A Large Extracellular Loop of TSPAN8 As a Novel Therapeutic Target for Metastatic Colorectal Cancer Cell Invasion

RESEARCH HIGHLIGHT

A large extracellular loop of TSPAN8 as a novel therapeutic target for metastatic colorectal cancer cell invasion

Taek-Keun Kim, Sukmook Lee

Laboratory of Molecular Cancer Therapeutics, Scripps Korea Antibody Institute, Hyoja-2-dong, Chuncheon-si, Gangwon-do, 200-701, South Korea

Correspondence: Sukmook Lee E-mail: [email protected] or [email protected] Received: January 21, 2016 Published online: March 08, 2016

We recently reported a study that identified the relevance of the large extracellular loop of tetraspanin 8 (TSPAN8-LEL) in metastatic colorectal cancer (mCRC) cell invasion. This study suggested a functional role for TSPAN8-LEL in mCRC cell invasion and the potential use of an antibody to inhibit the invasion of TSPAN8-expressing mCRCs. In this research highlight, we describe TSPAN8-LEL as a novel potential therapeutic target in mCRCs. TSPAN8 plays a key role in TSPAN8-expressing HCT116 mCRC cell invasion. Among its two extracellular loops, TSPAN8-LEL is the key domain that regulates HCT116 mCRC cell invasion. In addition, a fully human monoclonal antibody specific to TSPAN8-LEL (anti-TSPAN8-LEL HuMab) efficiently suppresses the invasion of HCT116 and LoVo mCRC cell lines more potently than HCT-8 and SW480 non-mCRC cell lines. These results indicate that targeting TSPAN8-LEL may suppress the invasion of TSPAN8-expressing mCRCs. Therefore, TSPAN8 may be a novel potential therapeutic target for antibody-based mCRC therapy. Keywords: TSPAN8; Therapeutic target; Cancer, Invasion; Human antibody To cite this article: Taek-Keun Kim, et al. A large extracellular loop of TSPAN8 as a novel therapeutic target for metastatic colorectal cancer cell invasion. Can Cell Microenviron 2016; 3: e1196. doi: 10.14800/ccm.1196.

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General features of TSPAN8 in cancers than 30 known members in humans [4]. Tetraspanins play key roles as facilitators that form molecular webs that bring Tetraspanins are a superfamily of small transmembrane together large complexes, allowing them to function more proteins that consist of four transmembrane domains, three efficiently. Depending on the interacting molecules, cytosolic domains, and two extracellular loops: a small tetraspanins are closely involved in a multitude of biological extracellular loop (SEL) and a large extracellular loop (LEL) processes, such as fertilization, parasite and viral infection, [1]. Highly conserved cysteines in the LEL are an essential synaptic contacts at neuromuscular junctions, platelet feature of tetraspanins [2]. The tetraspanin superfamily was aggregation, adhesion, and motility [5-8]. first recognized in 1990, when a comparison of the sequences of the newly cloned CD37, CD81, and sm23 genes with Tetraspanin 8 (TSPAN8), which is also known as CO-029 CD63 revealed sequence homology and a conserved and transmembrane 4 superfamily member 3 (TM4SF3), is a predicted structure [3]. The family has now grown to more tumor-associated antigen that is highly overexpressed in

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Figure 1. Schematic illustration of the role of TSPAN8-LEL and possible application of the anti-TSPAN8-LEL HuMab. TSPAN8-LEL interacts with specific binding partners on the surface of metastatic colorectal cancer (mCRC) cells and participates in TSPAN8-mediated invasion of mCRCs. The human antibody we have developed acts as a TSPAN8-blocking antibody that interrupts the molecular interactions between TSPAN8 and its binding partners, resulting in the inhibition of mCRC cell invasion. The antibody also has applications as a research tool for understanding the molecular mechanisms of TSPAN8 as well as for the in vivo imaging of TSPAN8-expressing mCRCs. several types of cancer, including colorectal, liver, 20]. Thus, increasing attention has been paid to identifying the pancreatic, and gastric cancers [1, 9, 10]. It associates with molecular mechanisms of tumor invasion and therapeutic diverse membrane binding partners, such as CD44, targets for cancer therapy. Our recent study showed that claudin-7, α6β4, and EpCAM [4, 11], and plays a role in a TSPAN8-LEL may be closely associated with mCRC variety of cancer cell functions, including cancer cell invasion. SiRNA-mediated TSPAN8 knockdown specifically survival, migration, metastasis, and tumor angiogenesis suppressed HCT116 mCRC cell invasion. Additionally, [12-18]. However, the importance of TSPAN8-LEL and its TSPAN8 overexpression increased the invasiveness of validation as a therapeutic target in metastatic colorectal COS-7 cells. Through competition assays with TSPAN8 cancers (mCRCs) has yet to be elucidated. extracellular loops, we found that TSPAN8-LEL is a critical domain for HCT116 mCRC cell invasion [21]. Collectively, Identification of TSPAN8-LEL as a key domain in mCRC these findings suggest that TSPAN8-LEL is likely a critical cell invasion domain necessary for TSPAN8-mediated mCRC cell invasion. Tumor invasion is a complex phenomenon that results from the coordinated action of many metastatic regulators [19, Generation and characterization of an

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Cancer Cell & Microenvironment 2016; 3: e1196. doi: 10.14800/ccm.1196; © 2016 by Taek-Keun Kim, et al. http://www.smartscitech.com/index.php/ccm anti-TSPAN8-LEL HuMab TSPAN8 interacts with many binding partners in cancer progression and metastasis [4, 11]. Our competitive blocking Traditionally, conventional antibody selection has used experiments, which showed that HCT116 mCRC cell whole extracellular domains as the target antigens and the invasion was potently blocked by TSPAN8-LEL protein and selected antibodies bind to the protein of interest but are not anti-TSPAN8-LEL antibody [21], lead us to speculate that the necessarily functional antibodies. Thus, the identification of LEL of TSPAN8 may act as a binding donor that interacts a functional domain that governs specific cellular functions with several binding proteins during mCRC cell invasion. of a molecular target has become a major challenge to Therefore, the blocking of their interaction may be critical overcome, particularly when considering the time-consuming for the specific inhibition of cell invasion. and labor-intensive functional antibody screening process. Our study presented an effective strategy to develop a We propose three possible uses of the anti-TSPAN8-LEL functional antibody by first identifying TSPAN8-LEL as a HuMab. First, the antibody may be a useful research tool for functional domain in mCRC cell invasion and subsequently gaining a better understanding of the interaction between selecting an anti-TSPAN8-LEL HuMab from a synthetic TSPAN8-LEL and its binding partners during human antibody library [21]. TSPAN8-mediated tumor cell invasion. Second, its specificity and high affinity to TSPAN8-LEL leads us to In particular, in order to isolate human monoclonal speculate that it may specifically target TSPAN8-expressing antibodies specific to TSPAN8-LEL (anti-TSPAN8-LEL mCRC cells in vivo and may also be used as an antibody HuMab), we performed bio-panning against purified platform for in vivo imaging of mCRC. Third, because the TSPAN8-LEL using a phage-displayed synthetic human antibody efficiently inhibits mCRC invasion in vitro and is a scFv library. Simultaneously, through several in vitro human antibody that is less immunogenic in vivo, the characterization analyses, the anti-TSPAN8-LEL HuMab anti-TSPAN8-LEL HuMab may efficiently suppress was demonstrated to have sub-nanomolar affinity (~0.6 nM) TSPAN8-mediated mCRC cell invasion when used in and specifically recognize a native, non-reduced form of combination with existing standard chemotherapies in TSPAN8-LEL, with the epitope residing within amino acids colorectal cancer therapy. 140-205 [21]. In summary, based on existing data and the Specific and potent suppression of mCRC cell invasion by spatiotemporal requirement of mCRC cells, we suggest that anti-TSPAN8-LEL HuMab TSPAN8-LEL may interact with specific binding partners and participate in TSPAN8-mediated invasion of mCRCs. Despite the remarkable development of anti-cancer drugs The antibody-based modulation of TSPAN8-LEL may over several decades, the molecular mechanisms and interrupt the molecular interactions between TSPAN8 and its therapeutic targets of cancer cell invasion have not been binding partners, resulting in the inhibition of mCRC cell clearly identified. Thus, cancer cell invasion still represents a invasion. Finally, TSPAN8-LEL is an attractive therapeutic major hurdle that must be overcome to improve patient target for antibody therapy in mCRCs. outcomes. Accordingly, there remains a need to identify a novel therapeutic target in cancer cell invasion and to Conflicting interests develop effective therapeutic interventions against it in cancer therapy. In our study, to validate the therapeutic value The authors have declared that no conflict of interest of TSPAN8-LEL in mCRC cell invasion, we compared the exists. effect of the anti-TSPAN8-LEL HuMab on mCRC cell invasion with that of non-mCRC cell invasion. We found that Acknowledgements the anti-TSPAN8-LEL HuMab prominently inhibited the invasion of mCRC cell lines, namely HCT116 and LoVo, This work was supported by a research grant (10TS03) compared with non-mCRC cell lines, such as HCT-8 and from Scripps Korea Antibody Institute and the Bio & SW480 [21]. Therefore, these findings support our notion that Medical Technology Development Program of the National TSPAN8-LEL may be a potential novel therapeutic target in Research Foundation (NRF) funded by the Korean mCRCs and that antibody-based modulation of government (MSIP) (2014M3A9 D9032525). TSPAN8-LEL may be an effective strategy for suppressing mCRC cell invasion in colorectal cancer therapy. Author contributions

Possible role of TSPAN8-LEL in mCRCs and usefulness Lee. S and Kim. T worked together to write the of anti-TSPAN8-LEL HuMab manuscript and make the schematic illustration.

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