Activation of the induced receptor for advanced glycation end products (RAGE) leads to initiation of NF- κ B and MAP kinase signaling pathways, resulting in propagation and perpetuation of inflammation. RAGE-knockout 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, high-mobility group protein 1 (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 mammalian target of rapamycin (mTOR) and increased Beclin-1/VPS34 autophagosome formation. These findings show that the inflammatory receptor, RAGE, has 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.

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 the unwinding and subsequent expulsion of DNA. NETs have 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 receptor for advanced glycation end products (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 mediated pathways.

Neutrophil extracellular traps (NETs) are generated when activated neutrophils release their intracellular contents containing DNA, histones and granule constituents into the tissue or circulation. While traditionally associated with sepsis, NETs also play a role in sterile inflammation and thrombosis. Recently, the process of autophagy has been implicated in NET formation. We previously reported that pancreatic ductal adenocarcinoma (PDA) is associated with increased autophagy in the tumor microenvironment and at distant organ sites. We examined NETs and autophagy in murine models and human PDA, as well as their potential role in hypercoagulability.

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.

Background and Aims: Recently altered cellular bioenergetics and inflammation have been added to the list of essential hallmarks of cancer. The precise mechanisms regulating these processes are not well characterized. We previously observed that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), promote early pancreatic carcinogenesis through an IL-6/STAT3-dependent pathway. Based on previous data suggesting a role for IL-6/STAT3 in mitochondrial function, we hypothesized that the RAGE/HMGB1 axis would play a role pancreatic tumor cell mitochondrial bioenergetics. Methods: The effects of HMGB1/RAGE on ATP generation were studied in cultured human and murine pancreatic tumor cell lines in vitro utilizing targeted knockdown with shRNA and site specific mutagenesis. Observations were confirmed in vivo utilizing orthotopic transplantation models of pancreatic cancer derived from RAGE wild-type or knockdown cell lines. Results: We found RAGE in the mitochondria of cultured tumor cells, as well as primary human tumor explants. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. There is a direct interaction between RAGE and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). The mitochondrial localization signal Ser377 of RAGE is phosphorylated by ERK1/2, which is required for HMGB1-mediated RAGE activation within the mitochondria. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. Conclusions: These findings link the HMGB1-RAGE pathway with changes in cellular bioenergetics. Moreover, our observations provide a novel mechanism by which necrosis and inflammation, within the tumor microenvironment, can promote tumor progression. Note: This abstract was not presented at the conference. Citation Format: Rui Kang, Daolin Tang, Nicole E. Schapiro, Michael T. Lotze, Herbert J. Zeh. The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B96.

Introduction: It is becoming increasingly clear that tumor cells utilize autophagy as a survival response when exposed to cytotoxic chemotherapy. We have previously demonstrated that targeted knockdown of the Receptor for Advanced Glycation End-products (RAGE) inhibits autophagy and increases susceptibility of cancer cells to chemotherapeutic toxicity. Chloroquine, a clinically available and relatively nontoxic drug currently used to treat rheumatologic disorders and malaria, is able to modulate autophagy by impeding fusion of the autophagosome with the lysosome, thus preventing an increase in autophagic flux. We hypothesized that inhibition of autophagy by chloroquine would increase the susceptibility of cancer cells to cytotoxic chemotherapy in an in vivo orthotopic model of mouse pancreatic cancer. Methods: C57/BL6 wild type (WT) mice underwent laparotomy and injection in the lesser sac with one million cells of the panc02 murine pancreatic cancer cell line transfected with a luciferase reporter gene. Luciferase readings were followed on a bi-weekly basis to assess tumor burden. On day seven after injection, mice (4-5/group) were treated with chloroquine (CQ) 25 mg/kg or PBS daily. Beginning day 9, oxaliplatin 5 mg/kg and gemcytabine 15 mg/kg (chemo) or PBS were given in two back to back doses each week for three weeks. Survival was defined as number of days between injection and either death of the animal or sacrifice of the animal upon meeting objective criteria using a body condition score. Survival is reported as mean number of days +/- standard deviation. One-tailed Student's t-test was used to compare survival data, with p<0.05 considered significant. Results: Mean survival in control mice was 34 +/- 4 days. Treatment with chemo or CQ alone resulted in a statistically nonsignificant trend towards increased survival in WT mice (48 +/- 11 days and 58 +/- 18 days respectively). Combined treatment with chemo and CQ in the WT resulted in statistically significant doubling of survival time (62 +/- 21 days, p<0.05). Conclusions: Autophagy in cancer cells may allow them to better proliferate in a hostile microenvironment. Inhibiting autophagy through use of chloroquine may decrease survival of cancer cells in response to chemotherapy and improve clinical cancer treatment outcomes.

The functional relationship and cross-regulation between autophagy and apoptosis is complex. In this study we show that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anticancer agents enhanced autophagy and apoptosis, as well as HMGB1 release. HMGB1 release may be a prosurvival signal for residual cells after various cytotoxic cancer treatments. Diminished HMGB1 by short hairpin RNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led predominantly to apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGEs), but not to Toll-like receptor 4, 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). On the contrary, oxidized HMGB1 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 links autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.