Introduction
Despite the diverse genetic mutations and the presence of HPV in head and neck cancer, the chemotherapy for this cancer has traditionally been based on cisplatin, and treatment outcomes have not improved for decades [
1,
2]. Cisplatin-based chemotherapy is non-specific and has significant side effects. Therefore, the development of targeted therapeutic agents with fewer side effects and related biomarkers is necessary.
Proteasomes are crucial intracellular proteins that regulate the degradation of ubiquitinated proteins [
3], and their complex composition of highly regulated proteins makes them attractive candidate for new targeted cancer therapy [
4]. PSMD1 is a suitable research target for drug repositioning since there is already an FDA-approved drug called proteasome inhibitor, which is mostly used to treat refractory cases of multiple myeloma [
5]. Indeed, researchers are currently investigating the potential of proteasome inhibitors like Bortezomib and Carfilzomib to be utilized in the treatment of different types of solid malignancies [
6]. Recent studies also have identified PSMD1, which encodes a subunit of the proteasome, as an upregulated factor and potential prognostic marker in several cancers including anaplastic thyroid carcinoma [
7], breast cancer [
8], gastric cancer [
9], colon cancer [
10], ovarian cancer [
11], among others [
12]. However, the significance of PSMD1 expression in head and neck cancer remains to be elucidated.
Therefore, the purpose of this study is to assess the prognostic significance of PSMD1 expression in oropharyngeal squamous cell carcinoma (OPSCC) patients using immunohistochemistry. Using clinicopathological data, we aimed to determine the clinical value of PSMD1 expression. Additionally, we investigated disease-specific survival (DSS) of OPSCC patients, considering both PSMD1 expression and clinicopathological information.
Materials and methods
Patients and tissue specimens
We included 64 individuals with OPSCC tissue confirmed by curative resection or diagnostic surgical biopsy at Seoul National University Bundang Hospital between April 2008 and August 2017. We excluded patients who were currently receiving or had previously received treatment for other types of squamous cell carcinoma (SCC) in the head and neck area, as well as those with histological findings that differed from SCC or its subtypes.
The clinicopathological data of the patients were obtained, including age, gender, tobacco and alcohol use, tumor subsite, surgical intent, tumor recurrence, postoperative treatment, and status at the last follow-up. Pathologic stages were classified based on the 8th edition AJCC staging system [
13]. The research protocol received approval from the Institutional Review Board of Seoul National University Bundang Hospital, and informed consent was waived (IRB No. B-2211–790-305).
Tissue microarrays (TMAs) were constructed from formalin-fixed paraffin-embedded blocks of specimens. Core tissue sections (with a diameter of 4 mm) were carefully extracted from individual OPSCC paraffin blocks (donor blocks) and organized in new TMA blocks using a trephine apparatus (Super-BioChips Laboratories, Seoul, Korea). To reduce the impact of heterogeneity of protein expression, three cores were sampled and incorporated into the TMA block from each patient.
HPV genotyping
HPV status was determined by HPV genotyping using the complete resected section and biopsy specimens. HPV genotyping was performed using peptic nucleic acid probe-based fluorescence melting curve analysis in a real-time PCR system (PANA RealTyper™ HPV Kit, PANAGENE, Daejeon, Republic of Korea) according to the manufacturer’s instructions as described in previous study [
14].
Assessment of PSMD1 protein expression
Immunohistochemistry for PMSD1 IHC analysis was conducted on the TMA sections (4 μm) using an automated platform (Benchmark Ultra; Ventana Medical Systems) following the manufacturer's instructions. For PSMD1 immunostaining, a rabbit polyclonal IgG specific to Human PSMD1 (Abcam, ab2941, 1:5000) was employed. At a random × 40 magnification field within the tumor, the nucleus, cytoplasm, and cellular DAB staining intensity in the malignant cells were quantified using an image analyzer (QuPath) for H-score interpretation. If more than 10% of the tumor cells were 1 + , 2 + , or 3 + , the tumor was graded as 1 + , 2 + , or 3 + , respectively. If less than 10% of tumor was 1 + or higher, the tumor was graded as 0 (negative). The total number of cells stained at each grade within the field was counted. Subsequently, the following formula was applied: H-score = (% of 1 + cells × 1) + (% of 2 + cells × 2) + (% of 3 + cells × 3). Consequently, an H-score ranging from 0 to 300 was obtained, where 300 represented 100% of tumor cells exhibiting strong staining (3 +).
Statistical analysis
All data analysis was conducted using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). The determination of appropriate cut-off values for the H-score was performed using receiver operating characteristic (ROC) curve analysis. Chi-squared test, Fisher’s exact test, t-test, and logistic regression were performed to compare clinicopathological data among groups. Pilot survival data were captured from The Cancer Genome Atlas (TCGA) [
15]. Survival data until 2021 were achieved from national statistical office. Kaplan–Meier method and the log-rank test were used to estimate disease-specific survival (DSS). Multivariate analysis was conducted using the Cox proportional hazards regression model. A
p-value less than 0.05 was considered statistically significant.
Discussion
We investigated the prognostic role of PSMD1 expression in patients with OPSCC. PSMD1 was highly expressed in advanced pathologic staging, HPV negative tumor, and non-tonsillar cancer. Especially location was significant factor in PSMD1 nuclear expression. In multivariate analysis, PSMD1 revealed as an independent poor prognostic factor in OPSCC.
Although several studies have also investigated clinical significance of PSMD1 expression in head and neck cancer [
12,
16], the survival analysis exhibited inconsistent results depending on subgroups. In a certain study, PSMD1 appeared even as a good prognostic factor in the survival analysis, which seems opposite from our results [
16]. Our study ensured internal validity by specifically focusing on the OPSCC subgroup, accurately defining disease-specific causes of death, and conducting multivariate analysis that included the specimen age. Moreover, we ensured the significance of survival analysis by accurately defining “high expression” based on the H score and ROC curve analysis. There may be another impact from adoption of the 8th edition AJCC which published after 2016, restaging most HPV + OPSCCs to stage I or II [
17].
There are various scientific explanations about PSMD1 as oncogene in literatures. PSMD1 gene encodes a subunit of the proteasome, 19S-RP, which regulates the degradation of various tumor suppressor and oncogenic proteins through the ubiquitin–proteasome pathway [
18]. For instance, it has been reported that knocking down PSMD1 in a breast cancer cell line resulted in a decrease in the S phase and an increase in the G2/M phase, indicating inhibition of the cell cycle. Also, knockdown of PSMD1 silencing has been shown to activate the p53 pathway including p21 [
8].
The role of p53 is already known to be associated with HPV infection in head and neck cancer. It has been observed that HPV-negative cases tend to have impaired p53 function particularly in the oropharynx [
19]. P53 itself is typically wild type in HPV-positive cancers while the viral protein E6 directly induces the ubiquitination and degradation of p53 [
20]. Indeed, if proteasome inhibitors such as bortezomib were used, proteasome is unable to degrade p53 even if E6 ubiquitinated it, thereby liberating p53 to resume its normal functions [
21].
In addition to the cell cycle and the p53 pathway, various signaling pathways can be influenced by the proteasome. One example is the NF-kB pathway. NF-kB is a protein that promotes cell survival and contributes to cancer growth. IkB, on the other hand, is a protein that inhibits NF-kB. The proteasome plays a role in degrading IkB, thereby allowing NF-kB to remain active and promote cancer growth. Such explanation has been observed in multiple myeloma [
22,
23]. These in vitro explanations support PMSD1 expression as a poor prognostic and oncogenic factor.
Our study may have inherent selection bias of IHC study towards early-stage cancers because of the requirement for surgical paraffin-embedded specimens. However, even in TCGA open data of head and neck cancer [
12], where there is no selection bias for gene targets and subsites, PSMD1 was confirmed as a poor prognostic factor. This finding, along with various studies about PSMD1 as a poor prognostic factor in other types of cancer [
5,
7‐
12], strengthens the external validity of our consistent study results. Also, survival rate of our study population decreased significantly in HPV negative group, with a
p-value of less than 0.001 in the log-rank test (Supplementary Fig.
2). This valid tendency indicates that our study population is representative sample group of OPSCC. However, due to the lower prevalence of HPV-negative cases as known, we couldn't conduct a separate analysis for the HPV-negative group, which is a limitation of small sample size.
Because of the retrospective nature of the study, another limitation of the study is the potential confounding effect of other clinical features. We try to overcome lack of uniformity between the groups by including previous treatment history and staging on cox-regression multivariate analysis. Year of pathology is another main confounding concern. Staining tended to lighten with increasing pathology year, even in separate linear regression analysis (
P < .001, Supplementary Fig.
3). To address this limitation, we incorporated specimen age when conducting Cox regression multivariate analysis. Nevertheless, significant
p-values were obtained for variables that were consistent with the hypothesis.
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