Abstract
Background/purpose
Heat shock factor 1 (HSF1) regulates heat shock proteins and stress-responsive genes and has been implicated in tumor progression and poor prognosis in several malignancies. However, the clinicopathological significance and biological role of HSF1 in oral squamous cell carcinoma (OSCC) remain unclear. This study investigated HSF1 expression in OSCC tissues, its association with clinicopathological parameters and survival, and its functional role in OSCC cell migration.
Materials and methods
HSF1 expression was evaluated by immunohistochemical staining in 80 OSCC specimens and 20 normal oral mucosa samples. HSF1 immunoreactivity was quantified using the labeling index (LI). Associations between HSF1 LI and clinicopathological parameters were analyzed. Kaplan–Meier survival analysis and Cox proportional hazards regression were performed. The effect of HSF1 downregulation on OSCC cell migration was examined in vitro.
Results
HSF1 expression was significantly increased in OSCC tissues compared with normal oral mucosa. Higher HSF1 LI was significantly associated with advanced T classification, nodal metastasis, and advanced clinical stage. Multivariate Cox regression analysis identified advanced T classification, positive nodal status, and high HSF1 LI as independent prognostic factors for overall survival. Patients with HSF1 LI > 53% showed increased mortality risk. In vitro experiments demonstrated that HSF1 downregulation reduced OSCC cell migration.
Conclusion
HSF1 is overexpressed in OSCC and is associated with tumor progression, advanced stage, and poor prognosis. HSF1 may also contribute to OSCC cell migration, suggesting its potential as a prognostic biomarker and therapeutic target in OSCC.
Keywords: Heat shock factor 1, Oral squamous cell carcinoma, Labeling index, Immunohistochemistry, Prognosis, Cell migration
Introduction
Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide and remains a major public health burden, particularly in regions with prevalent exposure to tobacco, alcohol, and betel quid.1 In Taiwan, OSCC is especially common among men.2 In 2020, the incidence of OSCC in Taiwanese males exceeded that of hepatocellular carcinoma, making it the third most common cancer in men. It is also the fourth leading cause of cancer-related death among males and the fifth leading cause of cancer mortality overall, regardless of sex.3
Although advances in diagnostic methods, surgical techniques, radiotherapy, chemotherapy, targeted therapy, and multidisciplinary care have improved the survival of patients with OSCC, treatment-related morbidity remains substantial.4 Survivors often experience long-term functional impairment and reduced quality of life, including xerostomia, tooth loss, malocclusion, trismus, dysphagia, speech dysfunction, and head and neck stiffness. 5 Patients undergoing extensive surgical resection may suffer from facial disfigurement and impaired mastication, swallowing, and articulation.6 In addition, those receiving adjuvant chemoradiotherapy frequently experience severe mucositis-related pain during treatment, followed by chronic complications such as osteoradionecrosis and radiation-induced fibrosis of the head and neck musculature.7 These complications may persist long after tumor control has been achieved and can profoundly affect daily function and psychosocial well-being.8
Another major challenge in OSCC management is local tumor recurrence and the development of second primary malignancies. The concept of field cancerization suggests that the clinically normal-appearing oral mucosa surrounding the tumor may harbor molecular alterations induced by carcinogenic exposure, thereby predisposing patients to recurrent disease or independent secondary tumors.9 Therefore, beyond improving conventional treatment modalities, identifying molecular biomarkers and therapeutic targets related to tumor aggressiveness, recurrence, and survival has become an important objective in OSCC research.10,11
Heat shock factors (HSFs) are transcription factors that regulate the expression of heat shock proteins (HSPs) and other stress-responsive genes by binding to heat shock elements in DNA.12–14 These factors coordinate cellular responses to various physiological and environmental stresses, including heat shock, oxidative stress, proteotoxic stress, hypoxia, and oncogenic stress.15 Among the HSF family members, heat shock factor 1 (HSF1) is the best-characterized regulator of the heat shock response and plays a central role in maintaining proteostasis and cellular survival under stress conditions.16
In recent years, HSF1 has attracted increasing attention in cancer biology. Elevated HSF1 expression has been reported in multiple malignancies, including prostate cancer, 17 hepatocellular carcinoma,18,19 breast cancer,20 esophageal cancer,21 pancreatic cancer,22 hematologic malignancies,23 gastric cancer,24 and OSCC.25 Although HSF1 is not a classical oncogene, accumulating evidence indicates that it can support malignant transformation, promote tumor cell survival, and facilitate cancer progression. 26 Experimental studies have shown that HSF1 deficiency suppresses chemically induced skin carcinogenesis, inhibits tumor formation driven by mutant p53, affects RAS-mediated transformation, and modulates cancer-associated metabolic reprogramming.27 These findings suggest that HSF1 functions as a key stress-adaptive regulator that enables cancer cells to tolerate hostile tumor microenvironments and therapeutic stress.
Despite the growing evidence linking HSF1 to tumor progression, its clinicopathological significance and prognostic value in OSCC remain insufficiently defined. In particular, whether HSF1 expression is associated with aggressive tumor behavior, lymph node metastasis, advanced clinical stage, and patient survival requires further clarification. In addition, the potential functional contribution of HSF1 to OSCC cell behavior, such as migration, has not been fully explored.
Therefore, the present study investigated HSF1 expression in OSCC tissues and normal oral mucosa using immunohistochemical analysis. We further evaluated the association between HSF1 expression and clinicopathological parameters, including tumor classification, nodal status, clinical stage, recurrence, histological differentiation, and overall survival. In addition, we examined the effect of HSF1 downregulation on OSCC cell migration. Through this study, we aimed to determine whether HSF1 may serve as a biomarker of tumor aggressiveness and prognosis in OSCC, as well as a potential therapeutic target for future translational investigation.
Materials and methods
Patients
The study cohort consisted of patients with oral squamous cell carcinoma (OSCC) who were treated at the Department of Oral and Maxillofacial Surgery, National Taiwan University Hospital. A total of 80 patients with OSCC were included in this study. The cohort comprised 65 men and 15 women. Thirty patients were aged 50 years or younger, whereas 50 patients were older than 50 years.
The primary tumor sites included the buccal mucosa in 35 cases, the tongue in 25 cases, and other oral mucosal sites in 20 cases. According to tumor classification, 35 patients had T1–T2 disease, and 45 patients had T3–T4 disease. Regarding nodal status, 49 patients were classified as N0, whereas 31 patients had N1–N3 disease. Clinical staging showed that 33 patients had stage I–II disease, while 47 patients had stage III–IV disease. Tumor recurrence was observed in 25 patients, whereas 55 patients had no recurrence. Histologically, 68 tumors were classified as well-differentiated OSCC, and 12 were classified as moderately differentiated OSCC, no poorly differentiated tumors were identified in this cohort. These clinicopathological characteristics are summarized in Table 1.
Table 1.
Correlation between the HSF1 expression and clinicopathological parameters in patients with oral squamous cell carcinoma.
| Mean HSF1 LI ± SD (%) | P-Value | |
|---|---|---|
| Patients’ age (year) | ||
| ≦ 50 (n = 30) | 59 ± 31 | 0.162 |
| >50 (n = 50) | 56 ± 28 | |
| Patients’ gender | ||
| Men (n = 65) | 61 ± 32 | 0.655 |
| Women (n = 15) | 56 ± 36 | |
| Cancer location | ||
| Buccal mucosa (n = 35) | 55 ± 27 | 0.633 |
| Tongue (n = 25) | 60 ± 30 | |
| Other oral mucosal sites (n = 20) | 58 ± 35 | |
| T status | ||
| T1 + T2 (n = 35) | 49 ± 28 | < 0.001 |
| T3 + T4 (n = 45) | 63 ± 26 | |
| N status | ||
| N0 (n = 49) | 49 ± 23 | 0.010 |
| N1 + N2 + N3 (n = 31) | 62 ± 28 | |
| Clinical staging | ||
| Stage 1 + 2 (n = 33) | 46 ± 25 | < 0.001 |
| Stage 3 + 4 (n = 47) | 64 ± 28 | |
| Recurrence | ||
| With (n = 25) | 59 ± 34 | 0.385 |
| Without (n = 55) | 55 ± 28 | |
| Histology of OSCC | ||
| Well-differentiated (n = 68) | 60 ± 30 | 0.587 |
| Moderately-differentiated (n = 12) | 55 ± 27 | |
P value < 0.05 indicates statistically significant. Abbreviations: HSF1, heat shock factor 1; LI, labeling index; SD, standard deviation; OSCC, oral squamous cell carcinoma.
In addition, 20 specimens of normal oral mucosa (NOM) were included as non-tumor controls. HSF1 expression was evaluated by immunohistochemical staining in OSCC and NOM specimens, and the degree of immunoreactivity was quantified using the HSF1 labeling index (LI). HSF1 positivity was evaluated primarily based on nuclear staining. The labeling index was defined as the percentage of HSF1-positive tumor cells among the total number of tumor cells counted in five randomly selected high-power fields. Immunohistochemical evaluation was performed by two experienced observers blinded to the clinicopathological data.
Cancer staging was determined according to the American Joint Committee on Cancer staging system applicable at the time of diagnosis. All diagnoses were confirmed histopathologically. Tissue specimens were obtained with written informed consent from the patients, and the study protocol was conducted in accordance with institutional ethical requirements.
Immunohistochemical staining
Immediately after surgical excision, all specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin-embedded tissues were sectioned at a thickness of 4 μm. Immunohistochemical staining was performed using an immunoperoxidase technique as previously described by our group.28
Briefly, paraffin sections were deparaffinized and subjected to antigen retrieval in 0.01 M sodium citrate buffer (Antigen Retrieval Citra; BioGenex, San Ramon, CA, USA) with heat treatment. Endogenous peroxidase activity was blocked by incubation in 3% methanolic hydrogen peroxide solution. To prevent nonspecific background staining, sections were incubated with 10% normal goat serum (DAKO, Glostrup, Denmark).
The sections were then incubated overnight at 4 °C with a diluted anti-HSF1 primary antibody (1:100 dilution; Epitomics, Burlingame, CA, USA; Catalog #2043-1) Antigen–antibody complexes were detected using the Super Sensitive™ Polymer-HRP detection system (BioGenex, San Ramon, CA, USA), followed by visualization with chromogenic substrate. Counterstaining was performed using Mayer’s hematoxylin to facilitate nuclear identification under light microscopy.
Stained sections were examined under a light microscope. Immunoreactivity was evaluated using the positive labeling index. For each specimen, five randomly selected high-power fields (100×magnification) were analyzed. The proportion of HSF1-positive cells was assessed within tumor cell nests of OSCC (including basal, parabasal, and spinous cell layers), as well as in dysplastic epithelial cells and normal mucosal epithelial cells.
Cell migration assay
A Boyden chamber assay was used to evaluate cell migration and invasion with 8.0-μm ThinCert inserts (Greiner Bio-One, Monroe, NC, USA) in a 24-well plate. Cells (5 × 104) with serum-free DMEM seeded in the upper layer and 2% fetal bovine serum medium in the lower layer were used to study the migration activity of OSCC cells. Inserts coated with 3 mg/mL of Matrigel Matrix (BD Biosciences, San Jose, CA, USA) were used to study the invasion activity of OSCC cells. An OSCC cell suspension (1 × 105) in serum-free DMEM was seeded in a Matrigel Invasion Chamber.
Western blotting
OSCC cells were seeded on 6-well plates at a density of 2 × 105/plate. After 2 days of growth, cells were drug-treated, collected, and lysed in radioimmunoprecipitation assay buffer containing IX Protease Inhibitor Cocktail (MedChem Express, Monmouth Junction, NJ, USA). Samples were denatured with Tris-Glycine SDS sample buffer, boiled at 95 °C for 10 min, separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, and electrophoretically transferred to nitrocellulose membranes (BioTrace™ NT, Pall Corporation, Port Washington, NY, USA). Non-specific binding was blocked via incubation in 5% (w/v) non-fat milk in tris-buffered saline (TBS) for 1 h at room temperature. Anti-human primary antibody was incubated at 4°C overnight. After washing with TBS with Tween, horseradish peroxidase-conjugated secondary antibody was incubated at room temperature for 1 h. Chemiluminescence was detected using a LAS-4000 system (Fujifilm Life Science, Cambridge, MA, USA).
Statistical analysis
Statistical analyses were performed using Statistica version 7.0 (StatSoft Inc., Tulsa, OK, USA) and SAS version 9.1 (SAS Institute Inc., Morrisville, NC, USA).
Differences in HSF1 expression among normal oral mucosa, oral epithelial dysplasia, and OSCC were analyzed using one-way analysis of variance (ANOVA). Associations between HSF1 expression in OSCC and clinicopathological parameters were evaluated using the chi-square test or Student’s t-test, as appropriate.
Overall survival was analyzed using the Kaplan–Meier method. The log-rank test was applied to determine the statistical significance of survival differences according to HSF1 expression status.
Univariate and multivariate survival analyses were performed using the Cox proportional hazards regression model. Univariate analysis was used to evaluate the associations between survival and individual clinicopathological parameters, including HSF1 expression. Multivariate analysis was conducted to identify independent prognostic factors and to determine the relative contribution of HSF1 expression to patient outcomes. A P-value <0.05 was considered statistically significant.
Results
HSF1 expression and functional role in OSCC cell migration
HSF1 expression was evaluated by immunohistochemical staining and quantified using the labeling index (LI). Compared with normal oral mucosa, HNSCC tissues showed markedly increased HSF1 immunoreactivity from TCGA data (Fig. 1A). HSF1 expression was weak or minimal in normal oral mucosal epithelium, whereas a significantly higher proportion of tumor cells demonstrated positive HSF1 staining in OSCC specimens, indicating that HSF1 is upregulated in OSCC tissues compared with normal oral mucosa (Fig. 1B).
Figure 1.
HSF1 expression and functional role in OSCC cell migration. (A) Analysis of TCGA data showed markedly increased HSF1 expression in HNSCC tissues compared with normal tissues. (B) Immunohistochemical staining demonstrated weak or minimal HSF1 expression in normal oral mucosal epithelium, whereas OSCC specimens showed a significantly higher proportion of HSF1-positive tumor cells, indicating upregulation of HSF1 in OSCC tissues. (C) Representative migration assay images and quantitative analysis showed that HSF1 knockdown reduced the migratory ability of Ca9-22 OSCC cells compared with control cells. (D) Consistent with the findings in Ca9-22 cells, HSF1 downregulation also attenuated the migratory phenotype of HSC3 OSCC cells. Abbreviations: HSF1, heat shock factor 1; OSCC, oral squamous cell carcinoma; TCGA, The Cancer Genome Atlas; HNSCC, head and neck squamous cell carcinoma.
To further examine the functional role of HSF1 in OSCC cells, HSF1 expression was downregulated using a knockdown approach. The efficiency of HSF1 downregulation was confirmed before functional analysis. Following HSF1 knockdown, OSCC cells of Ca9-22 showed reduced migratory ability compared with control cells, as demonstrated by representative migration assay images (Fig. 1C). Quantitative analysis further confirmed that the number of migrated OSCC cells was significantly decreased after HSF1 downregulation (Fig. 1C). Consistently, additional analysis of migration-related changes showed that reduced HSF1 expression attenuated the migratory phenotype of OSCC cells of HSC3 (Fig. 1D). These findings suggest that HSF1 may contribute to OSCC cell migration and aggressive tumor behavior.
Correlation between HSF1 expression and clinicopathological parameters in OSCC
The association between HSF1 expression and clinicopathological parameters in OSCC patients is summarized in Table 1. The mean HSF1 LI did not differ significantly according to patient age, sex, primary tumor location, recurrence status, or histological differentiation. Patients aged ≤50 years and >50 years showed comparable HSF1 LI values. No significant difference was observed between men and women, or among tumors arising from the buccal mucosa, tongue, or other oral mucosal sites (Table 1).
In contrast, elevated HSF1 expression was significantly associated with more aggressive tumor characteristics. Patients with advanced primary tumors had a significantly higher mean HSF1 LI than those with early primary tumors. Specifically, the mean HSF1 LI was 49 ± 28% in T1–T2 tumors and 63 ± 26% in T3–T4 tumors (P < 0.001; Table 1). Patients with cervical lymph node metastasis also showed higher HSF1 expression than those without nodal involvement, with mean HSF1 LI values of 62 ± 28% in N1–N3 tumors and 49 ± 23% in N0 tumors (P = 0.010; Table 1). In addition, HSF1 LI was significantly higher in advanced-stage disease than in early-stage disease, with mean values of 64 ± 28% in stage III–IV tumors and 46 ± 25% in stage I–II tumors (P < 0.001; Table 1).
These results indicate that increased HSF1 expression is significantly associated with larger tumor size or greater local invasiveness, nodal metastasis, and advanced clinical stage in OSCC.
Multivariate analysis of prognostic factors
Multivariate analysis using the Cox proportional hazards regression model was performed to identify independent prognostic factors for overall survival. As shown in Table 2, advanced T classification, positive N classification, and high HSF1 LI remained significantly associated with overall survival. Patients with T3–T4 tumors had a significantly increased risk of mortality compared with those with T1–T2 tumors, with a hazard ratio of 4.564 (95% CI, 1.754–6.767; P = 0.029; Table 2). Nodal metastasis was also an independent adverse prognostic factor, with a hazard ratio of 3.222 for N1–N3 disease compared with N0 disease (95% CI, 1.199–8.660; P = 0.020; Table 2). Importantly, high HSF1 expression, defined as HSF1 LI > 53%, was independently associated with poorer overall survival, with a hazard ratio of 2.249 (95% CI, 1.516–5.565; P = 0.019; Table 2).
Table 2.
Multivariate analyses of the HSF1 labeling index and clinicopathological parameters using the Cox proportional hazards regression model.
| Factor | Hazard ratio (95% CI) | P-Value |
|---|---|---|
| Multivariate | ||
| T classification (T1 + T2 vs. T3 + T4) | 4.564 (1.754–6.767) | 0.029 |
| N classification (N0 vs. N1 + N2 + N3) | 3.222 (1.199–8.660) | 0.020 |
| HSF1 LI (LI ≦ 53% vs. LI > 53%) | 2.249 (1.516–5.565) | 0.019 |
P value < 0.05 indicates statistically significant.
Abbreviations: HSF1, heat shock factor 1; LI, labeling index; CI, confidence interval.
These findings suggest that HSF1 expression provides prognostic information beyond conventional clinicopathological parameters.
Survival analysis according to HSF1 expression
Kaplan–Meier survival analysis was performed to evaluate the association between HSF1 expression and overall survival. Patients were stratified according to HSF1 LI using a cutoff value of 53%. The cutoff value was determined according to the median value of HSF1 LI in the OSCC cohort. Patients were stratified into low-expression and high-expression groups using HSF1 LI ≤ 53% and HSF1 LI > 53%, respectively. Survival analysis demonstrated a significant difference in overall survival between patients with HSF1 LI > 53% and those with HSF1 LI ≤ 53% (P = 0.036; Fig. 2).
Figure 2.
Kaplan–Meier survival analysis: correlation between HSF1 labeling index and overall survival. Kaplan–Meier survival curves illustrating the relationship between HSF1 positive labeling index and cumulative overall survival in patients with OSCC. Patients were stratified according to HSF1 expression levels (e.g., HSF1 LI > 53% vs. ≤ 53%). Individuals with higher HSF1 expression demonstrated significantly lower cumulative survival compared with those with lower HSF1 expression (log-rank test, P = 0.036). Abbreviations: HSF1, heat shock factor 1; LI, labeling index; OSCC, oral squamous cell carcinoma.
Together with the Cox regression results, these findings support the clinical significance of HSF1 as an adverse prognostic biomarker in OSCC. Elevated HSF1 expression was associated with advanced tumor status, nodal metastasis, advanced clinical stage, and reduced overall survival.
Discussion
In the present study, HSF1 expression was increased in OSCC compared with normal oral mucosa. Analysis of TCGA data showed markedly higher HSF1 expression in HNSCC tissues than in normal tissues, supporting the clinical relevance of HSF1 upregulation in head and neck malignancies. Consistently, immunohistochemical analysis demonstrated weak or minimal HSF1 staining in normal oral mucosal epithelium, whereas OSCC specimens exhibited a significantly higher proportion of HSF1-positive tumor cells. These findings indicate that HSF1 is upregulated in OSCC and may be involved in oral carcinogenesis and tumor progression.
Previous studies have reported HSF1 overexpression or functional involvement in tumorigenesis across multiple cancer types. Ishiwata et al. demonstrated prominent nuclear granular staining of HSF1 in surgical specimens from patients with OSCC,25 which is consistent with our findings. Collectively, these observations support the concept that HSF1 may play an important role in OSCC development and progression. Unlike previous studies that mainly described HSF1 expression in OSCC, the present study integrates tissue-based expression analysis, clinicopathological correlation, survival analysis, and in vitro functional assays, thereby providing both clinical and functional evidence supporting the prognostic relevance of HSF1 in OSCC.
Although HSF1 is not generally classified as a classical oncogene, accumulating evidence suggests that it functions as a key regulator of malignant phenotypes. HSF1 enables cancer cells to adapt to proteotoxic, metabolic, oxidative, and therapeutic stresses, thereby supporting tumor cell survival under hostile microenvironmental conditions.26,27 In this context, the increased HSF1 expression observed in OSCC tissues may reflect an adaptive mechanism that helps tumor cells tolerate oncogenic stress and maintain aggressive behavior.
In the present cohort, elevated HSF1 expression was significantly associated with more aggressive clinicopathological features. Patients with advanced primary tumors showed significantly higher HSF1 labeling index (LI) values than those with early primary tumors. Specifically, HSF1 LI was higher in T3–T4 tumors than in T1–T2 tumors. In addition, patients with cervical lymph node metastasis had higher HSF1 expression than those with N0 disease, and patients with stage III–IV disease showed significantly increased HSF1 LI compared with those with stage I–II disease.
These findings suggest that HSF1 expression is closely related to local tumor progression, nodal metastasis, and advanced clinical stage in OSCC. Dai et al. reported that HSF1 supports tumor cell survival by promoting ribosomal biogenesis and enhancing glucose metabolism.27 Similarly, Zhao et al. demonstrated that, in breast cancer, the oncogene ErbB2 positively regulates LDH-A through HSF1, thereby enhancing glycolysis.20 These mechanisms may partly explain why tumors with higher HSF1 expression demonstrate larger size, greater invasiveness, and more advanced clinical presentation.
Moreover, Mendillo et al., using ChIP-Seq analysis and comparison with the Molecular Signatures Database, showed that HSF1 regulates a broad cancer-specific transcriptional program.29 This program includes genes involved in protein folding, translation, mitosis, RNA binding, immune regulation, and apoptosis. Therefore, HSF1 may contribute to OSCC progression through multiple biological pathways, including enhanced proteostasis, increased proliferative capacity, altered metabolism, reduced apoptosis, and immune modulation.
In contrast, no statistically significant association was observed between HSF1 expression and patient age, sex, primary tumor location, recurrence status, or histological differentiation in our updated analysis. These findings suggest that HSF1 expression is more closely linked to tumor burden and metastatic potential than to demographic factors or tumor site. Although recurrence is clinically important, it is influenced by multiple factors, including surgical margin status, adjuvant therapy, molecular heterogeneity, immune status, lifestyle exposure, and treatment compliance. Therefore, the lack of a significant association between HSF1 expression and recurrence in this cohort does not exclude a possible role of HSF1 in recurrent disease, but indicates that further studies with larger cohorts and more detailed recurrence-related variables are needed.
In addition to the clinical association between HSF1 expression and aggressive tumor features, our in vitro findings further support a functional role of HSF1 in OSCC cell migration. Knockdown of HSF1 reduced the migratory ability of Ca9-22 OSCC cells, as shown by representative migration assay images and quantitative analysis. Similar findings were observed in HSC3 OSCC cells, indicating that the effect of HSF1 downregulation on cell migration was not limited to a single OSCC cell line.
These results are consistent with previous reports linking HSF1 to cancer cell motility and metastatic behavior. Hoang et al. reported HSF1 overexpression in the highly metastatic PC-3M prostate cancer cell line.17 O’Callaghan-Sunol et al. demonstrated that HSF1 participates in epidermal growth factor-induced cell migration, suggesting a functional relationship between HSF1 activation and enhanced invasive potential.30 Dai et al. also observed increased HSF1 expression in highly aggressive BPLER breast cancer cells with strong invasive and metastatic capabilities.26
Taken together, our clinical and functional findings suggest that HSF1 may contribute to OSCC aggressiveness not only by supporting tumor growth and survival but also by enhancing tumor cell migration. This may partly explain the significant association between high HSF1 expression and nodal metastasis observed in our cohort. Nevertheless, the precise molecular mechanisms by which HSF1 regulates OSCC cell motility, invasion, epithelial–mesenchymal transition, and lymphatic dissemination remain to be clarified.
Survival analysis demonstrated that increased HSF1 expression was significantly associated with poorer overall survival in patients with OSCC. Kaplan–Meier analysis showed a significant survival difference between patients with high and low HSF1 expression using an LI cutoff value of 53%. Furthermore, multivariate Cox proportional hazards regression analysis identified high HSF1 expression as an independent prognostic factor for overall survival, together with advanced T classification and positive N classification.
In the updated multivariate model, patients with T3–T4 tumors had a significantly increased mortality risk compared with those with T1–T2 tumors. Nodal metastasis was also independently associated with poor survival. Importantly, HSF1 LI >53% remained independently associated with worse outcome after adjustment for T and N classifications. These findings indicate that HSF1 provides prognostic information beyond conventional clinicopathological parameters.
The prognostic significance of HSF1 observed in our OSCC cohort is consistent with previous findings in other malignancies. Elevated HSF1 expression has been associated with poor survival in esophageal cancer,21 hepatocellular carcinoma, 19 and gastric cancer.24 Collectively, these data suggest that increased HSF1 expression may represent a common adverse prognostic feature across multiple tumor types. In OSCC, HSF1 may therefore serve as a clinically relevant biomarker for identifying patients with more aggressive disease and higher mortality risk.
Because HSF1 regulates heat shock proteins and other stress-responsive genes, the HSF1–HSP axis has attracted increasing attention as a potential therapeutic target in cancer. Among downstream effectors, heat shock protein 90 (HSP90) has been extensively investigated because of its role in stabilizing multiple oncogenic client proteins.31–34 Several HSP90 inhibitors, including 17-dimethylaminoethylamino-17-demethoxygeldanamycin, ganetespib,35 and NVP-AUY922,31 have shown inhibitory effects against OSCC cells in vitro.
Notably, pimitespib, another HSP90 inhibitor, was approved in Japan in 2022 following the Phase III CHAPTER-GIST-301 trial for gastrointestinal stromal tumor, in which it showed improved survival compared with placebo.36 Pimitespib has also entered phase I clinical development outside Japan, including studies in the United States and Europe; however, mature published results remain limited.37 Although these findings support the clinical feasibility of targeting HSP90 in selected cancers, clinical evidence for HSP90 inhibitors in head and neck cancer, including OSCC, remains limited.38
Given that high HSF1 expression was associated with advanced disease, lymph node metastasis, reduced overall survival, and increased migratory ability of OSCC cells in the present study, targeting the HSF1–HSP pathway may represent a promising therapeutic strategy. However, direct inhibition of HSF1 remains challenging, and the complex feedback regulation between HSF1 and HSPs should be carefully considered. Future studies should evaluate whether pharmacologic modulation of HSF1, HSP90, or related stress–response pathways can suppress OSCC progression and improve treatment response.
Several limitations should be acknowledged. First, this was a retrospective study with a relatively limited sample size. Although significant associations were observed between HSF1 expression and tumor progression, nodal metastasis, clinical stage, and overall survival, larger independent cohorts are required to validate these findings. Second, the normal oral mucosa control group was smaller than the OSCC group, which may limit statistical power for comparisons between non-neoplastic and malignant tissues. Third, although HSF1 knockdown reduced OSCC cell migration in vitro, the downstream molecular mechanisms responsible for this effect were not fully investigated. Additional studies are needed to determine whether HSF1 regulates OSCC migration through epithelial–mesenchymal transition, cytoskeletal remodeling, metabolic reprogramming, HSP-dependent signaling, or other pathways. Finally, in vivo studies are required to confirm whether HSF1 directly contributes to OSCC invasion and metastasis.
The present study demonstrates that HSF1 is upregulated in OSCC and is significantly associated with aggressive clinicopathological features, including advanced T classification, cervical lymph node metastasis, and advanced clinical stage. High HSF1 expression was also associated with poorer overall survival and remained an independent prognostic factor in multivariate analysis.
Functionally, HSF1 downregulation attenuated the migratory ability of OSCC cells, supporting a role for HSF1 in promoting OSCC aggressiveness. These findings suggest that HSF1 may serve as a useful prognostic biomarker and a potential therapeutic target in OSCC. Further mechanistic and translational studies are warranted to clarify the biological functions of HSF1 and to evaluate whether targeting the HSF1–HSP axis can provide therapeutic benefit for patients with OSCC.
Acknowledgments
There are no funding sources for this article.
Footnotes
Declaration of competing interest: There are no conflicts of interest to declare.
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