Intracellular Ca2+ is not essential for SHH signaling but is promoted by Shh ligand in embryonic fibroblasts.

Sonic Hedgehog Signaling Protects Human Hepatocellular Carcinoma Cells Against Ionizing Radiation in an Autocrine Manner

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Cell Surface Membrane Proteins Cdo and Boc Are Components and Targets of the Hedgehog Signaling Pathway and Feedback Network in Mice

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling from the primary cilium drives cerebellar granule cell precursor (GCP) proliferation. Mutations of hedgehog (Hh) pathway repressors commonly cause medulloblastoma, the most prevalent and malignant childhood brain tumor that arises from aberrant GCP proliferation. We demonstrate that Nestin Cre-driven conditional knock-out (CKO) of a Shh pathway repressor-Rab23 in the mouse brain of both genders caused mis-patterning of cerebellar folia and elevated GCP proliferation during early development, but with no prevalent occurrence of medulloblastoma at adult stage. Strikingly, Rab23-depleted GCPs exhibited upregulated basal level of Shh pathway activities despite showing an abnormal ciliogenesis of primary cilia. In line with the compromised ciliation, Rab23-depleted GCPs were desensitized against Hh pathway activity stimulations by Shh ligand and Smoothened (Smo) agonist-SAG, and exhibited attenuated stimulation of Smo-localization on the primary cilium in response to SAG. These results implicate multidimensional actions of Rab23 on Hh signaling cascade. Rab23 represses the basal level of Shh signaling, while facilitating primary cilium-dependent extrinsic Shh signaling activation. Collectively, our findings unravel instrumental roles of Rab23 in GCP proliferation and ciliogenesis. Furthermore, Rab23's potentiation of Shh signaling pathway through the primary cilium and Smo suggests a potential new therapeutic strategy for Smo/primary cilium-driven medulloblastoma.SIGNIFICANCE STATEMENT Primary cilium and Sonic hedgehog (Shh) signaling are known to regulate granule cell precursor (GCP) proliferation. Aberrant overactivation of Shh signaling pathway ectopically increases GCP proliferation and causes malignant childhood tumor called medulloblastoma. However, the genetic and molecular regulatory cascade of GCP tumorigenesis remains incompletely understood. Our finding uncovers Rab23 as a novel regulator of hedgehog (Hh) signaling pathway activity and cell proliferation in GCP. Intriguingly, we demonstrated that Rab23 confers dual functions in regulating Shh signaling; it potentiates primary cilium and Shh/Smoothened (Smo)-dependent signaling activation, while antagonizes basal level Hh activity. Our data present a previously underappreciated aspect of Rab23 in mediating extrinsic Shh signaling upstream of Smo. This study sheds new light on the mechanistic insights underpinning Shh signaling-mediated GCP proliferation and tumorigenesis.

Coordinated d-cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

Neural progenitor cells in the developing dorsal forebrain give rise to excitatory neurons, astrocytes, and oligodendrocytes for the neocortex. While we are starting to gain a better understanding about the mechanisms that direct the formation of neocortical neurons and astrocytes, far less is known about the molecular mechanisms that instruct dorsal forebrain progenitors to make oligodendrocytes. In this study, we show that Sonic hedgehog (Shh) signaling is required in dorsal progenitors for their late embryonic transition to oligodendrogenesis. Using genetic lineage-tracing in mice of both sexes, we demonstrate that most oligodendrocytes in the embryonic neocortex derive from Emx1+ dorsal forebrain progenitors. Deletion of the Shh signaling effector Smo specifically in Emx1+ progenitors led to significantly decreased oligodendrocyte numbers in the embryonic neocortex. Conversely, knock-out of the Shh antagonist Sufu was sufficient to increase neocortical oligodendrogenesis. Using conditional knock-out strategies, we found that Shh ligand is supplied to dorsal progenitors through multiple sources. Loss of Shh from Dlx5/6+ interneurons caused a significant reduction in oligodendrocytes in the embryonic neocortex. This phenotype was identical to that observed upon Shh deletion from the entire CNS using Nestin-Cre, indicating that interneurons migrating into the neocortex from the subpallium are the primary neural source of Shh for dorsal oligodendrogenesis. Additionally, deletion of Shh from migrating interneurons together with the choroid plexus epithelium led to a more severe loss of oligodendrocytes, suggesting that the choroid plexus is an important non-neural source of Shh ligand. Together, our studies demonstrate that the dorsal wave of neocortical oligodendrogenesis occurs earlier than previously appreciated and requires highly regulated Shh signaling from multiple embryonic sources.SIGNIFICANCE STATEMENT Most neocortical oligodendrocytes are made by neural progenitors in the dorsal forebrain, but the mechanisms that specify this fate are poorly understood. This study identifies Sonic hedgehog (Shh) signaling as a critical pathway in the transition from neurogenesis to oligodendrogenesis in dorsal forebrain progenitors during late embryonic development. The timing of this neuron-to-glia "switch" coincides with the arrival of migrating interneurons into the dorsal germinal zone, which we identify as a critical source of Shh ligand, which drives oligodendrogenesis. Our data provide evidence for a new model in which Shh signaling increases in the dorsal forebrain late in embryonic development to provide a temporally regulated mechanism that initiates the third wave of neocortical oligodendrogenesis.

The Mammalian Cos2 Homolog Kif7 Plays an Essential Role in Modulating Hh Signal Transduction during Development

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. The Hedgehog (Hh) signaling pathway is upregulated in PDAC. 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is active in early PanIN lesions and persists as the cancer progresses. Interestingly, Hh ligands are secreted by the tumor cells and function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor growth. PDAC is characterized by hypovascularity and a highly desmoplastic stroma. The downstream effects of Hh activation in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdon), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Gas1, Boc, and Cdon are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. During cancer progression, expression of all three co-receptors is prevalent throughout the desmoplastic stroma. Functional studies on Gas1-/-;Boc-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wildtype MEFs, indicating that these co-receptors are important for Hh signal transduction. Despite a reduced Hh-response, we found that Gas1-/-;Boc-/- MEFs promoted the growth of significantly larger, more vascularized tumors than their wildtype counterparts. A more physiologically relevant experiment with pancreatic fibroblasts and primary pancreatic tumor cells yielded the same counterintuitive result: removal of Gas1 and Boc from fibroblasts decreases Hh-response, and yet tumor-promoting potential was significantly increased. Moreover, chick chorioallantoic membrane (CAM) assays with tumor cells and fibroblasts revealed that Gas1-/-;Boc-/- MEFs stimulated blood vessel growth. We repeated these experiments with MEFs in which all three co-receptors were removed. Gas1-/-;Boc-/-;Cdon-/- MEFs displayed a more significantly abrogated Hh-response than both their Gas1-/-;Boc-/- and wildtype counterparts. However subcutaneous co-injection and CAM experiments revealed that these MEFs did not promote tumor growth, behaving similarly to Smo-/- MEFs in previously published findings. These results indicate the importance of Hh dosage in pancreatic cancer, which has important clinical implications. In particular, we found that intermediate levels of Hh signaling, such as that resulting from pharmaceutical inhibition, resulted in a marked angiogenesis response. This finding may partially account for the recent failure of Hh inhibition in a pancreatic cancer clinical trial. Citation Format: Esha Mathew, Yaqing Zhang, Alexander M. Holtz, Kevin T. Kane, Jane Song, Benjamin L. Allen, Marina Pasca di Magliano. Novel Hedgehog co-receptors in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A51.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. Currently there is inadequate understanding of the molecular mechanisms behind PDAC initiation and progression from pancreatic intraepithelial neoplasia (PanINs), the most common precursor lesions, to full blown carcinoma. Thus, there is a lack of effective treatment modalities. The Hedgehog (Hh) signaling pathway plays an important role in PDAC; 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is found in early PanIN lesions and promotes tumor growth, and persists as the cancer progresses. Interestingly, Hh ligands do not act on the tumor cells themselves, but instead function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor progression. PDAC is characterized by a highly desmoplastic stroma, however the mechanisms that activate Hh signaling in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdo), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Boc and Gas1 are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. We did not detect Cdo expression. Interestingly, during cancer progression, Gas1 and Boc expression is significantly increased throughout the cancer stroma. This finding is notable as expression of Gli2, a vital transcription factor in the Hh pathway, is also significantly increased in the stroma during tumorigenesis. Functional studies on Boc-/-;Gas1-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wild-type MEFs, indicating that these co-receptors are important for Hh signal transduction. To further functionally characterize these receptors, we performed co-injection experiments with wild-type pancreatic fibroblasts and tumor cells or Boc-/-;Gas1-/- fibroblasts and tumor cells to determine the effect of co-receptor loss on tumor growth. Strikingly, tumors co-injected with Boc-/-;Gas1-/- fibroblasts formed significantly larger tumors. Histological analysis revealed that these tumors had a higher degree of vascularity. Future studies include dissecting the manner in which mutant fibroblasts support increased vascularity and whether this increase renders these tumors more susceptible to chemotherapy. This abstract is also presented as poster B37. Citation Format: Esha Mathew, Benjamin L. Allen, Marina Pasca di Magliano. Novel hedgehog co-receptors in pancreatic cancer progression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR10.

Hedgehog (Hh) signaling regulates patterning, cell-fate and self-renewal in development. Hh proteins signal via Smoothened (Smo), a G-protein coupled receptor whose activity is dependent on translocation to the primary cilium, a single immotile tubulin-based structure present on most mammalian cells. Activation of Smo results in the stabilization of GLI activator transcription factors, which in turn induce Hh pathway gene expression. Aberrant activation of the Hh pathway is implicated in initiation and progression of a wide range of cancers, yet very few contain genetic mutations of Hh pathway components that account for increased signaling. Instead, the majority of Hh-driven tumors exhibit ligand-dependent signaling but the mechanisms governing this are unknown. We show that genetic inactivation of Trp53 and or Rb1 in mouse embryonic fibroblasts (MEFs) promotes ciliogenesis and cell responsiveness to Hh ligand. Pampliega et al. (Nature 2013) have previously described a functional interaction between autophagy and ciliogenesis. Interestingly, Trp53KO, Rb1KO and Trp53;Rb1KO MEFs exhibit defective autophagic flux and reduced expression of genes associated with autophagy. siRNA knockdown of Atg5, Atg9b, Ctsd and Pik3cg in C57Bl/6 wild-type MEFs resulted in increased Hh ligand responsiveness. To explore this further in a disease setting we assessed an extensive panel of mouse osteosarcoma (mOS) cell lines. In contrast to radiation-induced mOS cell lines that demonstrated variable sensitivity, all genetically induced mOS cell lines (OsxCre;Trp53fl/fl;Rb1fl/fl) were highly sensitive to Hh ligand. In all cases Hh pathway activation could be inhibited by the small-molecule Smo-inhibitor, LDE225. Hh responsiveness correlated to primary cilia frequency with responsive cell lines demonstrating high cilia frequency while nonresponsive cell lines exhibited few if any cilia, under both normal or serum-deprived conditions. Similarly, autophagic flux was significantly reduced in Hh responsive compared to nonresponsive mOS cell lines. Consistent with these findings, in a panel of human osteosarcoma (hOS) cell lines, those with p53 and/or RB-deficient pathways are associated with reduced autophagy and increased primary cilia frequency. Pathway inhibition by LDE225 in in vivo allograft and xenograft models of highly ciliated mOS and hOS cell lines leads to reduced tumor growth, increased survival and intratumoral bone deposition, but has no effect on xenografts of a cell line lacking primary cilia. These data suggest that p53 and Rb control of genes required for autophagy regulates ciliogenesis and ultimately Hh pathway responsiveness to ligand, implicating p53 and Rb mutation status and primary cilia frequency as biomarkers for Hh-ligand sensitivity and potential responsiveness to Hh-inhibitor therapy. Citation Format: Jason E. Cain, Catherine R. Cochrane, Vijesh Vaghjiani, Anette Szczepny, Andrew McCaw, Kirstyn Carey, Luciano Martelotto, Maya Kansara, David Thomas, Carl Walkley, William H. Matsui, David N. Watkins. p53 and RB regulate Hedgehog responsiveness via autophagy-mediated ciliogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4994.

Sonic hedgehog signaling regulates the mammalian cardiac regenerative response

BackgroundSonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.Methodology/Principal FindingsWe analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.Conclusions/SignificanceThe findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues.

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

PurposeSome patients undergoing thoracotomy may suffer from chronic post-thoracotomy pain (CPTP). Treatment of CPTP has been a clinical challenge and the underlying mechanisms of CPTP remain elusive. Recently, sonic hedgehog (Shh) signaling has been shown to be associated with various pain states but its role in the pathogenesis of CPTP is still unclear.MethodsCPTP was induced in rats by thoracotomy. Rats were divided into CPTP group and non-CPTP group based on the mechanical withdrawal threshold (MWT). Rats were administered with Shh signaling inhibitor cyclopamine and activator smoothened agonist (SAG), and then evaluated by MWT and cold allodynia testing. The expressions of Shh signaling (Shh ligand, patched and smoothened receptor, Gli transcription factors), brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase receptor B (Trk-B), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in rat T4-5 spinal cord dorsal horn (SDH) were detected by Western blotting and immunohistochemistry.ResultsThe expression of Shh signaling significantly increased and the BDNF/TrkB pathway was activated in T4-5 SDH of CPTP rats. Cyclopamine attenuated hyperalgesia and down-regulated the expressions of Gil1, BDNF, p-TrkB, p-PI3K and p-Akt in CPTP rats. SAG induced hyperalgesia in non-CPTP rats and elevated the expressions of Gil1, BDNF, p-TrkB, p-PI3K and p-Akt.ConclusionShh signaling may contribute to CPTP via activating BDNF/TrkB signaling pathway, and inhibition of Shh signaling may effectively alleviate CPTP.