Abstract
Disturbance in the proteolytic process is one of the malignant signs of tumors. Proteolysis is highly orchestrated by cysteine cathepsin and its inhibitors. Cystatin-B (CSTB) is a general cysteine cathepsin inhibitor that prevents cysteine cathepsin from leaking from lysosomes and causing inappropriate proteolysis. Our study found that CSTB was downregulated in both oral squamous cell carcinoma (OSCC) tissues and cells compared with normal controls. Immunohistochemical analysis showed that CSTB was mainly distributed in the epithelial structure of OSCC tissues, and its expression intensity was related to the grade classification. A correlation analysis between CSTB and clinical prognosis was performed using gene expression data and clinical information acquired from The Cancer Genome Atlas (TCGA) database. Patients with lower expression levels of CSTB had shorter disease-free survival times and poorer clinicopathological features (e.g., lymph node metastases, perineural invasion, low degree of differentiation, and advanced tumor stage). OSCC cell models overexpressing CSTB were constructed to assess the effects of CSTB on malignant biological behaviors and upregulation of CSTB inhibited cell proliferation, migration, and invasion in vitro. Weighted gene correlation network analysis (WGCNA) and gene set enrichment analysis (GSEA) were performed based on the TCGA data to explore potential mechanisms, and CSTB appeared to correlate with squamous epithelial proliferation-differentiation processes, such as epidermal cell differentiation and keratinization. Moreover, in WGCNA, the gene module most associated with CSTB expression (i.e., the brown module) was also the one most associated with grade classification. Upregulation of CSTB promoted the expression levels of markers (LOR, IVL, KRT5/14, and KRT1/10), reflecting a tendency for differentiation and keratinization in vitro. Gene expression profile data of the overexpressed CSTB cell line were obtained by RNA sequencing (RNA-seq) technology. By comparing the GSEA enrichment results of RNA-seq data (from the OSCC models overexpressing CSTB) and existing public database data, three gene sets (i.e., apical junction, G2/M checkpoint, etc.) and six pathways (e.g., NOTCH signaling pathway, glycosaminoglycan degradation, mismatch repair, etc.) were enriched in the data from both sources. Overall, our study shows that CSTB is downregulated in OSCC and might regulate the malignant characteristics of OSCC via the epithelial proliferation/differentiation program.
Highlights
As the sixth most common cancer in the world, head and neck squamous cell carcinoma (HNSCC) continues to rise yearly, and oral squamous cell carcinoma (OSCC) accounts for 90% of HNSCC cases in the region [1,2,3,4]
The roles of CSTB, a cysteine cathepsin inhibitor associated with various cancers, have been poorly understood, and few studies have focused on its association with HNSCC
The present study focused on OSCC and explored the regulatory roles of CSTB in vivo and in vitro with both experimental and bioinformatic data
Summary
As the sixth most common cancer in the world, head and neck squamous cell carcinoma (HNSCC) continues to rise yearly, and oral squamous cell carcinoma (OSCC) accounts for 90% of HNSCC cases in the region [1,2,3,4]. It is generally accepted that the initiation and development of OSCC is a complex process requiring the accumulation of genomic alterations, which is modified by individual genetic predisposition and environmental carcinogenic risk factors [7,8,9] It is necessary and challenging for clinical oncology and precision medicine science to understand the carcinogenic and progression mechanisms of OSCC, to identify biomarkers for early screening and to establish an accurate prognostic evaluation system, all of which would contribute to the reduction in the incidence and improvement of both the survival rate and living quality of tumor-bearing patients [10,11,12]
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