Summary Pancreatic cancer, a hypovascular and highly desmoplastic cancer, is characterized by tumor expression of Hedgehog (HH) ligands which signal to fibroblasts in the surrounding stroma that in turn promote tumor survival and growth. However, the mechanisms and consequences of stromal HH pathway activation are not well understood. Here we show that the HH co-receptors GAS1, BOC, and CDON are expressed in cancer-associated fibroblasts. Deletion of two co-receptors (Gas1 and Boc) in fibroblasts reduces HH-responsiveness. Strikingly, these fibroblasts promote greater tumor growth in vivo that correlates with increased tumor-associated vascularity. In contrast, deletion of all three co-receptors (Gas1, Boc and Cdon) results in the near complete abrogation of HH signaling and a corresponding failure to promote tumorigenesis and angiogenesis. Collectively, these data identify a novel role for HH-dosage in pancreatic cancer promotion and may explain the clinical failure of HH pathway blockade as a therapeutic approach in pancreatic cancer.

SUMMARYHedgehog (HH) signaling is essential for vertebrate and invertebrate embryogenesis. In Drosophila, feedback upregulation of the HH receptor Patched (PTC; PTCH in vertebrates), is required to restrict HH signaling during development. By contrast, PTCH1 upregulation is dispensable for early HH-dependent patterning in mice. Unique to vertebrates are two additional HH-binding antagonists that are induced by HH signaling, HHIP1 and the PTCH1 homologue PTCH2. Although HHIP1 functions semi-redundantly with PTCH1 to restrict HH signaling in the developing nervous system, a role for PTCH2 remains unresolved. Data presented here define a novel role for PTCH2 as a ciliary localized HH pathway antagonist. While PTCH2 is dispensable for normal ventral neural patterning, combined removal of PTCH2-and PTCH1-feedback antagonism produces a significant expansion of HH-dependent ventral neural progenitors. Strikingly, complete loss of PTCH2-, HHIP1-and PTCH1-feedback inhibition results in ectopic specification of ventral cell fates throughout the neural tube, reflecting constitutive HH pathway activation. Overall, these data reveal an essential role for liganddependent feedback inhibition of vertebrate HH signaling governed collectively by PTCH1, PTCH2 and HHIP1.

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HHIP1 acts as a secreted Hedgehog antagonist whose association with heparan sulfate–containing basement membranes regulates Hedgehog ligand distribution.

Proper levels of Hedgehog (HH) signaling are essential during embryonic development and adult tissue homeostasis. A central mechanism to control HH pathway activity is through the regulation of secreted HH ligands at the plasma membrane. Recent studies have revealed a collective requirement for the cell surface co-receptors GAS1, CDON and BOC in HH signal transduction. Despite their requirement in HH pathway function, the mechanisms by which these proteins act to promote HH signaling remain poorly understood. Here we focus on the function of the two structurally related co-receptors, CDON and BOC. We utilized an in vivo gain-of-function approach in the developing chicken spinal cord to dissect the structural requirements for CDON and BOC function in HH signal transduction. Notably, we find that although CDON and BOC display functional redundancy during HH-dependent ventral neural patterning, these molecules utilize distinct molecular mechanisms to execute their HH-promoting effects. Specifically, we define distinct membrane attachment requirements for CDON and BOC function in HH signal transduction. Further, we identify novel and separate extracellular motifs in CDON and BOC that are required to promote HH signaling. Together, these data suggest that HH co-receptors employ distinct mechanisms to mediate HH pathway activity.