Expression and functional roles of UNC13D in the progression of pancreatic cancer

다국어 초록 (Multilingual Abstract)

Pancreatic cancer (PC) is one of the most deadly cancers due to its late diagnosis and early metastasis, whose molecular mechanism is not fully understood. Recycling endosomal vesicles is fundamental to the migration of cancer cells, which in turn drives the metastasis of pancreatic cancer, a major contributor to cancer-related deaths. In this study, a comprehensive analysis of public resources revealed that higher expression of UNC13D was significantly associated with a poor prognosis in four independent pancreatic cancer cohorts. Besides, the expression level of UNC13D was markedly higher in the PC tissues than in the matched normal tissues. However, the role of UNC13D in PC remains unknown. Functional roles of UNC13D in the progression of PC, particularly as a key molecule promoting cancer cell migration were explored in this study. Biological studies have revealed that UNC13D plays a crucial role in the migration of pancreatic cancer cells by coupling the exocytosis of recycling endosomes with focal adhesion turnover via the regulation of FAK phosphorylation. Additionally, the formation of the RAB11-UNC13D-FAK axis in endosomes during integrin recycling was evidenced by immunoprecipitation and immunocytochemistry. Notably, co-immunoprecipitation and immunoblotting data indicated that UNC13D directly interacted with the FERM domain of FAK and regulated FAK phosphorylation in a calcium-dependent manner. Taken together, these results suggest that UNC13D, a novel prognostic factor, promotes pancreatic cancer progression by coupling integrin recycling with focal adhesion turnover via the RAB11-UNC13D-FAK axis for the migration of pancreatic cancer cells.

목차 (Table of Contents)

• Contents i
• List of Figures iii
• List of Tables iv
• Abbreviations v
• Abstract (in English) vi
• 1. Introduction 1
• 2. Materials and Methods 5
• 2.1. Public data sources and statistical analysis 5
• 2.2. Cell culture 5
• 2.3. Expression vectors and Mutagenesis 6
• 2.4. Transfection and stable cell line construction 6
• 2.5. Antibodies and reagents 7
• 2.6. Real-time PCR 8
• 2.7. Immunoblotting 8
• 2.8. Immunocytochemistry (ICC) 9
• 2.9. Co-immunoprecipitation (Co-IP) assay 9
• 2.10. Cell proliferation assay 10
• 2.11. Boyden chamber assay 10
• 2.12. Wound healing assay 11
• 2.13. Matrigel invasion assay 11
• 2.14. Focal adhesion dynamics analysis 12
• 2.15. Quantification and statistical analysis for the analysis of biological experiments 12
• 3. Results 17
• 3.1. UNC13D is a novel upregulated gene that is responsible for poor clinical outcomes in pancreatic cancer 17
• 3.2. UNC13D promoted the migration and invasion of pancreatic cancer cells in vitro 19
• 3.3. UNC13D modulates FA turnover during cell spreading 21
• 3.4. Phosphorylation of FAK is regulated by the RAB11-UNC13D-FAK axis located in recycling endosomes. 24
• 3.5. UNC13D directly interacts with FAK via the FERM domain 29
• 3.6. The interaction of UNC13D and FAK is a calcium-dependent manner 31
• 3.7. FAK inhibitor suppresses UNC13D-mediated cell migration and invasion 33
• 4. Discussion 37
• References 42
• 요 약 (Abstract in Korean) 50
• Acknowledgment 51