Implications of hedgehog signaling antagonists for cancer therapy

The hedgehog (Hh) pathway, initially discovered in Drosophila by two Nobel laureates, Dr. Eric Wieschaus and Dr. Christiane Nusslein-Volhard, is a major regulator for cell differentiation, tissue polarity and cell proliferation. Studies from many laboratories, including ours, reveal activation of this pathway in most basal cell carcinomas and in approximately 30% of extracutaneous human cancers, including medulloblastomas, gastrointestinal, lung, breast and prostate cancers. Thus, it is believed that targeted inhibition of Hh signaling may be effective in treating and preventing many types of human cancers. Even more exciting is the discovery and synthesis of specific signaling antagonists for the Hh pathway, which have significant clinical implications in novel cancer therapeutics. This review discusses the major advances in the current understanding of Hh signaling activation in different types of human cancers, the molecular basis of Hh signaling activation, the major antagonists for Hh signaling inhibition and their potential clinical application in human cancer therapy.     

Hedgehog signaling in skin cancers

An increasing progress on the role of Hedgehog (Hh) signaling for carcinogenesis has been achieved since the link of Hh pathway to human cancer was firstly established. In particular, the critical role of Hh signaling in the development of Basal cell carcinoma (BCC) has been convincingly demonstrated by genetic mutation analyses, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors. In addition, the Hh pathway activity is also reported to be involved in the pathogenesis of Squamous Cell Carcinoma (SCC), melanoma and Merkel Cell Carcinoma. These findings have significant new paradigm on Hh signaling transduction, its mechanisms in skin cancer and even therapeutic approaches for BCC. In this review, we will summarize the major advances in the understanding of Hh signaling transduction, the roles of Hh signaling in skin cancer development, and the current implications of “mechanism-based” therapeutic strategies.     

Hedgehog信号通路与脂肪形成

Hedgehog(Hh)信号通路是从果蝇到人类都非常保守的信号通路,在脊椎动物和非脊椎动物胚胎期多种组织器官的发育中发挥着重要作用。Hh信号通路的异常会导致疾病(先天性缺陷和癌症)的发生。近年的研究发现,Hh信号通路在脂肪生长发育中发挥重要作用,激活Hh信号通路能特异性地抑制白色脂肪组织细胞的分化,而对棕色脂肪组织细胞分化没有作用。该文综述了Hh信号通路在脂肪细胞分化中的作用及其分子机制,并对今后的研究和应用作了展望。     

Sonic hedgehog pathway activation regulates cervical cancer stem cell characteristics during epithelial to mesenchymal transition

Resistance to therapy and metastasis remains one of the leading causes of mortality due to cervical cancer despite advances in detection and treatment. The mechanism of epithelial to mesenchymal transition (EMT) provides conceptual explanation to the invasiveness and metastatic spread of cancer but it has not been fully understood in cervical cancer. This study aims to investigate the mechanism by which silencing of E-cadherin gene regulates EMT leading to proliferation, invasion, and chemoresistance of cervical cancer cells through the Hedgehog (Hh) signaling pathway. We developed an in vitro EMT model by the knockdown of E-cadherin expression in cervical cancer cell lines. To understand the role of developmental pathway like Hh in the progression of cervical cancer, we investigated the expression of Hh pathway mediators by array in E-cadherin low cervical cancer cells and observed upregulation of Hh pathway. This was further validated on low passage patient-derived cell lines and cervical carcinoma tissue sections from cervical cancer patients. Further, we evaluated the role of two inhibitors (cyclopamine and GANT58) of the Hh pathway on invasiveness and apoptosis in E-cadherin low cervical cancer cells. In conclusion, we observed that inhibition of Hh pathway with GANT58 along with current therapeutic procedures could be more effective in targeting drug-resistant EMT cells and bulk tumor cells in cervical cancer.     

Hedgehogs as Negative Regulators of the Cell Cycle

During the development of multicellular animals, cell proliferation must be precisely controlled, as deregulated proliferation can lead to overgrowth and cancer. In addition, proliferation must be tightly integrated with pattern formation and differentiation to generate the required number of cells in the right organs, and at the right time. All major signaling pathways employed during embryogenesis have been implicated in cell cycle regulation, indicating that no single pathway has been dedicated to this task. Also, the precise role of a particular signaling pathway in regulating proliferation is highly dependent on the cellular context, and may have opposite effects on cell-cycle progression in different cells and tissues. The Hedgehog (Hh) family of signaling proteins is known to control both differentiation and proliferation during development. So far, studies addressing the effect of Hh signaling on proliferation have shown it to have a stimulatory effect on cell-cycle progression. Here we review several recent studies indicating that Hh signaling can also have the opposite effect, directing cell-cycle exit in a number of cell types in vertebrate and in invertebrate embryos.     

CDO,an Hh-Coreceptor,Mediates Lung Cancer Cell Proliferation and Tumorigenicity through Hedgehog Signaling

Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling.     

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 signal transduction: from flies to vertebrates

The patterning and morphogenesis of multicellular organisms require a complex interplay of inductive signals which control proliferation, growth arrest, and differentiation of different cell types. A number of such signaling molecules have been identified in vertebrates and invertebrates. The molecular dissection of these pathways demonstrated that in vertebrates, mutations or abnormals function of these signaling pathways were often associated with developmental disorders and cancer formation. The Hedgehog (Hh) family of secreted proteins provides a perfect example of such signaling proteins. In the following review, we will not discuss in detail the role of Hh as a morphogen, but rather focus on its signal transduction pathway and its role in various human disorders.     

Purmorphamine activates the Hedgehog pathway by targeting Smoothened

Hedgehog (Hh) signaling is an important regulator of embryonic patterning, tissue regeneration, stem cell renewal and cancer growth. A purine derivative named purmorphamine was previously found to activate the Hh pathway and affect osteoblast differentiation through an unknown mechanism. We demonstrate here that purmorphamine directly targets Smoothened, a critical component of the Hh signaling pathway.     

The complexities of G-protein-coupled receptor kinase function in Hedgehog signaling

Hedgehog (Hh) signaling is essential for proper tissue patterning and maintenance and has a substantial impact on human disease. While many of the main components and mechanisms involved in transduction of the Hh signal have been identified, the details of how the pathway functions are continually being refined. One aspect that has attracted much attention recently is the involvement of G-protein-coupled receptor kinases (GRKs) in the pathway. These regulators of G-protein-coupled receptor (GPCR) signaling have an evolutionarily-conserved function in promoting high-threshold Hh target gene expression through regulation of Smoothened (Smo), a GPCR family member that activates intracellular Hh signaling. Several models of how GRKs impact on Smo to increase downstream signaling have been proposed. Recently, we demonstrated that these kinases have surprisingly complex and conflicting roles, acting to limit signaling through the pathway while also promoting Smo activity. In addition to the previously described direct effects of Gprk2 on Smo activation, Gprk2 also indirectly affects Hh signaling by controlling production of the second messenger cyclic AMP to influence Protein kinase A activity.     

Inhibition of the hedgehog pathway targets the tumor-associated stroma in pancreatic cancer

Purpose: The Hedgehog (Hh) pathway has emerged as an important pathway in multiple tumor types and is thought to be dependent on a paracrine signaling mechanism. The purpose of this study was to determine the role of pancreatic cancer-associated fibroblasts (human pancreatic stellate cells, HPSCs) in Hh signaling. In addition, we evaluated the efficacy of a novel Hh antagonist, AZD8542, on tumor progression with an emphasis on the role of the stroma compartment. Experimental Design: Expression of Hh pathway members and activation of the Hh pathway were analyzed in both HPSCs and pancreatic cancer cells. We tested the effects of Smoothened (SMO) inhibition with AZD8542 on tumor growth in vivo using an orthotopic model of pancreatic cancer containing varying amounts of stroma. Results: HPSCs expressed high levels of SMO receptor and low levels of Hh ligands, whereas cancer cells showed the converse expression pattern. HPSC proliferation was stimulated by Sonic Hedgehog with upregulation of downstream GLI1 mRNA. These effects were abrogated by AZD8542 treatment. In an orthotopic model of pancreatic cancer, AZD8542 inhibited tumor growth only when HPSCs were present, implicating a paracrine signaling mechanism dependent on stroma. Further evidence of paracrine signaling of the Hh pathway in prostate and colon cancer models is provided, demonstrating the broader applicability of our findings. Conclusion: Based on the use of our novel human-derived pancreatic cancer stellate cells, our results suggest that Hh-targeted therapies primarily affect the tumor-associated stroma, rather than the epithelial compartment. Mol Cancer Res; 10(9); 1147-57. ?2012 AACR.     

Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.     

Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it’s necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.     

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.     

Regulation of Hedgehog signaling by ubiquitination

The Hedgehog (Hh) signaling pathway plays crucial roles both in embryonic development and in adult stem cell function. The timing, duration and location of Hh signaling activity need to be tightly controlled. Abnormalities of Hh signal transduction lead to birth defects or malignant tumors. Recent data point to ubiquitination-related posttranslational modifications of several key Hh pathway components as an important mechanism of regulation of the Hh pathway. Here we review how ubiquitination regulates the localization, stability and activity of the key Hh signaling components.