patients with PDA. Citrullinated histone H3 expression, a marker of NET formation, was observed in pancreatic tumors obtained from murine models and patients with PDA. Inhibition of autophagy with chloroquine or genetic ablation of RAGE resulted in decreased propensity for NET formation, decreased serum DNA, and decreased citrullinated histone H3 expression in the pancreatic tumor microenvironment. We conclude that NETs are upregulated in pancreatic cancer through RAGE dependent/autophagy pathways.

Pancreatic cancer is an almost uniformly lethal disease, characterized by late diagnosis, early metastasis, resistance to chemotherapy, and early mutation of the Kras oncogene. Here we show that the receptor for advanced glycation endproducts (RAGE) is required for the activation of interleukin 6 (IL-6)-mediated mitochondrial signal transducers and activators of transcription 3 (STAT3) signaling in pancreatic carcinogenesis. RAGE expression correlates with elevated levels of autophagy in pancreatic cancer in vivo and in vitro, and this heightened state of autophagy is required for IL-6-induced STAT3 activation. To further explore the intersection of RAGE, autophagy, and pancreatic carcinogenesis, we created a transgenic murine model, backcrossing RAGE-null mice to a spontaneous mouse model of pancreatic cancer, Pdx1-Cre:Kras G12D/+ (KC). Targeted ablation of Rage in KC mice delayed neoplasia development, decreased levels of autophagy, and inhibited mitochondrial STAT3 activity and subsequent ATP production. Our results suggest a critical role for RAGE expression in the earliest stages of pancreatic carcinogenesis, potentially acting as the "autophagic switch," regulating mitochondrial STAT3 signaling.oncogenesis | bioenergetics | inflammation | metabolism | high-mobility group box 1 P ancreatic cancer ranks as the fourth leading cause of cancer death, accounting for 6-7% of all cancer-related deaths in the United States, in 2011 (1). Most pancreatic ductal adenocarcinomas (PDA) are thought to arise from well-defined precursor lesions, termed pancreatic ductal intraepithelial neoplasia (PanIN) (2). Many human PanIN lesions do not progress to invasive carcinomas, so defining the events that drive carcinogenesis in the emergent tumor microenvironment is of critical importance. Studies into human pancreatic carcinogenesis have been greatly facilitated by the development of a genetically engineered mouse model that expresses oncogenic Kras under a pancreatic promoter Pdx1-Cre:Kras G12D/+ (KC) (3). A more detailed understanding of how these pathways accelerate pancreatic carcinogenesis may allow improved therapeutic strategies.The receptor for advanced glycation endproducts (RAGE) is a member of the Ig superfamily. RAGE and its ligands, including high-mobility group box 1 (HMGB1) and S100, are linked to the development and progression of several cancers by facilitating the maintenance of a chronic inflammatory state (4) and/or by promotion of metastases (5). We previously observed that RAGE sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival during chemotherapy and oxidative stress in vivo and in vitro (6, 7). Autophagy is an essential catabolic process by which cells break down old or damaged organelles and proteins (8). Autophagy promotes cell survival and supports metabolism during cell stress (9). Conversely, apoptosis promotes tumor growth early in the development of cancer (10) during periods of inhibition of autophagy with a subsequent switch to suppressed apoptosis, acquired later...