Deciphering the roles of LGR6 in WNT and EMT signaling in pancreatic cancer

Wang, Jing (2019): Deciphering the roles of LGR6 in WNT and EMT signaling in pancreatic cancer. Dissertation, LMU München: Medizinische Fakultät

Abstract

The canonical WNT signal pathway has long been considered essential for regulating embryonic processes as well as tissue homeostasis. However, alterations in WNT signaling are also associated with diverse diseases including developmental defects as well as multiple cancers. The R-spondin (RSPO) family is a group of secreted factors that enhance previously activated WNT signaling activity by binding to LGR4/5/6 (leucine-rich repeat containing G-protein coupled receptors 4/5/6). Of note, one of the receptors - LGR5 - is a well-established WNT target gene and (cancer) stem cell marker. So far, little evidence exists about the function of LGR6 in PDAC. Epithelial–mesenchymal transition (EMT) is a process by which epithelial cells transdifferentiate into mesenchymal cells. The overlap between WNT signaling and EMT compelled us to investigate the potential role of LGR6 in EMT. According to open access data from the TCGA, we found that LGR6 positively correlated with typical published canonical WNT signal pathway signatures. Hence, we further hypothesized that LGR6 could be a novel WNT target gene in PDAC. WNT signaling was activated by stimulation with RSPO2 and/or WNT3a, which subsequently upregulated LGR6 expression in different PDAC cell lines. Moreover, inhibition of baseline WNT signaling activity by IWP2 led to downregulation of endogenous LGR6 expression. Taken together, we were able to show that LGR6 gene itself may be a direct target of the WNT signaling pathway in PDAC cells with LGR6 expression changes according to WNT activity. LGR6 is involved in regulating EMT processes in PDAC. Gene Set Enrichment Analysis (GSEA) showed that LGR6 was inversely correlated with EMT signatures inPDAC. PDAC cell lines of different morphological appearances were used to assess the distribution of LGR6 expression. We found that epithelial cell lines (BxPC3, Capan2) possessed a higher LGR6 expression compared to mesenchymal cell lines (MiaPaCa2, Panc1). IF showed that LGR6 was mostly located at the cell membrane in epithelial cell lines, while less or no expression was detected in mesenchymal cell lines. Furthermore, EMT induction was carried out and LGR6 expression was analyzed. We detected upregulated LGR6 expression upon EMT induction in Panc1. Additionally, Co-IP showed that LGR6 may constitute a part of the cell adhesion complex in epithelial PDAC cell lines. Cancer stemness in PDAC was assessed by surrogate assays in vitro including colony formation capacity in 2D and sphere formation capacity in 3D. We investigated the effects of LGR6 knock-down on cancer stemness. The numbers of colonies and spheres were smaller in the knock-down group compared with the vector group. Thus, we concluded that the deletion of LGR6 might have a negative impact on the growth of spheres and colonies. As gemcitabine resistant (GR) cells are more invasive and possessed increased CSC markers suggesting a higher proportion of CSC, we developed MiaPaCa2 GR and Panc1 GR cells by culturing them with increasing doses of gemcitabine for at least six months. LGR6 was detected overexpressed in Panc1 GR both on mRNA and protein level. In addition, we searched The Human Protein Atlas and showed that high LGR6 RNA expression in tissues correlates with poor survival in pancreatic cancer patients. This supported our hypothesis that LGR6 could serve as a marker of poor outcome in pancreatic cancer.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 27. Juni 2019
• 1. Bericht­erstatter:in: Werner, Jens