HtrA3: a promising prognostic biomarker and therapeutic target for head and neck squamous cell carcinoma

Abstract

Objective: The dysregulation of the human high-temperature requirement A (HtrA) family of serine proteases is associated with many malignancies. However, there are few reports on HtrAs in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the expression, prognostic value, and biological functions of HtrAs in HNSCC.

Methods: The RNA-sequencing data and clinical data of HNSCC were downloaded from The Cancer Genome Atlas (TCGA) database. The GSE30784 and GSE31056 datasets from the Gene Expression Omnibus (GEO) database were used for further verification. This study explored the differential expression of HtrAs and assessed their potential impact on the prognosis of HNSCC patients using a survival module. Correlations between clinical characteristics and HtrA expression levels were then explored using a Wilcoxon rank sum test. A Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed using "clusterProfile" in the R software. A Pearson/Spearman correlation test was applied to analyze the relationship between HtrAs and immune infiltration level/checkpoint genes. Validation of HtrA expression levels were carried out by RT-PCR and western blot in human squamous carcinoma cell lines (Fadu and Cal-27) and human non-tumorigenic bronchial epithelium cells (BEAS-2B). Finally, through cell transfection, CCK-8, Ki-67 immunofluorescence, and flow cytometry assays, the effect of HtrA3 knockdown on the malignant biological behavior of HNSCC cells was explored.

Results: The gene expression levels of HtrAs were significantly upregulated and associated with patient age, TNM stage, clinical stage, and TP53 mutation status in the TCGA-HNSCC cohort. High expressions of HtrA1/3 were associated with shorter overall survival, shorter progress-free interval, and lower disease-specific survival in HNSCC. A nomogram for HtrAs was constructed and validated. HtrA-related genes were significantly enriched in the immune response and cell apoptosis pathway. In addition, the expression of HtrAs showed significant correlations with B cells, M cells, DC cell infiltration, and immune infiltration checkpoint (CD276, TNFRSF14). Validation of HtrA expression was carried out by RT-PCR and western blot. Results of in vitro experiments indicated that HtrA3 gene knockdown inhibits the proliferation of FaDu and Cal-27 cells while concurrently promoting apoptosis.

Conclusions: HtrA3 shows significant potential as both a prognostic marker and a promising therapeutic target for HNSCC, highlighting its relevance and importance in future research and potential clinical applications.

Keywords: Head and neck squamous cell carcinoma; HtrA; Immune infiltration; Immunotherapy; Prognosis.

Figure 1. Flowchart of this study.

Figure 2. The expression levels of HtrAs in HNSCC tissues and normal tissues.

(A) mRNA expression of HtrAs in grouped samples between HNSCC tissue (n = 502) and normal tissue (n = 44) from the TCGA database; (B) mRNA expression of HtrAs in paired samples between HNSCC tissue (n = 43) and normal tissue (n = 43) from the TCGA database; (C) Expression differences of HtaA1-4 in the GSE30784 and GSE31056 datasets; (D–E) genetic alterations in differentially expressed HtrA1-4 in HNSCC.

Figure 3. Correlation between HtrA1-4 expression and clinical characteristics of HNSCC and survival curves.

The association between high and low expression levels of HtrA1-4 and overall survival (A), progress free interval (B), and disease-specific survival (C). The relevance of HtrAs and clinical stage (D), histologic grade (E), TP53 mutation status (F), pathologic T stage (G), and lymph node neck dissection (H). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4. Nomogram construction and validation.

(A) A nomogram to predict survival probability at 1, 3 and 5 years in patients with HNSCC; (B–D) the calibration curve of the nomogram; (E) receiver operating characteristic analysis (ROC) of HtrA1-4 in HNSCC patients.

Figure 5. Genomic alterations and interactions of HtrA1-4 in HNSCC.

(A) PPI network analysis of HtrA family members and their 20 co-regulated hub genes conducted by STRING; (B) HtrA-related gene enrichment, pathway analysis, and functional profiles; (C) Pearson correlation analysis of HtrA1-4; (D–H) significant gene set enrichment analysis (GSEA) results of HtrAs including the hallmark pathways.

Figure 6. Associations between HtrA1-4 and the tumor immune microenvironment.

(A) TIMER database association of HtrA1-4 expression with immune infiltration level; (B) correlation of tumor-infiltrating levels in HNSCC and the alterations of different somatic copy numbers in HtrA1-4; (C) the correlation of HtrA1-4 expression level with immune, stromal and ESTIMATE score; (D) heatmap of the 46 common immune checkpoint genes and HtrA1-4 gene expression.

Figure 7. The validation cohort and vitro experiment.

(A) Representative immunohistochemistry images of HtrA1-4 in HNSCC and normal tissues (HPA database); (B–C) protein expression of HtrA1 and HtrA3 in HNSCC and normal tissues with datasets from the CPTAC database; expression of HtrA1-4 in BEAS-2B, Cal-27 (D) and Fadu (E) cells obtained by western blot. Values associated with test proteins were normalized to standard β-actin for the relative expression measure; (F–G) column graphs of western blot analysis; (H) HtrA1-4 expression in BEAS-2B, FaDu, and Cal-27 cells by RT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 8. The effect of HtrA3 knockdown on the biological behavior of HNSCC cells.

(A–B) RT-PCR after cell transfection; (C–D) CCK8 experiment results of FaDu and Cal-27 cells after cell transfection. The OD values measured at 450 nm wavelength at 0 h, 12 h, 24 h, 48 h, and 72 h are displayed; (E) Ki-67 immunofluorescence staining of FaDu and Cal-27 cells after cell transfection. Ki67 red staining indicates cells in a state of proliferation, and DAPI blue nuclear staining represents the total number of living cells. The “Merge” image displays the combined view of both images. Relevant statistical graphs are also presented; (F–H) flow cytometry results and statistics related to apoptosis of FaDu and Cal-27 cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.