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Regulation of cellular behaviors via a cross-talk between TM4SF5 and protein tyrosine phosphatase receptor type-F (PTPRF) in liver cancer cells.

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Authors
조창연
Advisor
이정원
Major
약학대학 약학과
Issue Date
2017-08
Publisher
서울대학교 대학원
Keywords
3D cell cultureprotein interactioncellular metastasisextracellular matrixTM4SF5PTPRFfocal adhesiontumor microenvironment
Description
학위논문 (석사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. 이정원.
Abstract
Transmembrane 4 L six family member 5 (TM4SF5) is a glycoprotein on cell surface and highly expressed in various cancers including hepatocarcinoma. In previous studies, it is shown that TM4SF5 induces Epithelial-Mesenchymal Transition (EMT), as a regulation factor of cell morphological change, cell migration, invasion, and proliferation. In this study, I found protein tyrosine phosphatase receptor type-F (PTPRF) as a new binding partner of TM4SF5 through mass spectrometry. PTPRF is a transmembrane tyrosine phosphatase with one transmembrane domain. PTPRF is known to regulate cell proliferation, differentiation, and mitotic cell cycle, being associated with cell death-related protein activation. Here, I investigated how this interaction affected cellular behaviors using cell lines with overexpression or suppression of TM4SF5 and/or PTPRF. First, I could confirm their interaction by co-immunoprecipitation and immunofluorescence. Overexpression or down-regulation of either TM4SF5 or PTPRF did not affect their mRNA or protein levels. Further, the interaction between TM4SF5 and PTPRF decreased in the suspension state of the cells and recovered significantly when the cells became reattached to the extracellular matrix (ECM). I could thus rationalize that TM4SF5 and PTPRF interaction was correlated with and affect PTPRFs tyrosine-phosphatase activity. In a previous study, it was reported that TM4SF5 regulates activities of focal adhesion (FA) molecules and formation of FA enriched with paxillin. So, I focused on the influence of PTPRF expression on activities of FA molecules in the presence or absence of TM4SF5 expression. PTPRF co-localized with Paxillin during immunofluorescence studies, and overexpression of PTPRF led to dephosphorylation of FAK, Src, and Paxillin. When TM4SF5 and PTPRF were co-overexpressed, PTPRFs effects against phosphorylation of FAK, Src, and Paxillin were inhibited, indicating that TM4SF5 could antagonize PTPRF. To investigate such effects of PTPRF on cellular behaviors, I performed cell adhesion assay, cell migration assay, and sphere formation assay for the purposes of understanding the roles of the both in cancer metastasis. During the cell adhesion assay, cells expressing PTPRF inhibited tyrosine phosphorylation of paxillin in the suspended state, whereas cells lacking PTPRF retained tyrosine phosphorylation of paxillin in the suspended state. When those cells were attached to ECM fibronectin, PTPRF-lacking cells formed FAs faster than control PTPRF-expressing cells. However, cells lacking both PTPRF and TM4SF5 showed paxillin phosphorylation higher than TM4SF5 alone-lacking cells. In the cell migration assay, migration ability of PTPRF-lacking cells increased but cells lacking both PTPRF and TM4SF5 showed reduced migration ability than PTPRF alone-lacking cells. In case of the spheroid formation assay, PTPRF-lacking cells increased sphere formation but cells lacking both PTPRF and TM4SF5 showed a lower sphere formation capacity than PTPRF-alone lacking cells.
Altogether, these observations suggest that the role of PTPRF in cells to inhibit molecules at FAs, such as FAK, c-Src, and Paxillin, would be controlled by TM4SF5, especially when the cells were attached to the ECMs. In HCC that TM4SF5 is overexpressed, however, TM4SF5 can promote tyrosine phosphorylation of FA molecules that would be negatively targeted by PTPRF. Thus, the coordinated cross-talks between TM4SF5 and PTPRF can play roles in successful cancer metastasis, which further can be a promising target to deals with TM4SF5-dependent metastasis.
Language
English
URI
https://hdl.handle.net/10371/137932
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Master's Degree_약학과)
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