Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment.Experimental Design: We used a combination of pharmacologic inhibition, gain-and loss-of-function genetic experiments, cyto-metry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC.Results: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression.Conclusions: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers. Neuropilins (NRPs), which have well-defined roles in Semaphorin and VEGF signaling, positively regulate HH pathway function, although their mechanism of action in HH signaling remains unclear. Here, using luciferase-based reporter assays, we provide evidence that NRP1 regulates HH signaling specifically at the level of GLI transcriptional activator function. Moreover, we show that NRP1 localization to the primary cilium, a key platform for HH signal transduction, does not correlate with HH signal promotion. Rather, a structure-function analysis suggests that the NRP1 cytoplasmic and transmembrane domains are necessary and sufficient to regulate HH pathway activity. Furthermore, we identify a previously uncharacterized, 12-amino acid region within the NRP1 cytoplasmic domain that mediates HH signal promotion. Overall, our results provide mechanistic insight into NRP1 function within and potentially beyond the HH signaling pathway. These insights have implications for the development of novel modulators of HH-driven developmental disorders and diseases.

Background: Hedgehog (HH) signaling is essential for homeostasis in gustatory fungiform papillae (FP) and taste buds. However, activities of HH antagonists in these tissues remain unexplored. We investigated a potential role for HH-interacting protein (HHIP), an endogenous pathway antagonist, in regulating HH signaling during taste organ homeostasis. We found a restricted pattern of Hhip-expressing cells in the anterior epithelium of each nongustatory filiform papilla (FILIF) only. To test for roles in antagonism of HH signaling, we investigated HHIP after pathway inhibition with SMO inhibition via sonidegib and Smo deletion, Gli2 deletion/suppression, or with chorda tympani/lingual nerve cut.Results: In all approaches, the HHIP expression pattern was retained in FILIF suggesting HH-independent regulation of HHIP. Remarkably, after pathway inhibition, HHIP expression was detected also in the conical, FILIF-like atypical FP. We found a close association of de novo expression of HHIP in atypical FP with loss of Gli1+, HH-responding cells. Further, we report that PTCH1 is another potential HH antagonist in FILIF that co-localizes with HHIP.Conclusions: After HH pathway inhibition the ectopic expression of HHIP correlates with a FILIF-like morphology in atypical FP and we propose that localized expression of the HH antagonist HHIP regulates pathway inhibition to maintain FILIF during tongue homeostasis.

Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide-ranging functions. Previous work has identified Neuropilins, established Semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require Plexin co-receptors to mediate Semaphorin signaling, but a role for Plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple Plexins promote Hedgehog signaling in NIH/3T3 mouse fibroblasts and that Plexin loss-of-function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the Plexin GTPase activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that Plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia. Finally, we find that Plexin loss-of-function significantly reduces the response to Hedgehog pathway activation in the mouse dentate gyrus. Together, these data identify Plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.

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