Identification and characterization of galectin-3 interacting proteins in colon cancer initiating cells

• Main Authors: Shih-YinHuang, 黃詩尹
• Other Authors: CHUAN-FA CHANG
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/42h8w2

碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 106 === Colorectal cancer (CRC) is the third most frequent cancers with high incidence rate reported in many countries. Recent years, CRC survival rate is improved due to advances in diagnosis and treatment. However, drug resistance and tumor recurrence caused by Colon cancer initiating cells (CCICs) are urgent to be solved. In our previous studies, we found galectin-3 (Gal-3) should play very key roles in multidrug-resistant and CCICs properties. Gal-3 is a β-Galactoside-binding protein that interacts with glycoproteins to affect cell properties by either glycan-dependent interaction or protein-protein interaction. In this study, we tried to find Gal-3 glycan-dependent interacting proteins which worked together with Gal-3 by Gal-3 carbohydrate recognition domain (CRD) to regulate CCICs properties. Immunoprecipitation with anti-Gal-3 antibody and further treated with or without TD139, a novel Gal-3 CRD competitive inhibitor, was performed at the first step. The candidate proteins were separated by electrophoresis, visualized by Coomassie blue and then preparation by in-gel digestion for mass spectrometry-based proteomics. Finally, 15 Gal-3 interacting proteins were identified and considered to be involved in the regulation of colorectal cancer initiating cells (CCICs) via Gal-3 carbohydrate recognition activities. Among this, Annexin A2 and Annexin A13 (ANXs) were then verified by co-immunoprecipitation and immunofluorescence and further investigated the function of Gal-3/ANXs glycan-dependent interaction. Inhibition of GAL-3 carbohydrate binding ability not significantly affected the expression of Gal-3, ANXA2, and ANXA13. However, Gal-3 translocation from cytosol to plasma membrane was decreased in a dose-dependent manner, and interaction of p-ANXA2, ANXA2, ANXA13 with Gal-3 on the membrane was decreased. Inhibition of Gal-3/ANXs glycan-dependent interaction also suppressed the colon cancer spheres formation by SRC-mediated signaling. Based on these results, we suggested that Annexin A2 and Annexin A13 may work as Gal-3 glycan-dependent interacting proteins to involve in Gal-3 translocation to the plasma membrane. in addition, Gal-3 glycan-dependent interactions with ANXA2 and ANXA13 regulate CCICs characteristic through Src-dependent Akt / Erk signaling on the plasma membrane and have the potential to establish a novel therapeutic target for colon cancer treatment.