Roles for GLI Transcription Factors in Pancreas Development and Disease

Abstract

The Hedgehog (HH) signaling pathway plays a fundamental role in patterning numerous developing tissues, and dysregulated HH signaling has been linked to malignant human diseases. HH signaling is regulated on a transcriptional level by the GLI family of transcription factors (GLI1, GLI2, GLI3). While GLI1 functions exclusively as a transcriptional activator, GLI2 and GLI3 have dual roles as both activators and repressors. Importantly, the functions of GLI proteins are highly dynamic, and can vary dramatically based on context. The development and healthy function of the pancreas relies on tightly regulated HH signaling. HH signaling must be actively suppressed during pancreas specification, as ectopic activation leads to impaired organ formation and disrupted endocrine development. In addition, aberrant HH signaling is frequently detected in pancreatic ductal adenocarcinoma (PDA), one of the deadliest forms of cancer. While this pathway has been studied extensively at the level of ligands and receptors, the roles of GLI1-3 in pancreas development and disease remain largely unknown. In this dissertation, I investigate: 1) The role of GLI1-3 in PDA progression and 2) the contribution of GLI1-3 to pancreas development.

In the context of PDA, the role of HH signaling in PDA has been controversial, with both tumor-promoting and tumor-restricting roles reported. The controversy stems, in part from an incomplete understanding of the mechanisms of HH signal transduction in PDA. To determine the role of GLI1-3 in PDA, I utilized a combination of mouse genetics, ex vivo assays, and bioinformatic analysis. Expression analysis of both human and mouse tissue revealed that all three Gli genes are expressed by pancreatic fibroblasts in the healthy pancreas, and that the expression of Gli1-3 expands in PDA. Deleting Gli2/Gli3 in pancreatic fibroblasts reduces immunosuppressive macrophage infiltration, promotes the recruitment of T cells, and restrains tumor growth. In contrast, combined deletion of Gli1/Gli2/Gli3 in vivo promotes macrophage infiltration and supports tumor growth. RNA sequencing analysis revealed that the loss of Gli alters the expression of cytokines in pancreatic fibroblasts. Further, fibroblasts directly regulate the migration of macrophages and T cells in a Gli-dependent manner. These data indicate that Gli expression in fibroblasts directly regulates the tumor microenvironment of pancreatic cancer, and that Gli-mediated changes in fibroblast function regulates tumor growth.

In this dissertation, I also investigated the role of GLI1-3 in pancreas development. My data reveal that Gli2 and Gli3 are expressed broadly throughout the mesenchyme during pancreas development, while Gli1 is excluded from this tissue. Conditional deletion of Gli2 has no significant impacts on pancreas development. In contrast, deleting mesenchymal Gli3 leads to increased proliferation of pancreatic epithelial cells, abnormal organ morphogenesis, and reduced B-cells. Interestingly, the phenotypes observed depend on the timing of Gli3 deletion, suggesting that GLI3 plays distinct roles at different stages of pancreas development. Together, the data presented in this thesis reveal that GLI proteins regulate pancreas development and disease progression by modifying the pancreatic microenvironment. These data also reveal new insights into the role of fibroblasts in PDA, and open new opportunities for the development of novel therapies.