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 cell-cell 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.

FIGURE 1.

Immunohistochemical analysis of Dsg1 expression in normal tongue and OSCC. Sections of (A) normal tongue (n = 8) and (B) OSCC of the tongue (n = 25) were processed for immunostaining using antibodies directed against Dsg1 (1:25 dilution) followed by a biotinylated secondary antibody. Although the sample size was not sufficiently large for robust statistical power, staining trends indicate strong junctionally localized staining in normal tongue (scores: 1 = 12%, 2 = 24%, 3 = 63%), and weaker cytoplasmic staining in OSCC (scores: 1 = 56%, 2 = 35%, 3 = 8%). Arrows denote strong junctional Dsg1 staining. Magnification ×100.

FIGURE 2.

Proteinase inhibitors block Dsg1 processing. SCC25 cells were cultured in the absence (lanes 1, 3, 5) or presence (lanes 2, 4, 6) of leupeptin (100 μm) and chymostatin (100 μm) for 1, 2, or 7 days prior to lysis and electrophoresis on 9% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane and immunoblotted with anti-Dsg1 (1:1000) followed by peroxidase-conjugated secondary antibody (1:4000) and SuperSignal West Dura Extended Duration substrate. Arrows denote migration position of full-length Dsg1 (165 kDa); arrowheads denote migration position of cleavage product (∼130 kDa).

FIGURE 3.

Knockdown of KLK-5 expression reduces processing of Dsg1. A, immunocytochemical analysis of KLK5 expression in control (left panel) or KLK5-KD (right panel) SCC25 cells. Cells were cultured on glass coverslips, fixed and incubated with antibodies against KLK5 (1:50 dilution) followed by Alexa-Fluor-labeled secondary antibodies and counterstained with DAPI. Yellow scale bar, 100 μm. B, quantification of immunofluorescent staining with NIH ImageJ for average intensity. C, quantitative real time PCR analysis of KLK5 levels in control and KLK5-KD SCC25 cells. Relative quantification normalized against the housekeeping gene PGK-1 mRNA levels. Graph depicts KLK5 levels in SCC25-KLK5-KD cells normalized relative to SCC25 parental cells (designated as 100). D, analysis of Dsg1 processing in SCC25-KLK5-KD cells. Lysates from duplicate cultures of parental SCC25 cells (lanes 1 and 2) or two clones of SCC25-KLK5-KD cells (lanes 3 and 4) were electrophoresed on 9% SDS-polyacrylamide gels, transferred to PVDF membrane and immunoblotted with anti-Dsg1 (1:1000; upper panel), anti-E-cadherin (1:1000; middle panel), or anti-GAPDH (1:4000; lower panel) followed by peroxidase-conjugated secondary antibody (1:4000) and peroxidase substrate. Arrow denotes migration position of full-length Dsg1 (165 kDa); arrowhead denotes migration position of cleavage product (∼130 kDa). E, densitometric quantitation of band density of Dsg1 cleavage product denoted by arrowhead in D. F, densitometric quantitation of band density of corresponding blots shown in D.

FIGURE 4.

Ultrastructural analysis of desmosomes. SCC25 or SCC25-KLK5-KD cells were grown on coverslips to confluence, then fixed and processed for transmission electron microscopy. Ultrathin sections were examined with a JEOL 1400 Transmission Electron Microscope. Representative TEM image from (A) SCC25 and (B) SCC25-KLK5-KD cells. Scale bar, 0.2 μm. C, quantitation of desmosome number/field from a minimum of 70 images each of SCC25 and SCC25-KLK5-KD cells.

FIGURE 5.

Overexpression of KLK-5 expression induces processing of Dsg1. A, immunocytochemical analysis of KLK5 expression in control (left panel) OKF/6 cells or OKF/6 cells transfected with a KLK5 expression vector to generate OKF/6-KLK5+ cells (right panel). Cells were cultured on glass coverslips, fixed, and incubated with antibodies against KLK5 (1:50 dilution) followed by Alexa-Fluor-labeled secondary antibodies and counterstained with DAPI. Scale bar 100 μm. B, quantification of immunofluorescent staining with NIH ImageJ for average intensity. C, quantitative real time PCR analysis of KLK5 levels in control and KLK5+ OKF/6 cells. Relative quantification normalized against the housekeeping gene PGK-1 mRNA levels. Graph depicts KLK5 levels in OKF/6-KLK5+ cells normalized relative to OKF/6 parental cells (designated as 100). D, analysis of Dsg1 processing in OKF/6-KLK5+ cells. Lysates from parental OKF/6 cells (lane 1), vector-transfected OKF/6 cells (lane 2), and OKF/6-KLK5+ cells (lane 3) were electrophoresed on 9% SDS-polyacrylamide gels, transferred to PVDF membrane, and immunoblotted with anti-Dsg1 (1:1000; upper panel), anti-E-cadherin (1:1000; middle panel) or anti-GAPDH (1:4000; lower panel) followed by peroxidase-conjugated secondary antibody (1:4000) and peroxidase substrate. The arrow denotes migration position of full-length Dsg1 (165 kDa); arrowhead denotes migration position of cleavage product (∼130 kDa). E, densitometric quantitation of band density of Dsg1 cleavage product denoted by arrowhead in D. F, densitometric quantitation of band density of corresponding blots shown in D.

FIGURE 6.

Effect of KLK5 expression on cell-cell aggregation dynamics. Single cell suspensions of (A) SCC25, (B) SCC25-KLK5-KD, (C) OKF/6, or (D) OKF/6-KLK5+ were incubated in culture medium containing 0.5% BSA and rotated for 7 h. At the indicated time points, aliquots were removed and photographed to visualize cell-cell aggregation. E and H, quantitation of aggregation kinetics. The number of single cells remaining in the suspension at each time point was enumerated and is shown relative to time 0 (100% single cells). (Closed circle) SCC25, (open circle) SCC25-KLK5-KD, (closed triangle) OKF/6, (open triangle) OKF/6-KLK5+. F, G, I, J, distribution of cellular aggregates. Within a high-powered field, the number of cellular clusters comprised of (black bar) <10 cells, (white bar) 10–50 cells, and (gray bar) >50 cells was enumerated at the designated time points. (F) SCC25, (G) SCC25-KLK5-KD, (I) OKF/6, (J) OKF/6-KLK5+.

FIGURE 7.

Effect of KLK5 expression on monolayer cohesion. Cell-cell adherent monolayers were separated from culture dishes by pulsing with dispase as described under “Experimental Procedures,” transferred to conical tubes affixed to a rocking platform, and subjected to 50 inversion cycles. A, B, D, E, aliquots were photographed to visualize relative monolayer cohesion or dissociation. C and F, total number of fragments present following mechanical disruption was quantified.