Cathepsin B Expression and the Correlation with Clinical Aspects of Oral Squamous Cell Carcinoma

Abstract

Background: Cathepsin B (CTSB), a member of the cathepsin family, is a cysteine protease that is widely distributed in the lysosomes of cells in various tissues. It is overexpressed in several human cancers and may be related to tumorigenesis. The main purpose of this study was to analyze CTSB expression in oral squamous cell carcinoma (OSCC) and its correlation with patient prognosis.

Methodology/principal findings: Tissue microarrays were used to detect CTSB expression in 280 patients and to examine the association between CTSB expression and clinicopathological parameters. In addition, the metastatic effects of the CTSB knockdown on two oral cancer cell lines were investigated by transwell migration assay. Cytoplasmic CTSB expression was detected in 34.6% (97/280) of patients. CTSB expression was correlated with positive lymph node metastasis (p = 0.007) and higher tumor grade (p = 0.008) but not with tumor size and distant metastasis. In addition, multivariate analysis using a Cox proportional hazards model revealed a higher hazard ratio, demonstrating that CTSB expression was an independent unfavorable prognostic factor in buccal mucosa carcinoma patients. Furthermore, the Kaplan-Meier curve revealed that buccal mucosa OSCC patients with positive CTSB expression had significantly shorter overall survival. Moreover, treatment with the CTSB siRNA exerted an inhibitory effect on migration in OC2 and CAL27 oral cancer cells.

Conclusions: We conclude that CTSB expression may be useful for determining OSCC prognosis, particularly for patients with lymph node metastasis, and may function as a biomarker of the survival of OSCC patients in Taiwan.

Fig 1. Immunohistochemically staining for the expression of cytoplasmic Cathpesin B (CTSB) in oral cancer.

Tissue microarrays of primary oral squamous cell carcinoma (OSCC) (280 cases) were analyzed. (A) No detectable CTSB (0). (B) Weak expression levels (1+). (C) Strong expression levels (2+). (D) The filed had both normal and tumor regions. (E) and (F) show higher magnification of normal and tumor region in the black boxed area of (D) respectively. A–D Low-power field (×100). E and F High-power field (×200).

Fig 2

Kaplan-Meier survival curve showing the relation between cytoplasmic CTSB expression in primary tumors and survival in 280 oral squamous cell carcinoma (OSCC) patients (A) and 109 buccal mucosa squamous cell carcinoma patients (B). (C) The CTSB expression was not correlated with progression free survival in OSCC patients (p = 0.097). (D) The progression free survival of buccal mucosa squamous cell carcinoma patients with positive CTSB staining was significantly lower than that of patients with negative CTSB staining (p = 0.011). (E) From TCGA (The Cancer Genome Atlas) database (https://tcga-data.nci.nih.gov/tcga/), the Kaplan-Meier curve showed that higher expression of CTSD had a significantly poor survival in 107 oral cancer patients (p = 0.048). (F) The CTSL and (G) CTSS mRNA expression were not correlated with overall survival in oral cancer patients.

Fig 3. Cathepsin B knockdown in OC2 and CAL27 cells reduce cell migration.

Western blot analysis showing the cathepsin B Knockdown efficiency in (A) OC2 cell and (B) CAL27 oral cancer cell lines. Detection of cell migration ability by transfected with control siRNA or CTSB siRNA in (C) OC2 cell and (D) CAL27 cell. Results shown that cathepsin B siRNA significantly reduced the migration of OC2 and CAL27 cells. *p<0.05.