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Combinatorial Gli activity directs immune infiltration and tumor growth in pancreatic cancer [1]

['Michael K. Scales', 'Department Of Cell', 'Developmental Biology', 'University Of Michigan', 'Ann Arbor', 'Michigan', 'United States Of America', 'Ashley Velez-Delgado', 'Nina G. Steele', 'Hannah E. Schrader']

Date: 2022-10

Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.

Throughout life, proper organ function relies on tightly regulated communication between diverse cell types. Disruptions to this intercellular communication can lead to disease, including pediatric and adult cancers. In pancreatic cancer, abnormal communication between tumor cells, fibroblasts and immune cells prevents the immune system from effectively recognizing and destroying malignant cells. Finding a way to correct this deficiency could create new therapeutic opportunities for this deadly disease. In this study, we investigated the role of Hedgehog signaling in fibroblasts, a central cell type in the pancreatic tumor microenvironment. Specifically, we explored the contribution of the GLI family of transcription factors (which mediate the cellular responses to Hedgehog signaling) to pancreatic cancer progression. We found that Gli genes are expressed in pancreatic fibroblasts and that the proteins encoded by these genes impact pancreatic tumor growth and directly control immune cell migration. This work identifies GLI transcription factors as key regulators of fibroblast-immune cell communication in pancreatic cancer.

Funding: This work was supported by: National Institutes of Health training grant T32 HD007505 (MKS), National Institutes of Health fellowship F31 CA232655 (MKS), National Institutes of Health training grant T32 GM008353 (AVD), National Institutes of Health fellowship F31 CA247037 (AVD), National Institutes of Health training grant T32 CA009676 (NGS), National Institutes of Health award K99 CA263154 (NGS), National Institutes of Health award R50 CA232985 (YZ), National Institutes of Health training grant T32 GM007315 (REM), National Institutes of Health fellowship F31 CA257533 (REM), National Institutes of Health award U01 CA224145 (MPM, HCC), National Institutes of Health award R01 CA151588 (MPM), National Institutes of Health award R01 CA198074 (MPM, BLA), National Institutes of Health award R01 DC014428 (BLA), National Institutes of Health award R01 GM118751 (BLA), University of Michigan Bradley Merrill Patten Memorial Scholarship (MKS), University of Michigan Rackham Merit Fellowship (AVD, REM), University of Michigan Michigan Institute for Clinical and Health Research Postdoctoral Translational Scholar Program fellowship (NGS), University of Michigan Medical School Michigan Postdoctoral Pioneer Program award (ZCN), American Cancer Society postdoctoral award PF-19-096-01 (NGS), Association of Academic Surgery Joel Roslyn Award (FB), CTSA Precision Medicine Pilot award (KPO), and the Pancreas Center at NY Presbyterian Hospital (KPO). More information about the funding sources can be found at the following web addresses: National Institutes of Health ( https://www.nih.gov/ ), University of Michigan Bradley Merrill Patten Memorial Scholarship ( https://medicine.umich.edu/dept/cdb/giving ), University of Michigan Rackham Merit Fellowship ( https://rackham.umich.edu/funding/funding-types/rackham-merit-fellowship-program/ ), University of Michigan - Michigan Institute for Clinical and Health Research Postdoctoral Translational Scholar Program ( https://michr.umich.edu/ ), University of Michigan Medical School - Michigan Postdoctoral Pioneer Program ( https://umichpioneerprogram.org/ ), American Cancer Society ( https://www.cancer.org/ ), Association of Academic Surgery ( https://www.aasurg.org/ ), Columbia University CTSA Precision Medicine Pilot Award ( https://www.irvinginstitute.columbia.edu/services/columbia-precision-medicine-joint-pilot-grants-program ), Pancreas Center at NY Presbyterian Hospital ( https://columbiasurgery.org/media-learning-center/pancreas-center-new-york-presbyterian-columbia-university-medical-center ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Scales et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we investigated the role of GLI1-3 in PDA progression. We have determined that Gli1, Gli2, and Gli3 are expressed in the healthy pancreas, and expand throughout PDA progression. At pre-cancerous stages, genetic deletion of Gli2 and Gli3 in fibroblasts reduces collagen deposition and dramatically alters immune infiltration. Specifically, stromal depletion of Gli2 and Gli3 leads to a decrease in immunosuppressive macrophage infiltration and an increase in T cells. However, deleting all three Glis in fibroblasts leads to an increase in macrophage infiltration and the exclusion of T cells. Further, mice lacking Gli1, Gli2, and Gli3 display a widespread loss of pancreatic tissue, suggesting that a baseline level of GLI activity is necessary to maintain tissue integrity during disease progression. In invasive tumors, we have determined that the loss of Gli2 and Gli3 in fibroblasts decreases myeloid-derived suppressor cells (MDSCs) and increases natural killer (NK) cells, which in turn antagonize tumor growth. In contrast, Gli1/Gli2/Gli3 KO fibroblasts recruit MDSCs and exclude NK cells, leading to sustained tumor growth. Together, our data demonstrate that the activities of all three GLIs regulate immune infiltration throughout PDA progression, and these GLI-driven changes determine tumor growth.

The GLI family of proteins (GLI1, GLI2, GLI3) are the transcriptional effectors of the HH pathway. GLI1 exclusively functions as a transcriptional activator, while GLI2 and GLI3 contain both activator and repressor domains [ 36 ]. In multiple tissues, GLI2 primarily acts as a transcriptional activator [ 37 ], while GLI3 functions as a transcriptional repressor [ 38 ]. Prior work from our group has demonstrated that GLI1 supports pancreatic tissue recovery following induction of acute pancreatitis or oncogenic Kras-driven injury [ 39 ]. However, the expression and function of GLI2 and GLI3 in PDA remain largely unknown. Further, the combined role of multiple GLIs during PDA progression has not been explored.

Previous mouse studies indicated that HH pathway inhibition improves chemotherapy delivery and extends survival [ 15 ]. However, clinical trials employing SMO inhibitors provided no clinical benefit or, in some cases, led to worse outcomes [ 32 – 34 ]. Further, genetic loss of Shh shortens survival in mouse models of PDA, suggesting that HH signaling has tumor-restraining roles [ 19 , 20 ]. One explanation for these contradictory results is that the level of HH pathway activity influences pancreatic tumor growth. Combinatorial targeting of HH pathway co-receptors revealed that partial reduction of HH signaling in fibroblasts promotes tumor growth, while near complete ablation of the stromal HH response fails to promote tumorigenesis [ 18 ]. More recent work demonstrated that pharmacologic HH pathway inhibition alters cancer-associated fibroblast (CAF) composition and immune infiltration in PDA, indicating that HH signaling impacts multiple cell types within the pancreatic TME [ 35 ]. However, the downstream consequences of HH pathway activity on pancreatic tumor growth, specifically the transcriptional outcomes of HH signal transduction in the pancreatic stroma, remain unexplored.

Work from our lab and others has identified aberrant HH signaling as a feature of PDA [ 25 – 27 ]. In the context of PDA, HH ligands (primarily sonic hedgehog [SHH] and indian hedgehog [IHH]) are secreted by tumor cells and bind to the canonical receptor patched 1 (PTCH1) on fibroblasts [ 28 – 30 ]. Following ligand binding, the repressive activity of PTCH1 is inhibited, leading to the activation of smoothened (SMO), which in turn modulates the GLI family of HH transcription factors [ 31 ]. Although this paracrine model of HH signaling in pancreatic cancer is well-established, the contribution of HH signaling to pancreatic cancer progression remains controversial.

Pancreatic ductal adenocarcinoma (PDA) remains a deadly malignancy, with a 5-year survival rate of 11% [ 1 ]. One contributing factor to this low survival rate is a lack of effective therapies. Although the mechanisms driving resistance to treatment are complex, one major barrier is the tumor microenvironment (TME). The TME of PDA is extremely heterogeneous, involving a complex network of endothelial cells, nerves, fibroblasts, and immune cells [ 2 ]. Within this network, fibroblasts function as critical nodes for intercellular signaling. Pancreatic fibroblasts contribute to pancreatic disease through a variety of means, including the production of extracellular matrix (ECM) and the secretion of pro-tumorigenic factors [ 3 – 7 ]. Fibroblasts also provide metabolic support [ 8 – 10 ], confer chemoresistance [ 7 , 11 , 12 ], facilitate immunosuppression [ 13 , 14 ], and restrict tumor perfusion via ECM deposition [ 15 – 17 ]. However, fibroblasts also have tumor-restricting roles [ 18 – 21 ]. These seemingly disparate functions could be explained by the observation that cancer-associated fibroblasts are heterogeneous [ 22 – 24 ], with different populations having different, potentially opposing functions. However, the mechanisms and signals used by fibroblast populations to affect disease progression are not fully understood.

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010315

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