gdc-0449 and cyclopamine

Basal cell carcinomas (BCCs) are the most frequent human cancer. Over 90% of all BCCs have a mutation in PTCH1 or smoothened, two conducting proteins of the Hedgehog pathway. They rarely progress deeply and metastasize; however, if they do, these advanced basal cell carcinoma become amenable to treatment by inhibiting the Hedgehog and the P13K-mTOR pathways. Such innovative drugs include vismodegib, cyclopamine, itraconazole, everolimus and a few other agents that are in early clinical development.

Topics: Anilides; Carcinoma, Basal Cell; Drug Resistance, Neoplasm; Everolimus; Hedgehog Proteins; Humans; Itraconazole; Molecular Targeted Therapy; Neoplasm Staging; Phosphatidylinositol 3-Kinases; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Skin Neoplasms; Smoothened Receptor; TOR Serine-Threonine Kinases; Veratrum Alkaloids

Since its discovery by C. Nüsslein-Volhard and E. F. Wieschaus, hedgehog (hh) signaling has come a long way. Today it is regarded as a key regulator in embryogenesis where it governs processes like cell proliferation, differentiation, and tissue patterning. Furthermore, in adults it is involved in the maintenance of stem cells, and in tissue repair and regeneration. But hh signaling has a second-much darker-face: it plays an important role in several types of human cancers where it promotes growth and enables proliferation of tumor stem cells. The etiology of medulloblastoma and basal cell carcinoma is tightly linked to aberrant hh activity, but also cancers of the prostate, the pancreas, the colon, the breasts, rhabdomyosarcoma, and leukemia, are dependent on irregular hh activity. Recent clinical studies have shown that hh signaling can be the basis of an important new class of therapeutic agents with far-reaching implications in oncology. Thus, modulation of hh signaling by means of small molecules has emerged as a valuable tool in combating these hh-dependent cancers. Cyclopamine, a unique natural product with a fascinating history, was the first identified inhibitor of hh signaling and its story is closely linked to the progress in the whole field. In this review we will trace the story of cyclopamine, give an overview on the biological modes of hh signaling both in untransformed and malignant cells, and finally present potent modulators of the hh pathway-many of them already in clinical studies. For more than 30 years now the knowledge on hh signaling has grown steadily-an end to this development is far from being conceivable.

Topics: Antineoplastic Agents; Hedgehog Proteins; Humans; Neoplasms; Signal Transduction; Veratrum Alkaloids

Sonic hedgehog (Shh) is the most widely characterized of the three vertebrate Hedgehog homologs, and is essential for proper embryonic development. Shh binds to its receptor, Patched (Ptch1), resulting in the de-repression of Smoothened (Smo). This leads to the activation of Gli2, which regulates the transcription of target genes that include Gli1 and Ptch1. Several synthetic and naturally occurring small-molecule modulators of Smo have been discovered. Shh-signaling antagonists that bind to Smo include cyclopamine, SANT1, and Cur-61414. Shh signaling agonists that bind to Smo include the synthetic small molecules purmorphamine and SAG. Small molecules that inhibit Shh signaling downstream of Smo, GANT58 and GANT61 have also been reported. Robotnikinin inhibits the Shh pathway by directly targeting Shh. Although progress has been made in understanding and modulating Shh signaling, fundamental aspects of Shh signal transduction remain obscure, including the mechanism(s) whereby Ptch1 regulates Smo activity. Small-molecule modulators of Shh signaling provide a means to regulate the activity of a pathway implicated in medulloblastoma, basal cell carcinoma (BCC), pancreatic cancer, prostate cancer and developmental disorders. Several Shh inhibitors have not succeeded in the clinic for unknown reasons, but clinical trials in BCC and pancreatic cancer with the promising Smo antagonists GDC-0449 and IPI-926 are currently underway.

Topics: Anilides; Animals; Dioxoles; Hedgehog Proteins; Humans; Models, Biological; Molecular Structure; Piperazines; Pyrazoles; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Thiophenes; Veratrum Alkaloids

Topics: Animals; Boron Compounds; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Discovery; Drug Evaluation, Preclinical; Fluorescent Dyes; Humans; Mice; Molecular Docking Simulation; Molecular Structure; NIH 3T3 Cells; Receptors, G-Protein-Coupled; Structure-Activity Relationship; Veratrum Alkaloids

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Topics: Anilides; Animals; Cell Line, Tumor; Cerebellar Neoplasms; Disease Models, Animal; Hedgehog Proteins; HEK293 Cells; Humans; Male; Medulloblastoma; Mice; Mice, Transgenic; Pyridines; Random Allocation; Small Molecule Libraries; SOXB1 Transcription Factors; Transfection; TRPC Cation Channels; Tumor Suppressor Protein p53; Veratrum Alkaloids; Wnt Proteins; Wnt Signaling Pathway

Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway.

Topics: Alkaptonuria; Anilides; Cells, Cultured; Chondrocytes; Cilia; Dose-Response Relationship, Drug; Hedgehog Proteins; Homogentisic Acid; Humans; Hyperpigmentation; Pyridines; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids; Zinc Finger Protein GLI1

Hedgehog signaling is a pivotal developmental pathway that comprises hedgehog, PTCH1, SMO, and GLI proteins. Mutations in PTCH1 are responsible for Gorlin syndrome, which is characterized by developmental defects and tumorigenicity. Although the hedgehog pathway has been investigated extensively in Drosophila and mice, its functional roles have not yet been determined in human cells. In order to elucidate the mechanism by which transduction of the hedgehog signal is regulated in human tissues, we employed human fibroblasts derived from three Gorlin syndrome patients and normal controls. We investigated GLI1 transcription, downstream of hedgehog signaling, to assess native signal transduction, and then treated fibroblasts with a recombinant human hedgehog protein with or without serum deprivation. We also examined the transcriptional levels of hedgehog-related genes under these conditions. The expression of GLI1 mRNA was significantly higher in Gorlin syndrome-derived fibroblasts than in control cells. Hedgehog stimulation and nutritional deprivation synergistically enhanced GLI1 transcription levels, and this was blocked more efficiently by vismodegib, a SMO inhibitor, than by the natural compound, cyclopamine. Messenger RNA profiling revealed the increased expression of Wnt signaling and morphogenetic molecules in these fibroblasts. These results indicated that the hedgehog stimulation and nutritional deprivation synergistically activated the hedgehog signaling pathway in Gorlin syndrome fibroblasts, and this was associated with increments in the transcription levels of hedgehog-related genes such as those involved in Wnt signaling. These fibroblasts may become a significant tool for predicting the efficacies of hedgehog molecular-targeted therapies such as vismodegib.

Topics: Anilides; Animals; Basal Cell Nevus Syndrome; Cells, Cultured; Cilia; Culture Media, Serum-Free; Fibroblasts; Hedgehog Proteins; Humans; Ligands; Mice; Pyridines; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Smoothened Receptor; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1

Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (koff) rates were determined by kinetic binding studies using [(3)H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [(3)H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.

Topics: Anilides; Binding, Competitive; Cell Line; Drug Resistance, Neoplasm; Humans; Kinetics; Mutation; Pyridines; Pyrroles; Radioligand Assay; Receptors, G-Protein-Coupled; Smoothened Receptor; Veratrum Alkaloids

Glioblastoma multiforme (GBM) is composed of heterogeneous and genetically different cells, which are highly invasive and motile. The standard chemotherapeutic agent, temozolomide, affects GBM cell proliferation but is generally unable to prevent tumor recurrence. Hedgehog pathway activation has been reported to be relevant in GBM and different pharmacological pathway modulators have been identified. We report that by growing a commercially available recurrent GBM cell line (DBTRG-05MG) without serum and in the presence of defined growth factors; we obtained a less differentiated cell population, growing in suspension as neurospheres, in which the Hedgehog pathway is activated. Furthermore, the expression profile of Hedgehog pathway components found in DBTRG-05MG neurospheres is similar to primary stem-like cells derived from recurrent GBM patients. We report the effect of our novel specific Smoothened receptor antagonist (SEN450) on neurosphere growing cells and compared its effect to that of well known benchmark compounds. Finally, we showed that SEN450 is both antiproliferative on its own and further reduces tumor volume after temozolomide pretreatment in a mouse xenograft model using DBTRG-05MG neurosphere cells. Altogether our data indicate that the Hedgehog pathway is not irreversibly switched off in adherent cells but can be reactivated when exposed to well-defined culture conditions, thus restoring the condition observed in primary tumor-derived material, and that pharmacological modulation of this pathway can have profound influences on tumor proliferation. Therefore, pharmacological inhibition of the Hedgehog pathway is a potentially useful therapeutic approach in GBM.

Topics: Anilides; Animals; Cell Line, Tumor; Cell Proliferation; Dacarbazine; Gene Expression Profiling; Glioblastoma; Hedgehog Proteins; Humans; Mice; Mice, Nude; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Temozolomide; Transcription Factors; Veratrum Alkaloids; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1

The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM).. The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice.. A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors.. An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Anilides; Animals; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; Mesothelioma; Mice; Middle Aged; NIH 3T3 Cells; Phosphoproteins; Pleural Effusion, Malignant; Pyridines; Receptors, G-Protein-Coupled; RNA, Small Interfering; Signal Transduction; Smoothened Receptor; Survivin; Tomatine; Transcription Factors; Transplantation, Heterologous; Veratrum Alkaloids; YAP-Signaling Proteins; Zinc Finger Protein GLI1

Hedgehog (Hh) signaling is involved in the pathogenesis of liver fibrosis. It has been previously shown that Hh-inhibitor cyclopamine (CYA) can reduce liver fibrosis in rats. However, CYA is not stable in vivo, which limits its clinical application. This study compares the antifibrotic potential of two known Hh antagonists, vismodegib (GDC-0449, abbreviated to GDC) and CYA. GDC is a synthetic molecule presently in clinical cancer trials and has been reported to be safe and efficacious. These drugs attenuated early liver fibrosis in common bile duct ligated rats, improved liver function, and prevented hepatic stellate cell (HSC) activation, thereby suppressing epithelial to mesenchymal transition (EMT). While both CYA and GDC increased the number of proliferating cell nuclear antigen positive liver cells in vivo, only CYA increased Caspase-3 expression in HSCs in rat livers, suggesting that while GDC and CYA effectively attenuate early liver fibrosis, their hepatoprotective effects may be mediated through different modes of action. Thus, GDC has the potential to serve as a new therapeutic agent for treating early liver fibrosis.

Topics: Anilides; Animals; Caspase 3; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Hedgehog Proteins; Hepatic Stellate Cells; Liver Cirrhosis, Experimental; Liver Function Tests; Proliferating Cell Nuclear Antigen; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids

Dependence of basal cell carcinomas and medulloblastomas on the Hedgehog pathway provides an opportunity for targeted or "personalized" therapy. The recent effectiveness and FDA approval of the first Smoothened inhibitors validates this class of agents, but has revealed drug-resistant tumor variants that bypass Smoothened inhibition. Here, we summarize the effectiveness of Hedgehog pathway inhibitors and highlight promising areas for the development of next generation drug antagonists for Hedgehog-dependent cancers.

Topics: Anilides; Antineoplastic Agents; Carcinoma, Basal Cell; Cerebellar Neoplasms; Drug Resistance, Neoplasm; Hedgehog Proteins; Humans; Medulloblastoma; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Skin Neoplasms; Smoothened Receptor; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

Topics: Anilides; Animals; Benzamides; Benzimidazoles; beta-Lactamases; Binding Sites; Binding, Competitive; Cell Line; Cell Membrane; Cyclohexylamines; Genes, Reporter; Humans; Kinetics; Mice; Molecular Structure; Morpholines; NIH 3T3 Cells; Piperazines; Purines; Pyrazoles; Pyridines; Radioligand Assay; Receptors, G-Protein-Coupled; Recombinant Proteins; Smoothened Receptor; Thiophenes; Tomatine; Transfection; Veratrum Alkaloids