Philip Vernon

HMGB1 Promotes Drug Resistance in Osteosarcoma

Osteosarcoma is the most commonly occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. In this study, we implicate the DNA-binding protein HMGB1, which also exerts immunoregulatory effects in its secreted form, in the development of drug resistance in osteosarcoma. Anticancer agents including doxorubicin, cisplatin, and methotrexate each induced HMGB1 upregulation in human osteosarcoma cells, and RNA interference-mediated knockdown of HMGB1 restored the chemosensitivity of osteosarcoma cells in vivo and in vitro. Mechanistic investigation revealed that HMGB1 increased drug resistance by inducing autophagy, an intracellular self-defense mechanism known to confer drug resistance. We found that HMGB1 bound to the autophagy regulator Beclin1 and regulated the formation of the Beclin1-PI3KC3 [PI3KC3, phosphatidylinositol 3-kinase class 3] complex that facilitates autophagic progression. In addition, we found that interaction between HMGB1 and Beclin1 relied upon the autophagic complex ULK1-mAtg13-FIP200. Therefore, through its role as a regulator of autophagy, HMGB1 is a critical factor in the development of chemoresistance, and it offers a novel target for improving osteosarcoma therapy.

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.

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.

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.

BID mediates selective killing of APC-deficient cells in intestinal tumor suppression by nonsteroidal antiinflammatory drugs

Significance Colorectal cancer is the third leading cause of cancer-related death in the United States, but treatment options for this disease are of limited effectiveness. Most human colorectal tumors begin with an inactivating mutation in the adenomatous polyposis coli (APC) gene. We demonstrate a mechanism by which nonsteroidal antiinflammatory drugs (NSAIDs) protect against colon cancer development by killing intestinal stem cells that have lost functional APC. NSAID treatment combines with APC loss-induced gene expression changes to selectively activate BID and induce apoptosis in these cells, while leaving normal cells unharmed. These results provide a rationale for developing more effective cancer prevention strategies and agents. Colorectal tumorigenesis is driven by genetic alterations in the adenomatous polyposis coli (APC) tumor suppressor pathway and effectively inhibited by nonsteroidal antiinflammatory drugs (NSAIDs). However, how NSAIDs prevent colorectal tumorigenesis has remained obscure. We found that the extrinsic apoptotic pathway and the BH3 interacting-domain death agonist (BID) are activated in adenomas from NSAID-treated patients. Loss of BID abolishes NSAID-mediated tumor suppression, survival benefit, and apoptosis in tumor-initiating stem cells in APCMin/+ mice. BID-mediated cross-talk between the extrinsic and intrinsic apoptotic pathways is responsible for selective killing of neoplastic cells by NSAIDs. We further demonstrate that NSAIDs induce death receptor signaling in both cancer and normal cells, but only activate BID in cells with APC deficiency and ensuing c-Myc activation. Our results suggest that NSAIDs suppress intestinal tumorigenesis through BID-mediated synthetic lethality triggered by death receptor signaling and gatekeeper mutations, and provide a rationale for developing more effective cancer prevention strategies and agents.

The myeloid response to pancreatic carcinogenesis is regulated by the receptor for advanced glycation end-products

We identified a critical role for receptor for advanced glycation end products (RAGE) in the intratumoral accumulation of myeloid-derived suppressor cells (MDSCs) during pancreatic carcinogenesis. The absence of RAGE markedly delayed neoplasia and limited MDSC accumulation in mice expressing an oncogenic variant of Kras. In spite of MDSCs, these mice accumulated non-immunosuppressive macrophages. Thus, RAGE regulates carcinogenesis and consequent myeloid responses.

Abstract B08: HMGB1 regulates natural killer (NK) cell metabolism and cross-talk with dendritic cells (DCs)

HMGB1 is an evolutionarily ancient, highly abundant and conserved chromatin-associated protein which regulates transcription factor access to DNA in the nucleus. Within the cytosol, it promotes autophagy, and when extracellular promotes chemotaxis and induces inflammation. Under conditions of cellular stress, it translocates to the cytosol where it enhances autophagy and can be actively or passively secreted and serve as a damage associated molecular pattern (DAMP) molecule. HMGB1 is a critical regulator of metabolism in murine embryonic fibroblasts and tumor cells lacking the receptor for advanced glycation endproducts (RAGE) by modulation of autophagy and promotion of mitochondrial quality control. To determine if HMGB1 also plays a role in NK and DC metabolic regulation, NKp46-Cre and CD11c-Cre mice were crossed with HMGB1 flox/flox mice to generate NK- or DC-specific HMGB1-KO mice (NKH and DCH respectively). NK and DC derived from such mice were evaluated for metabolic flux using real time assessment of oxidative phosphorylation (OCR) and glycolysis (ECR) at basal levels and following sequential addition of oligomycin, FCCP, 2-deoxyglucose, and rotenone. DCH demonstrated depressed basal and CD40L-stimulated OCR. NKH cells similarly were significantly less metabolically active in response to IL-2 stimulation. Since HMGB1 secreted from NK cells play a critical role in DC maturation, wildtype DCs were briefly co-cultured with NK cells, markedly increasing their metabolic activity. This was substantially less in DCs cocultured with NKH. These findings suggest that NK HMGB1 enhances DC metabolic activity, promoting NK:DC crosstalk. References. 1: Hou W, Zhang Q, Yan Z, Chen R, Zeh Iii HJ, Kang R, Lotze MT, Tang D. Strange attractors: DAMPs and autophagy link tumor cell death and immunity. Cell Death Dis. 2013 Dec 12;4:e966. 2: Rubartelli A, Lotze MT, Latz E, Manfredi A. Mechanisms of sterile inflammation. Front Immunol. 2013 Nov 22;4:398. doi: 10.3389/fimmu.2013.00398. 3: Li G, Tang D, Lotze MT. Menage a Trois in stress: DAMPs, redox and autophagy. Semin Cancer Biol. 2013 Oct;23(5):380-90. 4: Kang R, Tang D, Schapiro NE, Loux T, Livesey KM, Billiar TR, Wang H, Van Houten B, Lotze MT, Zeh HJ. The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics. Oncogene. 2013 Jan 14. doi: 10.1038/onc.2012.631 5: Tang D, Billiar TR, Lotze MT. A Janus tale of two active high mobility group box 1 (HMGB1) redox states. Mol Med. 2012 Dec 20;18:1360-2. 6: Tang D, Kang R, Coyne CB, Zeh HJ, Lotze MT. PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol Rev. 2012 Sep;249(1):158-75. 7: Kang R, Loux T, Tang D, Schapiro NE, Vernon P, Livesey KM, Krasinskas A, Lotze MT, Zeh HJ 3rd. The expression of the receptor for advanced glycation endproducts (RAGE) is permissive for early pancreatic neoplasia. Proc Natl Acad Sci U S A. 2012 May 1;109(18):7031-6. doi: 10.1073/pnas.1113865109 8: Buchser WJ, Laskow TC, Pavlik PJ, Lin HM, Lotze MT. Cell-mediated autophagy promotes cancer cell survival. Cancer Res. 2012 Jun 15;72(12):2970-9. doi: 10.1158/0008-5472.CAN-11-3396. 9: Liang X, De Vera ME, Buchser WJ, Romo de Vivar Chavez A, Loughran P, Beer Stolz D, Basse P, Wang T, Van Houten B, Zeh HJ 3rd, Lotze MT. Inhibiting systemic autophagy during interleukin 2 immunotherapy promotes long-term tumor regression. Cancer Res. 2012 Jun 1;72(11):2791-801. 10: Weiner LM, Lotze MT. Tumor-cell death, autophagy, and immunity. N Engl J Med. 2012 Mar 22;366(12):1156-8. 11: Livesey KM, Kang R, Vernon P, Buchser W, Loughran P, Watkins SC, Zhang L, Manfredi JJ, Zeh HJ 3rd, Li L, Lotze MT, Tang D. p53/HMGB1 complexes regulate autophagy and apoptosis. Cancer Res. 2012 Apr 15;72(8):1996-2005. 12: Kang R, Livesey KM, Zeh HJ 3rd, Lotze MT, Tang D. Metabolic regulation by HMGB1-mediated autophagy and mitophagy. Autophagy. 2011 Oct;7(10):1256-8. 13: Tang D, Kang R, Livesey KM, Kroemer G, Billiar TR, Van Houten B, Zeh HJ 3rd, Lotze MT. High-mobility group box 1 is essential for mitochondrial quality control. Cell Metab. 2011 Jun 8;13(6):701-11. Citation Format: Guanqiao Li, Philip J. Vernon, Bennett van Houten, Xiaoyan Liang, Michael T. Lotze. HMGB1 regulates natural killer (NK) cell metabolism and cross-talk with dendritic cells (DCs). [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B08. doi:10.1158/1538-7445.CHTME14-B08

Depletion of high mobility group box 1(HMGB1) in dendritic cells (DCs) suppresses tumorigenesis and promotes viral clearance

HMGB1, an evolutionarily ancient and abundant DNA-binding protein within the nucleus, acts as a Damage Associated Molecular Pattern (DAMP) molecule extracellularly to promote immunity. During both cellular stress and immune cell activation, it is translocated into the cytosol and enhances autophagic flux. DCs are critical to both initiating and maintaining T cell adaptive immunity in human cancers. As such, they have formed the basis of many anti-tumor immunotherapies aimed at directing responses to tumor-associated antigens. Vaccinia virus, a large, enveloped virus with dsDNA genome, can infect DCs and inhibit DC maturation. HMGB1 is required for DC functionality and chemotaxis but how HMGB1 regulates DC function in anti-tumor immune response and viral clearance remains unclear.