||Identification and characterization of galectin-3 interacting proteins in colon cancer initiating cells
||Department of Medical Laboratory Science and Biotechnology
Cancer initiating cells
大腸癌在許多國家都擁有相當高的發生率以及死亡率，近年來，由於早期診斷以及治療方法的進步，大腸直腸癌的存活率已經大幅改善。然而治療後的高復發率以及化療藥物的抗藥性仍然迫切的需要被解決。研究發現腫瘤中存在的癌症起始細胞 (cancer initiating cells, CICs or cancer stem cells, CSCs) 與癌症的復發以及抗藥性的產生有高度的相關性。在我們先前的研究中發現，半乳糖凝集素3 (galectin-3, Gal-3) 會參與癌症起始細胞的產生以及癌症幹細胞特性的維持。Gal-3是一種β-半乳糖苷結合蛋白，能夠藉由其C-末端的醣識別結構域（carbohydrate recognition domain, CRD）辨識其他蛋白質上特定的醣鏈結構並產生交互作用，也能夠藉由N-末端直接產生蛋白-蛋白交互作用去參與細胞特性的調控。因此，本篇研究試圖找出Gal-3相互作用蛋白 (Gal-3 interacting proteins) 能夠和Gal-3結合一同去調控癌症起始細胞。首先我們利用anti-Gal-3的免疫沉澱法，去捕獲癌症起始細胞中所有和Gal-3產生交互作用的蛋白，並額外加入Gal-3醣識別抑制劑TD139，去鎖定藉由醣鏈和Gal-3結合的Gal-3相互作用蛋白。以質譜進行分析後，15候選蛋白被辨識為Gal-3聚醣依賴性相互作用蛋白 (Gal-3 glycan-dependent interacting proteins)。其中膜聯蛋白A2與A13 (Annexin A2, ANXA2 and Annexin A13, ANXA13) 被進一步地進行驗證與分析，我們的實驗結果發現當Gal-3的醣識別能力被抑制時，不會改變癌症起始細胞中Gal-3、ANXA2及ANXA13的表現量，然而卻使Gal-3從細胞質被轉運到細胞膜的數量下降，這造成Gal-3在細胞膜上和ANXA2及ANXA13的交互作用減少。我們同時觀察到癌症起始細胞的生成速率及細胞大小會被Gal-3醣識別抑制劑所抑制，而調控細胞生長的Src-dependent Akt / Erk 信號通路也會被抑制。根據以上的研究結果，我們認為ANXA2及ANXA13會藉由聚醣依賴性相互作用參與Gal-3的細胞膜轉運; 同時，ANXA2及ANXA13與Gal-3之間的交互作用能藉由Src-dependent Akt / Erk 信號通路來參與癌症起始細胞的生成與幹細胞特性的維持。因此針對Gal-3交互作用的抑制或許可以做為大腸直腸癌的潛在治療方法。
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.
TABLE OF CONTENTS
TABLE OF CONTENTS V
Colorectal cancer 1
Cancer initiating cells 2
Galectin-3 interacting proteins 4
Galectin-3 in cancer initiating cells 4
Study objective and Specific Aim 6
Experimental flow chart 7
MATERIALS AND METHODS 8
Cell culture and experimental conditions 8
Sphere formation assay 8
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 9
In-gel digestion of proteins separated by SDS-PAGE for mass spectrometry (MS) analysis 9
Liquid chromatography-mass spectrometry (nanoflow LC-MS/MS) 10
Cellular fractionation 12
Statistical analysis 12
Capturing of Gal-3 interacting proteins in colorectal cancer cells by immunoprecipitation. 13
Further screening of Gal-3 interacting proteins based on glycan-dependent interactions. 13
Identification of Gal-3 interacting proteins by mass spectrometry. 14
Validation of Annexin A2 and Annexin A13 16
Inhibition of GAL-3 carbohydrate binding ability decreases cell surface Annexin A2, Annexin A13, and Galectin-3 levels in CCICs. 17
Gal-3/ANXs glycan-dependent interaction is critical for sphere formation by SRC-mediated signaling. 18
Figure 1. CRC cell lysates were immunoprecipitated (IP) with anti-galectin3 Ab and followed by SDS-PAGE separation in Coomassie staining. 35
Figure 2. Galectin-3 / protein interactions though carbohydrate binding ability was recognized by incubating galectin-3 CRD inhibitor, TD139. 36
Figure 3. Interactions between ANXA2 and Gal-3 determined by co-immunoprecipitation. 37
Figure 4. Interactions between ANXA2 and Gal-3 were verified by co-immunoprecipitation. 38
Figure 5. Interactions between ANX2 and Gal-3 verified by immunofluorescence. 39
Figure 6. Interactions between ANX13 and Gal-3 verified by immunofluorescence. 40
Figure 7. Western blot analysis shows expression of ANXA2, ANXA13, Gal-3 increased in cancer spheres compare to adherent cells in CRC. 41
Figure 8. Effects of lactose on the expression of ANXA2, ANXA13, and Gal-3 in cancer sphere cells. 42
Figure 9. Gal-3 CRD inhibitor lactose decreased the translocation of Gal-3 to the plasma membrane but not affected ANXA2 and A13 in cancer spheres. 43
Figure 10. Gal-3 CRD inhibitor lactose decreased the translocation of Gal-3 from cytosol to the plasma membrane in a dose-dependent manner. 44
Figure 11. Lactose treatment suppressed Gal-3 interaction with ANXA2, p-ANXA2, ANXA13 on the plasma membrane. 45
Figure 12. Lactose treatment inhibits spheres formation. 46
Figure 13. Src-dependent Akt / Erk signaling was inhibited by Gal-3 CRD inhibitor. 47
Figure 14. The interaction between Gal-3 and Gal-3 interacting proteins regulates cancer initiating cell property and cancer stemness through Src-dependent Akt / Erk signaling. 48
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