Kallikrein-5 Promotes Cleavage of Desmoglein-1 and Loss of Cell-Cell Cohesion in Oral Squamous Cell Carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) ranks among the top 8 causes of cancer death worldwide, with only a 60% 5-year survival rate, highlighting the need for discovery of novel biomarkers and therapeutic targets. We have previously reported that expression of a panel of serine proteinase kallikreins (KLK 5, 7, 8, and 10) is correlated with formation of more aggressive OSCC tumors in a murine orthotopic OSCC model and is elevated in human OSCC. Current studies focus on understanding the potential role of KLK5 in OSCC progression. In initial studies, KLK levels in malignant OSCC cells (SCC25) were compared with cells from normal oral mucosa (OKF/6) and pre-malignant oral keratinocytes (pp126) using qPCR. A marked elevation of all KLKs was observed in aggressive SCC25 cells relative to OKF/6 cells. In normal skin, KLKs are involved in desquamation during epidermal differentiation via proteolytic cleavage of the desmosomal cadherin component desmoglein 1 (Dsg1). As loss of cell-cell cohesion is prevalent in tumor metastasis, Dsg1 integrity was evaluated. Results show that SCC25 cells exhibit cleavage of Dsg1, which is blocked by proteinase inhibitor treatment as well as by siRNA silencing of KLK5 expression. Furthermore, cell-cell aggregation assays demonstrate that silencing of KLK5 enforces cellcell adhesion; conversely, overexpression of KLK5 in normal oral mucosal cells (OKF/6) enhances cell dispersal. These data suggest that KLK5 may promote metastatic dissemination of OSCC by promoting loss of junctional integrity through cleavage of desmoglein 1.Oral cavity cancer results in over 200,000 deaths annually and is one of the top eight most frequently diagnosed cancers worldwide (1). The most common malignancy of the oral cavity is oral squamous cell carcinoma (OSCC), 3 including tumors affecting the tongue, floor of the mouth, buccal mucosa, lips, palate, and gingiva, causing more deaths than any other oral disease (2-3). According to National Cancer Institute statistics, about 23,110 new cases of oral cancer were diagnosed in 2009 in the United States (4). Unfortunately, despite advancements in surgery, chemotherapy, and radiation, only 60% of these individuals will survive for five years. The high mortality associated with OSCC is due primarily to the detection of late-stage disease after the primary tumor has metastasized. Treatment of advanced OSCC is also associated with high morbidity and mortality, resulting from local, regional, and distant metastasis (2-3). The cellular and biochemical factors that underlie locoregional and distant spread of the disease are poorly understood. Invasion and metastasis of OSCC require multiple cellular events including disruption of cell-cell adhesive contacts, cytoskeletal alterations, and basement membrane attachment, matrix protein proteolysis, and migration (5-6). Thus a more detailed analysis of the molecular events that contribute to OSCC metastasis is a necessary prerequisite for the development of novel early detection and treatment strategies that have a favorable im...

Hepatocyte Growth Factor Activator Inhibitor Type 1 Maintains the Assembly of Keratin into Desmosomes in Keratinocytes by Regulating Protease-Activated Receptor 2–Dependent p38 Signaling

“…For example, desmoglein 2 is a functional and physiological substrate of matriptase and KLK-7, 10 and KLK-5 promotes cleavage of desmoglein-1. 11 In the present study, we found that the desmoglein-3 protein level was reduced in HAI-1 KD HaCaT cells without significant alteration of its mRNA level, and treatment with the p38 inhibitor, SB203580, abrogated the reduction of desmoglein-3. However, it remains undetermined whether the decreased desmoglein 3 level simply reflects the decreased number of desmosomes or was caused by enhanced degradation in the absence of HAI-1, leading to a decreased number of desmosomes.…”

“…For example, matriptase, a type II transmembrane serine protease on the epithelial cell surface, degrades desmoglein-2, and kallikrein 1erelated peptidase (KLK)-5 promotes cleavage of desmoglein-1. 10,11 Hepatocyte growth factor activator inhibitor type 1 (HAI-1; official symbol SPINT1), encoded by the SPINT1 gene, is a serine protease inhibitor abundantly expressed in the placenta and in epithelial tissues. 12,13 HAI-1 regulates several trypsinlike serine proteinases, such as hepatocyte growth factor activator, matriptase, prostasin, hepsin, TMPRSS13, human airway trypsin-like protease, and KLKs 4 and 5.…”

Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma

“…SCC25-initiated tumors invade tongue muscle, nerve bundles, and blood and lymphatic vessels (35,36); however, distant metastasis is not observed prior to sacrifice for ethical considerations due to difficulty with feeding. Generation of an SCC25 cell line with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD) and empty vector control cells (SCC25-Vec) was previously described (18). KLK5 mRNA expression in KLK5-KD cells is reduced by 90% relative to corresponding vector controls, as determined by qPCR, and loss of KLK5 protein was shown by immunocytochemistry (18).…”

“…Generation of an SCC25 cell line with reduced KLK5 expression (KLK5 knockdown, designated SCC25-KLK5-KD) and empty vector control cells (SCC25-Vec) was previously described (18). KLK5 mRNA expression in KLK5-KD cells is reduced by 90% relative to corresponding vector controls, as determined by qPCR, and loss of KLK5 protein was shown by immunocytochemistry (18). SCC25-derived cell lines were routinely maintained in DMEM/F-12 1:1 media (Corning) containing 10% fetal calf serum and supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 0.2 g/ml puromycin.…”

“…We have previously reported that KLK5 is up-regulated at the mRNA and protein levels in oral cancer cell lines and human OSCC tissues (17) and catalyzes cleavage of the cell-cell adhesion molecule desmoglein-1, promoting loss of junctional integrity and enhanced invasive activity in vitro (18). Activity of KLK5 is tightly controlled via expression of the proteinase inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI, encoded by SPINK5).…”