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

doi:10.1016/j.oraloncology.2011.12.009

Oral Oncology

Volume 48, Issue 5, May 2012, Pages 409-416

https://doi.org/10.1016/j.oraloncology.2011.12.009 Get rights and content

Summary

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.

Introduction

It is generally accepted that chronic inflammation may eventually result in development of cancer, as it was already hypothesized by Virchow in 1863.¹ Head and neck squamous cell carcinoma (HNSCC) is a cancer entity that serves as a model of inflammation-associated carcinogenesis and tumor progression.² Surgery, chemotherapy, and radiation are currently the major options for HNSCC treatment. All three induce local or systemic inflammation triggered by tissue injury and cancer cell death.³

Accumulating evidence indicates that tumor cells release endogenous danger signals, called damage-associated molecular pattern molecules (DAMPs), which have cytokine-like properties when released from the cell. These signals are typically released following necrotic and late apoptotic tumor death. DAMPs include the small calcium-binding S100 family of molecules, inflammation-associated metabolites such as prostaglandins, purine metabolites such as uric acid and ATP, hyaluronan, heat shock proteins, heparan sulfate and syndecan.⁴ An interesting member of the DAMP family is the evolutionarily conserved nuclear protein, high mobility group box 1 (HMGB1).⁵ HMGB1 is present in the nucleus and cytoplasm of nearly all cell types. Extracellular HMGB1 acts in two fundamentally different manners: as a danger signal by active secretion from living inflammatory cells, or as an inflammatory mediator by passive release from necrotic or stressed cells. Anticancer treatments, such as radiotherapy and chemotherapy, cause cell death and promote passive HMGB1 release.6, 7, 8

Overexpression as well as cytoplasmic localization of HMGB1 has been observed in several tumor entities including gastrointestinal stromal tumors⁹, colon tumors¹⁰, and nasopharyngeal carcinoma.¹¹ HMGB1 mRNA expression in prostate cancer is increased compared with normal prostate tissue.¹² Consistently, serum levels of HMGB1 in patients with lung cancer were found to be increased compared to healthy individuals.¹³ Recently, Liu et al. provided evidence that the HMGB1 protein may contribute to the malignant progression of HNSCC, and multivariate analysis further demonstrated that HMGB1 was an independent prognostic factor for patients with HNSCC.¹⁴ Further, Choi et al. just recently reported on several HNSCC cell lines for the expression of HMGB1 and one of its receptors, receptor of advanced glycolisation end products (RAGE).¹⁵ Several members of the Toll-like receptor (TLR) family have been reported to recognize HMGB1, including TLR2, TLR4 and RAGE. TLRs are the archetypal pattern-recognition receptors that have emerged as key mediators of immune functions and are expressed on several subtypes of immune cells, including macrophages, monocytes, dendritic cells and T cells. We have previously reported that Treg overexpress the HMGB1-recognizing receptor TLR4 in patients with head and neck cancer.¹⁶

Here, we provide first evidence of interaction of HMGB1 with Treg in patients with HNSCC. We show that HMGB1 is clearly elevated in HNSCC and significant levels of released HMGB1 are detectable in patients’ sera. Tumor-infiltrating Treg express the HMGB1-recognizing receptors TLR4 and RAGE. Further, HMGB1 acts as a chemoattractant on Treg and enhances their immunosuppressive capacity. Our data provide new molecular insights into strategies of tumors to escape from efficient antitumor immune responses.

Section snippets

Patients and controls

This study was approved by the Institutional Review Board of the University Hospital Essen and all individuals signed informed consent for research use of the blood and for participating in the research project. The study was conducted according to the Declaration of Helsinki. All patients were seen at the outpatient clinic of the Department of Otorhinolaryngology of the University Hospital of Essen between 2007 and 2011. All patients had non-pretreated primary tumors, no history of previous

HMGB1 is strongly elevated in tumor tissue and patients’ sera

HMGB1 is known to be expressed in all eukaryotic cells and to be overexpressed in tumor tissues.⁸ Therefore, primary tumor tissues obtained from patients with HNSCC were evaluated for expression of this “danger signal”. Analysis of tumor tissues using immunofluorescence microscopy revealed that HMGB1 is expressed stronger in tumor islands than in tumor-surrounding stroma (Fig. 1A). mRNA content of HMGB1 was 1.57 ± 0.97-fold higher in tumor tissues from 32 patients with HNSCC compared to normal

Discussion

Damage-associated molecular patterns (DAMPs) such as HMGB1 have been described to act as “danger signals” that have a significant role in chronic inflammation and cancer progression.⁶ Tumor cells release inflammation-associated molecules to impair tumor-directed immune activity which induces tolerance in order to foster tumor-escape mechanisms. Elevated levels of HMGB1 have been reported for several tumor entities including nasopharyngeal, laryngeal, gastric and colorectal cancers9, 11, 14, 22,

Acknowledgements

We thank Michael T. Lotze (University of Pittsburgh) for discussion and advice. We thank Kirsten Bruderek, Anne-Marie Heider and Petra Altenhoff for their excellent technical assistance. We thank the WTZ Research Support Service (supported in part by the Deutsche Krebshilfe Comprehensive Cancer Center financing) for editing of the manuscript. The research described in this article was supported in part by the Deutsche Forschungsgemeinschaft (DFG BE/4190).

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HMGB1 aggravates lipopolysaccharide-induced acute lung injury through suppressing the activity and function of Tregs
2020, Cellular Immunology
Citation Excerpt :
Studies have revealed that Tregs overexpress the HMGB1-recognizing receptor TLRs and RAGE, that regulate the suppressive function and activity of Tregs in cancers, autoimmune diseases, infectious diseases, and graft rejection [18,19]. Furthermore, the extracellular HMGB1 could stimulate Tregs via binding TLRs and RAGE on the surface of Tregs and mediate their immune suppression function [19,20]. In our previous studies, we showed that HMGB1 could inhibit Tregs differentiation in the pathogenesis of asthma by activating NF-κB signaling pathway through TLR2, TLR4, and RAGE receptors [21].
CD4⁺CD25⁺FoxP3⁺ T helper cells (Tregs), a subgroup of CD4⁺ T helper cells, are critical effectors that protect against acute lung injury (ALI) by contact-dependent suppression or releasing anti-inflammatory cytokines including interleukin-10 (IL-10), and transforming growth factor (TGF-β). HMGB1 (High mobility group box 1 protein) was identified as a nuclear non-histone DNA-binding chromosomal protein, which participates in the regulation of lung inflammatory response and pathological processes in ALI. Previous studies have suggested that Tregs overexpresses the HMGB1-recognizing receptor. However, the interaction of HMGB1 with Tregs in ALI is still unclear.
To investigate whether HMGB1 aggravates ALI by suppressing immunosuppressive function of Tregs.
Anti-HMGB1 antibody and recombinant mouse HMGB1 (rHMGB1) were administered in lipopolysaccharide (LPS)-induced ALI mice and polarized LPS-primed Tregs in vitro. The Tregs pre-stimulated with or without rHMGB1 were adoptively transferred to ALI mice and depleted by Diphtheria toxin (DT). For coculture experiment, isolated Tregs were first pre-stimulated with or without rHMGB1 or anti-HMGB1 antibody, then they were cocultured with bone marrow-derived macrophages (BMMs) under LPS stimulation.
Tregs protected against acute lung pathological injury. HMGB1 modulated the suppressive function of Tregs as follows: reduction in the number of the cells and the activity of Tregs, the secretion of anti-inflammatory cytokines (IL-10, TGF-β) from Tregs, the production of IL-2 from CD4⁺ T cells and CD11c⁺ DCs, and the M2 polarization of macrophages, as well as inducing proinflammatory response of macrophages.
HMGB1 could aggravate LPS induced-ALI through suppressing the activity and function of Tregs.
Evaluating high-mobility group box 1 protein serum levels amongst Iranian patients with oral squamous cell carcinoma
2020, Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology
Citation Excerpt :
This is the only data set indicating that serum HMGB1 level may serve as a promising biomarker in diagnosis of OSCC. In line with the current findings, almost all published studies, such as those conducted on patients with head and neck squamous cell carcinoma (HNSCC) (3.93 ng/mL vs. 1.15 ng/mL, p = 0.002) [33], laryngeal squamous cell carcinoma (LSCC) (4.81 ± 2.33 ng/mL vs. 3.21 ± 1.08 ng/mL, p < 0.001) [34], recurrent cervical squamous cell carcinoma (50.8 ng/mL vs. 7.6 ng/mL, p = 0.027) [35], breast cancer (4.64 ± 2.50 ng/mL vs. 1.36 ± 0.75 ng/mL, p < 0.05) [1], advanced gastric cancer (16.5 ± 27.4 ng/mL vs. 3.9 ± 3.4 ng/mL, p < 0.05) [26], lung cancer (76.1 ± 37.0 ng/mL vs. 7.7 ± 6.1 ng/mL, p < 0.0001) [36], hepatocellular carcinoma (84.2 ± 50.4 ng/mL vs. 7.0 ± 5.9 ng/mL, p < 0.0001) [37], and colorectal carcinoma (58.8 ± 126.2 ng/mL vs. 39.7 ± 16.2 ng/mL, p = 0.03) [38] showed a higher level of HMGB1 in patients group in comparison with the healthy controls. Therefore, HMGB1 is capable of displaying a wide range of expression patterns in different type of malignancies, even in a specific cancer type [39].
Most patients with oral squamous cell carcinoma (OSCC) are diagnosed during advanced stages; hence, early detection programs might help the prognosis and survival rate. High mobility group box 1 protein (HMGB1) is a new biomarker, with possible association with the pathogenesis of human cancers. We aimed to investigate the diagnostic value of serum HMGB1 level amongst Iranian patients with OSCC.
In this cross-sectional study, HMGB1 serum activity was measured in 60 patients with OSCC and 60 eligible healthy individuals, using enzyme-linked immunosorbent assay (ELISA). The possible associations between the serological activity of HMGB1 and the clinicopathologic features of patients were also assessed.
The HMGB1 serum level in patients group (2514.1 ± 935.6 pg/mL) was significantly higher than the controls (508.7 ± 370.5 pg/mL) (p < 0.0001). This study demonstrated that the serum levels of HMGB1 were correlated with some of the features associated with a clinical advanced disease. A positive correlation was observed between HMGB1 level and tumor size (p = 0.038). HMGB1 level was significantly elevated in those with the lymph node involvement (p = 0.001), but no association was found with other clinicopathologic characteristics of OSCC patients, such as age, gender and different clinical stages (p > 0.05).
The HMGB1 levels in patient’s sera can be used as a diagnostic marker to detect OSCC. However, further studies with the larger sample size and longer follow-up are warranted to assess the potential of HMGB1 for the early detection of OSCC.
MiR-142-3p enhances chemosensitivity of breast cancer cells and inhibits autophagy by targeting HMGB1
2020, Acta Pharmaceutica Sinica B
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By bioinformatics analysis and luciferase reporter experiment, we found that HMGB1 was identified to act as a direct target of miR-142-3p. HMGB1 is overexpressed in many types of cancer40,41 including breast cancer42. In the current study, HMGB1 was highly expressed at both mRNA and protein levels in MCF-7/DOX cells compared with their parental MCF-7 cells.
MiR-142-3p has been reported to act as a tumor suppressor in breast cancer. However, the regulatory effect of miR-142-3p on drug resistance of breast cancer cells and its underlying mechanism remain unknown. Here, we found that miR-142-3p was significantly downregulated in the doxorubicin (DOX)-resistant MCF-7 cell line (MCF-7/DOX). MiR-142-3p overexpression increased DOX sensitivity and enhanced DOX-induced apoptosis in breast cancer cells. High-mobility group box 1 (HMGB1) is a direct functional target of miR-142-3p in breast cancer cells and miR-142-3p negatively regulated HMGB1 expression. Moreover, overexpression of HMGB1 dramatically reversed the promotion of apoptosis and inhibition of autophagy mediated by miR-142-3p up-regulation. In conclusion, miR-142-3p overexpression may inhibit autophagy and promote the drug sensitivity of breast cancer cells to DOX by targeting HMGB1. The miR-142-3p/HMGB1 axis might be a novel target to regulate the drug resistance of breast cancer patients.
SNHG14 silencing suppresses the progression and promotes cisplatin sensitivity in non-small cell lung cancer
2019, Biomedicine and Pharmacotherapy
Citation Excerpt :
Then, online software Starbase was employed to predict the targets of miR-34a. Among these candidates, HMGB1 was of interest in the study, considering the vital roles of HMGB1 in the development and progression of human cancers, such as malignant mesotelioma [30], papillary thyroid cancer [31] and head and neck cancer [32]. It was reported that anti-tumor agents increased HMGB1 release and HMGB1 knockdown promoted cell apoptosis and repressed autophagy in stressed cancer cells [33].
Long non-coding RNAs (lncRNAs) small nucleolar RNA host gene 14 (SNHG14) has been identified as an oncogene involved in the progression of various human cancers. Nevertheless, the functional role and molecular mechanism of SNHG14 on NSCLC remain largely elusive. qRT-PCR assay was performed to detect the levels of SNHG14, miR-34a and high mobility group box 1 (HMGB1) mRNA. HMGB1 protein level was assessed by western blot analysis. CCK-8 assay was used to determine the IC50 value of cisplatin (CDDP), and transwell assays were employed to detect cell migration and invasion abilities. Cell apoptosis was determined by flow cytometric analysis. Dual-luciferase reporter assay, RNA immuoprecipitation assay and RNA pull-down assay were performed to confirm the interaction between SNHG14 and miR-34a, or miR-34a and HMGB1. Our data demonstrated that SNHG14 was upregulated in NSCLC cells, and SNHG14 silencing repressed the migration, invasion while accelerated the apoptosis of NSCLC cells. Moreover, we manifested that SNHG14 silencing promoted NSCLC cell sensitivity to CDDP. SNHG14 repressed miR-34a expression by binding to miR-34a. Additionally, SNHG14 regulated HMGB1 expression by sponging miR-34a. SNHG14 silencing exerted its regulatory effect by miR-34a and HMGB1 mediated the regulatory effect of miR-34a on NSCLC cells. In conclusion, SNHG14 silencing suppressed NSCLC progression at least partly by miR-34a/HMGB1 axis in vitroand promoted NSCLC cell sensitivity to CDDP, highlighting that SNHG14 might be a potential target for NSCLC therapy.
The potentially conflicting cell autonomous and cell non-autonomous functions of autophagy in mediating tumor response to cancer therapy
2018, Biochemical Pharmacology
Citation Excerpt :
As an example, kidney and liver tumors treated with high doses of IL-2 showed regression upon inhibition of autophagy with chloroquine [42]. Although autophagy mediated release of HMGB1 can attract cytotoxic T-lymphocytes, under conditions of constitutively elevated levels promoted by systemic autophagy, HMGB1 also attracts T-regs [43] and MDSC [44], resulting in immunosuppression. One argument for the potential involvement of the immune system in the response to conventional therapeutics is the abscopal effect in radiation therapy [45,46].
Autophagy, a virtually uniform response to external stress such as that induced by chemotherapy and radiation, is generally considered to be cytoprotective in function, providing a foundation for multiple clinical trials designed to enhance therapeutic response via autophagy inhibition. However, this cell autonomous response can also be cytotoxic or nonprotective, with the consequence that autophagy inhibition would be counterproductive or ineffective, respectively. The non-cell autonomous function of autophagy remains quite controversial, with evidence both for and against autophagy-mediated activation of the immune system. If autophagy inhibition antagonizes the immune response, this would likely interfere with the potential sensitization resulting from suppression of the cell autonomous protective function. An additional complication, which has rarely been considered, is the nature of the contribution of therapy-induced autophagy in the tumor microenvironment, particularly the tumor stroma. Taken together, it is likely that the outcome of the current clinical trials, whether positive or negative, will be difficult to interpret given the complexity of the role of autophagy relating to the tumor cell (cell autonomous), the immune system (cell non-autonomous) and the tumor microenvironment.
HMGB1 regulates T helper 2 and T helper17 cell differentiation both directly and indirectly in asthmatic mice
2018, Molecular Immunology
The Th (T helper) 2 response is characteristic of allergic asthma, and Th17 cells are involved in more severe asthma. Recent studies demonstrated that HMGB1 (High mobility group box 1 protein) regulates airway inflammation and the Th2, Th17 inflammatory response in asthma. HMGB1 can interact with Toll-like receptors (TLR) 2 and 4, and the receptor for advanced glycation end products (RAGE), activating the NF-κB (nuclear factor kappa B) signaling pathway and inducing the release of downstream inflammatory mediators. Both Th cells and dendritic cells express TLR2, TLR4, and RAGE receptors. Therefore, we speculate that HMGB1 could regulate the differentiation of Th2, Th17 cells in asthma through direct and indirect mechanisms. An ovalbumin (OVA)-induced mouse asthmatic model was established. Anti-HMGB1 antibody or rHMGB1 was administered to OVA-sensitized mice 30 min prior to each challenge. For in vitro studies, magnetically separated CD4⁺ naive T cells were stimulated with or without rHMGB1 and/or anti-HMGB1 antibody. BMDCs (bone marrow-derived dendritic cells)-stimulated with or without rHMGB1 and/or anti-HMGB1 antibody were cocultured with CD4⁺ naive T cells. Our study showed that administration of rHMGB1 aggravated airway inflammation and mucus production, and induced Th2, Th17 polarization in asthmatic mice, and that anti-HMGB1 antibody weakened characteristic features of asthma and blocked the Th2, Th17 inflammatory responses. HMGB1 could directly act on naive T cells to induce differentiation of Th2, Th17 cells in vitro through activating the TLR2, TLR4, RAGE-NF-κB signal pathway in CD4⁺ naive T cells. HMGB1 could also indirectly promote Th2, Th17 differentiation via activating the TLR2, TLR4, RAGE-NF-κB signal pathway in DCs to mediate their maturation and antigen-presenting ability in vitro.

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

Summary

Introduction

Section snippets

Patients and controls

HMGB1 is strongly elevated in tumor tissue and patients’ sera

Discussion

Acknowledgements

Lancet

Respir Med

Eur J Cancer

Methods

Curr Opin Immunol

Neoplasia

Biochim Biophys Acta

Biochim Biophys Acta

Immunobiology of head and neck cancer

Cancer Metastasis Rev

Inflammation and necrosis promote tumour growth

Nat Rev Immunol

High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal

Nat Rev Immunol

Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine

J Immunol

HMGB1 and RAGE in inflammation and cancer

Annu Rev Immunol

Masquerader: high mobility group box-1 and cancer

Clin Cancer Res

Overexpression of high mobility group box 1 in gastrointestinal stromal tumors with KIT mutation

Cancer Res

Co-expression of receptor for advanced glycation end products and the ligand amphoterin associates closely with metastasis of colorectal cancer

Oncol Rep

Increased expression of high mobility group box 1 (HMGB1) is associated with progression and poor prognosis in human nasopharyngeal carcinoma

J Pathol

Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer

Oncol Rep