Multiple kallikrein (KLK 5, 7, 8, and 10) expression in squamous cell carcinoma of the oral cavity

Abstract

Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked one of the top 10 cancers worldwide. The 5-year survival rate has remained at a low 50% for the past several decades, necessitating discovery of novel biomarkers of aggressive disease and therapeutic targets. As overexpression of urinary type plasminogen activator and receptor (uPA/R) in OSCC is associated with malignant progression and poor outcome, cell lines were generated with either overexpression (SCC25-uPAR+) or silencing (SCC25-uPAR-KD) of uPAR. As SCC25-uPAR+ tumors behaved more aggressively both in vitro and in vivo, comparative cDNA microarray analysis was used to identify additional genes that may be associated with aggressive tumors. Four members of the human tissue kallikrein family (KLK 5, 7, 8, and 10) were identified and real-time RT-PCR (qPCR) was used to verify and quantify gene expression. qPCR analysis revealed 2.8-, 5.3-, 4.0-, and 3.5-fold increases in gene expression for KLK5, 7, 8, and 10, respectively, in SCC25-uPAR+ versus SCC25-uPAR-KD. Immunohistochemical analysis demonstrated strong reactivity for KLKs 5, 7, 8 and 10 in both orthotopic murine tumors and human OSCC tissues. Control experiments show lack of reactivity against KLK3 (prostate specific antigen). These results demonstrate that kallikreins 5, 7, 8, and 10 are abundantly expressed in human OSCC and may be implicated in malignant progression.

Fig. 1. Histology of SCC25-uPAR-KD and SCC25-uPAR+ murine oral tongue tumors

Tumors generated from pooled clones of SCC25-uPAR-KD and SCC25-uPAR+ cells were (A-D) stained with H&E (E-H) or immunostained with anti-cytokeratin AE1/AE3 (M3515, 1:25 dilution) followed by a biotinylated secondary antibody and detection of avidin-biotin with DAB chromagen and substrate as in Experimental Procedures. Panels A, C, E, G are 40× magnification; panels B,D, F, H are 400×. (T) – designates areas of tumor cells; (M) designates host tongue muscle; (arrowheads) – focal keratinization (‘keratin pearls’ are highlighted); (arrows) – example of well-circumscribed tumor nests.

Fig. 1. Histology of SCC25-uPAR-KD and SCC25-uPAR+ murine oral tongue tumors

Tumors generated from pooled clones of SCC25-uPAR-KD and SCC25-uPAR+ cells were (A-D) stained with H&E (E-H) or immunostained with anti-cytokeratin AE1/AE3 (M3515, 1:25 dilution) followed by a biotinylated secondary antibody and detection of avidin-biotin with DAB chromagen and substrate as in Experimental Procedures. Panels A, C, E, G are 40× magnification; panels B,D, F, H are 400×. (T) – designates areas of tumor cells; (M) designates host tongue muscle; (arrowheads) – focal keratinization (‘keratin pearls’ are highlighted); (arrows) – example of well-circumscribed tumor nests.

Fig. 2. Immunohistochemical analysis of multiple KLK expression in SCC25-uPAR+ murine oral tongue tumors

Serial sections of tumors generated from pooled clones of SCC25-uPAR+ cells were immunostained with antibodies against (a,b) KLK5, (c,d) KLK7, (e,f) KLK8, (g,h) KLK10, or (i,j) KLK3 at the dilutions provided in Experimental Procedures followed by a biotinylated secondary antibody and detection of avidin-biotin with DAB chromagen and substrate. Panels a,c,e,g,I − 40× magnification; panels b,d,f,g,j − 400× magnification. Arrows denote tumor cell nests staining positive for the respective KLK. Identical immunoreactivity in serial sections stained for KLK5, KLK7, KLK8 and KLK10 indicates expression of multiple KLKs by the same tumor cell nest.

Fig. 3. Immunohistochemical analysis of multiple KLK expression in SCC25-uPAR-KD murine oral tongue tumors

Serial sections of tumors generated from pooled clones of SCC25-uPAR-KD cells were immunostained with antibodies against (a,b) KLK5, (c,d) KLK7, (e,f) KLK8, (g,h) KLK10, or (i,j) KLK3 at the dilutions provided in Experimental Procedures followed by a biotinylated secondary antibody and detection of avidin-biotin with DAB chromagen and substrate. Panels a, c, e, g, i − 40× magnification; panels b, d, f, g, j − 400× magnification. Identical immunoreactivity in serial sections stained for KLK5, KLK7, KLK8 and KLK10 indicates expression of multiple KLKs by the same tumor cell nest, although staining intensity is significantly reduced relative to uPAR+ tumors (Fig. 2).

Fig. 4. Immunohistochemical analysis of multiple KLK expression in normal human oral mucosa

Serial sections of normal tongue were immunostained with antibodies against (a,b) KLK5, (c,d) KLK7, (e,f) KLK8, (g,h) KLK10, or (i,j) KLK3 at the dilutions provided in Experimental Procedures followed by a biotinylated secondary antibody and detection of avidin-biotin with DAB chromagen and substrate. Panels a, c, e, g, i − 40× magnification. Panels b, d, f, g, j − 400× magnification of the lower portion of the stratum superficiale.

Fig. 5. Immunohistochemical analysis of multiple KLK expression in human oral tongue tumors

Sections of human OSCC of the oral tongue were immunostained with antibodies against (a,b) KLK5, (c,d) KLK7, (e,f) KLK8, (g,h) KLK10, or (i,j) KLK3 at the dilutions provided in Experimental Procedures followed by a biotinylated secondary antibody and detection of avidin-biotin with DAB chromagen and substrate. Panels a,c,e,g,I − 40× magnification; panels b,d,f,g,j − 400× magnification. Arrows denote tumor cell nests staining positive for the respective KLK. Identical immunoreactivity in serial sections (a-h) stained for KLK5, KLK7, KLK8 and KLK10 indicates expression of multiple KLKs by the same tumor cell nest. KLK3 immunoreactivity is not observed.