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LEKTI, a Physiological Inhibitor of Multiple Serine Proteinases, Blocks Migration and Invasion of Head and Neck Squamous Cell Carcinoma (HNSCC) Cells

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LEKTI, a Physiological Inhibitor of Multiple Serine Proteinases, Blocks Migration and Invasion of Head and Neck Squamous Cell Carcinoma (HNSCC) Cells MOJ Proteomics & Bioinformatics Research Article Open Access LEKTI, a physiological inhibitor of multiple serine proteinases, blocks migration and invasion of head and neck squamous cell carcinoma (HNSCC) cells Abstract Volume 1 Issue 3 - 2014 Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different Lympho- Arumugam Jayakumar,1,2 Chandrani Epithelial Kazal-Type-Inhibitor (LEKTI) isoforms which are organized into longer Chattopadhyay,1 Hua-Kang Wu,1 Katrina than 15, 15, and 13 inhibitory domains. We identified LEKTI by its constitutive 1 1 1 expression in normal oral mucosa and lost or down regulated expression in matched Briggs, Ying Henderson, Yaan Kang, 3 1 tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Latha Ramdas, Shikha Sharma, Venugopal Previously, we showed that recombinant full-length LEKTI and rLEKTI fragments Radjendirane,2 Thomas D Shellenberger,1 inhibit the activity of plasmin, subtilisin A, cathepsin G, neutrophil elastase, trypsin, Gary L Clayman1 caspase 14, and kallikreins (KLK) 5, 6, 7, 13, and 14 to varied extents. Here, we 1Department of Head and Neck Surgery, University of Texas show that LEKTI protein is absent in HNSCC OSC19, Tu138, Tu177, and UMSCC1 MD Anderson Cancer Center, USA lines. We then determined the consequences of LEKTI re-expression on migration 2Department of Experimental Therapeutics, University of Texas and invasion, adhesion and gene expression profile of HNSCCOSC19 and UMSCC1 MD Anderson Cancer Center, USA lines. We demonstrate that LEKTI expressing OSC19 and UMSCC1 clones show 3Department of Experimental Radiation Oncology, University of markedly reduced migration and invasion. Moreover, LEKTI expressing OSC19 Texas MD Anderson Cancer Center, USA clones show striking morphological changes and enhanced adhesionon type I, III, Arumugam Jayakumar, Department of Head IV, and V collagens, fibronectin, and laminin5.In addition, we show that exogenous Correspondence: and Neck Surgery, University of Texas MD Anderson Cancer r-LEKTI blocks migration of OSC19-parental cells in a dose and time dependent Center, Houston, Texas 77030, USA, manner. Microarray analysis identified 186 genes which are differentially regulated Email [email protected] in both OSC19 LEKTI clones.mMP-14, KLK5, and ADAM8 are down regulated whilemMP-3, LEKTI, DSC2 and DSC3are up-regulated in OSC19 LEKTI clones. RT- Received: July 01, 2014 | Published: July 08, 2014 PCR and Western blot results confirmed microarray results formMP-14 andmMP-3in OSC19 LEKTI clones. In addition we discover thatmMP-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. Keywords: SPINK5, LEKTI, invasion, migration, adhesion, mMPs, HNSCC, KLK Abbreviations: LEKTI, lympho-epithelial kazal-type- We and others identified SPINK5 as one of the genes down inhibitor; SPINK5, serine protease inhibitor kazal-type 5; KLK, regulated in head and neck squamous cell carcinoma (HNSCC).16,17 kallikreins; kDa, kilodaltons; RT-PCR, reverse-transcriptase We cloned the cDNA encoding the 125-kDa isoform and established polymerase chain reaction; SD, standard deviation of the mean that recombinant pro-LEKTI is a potent inhibitor of multiple serine proteinases implicated in metastasis and angiogenesis. Moreover, Introduction rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. We further showed that recombinant pro-LEKTI was very Lympho-epithelial kazal-type-inhibitor (LEKTI)1 was named by efficiently cleaved in vitro by furin into five major and thirteen minor one of the original groups who cloned this protein’s gene to reflect proteolytic fragments.18 In the course of studies aimed at understanding the observed pattern of its expression in both epithelial tissue and 1 the structure and function of some of these domains, we demonstrated leukocytes. It was later identified as the same gene as the defective gene that recombinant LEKTI6-9´ inhibited trypsin and subtilisin A but in Netherton’s syndrome, SPINK5 (serine protease inhibitor Kazal- 19 2 not plasmin, cathepsin G, or elastase. We also produced a battery of type 5). Netherton’s syndrome is a genetic disorder characterized by LEKTI monoclonal antibodies and demonstrated that several of these congenital ichthyosis, hair shaft abnormalities, immune deficiency, LEKTI antibodies including 1C11G6 reacted specifically with pro- elevated immunoglobulin E (IgE) concentration, and failure to 20 2–13 LEKTI, LEKTI domains 1-6, 6-9, 9-12, and 12-15. We demonstrated thrive. SPINK5 encodes the LEKTI protein which consists of 1064 that the N-terminal signal peptide is required for LEKTI import into amino acids organized into 15 potential inhibitory domains on the the ER and ordered the cleavage products on the 125kDa pro-LEKTI basis of the furin cleavage sites found within the full-length molecule. from the amino- to carboxy-terminal as follows: 37-, 40-, and 60kDa.21 At the N-terminus is a secretory signal peptide sequence consisting 14 In our subsequent work, we characterized the interaction of two of 22 amino acids. Two of the 15 LEKTI domains (domains 2 and recombinant LEKTI domains 6-8 and 9-12 with recombinant rhK5 15) resemble typical Kazal-type serine proteinase inhibitors; the and recombinant rhK7.22 We showed that both fragments inhibited remaining 13 domains share partial homology to Kazal-type inhibitors 15 rhK5 similarly and established that LEKTI, at least in fragment form, but lack one of the three conserved Kazal-type disulfide bridges. is a potent inhibitor of rhK5 and that this protease may be a target of Submit Manuscript | http://medcraveonline.com MOJ Proteomics Bioinform. 2014;1(3):57‒71. 57 © 2014 Jayakumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: LEKTI, a physiological inhibitor of multiple serine proteinases, blocks migration and invasion of head and 58 neck squamous cell carcinoma (HNSCC) cells ©2014 Jayakumar et al. LEKTI in human skin. In our later studies we discovered that KLK5, 95% air. All cells were cultured at 37°C in humidified incubator with KLK6, KLK13 and KLK14 were potently inhibited by rLEKTI (1- 5% CO2 and 95% air. 6), rLEKTI (6-9’) and rLEKTI (9-12).23,24 We also assessed the basis To clone the pro-LEKTI expression plasmid, 3.24Kb BamHI- for phenotypic variations in patients with “mild”, “moderate” and KpnI fragment from LEKTI/pFASTBAC1, clone #4 was sub cloned “severe” NS.25 We observed that the magnitude of KLK activation into pcDNA 3.1. The pro-LEKTI expression plasmid encodes the correlated with both the barrier defect and clinical severity, and entire full-length LEKTI polypeptide and has a hexahistidine tag at inversely with residual LEKTI expression and LEKTI co-localizes its C-terminus. 293T cells transfected with this construct expressed within the stratum corneum (SC) with kallikreins 5 and 7 and inhibits LEKTI. At 24h and 48h post transfection, LEKTI is detected within both KLKs. Recently, we demonstrated that caspase 14 is inhibited by the cells and in the medium. In the medium LEKTI processed LEKTI full-length LEKTI and 5 recombinant fragments of LEKTI to varied fragments are also detected with LEKTI mAb 1C11G6. In order extents.26 to delete the signal sequence, this clone is digested with NotI and In the present study, we stably re-expressed LEKTI in HNSCC EcoRI and ligated to a PCR fragment lacking this signal sequence. cells and evaluated the effects of LEKTI re-expression on cellular The pro-LEKTI-∆1-22 expression plasmid encodes the full-length proliferation, morphology, adhesion, invasion and expression of LEKTI polypeptide without the N-terminus secretory signal sequence key mMPs involved in tumor progression. LEKTI re-expression and has a hexahistidine tag at its C-terminus. When transfected into in OSC19 cells causes striking morphological changes, strongly 293T cells, very little intracellular protein is observed. Following the enhances adhesion, markedly decreased migration and invasion. verification of these expression clones, HNSCC cells were transfected Stable re-expression of LEKTI in OSC19 cells resulted in markedly with these constructs and stable clones were selected after G418 decreased levels of mMP-9 and mMP-14. Furthermore, these results (0.5mg/ml) selection. We also performed transient transfection of demonstrate a novel negative regulatory role for LEKTI in modulating OSC-19 and HEK 293T cells with these constructs. Cells were plated the production of keymMPs involved in ECM degradation and suggest in 60×15-mm tissue culture dishes grown to 70% confluence, and that loss of LEKTI in HNSCC tumor cells could have a pivotal role in transiently transfected with 2.0µg each of pro-LEKTI expression HNSCC progression. plasmid DNA or pro-LEKTI-∆1-22 expression plasmid DNA or control vector plasmid DNA using Lipofectamine 2000 according Materials and methods to the manufacturer’s instructions. Approximately 50% transfection efficiency was achieved as determined by transfection with a GFP Materials control plasmid. The following reagents were obtained commercially as indicated: Human embryonic kidney cells (HEK 293T) (American Type Culture LEKTI secretion assays Collection, Manassas, VA); primary normal epidermal
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