Hh Pathway Antagonists Research Articles

Smoothened antagonist sonidegib affects the development of D. melanogaster larvae via suppression of epidermis formation

Hedgehog (Hh) signaling is essential for the regulation of embryonic growth and development, the maintenance of stem cell autostasis, and tissue formation, whether in vertebrates or invertebrates. However, exploration into the Hh pathway antagonists in Drosophila or other pests of agricultural importance has been scant. In order to gain a better understanding of the potential utility of the antagonists in insect investigations, a conventional Hh antagonist, sonidegib, was used to evaluate the effects on the development of Drosophila larvae. The results showed that early instar larvae exposed to sonidegib exhibited new epidermal abnormalities and decreased motility after molting. Transcriptome analysis revealed that Sonidegib had a profound effect on chitin-based cuticle development throughout all stages of larvae. Physiological experiments revealed that sonidegib suppressed the epidermis formation and decreased the chitin content. The results of this study shed new light on the potential use of Hh antagonists in agricultural pest management.

Inhibition of hedgehog signaling by stereochemically defined des-triazole itraconazole analogues

Dysregulation of the hedgehog (Hh) signaling pathway is associated with cancer occurrence and development in various malignancies. Previous structure-activity relationships (SAR) studies have provided potent Itraconazole (ITZ) analogues as Hh pathway antagonists. To further expand on our SAR for the ITZ scaffold, we synthesized and evaluated a series of compounds focused on replacing the triazole. Our results demonstrate that the triazole region is amenable to modification to a variety of different moieties; with a single methyl group representing the most favorable substituent. In addition, nonpolar substituents were more active than polar substituents. These SAR results provide valuable insight into the continued exploration of ITZ analogues as Hh pathway antagonists.

Steroidal alkaloid variation in Veratrum californicum as determined by modern methods of analytical analysis

Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.

Posaconazole, a Second-Generation Triazole Antifungal Drug, Inhibits the Hedgehog Signaling Pathway and Progression of Basal Cell Carcinoma.

Deregulation of Hedgehog (Hh) pathway signaling has been associated with the pathogenesis of various malignancies, including basal cell carcinomas (BCC). Inhibitors of the Hh pathway currently available or under clinical investigation all bind and antagonize Smoothened (SMO), inducing a marked but transient clinical response. Tumor regrowth and therapy failure were attributed to mutations in the binding site of these small-molecule SMO antagonists. The antifungal itraconazole was demonstrated to be a potent SMO antagonist with a distinct mechanism of action from that of current SMO inhibitors. However, itraconazole represents a suboptimal therapeutic option due to its numerous drug-drug interactions. Here, we show that posaconazole, a second-generation triazole antifungal with minimal drug-drug interactions and a favorable side-effect profile, is also a potent inhibitor of the Hh pathway that functions at the level of SMO. We demonstrate that posaconazole inhibits the Hh pathway by a mechanism distinct from that of cyclopamine and other cyclopamine-competitive SMO antagonists but, similar to itraconazole, has robust activity against drug-resistant SMO mutants and inhibits the growth of Hh-dependent BCC in vivo Our results suggest that posaconazole, alone or in combination with other Hh pathway antagonists, may be readily tested in clinical studies for the treatment of Hh-dependent cancers. Mol Cancer Ther; 15(5); 866-76. ©2016 AACR.

A Group of ent-Kaurane Diterpenoids Inhibit Hedgehog Signaling and Induce Cilia Elongation.

The Hedgehog (Hh) signaling pathway plays important roles in the tumorigenesis of multiple cancers and is a key target for drug discovery. In a screen of natural products extracted from Chinese herbs, we identified eight ent-Kaurane diterpenoids and two triterpene dilactones as novel Hh pathway antagonists. Epistatic analyses suggest that these compounds likely act at the level or downstream of Smoothened (Smo) and upstream of Suppressor of Fused (Sufu). The ent-Kauranoid-treated cells showed elongated cilia, suppressed Smo trafficking to cilia, and mitotic defects, while the triterpene dilactones had no effect on the cilia and ciliary Smo. These ent-Kaurane diterpenoids provide new prototypes of Hh inhibitors, and are valuable probes for deciphering the mechanisms of Smo ciliary transport and ciliogenesis.

Histone deacetylase 6 represents a novel drug target in the oncogenic Hedgehog signaling pathway.

Uncontrolled Hedgehog (Hh) signaling is the cause of several malignancies, including the pediatric cancer medulloblastoma, a neuroectodermal tumor affecting the cerebellum. Despite the development of potent Hh pathway antagonists, medulloblastoma drug resistance is still an unresolved issue that requires the identification of novel drug targets. Following up on our observation that histone deacetylase 6 (HDAC6) expression was increased in Hh-driven medulloblastoma, we found that this enzyme is essential for full Hh pathway activation. Intriguingly, these stimulatory effects of HDAC6 are partly integrated downstream of primary cilia, a known HDAC6-regulated structure. In addition, HDAC6 is also required for the complete repression of basal Hh target gene expression. These contrasting effects are mediated by HDAC6's impact on Gli2 mRNA and GLI3 protein expression. As a result of this complex interaction with Hh signaling, global transcriptome analysis revealed that HDAC6 regulates only a subset of Smoothened- and Gli-driven genes, including all well-established Hh targets such as Ptch1 or Gli1. Importantly, medulloblastoma cell survival was severely compromised by HDAC6 inhibition in vitro and pharmacologic HDAC6 blockade strongly reduced tumor growth in an in vivo allograft model. In summary, our data describe an important role for HDAC6 in regulating the mammalian Hh pathway and encourage further studies focusing on HDAC6 as a novel drug target in medulloblastoma.

Molecular Pathways: The Hedgehog Signaling Pathway in Cancer

The Hedgehog (Hh) signaling pathway regulates embryonic development and may be aberrantly activated in a wide variety of human cancers. Efforts to target pathogenic Hh signaling have steadily progressed from the laboratory to the clinic, and the recent approval of the Hh pathway inhibitor vismodegib for patients with advanced basal cell carcinoma represents an important milestone. On the other hand, Hh pathway antagonists have failed to show significant clinical activity in other solid tumors. The reasons for these negative results are not precisely understood, but it is possible that the impact of Hh pathway inhibition has not been adequately measured by the clinical endpoints used thus far or that aberrancies in Hh signal transduction limits the activity of currently available pathway antagonists. Further basic and correlative studies to better understand Hh signaling in human tumors and validate putative antitumor mechanisms in the clinical setting may ultimately improve the success of Hh pathway inhibition to other tumor types.

Abstract SY05-01: Biological and clinical activities of FDA-approved drugs that antagonize Hh signaling

Abstract The inappropriate activity of the Hedeghog (Hh) signaling pathway noted in a variety of tumor types has prompted efforts to develop small molecule antagonists of Hh pathway signaling. The use of FDA-approved drugs as Hh pathway antagonists may avoid some of the expense and delay associated with de novo development of Hh pathway antagonists. We have identified two FDA-approved drugs, itraconazole and arsenic trioxide, as potent inhibitors of the Hh pathway in vitro and in vivo at the level of Smoothened (Smo) and Gli, respectively. In addition, both itraconazole and arsenic trioxide have inhibitory activity against a mutant Smoothened resistant to GDC-0449, a potent Smo antagonist. These preclinical studies have led to a pilot clinical trial (ID: NCT01108094) using itraconazole for patients with basal cell carcinoma, a skin tumor that is driven by the Hh pathway. We have treated 13 subjects with 200mg of itraconazole twice daily and found -32% (95% CI: -19 to -42%) reduction in tumor diameter in 1 month. These studies suggest that FDA-approved drugs show promise as Hh pathway antagonists for the treatment of Hh-dependent cancers and warrant further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY05-01. doi:10.1158/1538-7445.AM2011-SY05-01

In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila

The recent establishment of high-throughput methods for culturing Drosophila provided a unique ability to screen compound libraries against complex disease phenotypes in the context of whole animals. However, as compound studies in Drosophila have been limited so far, the degree of conservation of compound activity between Drosophila and vertebrates or the effectiveness of feeding as a compound delivery system is not well known. Our comprehensive in vivo analysis of 27 small molecules targeting seven signaling pathways in Drosophila revealed a high degree of conservation of compound activity between Drosophila and vertebrates. We also investigated the mechanism of action of AY9944, one of the Hh pathway antagonists that we identified in our compound feeding experiments. Our epistasis analysis of AY9944 provided novel insights into AY9944's mechanism of action and revealed a novel role for cholesterol transport in Hh signal transduction. The online version of this article (doi:10.1007/s12154-010-0051-5) contains supplementary material, which is available to authorized users.

New developments in the discovery of small molecule Hedgehog pathway antagonists

The Hedgehog (Hh) signaling pathway is crucial for normal embryonic development. Aberrant Hh signaling is implicated in numerous pathologic conditions including proliferative diseases such as cancer. During the past decade, academic and industrial research efforts have resulted in the discovery of a variety of Hh pathway antagonists. This review focuses on the most recent advances in this field with particular emphasis on the medicinal chemistry approaches used to discover these Hh antagonists. While most of the small molecule modulators of the Hh pathway were discovered through screening and subsequent medicinal chemistry, a number of them originated from rational design or natural products.

Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Hedgehog pathway activity is required for the lethality and intestinal phenotypes of mice with hyperactive Wnt signaling

Several lines of evidence point to the central role of WNT signaling in the initiation of intestinal tumorigenesis, most often due to loss of APC, a negative regulator of the WNT-βCATENIN/TCF pathway. Modeling human colon cancers in mice through loss of Apc has shown that inappropriate activation of Wnt signaling is sufficient to induce adenoma formation. More recent analyses have also demonstrated a key role for HEDGEHOG-GLI (HH-GLI) signaling in human colon cancers. However, how the WNT and HH pathways interact during intestinal development, homeostasis and cancer is not clear. Marker analyses suggest predominant paracrine signaling from rare Shh producing cells in the crypt’s bottom to adjacent Gli1 + mesenchymal cells in normal adult mice. Using conditional KO models, we show that inhibition of the function of the critical Hh mediator Smoothened (Smo) rescues the lethality and intestinal phenotypes of loss of Apc. The results uncover an essential role of the Hh pathway in tumors induced by hyperactive Wnt signaling, suggest the action of the Hh pathway in parallel or downstream of Wnt signaling, and validate this model for its use in preclinical work testing Hh pathway antagonists.

Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade

Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.

Reply: molecular determinants of prognosis in oesophageal cancer patients treated with chemoradiation

Sir, We thank Dr Ajani and colleagues for their interest in our study and apologize for not citing their earlier study (Sims-Mourtada et al, 2006) in our original paper. From the aspect of management for metastasis in treatment-failure patients, we designed our study to evaluate the clinical implications of Hedgehog (Hh) signal activation for oesophageal squamous cell carcinoma (ESCC) patients who underwent pre-operative chemoradiotherapy (CRT) and radical surgery. It is unfortunate that we did not cover their experimental study, because they also revealed that Hh signalling activation mechanism is partly dependent on the ATP-binding cassette transporter family and that chemoresistance can be overcome by inhibitors of Hh pathways (Sims-Mourtada et al, 2007). Their papers have revealed the basic mechanism involved from experimental study seeking to overcome CRT resistance; whereas, we looked at the issue from a clinical perspective focusing on metastasis. In our paper, we have shown that persistent Gli-1 nuclear expression after CRT can predict very much earlier recurrence and poorer prognosis in ESCC patients; thus this a potential diagnostic biomarker and therapeutic target for ‘more aggressive' cancer cells that can initiate relapse and maintain disease (Yoshikawa et al, 2008). This is of importance in clinical management, because high-risk patients can easily be screened by Gli-1 evaluation. Management of metastasis has been an important challenge to us, although since 1996 neoadjuvant CRT has shown improved resectability and a better prognosis in ESCC (Fujiwara et al, 2005). Hedgehog pathway antagonists have already been studied in phase I clinical trials in advanced or metastatic skin basal cell carcinomas. A combination modality of conventional anti-cancer agents plus Hh pathway antagonists could potentially abrogate both primary ‘bulk' tumours and metastases in solid tumours (Feldmann et al, 2007). It was pointed out that we did not carry out multivariate analysis. This was because the number of parameters was more than one-tenth the number of patients included in this study; therefore, we considered that a multivariate analysis might result in misleading conclusions. Thus, we performed a univariate analysis because of the small number of patients available for this study. Our findings will need to be confirmed in a larger more detailed study using multivariate analysis.

A paracrine requirement for hedgehog signalling in cancer

Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.

New developments in medulloblastoma treatment: the potential of a cyclopamine–lovastatin combination

Pediatric brain tumors are the most common cause of cancer-related death in children. Despite advances in neuroimaging, surgical techniques, radiotherapy and the introduction of new chemotherapeutic agents, up to 30% of all patients diagnosed with medulloblastoma will die from their disease, thus underscoring the need for more effective treatments. This review explores the relationship between cholesterol and cancer in light of a recent study that demonstrated that the hedgehog (Hh) antagonist cyclopamine blocked expression of the Hh pathway targets PTCH1 and GLI1, lowered Bcl2 levels and increased apoptosis in medulloblastoma cells. In addition, Hh pathway blockade sensitized medulloblastoma to the effects of the proapoptotic, cholesterol-lowering agent lovastatin. The use of Hh pathway antagonists and cholesterol synthesis inhibitors in medulloblastoma is a novel and exciting approach as it targets pathways crucial for tumor survival. The safety of such approach, however, still needs to be tested in animal models of the disease before its application in human patients.