Abstract
Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different Lympho- Epithelial Kazal-Type-Inhibitor (LEKTI) isoforms which are organized into longer than 15, 15, and 13 inhibitory domains. We identified LEKTI by its constitutive expression in normal oral mucosa and lost or down regulated expression in matched tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Previously, we showed that recombinant full-length LEKTI and rLEKTI fragments inhibit the activity of plasmin, subtilisin A, cathepsin G, neutrophil elastase, trypsin, caspase 14, and kallikreins (KLK) 5, 6, 7, 13, and 14 to varied extents. Here, we show that LEKTI protein is absent in HNSCC OSC19, Tu138, Tu177, and UMSCC1 lines. We then determined the consequences of LEKTI re-expression on migration and invasion, adhesion and gene expression profile of HNSCCOSC19 and UMSCC1 lines. We demonstrate that LEKTI expressing OSC19 and UMSCC1 clones show markedly reduced migration and invasion. Moreover, LEKTI expressing OSC19 clones show striking morphological changes and enhanced adhesionon type I, III, IV, and V collagens, fibronectin, and laminin5.In addition, we show that exogenous r-LEKTI blocks migration of OSC19-parental cells in a dose and time dependent manner. Microarray analysis identified 186 genes which are differentially regulated in both OSC19 LEKTI clones. MMP-14, KLK5, and ADAM8 are down regulated while MMP-3, LEKTI, DSC2 and DSC3are up-regulated in OSC19 LEKTI clones. RT-PCR and Western blot results confirmed microarray results for MMP-14 and MMP-3in OSC19 LEKTI clones. In addition we discover that MMP-9 protein expression and pro-MMP-9 activity are severely reduced in LEKTI expressing clones as shown by WB and zymogram. Together, this work provides mechanistic insights into how loss of LEKTI protein expression promotes an invasive phenotype in HNSCC tumors.
Highlights
Lympho-epithelial kazal-type-inhibitor (LEKTI)1 was named by one of the original groups who cloned this protein’s gene to reflect the observed pattern of its expression in both epithelial tissue and leukocytes.1 It was later identified as the same gene as the defective gene in Netherton’s syndrome, Serine Protease Inhibitor Kazal-type 5 (SPINK5).2 Netherton’s syndrome is a genetic disorder characterized by congenital ichthyosis, hair shaft abnormalities, immune deficiency, elevated immunoglobulin E (IgE) concentration, and failure to thrive.2–13 SPINK5 encodes the LEKTI protein which consists of 1064 amino acids organized into 15 potential inhibitory domains on the basis of the furin cleavage sites found within the full-length molecule
Anti-LEKTI antibody 1C11G6 recognized a major protein of ∼125kDa and amine or protein of 110kDa in HNEKs indicating robust expression of LEKTI protein; in contrast both bands were absent in all four head and neck squamous cell carcinoma (HNSCC) tumor lines (Figure 1B)
The results were consistent with the patterns of LEKTI protein expression reported for normal kidney and colon tissues.30 recombinant LEKTI in these three clones is processed and secreted into the medium similar to what we reported for HNEK cells
Summary
Lympho-epithelial kazal-type-inhibitor (LEKTI) was named by one of the original groups who cloned this protein’s gene to reflect the observed pattern of its expression in both epithelial tissue and leukocytes. It was later identified as the same gene as the defective gene in Netherton’s syndrome, SPINK5 (serine protease inhibitor Kazaltype 5). Netherton’s syndrome is a genetic disorder characterized by congenital ichthyosis, hair shaft abnormalities, immune deficiency, elevated immunoglobulin E (IgE) concentration, and failure to thrive. SPINK5 encodes the LEKTI protein which consists of 1064 amino acids organized into 15 potential inhibitory domains on the basis of the furin cleavage sites found within the full-length molecule. Lympho-epithelial kazal-type-inhibitor (LEKTI) was named by one of the original groups who cloned this protein’s gene to reflect the observed pattern of its expression in both epithelial tissue and leukocytes.. Lympho-epithelial kazal-type-inhibitor (LEKTI) was named by one of the original groups who cloned this protein’s gene to reflect the observed pattern of its expression in both epithelial tissue and leukocytes.1 It was later identified as the same gene as the defective gene in Netherton’s syndrome, SPINK5 (serine protease inhibitor Kazaltype 5).. Stable re-expression of LEKTI in OSC19 cells resulted in markedly decreased levels of mMP-9 and mMP-14 These results demonstrate a novel negative regulatory role for LEKTI in modulating the production of keymMPs involved in ECM degradation and suggest that loss of LEKTI in HNSCC tumor cells could have a pivotal role in HNSCC progression
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