Hh Receptor Research Articles

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer.

The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

Hedgehog receptors exert immune-surveillance roles in the epidermis across species

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.

Hedgehog-induced ZFYVE21 promotes chronic vascular inflammation by activating NLRP3 inflammasomes in T cells.

The zinc finger protein ZFYVE21 is involved in immune signaling. Using humanized mouse models, primary human cells, and patient samples, we identified a T cell-autonomous role for ZFYVE21 in promoting chronic vascular inflammation associated with allograft vasculopathy. Ischemia-reperfusion injury (IRI) stimulated endothelial cells to produce Hedgehog (Hh) ligands, which in turn induced the production of ZFYVE21 in a population of T memory cells with high amounts of the Hh receptor PTCH1 (PTCHhi cells, CD3+CD4+CD45RO+PTCH1hiPD-1hi), vigorous recruitment to injured endothelia, and increased effector responses in vivo. After priming by interferon-γ (IFN-γ), Hh-induced ZFYVE21 activated NLRP3 inflammasome activity in T cells, which potentiated IFN-γ responses. Hh-induced NLRP3 inflammasomes and T cell-specific ZFYVE21 augmented the vascular sequelae of chronic inflammation in mice engrafted with human endothelial cells or coronary arteries that had been subjected to IRI before engraftment. Moreover, the population of PTCHhi T cells producing high amounts of ZFYVE21 was expanded in patients with renal transplant-associated IRI, and sera from these patients expanded this population in control T cells in a manner that depended on Hh signaling. We conclude that Hh-induced ZFYVE21 activates NLRP3 inflammasomes in T cells, thereby promoting chronic inflammation.

The effects of Hh morphogen source movement on signaling dynamics.

Morphogens of the Hh family trigger gene expression changes in receiving cells in a concentration-dependent manner to regulate their identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its logic includes a negative-feedback loop involving the Hh receptor Ptc. Here, using experiments and computational models we study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distributions of Ptc and the activator transcription factor CiA in wing, antenna and ocellus show similar features, but are markedly different from that in the compound eye. We propose that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the compound eye and the negative-feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results show how the interaction between spatial and temporal dynamics of signaling and differentiation processes may contribute to the informational versatility of the conserved Hh signaling pathway.

Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program.

Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurpose multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitive short linear motif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early upregulation of Hh transcription factor GLI-1 and regulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120-Hh-SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an α-TRP120-Hh-SLiM antibody. TRP120-Hh-SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection. Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

Hedgehog Signalling Modulates Immune Response and Protects against Experimental Autoimmune Encephalomyelitis

The Hedgehog (Hh) pathway is essential for the embryonic development and homeostatic maintenance of many adult tissues and organs. It has also been associated with some functions of the innate and adaptive immune system. However, its involvement in the immune response has not been well determined. Here we study the role of Hh signalling in the modulation of the immune response by using the Ptch-1-LacZ+/− mouse model (hereinafter referred to as ptch+/−), in which the hemizygous inactivation of Patched-1, the Hh receptor gene, causes the constitutive activation of Hh response genes. The in vitro TCR stimulation of spleen and lymph node (LN) T cells showed increased levels of Th2 cytokines (IL-4 and IL-10) in ptch+/−cells compared to control cells from wild-type (wt) littermates, suggesting that the Th2 phenotype is favoured by Hh pathway activation. In addition, CD4+ cells secreted less IL-17, and the establishment of the Th1 phenotype was impaired in ptch+/− mice. Consistently, in response to an inflammatory challenge by the induction of experimental autoimmune encephalomyelitis (EAE), ptch+/− mice showed milder clinical scores and more minor spinal cord damage than wt mice. These results demonstrate a role for the Hh/ptch pathway in immune response modulation and highlight the usefulness of the ptch+/− mouse model for the study of T-cell-mediated diseases and for the search for new therapeutic strategies in inflammatory diseases.

Microscale Thermophoresis to Evaluate the Functionality of Heterologously Overexpressed Membrane Proteins in Membrane Preparations.

Transmembrane proteins are challenging to express in heterologous systems and to purify, thus any technique enabling to evaluate the functionality of the protein produced prior purification provides a huge step forward. Furthermore, the membrane environment may be critical for the activity of the target protein and accessing information in the membrane fragments instead of solubilizing the target into a detergent that may be unsuitable for its function is key to study and evaluate its activity. Herein, we describe how microscale thermophoresis (MST) was used to evaluate the functionality of membrane proteins directly in host membrane preparation before purification. We give a protocol to measure the affinity between the human Hedgehog (Hh) receptor Ptch1 in yeast plasma membrane and the small molecule PAH, which was shown to inhibit its drug efflux activity.

Integral Membrane Protein 2A Is a Negative Regulator of Canonical and Non-Canonical Hedgehog Signalling.

The Hedgehog (Hh) receptor PTCH1 and the integral membrane protein 2A (ITM2A) inhibit autophagy by reducing autolysosome formation. In this study, we demonstrate that ITM2A physically interacts with PTCH1; however, the two proteins inhibit autophagic flux independently, since silencing of ITM2A did not prevent the accumulation of LC3BII and p62 in PTCH1-overexpressing cells, suggesting that they provide alternative modes to limit autophagy. Knockdown of ITM2A potentiated PTCH1-induced autophagic flux blockade and increased PTCH1 expression, while ITM2A overexpression reduced PTCH1 protein levels, indicating that it is a negative regulator of PTCH1 non-canonical signalling. Our study also revealed that endogenous ITM2A is necessary for timely induction of myogenic differentiation markers in C2C12 cells since partial knockdown delays the timing of differentiation. We also found that basal autophagic flux decreases during myogenic differentiation at the same time that ITM2A expression increases. Given that canonical Hh signalling prevents myogenic differentiation, we investigated the effect of ITM2A on canonical Hh signalling using GLI-luciferase assays. Our findings demonstrate that ITM2A is a strong negative regulator of GLI transcriptional activity and of GLI1 stability. In summary, ITM2A negatively regulates canonical and non-canonical Hh signalling.

Abstract 2421: PTCH1 C-terminal domain truncations in colorectal cancer increase mitogenic signalling and autophagy

Abstract The canonical Hedgehog (Hh) signaling pathway, driving activation of SMO and GLI1, has been implicated in colorectal cancer (CRC) stemness and resistance to chemotherapy. We recently reported that the C-terminal domain (CTD) of the Hh receptor PTCH1 limits autophagic flux and ERK signaling by non-canonical mechanisms (independently of SMO/GLI1). Given that those novel functions are implicated in tumorigenesis, we explored the existence of PTCH1 CTD mutations in cancer specimens. Analysis of the TCGA Cancer Atlas database and the Genomics England 100,000 Genomes Project revealed relatively frequent somatic mutations in the CTD of PTCH1 in CRC: S1203(fs) and R1308(fs), which result in premature truncations of the CTD in about 4% of cases. PTCH1 CTD mutations were strongly associated with BRAF mutations (61% samples) and with right-sided disease (10/10 of samples with sidedness information). Co-immunoprecipitation analysis of truncated PTCH1(S1203*) and PTCH1(R1308*) showed loss of interaction with ATG101 and GRB2, required for regulation of autophagic flux and ERK signaling, respectively. To determine the pathogenic role of those mutations, we engineered SW620 metastatic CRC cell lines using CRISPR/Cas9 to create two clones with indel mutations between S1203 and R1308. Truncation of the CTD increased basal ERK and AKT phosphorylation levels, proliferation, invasion and migration compared to isogenic cells expressing wild type PTCH1. The mutant clones also showed persistent proliferation in the presence of glycolysis inhibitors and of the autophagy inducer rapamycin. Moreover, both clones displayed an enhanced autophagic flux by analysis of the LC3BI/II and p62 markers in the presence and absence of Bafilomycin A1. RNA-seq analysis revealed that the CTD truncation resulted in increase in GO terms of metabolic pathways, pathways in cancer, mTOR signaling pathway, PI3K-AKT pathway, MAPK signaling pathway and regulation of actin cytoskeleton, among others. The clones morphology changed together with a remarkable cadherin switch and loss of vimentin at RNA and protein level, suggestive of mesenchymal-epithelial transition, a process necessary for establishment of metastasis following cancer cell spread. Interestingly, one clone showed enhanced apoptosis in response to FOLFOX (5-fluorouracil/lecovorin/oxaliplatin), the most effective chemotherapy for metastatic CRC, as determined by cleaved PARP and Caspase 7 activity, compared to the parental cells. These findings reveal important tumor suppressor functions of the CTD of PTCH1 acting to suppress autophagy and mitogenic signaling, which are dysregulated by CTD mutations in a subset of colon cancer patients. Our results provide mechanistic insights of the effect of PTCH1 mutations in CRC and identify potential therapeutic vulnerabilities. Citation Format: Begoña Caballero-Ruiz, Cintli C. Morales-Alcala, Henry M. Wood, Gianluca Canettieri, Natalia A. Riobo-Del Galdo. PTCH1 C-terminal domain truncations in colorectal cancer increase mitogenic signalling and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2421.

Competitive coordination of the dual roles of the Hedgehog co-receptor in homophilic adhesion and signal reception.

Hedgehog (Hh) signaling patterns embryonic tissues and contributes to homeostasis in adults. In Drosophila, Hh transport and signaling are thought to occur along a specialized class of actin-rich filopodia, termed cytonemes. Here, we report that Interference hedgehog (Ihog) not only forms a Hh receptor complex with Patched to mediate intracellular signaling, but Ihog also engages in trans-homophilic binding leading to cytoneme stabilization in a manner independent of its role as the Hh receptor. Both functions of Ihog (trans-homophilic binding for cytoneme stabilization and Hh binding for ligand sensing) involve a heparin-binding site on the first fibronectin repeat of the extracellular domain. Thus, the Ihog-Ihog interaction and the Hh-Ihog interaction cannot occur simultaneously for a single Ihog molecule. By combining experimental data and mathematical modeling, we determined that Hh-Ihog heterophilic interaction dominates and Hh can disrupt and displace Ihog molecules involved in trans-homophilic binding. Consequently, we proposed that the weaker Ihog-Ihog trans interaction promotes and stabilizes direct membrane contacts along cytonemes and that, as the cytoneme encounters secreted Hh ligands, the ligands trigger release of Ihog from trans Ihog-Ihog complex enabling transport or internalization of the Hh ligand-Ihog-Patched -receptor complex. Thus, the seemingly incompatible functions of Ihog in homophilic adhesion and ligand binding cooperate to assist Hh transport and reception along the cytonemes.

Inhibition of TGF-β1 on Gli2 expression was promoted by TNF-α in primary leukemia cells

Purpose Hedgehog (Hh) signaling pathway regulates a variety of tumors-related diseases including leukemia. Whether inhibition of TGF-β1 on Gli2 expression is promoted by TNF-α in primary leukemia cells remains to be determined. Methods Primary leukemia cells were treated with TGF-β1, TNF-α or SIS3 at different concentrations. Gli2 expression was detected by quantitative real-time PCR and western blot analyses. Results We found that TGF-β significantly decreased Gli2 expression, and co-treatment with TNF-αfurther decreased Gli2 expression in primary leukemia cells. TNF-α can increased TGF-βRI and TGF-βRII protein expression in primary leukemia cells, while SIS3 inhibited the effect of TGF-β. Conclusion Our results suggest that Gli2 expression in primary leukemia cells is induced by TGF-β in a Smad3-dependent manner, and independent of Hh receptor signaling.

Itraconazole exerts anti-liver cancer potential through the Wnt, PI3K/AKT/mTOR, and ROS pathways

Hepatocellular carcinoma (HCC) is one of the most common cancers with the highest morbidity and mortality. It is necessary to develop new anti-liver cancer drugs. Itraconazole is a popular systemic anti-fungal drug with a strong anti-tumor effect. However, so far, it is not clear whether itraconazole has specific anti-tumor effect on liver cancer. The purpose of this study was to investigate itraconazole resistant effect of liver cancer and to explore its potential anti-cancer mechanism. The effect of itraconazole on the proliferation of liver cancer cells was studied with MTT assay. Flow cytometry was used to determine the effect of itraconazole on apoptosis, cell cycle distribution, changes in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). In addition, after DAPI staining, nuclear morphological changes were observed under the fluorescent microscope, and the release of lactate dehydrogenase (LDH) was measured using the microplate reader. Finally, the expressions of proteins related to the anti-tumor signaling pathway were determined by Western blotting. The results showed that itraconazole significantly inhibited the proliferation of HepG2 and Bel-7405 cells. In addition, the data showed that itraconazole induced apoptosis in HepG2 cells, increased the production of ROS, blocked cell cycle, and decreased MMP. Furthermore, itraconazole inhibited HCC cell growth and promoted apoptosis through the Hh, Wnt/catenin, AKT/mTOR/S6K, ROS and death receptor pathways. Finally, we come to the conclusion that itraconazole exerts anti-liver cancer effect, and has potential for use as a new drug for liver cancer in clinic.

Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc.

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.

Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhNp) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhNp through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhNp and the other through the Ca2+-mediated interface on ShhNp. Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.

Sonic Hedgehog Signaling is Blue: Insights from the Patched Mutant Mice

The Hedgehog (Hh) pathway is a highly conserved signaling system regulating a range of developmental processes. A 1997 paper by Goodrich and colleagues provided major contributions to understanding the Hh pathway by mutating the gene encoding the Hh receptor, Patched, and thereby developing a mouse model for a human cancer predisposition syndrome, known as Gorlin syndrome. These studies provided one of the first genetically engineered mouse models for brain tumors.

Abstract 5808: Hedgehog pathway inhibitors itraconazole and cyclopamine produce synergistic suppression of Pten deficient prostate cancer

Abstract Hedgehog (Hh) signaling plays an important role in prostate development and is also a characteristic feature of prostate cancer (PCa). The ability of Hh pathway activation to promote cell invasiveness, epithelial to mesenchymal transition and metastasis suggest that targeting Hh pathway may provide an important clinical avenue for the treatment of advanced PCa. Loss of PTEN function is considered a characteristic feature of advanced PCa. To gain insight into the status of Hh signaling in PCa in relation to PTEN status we selected three cell lines based on their PTEN expression. Gli1 mRNA was upregulated 2000 fold in PC3 (PTEN-/- null) cells, 800 fold in DU145 (PTEN+/-) cells when compared with normal prostate epithelial cells suggesting that PTEN loss is associated with increased Hh signaling. We also observed significant increase in the mRNA levels of Hh ligand (Shh), Hh receptors (PTCH2 and SMO) and effectors of Hh signaling (Gli1 and Gli2) in the prostate tissues of PTEN knockout mice. A 400 fold increase in the mRNA levels of Gli1 and 15 fold increase in the levels of Gli2 mRNA levels was also observed. We also observed significant decrease in the mRNA levels of the negative regulators of Hh signaling, HHIP and SuFu in the prostate tissues of PTEN knockout mice with similar inhibition in the transcript levels of GAS1. We next investigated the effect on Hh signaling in PCa cells treated with itraconazole and cyclopamine alone and in combination. PCa 22Rν1 and PC-3 cells transfected with Gli-dependent luciferase reporter construct were treated with itraconazole (1 and 2 μM), cyclopamine (5 and 10 nM) and combination of itraconazole (1 μM) and cyclopamine (5 nM) for 40 hours. Gli-dependent luciferase reporter activity was inhibited by itraconazole and cyclopamine treatments; however the inhibition was significantly greater in combination. Treatment of PCa cells with the combination of itraconazole and cyclopamine led to increased cell growth inhibition that was greater than the sum of the individual agents alone suggesting synergism. We next analyzed the effect of the combination on the growth of CWR22Rν1 tumors under in vivo conditions using athymic nude mice. In control animals, targeted tumor volume of 1200 mm3 was reached at 6 weeks post cell inoculation and significantly later time in animals treated with itraconazole alone (9 weeks) and cyclopamine alone (8 weeks). Notably, tumor volumes stayed under 165 mm3 in mice that received both itraconazole and cyclopamine. In the prostate specific Pten knockout mice combination of itraconazole and cyclopamine produced 57% inhibition of tumor growth compared to 30% in cyclopamine alone and 45% in itraconazole alone treated mice. These data show that a combination of itraconazole and cyclopamine is considerably more efficient in inhibiting tumor growth highlighting the synergistic efficacy of this combination for the treatment of PCa. Citation Format: Vaqar M. Adhami, Imtiaz A. Siddiqui, Mohammad Imran Khan, Islam Rady, Leanna Sako, Hasan Mukhtar. Hedgehog pathway inhibitors itraconazole and cyclopamine produce synergistic suppression of Pten deficient prostate cancer [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 5808.

Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED1 and ATG101

The Hedgehog (Hh) receptor Patched1 (PTCH1) is a well-known tumor suppressor that in its active form represses Smoothened (SMO) activity, inhibits proliferation, and induces apoptosis. The cytoplasmic C-terminal domain (CTD) regulates PTCH1 turnover and nucleates a proapoptotic complex. In this study, it was mechanistically determined that Autophagy-related 101 (ATG101), essential for mammalian autophagy, physically interacts with the CTD of PTCH1 and connects it to the ULK complex, which stimulates the autophagy machinery in response to changes in nutrient availability. This interaction results in a blockade of basal autophagic flux and accumulation of autophagosomes with undegraded cargo. Remarkably, this function of PTCH1 is independent of its repressive activity on SMO, as shown in SMO-deficient cells or in the presence of a SMO inhibitor, but is opposed by Sonic Hedgehog (SHH). These findings reveal a novel noncanonical function of PTCH1 that limits autophagy, mediated by ATG101, which could have therapeutic implications in Hh-dependent cancers.Implications: Loss-of-function of the tumor suppressor Patched1 might promote cancer cell fitness by increasing autophagic flux in response to metabolic or environmental stresses. Mol Cancer Res; 16(5); 909-19. ©2018 AACR.

Hedgehog signaling regulates cell motility and optic fissure and stalk formation during vertebrate eye morphogenesis.

ABSTRACTEstablishment of precise three-dimensional tissue structure is vital for organ function. In the visual system, optic fissure and stalk morphogenesis is a crucial yet poorly understood process, disruptions of which can lead to coloboma, a birth defect causing visual impairment. Here, we use four-dimensional imaging, cell tracking, and molecular genetics in zebrafish to define the cell movements underlying normal optic fissure and stalk formation. We determine how these events are disrupted in a coloboma model in which the Hedgehog (Hh) receptor ptch2 is lost, resulting in overactive Hh signaling. In the ptch2 mutant, cells exhibit defective motile behaviors and morphology. Cells that should contribute to the fissure do not arrive at their correct position, and instead contribute to an ectopically large optic stalk. Our results suggest that overactive Hh signaling, through overexpression of downstream transcriptional targets, impairs cell motility underlying optic fissure and stalk formation, via non-cell-autonomous and cell-autonomous mechanisms. More broadly, our cell motility and morphology analyses provide a new framework for studying other coloboma-causing mutations that disrupt optic fissure or stalk formation.