EMMPRIN Reduction Via Scfv-M6-1B9 Intrabody Affects &Alpha
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Cancer Gene Therapy (2014) 21, 246–255 & 2014 Nature America, Inc. All rights reserved 0929-1903/14 www.nature.com/cgt ORIGINAL ARTICLE EMMPRIN reduction via scFv-M6-1B9 intrabody affects a3b1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2 S Sangboonruang1, P Thammasit1, N Intasai2, W Kasinrerk3,4, C Tayapiwatana3,4,5 and K Tragoolpua1 Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, a3b1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased a3b1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy. Cancer Gene Therapy (2014) 21, 246–255; doi:10.1038/cgt.2014.24; published online 13 June 2014 INTRODUCTION are highly expressed in various cancers and enhance the degra- Colorectal cancer (CRC) is the most common cancer worldwide. dation of basement membranes and cell migration to distant 9 It is a significant cause of cancer-related deaths that mostly results organs. There are several lines of evidence demonstrating that from its poor prognosis and the advanced ability of cancer in EMMPRIN can induce the expression of several MMPs, particularly 11 metastasis to distant organs.1,2 Although there are several methods MMP-2 and MMP-9. We therefore hypothesize that EMMPRIN for cancer treatment, the successful treatment of metastatic CRC suppression affects its associated molecules, including a3b1- has been limited. Extracellular matrix metalloproteinase inducer integrin, MCT1, MMP-2 and MMP-9 expression, and inhibits cell (EMMPRIN), also known as CD147, is a potential candidate for proliferation, migration and invasion. cancer-targeted therapy because it has an important role in Intracellular antibody or intrabody is an antibody molecule that various processes in cancer progression. Many previous studies functions inside a cell that blocks or modulates the function of a 12 on the function of EMMPRIN have demonstrated its role in cell target protein or antigen. Intrabody can achieve its effect in proliferation, invasion, metastasis, angiogenesis, glycolysis and different cellular compartments by adding localization signal multidrug-resistant (MDR) phenotypes mediated by many sequences. Intrabody with an endoplasmic reticulum (ER) reten- associated molecules.3–5 It has been reported that EMMPRIN is tion signal retains the target protein in the ER and prevents the 13 associated with cell adhesion molecules, especially a6b1-integrin expression of the target protein on the cell surface. Compared and a3b1-integrin, thus promoting invasion and metastasis. It was with RNAi-based approaches, intrabodies are more specific for an found that EMMPRIN and a3b1-integrin interaction activates focal intracellular-targeted protein, have a longer half-life and are 13,14 adhesion kinase (FAK)–paxillin and FAK–phosphoinositide 3 kinase therefore more effective. Most intrabodies are derived from (FAK-PI3K)–Ca2 þ signaling pathways, leading to the enhancement single-chain variable fragments (scFvs), which are expressed after of metastasis of cancer cells.6 EMMPRIN is also involved in tumor gene transfer in target cells. Adenovirus vectors have been used 12 cell glycolysis through the proton-coupled monocarboxylate for transfer of intrabody genes. We previously showed that an transporter (MCT) family, the key regulator of glycolysis and adenovirus serotype 5 vector encoding scFv-M6-1B9 intrabody intracellular pH (pHi) homeostasis in cancer cells. EMMPRIN acts gene against EMMPRIN (Ad5-scFv-M6-1B9) efficiently decreased 15 as a chaperone for MCT1 and facilitates MCT1 expression on the the surface expression of EMMPRIN on 293A cells and HeLa 16 cell membrane leading to transportation of lactate that promotes cells. It retained EMMPRIN in the ER with an ER retention signal cancer cell survival and disease progression.7,8 In addition, (KDEL peptide), and intrabody–antigen complexes are considered 17,18 EMMPRIN also stimulates the production of matrix metallopro- to be degraded via different mechanisms. Serotype 5 Ad teinases (MMPs), a group of zinc-dependent enzymes with vectors infect cells through the coxsackie and adenovirus receptor proteolytic activity.3,9,10 Many studies have revealed that MMPs (CAR). However, many human tumors often lack CAR, which makes 1Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand; 2Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand; 3Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand; 4Biomedical Technology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand and 5BioMedical Engineering Center, Chiang Mai University, Chiang Mai, Thailand. Correspondence: Dr K Tragoolpua, Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. E-mail: [email protected] Received 9 January 2014; revised 30 April 2014; accepted 30 April 2014; published online 13 June 2014 scFv-M6-1B9 intrabody affects EMMPRIN, a3b1-integrin in Caco-2 S Sangboonruang et al 247 them refractory to Ad5 infection.19,20 To overcome the disadvan- Enzyme-linked immunosorbent assay tages of Ad5-based vector, chimeric adenovirus 5/F35 (Ad5/F35), The binding activity of the scFv-M6-1B9 intrabody to EMMPRIN was containing the fibers from Ad serotype 35, has been generated. evaluated by enzyme-linked immunosorbent assay. Avidin-carbonate/ These vectors use CD46, a membrane protein that is upregulated bicarbonate buffer pH 8.6 (10 mgmlÀ 1) was coated on a microtiter plate in most tumors as a receptor.21,22 and incubated overnight at 4 1C. The plates were then blocked with 2% In this study, we transferred the gene encoding scFv-M6-1B9 bovine serum albumin (BSA) in PBS pH 7.2 at room temperature for 1 h. intrabody specific to EMMPRIN via a chimeric adenovirus 5/F35 The wells were washed with 0.05% Tween-20 in PBS pH 7.2. After the washing step, 100 mgmlÀ 1 recombinant biotinylated CD147-BCCP or (Ad5/F35-scFv-M6-1B9) into the CRC cell line Caco-2, which has a survivin-BCCP (SVV-BCCP, an irrelevant protein) fusion proteins in 2% low expression level of CAR. EMMPRIN surface expression and a BSA in PBS were added and incubated at room temperature for 1 h. The subsequent effect of scFv-M6-1B9 intrabody on related molecules, proteins extracted from Ad5/F35-scFv-M6-1B9-, Ad5/F35-scFv-irrelevant- or that is, a3b1-integrin, MCT1, MMP-2 and MMP-9, on Caco-2 cells Ad5/F35-GFP-transduced cell lysates were diluted at 1:50 and 1:100. After were investigated. washing, the protein lysates were added and incubated for 1 h at room temperature. The plate was washed with 0.05% Tween-20 in PBS pH 7.2 and followed by incubation with peroxidase-conjugated goat anti-HA mAb MATERIALS AND METHODS (Genscript, Piscataway, NJ, USA). The wells were then washed and followed by adding 100 ml of TMB substrate. The optical density at 450 nm was Cell culture measured after adding 1 N HCl to stop the reaction. Two sets of triplicates The human embryonic kidney cell line (293A) (Invitrogen, Grand Island, NY, were performed for each assay. USA) and the human CRC cell line (Caco-2) (a kind gift from Dr Samlee Mankhetkorn, Chiang Mai University, Chiang Mai, Thailand) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, Flow cytometry analysis NY, USA) supplemented with 10% fetal bovine serum (FBS), penicillin Caco-2 cells were transduced with Ad5/F35-scFv-M6-1B9, Ad5/F35-scFv- (100 units ml À 1) and streptomycin (100 mgmlÀ 1). The cell lines were irrelevant or Ad5/F35-GFP at MOI of 100 and harvested after 48 h of maintained in a humidified atmosphere of 5% CO at 37 1C. 2 transduction. Cells were then blocked with human AB serum for 30 min and incubated with mouse anti-CD147 mAb (clone M6-1B9) or mouse anti- The generation of Ad5/F35-scFv-M6-1B9 a3b1-integrin mAb (Abcam, Cambridge, MA, USA). Phycoerythrin-con- jugated sheep anti-mouse Ig pAb was used as a secondary antibody. For For construction of the pAdE/F35-scFv-M6-1B9 vector, the PmeI-linearized MCT1 staining, the cells were fixed and permeabilized with ice-cold and -purified pAdT-scFv-M6-1B9 vector (a shuttle vector plasmid) was methanol: acetone (1:1) at À 20 1C for 20 min and followed by washing co-electroporated with pAdEasy-1/35 (kindly provided by Prof. Andre with 1% BSA–PBS–NaN . Cells were stained with rabbit anti-MCT1 pAb. Lieber, University of Washington, Seattle, WA, USA) in E. coli BJ5183.15,22 3 Phycoerythrin-conjugated goat anti-rabbit IgG pAb was used as secondary Ad5/F35-scFv-M6-1B9 and Ad5/F35-scFv-irrelevant were generated and antibody. Finally, the cells were washed with 1% BSA–PBS–NaN , fixed with propagated in 293A cells and purified as described elsewhere.15,23 3 1% paraformaldehyde in PBS and analyzed by flow cytometry.