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During their biosynthesis, Sonic hedgehog (Shh) morphogens are covalently modified by cholesterol at the C-terminus and palmitate at the N-terminus. Although both lipids initially anchor Shh to the plasma membrane of producing cells, it later translocates to the extracellular compartment to direct developmental fates in cells expressing the Patched (Ptch) receptor. Possible release mechanisms for dually lipidated Hh/Shh into the extracellular compartment are currently under intense debate. In this paper, we describe the serum-dependent conversion of the dually lipidated cellular precursor into a soluble cholesteroylated variant (ShhC) during its release. Although ShhC is formed in a Dispatched- and Scube2-dependent manner, suggesting the physiological relevance of the protein, the depalmitoylation of ShhC during release is inconsistent with the previously postulated function of N-palmitate in Ptch receptor binding and signaling. Therefore, we analyzed the potency of ShhC to induce Ptch-controlled target cell transcription and differentiation in Hh-sensitive reporter cells and in the Drosophila eye. In both experimental systems, we found that ShhC was highly bioactive despite the absence of the N-palmitate. We also found that the artificial removal of N-terminal peptides longer than eight amino acids inactivated the depalmitoylated soluble proteins in vitro and in the developing Drosophila eye. These results demonstrate that N-depalmitoylated ShhC requires an N-peptide of a defined minimum length for its signaling function to Ptch. [ABSTRACT FROM AUTHOR]

In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors. Further analysis of Shh mRNA in cerebral cortical and hypothalamic neurons suggests that Shh is also expressed by glutamatergic neurons. Interestingly, we did not observe substantial Desert Hedgehog and Indian Hedgehog mRNA signals, nor Shh signals in S100β+ astrocytes and Iba1+ microglial cells. Collectively, the present work provides the most robust central map of Shh-expressing cells to date and underscores the importance of nitrergic neurons in regulating Shh availability to brain cells. Thus, our study provides a framework for future experiments aimed at better understanding of the functions of Shh signaling in the brain in normal and pathological states, and the characterization of novel regulatory mechanisms of the signaling pathway. [ABSTRACT FROM AUTHOR]

Both hedgehog (Hh) and target of rapamycin complex 2 (TORC2) are central, evolutionarily conserved signaling pathways that regulate development and metabolism. In C. elegans, loss of the essential TORC2 component RICTOR (rict-1) causes delayed development, shortened lifespan, reduced brood, small size and increased fat. Here, we report that knockdown of both the hedgehog-related morphogen grd-1 and its patched-related receptor ptr-11 rescues delayed development in TORC2 loss-of-function mutants, and grd-1 and ptr-11 overexpression delays wild-type development to a similar level to that in TORC2 loss-of-function animals. These findings potentially indicate an unexpected role for grd-1 and ptr-11 in slowing developmental rate downstream of a nutrient-sensing pathway. Furthermore, we implicate the chronic stress transcription factor pqm-1 as a key transcriptional effector in this slowing of whole-organism growth by grd-1 and ptr-11. We propose that TORC2, grd-1 and ptr-11 may act linearly or converge on pqm-1 to delay organismal development. [ABSTRACT FROM AUTHOR]

During their biosynthesis, Sonic hedgehog (Shh) morphogens are covalently modified by cholesterol at the C-terminus and palmitate at the N-terminus. Although both lipids initially anchor Shh to the plasma membrane of producing cells, it later translocates to the extracellular compartment to direct developmental fates in cells expressing the Patched (Ptch) receptor. Possible release mechanisms for dually lipidated Hh/Shh into the extracellular compartment are currently under intense debate. In this paper, we describe the serum-dependent conversion of the dually lipidated cellular precursor into a soluble cholesteroylated variant (ShhC) during its release. Although ShhC is formed in a Dispatched- and Scube2-dependent manner, suggesting the physiological relevance of the protein, the depalmitoylation of ShhC during release is inconsistent with the previously postulated function of N-palmitate in Ptch receptor binding and signaling. Therefore, we analyzed the potency of ShhC to induce Ptch-controlled target cell transcription and differentiation in Hh-sensitive reporter cells and in the Drosophila eye. In both experimental systems, we found that ShhC was highly bioactive despite the absence of the N-palmitate. We also found that the artificial removal of N-terminal peptides longer than eight amino acids inactivated the depalmitoylated soluble proteins in vitro and in the developing Drosophila eye. These results demonstrate that N-depalmitoylated ShhC requires an N-peptide of a defined minimum length for its signaling function to Ptch.

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

Clear cell renal cell carcinoma (ccRCC) is the deadliest neoplasm of the urinary tract, and we are still far from completely understanding ccRCC development and treatment. The renal tissue paraffin blocks (20) of patients with ccRCC were collected at the University Hospital in Split from 2019 to 2020, and tissue sections were stained with patched (PTCH), anti-smoothened (SMO) and anti-Sonic Hedgehog (SHH) antibodies. SHH was highly expressed (31.9%) in grade 1 tumour, it being higher than all other grades and the control (p < 0.001–p < 0.0001). The trend of a linear decrease in the expression of SHH was observed with the progression of the tumour grade (p < 0.0001). PTCH expression was significantly lower in grades 1 and 2 in comparison to the control (p < 0.01) and grade 4 (p < 0.0001). A significant increase in the expression of SMO was found in grade 4 compared to all other grades (p < 0.0001) and the control (p < 0.001). The strong expression of SHH was observed in carcinoma cells of the G1 stage with a diffuse staining pattern (>50% of neoplastic cells). Stroma and/or inflammatory infiltrate display no staining and no expression of SHH in G1 and G2, while mild focal staining (10–50% of neoplastic cells) was observed in G3 and G4. Patients with high PTCH and low SMO expression had significant time survival differences (p = 0.0005 and p = 0.029, respectively). Therefore, high levels of PTCH and low levels of SMO expression are important markers of better survival rates in ccRCC patients. [ABSTRACT FROM AUTHOR]

Primary cilia are sensory antennae located at the cell surface which mediate a variety of extracellular signals involved in development, tissue homeostasis, stem cells and cancer. Primary cilia are found in an extensive array of vertebrae cells but can only be generated when cells become quiescent. The small intestinal epithelium is a rapidly self-renewing tissue organized into a functional unit called the crypt–villus axis, containing progenitor and differentiated cells, respectively. Terminally differentiated villus cells are notoriously devoid of primary cilia. We sought to determine if intestinal crypts contain a quiescent cell population that could be identified by the presence of primary cilia. Here we show that primary cilia are detected in a subset of cells located deep in the crypts slightly above a Paneth cell population. Using a normal epithelial proliferative crypt cell model, we show that primary cilia assembly and activity correlate with a quiescent state. These results provide further evidence for the existence of a quiescent cell population in the human small intestine and suggest the potential for new modes of regulation in stem cell dynamics. [ABSTRACT FROM AUTHOR]

Although much attention has been devoted to a detailed genomic exposition of cutaneous melanoma, other nonmelanoma skin cancers have also recently been subjected to similar analytical scrutiny. Chief among these are the most common malignancies worldwide: basal cell carcinomas and cutaneous squamous cell carcinomas. In this review, the authors summarize their latest knowledge about the molecular pathways and therapeutic opportunities attendant to these keratinocytic skin cancers. Plain Language Summary: The most common cancers in the United States arise from skin cells called keratinocytes.Although these tumors are not formally tracked by the National Cancer Institute, it is estimated that there are millions of skin cancers called basal cell carcinomas and squamous cell carcinomas.This article reviews the current recent genetic insights into these tumors and therapeutic opportunities. Although the genomic landscape of cutaneous melanoma has received much attention, other nonmelanoma skin cancers also have recently been subjected to similar analytical scrutiny. Chief among these are basal cell carcinomas and cutaneous squamous cell carcinomas, which are the most common malignancies worldwide. The authors summarize recent genetic insights into these tumors and therapeutic opportunities attendant to these keratinocytic skin cancers. [ABSTRACT FROM AUTHOR]

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Mechanisms underlying anteroposterior body axis differences during adult tissue maintenance and regeneration are poorly understood. Here, we identify that post-translational modifications through the SUMO (Small Ubiquitin-like Modifier) machinery are evolutionarily conserved in the Lophotrocozoan Schmidtea mediterranea. Disruption of SUMOylation in adult animals by RNA-interference of the only SUMO E2 conjugating enzyme Ubc9 leads to a systemic increase in DNA damage and a remarkable regional defect characterized by increased cell death and loss of the posterior half of the body. We identified that Ubc9 is mainly expressed in planarian stem cells (neoblasts) but it is also transcribed in differentiated cells including neurons. Regeneration in Ubc9(RNAi) animals is impaired and associated with low neoblast proliferation. We present evidence indicating that Ubc9-induced regional cell death is preceded by alterations in transcription and spatial expression of repressors and activators of the Hedgehog signaling pathway. Our results demonstrate that SUMOylation acts as a regional-specific cue to regulate cell fate during tissue renewal and regeneration.

Abstract Objective The Hedgehog pathway is a fundamental signaling pathway in organogenesis. The expression patterns of the ligand Sonic Hedgehog (Shh) and key pathway components have been studied in many tissues but direct spatial comparisons across tissues with different cell compositions and structural organization are not common and could reveal tissue-specific differences in pathway dynamics. Results We directly compared the expression characteristics of Shh, and four genes with functional roles in signaling and whose expression levels serve as readouts of pathway activity in multiple tissues of the embryonic mouse head at embryonic day 15.5 by serial in situ hybridization. The four readout genes were the positive feedback regulator Gli1, and three negative feedback regulators, Patched1, Patched2, and Hedgehog Interacting Protein. While the relative abundance of Gli1 was similar across tissues, the relative expression levels and spatial distribution of Shh and the negative feedback regulators differed, suggesting that feedback regulation of hedgehog signaling is context dependent. This comparative analysis offers insight into how consistent pathway activity could be achieved in tissues with different morphologies and characteristics of ligand expression.

Clear cell renal cell carcinoma (ccRCC) is the deadliest neoplasm of the urinary tract, and we are still far from completely understanding ccRCC development and treatment. The renal tissue paraffin blocks (20) of patients with ccRCC were collected at the University Hospital in Split from 2019 to 2020, and tissue sections were stained with patched (PTCH), anti-smoothened (SMO) and anti-Sonic Hedgehog (SHH) antibodies. SHH was highly expressed (31.9%) in grade 1 tumour, it being higher than all other grades and the control (p < 0.001–p < 0.0001). The trend of a linear decrease in the expression of SHH was observed with the progression of the tumour grade (p < 0.0001). PTCH expression was significantly lower in grades 1 and 2 in comparison to the control (p < 0.01) and grade 4 (p < 0.0001). A significant increase in the expression of SMO was found in grade 4 compared to all other grades (p < 0.0001) and the control (p < 0.001). The strong expression of SHH was observed in carcinoma cells of the G1 stage with a diffuse staining pattern (>50% of neoplastic cells). Stroma and/or inflammatory infiltrate display no staining and no expression of SHH in G1 and G2, while mild focal staining (10–50% of neoplastic cells) was observed in G3 and G4. Patients with high PTCH and low SMO expression had significant time survival differences (p = 0.0005 and p = 0.029, respectively). Therefore, high levels of PTCH and low levels of SMO expression are important markers of better survival rates in ccRCC patients.

Despite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases. [ABSTRACT FROM AUTHOR]

The sterol-sensing domain (SSD) is present in several membrane proteins that function in cholesterol metabolism, transport, and signaling. Recent progress in structural studies of SSD-containing proteins, such as sterol regulatory element-binding protein (SREBP)-cleavage activating protein (Scap), Patched, Niemann-Pick disease type C1 (NPC1), and related proteins, reveals a conserved core that is essential for their sterol-dependent functions. This domain, by its name, 'senses' the presence of sterol substrates through interactions and may modulate protein behaviors with changing sterol levels. We summarize recent advances in structural and mechanistic investigations of these proteins and propose to divide them to two classes: M for 'moderator' proteins that regulate sterol metabolism in response to membrane sterol levels, and T for 'transporter' proteins that harbor inner tunnels for cargo trafficking across cellular membranes. Sterol-sensing domain (SSD)-containing proteins play a key role in sterol sensing, transport, and signaling. Structural comparison redefines the elements that constitute the SSD. Conformational changes of SSD are crucial for sterol sensing and transport. Structures of SSD-containing transporters reveal the transport path of sterols. [ABSTRACT FROM AUTHOR]

The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance-nodulation-division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control. [ABSTRACT FROM AUTHOR]

Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor.

Objectives: Sonic hedgehog (SHH) signaling is important in bladder development. Mice with defective hedgehog signaling develop bladder anomalies. Clinically, urinary tract malformations are reported in human fetuses and infants with mutations of SHH and related signaling pathway genes. Information on the expression of SHH and associated signaling genes in normal human bladder development is fragmentary. This study determined the temporal and spatial expression patterns of SHH signaling pathway components in human fetal bladders by immunohistochemistry (IHC).Material and Methods: Twenty-four bladder specimens from 16 male and 8 female human fetuses aged 12- to 36-week (wk) were obtained from the First Affiliated Hospital of Xi'an Jiaotong University. The tissue slides were processed for IHC staining with SHH, Patched1 (PTC-1), Patched2 (PTC-2), Smoothened (SMO), GLI1 and proliferating cell nuclear antigen (PCNA). The expression levels of each gene were analyzed by semi-quantitative histological scoring system.Results: High intensity of SHH and SMO expression was detected in developing bladder urothelial cells, with no staining in lamina propria (LP), but with minimal expression of SMO in differentiating smooth muscle (SM) layers. The spatial distribution pattern of PTC1 and GLI1 was more complex with minimal expression in the LP layer, moderate expression in the SM layer, and high expression in the urothelium. PTC2 expression was mainly localized in the urothelium and LP, but no expression in the SM layer. All of the SHH signaling components were detected in fetal bladder tissues throughout the development, with expression peaks at 12- and 23-wk, coinciding with high cell proliferation as indicated by PCNA staining in the cell nuclei of urothelium and SM.Conclusions: The autocrine SHH signaling in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC-1 and GLI1 were demonstrated during human bladder development. Expression of SHH signaling components peaked at 12-and 23-wk. The first expression peak at 12-wk may relate to urothelium growth, SM induction, and dilation of the bladder cavity. The second expression peaked at 23-wk may relate to urothelium and SM layer differentiation.

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.

Objective: The Hedgehog pathway is a fundamental signaling pathway in organogenesis. The expression patterns of the ligand Sonic Hedgehog (Shh) and key pathway components have been studied in many tissues but direct spatial comparisons across tissues with different cell compositions and structural organization are not common and could reveal tissue-specific differences in pathway dynamics. Results: We directly compared the expression characteristics of Shh, and four genes with functional roles in signaling and whose expression levels serve as readouts of pathway activity in multiple tissues of the embryonic mouse head at embryonic day 15.5 by serial in situ hybridization. The four readout genes were the positive feedback regulator Gli1, and three negative feedback regulators, Patched1, Patched2, and Hedgehog Interacting Protein. While the relative abundance of Gli1 was similar across tissues, the relative expression levels and spatial distribution of Shh and the negative feedback regulators differed, suggesting that feedback regulation of hedgehog signaling is context dependent. This comparative analysis offers insight into how consistent pathway activity could be achieved in tissues with different morphologies and characteristics of ligand expression. [ABSTRACT FROM AUTHOR]

Background: Basal cell carcinoma (BCC) is a common skin cancer often curable by excision; however, for patients with BCC around the eye, excision places visual organs and function at risk. In this article, we test the hypothesis that use of the hedgehog inhibitor vismodegib will improve vision‐related outcomes in patients with orbital and extensive periocular BCC (opBCC). Materials and Methods: In this open‐label, nonrandomized phase IV trial, we enrolled patients with globe‐ and lacrimal drainage system–threatening opBCC. To assess visual function in the context of invasive periorbital and lacrimal disease, we used a novel Visual Assessment Weighted Score (VAWS) in addition to standard ophthalmic exams. Primary endpoint was VAWS with a score of 21/50 (or greater) considered successful, signifying globe preservation. Tumor response was evaluated using RECIST v1.1. Surgical specimens were examined histologically by dermatopathologists. Results: In 34 patients with opBCC, mean VAWS was 44/50 at baseline, 46/50 at 3 months, and 47/50 at 12 months or postsurgery. In total, 100% of patients maintained successful VAWS outcome at study endpoint. Compared with baseline, 3% (95% confidence interval [CI], 0.1–15.3) experienced major score decline (5+ points), 14.7% (95% CI, 5 to 31.1) experienced a minor decline (2–4 points), and 79.4% experienced a stable or improved score (95% CI, 62.1–91.3). A total of 56% (19) of patients demonstrated complete tumor regression by physical examination, and 47% (16) had complete regression by MRI/CT. A total of 79.4% (27) of patients underwent surgery, of which 67% (18) had no histologic evidence of disease, 22% (6) had residual disease with clear margins, and 11% (3) had residual disease extending to margins. Conclusion: Vismodegib treatment, primary or neoadjuvant, preserves globe and visual function in patients with opBCC. Clinical trail identification number.NCT02436408. Implications for Practice: Use of the antihedgehog inhibitor vismodegib resulted in preservation of end‐organ function, specifically with regard to preservation of the eye and lacrimal apparatus when treating extensive periocular basal cell carcinoma. Vismodegib as a neoadjuvant also maximized clinical benefit while minimizing toxic side effects. This is the first prospective clinical trial to demonstrate efficacy of neoadjuvant antihedgehog therapy for locally advanced periocular basal cell carcinoma, and the first such trial to demonstrate end‐organ preservation. This article reports the results of a prospective clinical trial of vismodegib for patients with basal cell carcinoma occurring in the orbital and periocular regions to assess whether such treatment helps to preserve visual organs and function. [ABSTRACT FROM AUTHOR]

Despite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases.

Activation of the Hedgehog pathwaymay have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs. [ABSTRACT FROM AUTHOR]

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia‐like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient. Synopsis: Hedgehog (Hh) gradient formation in the Drosophila wing disc is enabled by Hh exosomal transport along filopodia‐like structures called cytonemes at the epithelial cell basolateral surface. Here, Hh receptor Patched (Ptc) is shown to undergo similar trafficking process to interact with Hh at discrete, synapse‐like cytoneme contact sites. Ptc internalization from apical surfaces is required for its ESCRT‐mediated sorting into multi‐vesicular bodies.Tetraspanin Tsp96F directly interacts with Ptc and regulates its exosomal sorting.SNARE complex components are required for Ptc‐containing exosome fusion at cytonemes. [ABSTRACT FROM AUTHOR]

Cell differentiation and proliferation require Hedgehog (HH) signaling and aberrant HH signaling causes birth defects or cancers. In this signaling pathway, the N-terminally palmitoylated and C-terminally cholesterylated HH ligand is secreted into the extracellular space with help of the Dispatched-1 (DISP1) and Scube2 proteins. The Patched-1 (PTCH1) protein releases its inhibition of the oncoprotein Smoothened (SMO) after binding the HH ligand, triggering downstream signaling events. In this review, we discuss the recent structural and biochemical studies on four major components of the HH pathway: the HH ligand, DISP1, PTCH1, and SMO. This research provides mechanistic insights into how HH signaling is generated and transduced from the cell surface into the intercellular space and will aid in facilitating the treatment of HH-related diseases. The Hedgehog (HH) signaling pathway is essential for the development of animals and is involved in many human cancers. The secreted ligand HH binds its receptor Patched-1 (PTCH1) and releases the inhibition of Smoothened (SMO) by PTCH1; PTCH1 regulates SMO catalytically via sterols. How HH recognizes and inhibits PTCH1, how SMO is regulated, and how the HH signal is transduced into cells remain unknown and controversial. Recent structural studies on HH–PTCH1 complexes and SMO in different conformations reveal the mechanism of PTCH1 recognition and inhibition by HH and provide insights into the mechanism of SMO activation. [ABSTRACT FROM AUTHOR]

Signaling pathways that mediate cell-cell communication are essential for collective cell behaviors in multicellular systems. The hedgehog (HH) pathway, first discovered and elucidated in Drosophila, is one of these iconic signaling systems that plays many roles during embryogenesis and in adults; abnormal HH signaling can lead to birth defects and cancer. We review recent structural and biochemical studies that have advanced our understanding of the vertebrate HH pathway, focusing on the mechanisms by which the HH signal is received by patched on target cells, transduced across the cell membrane by smoothened, and transmitted to the nucleus by GLI proteins to influence gene-expression programs. [ABSTRACT FROM AUTHOR]

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides. [ABSTRACT FROM AUTHOR]

Hedgehog signaling plays pivotal roles in the development and homeostasis of epithelial barrier tissues. However, whether and how Hedgehog signaling directly regulates innate immunity in epithelial cells remains unknown. By utilizing C. elegans epidermis as the model, we found that several Hedgehog receptors are involved in cell-autonomous regulation of the innate immune response in the epidermis. Particularly, loss of the Patched family receptor induces aberrant up-regulation of epidermal antimicrobial peptides in a STAT-dependent manner. External or internal insult to the epidermis triggers rapid rearrangement of Patched distribution along the plasma membrane, indicating that the Hedgehog (Hh) receptor is likely involved in recognition and defense against epidermal damage. Loss of PTCH1 function in primary human keratinocytes and intact mouse skin also results in STAT-dependent immune activation. These findings reveal an evolutionally conserved immune-surveillance function of Hedgehog receptors and an insult-sensing and response strategy of epithelial tissues. [Display omitted] • The Patched family Hh receptors regulate epidermal innate immunity in C. elegans • C. elegans Patched regulates epidermal immunity via the STAT family protein STA-2 • Epidermal disturbance triggers disaggregation/degradation of Patched molecules • Mammalian Hh receptors regulate epidermal innate immunity in vitro and in vivo Wang et al. show that Patched family Hh receptors suppress the expression of innate immune effectors cell autonomously in epithelial cells of nematode and mammalian skin. This immune-regulating function of Hh receptors relies on STAT family transcriptional factors but is independent of canonical Hh signaling. [ABSTRACT FROM AUTHOR]

Cell migration and guidance are complex processes required for morphogenesis, the formation of tumor metastases, and the progression of human cancer. During migration, guidance molecules induce cell directionality and movement through complex intracellular mechanisms. Expression of these molecules has to be tightly regulated and their signals properly interpreted by the receiving cells so as to ensure correct navigation. This molecular control is fundamental for both normal morphogenesis and human disease. The Hedgehog (Hh) signaling pathway is evolutionarily conserved and known to be crucial for normal cellular growth and differentiation throughout the animal kingdom. The relevance of Hh signaling for human disease is emphasized by its activation in many cancers. Here, I review the current knowledge regarding the involvement of the Hh pathway in cell migration and guidance during Drosophila development and discuss its implications for human cancer origin and progression.

Adrenocortical carcinoma (ACC) presents a high risk of relapse and metastases with outcomes not improving despite extensive research and new targeted therapies. We recently showed that the Hedgehog receptor Patched is expressed in ACC, where it strongly contributes to doxorubicin efflux and treatment resistance. Here, we report the identification of a new inhibitor of Patched drug efflux, the anti-histaminergic drug astemizole. We show that astemizole enhances the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells at concentrations of astemizole or doxorubicin that are not effective by themselves. Our results suggest that a low concentration of astemizole sensitizes ACC cells to doxorubicin, which is a component of the standard treatment for ACC composed of etoposide, doxorubicin, cisplatin and mitotane (EDPM). Patched uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that the use of astemizole or a derivative in combination with EDPM could be a promising therapeutic option for ACC by increasing the treatment effectiveness at lower doses of EDPM, which would reduce the severe side effects of this regimen.

Melanoma patients harboring the BRAFV600E mutation are treated with vemurafenib. Almost all of them ultimately acquire resistance, leading to disease progression. Here, we find that a small molecule from a marine sponge, panicein A hydroquinone (PAH), overcomes resistance of BRAFV600E melanoma cells to vemurafenib, leading to tumor elimination in corresponding human xenograft models in mice. We report the synthesis of PAH and demonstrate that this compound inhibits the drug efflux activity of the Hedgehog receptor, Patched. Our SAR study allowed identifying a key pharmacophore responsible for this activity. We showed that Patched is strongly expressed in metastatic samples from a cohort of melanoma patients and is correlated with decreased overall survival. Patched is a multidrug transporter that uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that PAH is a highly promising lead for the treatment of vemurafenib resistant BRAFV600E melanoma.

Aim: To investigate export of Hedgehog pathway (Hh) proteins Patched1, Smoothened, Sonic hedgehog and Indian hedgehog in cervical cancer cell line (CaCx) exosomes. Methods: Exosomes were isolated and characterized by Western blotting, scanning electron microscopy and in a colorimetric assay. Nucleic acids (RNA, DNA) and protein content of exosomes were analyzed. Hh pathway proteins in exosomes were detected using Western blotting. Results: CaCx secrete bio-macromolecule (DNA, RNA and proteins) enriched exosomes. CaCx exosomes contained higher amount of RNA with respect to DNA. CaCx preferentially exported Hh proteins (Patched1, Smoothened, Sonic hedgehog, Indian hedgehog) in their exosomes. Cellular uptake assay revealed rapid internalization of CaCx exosomes in human umbilical vein endothelial cells. Conclusion: Our study showed that Hh proteins are exported in CaCx exosomes. [ABSTRACT FROM AUTHOR]

Purpose: Basal cell carcinoma (BCC) is one of the most common skin cancers, and is typically driven by an aberrantly activated Hedgehog (Hh) pathway. The Hh pathway is regulated by interactions between the Patched-1 (Ptch1) and Smoothened (Smo) receptors. Smo is an activating receptor and is subject to inhibition by Ptch1. Following ligand binding to Ptch1, its inhibitory action is relieved and pathway activation occurs. This receptor interaction is pivotal to restraining uncontrolled cellular growth. Both receptors have been found to be frequently mutated in BCCs. Ptch2 is a Ptch1 paralog that exhibits overlapping functions in both normal development and tissue homeostasis. As yet, its contribution to cancer growth is poorly defined. Here we set out to assess how Ptch2 inhibits BCC growth.Methods: We used several in vitro readouts for transcriptional and chemotactic Hh signaling in BCC-derived ASZ001 cells, and a novel xenograft model to assess in vivo BCC tumor growth. Gene editing by TALEN was used to untangle the different Ptch2-dependent responses to its ligand sonic hedgehog (Shh).Results: We first defined the signaling competence of Ptch2 in Ptch1-deficient ASZ001 cells in vitro, and found that Ptch2 ligand binding drives their migration rather than eliciting a transcriptional response. We found that subsequent targeting of Ptch2 abrogated the chemotaxic effect. Next, we tested the contribution of Ptch2 to in vivo tumor growth using a xenograft model and found that reduced Ptch function results in increased tumor growth, but that selective pressure appatently acts against complete Ptch2 ablation.Conclusions: We conclude that like Ptch1, Ptch2 exerts a tumor-suppressive function in BCC cells, and that after targeting of both paralogs, ligand-independent activation of the Hh pathway contributes to tumor growth. [ABSTRACT FROM AUTHOR]

One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug‐like molecule enhances the cytotoxic, pro‐apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched‐expressing cancers. [ABSTRACT FROM AUTHOR]

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation. [ABSTRACT FROM AUTHOR]