Nicole E. Schapiro

HMGB1 Release and Redox Regulates Autophagy and Apoptosis in Cancer Cells

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.

The Receptor for Advanced Glycation End-products (RAGE) Sustains Autophagy and Limits Apoptosis, Promoting Pancreatic Tumor Cell Survival

Activation of the induced receptor for advanced glycation end products (RAGE) leads to initiation of NF-κB 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.

The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics

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.

The expression of the receptor for advanced glycation endproducts (RAGE) is permissive for early pancreatic neoplasia

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:KrasG12D/+ (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.

Ethyl pyruvate administration inhibits hepatic tumor growth

EP is a potent inhibitor of HMGB1 release that has significant anti–inflammatory activities and exerts a protective effect in animal models of inflammation. As inflammation is linked to cancer growth, we hypothesized that EP would have anti–tumor activity and explored its effects in a liver tumor model. Mice injected intraportally with MC38 colorectal cancer cells led to the growth of visible hepatic tumors within 2 weeks. Pretreatment with EP 30 min prior to infusion of tumor cells and continuing daily for 9 days inhibited tumor growth significantly in a dose–dependent manner, with 80 mg/kg EP achieving >70% reduction in the number of tumor nodules when compared with untreated animals. Delayed treatment with EP also suppressed tumor growth significantly, although to a lesser extent. Tumors had early, marked leukocytic infiltrates, and EP administration decreased innate (NK cells, monocytes) and adaptive (T and B cell lymphocytic) immune cell infiltrates acutely and significantly in the liver. Serum IL–6 and HMGB1 levels, which were elevated following tumor injection, were decreased significantly in EP–treated animals. Tumors showed an increase in apoptosis in EP–treated mice, and tumor cells treated in vitro with EP had marked increases in LC3–II and cleaved PARP, consistent with enhanced autophagic flux and apoptosis. Thus, EP inhibition of tumor growth in the liver was mediated by tumor (induction of apoptosis) and host (decreased inflammation) effects. EP administration may have a therapeutic role in the treatment of cancer in conjunction with other therapeutic agents.

The Receptor for Advanced Glycation End-Products (RAGE) Protects Pancreatic Tumor Cells Against Oxidative Injury

Reactive oxygen species, including hydrogen peroxide (H(2)O(2)), can cause toxicity and act as signaling molecules in various pathways regulating both cell survival and cell death. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated the effect of oxidative stress on activation of the Receptor for Advanced Glycation End-products (RAGE) and subsequent protection against H(2)O(2)-induced pancreatic tumor cell damage. We found that exposure of pancreatic tumor cells to H(2)O(2) provoked a nuclear factor kappa B (NF-κB)-dependent increase in RAGE expression. Further, suppression of RAGE expression by RNA interference increased the sensitivity of pancreatic tumor cells to oxidative injury. Furthermore, targeted knockdown of RAGE led to increased cell death by apoptosis and diminished cell survival by autophagy during H(2)O(2)-induced oxidative injury. Moreover, we demonstrate that RAGE is a positive feedback regulator for NF-κB as knockdown of RAGE decreased H(2)O(2)-induced activity of NF-κB. Taken together, these results suggest that RAGE is an important regulator of oxidative injury.

The receptor for advanced glycation end products (RAGE) enhances autophagy and neutrophil extracellular traps in pancreatic cancer.

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.

The Receptor for Advanced Glycation End Products Promotes Pancreatic Carcinogenesis and Accumulation of Myeloid-Derived Suppressor Cells

Pancreatic ductal adenocarcinoma (PDA) has an aggressive natural history and is resistant to therapy. The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor for many damage-associated molecular pattern molecules. RAGE is overexpressed in both human and murine models of PDA as well as most advanced epithelial neoplasms. The immunosuppressive nature of the PDA microenvironment is facilitated, in part, by the accumulation of regulatory immune cell infiltrates such as myeloid-derived suppressor cells (MDSCs). To study the role of RAGE expression in the setting of mutant Ras-promoted pancreatic carcinogenesis (KC), a triple-transgenic model of spontaneous murine PDA in a RAGE-null background (KCR) was generated. KCR mice had markedly delayed pancreatic carcinogenesis and a significant diminution of MDSCs compared with KC mice at comparable time points postweaning. Although RAGE was not required for the development or suppressor activity of MDSCs, its absence was associated with temporally limited pancreatic neoplasia and altered phenotype and function of the myeloid cells. In lieu of MDSCs, KCR animals at comparable time points exhibited mature CD11b+Gr1−F4/80+ cells that were not immunosuppressive in vitro. KCR mice also maintained a significantly less suppressive milieu evidenced by marked decreases in CCL22 in relation to CXCL10 and diminished serum levels of IL-6.

Abstract B96: The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics.

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.