High P4HA1 expression is an independent prognostic factor for poor overall survival and recurrent‐free survival in head and neck squamous cell carcinoma

Abstract Background Prolyl 4‐hydroxylase subunit alpha 1 (P4HA1) plays a critical role in modulating the extracellular matrix and promoting tumor progression in various cancers. However, the association between P4HA1 and head and neck squamous cell carcinomas (HNSCC) has not been thoroughly elucidated to date. Methods P4HA1 mRNA and protein expression in cancer and normal tissues were analyzed using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus, and Human Protein Atlas databases. Quantitative PCR was applied to determine P4HA1 mRNA expression levels in 162 paired HNSCC and adjacent normal tissues. The cBioPortal for Cancer Genomics was utilized to explore P4HA1 genetic alterations in HNSCC. Then, KEGG analysis of P4HA1 co‐expressed genes in HNSCC was conducted using ClueGo in Cytoscape. Results P4HA1 mRNA and protein levels were significantly increased in HNSCC tissues compared with normal tissues. High P4HA1 expression in HNSCC tissues was significantly associated with tumor category, lymphatic metastasis and pathological stage. The area under summary receiver operating characteristic curve of TCGA and validation cohort was 0.887 and 0.883, respectively. Moreover, elevated P4HA1 expression was associated with unfavorable OS (HR: 1.728, P = .001) and RFS (HR: 2.025, P = .002) in HNSCC patients. Conclusions This integrated analysis provides strong evidence that increasing P4HA1 expression is significantly associated with the carcinogenesis of HNSCC. Additionally, high P4HA1 expression serves as both diagnostic biomarker and independent prognostic factor for poor OS and RFS in HNSCC patients.


| INTRODUC TI ON
Head and neck cancers represent the sixth most common cancer worldwide. The vast majority (greater than 90%) are head and neck squamous cell carcinomas (HNSCC), such that the term head and neck cancer refers to cancer arising from the epithelium lining the of the upper aerodigestive tract (lip, oral cavity, pharynx, and larynx) and exhibiting microscopic evidence of squamous differentiation. 1 According to the latest report of the International Agency for Research on Cancer, approximately one million new HNSCC patients were estimated to be clinically diagnosed in 2018 with greater than 542 943 deaths worldwide. 2 The risk for developing HNSCC is associated with several traditional etiological factors, including cigarette smoking and alcohol abuse. 3 Increasing evidence also demonstrates that infection with a high-risk human papillomavirus (HPV) strain is associated with HNSCC and is an important favorable prognostic factor, especially for oral cavity and oropharynx cancer. 4,5 Although the recent diagnostic and therapeutic strategies have yielded some significant improvements, the 5-year survival rate for HNSCC patients over the last decade remained at approximately 50%. 6 Although multiple molecular mechanisms are associated with HNSCC initiation, growth, invasion, and metastasis, the exact pathogenesis of tumorigenesis remains unclear. Development of new technologies, such as microarray technology and next-generation sequencing, has allowed for collection of large amounts of data to explore the key genes in the pathogenesis of HNSCC, 7,8 such as CDKN2A, 9 CDH1, 10 and EGFR. 11 Therefore, the identification of oncogenic drivers and potential therapeutic targets is crucial for both early diagnosis and effective treatment for HNSCC.
Tumor hypoxia is an essential characteristic of the neoplastic microenvironment that may be correlated with cell proliferation, apoptosis, differentiation, vascularization/angiogenesis, genetic instability, tumor metabolism, tumor immune responses, and invasion and metastasis. 12 Under hypoxic microenvironments, hypoxia inducible factor-1 (HIF-1) promotes extracellular matrix (ECM) remodeling by inducing prolyl 4-hydroxylase subunit alpha 1 (P4HA1), prolyl 4-hydroxylase subunit alpha 2 (P4HA2), and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, leading to changes in cancer cell morphology, adhesion and motility that enhance invasion and metastasis. 13 Located at 10q22.1, P4HA1 encodes an active catalytic subunit of prolyl 4-hydroxylase that catalyzes the formation of 4-hydroxyproline in collagen, which is essential to the formation and stabilization of the triple helical domain of newly synthesized procollagen chains. 14 P4HA1 was identified as hypoxia-responsive gene and plays a critical role in regulating collagen biosynthesis. 15 Previous evidence suggested that P4HA1 overexpression plays a critical role in cancer progression. Hu et al 16 reported that high P4HA1 expression is correlated with the malignancy of gliomas and could serve as a prognostic indicator for patients with high-grade gliomas. In human breast cancer, P4HA1 plays an essential role in enhancing invasion and metastasis and is significantly associated with decreased patient survival. 17 However, until now, the association between P4HA1 and HNSCC as well as its clinical value was not clearly delineated.
In this study, we evaluated the expression of P4HA1 in HNSCC and its clinical value. In addition, we also investigated the enrichment of P4HA1 co-expressed genes in KEGG pathways to explore its underlying mechanism in HNSCC.

| Comparison of P4HA1 gene expression between tumor vs non-tumor samples using Gene Expression Omnibus database
P4HA1 mRNA expression in HNSCC samples compared with normal tissue was also analyzed using published databases (GSE6631) 18 downloaded from Gene Expression Omnibus (GEO). In the GSE6631 database, data for gene expression profiling of 22 paired HNSCC samples and corresponding adjacent normal tissues were obtained.

| Total RNA extraction and quantitative realtime PCR
Total RNA was extracted from 162 paired HNSCC and normal tissues using the TRIzol reagent (Invitrogen), then reverse transcribed into cDNA by GoScript Reverse Transcription (RT) System (Promega) following the manufacturer's instructions. Real-time quantitative reverse transcription-polymerase chain reaction quantitative realtime PCR (qRT-PCR) was performed as previously described. 19 The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalize control.

| Immunohistochemistry staining
P4HA1 protein expression levels in HNSCC tissues and in normal tissues were explored using immunohistochemistry (IHC) staining data from the Human Protein Atlas (HPA; http://www.prote inatl as.org/). and |Spearman's r| ≥ .4). Then, the co-expressed genes were loaded into ClueGo in Cytoscape for analysis of KEGG pathways. Only pathways with a P-value ≥.05 were included. and recurrent-free survival (RFS) after initial therapy were generated, and log-rank tests were performed to evaluate the difference between the survival curves. Univariate and multivariate Cox F I G U R E 1 P4HA1 expression levels are significantly elevated in HNSCC tissues compared with normal tissues using public databases. A-B, Heatmap (A) and plot (B) showing P4HA1 expression in HNSCC tissue and normal tissue using TCGA database. C, P4HA1 expression in HNSCC tissue and normal tissue using GEO database. N, Sample number regression analyses were performed to determine the independent prognostic value of P4HA1 expression in terms of OS and RFS in HNSCC patients. P-value <.05 was considered to be statistically significant.

| P4HA1 expression is significantly elevated in HNSCC tissues
By comparing P4HA1 mRNA expression using the RNA-Seq data of 520 HNSCC tissues and normal tissues in TCGA, we revealed that HNSCC tissues exhibited significantly elevated P4HA1 mRNA expression compared with normal tissues (P = 1.64E-23; Figure 1A,B), consistent with our findings using GEO data (P = 6.16E-04; Figure 1C). The qRT-PCR analysis using the 162 paired HNSCC samples confirmed that P4HA1 mRNA expression levels were significantly upregulated in HNSCC tissues compared with adjacent normal tissues (P = 1.41E-40, Figure 2). We further explored P4HA1 protein expression in HNSCC tissues and normal tissues using the HPA database. Immunohistochemical staining images revealed that P4HA1 exhibited high expression in HNSCC tissues ( Figure 3A). In comparison, oral mucosa exhibited medium P4HA1 expression ( Figure 3B).

| Association of P4HA1 expression with some clinical features of HNSCC
Then, we analyzed the association between P4HA1 mRNA expression levels and clinicopathological characteristics of patients with HNSCC. As shown in Table 1, high P4HA1 expression in HNSCC tissues was significantly associated with alcohol consumption (P = .019), tumor location (P = .017), HPV infection (P = .011), tumor category (P = .006), lymphatic metastasis (P = .006), and pathological stage (P = .002).

| Diagnostic value of P4HA1 expression for HNSCC
We examined the diagnostic value of P4HA1 expression in HNSCC using ROC curves. An area under the ROC curve (AUC) closer to 1.0 signifies that the test exhibits more perfect discrimination. The maximum Youden index was used as a cut-off point. The result suggested that P4HA1 expression yielded an AUC of 0.887, a sensitivity of 88.8%, and a specificity of 78.1% using TCGA cohort ( Figure 4A) and yielded an AUC of 0.883, a sensitivity of 78.4% and a specificity of 83.3% using our validation cohort ( Figure 4B).

| High P4HA1 expression was an independent prognostic predictor of unfavorable OS and RFS in HNSCC patients
Using the maximum Youden index as cut-off point (10.665), we classi-

| P4HA1 genetic alteration was associated with worse OS and DFS in HNSCC patients
The cBioPortal for Cancer Genomics was utilized to explore P4HA1 genetic alterations in HNSCC P4HA1 was only altered in 8 samples, including 504 sequenced HNSCC patients from the Cancer Genome Atlas Research ( Figure 6). Then, we also evaluated the association between P4HA1 genetic alteration and survival in HNSCC patients.
Survival curves indicated that HNSCC patients with P4HA1 genetic alterations exhibited significantly worse OS (log-rank P = .025) and DFS (log-rank P = .007).

| KEGG analysis based on P4HA1 coexpressed genes
By data mining using cBioPortal for Cancer Genomics, we identi-

| D ISCUSS I ON
Collagens are the major structural extracellular matrix (ECM) proteins and form fibers or networks in tumor tissues to support the tumor microenvironment and play crucial roles in carcinogenesis. 20,21 P4HA1 is a key intracellular enzyme to catalyze the formation of 4-hydroxyproline that is essential for proper three-dimensional folding of newly synthesized procollagen chains, maintaining ECM homeostasis. 14 Accumulating evidence indicates that increased P4HA1 is associated with the initiation, invasion, and metastasis of many human cancers, including hepatocellular carcinoma, 22 breast cancer, 17 and prostate cancer. 23 However, the association of P4HA1 with HNSCC remains uninvestigated. In the present study, significantly increased P4HA1 mRNA levels were observed in HNSCC tissues compared with nontumor tissues using TCGA database, which is consistent with the analysis results of the GEO database.
Furthermore, the HPA database validated that P4HA1 protein levels were elevated in HNSCC compared with surrounding normal tissue and demonstrated that P4HA1 was mainly localized to the endoplasmic reticulum and slightly localized to the mitochondria and vesicles.
All these results suggested that P4HA1 plays an important role in HNSCC transformation.
The history of high alcohol consumption is a crucial factor for increased the risk of HNSCC. 24 In this study, using RNA-seq data in TCGA-HNSC, we found that increased P4HA1 expression was significantly correlated with alcohol consumption, suggesting that alcohol might contribute to HNSCC by inducing P4HA1 expression.
Accumulating evidence indicates that HPV infection is an important risk factor for HNSCC. HPV-positive HNSCCs and HPV-negative HNSCCs differ with respect to the molecular mechanisms underlying their oncogenic processes. 4 HPV-positive cancers are more susceptible to chemotherapy and radiation with better prognosis compared with HPV-negative patients. 25,26 Studies have consistently demonstrated that most HNSCCs with HPV detected in the tumor are from the oral cavity and oropharynx, 27 and HPV is driving the increasing incidence of oral cavity and oropharyngeal cancer over the past 30 years. 28,29 Consistent with prior reports that integration of HPV into the genome results in altered DNA copy number and mRNA transcript abundance and splicing, 30   Despite current treatment regimens with curative intent, including surgery, radiotherapy and chemotherapy, local or distant recurrence rates remain high, and the 5-year overall survival rate of HNSCC patients is less than 50%. 33 Emerging therapeutic strategies, such as anti-EGFR antibody (cetuximab) and anti-PD-1 antibodies (pembrolizumab and nivolumab) that have recently been approved for the

| CON CLUS IONS
This integrated bioinformatics analysis provides strong evidence that increasing P4HA1 is significantly associated with HNSCC carcinogenesis and metastasis. Additionally, high P4HA1 expression is both a diagnostic biomarker and an independent prognostic factor for poor OS and RFS in HNSCC patients. Thanks are due to the professional English language copy editing provided by American Journal Experts Company.

CO N FLI C T S O F I NTE R E S T
None of the authors have any commercial or other associations that might pose a conflict of interest. F I G U R E 7 KEGG pathway analysis of the genes co-expressed with P4HA1 in HNSCC