ABSTRACT
The prognosis of elderly AML patients is still poor due to chemotherapy resistance. The Hedgehog (HH) pathway is important for leukemic transformation because of aberrant activation of GLI transcription factors. MBZ is a well-tolerated anthelmintic that exhibits strong antitumor effects. Herein, we show that MBZ induced strong, dose-dependent anti-leukemic effects on AML cells, including the sensitization of AML cells to chemotherapy with cytarabine. MBZ strongly reduced intracellular protein levels of GLI1/GLI2 transcription factors. Consequently, MBZ reduced the GLI promoter activity as observed in luciferase-based reporter assays in AML cell lines. Further analysis revealed that MBZ mediates its anti-leukemic effects by promoting the proteasomal degradation of GLI transcription factors via inhibition of HSP70/90 chaperone activity. Extensive molecular dynamics simulations were performed on the MBZ-HSP90 complex, showing a stable binding interaction at the ATP binding site. Importantly, two patients with refractory AML were treated with MBZ in an off-label setting and MBZ effectively reduced the GLI signaling activity in a modified plasma inhibitory assay, resulting in a decrease in peripheral blood blast counts in one patient. Our data prove that MBZ is an effective GLI inhibitor that should be evaluated in combination to conventional chemotherapy in the clinical setting.
Subject(s)
Leukemia, Myeloid, Acute/metabolism , Mebendazole/pharmacology , Proteasome Endopeptidase Complex/metabolism , Tubulin Modulators/pharmacology , Zinc Finger Protein GLI1/metabolism , Case-Control Studies , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Humans , Leukemia, Myeloid, Acute/etiology , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Proteolysis , Signal Transduction/drug effects , Structure-Activity Relationship , Transcription Factors/chemistry , Transcription Factors/metabolism , Zinc Finger Protein GLI1/antagonists & inhibitors , Zinc Finger Protein GLI1/chemistryABSTRACT
The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.
Subject(s)
Chromatin/genetics , Regulatory Elements, Transcriptional , Transcription Factors/metabolism , Transcription Initiation Site , Zinc Finger Protein GLI1/metabolism , Cell Line, Tumor , Chromatin/metabolism , Chromatin Assembly and Disassembly , DNA/genetics , DNA/metabolism , HEK293 Cells , Humans , Protein Domains , Protein Interaction Maps , Transcription Factors/chemistry , Transcriptional Activation , Zinc Finger Protein GLI1/chemistryABSTRACT
The Glioma-associated homologue-1 (GLI-1) gene was first discovered to be amplified in glioblastoma multiforme. It encodes for a zinc-finger transcription factor in the Kruppel family of proteins and is important in the sonic hedgehog signalling pathway. GLI-1 also plays a role in several other pathways and is important for proliferation, migration, invasion, growth and angioinvasion, and cancer stem cell self-renewal in a variety of malignancies. GLI-1 is amplified in several malignancies, including an epithelioid, pericytomatous soft tissue neoplasm that can exhibit malignant behaviour. More recently, GLI-1 fusions with other partner genes have been found in three rare tumours: a pericytomatous tumour with a t(7;12) translocation, where it partners with Actin beta 1, and gastroblastoma and plexiform fibromyxoma, where the partner gene is metastasis-associated lung adenocarcinoma transcript 1, respectively.
Subject(s)
Zinc Finger Protein GLI1/genetics , Gene Amplification , Gene Fusion/genetics , Hemangiopericytoma/genetics , Humans , Neurofibroma, Plexiform/genetics , Stomach Neoplasms/genetics , Zinc Finger Protein GLI1/chemistry , Zinc Finger Protein GLI1/physiologyABSTRACT
Hypoxia, or the deficiency of oxygen, in solid tumors is majorly responsible for the progression of cancer and remains unaffected by chemotherapy, but still requires definitive definition of the hypoxia signaling. Hypoxia disrupts the complete folding of mitochondrial proteins, leading to several diseases. The present study confirms that hypoxia activates the Hedgehog pathway in colorectal cancer (CRC), considering its role in cancer epithelial to mesenchymal transition, migration, and invasion. The activity of hypoxia-mediated Gli-1, a Hedgehog signaling factor in hypoxia, was confirmed by in vitro western blotting, immunofluorescence staining, wound-healing assay, and matrigel invasion assay, as well as by in vivo xenograft models (n = 5 per group). The Gli-1 mechanism in hypoxia was analyzed via mass spectrometry. Hypoxia enhanced the interaction of Gli-1 and T-complex protein 1 subunit beta (CCT2), as observed in the mass spectrometric analysis. We observed that reduction in CCT2 inhibits tumor induction by Gli-1. Ubiquitination-mediated Gli-1 degradation by ß-TrCP occurs during incomplete folding of Gli-1 in hypoxia. The human CRC tissues revealed greater CCT2 expression than did the normal colon tissues, indicating that higher CCT2 expression in tumor tissues from CRC patients reduced their survival rate. Moreover, we suggest that CCT2 correlates with Gli-1 expression and is an important determinant of survival in the CRC patients. The results reveal that CCT2 can regulate the folding of Gli-1 in relation to hypoxia in CRC.
Subject(s)
Chaperonin Containing TCP-1/genetics , Colorectal Neoplasms/genetics , Tumor Hypoxia/genetics , Zinc Finger Protein GLI1/genetics , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Chaperonin Containing TCP-1/chemistry , Colorectal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , Hedgehog Proteins/genetics , Heterografts , Humans , Male , Mice , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Protein Folding , Proteolysis/drug effects , Signal Transduction/genetics , Survival Rate , Ubiquitination/genetics , Zinc Finger Protein GLI1/chemistry , beta-Transducin Repeat-Containing Proteins/pharmacologyABSTRACT
The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Papillomaviridae/physiology , Repressor Proteins/metabolism , Zinc Finger Protein GLI1/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , DNA/metabolism , Humans , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Repressor Proteins/chemistry , Sequence Alignment , Transcriptional Activation , Virus Replication , Zinc Finger Protein GLI1/chemistry , Zinc Finger Protein GLI1/geneticsABSTRACT
Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.
Subject(s)
Bone Neoplasms/genetics , Chondrosarcoma/genetics , Gene Expression Profiling/methods , Nuclear Proteins/metabolism , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein Gli2/metabolism , Animals , Bone Neoplasms/metabolism , Chondrosarcoma/metabolism , Chromatin Immunoprecipitation , Gene Expression Regulation, Neoplastic , Hedgehog Proteins/genetics , Humans , Mice , Nuclear Proteins/chemistry , Oligonucleotide Array Sequence Analysis , Protein Binding , Signal Transduction , Tumor Cells, Cultured , Zinc Finger Protein GLI1/chemistry , Zinc Finger Protein Gli2/chemistryABSTRACT
GLI1 is a key downstream transcription effector of the Hedgehog (Hh) signaling pathway that is involved in promoting cell growth, differentiation and tissue patterning in embryonic development. GLI1 over-activation and its nuclear localization has also been linked to the increased aggressiveness of a number of cancers. It has previously been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate GLI1 and promote GLI1 nuclear localization and its transcriptional activity. Utilizing recombinant human GLI1 and DYRK1A proteins and phospho-peptide mass spectrometry, we demonstrated that GLI1 is phosphorylated by DYRK1A at Ser408, a phospho-site that falls within the putative nuclear localization sequence (NLS) of GLI1, suggesting a possible mechanistic role in modulating its translocation. Further, we showed that the Ser408 site on GLI1 was not phosphorylated in the presence of the selective DYRK1A inhibitor harmine. The data described herein provide the first identification of a DYRK1A-mediated site of phosphorylation on GLI1 within its NLS and may serve as a valuable mechanism for further understanding Hh signaling modulation.