HMGB1 converts cellular damage into inflammatory and chemotactic signals and regulates tumor immunity, tumor stemness, and therapy tolerance through TLRs, RAGE, and CXCR4.

Keywords: HMGB1; damage-associated molecular pattern; DAMP; TLR4; RAGE; CXCL12; CXCR4; NF-κB; MAPK; tumor immunity; tumor stemness; chemoresistance

1 Molecular States of HMGB1 and Its Damage-Associated Molecular Pattern Function

1.1 Structure and Intracellular Function of HMGB1

High mobility group box 1 (HMGB1) is a non-histone chromatin-binding protein composed of an A box, a B box, and an acidic C-terminal tail enriched in acidic amino acids.

(1) A box

The A box participates in DNA structure recognition and protein interactions. Free A box can competitively inhibit the extracellular pro-inflammatory activity of full-length HMGB1 in some experimental systems.

(2) B box

The B box contains an important pro-inflammatory functional region of HMGB1 and can participate in activation of receptor complexes such as TLR4/MD-2.

(3) Acidic C-terminal tail

The acidic C-terminal tail regulates HMGB1 conformation and its binding ability to DNA and other proteins.

In the nucleus, HMGB1 can bind bent or damaged DNA and participate in chromatin conformation regulation, transcription, and DNA damage response. After HMGB1 translocates to the cytoplasm or is released into the extracellular space, its functional focus shifts from chromatin regulation to inflammatory, chemotactic, and tissue injury signal transmission.

1.2 Redox State Determines the Extracellular Function of HMGB1

HMGB1 contains three cysteine residues, Cys23, Cys45, and Cys106. Different oxidation states correspond to different receptor preferences and biological functions.

Table 1. HMGB1 Redox States and Main Functions

1.3 DAMP Function of HMGB1

After cellular injury, extracellular HMGB1 can act as a damage-associated molecular pattern, converting cellular and tissue damage into innate immune signals.

(1) Activates macrophages, dendritic cells, and neutrophils.

(2) Promotes the expression of inflammatory cytokines, chemokines, and adhesion molecules.

(3) Regulates immune cell migration toward injured regions or the tumor microenvironment.

(4) Participates in chronic inflammation, vascular changes, and tissue remodeling when persistently released.

2 HMGB1 Release Mechanisms and Extracellular Inflammatory Effects

2.1 Passive Release Caused by Injury and Lytic Cell Death

Necrosis, pyroptosis, and other forms of cell death accompanied by plasma membrane rupture can release nuclear HMGB1 into the extracellular space. Tumor hypoxia, mechanical injury, chemotherapy, and radiotherapy may all increase passive HMGB1 release.

Early apoptotic cells usually still maintain plasma membrane integrity, and HMGB1 does not immediately leak out in large amounts. If apoptotic cells are not cleared in time and undergo secondary membrane rupture, HMGB1 can also be released extracellularly. Different modes of cell death and oxidative environments can alter the molecular state and receptor activity of HMGB1.

2.2 Active Release from Immune Cells

After stimulation by pathogen components, inflammatory cytokines, or tissue injury signals, monocytes, macrophages, and dendritic cells can promote HMGB1 translocation from the nucleus to the cytoplasm and release it through non-classical secretory pathways.

(1) Post-translational modifications

Modifications such as lysine acetylation can reduce the nuclear localization capacity of HMGB1 and promote its accumulation in the cytoplasm.

(2) Cellular stress

Oxidative stress, DNA damage, and inflammatory signaling can regulate HMGB1 nuclear-cytoplasmic translocation and extracellular release.

(3) Non-classical secretory pathways

Secretory lysosomes, extracellular vesicles, and changes in membrane permeability can all participate in HMGB1 release.

2.3 Main Effector Cells of Extracellular HMGB1

(1) Macrophages

HMGB1 can induce the expression of inflammatory cytokines and chemokines and alter macrophage functional states in the tumor microenvironment.

(2) Dendritic cells

Therapy-induced HMGB1 release can influence dendritic cell uptake, processing, and presentation of tumor antigens and participate in adaptive immune initiation after immunogenic cell death.

(3) Vascular endothelial cells

HMGB1 can promote endothelial cell activation, adhesion molecule expression, and changes in vascular permeability, providing conditions for immune cell recruitment and tumor vascular remodeling.

(4) Tumor cells and stromal cells

Tumor cells, fibroblasts, and other stromal cells can respond to HMGB1 through RAGE-, TLR-, or CXCR4-related signaling, altering proliferation, migration, and therapy sensitivity.

3 HMGB1-TLRs/RAGE Signaling and Activation of NF-κB and MAPK Pathways

3.1 TLR4/MD-2-Mediated Inflammatory Response

Disulfide HMGB1 can interact with MD-2 and activate the TLR4 receptor complex. TLR4 transmits signals downstream through molecules such as MyD88, IRAK, and TRAF6, further activating IKK/NF-κB and MAPK pathways.

(1) Promotes the expression of inflammatory cytokines such as TNF, IL-1β, and IL-6.

(2) Enhances dendritic cell maturation and antigen presentation.

(3) Increases chemokine and adhesion molecule expression.

(4) Promotes inflammatory cell recruitment into injured tissues or tumor tissues.

TLR2 may also participate in cellular responses induced by some HMGB1 complexes. Endotoxin, nucleic acids, or other bound molecules in recombinant HMGB1 can interfere with TLR experiments, so endotoxin controls and receptor blockade groups should be included.

3.2 RAGE-Mediated Sustained Signaling

The receptor for advanced glycation end products, RAGE, is encoded by the AGER gene and can be expressed in myeloid cells, vascular endothelial cells, and many tumor cells. After HMGB1 binds RAGE, it can regulate NF-κB, ERK, p38, PI3K/AKT, and other signaling pathways.

(1) Promotes survival, proliferation, migration, and invasion of some tumor cells.

(2) Regulates the function of tumor-associated macrophages and stromal cells.

(3) Participates in angiogenesis, lymphangiogenesis, and tumor tissue remodeling.

(4) Affects autophagy, mitochondrial dynamics, and therapy tolerance.

3.3 NF-κB and MAPK Signal Output

(1) NF-κB pathway

IKK activation promotes IκBα phosphorylation and degradation, allowing p65/p50 to enter the nucleus and regulate the expression of inflammatory cytokines, chemokines, anti-apoptotic proteins, and immunoregulatory molecules.

(2) p38 MAPK pathway

The p38 MAPK pathway participates in inflammatory cytokine transcription, mRNA stability, and cellular stress responses and is closely related to inflammatory activation of myeloid cells.

(3) ERK pathway

The ERK pathway participates in tumor cell proliferation, migration, mitochondrial fission, and some therapy tolerance processes.

(4) JNK/AP-1 pathway

The JNK/AP-1 pathway participates in stress responses and inflammatory gene transcription and can cooperate with NF-κB to regulate HMGB1-mediated inflammatory output.

4 HMGB1-CXCL12-CXCR4 Axis Regulates Cell Migration

4.1 HMGB1 Forms a Heterocomplex with CXCL12

Fully reduced HMGB1 can form a heterocomplex with CXCL12 and enhance the interaction between CXCL12 and CXCR4, allowing cells to generate stronger chemotactic responses at lower CXCL12 concentrations.

The HMGB1-CXCL12 complex mainly enhances CXCR4-dependent cell migration, which differs from the induction of inflammatory cytokines by disulfide HMGB1 through TLR4. In experiments, cell migration and inflammatory cytokine release should be evaluated separately.

4.2 Migration of Immune Cells and Repair Cells

(1) Recruits monocytes and other immune cells into injured regions.

(2) Regulates lymphocyte localization in inflammatory and tumor tissues.

(3) Recruits mesenchymal stem cells and progenitor cells related to tissue repair.

(4) Enhances chemotactic migration of CXCR4-positive tumor cells.

4.3 Tumor Migration and Spatial Distribution of Immune Cells

CXCR4-positive tumor cells can respond to CXCL12 concentration gradients and migrate toward tissues rich in CXCL12, such as lymph nodes, bone marrow, lung, or liver. After HMGB1 enhances CXCL12/CXCR4 signaling, migration and invasion capacity may increase in some tumor cells.

The CXCL12/CXCR4 axis can also affect the distribution of regulatory T cells, myeloid-derived suppressor cells, macrophages, and effector T cells in tumor tissues. Related research can combine Transwell migration assays, chemotaxis assays, and analysis of immune cell infiltration in tumor tissues.

Figure 1. HMGB1 receptor signaling and its inflammatory and chemotactic effects

5 HMGB1 Regulates Tumor Inflammation, Tumor Stemness, and Therapy Tolerance

5.1 Dual Roles of HMGB1 in Tumor Immunity

(1) Promotion of antitumor immunity

Some chemotherapy, radiotherapy, and other forms of immunogenic cell death can induce HMGB1 release, promote dendritic cell processing of tumor antigens, and support tumor-specific T cell responses.

(2) Maintenance of pro-tumor inflammation

When tumor tissues persistently release HMGB1, RAGE and TLRs can maintain inflammatory cytokine expression, vascular changes, and stromal remodeling.

(3) Promotion of immunosuppression

Chronic HMGB1 signaling can alter the state of tumor-associated macrophages and other myeloid cells, limiting effector T cell infiltration and function.

5.2 Tumor Stemness and Self-Renewal

HMGB1 can affect survival, self-renewal, and tumorigenic capacity in some tumor cells through RAGE, TLR4, ERK, PI3K/AKT, and other signaling pathways. Its effects are tumor type- and cell context-dependent.

HMGB1-related tumor stemness research can detect:

(1) Stemness markers such as SOX2, NANOG, and OCT4.

(2) Tumor sphere formation and serial passaging capacity.

(3) Limiting dilution tumorigenic capacity.

(4) Changes in stemness phenotypes after HMGB1, TLR4, or RAGE intervention.

5.3 HMGB1-Mediated Chemoresistance

(1) Protective autophagy

Cytoplasmic or extracellular HMGB1 can promote autophagy-related responses, providing metabolic adaptation and organelle quality control for damaged tumor cells and reducing the sensitivity of some tumors to therapy.

(2) Nucleophagy

In some tumor models, HMGB1 can participate in nucleophagy and DNA damage handling, helping tumor cells tolerate chemotherapy-induced nuclear damage.

(3) Mitochondrial fission

HMGB1 released from dying tumor cells can regulate DRP1 activity and mitochondrial fission through RAGE-ERK signaling, supporting the survival and regrowth of residual tumor cells.

(4) Microenvironmental protection

HMGB1 can regulate macrophages and tumor-associated fibroblasts, providing support for tumor cell survival, repair, and therapy escape.

6 HMGB1-Targeted Intervention and Immune Checkpoint Therapy

6.1 Direct HMGB1 Intervention

(1) HMGB1 neutralization or pharmacological inhibition

Blocking extracellular HMGB1 activity can be used to verify whether inflammatory factor release, cell migration, and therapy tolerance depend on HMGB1.

(2) HMGB1 gene intervention

siRNA or gene knockout can be used to reduce overall HMGB1 expression and determine whether a phenotype depends on HMGB1.

(3) Inhibition of nuclear-cytoplasmic translocation and release

Regulating HMGB1 acetylation, oxidative stress, or non-classical secretion can reduce extracellular HMGB1 accumulation.

(4) Interference with the HMGB1-CXCL12 complex

Selective reduction of CXCR4-dependent chemotaxis can be used to evaluate the contribution of this complex to immune cell recruitment and tumor migration.

6.2 Receptor and Downstream Signal Intervention

(1) Blocking TLR4/MD-2 to inhibit inflammatory cytokine expression mediated by disulfide HMGB1.

(2) Blocking RAGE to reduce sustained NF-κB, MAPK, tumor cell survival, and therapy tolerance signaling.

(3) Inhibiting CXCR4 to restrict directional migration of tumor cells or immunosuppressive cells.

(4) Inhibiting IKK/NF-κB, ERK, or p38 to verify the role of specific downstream branches in inflammatory and tumor phenotypes.

6.3 Combined Mechanisms of HMGB1 and Immune Checkpoint Therapy

(1) Immune activation during early treatment

After chemotherapy or radiotherapy induces tumor cell death, HMGB1 release can promote dendritic cell antigen processing and provide a basis for antigen release and innate immune activation for immune checkpoint blockade such as PD-1/PD-L1 targeting.

(2) Immunosuppression mediated by chronic signaling

Persistent HMGB1/RAGE or HMGB1/TLR signaling can maintain myeloid cell accumulation, chronic inflammation, and tumor cell survival, limiting the efficacy of immune checkpoint therapy.

(3) Intervention window

The timing and intensity of HMGB1 blockade need to be determined according to treatment modality, HMGB1 redox state, dominant receptor, and major effector cells in the tumor microenvironment.

7 Products Related to HMGB1 Damage-Associated Inflammation and Tumor Immunity Research

The key to HMGB1 research is to distinguish its intracellular and extracellular localization, redox state, and dominant receptor, thereby determining whether it mainly participates in damage recognition, immune activation, tumor migration, or therapy tolerance.

For more related articles, please see below:

[1] Interleukin Receptor Signaling: Mechanistic Analysis of the JAK-STAT and NF-κB Axes with an Overview of Assays, Experimental Models, and Translational Boundaries

[2] How to Map the NF-κB Pathway and Choose Inhibitors: Bringing Inflammatory Transcriptional Output into a “Controllable Range” (Tables A–F)

[3] Ras-Raf-MEK-ERK Signaling

[4] ALK Inhibitors

[5] PI3K-Akt-mTORC Signaling