Intracellular HMGB1 as a novel tumor suppressor of pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) driven by oncogenic K-Ras remains among the most lethal human cancers despite recent advances in modern medicine. The pathogenesis of PDAC is partly attributable to intrinsic chromosome instability and extrinsic inflammation activation. However, the molecular link between these two events in pancreatic tumorigenesis has not yet been fully established. Here, we show that intracellular high mobility group box 1 (HMGB1) remarkably suppresses oncogenic K-Ras-driven pancreatic tumorigenesis by inhibiting chromosome instability-mediated pro-inflammatory nucleosome release. Conditional genetic ablation of either single or both alleles of HMGB1 in the pancreas renders mice extremely sensitive to oncogenic K-Ras-driven initiation of precursor lesions at birth, including pancreatic intraepithelial neoplasms, intraductal papillary mucinous neoplasms, and mucinous cystic neoplasms. Loss of HMGB1 in the pancreas is associated with oxidative DNA damage and chromosomal instability characterized by chromosome rearrangements and telomere abnormalities. These lead to inflammatory nucleosome release and propagate K-Ras-driven pancreatic tumorigenesis. Extracellular nucleosomes promote interleukin 6 (IL-6) secretion by infiltrating macrophages/neutrophils and enhance oncogenic K-Ras signaling activation in pancreatic lesions. Neutralizing antibodies to IL-6 or histone H3 or knockout of the receptor for advanced glycation end products all limit K-Ras signaling activation, prevent cancer development and metastasis/invasion, and prolong animal survival in Pdx1-Cre;K-Ras^G12D/+;Hmgb1^-/- mice. Pharmacological inhibition of HMGB1 loss by glycyrrhizin limits oncogenic K-Ras-driven tumorigenesis in mice under inflammatory conditions. Diminished nuclear and total cellular expression of HMGB1 in PDAC patients correlates with poor overall survival, supporting intracellular HMGB1 as a novel tumor suppressor with prognostic and therapeutic relevance in PDAC.

Figure 1

HMGB1 depletion accelerates initiation and progression of K-Ras-driven PDAC. (A) Kaplan-Meier survival analysis was performed in wild type (WT), CH (Pdx1-Cre;Hmgb1^−/−), KC (Pdx1-Cre;K-Ras^G12D/+;Hmgb1^+/+), KCH (Pdx1-Cre;K-Ras^G12D/+;Hmgb1^−/−), and KCH^+/− (Pdx1-Cre;K-Ras^G12D/+;Hmgb1^+/−) mice (^**P < 0.01, ^***P < 0.001, log-rank test). (B) Incidence of pancreatic lesions, including pancreatic intraepithelial neoplasms (PanINs), intraductal papillary mucinous neoplasms (IPMNs), and mucinous cystic neoplasms (MCNs) in KC, KCH, and KCH^+/− mice at indicated ages (n = 5 mice/genotype /age). (C) Percentages of ductal structures exhibiting normal morphology and indicated neoplastic ducts in KC, CH, KCH, and KCH^+/− mice (n = 5 mice/genotype /age). (D) Representative histologic progression of pancreata in KC, CH, KCH, and KCH^+/− mice at indicated ages shown by hematoxylin and eosin (H&E) staining (high resolution images shown in Supplementary information, Figure S2). (E) Percentages of intact acinar structures in pancreata from KC, CH, KCH, and KCH^+/− mice at six weeks of age (n = 5 mice/genotype). (F) Incidence of PDAC in pancreata from KC, KCH, and KCH^+/− mice (n = 5 mice/genotype/age). Representative samples H&E-stained for PDAC with stromal structure shown in right panel. (G) Incidence of tumor metastasis/invasion in KC, KCH, and KCH^+/− mice at six to 24 weeks of age. Representative samples H&E-stained for tumor metastasis/invasion to the liver, lung, and kidney shown in upper panels. (H) Western blot analysis of HMGB1 expression in isolated tumor (T) or normal (N) tissue from KCH mice. (I) Cell proliferation analysis of indicated isolated tumor cells from KCH mice (n = 3, ^**P < 0.01, unpaired t-test).

Figure 2

Oxidative DNA damage promotes pancreatic tumorigenesis in KCH mice. (A) Percentage of abnormal telomeres in ductal cells from KC and KCH mice at six weeks of age (n = 5 mice/genotype). CF = chromosome fusions and concatenation; SFE = telomere signal-free end; STF = sister telomere fusion. Total = CF + SFE + STF. (B-C) Immunohistochemical staining of DNA damage marker (γ-H2AX) and oxidative DNA damage marker (8-OHDG) in KC and KCH mice. (D) ELISA analysis of serum nucleosome levels in KC and KCH mice (n = 5 mice/genotype/age). (E-I) Antioxidant N-acetylcysteine (NAC, 300 mg/kg i.p., five times per week, started at 4 weeks of age for four weeks) treatment prolonged survival in KCH mice (E, ^**P < 0.01, log-rank test) with decreased telomere defects (F), histone H2AX phosphorylation (G), oxidative DNA damage (H), and serum nucleosome levels (I). ^*P < 0.05, ^**P< 0.01 (n = 5 mice/genotype, unpaired t-test).

Figure 3

Extracellular histone promotes pancreatic tumorigenesis in KCH mice. (A) H3 Ab or IL-6 Ab treatment (10 mg/kg i.p., twice per week, started at four weeks of age for four weeks) prolonged survival in KCH mice at 12 weeks of age (n = 10 mice/treatment, ^**P < 0.01, ^***P < 0.001, log-rank test). The median survival of untreated, IgG-treated, H3 Ab-treated, and IL-6 treated KCH mice were nine, nine, 12, and 12 weeks, respectively, in this setting. (B-I) In parallel, pancreatic lesion formation (B), incidence of tumor metastasis/invasion (C), serum IL-6 level (D), percentage of tumor infiltration of macrophages (E) and neutrophils (F), and relative expression of p-ERK1/2 (G), p-AKT (H), and p-STAT3 (I) in the pancreas were assayed. Graphs show means ± sem, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (n = 5 mice/treatment, unpaired t-test).

Figure 4

Deletion of RAGE protects against pancreatic tumorigenesis in KCH mice. (A) Kaplan-Meier survival analysis was performed in KCH (Pdx1-Cre;K-Ras^G12D/+;Hmgb1^−/−), KCHR (Pdx1-Cre;K-Ras^G12D/+;Hmgb1^−/−;Rage^−/−), and KCHT9 (Pdx1-Cre;K-Ras^G12D/+;Hmgb1^−/−;Tlr9^−/−) mice (n = 5 mice/treatment, ^**P < 0.01, log-rank test). (B-G) In parallel, pancreatic lesion formation (B), tumor metastasis/invasion incidence (C), serum IL-6 level (D), and relative expressions of p-ERK1/2 (E), p-AKT (F), and p-STAT3 (G) in the pancreas were assayed. Graphs show means ± sem, ^*P < 0.05, ^**P < 0.01 (n = 5 mice/treatment, unpaired t-test).

Figure 5

Inhibition of nuclear HMGB1 loss and release by glycyrrhizin limits K-Ras-induced PanIN formation. (A, B) Effects of K-Ras^G12D transfection on HMGB1 expression (A) and release (B) in primary mouse pancreatic acinar cells (n = 3, ^***P < 0.001, data are expressed as means ± sem, unpaired t-test). (C, D) Effects of glycyrrhizin (500 μM) on HMGB1 expression (C) and release (D) in primary mouse pancreatic acinar cells after K-Ras^G12D transfection for 48 h (n = 3, ^***P < 0.001, data are expressed as means ± sem, unpaired t-test). (E-G) Effects of glycyrrhizin (10 mg/kg) on PanIN formation (E) and HMGB1 expression (F) and release (G) on day 21 in a cerulein-mediated accelerated oncogenic K-Ras mouse model of pancreatic cancer (n = 5 mice/treatment, ^***P < 0.001, data are expressed as means ± sem, unpaired t-test).

Figure 6

Reduced HMGB1 expression in pancreatic tissue compared with normal adjacent tissue (NAT) in pancreatic cancer patients (A) with poor survival outcomes (B) (^*P < 0.05, log-rank test).

Figure 7

Schematic depicting HMGB1 deficiency-mediated nucleosome release linking chromosomal instability to the inflammatory response in K-Ras-driven PDAC. Oncogenic K-Ras^G12D leads to HMGB1 translocation and release into the extracellular space. Loss of intracellular HMGB1 increases chromosomal instability and promotes nucleosome release (current study). In addition, we previously demonstrated that extracellular nucleosome activates innate immune cells (e.g., macrophages) to secrete HMGB1 into circulation. Both extracellular nucleosome and HMGB1 exacerbates PDAC development by enhancing RAGE-dependent inflammatory responses.