Assessment of tissue homogenate levels of TGM1, PPL and KRT8 in a group of patients with HNSCC tumors and matched surgical margin samples

Introduction: Head and neck squamous cell carcinoma (HNSCC) is a group of malignancies with significantly increasing incidence and mortality. Associated TGM1, PPL, and KRT proteins are involved in epithelial cell structure, adhesion, and differentiation.

Objective, patients and methods: This study aimed to evaluate TGM1, PPL, and KRT8 levels in tumors and matched surgical margin samples from 52 HNSCC patients and assess correlations with clinical and demographic variables using ELISA.

Results: No significant differences in TGM1, PPL, and KRT8 levels were found between tumor and margin samples. However, in tumor tissue, TGM1 and KRT8 levels showed a statistically significant association with T status. In margins, PPL and KRT8 levels were also associated with T status. Additionally, PPL and TGM1 levels were correlated with N status in both tumor and margin samples, respectively. A significantly higher level of PPL was observed in OSCC tumors compared to HPSCC+LSCC. TGM1 levels in tumor and margin samples were correlated in patients with concomitant diseases. Analysis of HPV and p16 status revealed differences in PPL and KRT8 levels between tumor and margin samples. Furthermore, differences in PPL, TGM1, and KRT8 levels were observed in relation to smoking and alcohol use, distinguishing regular or occasional users from abstinent patients.

Conclusions: Our results suggest that impaired TGM1, PPL, and KRT8 signaling pathways might play a role in HNSCC, indicating their potential relevance for future diagnostic and therapeutic investigations. Further studies are needed to confirm our findings, clarify the mechanistic role of these proteins in disease progression, and assess their clinical utility.

Introduction

Head and neck squamous cell carcinoma (HNSCC) comprises a heterogeneous group of malignancies in the upper respiratory and gastrointestinal tracts (1). Globally, HNSCC incidence and mortality have increased significantly, with an estimated 500,000 new cases annually (2). Major risk factors include tobacco, alcohol, poor oral hygiene, missing teeth, dental caries, and untreated periodontal disease (3). In recent years, viral infections, particularly HPV, have gained prominence in the etiopathogenesis of HNSCC, especially in oral squamous cell carcinomas (OSCC) and oropharyngeal carcinomas (OPSCC) (4). HNSCCs also show diverse molecular abnormalities, and advances in molecular biology have improved our understanding of the pathways involved. Despite progress in diagnosis and treatment, the 5-year progression-free survival remains at 40–50%, with limited improvement in metastatic cases (5). This is likely due to the absence of effective screening tools and diagnostic biomarkers, leading to late-stage diagnoses. Biomarkers, classified as diagnostic, prognostic, or predictive, are crucial for early detection, disease monitoring, and therapy response. Although many potential biomarkers have been proposed, few have reached clinical implementation (6).

Transglutaminase 1 (TGM1) belongs to the transglutaminase enzyme group and is involved in the processes of forming the keratinized cell envelope, which is important for maintaining the dermal barrier against physical, chemical, and microbiological agents. TGM1 forms strong crosslinks between structural proteins that form the keratinized cell envelope, providing strength and stability to the epidermis (7). Although dysregulated transglutaminase activity has been described in several malignancies (8, 9), the specific role of TGM1 in HNSCC remains largely unknown. Considering that HNSCC arises from highly differentiated squamous epithelium and often exhibits profound disturbances in keratinocyte maturation and barrier integrity, the contribution of TGM1 to tumor biology represents a notable gap in our current knowledge.

Periplakin (PPL) is a member of the plakin family of cytolytic proteins and is a major component of the precursor of the cornified envelope found beneath the plasma membrane of differentiating keratinocytes. Furthermore, PPL is also found in the desmosomes of keratinocytes, other epithelial cells, and tissues exposed to mechanical dynamics, such as the heart, lung, and skeletal muscle (10). PPL interacts with other proteins, taking part in the regulation of signaling pathways modulating cell adhesion, migration, and differentiation processes, among others. In turn, abnormalities in the proteins of the plakin family are associated with numerous abnormalities in tissue integrity, the stability of the keratinized epidermal envelope of the skin, and the functioning of the nervous and muscular systems (11). Importantly, it has been shown that PPL, by regulating invasion, proliferation, metastasis, and immunity processes, can correlate with the development and progression of several types of cancer, including bladder cancer, triple negative breast cancer, colon cancer, and esophageal cancer (12). The involvement of PPL in HNSCC has not been systematically assessed. Given the central role of desmosomal remodeling and loss of epithelial cohesion in HNSCC pathogenesis, the lack of data on PPL represents a clear research gap that warrants investigation.

Keratin 8 (KRT8) is a member of the type II keratin family and serves as a major component of the cytoskeleton. Together with KRT18, it forms intermediate filaments in the cytoplasm of monolayers of epithelial cells (13). KRT8 is involved in the processes of maintaining the structural integrity of cells and in signal transduction and cell differentiation (14). Abnormalities in KRT8 expression may be associated with the occurrence of various types of cancer (15). In addition, some studies have shown the likely involvement of KRT8 in key tumor processes such as cell migration, adhesion and drug resistance (16). Studies specifically addressing KRT8 in HNSCC are scarce. This is noteworthy because cytoskeletal reprogramming and partial epithelial–mesenchymal transition are critical events in HNSCC progression and metastasis.

Taken together, TGM1, PPL, and KRT8 represent biologically plausible but underexplored proteins whose roles in HNSCC remain insufficiently characterized. Their involvement in epithelial integrity, cytoskeletal dynamics, and signaling pathways relevant to tumor behavior highlights the need for focused studies addressing their expression patterns and potential clinical significance in HNSCC. Our study aims to fill this apparent gap in the available literature.

It is hypothesized that the expression levels of TGM1, PPL, and KRT8 will be significantly altered in tumor and margin samples from patients with HNSCC, reflecting their potential involvement in epithelial cell structure, adhesion, and differentiation. We hypothesize that the expression levels of these proteins will be correlated with selected clinical and sociodemographic factors, which may indicate their relevance as potential biomarkers for prognosis or therapeutic targeting in HNSCC.

This study aimed to analyze the TGM1, PPL and KRT8 protein level in tumors and matched surgical margin samples from patients with HNSCC. The level of selected proteins was also evaluated in connection with clinical and demographic variables, smoking status, alcohol consumption, p16, and HPV status.

Patients and methods

Patients

Patients for this study were recruited from the Department of Otolaryngology and Maxillary Surgery of St. Vincent De Paul Hospital, Gdynia, Poland (Regional Medical Chamber in Gdansk, Poland no. KB-42/21 and KB-9/25). A total of 52 individuals participated in the study. The main inclusion criteria for the HNSCC group were age ≥18, diagnosis of a primary tumor and no preoperative radio/chemotherapy and signed consent to participate in the study. Details are presented in Table 1. The tumor and the corresponding margins were collected after surgical resection. The collected samples were histologically examined and were classified as primary HNSCC. Distant metastases were not present in any of the patients. Marginal samples were histologically confirmed to be cancer-free and were taken from the surgical margin at least 10 mm from the tumor margin. The classification and staging of the tumor specimens were in accordance with the 8th edition of the AJCC cancer staging manual (17). The clinical characteristics of the study group are presented in Table 2. All samples were secured and transported on dry ice to the Department of Medical and Molecular Biology, Faculty of Medical Science in Zabrze, Medical University of Silesia, Katowice, Poland, and stored at -80°C until further analysis.

Table 1

Table 1. Characteristics of the study participants.

Table 2

Table 2. Clinical data of the HNSCC group.

Experimental procedures

Tumor and corresponding surgical margin samples were weighed and mechanically homogenized in nine volumes of phosphate-buffered saline (PBS; Eurx, Gdańsk, Poland) using a PRO 200 homogenizer (PRO Scientific Inc., Oxford, CT, USA) operating at 10,000 rpm for approximately 30–40 seconds, with the tubes maintained on ice to prevent protein degradation. The resulting homogenates were subsequently subjected to sonication with a UP100H ultrasonic cell disruptor (Hielscher Ultrasonics GmbH, Teltow, Germany) at 100% amplitude, applying three 10-second pulses separated by 20-second intervals, while keeping the samples on ice to control temperature during processing. Total protein concentration was assessed with the AccuOrange™ Protein Quantitation Kit (Biotium, Fremont, CA, USA). Fluorescence intensity was recorded at an excitation wavelength of 480 nm and an emission of 598 nm using a SYNERGY H1 microplate reader (BIOTEK, Winooski, VT, USA) and Gen5 2.06 software.

The concentrations of TGM1, PPL, and KRT8 proteins were assessed by ELISA in tissue homogenates, following the procedures described by the manufacturer’s guidelines outlined in the technical manuals provided with the kits. To determine the concentrations of the tested samples, a standard curve was prepared using the standards from the kits. All standards and tissue homogenates were run in duplicates; additionally, samples for PPL were diluted 5× in phosphate-buffered saline (PBS; Eurx, Gdańsk, Poland). For all ELISA tests, plates were read by SYNERGY H1 microplate reader (BIOTEK, Winooski, VT, USA) using 450 nm as the primary wavelength. Data analysis software Gen5 2.06 was used. The concentration of the measured proteins was normalized to the total protein concentration and reported in pg/µg for all three proteins. The following tests were used: Cloud-Clone Corp., Katy, TX, USA; assay ID: SEH334Hu for PPL (with a sensitivity of 0.122 ng/mL), SEB773Hu for TGM1 (with a sensitivity of 0.121 ng/mL) and SEC025Hu for KRT8 (with a sensitivity of 0.061 ng/mL). The intra-assay variation was below 10% and the inter-assays were below 12% for the evaluated proteins.

p16 and Ki-67 was assessed by immunohistochemical staining and evaluated in the Department of Pathomorphology, Gdynia Center of Oncology, Pomeranian Hospitals. The p16 status was assessed according to widely accepted criteria used in HPV-related head and neck cancers: tumors were considered p16-positive when strong and diffuse nuclear and cytoplasmic staining was present in ≥70% of tumor cells. Cases not meeting this threshold were classified as p16-negative. DNA-HPV confirmation was performed using a PCR and “Flow-through”hybridization with GeneFlow™ HPV Array Test Kit (DiagCor Bioscience Ltd., Kowloon Bay, Hongkong) with FTPRO Flow-through System (DiagCor Bioscience Ltd., Kowloon Bay, Hongkong) and FTPRO Auto System (DiagCor Bioscience Ltd., Kowloon Bay, Hongkong) in the laboratory of the Department of Medical and Molecular Biology, Faculty of Medical Sciences in Zabrze, Medical University of Silesia in Katowice, Poland in accordance with the methodology described in our previous study (18). The extracted DNA served as a template for PCR amplification carried out on a Mastercycler Personal Thermal Cycler (Eppendorf, Hamburg, Germany). The resulting amplicons were then subjected to denaturation and hybridization, followed by enzymatic conjugation and color-development procedures. Final readouts were acquired using the FTPRO Auto System (DiagCor Bioscience Ltd., Kowloon Bay, Hong Kong). Each assay run included both manufacturer-supplied positive and negative controls to ensure result validity. In the study, we analyzed 33 HPV subtypes, including high-risk subtypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and low-risk subtypes (6, 11, 26, 40, 42, 43, 44, 54, 55, 57, 61, 70, 71, 72, 81, 84).

Statistical analysis

The results were evaluated for normal distribution with the Shapiro-Wilk test. Student’s t-test, U Mann-Whitney test or Kruskal Wallis with Dun-Sidak post hoc were used to verify significance of differences in the means or medians between the groups. Correlation was established with Spearman’s rank correlation coefficient. For survival analyses, we used the Kaplan – Meier method with Log-Rank test where patients were divided into two groups according to protein concentration as follows: one group had protein concentration lower than median concentration and other group had protein concentration above or equal to the median concentration. Survival analysis was done for 1- and 2-years post-surgery with survival rate on the end of 1- or 2 years post surgery. Results with a p-value <0.05 were considered significant. STATISTICA version 13 software (TIBCO Software Inc., Palo Alto, CA, USA) was used to perform all the analyses. To avoid small groups of less than 6 cases, we combined some of them with their neighbor group as in case of T classification. But in some cases, this was impossible to achieve as in the abstinent group case. Results in the text and in the tables are presented as median with interquartile range (M (Q1-Q3)) or mean ± standard deviation (M ± SD). Survival rate is presented as ratio alive patients to all patients at the end of 1^st or 2^nd year post surgery. The study design is shown in the Figure 1.

Figure 1

Figure 1. General study design.

Results

Proteins level, clinical parameters and localization of primary tumor

No significant differences were found in the levels of TGM1 (0.46 (0.15 – 2.40) vs. 0.53 (0.15 – 2.61); p = 0.8419), PPL (44.28 (14.91 – 88.73) vs. 60.84 (22.53 – 120.25); p = 0.1194) and KRT8 (0.19 (0.07 – 0.59) vs. 0.22 (0.07 – 0.47); p = 0.7054) in HNSCC tumor samples compared to the margin samples. There was a higher level of TGM1 protein in tumor samples of patients with a lower T status, T1+T2 vs. T4 (2.63 (1.16 - 3.01) vs. 0.17 (0.12 - 0.58); p = 0.0162). PPL concentration in the margin samples was lower in the patients with clinical stage T1+T2 than in patients with T3 (32.56 (18.40 - 54.83) vs. 122.71 (51.43 - 193.15); p = 0.0276) and between patients with clinical stage T4 than in patients with T3 (54.63 (18.42 - 86.02) vs. 122.71 (51.43 - 193.15); p = 0.0247). Moreover, patients with status T1+T2 showed significantly higher levels of KRT8 protein in the tumor samples than patients with T4 status (0.81 (0.60 - 2.80) vs. 0.17 (0.05 - 0.37); p = 0.0088). Similarly, KRT8 concentration was significantly higher in the group of patients with T1+T2 status than in patients with T4 status in margin (0.56 (0.40 - 0.71) vs.; p = 0.033). The results are presented in Figure 2A. The group with T1 and T2 status were combined due to the small number of T1 cases.

Figure 2

Figure 2. The level of TGM1, PPL, and KRT8 proteins in tumor and margin samples according to (A) the patient’s T status; (B) the patient’s N status; (C) tumor status G. *p<0.05.

We observed higher levels of TGM1 protein in margin samples of patients with higher nodal status, N0 vs N2+N3 (0.18 (0.14 - 0.58) vs. 2.61 (0.278-3.81); p = 0.0049). The higher concentrations of PPL in tumor samples showed a group of patients with N0 nodal status, which was statistically significant, N0 vs. N1 (58.69 (35.09 - 100.39) vs. 10.29 (1.55 - 52.93); p = 0.0356). These results are shown in Figure 2B. The group with N2 and N3 status was combined due to the small number of N3 cases.

Tumors with G1 had lower concentration of KRT8 protein than G2 tumors (0.11 (0.06 – 0.19) vs. 0.60 (0.16 – 2.31); p = 0.0415). No other connections were observed between protein concentration with G status in tumor and margin samples.

When classifying cancers according to location, only two subgroups were studied: OSCC (28 samples) and the combined group composed of the LSCC and HPSCC subtypes (21 samples). Significant differences in PPL concentration were observed between OSCC and LSCC+HPSCC in tumor (58.69 (16.83 - 105.48) vs. 37.00 (8.15 - 48.39); p = 0.0429). The results are presented in Figure 3.

Figure 3

Figure 3. The level of TGM1, PPL, and KRT8 proteins in the tumor and margin samples according to the localization of tumor. *p<0.05.

Correlation of analyzed proteins in a group of patients with concomitant diseases

We found that the TGM1 concentration was significantly lower in tumor and margin samples in patients with any concomitant disease (cardiovascular, endocrine, digestive system or kidney diseases), compared to patients without concomitant diseases (0.21 (0.12-0.50) vs 2.12 (0.46 - 3.19); p=0.0010)(0.20 (0.12 - 1.45) vs. 1.25 (0.70 - 3.84); p = 0.0027), as presented in Figure 4.

Figure 4

Figure 4. The level of TGM1, PPL, and KRT8 proteins in tumor and margin samples according to the presence of concomitant diseases. *p<0.05.

Concentration of analyzed proteins and Ki-67, p16, and HPV status

No significant difference in protein concentration was observed in Ki-67 positive and negative groups.

Patients with p16 positive status (p16(+)) had a lower PPL concentration than patients with p16 negative status (p16(-)) in margin samples (22.64 (16.88 - 33.57) vs. 76.85 (43.57 - 137.23); p = 0.0051) as presented in Figure 5A.

Figure 5

Figure 5. The level of TGM1, PPL and KRT8 proteins in tumor and margin samples according to the status of (A) p16; (B) HPV. *p<0.05.

The tumor samples from HPV-positive (HPV(+)) patients showed a higher level of PPL compared to those with negative HPV status (HPV(-)); (93.96 (34.48 - 169.78) vs. 35.15 (3.66 - 87.60); p = 0.0401). Tumor and margin samples from HPV(-) patients showed a higher level of KRT8 compared to patients with HPV(+) status; (0.49 (0.15 - 1.76) vs. 0.08 (0.04 - 0.12); p = 0.0124) (0.23 (0.08 - 0.49) vs. 0.06 (0.04 - 0.19); p = 0.0289) (Figure 5B). Five of our patients were both p16(+) and HPV(+).

Concentration of analyzed proteins and alcohol and tobacco use

Results for protein concentrations and alcohol or tobacco use are presented in Figure 6. Exact values are presented in Table 3 with significant results in bold. For the TGM1 and KRT8 proteins, a higher level was observed in patients who drink occasionally than in those who drink regularly in tumor and margin samples (Figure 6B, Table 3). In case of smoking, TGM1 and KRT8 were higher in smokers than in patients who don’t smoke in both tumor and margin samples (Figure 6A, Table 3). PPL protein was higher in the abstinent group than in patients who regularly drink in margin samples (Figure 6B, Table 3), but it had a lower level in smokers than in non-smokers in tumor samples (Figure 6A, Table 3).

Figure 6

Figure 6. The levels of TGM1, PPL, and KRT8 proteins in tumor and margin samples according to (A) smoking status; (B) alcohol drinking status. *p<0.05.

Table 3

Table 3. The median TGM1, PPL, and KRT8 protein level and p-value according to smoking and drinking status.

Survival analysis

We were able to obtain follow-up information for 43 of the 52 patients. Complete information was available for one year after tumor removal and half of the patients were operated upon during the last two years. The group was divided according to protein concentration into 2 groups: one group had protein concentrations lower than the median and other group had protein concentrations above or equal to the median concentration. The division was done for all proteins in tumors and in margins. There were no differences in survival between 1- and 2-years posttreatment (p>0.05), with mean survival rate 0.88 ± 0.04 in lower concentration groups and 0.89 ± 0.04 in higher concentration groups after 1 year and mean survival rate 0.76 ± 0.08 in lower concentration groups and 0.85 ± 0.08 in higher concentration groups after 2 years, detailed results are presented in Table 4.

Table 4

Table 4. Survival rate of groups characterized with lower or higher concentration of TGM1, PPL or KRT8 proteins in tumor or margin samples.

Discussion

Head and neck squamous cell carcinoma (HNSCC) accounts for the majority of malignant tumors in this area, and its incidence is increasing globally (2). Despite advances in diagnosis and therapy, the disease is often detected at an advanced stage, resulting in a high mortality rate with a high rate. In recent years, interest in the molecular mechanisms of the development of HNSCC has increased, but effective diagnostic and prognostic markers are still lacking (5). Analysis of the expression of PPL, TGM1, and KRT8 proteins involved in maintaining epithelial cell structure and adhesion may provide new data on the mechanisms of tumor progression, as well as a potential source of useful biomarkers in HNSCC.

Our analysis showed no statistical differences in the expression levels of PPL, TGM1, and KRT8 proteins in the tumor samples as compared to the surgical margin samples. One possible explanation is the existence of the “field cancerization,” i.e. the presence of molecular changes in the surrounding tissue that are not yet visible histopathologically (19). Alternatively, these results may indicate that the proteins analyzed do not play a key role in differentiating tumor samples from margins in HNSCC, or that their importance may be more related to other aspects of disease progression. However, significantly higher levels of TGM1 protein were observed in patients with T1+T2 compared to T4 status in the tumor. In the context of cancer, research on the role of TGM1 is limited, and TGM1 is suspected to play a role in the early stages of HNSCC development with decreased expression with tumor progression. This may suggest involvement of TGM1 in maintaining epithelial differentiation, a characteristic that is lost in more advanced and aggressive forms of cancer. The decrease in TGM1 expression at higher clinical stages may also reflect a shift toward a more invasive cellular phenotype (20, 21). On the other hand, TGM1, by forming a network of cross-linked proteins, may impede tumor cell access to mechanisms that enable degradation of the extracellular matrix, which is crucial for invasion (22). Some studies suggest that TGM1 may interact with pathways involved in cell cycle control, apoptosis, and cell migration. By modulating these pathways, TGM1 can contribute to inhibiting tumor cell proliferation and increasing their susceptibility to apoptotic signals, which will slow tumor growth (8, 23).

Additionally, we found elevated levels of PPL in the margin samples of patients with T3 status, compared to those with T1+T2 status, as well as in patients with T3 status compared to T4. In the study by Li et al. (12), PPL expression was down-regulated in colon cancer and positively correlated with tumor size. However, direct studies investigating PPL in the context of primary tumor T status are limited, and therefore our findings can only be cautiously interpreted in light of proposed biological and adaptive mechanisms that accompany tumor progression (24). The observed increase in PPL levels in margin samples in patients with T3 status compared to T1+T2 may represent an adaptive cellular response to increasing microenvironmental stress, potentially associated with a partial epithelial-mesenchymal transition (EMT), during which cells strive to preserve adhesive properties (25). Conversely, the higher PPL levels in T3 compared to T4 may reflect an intermediate stage, wherein epithelial characteristics are still partially retained, while the T4 stage may be associated with a more complete EMT, accompanied by a decline in adhesion-related proteins, including PPL (26).

We observed a statistically significant association between higher expression of KRT8 in patients with T1+T2 stage compared to T4 in the margin and tumor. On the other hand, an immunohistochemical study by Fillies et al. in patients with oral squamous cell carcinoma found no correlation between KRT8 and tumor size (27). Another study by Alam et al. analyzed the significance of the loss of KRT8 phosphorylation at Ser73 and Ser431 residues in OSCC and found that high expression of KRT8 was correlated with tumor size (28). In the case of gastric cancer, no differences were found between tumor size and KRT8 expression using an immunohistochemical method (29). Our results can be explained as an effect of maintaining the characteristics of epithelial differentiation in the early stages of T1+T2, where tumor cells have higher expression of proteins typical of tissue of epithelial origin (30, 31). In the advanced T4 stage, EMT and cytoskeletal reorganization occurs, leading to a decrease in the expression of epithelial proteins, including KRT8 (32, 33).

We observed higher levels of TGM1 protein in the margin samples of patients with N0 vs. N2+N3 status. On the contrary, higher concentrations of PPL in tumor samples in a group of patients with lower nodal status was observed to be statistically significant (N0 vs. N1). Higher levels of TGM1 in the margins of patients with advanced nodal status may be due to the induction of mechanisms that strengthen the barrier surrounding the tumor in response to increasing tumor aggressiveness (22). In contrast, higher levels of PPL in tumor tissues in patients with N0 nodal status may indicate that stronger intercellular adhesion is maintained in the absence of tumor spread to the lymph nodes (24). The decrease in PPL expression in tumors from N1 patients may reflect the onset of the EMT process, facilitating invasion and metastasis (34). Such opposing dynamics of TGM1 in the margins and PPL within the tumor indicate different adaptive mechanisms of the tumor and surrounding tissues depending on the stage.

In this study, we observed lower KRT8 protein levels in the group of patients with G1 status when compared to patients with G2 status in tumors. This result may reflect the role of KRT8 in tumor progression and loss of cellular differentiation (35). The increase in KRT8 expression in G2 tumors may indicate a transition toward a more aggressive phenotype, which is consistent with reports suggesting that elevated KRT8 levels correlate with tumor progression, poor differentiation, and the ability to form metastases in colorectal, lung, and breast epithelial cancers (30, 36, 37).

Furthermore, we reported statistically higher PPL concentrations in the OSCC group compared to the combined group consisting of patients with LSCC and HPSCC. This may be due, among other things, to tissue specificity, as OSCC is derived from oral epithelial cells, which are strongly associated with the maintenance of desmosomal structures and thus have higher expression of PPL to maintain tissue integrity (25). In addition, OSCC may exhibit a higher degree of epithelial differentiation compared to cells in LSCC and HPSCC, and the specific oral environment may induce compensatory mechanisms, which is associated with higher expression of adhesion proteins, including PPL (12, 26). Different signaling pathways, as well as genetic and epigenetic modifications, may lead to specific regulation of PPL in different types of head and neck region cancers (38).

Moreover, we report a decreased concentration of TGM1 in patients with concomitant diseases (including cardiovascular diseases, kidney diseases, gastrointestinal diseases, endocrine diseases and others), compared to patients without concomitant disease. Our results may indicate a broader impact of systemic disorders on molecular pathways involved in epithelial homeostasis and cell differentiation (20). TGM1, as an enzyme involved in the processes of terminal differentiation and stabilization of epithelial cell structure, may be regulated by metabolic and hormonal factors, the balance of which is often disturbed in the course of chronic diseases (39). Reduced TGM1 expression in this group of patients may therefore reflect an altered intracellular and systemic environment that affects the tumor phenotype and its microenvironment, independent of classical inflammatory mechanisms (8).

Our study showed that in margin samples, the median protein concentrations of PPL were higher in p16 (–) patients than in the p16(+) group. Moreover, we found that in the case of HPV status, we observe a higher level of PPL proteins in HPV(+) tumor samples. In turn, a higher KRT8 protein level was noted in the tumor and margin in group of patients with HPV(-) status compared to HPV(+). The observed differences may reflect the different pathogenetic pathways of HPV-dependent and HPV-independent cancers. HPV(+) tumors, often p16(+), are characterized by activation of the E6/E7 pathway which disrupts the functions of p53 and Rb proteins, leading to changes in cell cycle, adhesion, and epithelial differentiation (40). In this context, higher PPL levels in HPV(+) tumors may be related to a compensatory mechanism for maintaining intercellular adhesion in response to virus-induced disruption of epithelial homeostasis. In contrast, lower levels of PPL in p16(+) margins may be indicative of a wider range of molecular changes in surrounding tissues, which may be the result of local viral or tumor field activity. In contrast, higher KRT8 levels in HPV(-) patients may result from activation of alternative signaling pathways typical of HPV-independent HNSCC, such as EGFR/PI3K/AKT and EMT (epithelial-mesenchymal transition), in which KRT8 may be involved as a marker of a transitional or more aggressive phenotype (41–43). KRT8, typical of monolayered or bilayer epithelia cytokeratin, may also reflect changes toward a less differentiated and more invasive phenotype in HPV(–) tumors.

In case of smoking, TGM1 and KRT8 were higher in smokers than in patients who don’t smoke in both tumor and margin samples. PPL had a lower level in smokers than in patients who are nonsmokers in tumor samples. Smoking, whose components include harmful, toxic and carcinogenic compounds, is one of the exogenous factors that influence changes in gene and protein expression that are crucial in cancer transformation (44). Smoking has been shown to lead to squamous cell metaplasia and epithelial hyperkeratosis and generate severe oxidative stress and chronic inflammation, activating the NF κB and AP-1 pathways that stimulate the transcription of the KRT8 and TGM1 genes (45, 46). In addition, chronic exposure to tobacco smoke toxins leads to cytoskeletal reorganization, activation of EMT, which is associated with degradation of desmosomal adhesion proteins, including PPL (47, 48).

In our study, we observed a higher level of TGM1 and KRT8 in patients who drink occasionally than in those who drink regularly in both tumor and margin samples. PPL protein was higher in the abstinent group than in patients who drink regularly in margin samples. Similarly to smoking, regular alcohol consumption leads to ongoing oxidative stress and inflammation, which can epigenetically suppress the expression of epithelial differentiation genes, including TGM1 and KRT8 (39). In occasional drinkers, exposure to ethanol is short-lived, which preserves higher levels of TGM1 and KRT8, consistent with a normal epithelial differentiation program (15). Lorand and Graham described that occasional contact of epithelial cells with alcohol can temporarily stimulate transglutaminase activity as a cytoprotective mechanism during short-term stress (22). Moreover, higher levels of PPL are maintained in nondrinkers, which may reflect preserved desmosomal integrity and cellular barrier (49). On the contrary, regular alcohol consumption can promote DNA hypermethylation in regions that encode adhesion proteins such as PPL, leading to reduced expression (50).

Conclusions

We found no significant differences in the levels of PPL, TGM1, and KRT8 proteins between tumor tissue and surgical margins. While this pattern may be consistent with the concept of “field cancerization,” we acknowledge that this interpretation remains tentative in the absence of supporting molecular data such as mutational or methylation profiling. Alternative explanations for our results could be associated with protein stability, technical factors related to sample processing, or biological redundancy in epithelial tissues. Moreover, we showed significant correlation in selected protein concentrations according to tumor stage, nodal status, tumor location, HPV/p16 status, and alcohol and smoking habits, which may indicate the role of selected proteins in tumor processes, including differentiation, adhesion and EMT. In view of the results, PPL, TGM1, and KRT8 may reflect a complex interaction between epithelial differentiation, cell adhesion and micro- and macro-environmental factors affecting the development of HNSCC. The lack of extensive research on these proteins in HNSCC underscores the need for further studies to elucidate their precise roles. Further studies integrating genomic, epigenetic, and proteomic analyses will be necessary to clarify the mechanisms of PPL, TGM1 and KRT8 in HNSCC. Understanding these mechanisms could lead to novel therapeutic targets and improve patient outcomes. Overall, this research provides a valuable foundation for advancing the molecular biology of HNSCC.

The main limitation of the study was the small number of samples collected. Accordingly, some groups were combined for statistical analysis. In order to more accurately confirm the results of our analyses, further studies should be conducted on larger patient cohorts. In addition, it is necessary to conduct tests on cell lines and animal models, which could provide valuable information to better understand the role of these proteins in the development of HNSCC.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The studies involving humans were approved by Regional Medical Chamber in Gdansk, Poland (no. KB-42/21 and KB-9/25). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Author contributions

DN: Conceptualization, Data curation, Formal Analysis, Methodology, Project administration, Visualization, Writing – original draft. AŚ: Investigation, Writing – original draft. DH: Visualization, Writing – original draft. ZZ: Resources, Writing – review & editing. JO: Resources, Writing – review & editing. RL: Resources, Writing – review & editing. MD: Resources, Writing – original draft. DA: Writing – review & editing. JKS: Conceptualization, Methodology, Supervision, Writing – review & editing.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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References

1. Mody MD, Rocco JW, Yom SS, Haddad RI, and Saba NF. Head and neck cancer. Lancet. (2021) 398:2289–99. doi: 10.1016/S0140-6736(21)01550-6

PubMed Abstract | Crossref Full Text | Google Scholar

2. Cohen N, Fedewa S, and Chen AY. Epidemiology and demographics of the head and neck cancer population. Oral Maxillofac Surg Clin N Am. (2018) 30:381–95. doi: 10.1016/j.coms.2018.06.001

PubMed Abstract | Crossref Full Text | Google Scholar

3. Yan F, Knochelmann HM, Morgan PF, Kaczmar JM, Neskey DM, Graboyes EM, et al. The evolution of care of cancers of the head and neck region: State of the science in 2020. Cancers. (2020) 12:1543. doi: 10.3390/cancers12061543

PubMed Abstract | Crossref Full Text | Google Scholar

4. Mahal BA, Catalano PJ, Haddad RI, Hanna GJ, Kass JI, Schoenfeld JD, et al. Incidence and demographic burden of HPV-associated oropharyngeal head and neck cancers in the United States. Cancer Epidemiol Biomarkers Prev. (2019) 28:1660–7. doi: 10.1158/1055-9965.EPI-19-0038

PubMed Abstract | Crossref Full Text | Google Scholar

5. Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR, et al. Head and neck squamous cell carcinoma. Nat Rev Dis Primers. (2020) 6:92. doi: 10.1038/s41572-020-00224-3

PubMed Abstract | Crossref Full Text | Google Scholar

6. Leemans C, Snijders P, and Brakenhoff R. The molecular landscape of head and neck cancer. Nat Rev Cancer. (2018) 18:269–82. doi: 10.1038/nrc.2018.11

PubMed Abstract | Crossref Full Text | Google Scholar

7. Surbek M, Van de Steene T, Sachslehner AP, Golabi B, Griss J, Eyckerman S, et al. Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1. Sci Rep. (2023) 13:21550. doi: 10.1038/s41598-023-48856-1

PubMed Abstract | Crossref Full Text | Google Scholar

8. Huang H, Chen Z, and Ni X. Tissue transglutaminase-1 promotes stemness and chemoresistance in gastric cancer cells by regulating Wnt/β-catenin signaling. Exp Biol Med (Maywood). (2017) 242:194–202. doi: 10.1177/1535370216670541

PubMed Abstract | Crossref Full Text | Google Scholar

9. Wu R, Li D, Zhang S, Wang J, Chen K, Tuo Z, et al. A pan-cancer analysis of the oncogenic and immunological roles of transglutaminase 1 (TGM1) in human cancer. J Cancer Res Clin Oncol. (2024) 150:123. doi: 10.1007/s00432-024-05640-6

PubMed Abstract | Crossref Full Text | Google Scholar

10. Boczonadi V and Määttä A. Functional analysis of periplakin and envoplakin, cytoskeletal linkers, and cornified envelope precursor proteins. Methods Enzymol. (2016) 569:309–29. doi: 10.1016/bs.mie.2015.06.019

PubMed Abstract | Crossref Full Text | Google Scholar

11. Gujrati M, Mittal R, Ekal L, and Mishra RK. SUMOylation of periplakin is critical for efficient reorganization of keratin filament network. Mol Biol Cell. (2019) 30:357–69. doi: 10.1091/mbc.E18-04-0244

PubMed Abstract | Crossref Full Text | Google Scholar

12. Li X, Zhang G, Wang Y, Elgehama A, Sun Y, Li L, et al. Loss of periplakin expression is associated with the tumorigenesis of colorectal carcinoma. BioMed Pharmacother. (2017) 87:366–74. doi: 10.1016/j.biopha.2016.12.103

PubMed Abstract | Crossref Full Text | Google Scholar

13. Jacob JT, Coulombe PA, Kwan R, and Omary MB. Types I and II keratin intermediate filaments. Cold Spring Harb Perspect Biol. (2018) 10:10. doi: 10.1101/cshperspect.a018275

PubMed Abstract | Crossref Full Text | Google Scholar

14. Lim Y, Kim S, Yoon HN, and Ku NO. Keratin 8/18 regulate the Akt signaling pathway. Int J Mol Sci. (2021) 22:9227. doi: 10.3390/ijms22179227

PubMed Abstract | Crossref Full Text | Google Scholar

15. Matthias C, Mack B, Berghaus A, and Gires O. Keratin 8 expression in head and neck epithelia. BMC Cancer. (2008) 8:267. doi: 10.1186/1471-2407-8-267

PubMed Abstract | Crossref Full Text | Google Scholar

16. Busch T, Armacki M, Eiseler T, Joodi G, Temme C, Jansen J, et al. Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells. J Cell Sci. (2012) 125:2148–59. doi: 10.1242/jcs.080127

PubMed Abstract | Crossref Full Text | Google Scholar

17. Amin MB, Greene FL, Edge SB, Compton CC, Gershenwald JE, Brookland RK, et al. The eighth edition AJCC cancer staging manual: continuing to build a bridge from a population-based to a more “personalized” approach to cancer staging. CA Cancer J Clin. (2017) 67:93–9. doi: 10.3322/caac.21388

PubMed Abstract | Crossref Full Text | Google Scholar

18. Nałęcz D, Świętek A, Hudy D, Wiczkowski K, Złotopolska Z, and Strzelczyk JK. Assessment of concentration KRT6 proteins in tumor and matching surgical margin from patients with head and neck squamous cell carcinoma. Int J Mol Sci. (2024) 25:7356. doi: 10.3390/ijms25137356

PubMed Abstract | Crossref Full Text | Google Scholar

19. Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, and Brakenhoff RH. A genetic explanation of Slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res. (2003) 63:1727–30.

PubMed Abstract | Google Scholar

20. Candi E, Schmidt R, and Melino G. The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol. (2005) 6:328–40. doi: 10.1038/nrm1619

PubMed Abstract | Crossref Full Text | Google Scholar

21. Akhmetkaliyev A, Alibrahim N, Shafiee D, and Tulchinsky E. EMT/MET plasticity in cancer and Go-or-Grow decisions in quiescence: the two sides of the same coin? Mol Cancer. (2023) 22:90. doi: 10.1186/s12943-023-01793-z

PubMed Abstract | Crossref Full Text | Google Scholar

22. Lorand L and Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol. (2003) 4:140–56. doi: 10.1038/nrm1014

PubMed Abstract | Crossref Full Text | Google Scholar

23. Tabolacci C, De Martino A, Mischiati C, Feriotto G, and Beninati S. The role of tissue transglutaminase in cancer cell initiation, survival and progression. Med Sci (Basel). (2019) 7:19. doi: 10.3390/medsci7020019

PubMed Abstract | Crossref Full Text | Google Scholar

24. Yamada K, Hagiwara T, Inazuka F, Sezaki T, Igari T, Yokoi C, et al. Expression of the desmosome-related molecule periplakin is associated with advanced stage and poor prognosis of esophageal squamous cell carcinoma. Transl Cancer Res. (2019) 8:3820–30. doi: 10.21037/tcr.2018.01.03

Crossref Full Text | Google Scholar

25. Tonoike Y, Matsushita K, Tomonaga T, Katada K, Tanaka N, Shimada H, et al. Adhesion molecule periplakin is involved in cellular movement and attachment in pharyngeal squamous cancer cells. BMC Cell Biol. (2011) 12:41. doi: 10.1186/1471-2121-12-41

PubMed Abstract | Crossref Full Text | Google Scholar

26. Liu YQ, Zou HY, Xie JJ, and Fang WK. Paradoxical roles of desmosomal components in head and neck cancer. Biomolecules. (2021) 11:914. doi: 10.3390/biom11060914

PubMed Abstract | Crossref Full Text | Google Scholar

27. Fillies T, Werkmeister R, Packeisen J, Brandt B, Morin P, Weingart D, et al. Cytokeratin 8/18 expression indicates a poor prognosis in squamous cell carcinomas of the oral cavity. BMC Cancer. (2006) 13:6–10. doi: 10.1186/1471-2407-6-10

PubMed Abstract | Crossref Full Text | Google Scholar

28. Alam H, Gangadaran P, Bhate AV, Chaukar DA, Sawant SS, Tiwari R, et al. Loss of keratin 8 phosphorylation leads to increased tumor progression and correlates with clinico-pathological parameters of OSCC patients. PloS One. (2011) 6:27767. doi: 10.1371/journal.pone.0027767

PubMed Abstract | Crossref Full Text | Google Scholar

29. Fang J, Wang H, Liu Y, Ding F, Ni Y, Shao S, et al. High KRT8 expression promotes tumor progression and metastasis of gastric cancer. Cancer Sci. (2017) 108:178–86. doi: 10.1111/cas.13120

PubMed Abstract | Crossref Full Text | Google Scholar

30. Chu PG and Weiss LM. Keratin expression in epithelial neoplasms: an update. Histopathology. (2002) 41:403–9. doi: 10.1046/j.1365-2559.2002.01387.x

PubMed Abstract | Crossref Full Text | Google Scholar

31. Wesley T, Escalona RM, Kannourakis G, and Ahmed N. Plakin expression in serous epithelial ovarian cancer has the potential to impede metastatic spread and epithelial-mesenchymal transition: a comparative expression analysis of immunohistochemical and in silico datasets. Cancers (Basel). (2024) 16:4087. doi: 10.3390/cancers16234087

PubMed Abstract | Crossref Full Text | Google Scholar

32. Moll R, Divo M, and Langbein L. The human keratins: biology and pathology. Histochem Cell Biol. (2008) 130:705–33. doi: 10.1007/s00418-008-0435-6

PubMed Abstract | Crossref Full Text | Google Scholar

33. Fortier AM, Asselin E, and Cadrin M. Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation. J Biol Chem. (2013) 288:11555–71. doi: 10.1074/jbc.M112.428920

PubMed Abstract | Crossref Full Text | Google Scholar

34. Thiery JP. Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer. (2002) 2:442–54. doi: 10.1038/nrc822

PubMed Abstract | Crossref Full Text | Google Scholar

35. Ku NO, Strnad P, Bantel H, and Omary MB. Keratins: biomarkers and modulators of apoptotic and necrotic cell death in the liver. Hepatology. (2016) 64:966–76. doi: 10.1002/hep.28493

PubMed Abstract | Crossref Full Text | Google Scholar

36. Karantza V. Keratins in health and cancer: more than mere epithelial cell markers. Oncogene. (2011) 30:127–38. doi: 10.1038/onc.2010.456

PubMed Abstract | Crossref Full Text | Google Scholar

37. Arentz G, Chataway T, Condina MR, Price TJ, Hoffmann P, Hardingham JE, et al. Increased phospho-keratin 8 isoforms in colorectal tumors associated with EGFR pathway activation and reduced apoptosis. ISRN Mol Biol. (2012) 2012:706545. doi: 10.5402/2012/706545

PubMed Abstract | Crossref Full Text | Google Scholar

38. Adorno-Farias D, Morales-Pisón S, Gischkow-Rucatti G, Margarit S, and Fernández-Ramires R. Genetic and epigenetic landscape of early-onset oral squamous cell carcinoma: insights of genomic underserved and underrepresented populations. Genet Mol Biol. (2024) 47:20240036. doi: 10.1590/1678-4685-GMB-2024-0036

PubMed Abstract | Crossref Full Text | Google Scholar

39. Eckert RL, Kaartinen MT, Nurminskaya M, Belkin AM, Colak G, Johnson GV, et al. Transglutaminase regulation of cell function. Physiol Rev. (2014) 94:383–417. doi: 10.1152/physrev.00019.2013

PubMed Abstract | Crossref Full Text | Google Scholar

40. Doorbar J, Egawa N, Griffin H, Kranjec C, and Murakami I. Human papillomavirus molecular biology and disease association. Rev Med Virol. (2015) 25:2–23. doi: 10.1002/rmv.1822

PubMed Abstract | Crossref Full Text | Google Scholar

41. Baumeister P, Zhou J, Canis M, and Gires O. Epithelial-to-mesenchymal transition-derived heterogeneity in head and neck squamous cell carcinomas. Cancers. (2021) 13:5355. doi: 10.3390/cancers13215355

PubMed Abstract | Crossref Full Text | Google Scholar

42. Schinke H, Shi E, Lin Z, Quadt T, Kranz G, Zhou J, et al. A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer. Mol Cancer. (2022) 21:178. doi: 10.1186/s12943-022-01646-1

PubMed Abstract | Crossref Full Text | Google Scholar

43. Chen H, Chen X, Pan B, Zheng C, Hong L, and Han W. KRT8 serves as a novel biomarker for LUAD and promotes metastasis and EMT via NF-κB signaling. Front Oncol. (2022) 12:875146. doi: 10.3389/fonc.2022.875146

PubMed Abstract | Crossref Full Text | Google Scholar

44. Li Y and Hecht SS. Carcinogenic components of tobacco and tobacco smoke: a 2022 update. Food Chem Toxicol. (2022) 165:113179. doi: 10.1016/j.fct.2022.113179

PubMed Abstract | Crossref Full Text | Google Scholar

45. Cipollina C, Bruno A, Fasola S, Cristaldi M, Patella B, Inguanta R, et al. Cellular and molecular signatures of oxidative stress in bronchial epithelial cell models injured by cigarette smoke extract. Int J Mol Sci. (2022) 23:1770. doi: 10.3390/ijms23031770

PubMed Abstract | Crossref Full Text | Google Scholar

46. Ebrahimi Samani S, Tatsukawa H, Hitomi K, and Kaartinen MT. Transglutaminase 1: emerging functions beyond skin. Int J Mol Sci. (2024) 25:10306. doi: 10.3390/ijms251910306

PubMed Abstract | Crossref Full Text | Google Scholar

47. Chidgey M and Dawson C. Desmosomes: a role in cancer? Br J Cancer. (2007) 96:1783–7. doi: 10.1038/sj.bjc.6603808

PubMed Abstract | Crossref Full Text | Google Scholar

48. Xin Z, Yamaguchi A, and Sakamoto K. Aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral squamous cell carcinoma. Virchows Arch. (2014) 465:35–47. doi: 10.1007/s00428-014-1594-6

PubMed Abstract | Crossref Full Text | Google Scholar

49. Nishimori T, Tomonaga T, Matsushita K, Oh-Ishi M, Kodera Y, Maeda T, et al. Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin. Proteomics. (2006) 6:1011–8. doi: 10.1002/pmic.200500262

PubMed Abstract | Crossref Full Text | Google Scholar

50. Otsubo T, Hagiwara T, Tamura-Nakano M, Sezaki T, Miyake O, Hinohara C, et al. Aberrant DNA hypermethylation reduces the expression of the desmosome-related molecule periplakin in esophageal squamous cell carcinoma. Cancer Med. (2015) 4:415–25. doi: 10.1002/cam4.369

PubMed Abstract | Crossref Full Text | Google Scholar