Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro.

BackgroundOligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals.MethodsOur protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq).ResultsWe demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice.ConclusionsThis is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs

Investigating the role of Hedgehog/GLI1 signaling in glioblastoma cell response to temozolomide.

Resistance to chemotherapy substantially hinders successful glioblastoma (GBM) treatment, contributing to an almost 100% mortality rate. Resistance to the frontline chemotherapy, temozolomide (TMZ), arises from numerous signaling pathways that are deregulated in GBM, including Hedgehog (Hh) signaling. Here, we investigate suppression of Hh signaling as an adjuvant to TMZ using U87-MG and T98G cell lines as in vitro models of GBM. We found that silencing GLI1 with siRNA reduces cell metabolic activity by up to 30% in combination with TMZ and reduces multidrug efflux activity by 2.5-fold. Additionally, pharmacological GLI inhibition modulates nuclear p53 levels and decreases MGMT expression in combination with TMZ. While we surprisingly found that silencing GLI1 does not induce apoptosis in the absence of TMZ co-treatment, we discovered silencing GLI1 without TMZ co-treatment induces senescence as evidenced by a significant 2.3-fold increase in senescence associated β-galactosidase staining, and this occurs in a loss of PTEN-dependent manner. Finally, we show that GLI inhibition increases apoptosis in glioma stem-like cells by up to 6.8-fold in combination with TMZ, and this reduces the size and number of neurospheres grown from glioma stem-like cells. In aggregate, our data warrant the continued investigation of Hh pathway inhibitors as adjuvants to TMZ chemotherapy and highlight the importance of identifying signaling pathways that determine whether co-treatment will be successful

Targeting GLI factors to inhibit the Hedgehog pathway

Hedgehog (Hh) signaling has emerged in recent years as an attractive target for anticancer therapy because its aberrant activation is implicated in several cancers. Major progress has been made in the development of SMOOTHENED (SMO) antagonists, although they have shown several limitations due to downstream SMO pathway activation or the occurrence of drug-resistant SMO mutations. Recently, particular interest has been elicited by the identification of molecules able to hit glioma-associated oncogene (GLI) factors, the final effectors of the Hh pathway, which provide a valid tool to overcome anti-SMO resistance. Here, we review results achieved in developing GLI antagonists, explaining their mechanisms of action and highlighting their therapeutic potential. We also underline the relevance of structural details in their discovery and optimization

HCV derived from sera of HCV-infected patients induces pro-fibrotic effects in human primary fibroblasts by activating GLI2

Hepatitis C virus (HCV) infection is a leading cause of liver fibrosis, especially in developing countries. The process is characterized by the excess accumulation of ECM that may lead, over time, to hepatic cirrhosis, liver failure and also to hepatocarcinoma. The direct role of HCV in promoting fibroblasts trans-differentiation into myofibroblasts, the major fibrogenic cells, has not been fully clarified. In this study, we found that HCV derived from HCV-infected patients infected and directly induced the trans-differentiation of human primary fibroblasts into myofibroblasts, promoting fibrogenesis. This effect correlated with the activation of GLI2, one of the targets of Hedgehog signaling pathway previously reported to be involved in myofibroblast generation. Moreover, GLI2 activation by HCV correlated with a reduction of autophagy in fibroblasts, that may further promoted fibrosis. GLI2 inhibition by Gant 61 counteracted the pro-fibrotic effects and autophagy inhibition mediated by HCV, suggesting that targeting HH/GLI2 pathway might represent a promising strategy to reduce the HCV-induced fibrosis

Hedgehog signaling pathway and its targets for treatment in basal cell carcinoma

Basal cell carcinoma (BCC) of the skin is the most common type of cancer, and accounts for up to 40% of all cancers in the United States with a growing incidence rate in the last decades in all developed countries. Surgery is curative for most patients, although it leaves unaesthetic scars, but those that develop locally advanced or metastatic BCC require different therapeutical approaches. Furthermore, patients with BCC present an high risk of developing additional tumors. The increasing economic burden and the morbidity of BCC render of primary interest the development of targeted treatments for this disease. Among the molecular signals involved in the development of BCC, it has become evident the critical role of the morphogenic Hedgehog (Hh) pathway. This pathway is found altered and activated in almost all the BCC, both sporadic or inherited. Given the centrality of the Hh pathway in the pathophysiology of BCC, the primary efforts to identify molecular targets for the topical or systemic treatment of this cancer have focused on the Hedgehog components. Several Hh inhibitors have been so far identified, from the first, the natural cyclopamine to the recently FDA-approved synthetic Vismodegib, most targeting the Hh receptor Smo (either its function or its translocation to the primary cilium). Other molecules await further characterization (Bisamides compounds), while drugs currently approved for other diseases such as Itraconazole (a antimicotic agent) and Vitamin D3 have been tested on BCC with encouraging results. The outcome of the numerous ongoing clinical trials is expected to expand the field in short time. Further research is needed to obtain drugs targeting downstream components of the Hh pathway (eg Gli) or to exploit combinatorial therapies (eg with PI3K inhibitors, or retinoids) in order to overcome potential drug resistance

Mitochondria-derived reactive oxygen species drive GANT61-induced mesothelioma cell apoptosis

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe

Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients’ prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients

Hedgehog pathway dysregulation contributes to the pathogenesis of human gastrointestinal stromal tumors via GLI-mediated activation of KIT expression.

Gastrointestinal stromal tumors (GIST) arise within the interstitial cell of Cajal (ICC) lineage due to activating KIT/PDGFRA mutations. Both ICC and GIST possess primary cilia (PC), which coordinate PDGFRA and Hedgehog signaling, regulators of gastrointestinal mesenchymal development. Therefore, we hypothesized that Hedgehog signaling may be altered in human GIST and controls KIT expression. Quantitative RT-PCR, microarrays, and next generation sequencing were used to describe Hedgehog/PC-related genes in purified human ICC and GIST. Genetic and pharmacologic approaches were employed to investigate the effects of GLI manipulation on KIT expression and GIST cell viability. We report that Hedgehog pathway and PC components are expressed in ICC and GIST and subject to dysregulation during GIST oncogenesis, irrespective of KIT/PDGFRA mutation status. Using genomic profiling, 10.2% of 186 GIST studied had potentially deleterious genomic alterations in 5 Hedgehog-related genes analyzed, including in the PTCH1 tumor suppressor (1.6%). Expression of the predominantly repressive GLI isoform, GLI3, was inversely correlated with KIT mRNA levels in GIST cells and non-KIT/non-PDGFRA mutant GIST. Overexpression of the 83-kDa repressive form of GLI3 or small interfering RNA-mediated knockdown of the activating isoforms GLI1/2 reduced KIT mRNA. Treatment with GLI1/2 inhibitors, including arsenic trioxide, significantly increased GLI3 binding to the KIT promoter, decreased KIT expression, and reduced viability in imatinib-sensitive and imatinib-resistant GIST cells. These data offer new evidence that genes necessary for Hedgehog signaling and PC function in ICC are dysregulated in GIST. Hedgehog signaling activates KIT expression irrespective of mutation status, offering a novel approach to treat imatinib-resistant GIST

Mechanisms acting on Hedgehog-GLI pathway and their therapeutic potential

Hedgehog signaling is crucial for diverse aspects of animal development, essential in regulating many cellular processes and is largely implicated in various forms of human cancer. However, many aspects of Hedgehog signaling are not completely understood. This thesis aims to contribute towards a better understanding of the mechanisms acting on Hedgehog-GLI signaling and explore their possible therapeutic potential. PAPER I. We demonstrate that the small molecule RITA, a p53 activator, downregulates Hedgehog signaling in human medulloblastoma and rhabdomyosarcoma cells via JNK kinase and irrespective of p53. In vitro RITA enhanced the anti-proliferative effects of the GLI antagonist GANT61. RITA was more potent than GANT61 in downregulating Hedgehog-GLI signaling in rhabdomyosarcoma subcutaneous xenograft tumors with the dual drug administration almost completely blocking the Hedgehog signaling response in vivo, suggesting a certain antagonism of the two drugs. Notably, RITA and GANT61 co-administration decreased cell proliferation and elicited a broader response of pathways involved in cancer cell growth, providing a plausible interpretation for tumor reduction in the absence of Hedgehog signaling downregulation. PAPER II. We address the possible therapeutic role of Hedgehog-GLI1 signaling for targeting and prognosis of ER-alpha positive breast cancer. We showed that expression of the Hedgehog signaling effector protein GLI1 is higher in tamoxifen resistant relative to tamoxifen sensitive cells. In both cell types GLI1 depletion mitigated cell proliferation and ER-alpha activity, irrespective of estrogen stimulation. Tamoxifen cytotoxicity was enhanced by GANT61 co-treatment, both in tamoxifen resistant and sensitive breast cancer cells, reflecting a crosstalk between ER-alpha and Hedgehog-GLI1 signaling. We have observed a positive correlation between GLI1 and ER-alpha/ER-alpha target gene expression, while high GLI1 expression was associated with poor distant metastasis-free survival in breast cancer patients. PAPER III. We identified a signature of GLI1 target genes via a combination of RNA-seq analyses of GLI1 overexpression and depletion datasets supplemented with in-depth validation in human cancer cell lines. Additionally, we found that RNA editing of GLI1 can modulate its effects on GLI1 target genes. Markedly, one of the highly upregulated targets, FOXS1, was found to engage in feedback mechanisms limiting the capacity of GLI1 to act as a proliferation factor in medulloblastoma and rhabdomyosarcoma cells. FOXS1 was both highly expressed and positively correlated with GLI1 in SHH medulloblastoma, further arguing for the existence of a FOXS1-GLI1 interplay in human tumors. PAPER IV (Manuscript). In this ongoing work we address the role of circRNAs in the context of Hedgehog signaling activation and Hedgehog-linked SHH medulloblastoma tumors. Via modified RNA-seq protocols we have determined the circRNA transcriptome of Daoy medulloblastoma and human embryonic palatal mesenchyme HEPM cells, following activation of Hedgehog pathway with SHH ligand or Smoothened agonist SAG. In total, 29 selected circRNAs were independently validated by Sanger sequencing and RT-PCR assays. Of these circRNAs, 10 were apparently regulated by Hedgehog signaling activation, however to a much lesser extent compared with known target genes of the pathway, e.g. GLI1 and HHIP. 7 circRNAs had reduced expression in human medulloblastoma tumors in comparison to normal cerebellum, while the linear mRNAs originating from the same genes did not exhibit a reduced expression. These findings highlight distinct regulatory mechanisms acting on the BACH1, CDYL, FKBP8, GLIS1, OGDH, SMARCA5 and ZKSCAN1 circRNAs and deserve further analysis for possible contribution to the development of medulloblastoma

Exploring new therapeutic targets and novel therapies for resistant colorectal cancer subtypes

Colorectal cancer (CRC) represents the fourth leading cause of death by cancer in the world. CRC treatment is determined according to disease stage. 5-fluoruracil (5-FU), oxaliplatin and Irinotecan are the main chemotherapeutic compounds used in CRC treatment in different therapeutic strategies. However, in most aggressive CRCs, cells often develop resistance mechanisms leading to ineffectiveness of these therapies. Thus, it is of great importance the better understanding of molecular mechanisms underlying CRC in order to find new therapeutic targets and novel therapeutic strategies for treating advanced and resistant CRC. Deregulation of Wnt/β-catenin and Sonic hedgehog/Gli (Shh/Gli) signaling pathways, among many others, has been implicated in CRC carcinogenesis and metastasis. Excessive activation of Wnt/β-catenin signaling stimulates CRC development through activation of downstream cancer-related targets. TCF7L2 gene, encoding the main transcriptional activator of this pathway, originates different TCF7L2 isoforms that have been implicated in CRC. Moreover, defective regulation of Shh/Gli pathway have been denoted for its significant role in CRC progression, affecting the regulation of a diversity of cell processes involved in CRC carcinogenesis. During metastasis, cells develop mesenchymal characteristics, through the epithelial-to-mesenchymal transition (EMT) process. Furthermore, there is an established link between cancer stem cells (CSCs) and metastasis. Therefore, the aim of this project was to explore new therapeutic targets and novel therapies for resistant colorectal cancer subtypes. By studying TCF7L2 gene expression in a cohort of 38 CRC patients and CRC representative cell lines, we identified differential expression of specific TCF7L2 isoforms, varying in exon inclusion within exons 1-5 and exons 11-17, that appear to differ among CRC patients’ samples and between normal and tumor tissues. Therefore, this opens the door for further investigation on differential expression of TCF7L2 isoforms and its relation to CRC tumorigenesis and eventually to CRC risk. In a second phase of the project we tested a panel of 10 different compounds (cytostatic drugs used in conventional CRC treatment, epigenetic modulators, targeted therapies of specific signaling pathways and nutraceuticals) and the most promising combinations between them, for anti-proliferative and anti-migratory activities, in two CRC cell lines representative of mucinous tumors resistant to therapy (HT-29 and LS174T). The most promising combinations were evaluated for their effect in the expression of gene markers involved in cell-cycle, CRC stemness, epithelial-mesenchymal transition and Wnt/β-catenin and Sonic hedgehog/Gli (Shh/Gli) signaling pathways. With this work we were able to identify promising therapy combinations for the treatment of resistant CRC subtypes and explore the potential of new compounds as complement for conventional therapy in treatment of aggressive forms of CRC