HMGB1 as an autocrine stimulus in human T98G glioblastoma cells: role in cell growth and migration

HMGB1 (high mobility group box 1 protein) is a nuclear protein that can also act as an extracellular trigger of inflammation, proliferation and migration, mainly through RAGE (the receptor for advanced glycation end products); HMGB1–RAGE interactions have been found to be important in a number of cancers. We investigated whether HMGB1 is an autocrine factor in human glioma cells. Western blots showed HMGB1 and RAGE expression in human malignant glioma cell lines. HMGB1 induced a dose-dependent increase in cell proliferation, which was found to be RAGE-mediated and involved the MAPK/ERK pathway. Moreover, in a wounding model, it induced a significant increase in cell migration, and RAGE-dependent activation of Rac1 was crucial in giving the tumour cells a motile phenotype. The fact that blocking DNA replication with anti-mitotic agents did not reduce the distance migrated suggests the independence of the proliferative and migratory effects. We also found that glioma cells contain HMGB1 predominantly in the nucleus, and cannot secrete it constitutively or upon stimulation; however, necrotic glioma cells can release HMGB1 after it has translocated from the nucleus to cytosol. These findings provide the first evidence supporting the existence of HMGB1/RAGE signalling pathways in human glioblastoma cells, and suggest that HMGB1 may play an important role in the relationship between necrosis and malignancy in glioma tumours by acting as an autocrine factor that is capable of promoting the growth and migration of tumour cells. Rosaria Bassi and Paola Giussani contributed equally to this study.     

HMGB1/TLR4通路参与Fibulin-5对肺癌细胞增殖和转移的抑制作用

背景与目的：Fibulin-5在肺癌组织中低表达,具有抑癌作用。高迁移率族蛋白B1(high mobility group box 1,HMGB1)在肺癌中高表达,能够促进肿瘤的侵袭转移。该研究旨在探讨Fibulin-5抑制肺癌细胞增殖和转移的分子机制。方法：该研究首先检测了肺上皮细胞和肺癌细胞中Fibulin-5和HMGB1的表达,然后利用转染试剂将Fibulin-5过表达质粒和HMGB1的siRNA转染人A549细胞。实现Fibulin-5过表达和HMGB1低表达后,采用MTT实验检测细胞增殖情况,Transwell实验检测细胞的侵袭和迁移能力。本研究采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测A549细胞中HMGB1 mRNA表达变化,采用酶联免疫吸附剂测定实验(enzyme-linked immunosorbent assay,ELisa)检测HMGB1蛋白的分泌;采用蛋白［质］印迹法(Western blot)检测HMGB1、cyclin D1、基质金属蛋白酶(matrix metalloproteinases,MMPs)和TLR4/NF-κB通路相关蛋白的表达变化。结果：在肺癌细胞A549中,Fibulin-5低表达,HMGB1高表达。过表达Fibulin-5和低表达HMGB1后,HMGB1、cyclin D1、MMP2、MMP7和MMP9的表达均明显降低,A549细胞的增殖、侵袭和迁移能力明显减弱(P<0.05);此外,过表达Fibulin-5下调了TLR4、MyD88、p-p65的表达,上调了IκBα的表达(P<0.05)。结论：Fibulin-5可能是通过抑制HMGB1的表达以及其下游的TLR4/NF-κB通路,抑制肺癌细胞的增殖、侵袭和迁移的过程。     

RAGE和HMGB1在乳腺癌中表达及其临床意义

目的探讨乳腺癌组织中RAGE和HMGB1基因表达及其临床意义。方法各对50例早期、晚期乳腺癌组织及正常乳腺组织蜡块标本,运用real-time PCR技术检测RAGE和HMGB1基因的表达情况。并分析各基因表达与乳腺癌组织的分化程度、浸润深度、淋巴结转移、TNM分期之间的关系。结果 RAGE和HMGB1基因表达荧光实时定量PCR法上调分别为73%、79%。乳腺癌TNM分期、淋巴结转移与基因高表达有密切关系,RAGE基因表达与HMGB1表达呈正相关(P<0.05)。结论 RAGE和HMGB1基因表达对于乳腺癌早期诊断和预后分析有重要指导意义,根据其表达开展有选择性地基因治疗应有良好的应用前景。     

HMGB1 is overexpressed in tumor cells and promotes activity of regulatory T cells in patients with head and neck cancer

HMGB1 has gained a prominent role in cancer development and is implicated in tumor escape phenomena. To date, only few data are available on effects of HMGB1 on regulatory T cells (Treg) in cancer patients. This study evaluates the prevalence of HMGB1 and its effects on Treg in patients with head and neck squamous cell carcinoma (HNSCC). Sixty-seven patients with HNSCC and seventeen healthy donors were included in this study. Tumor tissues of patients were analyzed for expression of HMGB1 employing immunofluorescence and qRT-PCR. HMGB1 serum levels were assessed using ELISA. Tumor-infiltration and Treg from peripheral blood were phenotyped with flow cytometry and immunofluorescence microscopy. Migration and suppressive function of Treg upon HMGB1 stimulation was analyzed in chemotaxis assays and CFSE assays. HMGB1 is overexpressed in tumor cells of HNSCC, and serum levels are significantly elevated. Tumor-infiltrating Treg express HMGB1-recognizing receptors, TLR4 and RAGE. HMGB1 is a chemoattractant for Treg and promotes their suppressive function. Our data provide new aspects how the HMGB1 tumor-derived danger signal augments function of Treg in patients with HNSCC.     

Low molecular weight heparin suppresses receptor for advanced glycation end products‐mediated expression of malignant phenotype in human fibrosarcoma cells

The receptor for advanced glycation end products (RAGE) is a pattern‐recognition receptor and its engagement by ligands such as high mobility group box 1 (HMGB1) is implicated in tumor growth and metastasis. Low molecular weight heparin (LMWH) has an antagonistic effect on the RAGE axis and is also reported to exert an antitumor effect beyond the known activity of anticoagulation. However, the link between the anti‐RAGE and antitumor activities of LMWH has not yet to be fully elucidated. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, invasion, and metastasis by blocking the RAGE axis using in vitro and in vivo assay systems. Stably transformed HT1080 human fibrosarcoma cell lines were obtained, including human full‐length RAGE‐overexpressing (HT1080^RAGE), RAGE dominant‐negative, intracellular tail‐deleted RAGE‐overexpressing (HT1080^dnRAGE), and mock‐transfected control (HT1080^mock) cells. Confocal microscopy showed the expression of HMGB1 and RAGE in HT1080 cells. The LMWH significantly inhibited HMGB1‐induced NFκB activation through RAGE using an NFκB‐dependent luciferase reporter assay and the HT1080 cell lines. Overexpression of RAGE significantly accelerated, but dnRAGE expression attenuated HT1080 cell proliferation and invasion in vitro, along with similar effects on local tumor mass growth and lung metastasis in vivo. Treatment with LMWH significantly inhibited the migration, invasion, tumor formation, and lung metastasis of HT1080^RAGE cells, but not of HT1080^mock or HT1080^dnRAGE cells. In conclusion, this study revealed that RAGE exacerbated the malignant phenotype of human fibrosarcoma cells, and that this exacerbation could be ameliorated by LMWH. It is suggested that LMWH has therapeutic potential in patients with certain types of malignant tumors.     

2-deoxyglucose inhibits chemotheapeutic drug-induced apoptosis in human monocytic leukemia U937 cells ith inhibition of c-Jun N-terminal kinase 1/stress-activated protein kinase activation

Human monocytic leukemia U937 cells undergo apoptosis when treated with antitumor drugs, such as etoposide, camptothecin and mitomycin C. The molecular mechanism of the drug-induced apoptosis is not well understood. In this study, we found that 2-deoxyglucose (2DG), an analog of D-glucose and an inducer of glucose-regulated stress, inhibited anticancer drug-induced but not tumor necrosis factor-alpha-induced apoptosis of U937 cells. 2DG did not reduce initial cellular damage caused by etoposide, an inhibitor of topoisomerase II, suggesting that 2DG affected subsequent cellular responses involved in apoptosis. 2DG inhibited the etoposide-induced activation of c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK) and the subsequent activation of CPP32, both of which are positive regulators for etoposide-induced apoptosis of U937 cells. Our results indicate that 2DG inhibits apoptosis by blocking the signals from cellular DNA damage for JNK1/SAPK activation. Int. J. Cancer 76:86–90, 1998.© 1998 Wiley-Liss, Inc.     

E-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances HMGB1 release

Extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. Extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. The present study shows that the interaction between recombinant E-selectin (rE-selectin) and colorectal cancer (CRC) cells alters the gene expression profile of the cancer cells. A DNA microarry analysis indicated that E-selectin-mediated alterations were significantly more pronounced in the metastatic CRC variants SW620 and KM12SM than in the corresponding non-metastatic local SW480 and KM12C variants. The number of genes altered by E-selectin in the metastatic variants was about 10-fold higher than the number of genes altered in the corresponding local variants. Aiming to identify genes involved in CRC metastasis, we focused, by using a DNA microarry analysis, on genes that were altered by E-selectin in a similar fashion exclusively in both metastatic variants. This analysis indicated that E-selectin down regulated (at least by 1.6-folds) the expression of 7 genes in a similar fashion, in both metastatic cells. The DNA microarry analysis was validated by real time PCR or by RT-PCR. HMGB1 was among these genes. Confocal microscopy indicated that E-selectin down regulated the cellular expression of the HMGB1 protein and enhanced the release of HMGB1 into the culture medium. The released HMGB1 in turn, activated endothelial cells to express E-selectin.     

高迁移率族蛋白1及晚期糖基化终产物受体在宫颈鳞状细胞癌中的表达及临床意义

目的 研究高迁移率族蛋白1(HMGB1)和晚期糖基化终产物受体(RAGE)在宫颈鳞状细胞癌(CSCC)组织中的表达及其与临床病理间的关系,探讨HMGB1/RAGE信号通路在CSCC转移中的作用.方法 采用荧光实时定量PCR、免疫组化及Western blot方法分别检测CSCC组织和正常宫颈鳞状上皮组织中HMGB1、RAGE基因及蛋白的表达.结果HMGB1在CSCC中的表达水平高于正常宫颈鳞状上皮组织(P＜0.05),其在CSCC组织中的表达与肿瘤分期、侵袭及转移明显相关(P＜0.05),而与肿瘤大小、分化程度无关(P＞0.05).RAGE mRNA的表达与CSCC转移明显相关(P＜0.05).转移组中的HMGB1 mRNA和RAGE mRNA表达水平呈显著相关性.结论 HMGB-1与CSCC发生、侵袭和转移相关,RAGE与CSCC转移相关,HMGB1/RAGE信号通路在CSCC转移过程中可能发挥重要作用.     

Expression and Clinical Significance of HMGB1 and RAGE in Cervical Carcinoma

OBJECTIVE To study the expression level and clinical significance of HMGB1 and RAGE in cervical squamous epithelial carcinoma. METHODS Real time quantitative polymerase chain reaction(qRT-PCR) was employed to examine the expression of HMGB1(high mobility group box protein1),and RAGE(receptor for advanced glycation endproducts)in 60 cervical squamous epithelial carcinomas(CSEC),their paraneoplastic tissues(PS)and 30 normal cervix tissues(NCS). RESULTS The expression of HMGB1 in the CSEC samples and PS was similar(P>0.05),but higher compared to NCS(P<0.05).Overexpression of HMGB1 in the CESC tissues was significantly correlated with the tumor (P<0.05),and the presence of metastasis(P<0.01),but not correlated with the tumor diameter or tumor grade.RAGE expression was not significantly different among these tissue types,and showed no significant correlation with the the tumor stage,diameter or grade.But there was a significant positive correlation between RAGE expression and CSEC metastasis. CONCLUSION The results suggest that HMGB1 may be related to the proliferation,progression and metastasis of CSEC.The relationship of HMGB1/RAGE may be of importance for CSEC metastasis.HMGB1 presents a new potential gene target for prevention and treatment of CSEC. Study of HMGB1/RAGE expression will offer an experimental foundation for understanding the pathogenesis of CSES.     

RAGE mediates S100A4-induced cell motility via MAPK/ERK and hypoxia signaling and is a prognostic biomarker for human colorectal cancer metastasis

Survival of colorectal cancer patients is strongly dependent on development of distant metastases. S100A4 is a prognostic biomarker and inducer for colorectal cancer metastasis. Besides exerting intracellular functions, S100A4 is secreted extracellularly. The receptor for advanced glycation end products (RAGE) is one of its interaction partners. The impact of the S100A4-RAGE interaction for cell motility and metastasis formation in colorectal cancer has not been elucidated so far. Here we demonstrate the RAGE-dependent increase in migratory and invasive capabilities of colorectal cancer cells via binding to extracellular S100A4. We show the direct interaction of S100A4 and RAGE, leading to hyperactivated MAPK/ERK and hypoxia signaling. The S100A4-RAGE axis increased cell migration (P<0.005) and invasion (P<0.005), which was counteracted with recombinant soluble RAGE and RAGE-specific antibodies. In colorectal cancer patients, not distantly metastasized at surgery, high RAGE expression in primary tumors correlated with metachronous metastasis, reduced overall (P=0.022) and metastasis-free survival (P=0.021).In summary, interaction of S100A4-RAGE mediates S100A4-induced colorectal cancer cell motility. RAGE by itself represents a biomarker for prognosis of colorectal cancer. Thus, therapeutic approaches targeting RAGE or intervening in S100A4-RAGE-dependent signaling early in tumor progression might represent alternative strategies restricting S100A4-induced colorectal cancer metastasis.     

Co-expression of RAGE and HMGB1 is associated with cancer progression and poor patient outcome of prostate cancer

Receptor for advanced glycation end products (RAGE), along with its ligand high mobility group box 1 (HMGB1), is believed to play an important role in prostate cancer. The aim of this retrospective study was to investigate the expression of RAGE and HMGB1 and their clinical impact on prostate cancer progression and prognosis. The expression of RAGE and HMGB1 was assessed by immunohistochemistry in cancer lesions from 85 confirmed prostate cancer cases. We determined the potential association between the expression level of these two proteins and the clinicopathological features and overall patient survival. RAGE and HMGB1 were expressed in 78.8% (67/85) and 68.2% (58/85) cases of prostate cancer, respectively, and in the majority (54/85) of cases, these two proteins were co-expressed. There was a strong correlation between RAGE and HMGB1 expressions (P<0.001). The expression of RAGE, HMGB1 and their co-expression were all associated with advanced tumor clinical stage (P<0.05 for all). RAGE expression was also associated with the prostate specific antigen (PSA) level (P=0.014). However, neither the individual expression of those genes nor their co-expression was significantly related with age or Gleason score. The co-expression of RAGE and HMGB1 was associated with poor overall survival in patients with stage III and IV prostate cancer (P=0.047). These results suggest that the expression of RAGE and HMGB1 is associated with the progression and poor prognosis of prostate cancer. RAGE and HMGB1 could be new prognostic biomarkers for prostate cancer as well as molecular target for novel forms of therapies.     

High mobility group box-1-inducible melanoma inhibitory activity is associated with nodal metastasis and lymphangiogenesis in oral squamous cell carcinoma

Melanoma inhibitory activity (MIA) is an 11-kDa secretory protein isolated from malignant melanoma cells that is correlated with invasion and metastasis in various human malignancies. We examined MIA expression in 62 oral squamous cell carcinomas (OSCC) by immunohistochemistry. MIA expression was significantly associated with nodal metastasis ( P =  0.00018). MIA expression was also associated with expression of high mobility group box-1 (HMGB1) ( P <  0.0001) and lymph vessel density ( P <  0.0001). Expression levels of MIA, HMGB1, nuclear factor kB (NFkB) p65 and HMGB1–NFkB p65 binding were significantly higher in a metastatic human OSCC cell line (HSC3) than those in a non-metastatic OSCC cell line (HSC4). Treatment with receptor for advanced glycation end products (RAGE) antisense or small interfering RNA and human recombinant HMGB1 (hrHMGB1) did not affect MIA expression, whereas HMGB1 antisense or siRNA treatment decreased MIA expression in HSC3 cells. Then HMGB1 enhanced MIA expression as an NFkB cofactor but not as a RAGE ligand. MIA neutralization by MIA antibodies increased extracellular signal-related kinase 1/2 phosphorylation, but decreased p38 phosphorylation and the expression of vascular epithelial growth factor (VEGF)-C and -D. Treatment with p38 inihibitor decreased VEGF-C and -D expression in HSC3 cells. These results suggest that MIA expression is enhanced by the interaction of intracellular HMGB1 and NFkBp65 and MIA is closely involved in tumor progression and nodal metastasis by the increments of VEGF-C and VEGF-D in OSCC. ( Cancer Sci 2008; 99: 1806– 1812)     

Oxidized high mobility group B‐1 enhances metastability of colorectal cancer via modification of mesenchymal stem/stromal cells

High mobility group box‐1 (HMGB1) is known to be a chemotactic factor for mesenchymal stem/stromal cells (MSCs), but the effect of post‐translational modification on its function is not clear. In this study, we hypothesized that differences in the oxidation state of HMGB1 would lead to differences in the function of MSCs in cancer. In human colorectal cancer, MSCs infiltrating into the stroma were correlated with liver metastasis and serum HMGB1. In animal models, oxidized HMGB1 mobilized three‐fold fewer MSCs to subcutaneous tumors compared with reduced HMGB1. Reduced HMGB1 inhibited the proliferation of mouse bone marrow MSCs (BM‐MSCs) and induced differentiation into osteoblasts and vascular pericytes, whereas oxidized HMGB1 promoted proliferation and increased stemness, and no differentiation was observed. When BM‐MSCs pretreated with oxidized HMGB1 were co‐cultured with syngeneic cancer cells, cell proliferation and stemness of cancer cells were increased, and tumorigenesis and drug resistance were promoted. In contrast, co‐culture with reduced HMGB1‐pretreated BM‐MSCs did not enhance stemness. In an animal orthotopic transplantation colorectal cancer model, oxidized HMGB1, but not reduced HMGB1, promoted liver metastasis with intratumoral MSC chemotaxis. Therefore, oxidized HMGB1 reprograms MSCs and promotes cancer malignancy. The oxidized HMGB1–MSC axis may be an important target for cancer therapy.     

RAGE and Its Ligands: Molecular Interplay Between Glycation,Inflammation, and Hallmarks of Cancer—a Review

Risk of cancer especially of colon, breast, and pancreas is high in diabetic and obese patients, with potential involvement of augmented expression of RAGE (receptor for advanced glycation end products) and its ligands, namely AGEs (advanced glycation end products), HMGB1 (high-mobility group box 1 protein), and S100 group of proteins. Studies have reported the involvement of RAGE activation by its ligands in growth and survival of cancers, including metastasis and poor prognosis. We propose that this receptor-ligand axis provides the molecular link between certain pre-existing states as hypoxia, hyperglycemia, glycation, inflammation, oxidative stress, and onset of cancers. The chronic inflammatory, hyperglycemic milieu accompanied by glycoxidative stress as in diabetes and obesity, concomitant with the formation of RAGE ligands, instigates RAGE and cancer stem cells, leading to the oncogenic transformation of normal and pre-malignant tissues towards development of neoplasms. We have aimed to elucidate the complete signalling map initiated upon RAGE-ligand splicing, from oncogenesis to progression, epithelial-mesenchymal transition, invasion, cancer stem cell renewal, chemo-resistance, and cancer relapse. We have attributed the complex molecular functions of RAGE-ligand signalling cues to every aspect of cancer promotion, explaining the central network in bridging glycation, inflammation, oxidation, and the hallmarks of cancer. Underlining the substantial requisite for anti-neoplastic agents targeting RAGE and its ligands, we have explicitly discoursed RAGE and its allied components (AGEs, soluble RAGE, RAGE gene polymorphisms) as potential diagnostic and prognostic biomarkers for prompt detection of cancers and implication in impending RAGE-ligand directed, novel combinatorial, and targeted onco-therapeutics.     

HMGB1 promotes the synthesis of pro-IL-1β and pro-IL-18 by activation of p38 MAPK and NF-κB through receptors for advanced glycation end-products in macrophages

The high mobility group box-1 (HMGB1) protein and NALP3 inflammasome have been identified to play important roles in inflammation and cancer pathogenesis, but the relationships between the two and cancer remain unclear. The current study investigated the relationship between HMGB1 and the NALP3 inflammasome in THP-1 macrophages. HMGB1 was found unable to activate the NALP3 inflammasome and failed to induce the release of the IL-1β and IL-18 in THP-1 macrophages. HMGB1 was also found significantly enhanced the activity of ATP to induce IL-1β and IL-18 by the induction of increased expression of pro-IL-1β and pro-IL-18. This process was dependent on activation of RAGE, MAPK p38 and NF-κB signaling pathway. These results demonstrate that HMGB1 promotes the synthesis of pro-IL-1β and pro-IL-18 in THP-1 macrophages by the activation of p38 MAPK and NF-κB through RAGE. HMGB1 likely plays an important role in the first step of the release of the IL-1β and IL-18, preparing for other cytokines to induce excessive release of IL-1β and IL-18 which promote inflammation and cancer progression.