Smoothened Research Articles

The Loss of SMAD4/DPC4 Expression Associated with a Strongly Activated Hedgehog Signaling Pathway Predicts Poor Prognosis in Resected Pancreatic Cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) progression is mediated by mutations in driver genes and a complex stroma that is mainly dependent on the Sonic hedgehog (Shh) signaling pathway. However, the association between driver genes and Shh-pathway proteins and their potential prognostic significance remain unclear.Methods: We analyzed protein expressions of the KRAS, TP53, SMAD4, and CDKN2A/P16 driver genes and the Shh-pathway molecules, including Shh, glioma-associated oncogene (Gli) 1, Gli2, and smoothened (SMO) by immunohistochemistry using tissue microarrays in 237 patients with resectable PDAC and statistically determined their prognostic significance.Results: SMAD4lost mutation was associated with shorter survival outcomes [overall survival (OS): Hazard ratio (HR) 1.887, p < 0.001]; recurrence-free survival (RFS): HR 1.886, p < 0.001) and abnormal p53 immunolabeling was associated with poor OS (HR 1.436, p = 0.011) in patients with PDAC. The mutational status of p16 had no effect on patient survival. High levels of SMO and Gli1 expression were associated with poor survival outcomes in both univariate and multivariate analyses. Pearson's χ2 test showed a medium correlation between the SMAD4lost mutation and Shh (R = 0.343) and Gli1 (R = 0.505) expression levels (p < 0.001). Patients with the SMAD4lost mutation and high levels of Shh and Gli1 expression showed the poorest survival outcomes (RFS: HR 2.976; OS: HR 3.598; p < 0.001 for both) compared with other patients in the study.Conclusion: Loss of SMAD4 associated with a strongly activated Shh pathway resulted in poor survival outcomes in patients with resected PDAC.

Cholesterol Interaction Sites on the Transmembrane Domain of the Hedgehog Signal Transducer and Class F G Protein-Coupled Receptor Smoothened

SummaryTransduction of Hedgehog signals across the plasma membrane is facilitated by the class F G-protein-coupled-receptor (GPCR) Smoothened (SMO). Recent studies suggest that SMO is modulated via interactions of its transmembrane (TM) domain with cholesterol. We apply molecular dynamics simulations of SMO embedded in cholesterol containing lipid bilayers, revealing a direct interaction of cholesterol with the TM domain at regions distinct from those observed in class A GPCRs. In particular the extracellular tips of helices TM2 and TM3 form a well-defined cholesterol interaction site. Potential of mean force calculations yield a free energy landscape for cholesterol binding. Alongside analysis of equilibrium cholesterol occupancy, this reveals the existence of a dynamic “greasy patch” interaction with the TM domain of SMO, which may be compared with previously identified lipid interaction sites on other membrane proteins. These predictions provide molecular-level insights into cholesterol interactions with a class F GPCR, suggesting potential druggable sites.

Targeting the Oncoprotein Smoothened by Small Molecules: Focus on Novel Acylguanidine Derivatives as Potent Smoothened Inhibitors.

Hedgehog-GLI (HH) signaling was originally identified as a critical morphogenetic pathway in embryonic development. Since its discovery, a multitude of studies have reported that HH signaling also plays key roles in a variety of cancer types and in maintaining tumor-initiating cells. Smoothened (SMO) is the main transducer of HH signaling, and in the last few years, it has emerged as a promising therapeutic target for anticancer therapy. Although vismodegib and sonidegib have demonstrated effectiveness for the treatment of basal cell carcinoma (BCC), their clinical use has been hampered by severe side effects, low selectivity against cancer stem cells, and the onset of mutation-driven drug resistance. Moreover, SMO antagonists are not effective in cancers where HH activation is due to mutations of pathway components downstream of SMO, or in the case of noncanonical, SMO-independent activation of the GLI transcription factors, the final mediators of HH signaling. Here, we review the current and rapidly expanding field of SMO small-molecule inhibitors in experimental and clinical settings, focusing on a class of acylguanidine derivatives. We also discuss various aspects of SMO, including mechanisms of resistance to SMO antagonists.

Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability.

Spinocerebellar ataxia type 11 (SCA11) is a rare, dominantly inherited human ataxia characterized by atrophy of Purkinje neurons in the cerebellum. SCA11 is caused by mutations in the gene encoding the Serine/Threonine kinase Tau tubulin kinase 2 (TTBK2) that result in premature truncations of the protein. We previously showed that TTBK2 is a key regulator of the assembly of primary cilia in vivo. However, the mechanisms by which the SCA11-associated mutations disrupt TTBK2 function, and whether they interfere with ciliogenesis were unknown. In this work, we present evidence that SCA11-associated mutations are dominant negative alleles and that the resulting truncated protein (TTBK2SCA11) interferes with the function of full length TTBK2 in mediating ciliogenesis. A Ttbk2 allelic series revealed that upon partial reduction of full length TTBK2 function, TTBK2SCA11 can interfere with the activity of the residual wild-type protein to decrease cilia number and interrupt cilia-dependent Sonic hedgehog (SHH) signaling. Our studies have also revealed new functions for TTBK2 after cilia initiation in the control of cilia length, trafficking of a subset of SHH pathway components, including Smoothened (SMO), and cilia stability. These studies provide a molecular foundation to understand the cellular and molecular pathogenesis of human SCA11, and help account for the link between ciliary dysfunction and neurodegenerative diseases.

Sonic Hedgehog Signaling Pathway Mediates Proliferation and Migration of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via MAPK/ERK Signaling Pathway.

Fibroblast-like synoviocytes (FLSs) are the major effector cells that lead to rheumatoid arthritis (RA) synovitis and joint destruction. Our previous studies showed that Sonic Hedgehog (SHH) signaling pathway is involved in aberrant activation of RA-FLSs and inhibition of SHH pathway decreases proliferation and migration of RA-FLSs. The objective of this study was to investigate if the SHH pathway mediates proliferation and migration of RA-FLSs via the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. SHH signaling was studied by using SHH agonist (Purmorphamine) and antagonist (Cyclopamine) targeting the Smoothened (SMO) in FLSs. U0126-EtOH was used to inhibit the MAPK/ERK signaling pathway. The phosphorylation of ERK 1/2 (p-ERKl/2) was examined by western blot. Cell viability was detected using cell proliferation and cytotoxicity kit-8 (CCK8), and cell cycle distribution and proliferating cells were evaluated by the flow cytometry. Cell migration was examined by Transwell assay. Results showed that, compared with the control group, Purmorphamine increased the levels of p-ERK1/2 in concentration-and time-dependent manners (P < 0.01). Co-treated with Purmorphamine and U0126-EtOH or Cyclopamine both decreased the levels of p-ERK1/2 (P < 0.05). RA-FLSs treated with Purmorphamine resulted in alteration of cell cycle distribution, increasing of proliferating cells, cell viability, and migration cells compared to controls (P < 0.01). However, the above phenomenon can be abolished by U0126-EtOH (P < 0.05). The findings suggest that SHH signaling pathway mediates proliferation and migration of RA-FLSs via MAPK/ERK pathway and may contribute to progression of RA. Targeting SHH signaling may have a therapeutic potential in patients with RA.

Overcoming the emerging drug resistance of smoothened: an overview of small-molecule SMO antagonists with antiresistance activity.

Hedgehog (HH) signaling pathway plays vital roles in controlling embryonic cell fate and homeostatic, and becomes dormant in mature individuals, aberrant activation of HH signaling pathway is involved in a number of human cancers. Smoothened (SMO), a vital transducer of HH signaling pathway, attracts significant attentions in HH signaling pathway-related cancer therapy. The approval of SMO antagonists vismodegib proves that SMO is a promising therapeutic target, and a number of SMO antagonists are reported since then. However, high incidence of tumor recurrence with the clinical application of vismodegib urges exploring of novel drugs with antiresistance profiles. This review provides an overview of SMO mutations reported in the literature, crystal structures of SMO, as well as reported antagonists with antiresistance profiles.

Glasdegib in Addition to Intensive or Non-Intensive Chemotherapy in Patients with Acute Myeloid Leukemia: Safety Analysis of Glasdegib ‘On Target’ Adverse Events

Glasdegib in Addition to Intensive or Non-Intensive Chemotherapy in Patients with Acute Myeloid Leukemia: Safety Analysis of Glasdegib ‘On Target’ Adverse Events

Population Pharmacokinetic/Pharmacodynamic Evaluation of the Relationship between Glasdegib Treatment/ Exposure and Overall Survival in AML Patients

Introduction: Glasdegib is an inhibitor of the Hedgehog signaling pathway through binding to its target, Smoothened (SMO). B1371003 was a Phase 1b/2 study evaluating glasdegib in combination with chemotherapy in patients with acute myeloid leukemia (AML). In one part of the Phase 2 portion of B1371003, patients who were not candidates for more standard chemotherapy were randomized 2:1 to receive low-dose cytarabine (LDAC) with glasdegib or LDAC alone. The primary endpoint was overall survival (OS). A Cox regression analysis for overall survival (OS) was conducted, and median OS for AML patients treated with glasdegib plus LDAC was 8.3 months compared to 4.3 months with LDAC alone (hazard ratio [HR] 0.463). This analysis aimed to assess the potential relationship between glasdegib exposure and OS (exposure-response [ER]) and to evaluate the effect on OS of moderators/covariates, including treatment-response (TR) for glasdegib + LDAC vs. LDAC alone.Methods: The analyses included data from the Phase 2 non-intensive AML patients. Glasdegib was administered continuously at 100 mg once daily (QD) orally in 28-day cycles. LDAC was administered subcutaneously at 20 mg twice daily on Days 1-10 of each 28-day cycle. Glasdegib exposure from the glasdegib + LDAC treated arm was estimated utilizing a population pharmacokinetics (PK) model. Survival was assessed at every visit then every month for the first 2 months after discontinuation from study treatment and thereafter every 2 months until death or termination of the study.Parametric time-to-event survival models were used to assess the relationship between OS, treatment arm, and glasdegib exposure. Baseline characteristics (such as age, sex, body weight, and white blood cell count) and other prognostic factors (such as disease status and treatment arm) were evaluated as potential predictors of OS by forward inclusion and backward elimination. R statistical programming (version 3.2.2) and NONMEM software (version 7.3.0) were used for the analysis.Results: Glasdegib PK was adequately characterized by a 2-compartment model with first order absorption kinetics. Demographic and baseline characteristics such as age, sex, body weight, and renal and hepatic function were either non-significant or did not have a clinically relevant impact on glasdegib PK. The TR analysis included all Phase 2 AML patients with available OS data who were randomized to receive LDAC with (n=78) or without (n=38) glasdegib. Glasdegib-treated patients with at least one PK observation (n=75) were evaluated in the ER analysis. Exponential, Weibull, and log-logistic parametric distributions were explored, and the exponential distribution was selected as the model that best described the observed OS data (Figure). In the TR analysis, addition of glasdegib to LDAC resulted in longer OS with HR of 0.42 (95% confidence interval: 0.28-0.66). No ER relationship was identified in the analysis between glasdegib plasma exposures at 100 mg QD and OS. No other baseline characteristics were identified as potential predictors of survival in either the TR or ER analyses.Conclusions: Addition of glasdegib treatment to LDAC chemotherapy in AML patients who are not suitable for intensive chemotherapy significantly prolonged OS compared to LDAC chemotherapy alone. Across the range of plasma exposures for glasdegib 100-mg QD treatment, no ER relationship was identified. No other covariates were found to be predictors of survival, therefore supporting the broad use of glasdegib as an addition to chemotherapy in AML patients. [Display omitted] DisclosuresLin:Pfizer: Employment. Shaik:Pfizer Inc: Employment, Equity Ownership. Chan:Pfizer: Employment, Equity Ownership. Cortes:Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Arog: Research Funding. Ruiz-Garcia:Pfizer: Employment, Equity Ownership.

Design of Hedgehog pathway inhibitors for cancer treatment

Hedgehog (Hh) signaling is involved in the initiation and progression of various cancers and is essential for embryonic and postnatal development. This pathway remains in the quiescent state in adult tissues but gets activated upon inflammation and injuries. Inhibition of Hh signaling pathway using natural and synthetic compounds has provided an attractive approach for treating cancer and inflammatory diseases. While the majority of Hh pathway inhibitors target the transmembrane protein Smoothened (SMO), some small molecules that target the signaling cascade downstream of SMO are of particular interest. Substantial efforts are being made to develop new molecules targeting various components of the Hh signaling pathway. Here, we have discussed the discovery of small molecules as Hh inhibitors from the diverse chemical background. Also, some of the recently identified natural products have been included as a separate section. Extensive structure-activity relationship (SAR) of each chemical class is the focus of this review. Also, clinically advanced molecules are discussed from the last 5 to 7 years. Nanomedicine-based delivery approaches for Hh pathway inhibitors are also discussed concisely.

Relationship between spinal Sonic Hedgehog signaling pathway and inflammatory responses in rats with neuropathic pain

Objective To evaluate the relationship between spinal Sonic hedgehog(Shh) signaling pathway and inflammatory responses in rats with neuropathic pain(NP). Methods Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 250-300 g, were divided into 4 groups(n=12 each) using a random number table method: sham operation group(group S), group NP, dimethyl sulfoxide(DMSO) group(group D) and specific Shh signaling pathway inhibitor cyclopamine group(group CP). Spared nerve injury was produced by exposing the sciatic nerve and branches followed by ligation and transection of tibial and common fibular nerves in anesthetized rats. The mechanical paw withdrawal threshold(MWT) was measured at 1 day before establishing the model and 1 and 7 days after establishing the model., The animals were sacrificed after measurement of pain threshold at 7 days after operation, and the lumbar segment L4-6 of the spinal cord was obtained for determination of the expression of Shh, Patched homolog(Ptch), Smoothened(Smo) and zinc finger-containing transcription factors 1(Gli1)(by Western blot) and contents of tumor necrosis factor-alpha(TNF-α) and interleukin-1beta(IL-1β)(by enzyme-linked immunosorbent assay). Results Compared with group S, the MWT was significantly decreased, the expression of Shh, Patched, Smo and Gli1 was up-regulated, and the contents of TNF-α and IL-1β were increased in NP, D and CP groups(P<0.05). Compared with group NP, the MWT was significantly increased, the expression of Shh, Patched, Smo and Gli1 was down-regulated, and the contents of TNF-α and IL-1β were decreased in group CP(P<0.05), and no significant change was found in the parameters mentioned above in group D(P<0.05). Conclusion The mechanism by which Shh signaling pathway is involved in the development and maintenance of inflammatory responses is related to inhibiting inflammatory responses of the spinal cord in rats with NP. Key words: Neuralgia; Hedgehog proteins; Signal transduction; Inflammation; Spinal cord

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

Cells to Surgery Quiz: December 2018

Cells to Surgery Quiz: December 2018

Tumidulin, a Lichen Secondary Metabolite, Decreases the Stemness Potential of Colorectal Cancer Cells.

Lichens produce various unique chemicals that are used in the pharmaceutical industry. To screen for novel lichen secondary metabolites that inhibit the stemness potential of colorectal cancer cells, we tested acetone extracts of 11 lichen samples collected in Chile. Tumidulin, isolated from Niebla sp., reduced spheroid formation in CSC221, DLD1, and HT29 cells. In addition, mRNA expressions and protein levels of cancer stem markers aldehyde dehydrogenase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, and Musashi-1 were reduced after tumidulin treatment. Tumidulin decreased the transcriptional activity of the glioma-associated oncogene homolog zinc finger protein (Gli) promoter in reporter assays, and western blotting confirmed decreased Gli1, Gli2, and Smoothened (SMO) protein levels. Moreover, the tumidulin activity was not observed in the presence of Gli and SMO inhibitors. Together, these results demonstrate for the first time that tumidulin is a potent inhibitor of colorectal cancer cell stemness.

The hierarchical micro-/nanotextured topographies promote the proliferation and angiogenesis-related genes expression in human umbilical vein endothelial cells by initiation of Hedgehog-Gli1 signaling

The hierarchical microtextured/nanotextured topographies have been recognized to have better tissue integration properties, but the underlying mechanisms are only partially understood. Hedgehog signaling plays a pivotal role in developmental and homeostatic angiogenesis. We suppose that the Hedgehog-Gli1 signaling may play a significant role in the response of endothelial cells to microtextured/nanotextured topographies (MNTs). To confirm this hypothesis, we produced the MNTs decorated with TiO2 nanotubes of two different diameters (25 and 70 nm), and the proliferation, apoptosis, angiogenesis-related genes expression and Hedgehog signaling activity of human umbilical vein endothelial cells (HUVECs) grown onto these MNTs were measured. Our results showed that the MNTs induced significantly high expression of Sonic Hedgehog (SHH), Smoothened (SMO) and GLI1 in the HUVECs as well as high activation of Hedgehog-Gli1 signaling, compared to the smooth surface. The HUVECs grown on the MNTs showed significantly high levels of adhesion, proliferation and expression of angiogenesis-related genes, including angiopoietin-1 (ANG-1), vascular endothelial growth factor (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (ENOS); these enhancements were attenuated by siRNA-mediated depletion of SMO, which indicated a significant role of Hedgehog-Gli1 signaling in mediating the enhanced effect of the MNTs on the angiogenic potential of HUVECs. This study may contribute to the modification of biomaterial surfaces for better tissue integration and clinical performance.

Sonic hedgehog regulation of cavernous nerve regeneration and neurite formation in aged pelvic plexus

IntroductionErectile dysfunction (ED) is a significant health concern that greatly impacts quality of life, and is common in men as they age, impacting 52% of men between the ages of 40 and 70. A significant underlying cause of ED development is injury to the cavernous nerve (CN), a peripheral nerve that innervates the penis. CN injury also occurs in up to 82% of prostatectomy patients. We recently showed that Sonic hedgehog (SHH) protein delivered by peptide amphiphile (PA) nanofiber hydrogel to the CN and penis of a prostatectomy model of CN injury, is neuroprotective, accelerates CN regeneration, improves erectile function ~60%, preserves penile smooth muscle 56% and suppresses collagen deposition 30%. This regenerative potential is substantial in an adult prostatectomy model (P120). However prostatectomy patients are typically older (61.5 ± 9.6 years) and our models should mimic patient conditions more effectively when considering translation. In this study we examine regenerative potential in an aged prostatectomy model (P200–329). MethodsThe caudal portion of the pelvic ganglia (MPG) and CN were dissected from adult (n = 11), and aged (n = 13) Sprague Dawley rats, and were grown in organ culture 3 days. Uninjured and 2 day CN crushed MPG/CN were exposed to Affi-Gel beads containing SHH protein, PBS (control), or 5e1 SHH inhibitor. Neurites were quantified by counting the number of growth cones normalized by tissue perimeter (mm) and immunohistochemistry for SHH, patched1 (PTCH1), smoothened (SMO), GLI1-3, and GAP43 were performed. ResultsSHH treatment increased neurites 3.5-fold, in uninjured adult, and 5.7-fold in aged rats. Two days after CN crush, SHH treatment increased neurites 1.8-fold in adult rats and 2.5-fold in aged rats. SHH inhibition inhibited neurite formation in uninjured MPG/CN but not in 2 day CN crushed MPG/CN. PTCH1 and SMO (SHH receptors), and SHH transcriptional activators/repressors, GLI1-3, were abundant in aged MPG/CN with unaltered localization. ROCK1 was induced with SHH treatment. ConclusionsReintroduction of SHH protein in an aged prostatectomy model is even more effective in promoting neurite formation/CN regeneration than in the adult. The first 48 h after CN injury are a critical window when growth factors are released, that impact later neurite formation. These studies are significant because most prostatectomy patients are not young and healthy, as with adult rats, so the aged prostatectomy model will more accurately simulate ED patient response. Understanding how neurite formation changes with age is critical for clinical translation of SHH PA to prostatectomy patients.

Genetics of anophthalmia and microphthalmia. Part2: Syndromes associated with anophthalmia-microphthalmia.

As new genes for A/M are identified in the genomic era, the number of syndromes associated with A/M has greatly expanded. In this review, we provide a brief synopsis of the clinical presentation and molecular genetic etiology of previously characterized pathways involved in A/M, including the Sex-determining region Y-box 2 (SOX2), Orthodenticle Homeobox 2 (OTX2) and Paired box protein-6 (PAX6) genes, and the Stimulated by retinoic acid gene 6 homolog (STRA6), Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3), and RA Receptor Beta (RARβ) genes that are involved in retinoic acid synthesis. Less common genetic causes of A/M, including genes involved in BMP signaling [Bone Morphogenetic Protein 4 (BMP4), Bone Morphogenetic Protein 7 (BMP7) and SPARC-related modular calcium-binding protein 1 (SMOC1)], genes involved in the mitochondrial respiratory chain complex [Holocytochrome c-type synthase (HCCS), Cytochrome C Oxidase Subunit 7B (COX7B), and NADH:Ubiquinone Oxidoreductase subunit B11 (NDUFB11)], the BCL-6 corepressor gene (BCOR), Yes-Associated Protein 1 (YAP1) and Transcription Factor AP-2 Alpha (TFAP2α), are more briefly discussed. We also review several recently described genes and pathways associated with A/M, including Smoothened (SMO) that is involved in Sonic hedgehog (SHH) signaling, Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) and Solute carrier family 25 member 24 (SLC25A24), emphasizing phenotype-genotype correlations and shared pathways where relevant.

LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212

Activation of hepatic stellate cells (HSCs), a pivotal event in liver fibrosis, is considered as an epithelial–mesenchymal transition (EMT) process. Deregulation of long noncoding RNAs (lncRNAs) has been reported to be involved in a series of human diseases. LncRNA-maternally expressed gene 3 (MEG3) functions as a tumor suppressor in cancers and has been shown to play a vital role in EMT process. However, the biological role of MEG3 in liver fibrosis is largely unknown. In this study, MEG3 was reduced in vivo and in vitro during liver fibrosis. Restoring of MEG3 expression led to the suppression of liver fibrosis, with a reduction in α-SMA and type I collagen. Notably, MEG3 overexpression inhibited HSC activation through EMT, associated with an increase in epithelial markers and a reduction in mesenchymal markers. Further studies showed that Hedgehog (Hh) pathway-mediated EMT process was involved in the effects of MEG3 on HSC activation. Smoothened (SMO) is a member of Hh pathway. Using bioinformatic analysis, an interaction between MEG3 and SMO protein was predicted. This interaction was confirmed by the results of RNA immunoprecipitation and deletion-mapping analysis. Furthermore, MEG3 was confirmed as a target of microRNA-212 (miR-212). miR-212 was partly responsible for the effects of MEG3 on EMT process. Interestingly, MEG3 was also reduced in chronic hepatitis B (CHB) patients with liver fibrosis when compared with healthy controls. MEG3 negatively correlated with fibrosis stage in CHB patients. In conclusion, we demonstrate that MEG3 inhibits Hh-mediated EMT process in liver fibrosis via SMO protein and miR-212.

Dexamethasone and Fludrocortisone Inhibit Hedgehog Signaling in Embryonic Cells.

The hedgehog (Hh) pathway plays a central role in the development and repair of our bodies. Therefore, dysregulation of the Hh pathway is responsible for many developmental diseases and cancers. Basal cell carcinoma and medulloblastoma have well-established links to the Hh pathway, as well as many other cancers with Hh-dysregulated subtypes. A smoothened (SMO) receptor plays a central role in regulating the Hh signaling in the cells. However, the complexities of the receptor structural mechanism of action and other pathway members make it difficult to find Hh pathway inhibitors efficient in a wide range. Recent crystal structure of SMO with cholesterol indicates that it may be a natural ligand for SMO activation. Structural similarity of fluorinated corticosterone derivatives to cholesterol motivated us to study the effect of dexamethasone, fludrocortisone, and corticosterone on the Hh pathway activity. We identified an inhibitory effect of these three drugs on the Hh pathway using a functional assay in NIH3T3 glioma response element cells. Studies using BODIPY-cyclopamine and 20(S)-hydroxy cholesterol [20(S)-OHC] as competitors for the transmembrane (TM) and extracellular cysteine-rich domain (CRD) binding sites showed a non-competitive effect and suggested an alternative or allosteric binding site for the three drugs. Furthermore, the three steroids showed an additive effect on Hh pathway inhibition when tested in combination with cyclopamine. Our study reports the antagonistic effect of dexamethasone, fludrocortisone, and corticosterone on the Hh pathway using functional assay and confirmed that they do not bind to the CRD or adjacent TM binding cavities of SMO. The study also suggests that dexamethasone could be additionally beneficial as the adjuvant therapy for cancer patients with an established link to the dysregulated Hh pathway.

Different Response of Ptch Mutant and Ptch Wildtype Rhabdomyosarcoma Toward SMO and PI3K Inhibitors

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.

Smoothened stabilizes and protects TRAF6 from degradation: A novel non-canonical role of smoothened with implications in lymphoma biology

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a role in many biological processes and its activity is relevant in diffuse large B cell lymphoma (DLBCL) biology. Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 and/or enhance TRAF6 function remain largely unclear. We found that TRAF6 amplifies pAKT signaling in DLBCL. Moreover, TRAF6 activation and stabilization of its ubiquitination profile are facilitated by smoothened (SMO), signal transducer of canonical Hedgehog signaling. Here, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels, and that the SMO/TRAF6 axis contributes to doxorubicin resistance in DLBCL. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes and protects TRAF6 from degradation, an effect mediated by ubiquitin-specific protease-8. Moreover, this functional link between SMO and TRAF6 is reflected in DLBCL patients where high expression of both molecules correlates with poor prognosis. In summary, our study reveals a novel cell survival mechanism in which SMO stabilizes and protects TRAF6 from degradation. The axis SMO/TRAF6/AKT is highly relevant in the biology of DLBCL and is involved in doxorubicin resistance.