Hedgehog signaling activation in the development of squamous cell carcinoma and adenocarcinoma of esophagus

Hedgehog (Hh) signaling is frequently activated in human cancer, including esophageal cancer. Most esophageal cancers are diagnosed in the advanced stages, therefore, identifying the very alterations that drive esophageal carcinogenesis may help designing novel strategies to diagnose and treat the disease. Analysis of Hh signaling in precancerous lesions is a critical first step in determining the significance of this pathway for carcinogenesis. Here we report our data on Hh target gene expression in 174 human esophageal specimens [28 esophageal adenocarcinomas (EAC), 19 Barrett’s esophagus, 103 cases of esophageal squamous cell carcinoma (ESCC), and 24 of squamous dysplastic lesions], and in two rat models of esophageal cancer. We found that 96% of human EAC express Hh target genes. We showed that PTCH1 expression is the most reliable biomarker. In contrast to EAC, only 38% of ESCC express Hh target genes. We found activation of Hh signaling in precancerous lesions of ESCCs and EACs in different degrees (21% and 58% respectively). Expression of Hh target genes is frequently detected in severe squamous dysplasia/ carcinoma in situ (p=0.04) and Barrett’s esophagus (p=0.01). Unlike EAC, sonic hedgehog (Shh) expression was rare in ESCCs. Consistent with the human specimen data, we found a high percentage of Hh signaling activation in precancerous lesions in rat models. These data indicate that Hh signaling activation is an early molecular event in the development of esophageal cancer, particularly EAC.     

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012. © 2011 Wiley Periodicals, Inc.     

MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1

Dysexpression of microRNAs has been found in many tumors, including lung cancer. The hedgehog (Hh) signaling pathway plays an important role during normal development, and the abnormal regulation of its members has also been related to many tumors. However, little is known about the relationship between microRNA and the Hh pathway. In this paper, we report microRNA-212 (miR-212) playing a role in non-small cell lung cancer (NSCLC) and targeting PTCH1, a receptor of the Hh pathway. We found that miR-212 was up-regulated when cells were treated with 4ß-12-O-tetradecanoylphorbol-13-acetate (TPA). We ectopically expressed miR-212 in NSCLC cell lines to examine the influence of miR-212 overexpression. The results showed that overexpression of miR-212 in NSCLC cells promoted cell cycle progression and cell proliferation, migration, and invasion. The promoting effects of miR-212 on cell proliferation, migration, and invasion were partially reversed by the miR-212 inhibitor anti-miR-212. These results suggested that miR-212 might have tumor-promoting properties. Potential targets of miR-212 were predicted, and we showed tumor suppressor PTCH1 was a functional target of miR-212. PTCH1 may be responsible for the effect of miR-212 on cell proliferation. Altogether, our results indicated that miR-212 was involved in tumorigenesis, and the oncogenic activity of miR-212 in NSCLC cells was due, in part, to suppression of PTCH1.     

Pathways to chromothripsis

Chromothripsis is a recently recognized mode of genetic instability that generates chromosomes with strikingly large numbers of segmental re-arrangements. While the characterization of these derivative chromosomes has provided new insights into the processes by which cancer genomes can evolve, the underlying signaling events and molecular mechanisms remain unknown. In medulloblastomas, chromothripsis has been observed to occur in the context of mutational inactivation of p53 and activation of the canonical Hedgehog (Hh) pathway. Recent studies have illuminated mechanistic links between these 2 signaling pathways, including a novel PTCH1 homolog that is regulated by p53. Here, we integrate this new pathway into a hypothetical model for the catastrophic DNA breakage that appears to trigger profound chromosomal rearrangements.     

Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.     

Mutational Analysis of Hedgehog Signaling Pathway Genes in Human Malignant Mesothelioma

Background

The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown.

Methodology/Principal Findings

Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour.

Conclusions/Significance

We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133⁺ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.     

A molecular dynamics approach to identify an oxysterol-based hedgehog pathway inhibitor

BackgroundMultiple oxysterols (OHCs) have demonstrated the ability to act as agonists or antagonists of the hedgehog (Hh) signaling pathway, a developmental signaling pathway that has been implicated as a potential therapeutic target in a variety of human diseases. These OHCs are known to modulate Hh signaling through direct binding interactions with the N-terminal cysteine rich domain (CRD) of Smoothened, a key regulator of Hh signal transduction.MethodsHomology modeling, molecular dynamics simulations, and MM/GBSA energy calculations were utilized to explore binding interactions between the OHC scaffold and the human Smoothened CRD. Follow-up cellular assays explored the in vitro activity of potential Hh pathway modulators.ResultsStructural features that govern key molecular interactions between the Smoothened CRD and the OHC scaffold were identified. Orientation of the iso-octyl side chain as well as the overall entropy of the OHC-CRD complex are important for determining activity against the Hh pathway. OHC 9, which was previously thought to be inactive because it was not an Hh agonist, was identified as an inhibitor of Hh signal transmission.ConclusionsCalculated MM/GBSA binding energies for OHCs in complex with the CRD of Smoothened correlate well with in vitro Hh modulatory activity. Compounds with high affinity stabilize Smoothened and are antagonists, whereas compounds with reduced affinity allow a conformational change in Smoothened that results in pathway activation.General significanceComputational modeling and molecular dynamics simulations can be used to predict whether a small molecule that binds the Smoothened CRD will be an agonist or antagonist of the pathway.     

In vivo inhibition of endogenous brain tumors through systemic interference of Hedgehog signaling in mice

The full spectrum of developmental potential includes normal as well as abnormal and disease states. We therefore subscribe to the idea that tumors derive from the operation of paradevelopmental programs that yield consistent and recognizable morphologies. Work in frogs and mice shows that Hedgehog (Hh)-Gli signaling controls stem cell lineages and that its deregulation leads to tumor formation. Moreover, human tumor cells require sustained Hh-Gli signaling for proliferation as cyclopamine, an alkaloid of the lily Veratrum californicum that blocks the Hh pathway, inhibits the growth of different tumor cells in vitro as well as in subcutaneous xenografts. However, the evidence that systemic treatment is an effective anti-cancer therapy is missing. Here we have used Ptc1(+/-); p53(-/-) mice which develop medulloblastoma to test the ability of cyclopamine to inhibit endogenous tumor growth in vivo after tumor initiation through intraperitoneal delivery, which avoids the brain damage associated with direct injection. We find that systemic cyclopamine administration improves the health of Ptc1(+/-);p53(-/-) animals. Analyses of the cerebella of cyclopamine-treated animals show a severe reduction in tumor size and a large decrease in the number of Ptc1-expressing cells, as a readout of cells with an active Hu-Gli pathway, as well as an impairment of their proliferative capacity, always in comparison with vehicle treated mice. Our data demonstrate that systemic treatment with cyclopamine inhibits tumor growth in the brain supporting its therapeutical value for human HH-dependent tumors. They also demonstrate that even the complete loss of the well-known tumor suppressor p53 does not render the tumor independent of Hh pathway function.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.     

Leptin Promotes the Myofibroblastic Phenotype in Hepatic Stellate Cells by Activating the Hedgehog Pathway

Trans-differentiation of quiescent hepatic stellate cells (Q-HSCs), which exhibit epithelial and adipocytic features, into myofibroblastic-HSC (MF-HSCs) is a key event in liver fibrosis. Culture models demonstrated that Hedgehog (Hh) pathway activation is required for transition of epithelioid/adipocytic Q-HSCs into MF-HSCs. Hh signaling inhibits adiposity and promotes epithelial-to-mesenchymal transitions (EMTs). Leptin (anti-adipogenic, pro-EMT factor) promotes HSC trans-differentiation and liver fibrosis, suggesting that the pathways may interact to modulate cell fate. This study aimed to determine whether leptin activates Hh signaling and whether this is required for the fibrogenic effects of leptin. Cultures of primary HSCs from lean and fa/fa rats with an inherited ObRb defect were examined. Inhibitors of PI3K/Akt, JAK/STAT, and Hh signaling were used to delineate how ObRb activation influenced Hh signaling and HSC trans-differentiation. Fibrogenesis was compared in wild type and db/db mice (impaired ObRb function) to assess the profibrotic role of leptin. The results demonstrate that leptin-ObR interactions activate Hh signaling with the latter necessary to promote trans-differentiation. Leptin-related increases in Hh signaling required ObR induction of PI3K/Akt, which was sufficient for leptin to repress the epithelioid/adipocytic program. Leptin-mediated induction of JAK/STAT was required for mesenchymal gene expression. Leptin-ObRb interactions were not necessary for HSC trans-differentiation to occur in vitro or in vivo but are important because liver fibrogenesis was attenuated in db/db mice. These findings reveal that leptin activates Hh signaling to alter gene expression programs that control cell fate and have important implications for liver fibrosis and other leptin-regulated processes involving EMTs, including development, obesity, and cancer metastasis.