Protease-Activated Receptor 1 (PAR1) Expression Contributes to HPV-Associated Oropharyngeal Cancer Prognosis

Human papillomavirus (HPV)-associated oropharyngeal cancer occasionally has a poor prognosis, making prognostic risk stratification crucial. Protease-activated receptor-1 (PAR1) is involved in carcinogenesis, and its expression is regulated by alpha-arrestin domain-containing protein 3 (ARRDC3). It is also involved in the tumor microenvironment. We sought to evaluate the predictive ability of PAR1, ARRDC3, and tumor-infiltrating lymphocyte (TIL) scores in patients with oropharyngeal, hypopharyngeal, and uterine cervical cancers, serving as comparators for HPV-associated oropharyngeal cancer. Immunohistochemical analysis of p16, ARRDC3, and PAR1 expression was performed on 79 oropharyngeal, 44 hypopharyngeal, and 42 uterine cervical cancer samples. The TIL scores were assessed and classified into the following groups based on invasion: low: 0–10%, medium: 20–40%, and high: > 50%. For prognostic analysis, the three groups were evaluated by dividing them into low, medium, and high categories, or alternatively into two groups using the median value as the cutoff. p16 was expressed in 44 (56%) oropharyngeal, 8 (18%) hypopharyngeal, and all uterine cervical cancer samples. ARRDC3 was detected in 39 (49%) oropharyngeal, 25 (57%) hypopharyngeal, and 23 (55%) uterine cervical cancer samples. PAR1 was expressed in 45 (57%) oropharyngeal, 22 (50%) hypopharyngeal, and 22 (50%) uterine cervical cancer samples. Patients diagnosed with p16-positive oropharyngeal cancer had a substantially improved prognosis compared to those diagnosed with p16-negative cancer. The PAR1-negative cases had a considerably improved prognosis compared to the positive cases (disease-specific survival [DSS] and -negative cases (disease-free survival [DFS]). Multivariate analysis revealed that ARRDC3-positive cases had an appreciably better DSS prognosis than patients with p16-negative oropharyngeal cancers. PAR1-positive patients among patients with p16-positive oropharyngeal cancer had a poor prognosis. With respect to DFS, patients with PAR1-positive and p16-negative oropharyngeal cancer had a 35-fold higher recurrence rate than those with PAR1-negative and p16-negative oropharyngeal cancer. Our results suggest that PAR1 expression affects the prognosis and recurrence rate of HPV-associated oropharyngeal cancer.


Introduction
The number of cases of oropharyngeal cancers caused by human papillomavirus (HPV) infection has increased in recent years [1].In the 2000s, HPV was detected in approximately 50% of oropharyngeal cancers, with an increasing trend observed, particularly among younger age groups [2].In Japan, a multicenter collaborative study on HPV infection and oropharyngeal cancer [3] reported an infection rate 1 3 of approximately 50%, with HPV16 accounting for 90% of the HPV types.p16 expression, assessed through immunohistochemical staining, has served a proxy marker for HPV infection [4].HPV-associated oropharyngeal cancers exhibit greater sensitivity to radiation therapy and chemotherapy than non-HPV-associated cancers, consequently leading to a better prognosis [5][6][7].As a result, p16-positive and -negative oropharyngeal cancers are considered independent diseases based on the tumor, node, metastasis (TNM) classification (International Union Against Cancer [UICC]/ American Joint Committee on Cancer [AJCC], 8th edition).
Although HPV-associated oropharyngeal cancer has a good prognosis, clinical practice reveals that cases with advanced stages or poor prognoses arising from recurrence or metastasis are frequently encountered [8].Therefore, it is crucial to determine the prognostic risk stratification of HPV-associated oropharyngeal cancer in the histopathological examination stage, including biopsy, to aid in treatment selection and prognosis estimation.This objective can be accomplished through the utilization of appropriate histopathological biomarkers.
G protein-coupled receptors (GPCRs) are a class of cell surface receptors that receive external stimuli and regulate intracellular signaling.GPCRs are involved in physiological processes, particularly neurotransmission and immune responses [9].A GPCR, protease-activated receptor-1 (PAR1), is highly expressed on platelets and endothelial cells and is pivotal in mediating between coagulation and inflammation and in inflammatory and fibrotic lung disease development [10].In malignant tumors, PAR1 is crucial in carcinogenesis, angiogenesis, and metastasis [11].In triple negative breast carcinoma, α-arrestin domain-containing protein 3 (ARRDC3) is involved in PAR1 degradation and regulation through apoptosis linked gene-2 (ALG-2)-interacting protein X (ALIX) [12].Interestingly, genome-wide association studies reveal that uterine cervical cancer, similar to HPV-associated oropharyngeal cancer, is caused by HPV infection.Additionally, ARRDC3 is implicated in cell proliferation and susceptibility to HPV infection [13].
PAR1 is also involved in the tumor microenvironment (TME).Its expression in pancreatic adenocarcinoma contributes to the creation of a cellular microenvironment within the tumor, marked by the reduced number of cytotoxic T cells infiltrating the cancerous cells, as well as increased numbers of primitive immunosuppressor cell types [14].The tumor-infiltrating lymphocyte (TIL) is an important TME component and reflects the antitumor immune response of the host [15,16].In patients with breast cancer, TIL levels can be used to predict response to preoperative adjuvant and adjuvant chemotherapy [17,18].Additionally, PD-L1, CD8 + TIL, and HIF-1α levels are useful biomarkers for head and neck squamous cell carcinoma (HNSCC) [19].Therefore, evaluation of TILs is becoming progressively vital in routine pathology, primarily due to the strong correlation between TIL levels and quantitative results of immune gene expression [20].Thus, TIL assessments may serve as a valid, inexpensive, and easily obtainable substitute for analyzing immune gene expression [21].Recently, the International Immuno-Oncology Biomarker Working Group on Breast Cancer was established and international guidelines for the evaluation of TILs were developed [22].International guidelines for the evaluation of TILs have also been established for solid tumors, including metastatic disease, and they can be applied to head and neck cancer [23].
In this retrospective cohort study, we evaluated the expression of p16, PAR1, and ARRDC3 in patients with oropharyngeal and hypopharyngeal cancers and assessed TIL levels using the scoring method recommended by the International Immuno-Oncology Biomarker Working Group on Breast Cancer.In addition, patients with uterine cervical cancer were compared to those with oropharyngeal cancer caused by the same HPV infection.In addition, breast cancer cell lines were evaluated to validate the reliability of this study and establish the consistency of reports in breast cancer.This step was necessary as ongoing studies on PAR1 and ARRDC3 continue to evolve.

Patients
This study involved patients with oropharyngeal, hypopharyngeal, and uterine cervical cancers at Kawasaki Medical University Hospital and Kawasaki Medical University General Medical Center who consented to have their tumor tissues (stored specimens) used in the study.Between 2006 and 2021, 79 patients with oropharyngeal, 44 with hypopharyngeal, and 42 with cervical cancers who underwent surgical treatment or chemoradiation after tissue diagnosis were enrolled.Surgical cases were selected only if a 1-cm safety zone was maintained at the time of resection and if the resection margins were negative.The exclusion criteria included second primary cancer, distant metastasis, and prior irradiation.Patient data were retrospectively collected from electronic medical records.p16 status caused differences in staging; therefore, the histologic type and grade were determined based on the TNM classification (7th edition) defined by the AJCC and UICC [24].

Tissue Preparation and Staining
For pathologic diagnosis, paraffin-embedded sections were prepared and hematoxylin and eosin (H&E) staining and immunohistochemistry were performed.
Fig. 1 Immunohistochemical staining of p16.Specimens with a nuclear expression intensity of ≥ + 2/+ 3 and a positive distribution of ≥ 75% were p16-positive based on the American Joint Committee on Cancer (AJCC) (a).H-scores for alpha-arrestin domain-containing protein 3 (ARRDC3) and protease-activated receptor-1 (PAR1) were evaluated using four scores based on staining intensity (b) Negative controls were treated similarly, without the primary antibodies.
Patients with a nuclear expression intensity of ≥ + 2/+ 3 and a positive distribution of ≥ 75% were p16-positive, based on the AJCC classification.The staining intensities of ARRDC3 and PAR1 were graded into four levels (0, negative; 1, weakly positive [staining intensity lower than the positive control]; 2, moderately positive [staining intensity equal to the positive control]; 3, strongly positive [staining intensity higher than the positive control]).The staining intensity of the entire observed field of view was semi-quantified according to the following expression: H-score (0 to 300 = 0 × of the negative cells + l × of the weak positive cells + 2 × of the intermediate positive cells + 3 × of the strong positive cells).One section per patient was evaluated.The entire tissue section was examined in 200 × field of view, and the average H-score of three fields of view was used as the H-score for that case (Fig. 1).

Tumor-Infiltrating Lymphocytes
TIL scoring was performed according to the Immuno-Oncology International TILs Working Group's definitions [23].Tumor stromal areas were selected at a low magnification.The percentages of stromal areas and mononuclear cells were evaluated at magnifications ranging from ×200 to ×400.Granulocytes and polynuclear cells were excluded and evaluated in accordance with the guidelines of the TILs Working Group using light microscopy (BX53; Olympus, Tokyo, Japan).Tumors were classified into three groups based on TIL infiltration level: low: 0-10%, medium: 20-40%, and high: > 50%.Regarding prognostic analysis, the three groups were evaluated by dividing them into low, medium, and high categories, or alternatively into two groups using the median value as the cutoff (Fig. 2).

Breast Cancer Cell Lines
MCF-7 and MDA-MB-231 human breast cancer cell lines were provided by Dr. Robert Dickson (Lombardi Cancer Research Center, West Virginia, Washington DC, USA).The breast cancer cell line KPL-4 was established at the Department of Breast and Thyroid Surgery, Kawasaki Medical University, Kurashiki, Okayama, Japan [25].The cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 0.02% kanamycin (Meiji, Tokyo, Japan) for 3 or 4 days under normal oxygen (5% CO 2 ) conditions at 37 °C.

Cell Blocks
MCF-7, MDA-MB-231, and KPL-4 cells were cultured under normal oxygen conditions.The cells were then washed with phosphate-buffered saline (PBS), and 0.25% trypsin and 0.2% EDTA were added per 100 mm 2 of the culture dish to detach the breast cancer cells.After detachment, the cells were incubated in a CO 2 incubator at 37 °C for 7 min, and 7.5 mL of DMEM containing 10% FBS was added to the culture dish to inactivate trypsin.The detached breast cancer cells were transferred to 15-mL tubes and centrifuged at 1200 rpm for 5 min to form a pellet.The supernatant was removed, washed with PBS, and centrifuged at 1200 rpm for 5 min.After removing the supernatant, 500 μL of 10% neutral buffered formalin was added to the cell pellet, mixed well, and allowed to stand for 24 h for fixation.Formalinfixed cell masses were embedded in paraffin, and sections of MCF-7, MDA-MB-231, and KPL-4 breast cancer cell lines were prepared.Subsequently, hematoxylin and eosin staining and PAR1 and immunostaining of ARRDC3 were performed.

Western Blotting
For protein extraction, the cells were lysed in Pierce RIPA buffer (Thermo Fisher Scientific).Direct Detect tm Spectrometer (Merck, Darmstadt, Germany) was used to determine total protein concentration.All proteins were separated using 4-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Thermo Fisher Scientific).The membranes were blocked for 1 h at 15-25 °C and incubated with primary antibodies for 8-16 h at 4 °C in blocking buffer consisting of TBS plus 5% BSA and 0.2% Tween 20.Different dilutions of the same antibodies (ARRDC3 1:2000 and PAR1 1:1000) were used for immunohistochemistry. Subsequently, the cells were incubated with a secondary antibody, goat anti-rabbit IgG horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA; lot no.L2308; dilution 1:10,000), at 15-25 °C for 1 h.The membranes were then incubated for 1 h at 15-25 °C.An Amersham Imager 680 (Thermo Fisher Scientific) was used to visualize the protein bands transferred on to the membranes.As a loading control, β-actin (Sigma-Aldrich, dilution 1:1000) was used.The band signal intensities of PAR1 and ARRDC3 and the loading control β-actin were quantified, the amounts of target protein relative to the loading control protein were calculated, and histograms were generated.

Statistical Analysis
After dividing the patients into two groups based on p16 expression, the ARRDC3, PAR1, and TIL scores were combined with clinical data and pathological parameters for statistical analysis.ARRDC3 and PAR1 expression levels were evaluated in two groups: high (> median) and low (≤ median), using the median value as the cutoff.TILs were divided into three groups (low: 0-10%, medium: 20-40%, high: > 50%) and two groups: high (> median) and low (≤ median), using the median value as the cutoff.
The categorical variables are reported as number and percentage; they were evaluated using the chi-square or exact Fisher test.The median and 25th and 75th percentiles of continuous variables were calculated using the t-test if they showed a normal pattern of distribution.Otherwise, Mann-Whitney U test was used.The primary endpoint was disease-specific survival (DSS) and the secondary endpoint was disease-free survival (DFS).DSS and DFS were evaluated using Kaplan-Meier curves and log-rank tests, respectively.Cox regression analysis was used for univariate and multivariate analyses.Oropharyngeal and hypopharyngeal cancers were adjusted for TNM stage (stages I-II vs. III-IV), smoking status, alcohol consumption, venous invasion, lymphatic invasion, duplicate cancer diagnoses, site of origin, and histological differentiation.Cervical cancer was adjusted for TNM stage (stage I-II vs. III-IV), venous invasion, and lymphovascular invasion.The p16-positive and -negative cases were separately analyzed for differences in Fig. 3 Disease-specific survival (DSS) in oropharyngeal cancer.The 3-year DSS was 78%.p16-positive cases had a significantly better prognosis than the negative cases (p = 0.0488) (a).The p16-negative oropharyngeal cancer subgroup (n = 35, 44%) did not significantly differ in alpha-arrestin domain-containing protein 3 (ARRDC3), protease-activated receptor-1 (PAR1), or tumor-infiltrating lymphocyte (TIL) status (b-e).The p16-positive oropharyngeal cancer subgroup (n = 44, 56%) had no significant differences in ARRDC3 expression and TIL status (f, h, and i).(p = 0.32) (f).(p = 0.311) (i).However, PAR1-negative cases had significantly better prognosis than the positive cases (p = 0.0438) (g) association with the outcome.All tests were two-tailed, and statistical significance was set at p < 0.05.
All data were analyzed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interfaces for R (R Foundation for Statistical Computing, Vienna, Austria) [26].To be more precise, it is a customized version of the R program commander that is designed to add statistical functions that are often used in biostatistics.
A comparison of the clinicopathological parameters based on p16 expression revealed significant differences at the TNM stage (p = 0.0348), overlapping cancers (p = 0.005), and TIL scores (TIL3 group, p = 0.0135; TIL2 group, p = 0.006) for oropharyngeal cancer and age (p = 0.022) for hypopharyngeal cancer.
Fig. 5 Comparison of protease-activated receptor-1 (PAR1) and alpha-arrestin domain-containing protein 3 (ARRDC3) expression in human breast cancer tissues.From left to right, hematoxylin and eosin (H&E) staining, PAR1 expression, and ARRDC3 expression in breast cancer cell lines (MDA-MB-231, KPL-4, and MCF-7) were evaluated using cell block preparations (a).Western blotting (b and c).MDA-MB-231, an estrogen receptor (ER)-negative, human epidermal growth factor receptor 2 (HER2)-negative cell line with a poor prognosis, had the highest PAR1 staining intensity among the three lines and the lowest ARRDC3 staining intensity among the three cell lines.In ER-negative and HER2-positive KPL-4 cells, PAR1 staining intensity was weaker than that in MDA-MB-231 cells, and ARRDC3 had the strongest intensity among the three.MCF-7, an ER-positive, HER2-negative breast cancer cell line with a relatively good prognosis, had the lowest PAR1 staining intensity among the three strains and the higher ARRDC3 expression intensity compared to MDA-MB-231 (a).Western blotting showed that the PAR1 was most strongly expressed in MDA-MB-231 (b).ARRDC3 was weakly expressed and KPL-4 was most strongly expressed (c)

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To compare the prognostic significance of the combined immunostaining results and TIL status, univariate and multivariate analyses were performed.ARRDC3 expression correlated with DSS in p16-negative oropharyngeal cancer cases in the multivariate analysis and PAR1 expression correlated with DSS in p16-positive oropharyngeal cancer cases in the multivariate analysis.Furthermore, PAR1 correlated with DFS in p16-negative oropharyngeal cancer cases in the univariate analysis and multivariate analysis (Table 2).However, none of these factors were significant in hypopharyngeal cancer specimens (Table 2) and uterine cervical cancer specimens (Table S2).
To validate the immunohistochemical staining results, we performed immunostaining against PAR1 and ARRDC3 using blocks of the three breast cancer cell lines and compared the results with those of western blotting (Fig. 5).Both methods had consistent results, with PAR1 expressed decreased in the order of MDA-MB-231 > KPL-4 > MCF-7, and ARRDC3 expressed in the order of KPL-4 > MCF-7 > MDA-MB-231 (Fig. 5b).These results are consistent with those reported previously [12,27,28].

Discussion
To the best of our knowledge, this study is the first to demonstrate that p16-positive oropharyngeal cancer with high PAR1 expression is short-lived.HPV-associated oropharyngeal cancer has a better prognosis than squamous cell carcinomas of other head and neck sites.Clinical trials are underway to de-escalate treatment and spare these patients from the consequences of overtreatment [29][30][31][32], as they may present at a younger age than other head and neck squamous cell carcinomas [33].
However, an open-label, randomized, controlled phase III trial revealed that cetuximab in patients with HPVassociated oropharyngeal cancer was not more beneficial than standard cisplatin regimens in terms of reduced toxicity; rather, it displayed a prominent disadvantage in tumor control [34].Additionally, there was no evidence of a marked difference between radiotherapy, oral surgery (TOS), and neck dissection (ND).Enrollment was stopped due to excessive toxic effects in TOS and ND [35].Therefore, until alternative treatments based on the eighth edition staging system are validated in clinical trials, treatment decisions will be made based on the seventh edition staging system.Thus, we adopted the seventh edition system for our study.Furthermore, it is essential to clarify the prognostic risk stratification of HPV-associated positive oropharyngeal cancer and use it for treatment selection and prognostic estimation.Interestingly, even in DFS of p16-negative oropharyngeal cancer, cases with high PAR1 expression experienced earlier recurrence (univariate: HR 3.882 [1.1-13.7];p = 0.03503; multivariate: HR 35.68 [3.62-351.7];p = 0. 0022).
PAR1 is involved in the motility and metastasis of tumor cells in malignant tumors [10].Therefore, it may also be an important factor in HPV-unrelated oropharyngeal cancer.However, in uterine cervical cancer-also HPV-associated-no significance was found between the prognoses of both groups regarding PAR1 expression.This result could be attributed to the relatively early stage of uterine cervical cancer in the enrolled patients [TNM staging I: n = 25 (60%)].
ARRDC3 downregulates integrin β4 expression and inhibits breast cancer progression [27].It is specifically involved in the degradation and regulation of PAR1 expression via ALIX in triple-negative breast cancer [12].ARRDC3 also suppresses tumor metastasis in other cancers [36][37][38][39][40].In the present study, no significant results were obtained for p16-positive oropharyngeal cancer specimens.A genome-wide study of cervical cancer (HPV-associated) suggested that ARRDC3 is involved in the invasion of HPV into cells [13].We hope further studies will be conducted on ARRD3 in HPV-associated oropharyngeal cancer.In recent years, the evaluation of TILs has attracted attention in the head and neck cancer field.
HPV-positive oropharyngeal cancers reportedly have considerably higher numbers of CD4+ and CD8 + T cells [41,42].In addition, in both HPV-positive and HPVnegative tumors, high CD8 + T-cell infiltration has been correlated with a favorable clinical outcome [43,44].TIL is a prognostic factor in HPV-positive oropharyngeal cancers; one report revealed that patients with higher TIL levels had a 4.5-fold higher survival rate [45].Some other studies did not reveal favorable clinical outcomes [46,47].Unfortunately, in this study, we did not find notable prognostic differences based on the TIL score.Therefore, TIL invasion might have different characteristics depending on the tumor site, histology, or molecular subtype [48], and the differences in the prognostic value of TIL may reflect these different biological factors.TILs should be evaluated in resected specimens [23].In the present study, we evaluated 42 (53%) oropharyngeal and 31 (70%) hypopharyngeal cancer cases using biopsy tissue samples.Therefore, the TILs in the biopsy tissue may not be representative of the overall tumor immune infiltration and may have been inadequately evaluated.Radical cure using surgical resection is often challenging in head and neck squamous cell carcinoma, and the availability of biopsy specimens is often limited.Restricting case selection to surgical intervention may introduce selection bias, as it tends to favor cases with relatively good general health.Thus, the prognostic relevance of TILs remains controversial.Furthermore, randomized studies with larger samples are necessary to establish the correlation between TILs and HPV.In conclusion, PAR1 is a potential biomarker for HPV-associated oropharyngeal cancer.

Fig. 2
Fig. 2 Evaluating tumor-infiltrating lymphocytes.The density of tumor-infiltrating lymphocytes (TILs) was determined using hematoxylin and eosin (H&E)-stained sections.Tumor stromal areas were selected at a low magnification.The percentages of stromal areas and

Table 1
Baseline characteristics of patients with oropharyngeal and hypopharyngeal cancers Each marker was grouped into positive and negative expression groups based on median values Prognostic analysis was performed for three and two groups based on median valuesThe patients were classified into the following groups based on the TIL score: low: 0-10%; medium: 20-40%; high: > 50% N/E not evaluable, OPE operation, CRT chemoradiotherapy, RT radiotherapy, CT chemotherapy, PS pyriform sinus, PW posterior wall, PC postcricoid, WD well differentiated, MD moderately differentiated, PD poorly differentiated

Table 2
Cox regression analysis was used for univariate and multivariate analyses Univariate and multivariate time-to-event analyses for Disease-specific survival (DSS) and Disease-free survival (DFS) Multivariate models were adjusted for TNM stage, smoking status, alcohol consumption, venous invasion, lymphatic invasion, double cancers, site of origin, and histologic differentiation