Clinical significance of kallikrein-related peptidase-4 in oral cancer

Abstract

Kallikrein-related-peptidase-4 (KLK4), a serine protease originally discovered in developing tooth with broad target sequence specificity, serves vital functions in dental enamel formation. KLK4 is involved in degradation of extracellular matrix proteins and it is thought that this proteolytic activity could also promote tumor invasion and metastasis. Recent studies have associated KLK4 expression with tumor progression and clinical outcome, particularly in prostate and ovarian cancer. Very little is known in regard KLK4 involvement in oral squamous cell carcinomas (OSCCs). Our objective was to investigate KLK4 expression in OSCC pathogenesis and disease progression. KLK4 expression was evaluated by immunohistochemistry, western blots and zymograms in OSCC lines. Invasion assays using high versus low/undetectable KLK4-expressing OSCC cell lines were performed jointly with KLK4 siRNA inhibition. A large collection of OSCC specimens was evaluated for KLK4 expression and correlation with patients' characteristics and outcomes were determined. Our data indicate that KLK4 is differentially expressed in oral carcinomas. OSCC cell lines with high invasive and metastatic potential have the highest levels of KLK4 expression. KLK4 mRNA and protein expression correlated with enzyme activity detected by zymograms. Inhibition of KLK4 expression results in diminished invasive potential in OSCC cell lines. Consistently, KLK4 expression is stronger in primary tumors that later either recurred or developed metastases, suggesting that its preferential expression in OSCC might contribute to individual tumor biology. Therefore, this study provides supportive evidence in favor of a prognostic value for KLK4 in OSCC and suggests that KLK4 could serve as a potential therapeutic target in patients with oral cancer.

Keywords: Kallikrein-related-peptidase-4 (KLK4); Oral squamous cell carcinoma (OSCC); cell lines; clinical outcome; invasion; metastasis.

Figure 1. KLK4 detection in oral squamous cell carcinomas lines (SCC4 and Cal 27 comparatively) at RNA (A; quantitative real time PCR) and protein (B; immunofluorescence) levels. KLK4 RNA and protein expression was correlated with enzyme activity detected by zymograms (C; dental enamel was used as control). Inhibition of in vitro invasiveness of Cal27 cells with KLK4 specific small interfering RNA (siRNA, D)

Quantification of the cell invasion indicated that KLK4 expression was linked to OSCC propensity for invasion, whereas inhibition of KLK4 expression resulted in diminished invasive potential in OSCC cell lines. Transfections with control siRNA (Ambion, Grant Island, NY, USA) or KLK4 siRNA (SantaCruz Biotechnology, Dallas, TX, USA) was carried out in triplicate with Lipofectamine RNAi Max (Invitrogen, Grant Island, NY) according to the manufacturer’s instructions. Twenty-four hours post-transfection with the siRNA, invasion assay was carried out for another 24 hours using medium with 10% fetal bovine serum and epithelial growth factor (Lonza Basel, Switzerland) as chemo-attractants. Invading cells were fixed and stained with Diff-Quick stain. Representative phase micrograph of Cal27 cells invading the Matrigel matrix. Scale bar = 100 μm.

Figure 2. KLK4 expression in primary OSCC metastatic (A) versus non-metastatic (B) specimens – representative illustrations of the immunohistochemical staining

KLK4 is highly expressed in the metastatic tumor specimen with a cytoplasmic pattern of various intensities within the tumor and stromal cells (A). In contrast, in the non-metastatic tumor specimen, only few areas of tumor cells are positive for KLK4 with a cytoplasmic but less intense distribution (B). Magnification: 40 μm (A), 20 μm (B). The Kaplan-Meyer curve has identified two prognostic subgroups: patients with KLK4 high-expressing tumors versus those with KLK4 low-expressing tumors. There was a trend towards worse patient survival among the KLK4 high-expressing tumors (p = N.S).