The SMAD-Pathway Mediates HMGB1-Induced Proliferation and Metastatic Progression in Cutaneous Squamous Cell Carcinoma Cells

Abstract

Background

High-mobility group box protein 1 (HMGB1) is a chromatin-binding protein involved in arthritis, ischemia, sepsis, atherosclerosis, neurodegenerative disorders, meningitis, and cancer. HMGB1 exhibits dual roles in cancer, acting as either a tumor suppressor or oncoprotein depending on context.

Objective

This research aimed to elucidate HMGB1’s functional significance in cutaneous squamous cell carcinoma (cSCC).

Methods

We overexpressed HMGB1 in cSCC cell lines using recombinant adenovirus and examined its effects on cell proliferation, colony formation, and cell migration.

Results

Immunohistochemical analysis revealed elevated HMGB1 expression levels in cSCC tissue relative to normal epidermis. To assess the influence of HMGB1, we employed recombinant adenoviruses expressing HMGB1 to transduce SCC cell lines (SCC12 and SCC13). Enhanced HMGB1 expression significantly promoted cellular proliferation and colony formation capacity. Notably, HMGB1 overexpression elevated the levels of proliferation regulators, including P63, SOX2, CDK4 and CDK6. Furthermore, HMGB1 overexpression substantially enhanced tumor invasiveness, accompanied by upregulation of epithelial-mesenchymal transition (EMT) biomarkers. Mechanistically, overexpression of HMGB1 enhanced transforming growth factor-β signaling by increasing phosphorylation of SMAD2/3, the key mediators of EMT.

Conclusion

These data imply that HMGB1 acts as a tumor-promoting factor in cSCC.

INTRODUCTION

Cutaneous squamous cell carcinomas (cSCCs) represent malignant tumors derived from keratinocytes, specifically those situated in the epidermal layer or associated cutaneous appendage structures, which has become more common in recent years¹,². It ranks as the second most deadly form of skin cancer after melanoma, and accounts for most skin cancer-related deaths in individuals over age eighty-five². Its development is influenced by both environmental and intrinsic risk factors. Key intrinsic factors include advanced age, male gender, immunosuppression, β-human papilloma virus infection, tobacco use, genetic predisposition (such as fair skin and certain genetic syndromes), and prior actinic keratosis. Meanwhile, prolonged sun exposure is the most critical and well-documented environmental risk for cSCC³,⁴,⁵,⁶,⁷,⁸. Given the rising incidence of cSCC and its potential for recurrence and metastasis, increasing our understanding of the molecular mechanisms of this disease is critical to improving existing therapies. While the biological functions of numerous candidate genes have been identified, this knowledge remains inadequate for a complete understanding of pathogenic mechanisms of cSCC.

High-mobility group box protein 1 (HMGB1), a widely expressed nuclear-localized protein identified in mammalian species⁹,¹⁰, plays a critical role in maintaining genomic integrity through its DNA-binding motifs and participation in DNA repair mechanisms¹¹. The extracellular release of HMGB1 occurs either through granulocyte secretion or necrotic cell leakage, enabling its function as a damage-associated molecular pattern that mediates chemotaxis in various pathological states including cancer and ischemic events¹². Studies have confirmed HMGB1 overexpression and increased serum HMGB1 levels in various cancers, spanning lymphoid neoplasms (non-Hodgkin’s lymphoma), epithelial cancers (hepatobiliary, gastrointestinal, cervical, breast), mesenchymal tumors (osteosarcoma), and melanocytic (melanoma) as well as mesothelial malignancies¹³. Elevated serum HMGB1 level in colorectal cancer patients demonstrates a positive association with lymph node metastasis, and immunohistochemical profiling reveals concurrent upregulation of HMGB1, pERK, and c-IAP2 in colorectal cancer tissues, potentially indicating HMGB1-mediated suppression of apoptotic pathways in malignant cells¹⁴. HMGB1-mediated chronic inflammation serves as a pivotal mechanism underlying asbestos-induced tumorigenesis and the tumorigenesis of mesothelioma¹⁵. Comprehensive assessment of 18 research reports inculuding 11 cancer types shows a statistically significant association between HMGB1 overexpression and worse prognostic indicators¹⁶. Although HMGB1 has been implicated in many cancers, our understanding of how HMGB1 specifically influences the risk profile, disease progression, and metastatic mechanisms of cSCC remains incomplete. Our experimental paradigm combining adenoviral delivery systems with cSCC cell lines reveals novel insights into HMGB1-mediated progression of cSCC.

MATERIALS AND METHODS

Immunohistochemistry

Tissue specimens were fixed in 10% neutral buffered formalin and subsequently paraffin-embedded. Following deparaffinization, tissue sections were rehydrated through an ethanol series and rinsed 3 times with PBS. Immunostaining was performed using a primary antibody targeting HMGB1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) subjected to 4°C overnight incubation, followed by incubation in horseradish peroxidase-linked secondary antibody (Dako, Carpinteria, CA, USA) and visualization through the Dako ChemMate EnVision Detection System.

Cell culture

SCC12/SCC13 are authenticated human cell lines established from facial cSCC tumors¹⁷. Both cell lines were propagated in Dulbecco’s modified Eagle’s medium enriched with 5% fetal bovine serum (FBS) plus penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA).

Generation of recombinant adenovirus

The HMGB1 cDNA was amplified employing reverse transcription-polymerase chain reaction (PCR) amplification. Total RNA was harvested from cultured normal keratinocytes utilizing the easy-blue RNA extraction kit (Intron Biotechnology, Seongnam, Korea). Two micrograms of RNA were reverse translated using MMLV cDNA synthesis enzyme (Elpis Biotech, Daejeon, Korea). A portion mixture amplified by PCR cycles with primers for HMGB1: 5'-AATTGGATCCATGGGCAAAGGAGATCCTAA and 5'-AATTCTCGAGTTATTCATCATCATCATCTT. The resulting full-length cDNA amplicon of HMGB1 was cloned into pENT/CMV-green fluorescent protein (GFP) plasmid, duplication-deficient adenovirus was generated using the Virapower Adenoviral Expression System (Thermo Fisher Scientific).

Western blot

Cell lysis was performed in Proprep solution (Intron Biotechnology), and total protein were determined quantified by BCA assay (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose membranes. After incubating with 5% skim milk, membranes were exposed to primary antibodies, then incubated with horseradish peroxidase-conjugated secondary antibodies, finally analyzed by intensified chemiluminescence (Intron Biotechnology). Primary antibodies utilized included: HMGB1 (Santa Cruz Biotechnology); CDK4, CDK6, E-Cadherin, Vimentin, N-Cadherin, Snail, Twist, pSMAD2, SMAD2, pSMAD3, SMAD3 (Cell Signaling Technology, Danvers, MA, USA); P63, Slug (Abcam, Cambridge, UK); β-Actin (Sigma-Aldrich, St Louis, MO).

Cell growth assay

To assess cell growth, 2×10⁴ cells were plated in-millimeter culture dishes and transduced with adenovirus at an multiplicity of infection of 10 overnight. Following replacement with new medium, SCC12 and SCC13 cells were expanded for an supplementary 3 days. Cell counts were obtained by trypsinization and hemocytometer enumeration at specified time points.

Colony forming assay

Cells were first transduced with adenovirus for 24 hours before medium replacement. After 48 hours of additional culture, cells were trypsinized, then plated at low density (1×10³ cells per 100-mm dish). Following 14–21 days of undisturbed growth, colonies were fixed and visualized using 0.5% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA).

Invasion assay

The invasion assay was conducted via the Chemicon cell invasion assay kit (Merck KGaA, Darmstadt, Germany). Following adenovirus transduction, cells were suspended in serum-deprived medium and seeded in the apical compartment, while the lower compartment was filled with medium supplemented with 5% FBS. After 48–72 hours of incubation, upper chamber-retained cells and the ECMatrix were removed with cotton swabs. Transmigrated cells were then stained for visualization. For measurement, stained cells were solvated in 10% acetic acid and then subjected to colorimetric reading of optical density at 560 nm.

Statistical analysis

Statistical analysis was carried out with SPSS software v22.0 (IBM Corporate, Armonk, NY, USA), applying one-way analysis of variance or t-test. A p-value<0.05 was deemed statistically significant.

RESULTS

Expression of HMGB1 in SCC

To assess HMGB1 expression, we began by conducting immunohistochemical staining on skin samples collected from patients with SCC. While HMGB1 immunoreactivity was weak or patchy in the epidermis and vessels of adjacent normal tissue, it was markedly upregulated in SCC lesions (Fig. 1A). In agreement with our immunohistochemical results, cultured cSCC cells (SCC12 and SCC13) exhibited higher HMGB1 protein levels compared to normal skin cells (Fig. 1B).

Fig. 1. Detection of HMGB1 expression in cutaneous SCC. (A) Skin samples were collected from SCC patients and immunohistochemically stained with an anti-HMGB1 antibody. The normal regions of the skin samples exhibited weak immunoreactivity, whereas the adjacent SCC regions showed strong staining. (B) Expression of HMGB1 in cultured skin cells was assessed by Western blot.The study utilized SCC12 and SCC13, 2 established cutaneous SCC cell lines.

SCC: squamous cell carcinoma, HMGB1: high-mobility group box protein 1.

Influence of HMGB1 on cell growth and colony-forming activity of SCC cells

To assess HMGB1 function in SCC, we generated a recombinant adenovirus (Ad/GFP-HMGB1) for HMGB1 overexpression. Transduction of cSCC lines (SCC12 and SCC13) with Ad/GFP-HMGB1 substantially elevated HMGB1 levels compared to Ad/GFP control adenovirus (Fig. 2A). To evaluate the effect of HMGB1 on proliferation, we next performed cell growth assay. We found that HMGB1 overexpression significantly enhanced proliferation in both SCC cell lines (Fig. 2B). Then we evaluated colony forming activity, which can be an in vitro tumorigenicity indicator¹⁸. As a result, HMGB1 overexpression appreciably increased the colony forming activity relative to adenovirus-treated controls (Fig. 2C). Finally, we investigated whether HMGB1 affects proliferation regulators. HMGB1 overexpression significantly elevated proliferation regulators including P63, SOX2, CDK4 and CDK6 (Fig. 2D).

Fig. 2. Influence of HMGB1 on cellular proliferation and colony-forming capacity. (A) Adenoviral vectors expressing protein HMGB1 (Ad/GFP- HMGB1) were used to transduce SCC12 and SCC13 cells, while Ad/GFP served as the negative control. Western blotting demonstrated efficient HMGB1 overexpression in both cell types. (B) Impact of HMGB1 on cell proliferation. Elevated expression of HMGB1 promoted cell growth. Results are shown as mean ± standard deviation of triplicate measurements. (C) Colony forming assay. Elevated HMGB1 expression enhanced colony formation capacity. (D) Influence of HMGB1 on cellular proliferation markers. Elevated expression of HMGB1 upregulated the levels of P63, SOX2, CDK6 and CDK4.

SCC: squamous cell carcinoma, GFP: green fluorescent protein, HMGB1: high-mobility group box protein 1.

^*p<0.05.

Effect of HMGB1 on invasion and migration of SCC cells

Given that invasion and migration play important roles in tumor evolution, we analyzed the role of HMGB1 on SCC cell invasion. HMGB1 overexpression strongly enhanced invasive capacity (Fig. 3A). Likewise, HMGB1 overexpression enhanced cell migration (Fig. 3B). Then, we investigated the effect of HMGB1 on the protein levels of epithelial-mesenchymal transition (EMT) markers. E-Cadherin loss is a hallmark of EMT, while N-Cadherin, Vimentin, Snail, and Slug are typically upregulated¹⁹. Interestingly, HMGB1 overexpression decreased E-Cadherin, whereas mildly increased those EMT inducers (Fig. 3C). Collectively, these findings indicate that HMGB1 exerts tumor-promoting effects in cSCC cells.

Fig. 3. Impact of HMGB1 on cellular invasion and migration capacity. (A) After adenovirus-mediated gene delivery, Matrigel invasion assays demonstrated that HMGB1 overexpression significantly increased invasiveness. Results are shown as mean ± SD of triplicate measurements. (B) After adenovirus-mediated gene delivery, a uniform wound was created in the cell monolayer with a sterile pipette tip. Wound closure rate was quantified by measuring the percentage reduction in wound area at 12 hours relative to baseline. Results are shown as mean ± SD of triplicate measurements. (C) Influence of HMGB1 on key epithelial-mesenchymal transition markers. Elevated expression of HMGB1 upregulated EMT marker expression.

SCC: squamous cell carcinoma, GFP: green fluorescent protein, HMGB1: high-mobility group box protein 1, SD: standard deviation.

^*p<0.05.

Regulatory role of HMGB1 in modulating key intracellular signaling mediators

The transforming growth factor-β (TGF-β) signaling cascade is widely recognized as the primary trigger for EMT²⁰. To elucidate the molecular mechanisms underlying the tumor-promoting activity mediated by HMGB1, we examined the impact of TGF-β on EMT markers. TGF-β decreased E-Cadherin in a concentration-dependent manner, whereas increased EMT inducers including N-Cadherin, Vimentin, Slug, Twist, and Snail. This molecular profile is characteristic of enhanced EMT phenotype (Fig. 4A). The regulatory mechanisms of TGF-β signaling involve canonical SMAD-dependent pathways. Therefore, we investigated the potential effects of HMGB1 on SMAD signaling. Overexpression of HMGB1 increased the phosphorylation of SMAD2 and SMAD3 (Fig. 4B). Our data indicate that HMGB1 positively affects EMT process by stimulating SMAD signaling.

Fig. 4. (A) Impact of TGF-β on epithelial-mesenchymal transition Markers. TGF-β treatment downregulated E-cadherin expression while upregulating N-cadherin, vimentin, Slug, Twist, and Snail levels. (B) Impact of HMGB1 on cytoplasmic signaling cascades. Elevated expression of HMGB1 enhanced phosphorylation of SMAD2 and SMAD3.

SCC: squamous cell carcinoma, TGF-β: transforming growth factor-β, GFP: green fluorescent protein, HMGB1: high-mobility group box protein 1.

DISCUSSION

Our study revealed HMGB1 as a key oncogenic driver in cSCC. Comparative analysis revealed significant HMGB1 upregulation in SCC versus normal skin. Functional studies demonstrated that HMGB1 overexpression enhanced multiple malignant phenotypes, including proliferative capacity, clonogenic survival, invasive potential, and EMT progression. Mechanistically, HMGB1 activated SMAD signaling, thereby promoting tumorigenic behaviors in cSCC cells.

The nuclear protein HMGB1 exhibits both evolutionary conservation and universal expression across tissues, where it serves dual functions in maintaining nucleosome architecture and coordinating essential DNA transactions encompassing replication, transcriptional regulation, genetic recombination and damage repair²¹. HMGB1 in cancer biology present an intriguing paradox. Experimental data demonstrate that HMGB1 can paradoxically serve as either a tumor suppressor or oncogenic factor in cancer development and therapy, with its role contingent upon specific experimental conditions. Pro-tumorigenic effects are particularly evident under conditions of chronic inflammation, where sustained HMGB1 overexpression promotes malignant transformation. In the tumor microenvironment of developed malignancies, tumor-derived HMGB1 may amplify inflammation-mediated immunosuppressive responses. Experimental evidence demonstrates that HMGB1 mediates lipopolysaccharide-triggered secretion of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α), thereby establishing a permissive microenvironment for colorectal cancer advancement²². Paradoxically, HMGB1 also demonstrates protective anti-tumor effects, particularly in the context of cancer treatment. Within the nucleus, HMGB1 participates in telomere regulation and genomic stability preservation. Loss of HMGB1 function induces genomic instability and facilitates tumor development²³. Our data illustrate that HMGB1 enhances malignant phenotypes in cSCC cells, corroborating its oncogenic function in SCC progression.

EMT, a phylogenetically ancient developmental process, plays a pivotal role in tumorigenesis by endowing malignant cells with migratory and invasive capacities, alongside heightened resistance to programmed cell death triggers²⁴. During EMT, cells characteristically downregulate epithelial markers (e.g., ZO-1, E-Cadherin, occludin) while enhancing mesenchymal markers (e.g., N-Cadherin, vimentin, fibronectin), thereby acquiring migratory and invasive properties²⁵. To elucidate the hypothesized pathways via which HMGB1 drives tumor advancement, we systematically explored and demonstrated that HMGB1 promotes the progression of EMT by influencing the EMT marker molecules N-Cadherin, E-Cadherin, Snail, Vimentin, and Slug. The EMT process is regulated by several critical signaling networks, notably the TGF-β, Wnt, Notch, and Hedgehog pathways, which are well-documented mediators of this cellular transition²⁶. Activation of TGF-β receptors initiates 2 distinct signaling branches: a SMAD-mediated pathway and a SMAD-independent route. Within the SMAD-dependent cascade, the SMAD complex translocates to the nucleus upon receptor engagement, directly inducing transcription of mesenchymal markers, including vimentin, through targeted gene regulation²⁷. In our study, HMGB1 overexpression significantly enhanced phosphorylation of SMAD2 and SMAD3. These results suggest that HMGB1 promotes activation of TGF-β/SMAD signaling, thereby increasing the invasiveness and metastatic potential of cSCC. Several mechanisms can be considered for how HMGB1 overexpression can increase the phosphorylation of SMAD2 and SMAD3. First, when HMGB1 is overexpressed and its amount in the nucleus increases, HMGB1 can translocate to the cytoplasm and be secreted outside the cells. HMGB1 can then increase TGF-β1 expression through autocrine/paracrine signaling or promote the activation of the latent TGF-β complex. Consequently, the TGF-β → TGF-βRII/TGF-βRI → SMAD2/3 phosphorylation pathway can be activated. Second, activation of the Receptor for Advanced Glycation End-Products (RAGE) pathway can be considered. HMGB1 binds to RAGE and activates various mitogen-activated protein kinases (MAPKs) (ERK, JNK, p38) and NF-κB signaling. MAPK activation can amplify TGF-β receptor signaling or assist in the phosphorylation of SMAD2/3. In particular, ERK/p38 can regulate the transcriptional activity of SMAD by increasing the phosphorylation of the SMAD. Third, HMGB1 can bind to TLR2/4 and activate the MyD88–IRAK–TRAF6–NF-κB pathway. NF-κB activation can increase the expression of growth factors such as TGF-β1, TGF-βR, platelet-derived growth factor, and vascular endothelial growth factor, ultimately enhancing SMAD2/3 phosphorylation. Further research is needed to determine the precise mechanism by which HMGB1 overexpression increases the phosphorylation of SMAD2/3 in SCC cells.

In conclusion, our findings reveal that HMGB1 enhances the tumorigenic activity of cSCC cells by modulating SMAD signaling. These results offer novel perspectives for future research into the molecular mechanisms driving SCC progression.