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Waas B, Carpenter BS, Franks NE, Merchant OQ, Verhey KJ, Allen BL. Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development. SCIENCE ADVANCES 2024; 10:eade1650. [PMID: 38669326 PMCID: PMC11051677 DOI: 10.1126/sciadv.ade1650] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Accepted: 03/26/2024] [Indexed: 04/28/2024]
Abstract
While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.
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Affiliation(s)
- Bridget Waas
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Brandon S. Carpenter
- Department of Molecular and Cellular Biology, College of Science and Mathematics, Kennesaw State University, Kennesaw, GA, 30061, USA
| | - Nicole E. Franks
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Olivia Q. Merchant
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Kristen J. Verhey
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Benjamin L. Allen
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA
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Hamada T, Higashi M, Yokoyama S, Akahane T, Hisaoka M, Noguchi H, Furukawa T, Tanimoto A. MALAT1 functions as a transcriptional promoter of MALAT1::GLI1 fusion for truncated GLI1 protein expression in cancer. BMC Cancer 2023; 23:424. [PMID: 37165307 PMCID: PMC10173563 DOI: 10.1186/s12885-023-10867-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Accepted: 04/20/2023] [Indexed: 05/12/2023] Open
Abstract
BACKGROUND The long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a cancer biomarker. Furthermore, fusion of the MALAT1 gene with glioma-associated oncogene 1 (GLI1) is a diagnostic marker of plexiform fibromyxoma and gastroblastoma; however, the function of this fusion gene remains unexplored. METHOD In this study, we elucidate the structure and function of the MALAT1::GLI1 fusion gene. To this end, we determined a transcriptional start site (TSS) and promoter region for truncated GLI1 expression using rapid amplification of the 5' cDNA end and a luciferase reporter assay in cultured cells transfected with a plasmid harboring the MALAT1::GLI1 fusion gene. RESULTS We found that the TATA box, ETS1 motif, and TSS were located in MALAT1 and that MALAT1 exhibited transcriptional activity and induced expression of GLI1 from the MALAT1::GLI1 fusion gene. Truncated GLI1, lacking SUMOylation and SUFU binding sites and located in the nucleus, upregulated mRNA expression of GLI1 target genes in the hedgehog signaling pathway. CONCLUSIONS We demonstrate a distinct and alternative function of MALAT1 as a transcriptional promoter for expression of the MALAT1::GLI1 fusion gene. Our findings will aid future research on MALAT1 and its fusion gene partners.
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Affiliation(s)
- Taiji Hamada
- Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
| | - Michiyo Higashi
- Department of Surgical Pathology, Kagoshima University Hospital, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
| | - Seiya Yokoyama
- Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
| | - Toshiaki Akahane
- Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
- Center for Human Genome and Gene Analysis, Kagoshima University Hospital, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
| | - Masanori Hisaoka
- Department of Pathology and Oncology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi, Kitakyushu, 807-8556, Japan
| | - Hirotsugu Noguchi
- Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
| | - Tatsuhiko Furukawa
- Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan
| | - Akihide Tanimoto
- Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan.
- Center for Human Genome and Gene Analysis, Kagoshima University Hospital, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan.
- Center for the Research of Advanced Diagnosis and Therapy of Cancer, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan.
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Piteša N, Kurtović M, Bartoniček N, Gkotsi DS, Čonkaš J, Petrić T, Musani V, Ozretić P, Riobo-Del Galdo NA, Sabol M. Signaling Switching from Hedgehog-GLI to MAPK Signaling Potentially Serves as a Compensatory Mechanism in Melanoma Cell Lines Resistant to GANT-61. Biomedicines 2023; 11:1353. [PMID: 37239024 PMCID: PMC10216463 DOI: 10.3390/biomedicines11051353] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Revised: 04/18/2023] [Accepted: 04/24/2023] [Indexed: 05/05/2023] Open
Abstract
BACKGROUND Melanoma represents the deadliest skin cancer due to its cell plasticity which results in high metastatic potential and chemoresistance. Melanomas frequently develop resistance to targeted therapy; therefore, new combination therapy strategies are required. Non-canonical signaling interactions between HH-GLI and RAS/RAF/ERK signaling were identified as one of the drivers of melanoma pathogenesis. Therefore, we decided to investigate the importance of these non-canonical interactions in chemoresistance, and examine the potential for HH-GLI and RAS/RAF/ERK combined therapy. METHODS We established two melanoma cell lines resistant to the GLI inhibitor, GANT-61, and characterized their response to other HH-GLI and RAS/RAF/ERK inhibitors. RESULTS We successfully established two melanoma cell lines resistant to GANT-61. Both cell lines showed HH-GLI signaling downregulation and increased invasive cell properties like migration potential, colony forming capacity, and EMT. However, they differed in MAPK signaling activity, cell cycle regulation, and primary cilia formation, suggesting different potential mechanisms responsible for resistance occurrence. CONCLUSIONS Our study provides the first ever insights into cell lines resistant to GANT-61 and shows potential mechanisms connected to HH-GLI and MAPK signaling which may represent new hot spots for noncanonical signaling interactions.
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Affiliation(s)
- Nikolina Piteša
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
| | - Matea Kurtović
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
| | - Nenad Bartoniček
- The Garvan Institute of Medical Research, Genome Informatics, Genomics & Epigenetics Division, 384 Victoria St., Darlinghurst, NSW 2010, Australia;
- The Kinghorn Centre for Clinical Genomics, 370 Victoria St., Darlinghurst, NSW 2010, Australia
| | - Danai S. Gkotsi
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK; (D.S.G.); (N.A.R.-D.G.)
- Astbury Centre for Molecular Structural Biology, University of Leeds, Leeds LS2 9JT, UK
| | - Josipa Čonkaš
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
| | - Tina Petrić
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
| | - Vesna Musani
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
| | - Petar Ozretić
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
| | - Natalia A. Riobo-Del Galdo
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK; (D.S.G.); (N.A.R.-D.G.)
- Astbury Centre for Molecular Structural Biology, University of Leeds, Leeds LS2 9JT, UK
- Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, UK
- Leeds Cancer Research Centre, University of Leeds, Leeds LS2 9JT, UK
| | - Maja Sabol
- Ruđer Bošković Institute, Division of Molecular Medicine, 10 000 Zagreb, Croatia; (N.P.); (M.K.); (J.Č.); (T.P.); (V.M.); (P.O.)
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Choi S, Cho N, Kim KK. The implications of alternative pre-mRNA splicing in cell signal transduction. Exp Mol Med 2023; 55:755-766. [PMID: 37009804 PMCID: PMC10167241 DOI: 10.1038/s12276-023-00981-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Revised: 01/05/2023] [Accepted: 01/27/2023] [Indexed: 04/04/2023] Open
Abstract
Cells produce multiple mRNAs through alternative splicing, which ensures proteome diversity. Because most human genes undergo alternative splicing, key components of signal transduction pathways are no exception. Cells regulate various signal transduction pathways, including those associated with cell proliferation, development, differentiation, migration, and apoptosis. Since proteins produced through alternative splicing can exhibit diverse biological functions, splicing regulatory mechanisms affect all signal transduction pathways. Studies have demonstrated that proteins generated by the selective combination of exons encoding important domains can enhance or attenuate signal transduction and can stably and precisely regulate various signal transduction pathways. However, aberrant splicing regulation via genetic mutation or abnormal expression of splicing factors negatively affects signal transduction pathways and is associated with the onset and progression of various diseases, including cancer. In this review, we describe the effects of alternative splicing regulation on major signal transduction pathways and highlight the significance of alternative splicing.
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Affiliation(s)
- Sunkyung Choi
- Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, 34134, Republic of Korea
| | - Namjoon Cho
- Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, 34134, Republic of Korea
| | - Kee K Kim
- Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, 34134, Republic of Korea.
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Zhao L, Xiong X, Liu L, Liang Q, Tong R, Feng X, Bai L, Shi J. Recent research and development of DYRK1A inhibitors. CHINESE CHEM LETT 2022. [DOI: 10.1016/j.cclet.2021.10.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Higashi M, Hamada T, Sasaki K, Tsuruda Y, Shimonosono M, Kitazono I, Kirishima M, Tasaki T, Noguchi H, Tabata K, Hisaoka M, Fukukura Y, Ohtsuka T, Tanimoto A. Esophageal plexiform fibromyxoma: A case report with molecular analysis for MALAT1-GLI1 fusion. Pathol Res Pract 2022; 233:153878. [DOI: 10.1016/j.prp.2022.153878] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Revised: 03/30/2022] [Accepted: 04/01/2022] [Indexed: 02/07/2023]
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A novel promoter-associated non-coding small RNA paGLI1 recruits FUS/P65 to transactivate GLI1 gene expression and promotes infiltrating glioma progression. Cancer Lett 2022; 530:68-84. [DOI: 10.1016/j.canlet.2022.01.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 12/29/2021] [Accepted: 01/13/2022] [Indexed: 11/17/2022]
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Tarpley M, Oladapo HO, Strepay D, Caligan TB, Chdid L, Shehata H, Roques JR, Thomas R, Laudeman CP, Onyenwoke RU, Darr DB, Williams KP. Identification of harmine and β-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies. Eur J Pharm Sci 2021; 162:105821. [PMID: 33781856 PMCID: PMC8404221 DOI: 10.1016/j.ejps.2021.105821] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 02/25/2021] [Accepted: 03/21/2021] [Indexed: 12/16/2022]
Abstract
DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the β-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM) cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.
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Affiliation(s)
- Michael Tarpley
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - Helen O Oladapo
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA; INBS PhD Program, North Carolina Central University, Durham, NC 27707, USA
| | - Dillon Strepay
- Department of Biological and Biomedical Sciences, North Carolina Central University, Durham, NC 27707, USA
| | - Thomas B Caligan
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - Lhoucine Chdid
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - Hassan Shehata
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA; INBS PhD Program, North Carolina Central University, Durham, NC 27707, USA
| | - Jose R Roques
- Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27514, USA
| | - Rhashad Thomas
- Department of Pharmaceutical Sciences; North Carolina Central University, Durham, NC 27707, USA
| | - Christopher P Laudeman
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - Rob U Onyenwoke
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA; Department of Pharmaceutical Sciences; North Carolina Central University, Durham, NC 27707, USA
| | - David B Darr
- Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27514, USA
| | - Kevin P Williams
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA; Department of Pharmaceutical Sciences; North Carolina Central University, Durham, NC 27707, USA.
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9
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Avery JT, Zhang R, Boohaker RJ. GLI1: A Therapeutic Target for Cancer. Front Oncol 2021; 11:673154. [PMID: 34113570 PMCID: PMC8186314 DOI: 10.3389/fonc.2021.673154] [Citation(s) in RCA: 67] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Accepted: 04/30/2021] [Indexed: 12/11/2022] Open
Abstract
GLI1 is a transcriptional effector at the terminal end of the Hedgehog signaling (Hh) pathway and is tightly regulated during embryonic development and tissue patterning/differentiation. GLI1 has low-level expression in differentiated tissues, however, in certain cancers, aberrant activation of GLI1 has been linked to the promotion of numerous hallmarks of cancer, such as proliferation, survival, angiogenesis, metastasis, metabolic rewiring, and chemotherapeutic resistance. All of these are driven, in part, by GLI1’s role in regulating cell cycle, DNA replication and DNA damage repair processes. The consequences of GLI1 oncogenic activity, specifically the activity surrounding DNA damage repair proteins, such as NBS1, and cell cycle proteins, such as CDK1, can be linked to tumorigenesis and chemoresistance. Therefore, understanding the underlying mechanisms driving GLI1 dysregulation can provide prognostic and diagnostic biomarkers to identify a patient population that would derive therapeutic benefit from either direct inhibition of GLI1 or targeted therapy towards proteins downstream of GLI1 regulation.
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Affiliation(s)
- Justin T Avery
- Oncology Department, Drug Discovery Division, Southern Research, Birmingham, AL, United States
| | - Ruowen Zhang
- Department of Medicine, Stony Brook University, Stony Brook, NY, United States
| | - Rebecca J Boohaker
- Oncology Department, Drug Discovery Division, Southern Research, Birmingham, AL, United States
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10
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Prajapati M, Pettiglio MA, Conboy HL, Mercadante CJ, Hojyo S, Fukada T, Bartnikas TB. Characterization of in vitro models of SLC30A10 deficiency. Biometals 2021; 34:573-588. [PMID: 33713241 DOI: 10.1007/s10534-021-00296-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Accepted: 02/26/2021] [Indexed: 11/25/2022]
Abstract
Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of 54Mn import and export in mutant cells. While impaired 54Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired 54Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated 54Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.
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Affiliation(s)
- Milankumar Prajapati
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, 02912, USA.
| | - Michael A Pettiglio
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, 02912, USA.,Vor Biopharma, Cambridge, MA, USA
| | - Heather L Conboy
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, 02912, USA
| | - Courtney J Mercadante
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, 02912, USA.,Sanofi-Bioverativ, Waltham, MA, USA
| | - Shintaro Hojyo
- Deutsches Rheuma-Forschungszentrum Berlin, 10117, Berlin, Germany.,Division of Molecular Psychoimmunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0815, Japan
| | - Toshiyuki Fukada
- Molecular and Cellular Physiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, 770-8514, Japan
| | - Thomas B Bartnikas
- Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, 02912, USA
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11
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Iriana S, Asha K, Repak M, Sharma-Walia N. Hedgehog Signaling: Implications in Cancers and Viral Infections. Int J Mol Sci 2021; 22:1042. [PMID: 33494284 PMCID: PMC7864517 DOI: 10.3390/ijms22031042] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Revised: 01/11/2021] [Accepted: 01/14/2021] [Indexed: 12/14/2022] Open
Abstract
The hedgehog (SHH) signaling pathway is primarily involved in embryonic gut development, smooth muscle differentiation, cell proliferation, adult tissue homeostasis, tissue repair following injury, and tissue polarity during the development of vertebrate and invertebrate organisms. GLIoma-associated oncogene homolog (GLI) family of zinc-finger transcription factors and smoothened (SMO) are the signal transducers of the SHH pathway. Both SHH ligand-dependent and independent mechanisms activate GLI proteins. Various transcriptional mechanisms, posttranslational modifications (phosphorylation, ubiquitination, proteolytic processing, SUMOylation, and acetylation), and nuclear-cytoplasmic shuttling control the activity of SHH signaling pathway proteins. The dysregulated SHH pathway is associated with bone and soft tissue sarcomas, GLIomas, medulloblastomas, leukemias, and tumors of breast, lung, skin, prostate, brain, gastric, and pancreas. While extensively studied in development and sarcomas, GLI family proteins play an essential role in many host-pathogen interactions, including bacterial and viral infections and their associated cancers. Viruses hijack host GLI family transcription factors and their downstream signaling cascades to enhance the viral gene transcription required for replication and pathogenesis. In this review, we discuss a distinct role(s) of GLI proteins in the process of tumorigenesis and host-pathogen interactions in the context of viral infection-associated malignancies and cancers due to other causes. Here, we emphasize the potential of the Hedgehog (HH) pathway targeting as a potential anti-cancer therapeutic approach, which in the future could also be tested in infection-associated fatalities.
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12
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Kokkorakis N, Gaitanou M. Minibrain-related kinase/dual-specificity tyrosine-regulated kinase 1B implication in stem/cancer stem cells biology. World J Stem Cells 2020; 12:1553-1575. [PMID: 33505600 PMCID: PMC7789127 DOI: 10.4252/wjsc.v12.i12.1553] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 09/29/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023] Open
Abstract
Dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B), also known as minibrain-related kinase (MIRK) is one of the best functionally studied members of the DYRK kinase family. DYRKs comprise a family of protein kinases that are emerging modulators of signal transduction pathways, cell proliferation and differentiation, survival, and cell motility. DYRKs were found to participate in several signaling pathways critical for development and cell homeostasis. In this review, we focus on the DYRK1B protein kinase from a functional point of view concerning the signaling pathways through which DYRK1B exerts its cell type-dependent function in a positive or negative manner, in development and human diseases. In particular, we focus on the physiological role of DYRK1B in behavior of stem cells in myogenesis, adipogenesis, spermatogenesis and neurogenesis, as well as in its pathological implication in cancer and metabolic syndrome. Thus, understanding of the molecular mechanisms that regulate signaling pathways is of high importance. Recent studies have identified a close regulatory connection between DYRK1B and the hedgehog (HH) signaling pathway. Here, we aim to bring together what is known about the functional integration and cross-talk between DYRK1B and several signaling pathways, such as HH, RAS and PI3K/mTOR/AKT, as well as how this might affect cellular and molecular processes in development, physiology, and pathology. Thus, this review summarizes the major known functions of DYRK1B kinase, as well as the mechanisms by which DYRK1B exerts its functions in development and human diseases focusing on the homeostasis of stem and cancer stem cells.
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Affiliation(s)
- Nikolaos Kokkorakis
- Laboratory of Cellular and Molecular Neurobiology-Stem Cells, Hellenic Pasteur Institute, Athens 11521, Greece
| | - Maria Gaitanou
- Laboratory of Cellular and Molecular Neurobiology-Stem Cells, Hellenic Pasteur Institute, Athens 11521, Greece.
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Larsen LJ, Møller LB. Crosstalk of Hedgehog and mTORC1 Pathways. Cells 2020; 9:cells9102316. [PMID: 33081032 PMCID: PMC7603200 DOI: 10.3390/cells9102316] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Revised: 09/30/2020] [Accepted: 10/13/2020] [Indexed: 02/07/2023] Open
Abstract
Hedgehog (Hh) signaling and mTOR signaling, essential for embryonic development and cellular metabolism, are both coordinated by the primary cilium. Observations from cancer cells strongly indicate crosstalk between Hh and mTOR signaling. This hypothesis is supported by several studies: Evidence points to a TGFβ-mediated crosstalk; Increased PI3K/AKT/mTOR activity leads to increased Hh signaling through regulation of the GLI transcription factors; increased Hh signaling regulates mTORC1 activity positively by upregulating NKX2.2, leading to downregulation of negative mTOR regulators; GSK3 and AMPK are, as members of both signaling pathways, potentially important links between Hh and mTORC1 signaling; The kinase DYRK2 regulates Hh positively and mTORC1 signaling negatively. In contrast, both positive and negative regulation of Hh has been observed for DYRK1A and DYRK1B, which both regulate mTORC1 signaling positively. Based on crosstalk observed between cilia, Hh, and mTORC1, we suggest that the interaction between Hh and mTORC1 is more widespread than it appears from our current knowledge. Although many studies focusing on crosstalk have been carried out, contradictory observations appear and the interplay involving multiple partners is far from solved.
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Doheny D, Manore SG, Wong GL, Lo HW. Hedgehog Signaling and Truncated GLI1 in Cancer. Cells 2020; 9:cells9092114. [PMID: 32957513 PMCID: PMC7565963 DOI: 10.3390/cells9092114] [Citation(s) in RCA: 122] [Impact Index Per Article: 24.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2020] [Revised: 09/10/2020] [Accepted: 09/15/2020] [Indexed: 12/18/2022] Open
Abstract
The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform.
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Affiliation(s)
- Daniel Doheny
- Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA; (D.D.); (S.G.M.); (G.L.W.)
| | - Sara G. Manore
- Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA; (D.D.); (S.G.M.); (G.L.W.)
| | - Grace L. Wong
- Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA; (D.D.); (S.G.M.); (G.L.W.)
| | - Hui-Wen Lo
- Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA; (D.D.); (S.G.M.); (G.L.W.)
- Wake Forest Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA
- Correspondence: ; Tel.: +1-336-716-0695
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15
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Matissek SJ, Elsawa SF. GLI3: a mediator of genetic diseases, development and cancer. Cell Commun Signal 2020; 18:54. [PMID: 32245491 PMCID: PMC7119169 DOI: 10.1186/s12964-020-00540-x] [Citation(s) in RCA: 69] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Accepted: 02/27/2020] [Indexed: 02/07/2023] Open
Abstract
The transcription factor GLI3 is a member of the Hedgehog (Hh/HH) signaling pathway that can exist as a full length (Gli3-FL/GLI3-FL) or repressor (Gli3-R/GLI3-R) form. In response to HH activation, GLI3-FL regulates HH genes by targeting the GLI1 promoter. In the absence of HH signaling, GLI3 is phosphorylated leading to its partial degradation and the generation of GLI3-R which represses HH functions. GLI3 is also involved in tissue development, immune cell development and cancer. The absence of Gli3 in mice impaired brain and lung development and GLI3 mutations in humans are the cause of Greig cephalopolysyndactyly (GCPS) and Pallister Hall syndromes (PHS). In the immune system GLI3 regulates B, T and NK-cells and may be involved in LPS-TLR4 signaling. In addition, GLI3 was found to be upregulated in multiple cancers and was found to positively regulate cancerous behavior such as anchorage-independent growth, angiogenesis, proliferation and migration with the exception in acute myeloid leukemia (AML) and medulloblastoma where GLI plays an anti-cancerous role. Finally, GLI3 is a target of microRNA. Here, we will review the biological significance of GLI3 and discuss gaps in our understanding of this molecule. Video Abstract.
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Affiliation(s)
- Stephan J. Matissek
- Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, 46 College Rd Rudman 291, Durham, NH 03824 USA
| | - Sherine F. Elsawa
- Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, 46 College Rd Rudman 291, Durham, NH 03824 USA
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16
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Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors. Cells 2019; 9:cells9010010. [PMID: 31861467 PMCID: PMC7016899 DOI: 10.3390/cells9010010] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2019] [Revised: 12/13/2019] [Accepted: 12/15/2019] [Indexed: 12/21/2022] Open
Abstract
Brain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology.
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17
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Piirsoo A, Pink A, Kasak L, Kala M, Kasvandik S, Ustav M, Piirsoo M. Differential phosphorylation determines the repressor and activator potencies of GLI1 proteins and their efficiency in modulating the HPV life cycle. PLoS One 2019; 14:e0225775. [PMID: 31770404 PMCID: PMC6879148 DOI: 10.1371/journal.pone.0225775] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Accepted: 11/12/2019] [Indexed: 11/19/2022] Open
Abstract
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.
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Affiliation(s)
- Alla Piirsoo
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Anne Pink
- Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia
| | - Lagle Kasak
- Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia
| | - Martin Kala
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Sergo Kasvandik
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Mart Ustav
- Institute of Technology, University of Tartu, Tartu, Estonia
| | - Marko Piirsoo
- Institute of Technology, University of Tartu, Tartu, Estonia
- * E-mail:
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18
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Pietrobono S, Gagliardi S, Stecca B. Non-canonical Hedgehog Signaling Pathway in Cancer: Activation of GLI Transcription Factors Beyond Smoothened. Front Genet 2019; 10:556. [PMID: 31244888 PMCID: PMC6581679 DOI: 10.3389/fgene.2019.00556] [Citation(s) in RCA: 214] [Impact Index Per Article: 35.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2019] [Accepted: 05/24/2019] [Indexed: 12/16/2022] Open
Abstract
The Hedgehog-GLI (HH-GLI) pathway is a highly conserved signaling that plays a critical role in controlling cell specification, cell–cell interaction and tissue patterning during embryonic development. Canonical activation of HH-GLI signaling occurs through binding of HH ligands to the twelve-pass transmembrane receptor Patched 1 (PTCH1), which derepresses the seven-pass transmembrane G protein-coupled receptor Smoothened (SMO). Thus, active SMO initiates a complex intracellular cascade that leads to the activation of the three GLI transcription factors, the final effectors of the HH-GLI pathway. Aberrant activation of this signaling has been implicated in a wide variety of tumors, such as those of the brain, skin, breast, gastrointestinal, lung, pancreas, prostate and ovary. In several of these cases, activation of HH-GLI signaling is mediated by overproduction of HH ligands (e.g., prostate cancer), loss-of-function mutations in PTCH1 or gain-of-function mutations in SMO, which occur in the majority of basal cell carcinoma (BCC), SHH-subtype medulloblastoma and rhabdomyosarcoma. Besides the classical canonical ligand-PTCH1-SMO route, mounting evidence points toward additional, non-canonical ways of GLI activation in cancer. By non-canonical we refer to all those mechanisms of activation of the GLI transcription factors occurring independently of SMO. Often, in a given cancer type canonical and non-canonical activation of HH-GLI signaling co-exist, and in some cancer types, more than one mechanism of non-canonical activation may occur. Tumors harboring non-canonical HH-GLI signaling are less sensitive to SMO inhibition, posing a threat for therapeutic efficacy of these antagonists. Here we will review the most recent findings on the involvement of alternative signaling pathways in inducing GLI activity in cancer and stem cells. We will also discuss the rationale of targeting these oncogenic pathways in combination with HH-GLI inhibitors as a promising anti-cancer therapies.
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Affiliation(s)
- Silvia Pietrobono
- Tumor Cell Biology Unit - Core Research Laboratory, Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence, Italy
| | - Sinforosa Gagliardi
- Tumor Cell Biology Unit - Core Research Laboratory, Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence, Italy
| | - Barbara Stecca
- Tumor Cell Biology Unit - Core Research Laboratory, Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence, Italy
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19
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Montagnani V, Stecca B. Role of Protein Kinases in Hedgehog Pathway Control and Implications for Cancer Therapy. Cancers (Basel) 2019; 11:cancers11040449. [PMID: 30934935 PMCID: PMC6520855 DOI: 10.3390/cancers11040449] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2019] [Revised: 03/20/2019] [Accepted: 03/26/2019] [Indexed: 02/08/2023] Open
Abstract
Hedgehog (HH) signaling is an evolutionarily conserved pathway that is crucial for growth and tissue patterning during embryonic development. It is mostly quiescent in the adult, where it regulates tissue homeostasis and stem cell behavior. Aberrant reactivation of HH signaling has been associated to several types of cancer, including those in the skin, brain, prostate, breast and hematological malignancies. Activation of the canonical HH signaling is triggered by binding of HH ligand to the twelve-transmembrane protein PATCHED. The binding releases the inhibition of the seven-transmembrane protein SMOOTHENED (SMO), leading to its phosphorylation and activation. Hence, SMO activates the transcriptional effectors of the HH signaling, that belong to the GLI family of transcription factors, acting through a not completely elucidated intracellular signaling cascade. Work from the last few years has shown that protein kinases phosphorylate several core components of the HH signaling, including SMO and the three GLI proteins, acting as powerful regulatory mechanisms to fine tune HH signaling activities. In this review, we will focus on the mechanistic influence of protein kinases on HH signaling transduction. We will also discuss the functional consequences of this regulation and the possible implications for cancer therapy.
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Affiliation(s)
- Valentina Montagnani
- Core Research Laboratory⁻Institute for Cancer Research, Prevention and Clinical Network (ISPRO), 50139 Florence, Italy.
| | - Barbara Stecca
- Core Research Laboratory⁻Institute for Cancer Research, Prevention and Clinical Network (ISPRO), 50139 Florence, Italy.
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20
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The role of GLI-SOX2 signaling axis for gemcitabine resistance in pancreatic cancer. Oncogene 2018; 38:1764-1777. [PMID: 30382189 PMCID: PMC6408295 DOI: 10.1038/s41388-018-0553-0] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2017] [Revised: 08/03/2018] [Accepted: 09/14/2018] [Indexed: 12/21/2022]
Abstract
Pancreatic cancer, mostly pancreatic ductal adenocarcinomas (PDAC), is one of the most lethal cancers, with a dismal median survival around 8 months. PDAC is notoriously resistant to chemotherapy. Thus far, numerous attempts using novel targeted therapies and immunotherapies yielded limited clinical benefits for pancreatic cancer patients. It is hoped that delineating the molecular mechanisms underlying drug resistance in pancreatic cancer may provide novel therapeutic options. Using acquired gemcitabine resistant pancreatic cell lines, we revealed an important role of the GLI-SOX2 signaling axis for regulation of gemcitabine sensitivity in vitro and in animal models. Down-regulation of GLI transcriptional factors (GLI1 or GLI2), but not SMO signaling inhibition, reduces tumor sphere formation, a characteristics of tumor initiating cell (TIC). Down-regulation of GLI transcription factors also decreased expression of TIC marker CD24. Similarly, high SOX2 expression is associated with gemcitabine resistance whereas down-regulation of SOX2 sensitizes pancreatic cancer cells to gemcitabine treatment. We further revealed that elevated SOX2 expression is associated with an increase in GLI1 or GLI2 expression. Our ChIP assay revealed that GLI proteins are associated with a putative Gli binding site within the SOX2 promoter, suggesting a more direct regulation of SOX2 by GLI transcription factors. The relevance of our findings to human disease was revealed in human cancer specimens. We found that high SOX2 protein expression is associated with frequent tumor relapse and poor survival in stage II PDAC patients (all of them underwent gemcitabine treatment), indicating that reduced SOX2 expression or down-regulation of GLI transcription factors may be effective in sensitizing pancreatic cancer cells to gemcitabine treatment.
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21
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Diao Y, Rahman MFU, Vyatkin Y, Azatyan A, St Laurent G, Kapranov P, Zaphiropoulos PG. Identification of novel GLI1 target genes and regulatory circuits in human cancer cells. Mol Oncol 2018; 12:1718-1734. [PMID: 30098229 PMCID: PMC6166001 DOI: 10.1002/1878-0261.12366] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Revised: 07/03/2018] [Accepted: 07/26/2018] [Indexed: 01/12/2023] Open
Abstract
Hedgehog (HH) signaling is involved in many physiological processes, and pathway deregulation can result in a wide range of malignancies. Glioma‐associated oncogene 1 (GLI1) is a transcription factor and a terminal effector of the HH cascade. Despite its crucial role in tumorigenesis, our understanding of the GLI1 cellular targets is quite limited. In this study, we identified multiple new GLI1 target genes using a combination of different genomic surveys and then subjected them to in‐depth validation in human cancer cell lines. We were able to validate >90% of the new targets, which were enriched in functions involved in neurogenesis and regulation of transcription, in at least one type of follow‐up experiment. Strikingly, we found that RNA editing of GLI1 can modulate effects on the targets. Furthermore, one of the top targets, FOXS1, a gene encoding a transcription factor previously implicated in nervous system development, was shown to act in a negative feedback loop limiting the cellular effects of GLI1 in medulloblastoma and rhabdomyosarcoma cells. Moreover, FOXS1 is both highly expressed and positively correlated with GLI1 in medulloblastoma samples of the Sonic HH subgroup, further arguing for the existence of FOXS1/GLI1 interplay in human tumors. Consistently, high FOXS1 expression predicts longer relapse‐free survival in breast cancer. Overall, our findings open multiple new avenues in HH signaling pathway research and have potential for translational implications.
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Affiliation(s)
- Yumei Diao
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | | | - Yuri Vyatkin
- St. Laurent Institute, Cambridge, MA, USA.,AcademGene LLC, Novosibirsk, Russia
| | - Ani Azatyan
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
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22
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Sabol M, Trnski D, Musani V, Ozretić P, Levanat S. Role of GLI Transcription Factors in Pathogenesis and Their Potential as New Therapeutic Targets. Int J Mol Sci 2018; 19:2562. [PMID: 30158435 PMCID: PMC6163343 DOI: 10.3390/ijms19092562] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2018] [Revised: 08/17/2018] [Accepted: 08/25/2018] [Indexed: 02/05/2023] Open
Abstract
GLI transcription factors have important roles in intracellular signaling cascade, acting as the main mediators of the HH-GLI signaling pathway. This is one of the major developmental pathways, regulated both canonically and non-canonically. Deregulation of the pathway during development leads to a number of developmental malformations, depending on the deregulated pathway component. The HH-GLI pathway is mostly inactive in the adult organism but retains its function in stem cells. Aberrant activation in adult cells leads to carcinogenesis through overactivation of several tightly regulated cellular processes such as proliferation, angiogenesis, EMT. Targeting GLI transcription factors has recently become a major focus of potential therapeutic protocols.
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Affiliation(s)
- Maja Sabol
- Laboratory for Hereditary Cancer, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
| | - Diana Trnski
- Laboratory for Hereditary Cancer, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
| | - Vesna Musani
- Laboratory for Hereditary Cancer, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
| | - Petar Ozretić
- Laboratory for Hereditary Cancer, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
| | - Sonja Levanat
- Laboratory for Hereditary Cancer, Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia.
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23
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Singh R, Lauth M. Emerging Roles of DYRK Kinases in Embryogenesis and Hedgehog Pathway Control. J Dev Biol 2017; 5:E13. [PMID: 29615569 PMCID: PMC5831797 DOI: 10.3390/jdb5040013] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 11/17/2017] [Accepted: 11/18/2017] [Indexed: 12/19/2022] Open
Abstract
Hedgehog (Hh)/GLI signaling is an important instructive cue in various processes during embryonic development, such as tissue patterning, stem cell maintenance, and cell differentiation. It also plays crucial roles in the development of many pediatric and adult malignancies. Understanding the molecular mechanisms of pathway regulation is therefore of high interest. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) comprise a group of protein kinases which are emerging modulators of signal transduction, cell proliferation, survival, and cell differentiation. Work from the last years has identified a close regulatory connection between DYRKs and the Hh signaling system. In this manuscript, we outline the mechanistic influence of DYRK kinases on Hh signaling with a focus on the mammalian situation. We furthermore aim to bring together what is known about the functional consequences of a DYRK-Hh cross-talk and how this might affect cellular processes in development, physiology, and pathology.
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Affiliation(s)
- Rajeev Singh
- Philipps University Marburg, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor and Immune Biology (ZTI), Hans-Meerwein-Str. 3, 35043 Marburg, Germany.
| | - Matthias Lauth
- Philipps University Marburg, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor and Immune Biology (ZTI), Hans-Meerwein-Str. 3, 35043 Marburg, Germany.
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24
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Ehe BK, Lamson DR, Tarpley M, Onyenwoke RU, Graves LM, Williams KP. Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1. Biochem Biophys Res Commun 2017; 491:767-772. [PMID: 28735864 PMCID: PMC5594740 DOI: 10.1016/j.bbrc.2017.07.107] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Accepted: 07/19/2017] [Indexed: 12/21/2022]
Abstract
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.
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Affiliation(s)
- Ben K Ehe
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - David R Lamson
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - Michael Tarpley
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA
| | - Rob U Onyenwoke
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA; Department of Pharmaceutical Sciences, North Carolina Central University, Durham, NC 27707, USA
| | - Lee M Graves
- Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kevin P Williams
- Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA; Department of Pharmaceutical Sciences, North Carolina Central University, Durham, NC 27707, USA.
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25
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Ramsbottom SA, Pownall ME, Roelink H, Conway SJ. Regulation of Hedgehog Signalling Inside and Outside the Cell. J Dev Biol 2016; 4:23. [PMID: 27547735 PMCID: PMC4990124 DOI: 10.3390/jdb4030023] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The hedgehog (Hh) signalling pathway is conserved throughout metazoans and plays an important regulatory role in both embryonic development and adult homeostasis. Many levels of regulation exist that control the release, reception, and interpretation of the hedgehog signal. The fatty nature of the Shh ligand means that it tends to associate tightly with the cell membrane, and yet it is known to act as a morphogen that diffuses to elicit pattern formation. Heparan sulfate proteoglycans (HSPGs) play a major role in the regulation of Hh distribution outside the cell. Inside the cell, the primary cilium provides an important hub for processing the Hh signal in vertebrates. This review will summarise the current understanding of how the Hh pathway is regulated from ligand production, release, and diffusion, through to signal reception and intracellular transduction.
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Affiliation(s)
- Simon A. Ramsbottom
- Institute of Genetic Medicine, International Centre for Life, Newcastle University, NE1 3BZ Newcastle upon Tyne, UK
- Correspondence: ; Tel.: +44-(0)191-241-8612
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26
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Tamoxifen Treatment of Breast Cancer Cells: Impact on Hedgehog/GLI1 Signaling. Int J Mol Sci 2016; 17:308. [PMID: 26927093 PMCID: PMC4813171 DOI: 10.3390/ijms17030308] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2015] [Revised: 02/17/2016] [Accepted: 02/22/2016] [Indexed: 11/30/2022] Open
Abstract
The selective estrogen receptor (ER) modulator tamoxifen (TAM) has become the standard therapy for the treatment of ER+ breast cancer patients. Despite the obvious benefits of TAM, a proportion of patients acquire resistance to treatment, and this is a significant clinical problem. Consequently, the identification of possible mechanisms involved in TAM-resistance should help the development of new therapeutic targets. In this study, we present in vitro data using a panel of different breast cancer cell lines and demonstrate the modulatory effect of TAM on cellular proliferation and expression of Hedgehog signaling components, including the terminal effector of the pathway, the transcription factor GLI1. A variable pattern of expression following TAM administration was observed, reflecting the distinctive properties of the ER+ and ER− cell lines analyzed. Remarkably, the TAM-induced increase in the proliferation of the ER+ ZR-75-1 and BT474 cells parallels a sustained upregulation of GLI1 expression and its translocation to the nucleus. These findings, implicating a TAM-GLI1 signaling cross-talk, could ultimately be exploited not only as a means for novel prognostication markers but also in efforts to effectively target breast cancer subtypes.
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27
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Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance. Cancers (Basel) 2015; 7:2330-51. [PMID: 26633513 PMCID: PMC4695894 DOI: 10.3390/cancers7040894] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2015] [Revised: 11/12/2015] [Accepted: 11/20/2015] [Indexed: 12/18/2022] Open
Abstract
The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for aberrant GLI1 expression and discuss GLI1-mediated HH signaling on DNA damage responses, carcinogenesis and chemoresistance.
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Sadam H, Liivas U, Kazantseva A, Pruunsild P, Kazantseva J, Timmusk T, Neuman T, Palm K. GLI2 cell-specific activity is controlled at the level of transcription and RNA processing: Consequences to cancer metastasis. Biochim Biophys Acta Mol Basis Dis 2015; 1862:46-55. [PMID: 26459048 DOI: 10.1016/j.bbadis.2015.10.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2015] [Revised: 10/08/2015] [Accepted: 10/09/2015] [Indexed: 01/14/2023]
Abstract
High activity of GLI family zinc finger protein 2 (GLI2) promotes tumor progression. Removal of the repressor domain at the N terminus (GLI2∆N) by recombinant methods converts GLI2 into a powerful transcriptional activator. However, molecular mechanisms leading to the formation of GLI2∆N activator proteins have not been established. Herein we report for the first time that the functional activities of GLI2 are parted into different protein isoforms by alternative promoter usage, selection of alternative splicing, transcription initiation and termination sites. Functional studies using melanoma cells revealed that transcriptional regulation of GLI2 is TGFbeta-dependent and supports the predominant production of GLI2∆N and C-terminally truncated GLI2 (GLI2∆C) isoforms in cells with high migratory and invasive phenotype. Taken together, these results highlight the role of transcription and RNA processing as major processes in the regulation of GLI2 activity with severe impacts in cancer development.
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Affiliation(s)
- Helle Sadam
- Protobios Llc, Mäealuse 4, Tallinn 12618, Estonia; Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia
| | - Urmas Liivas
- Protobios Llc, Mäealuse 4, Tallinn 12618, Estonia
| | - Anna Kazantseva
- Protobios Llc, Mäealuse 4, Tallinn 12618, Estonia; Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia
| | - Priit Pruunsild
- Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia
| | | | - Tõnis Timmusk
- Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia
| | | | - Kaia Palm
- Protobios Llc, Mäealuse 4, Tallinn 12618, Estonia; Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
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Flemban A, Qualtrough D. The Potential Role of Hedgehog Signaling in the Luminal/Basal Phenotype of Breast Epithelia and in Breast Cancer Invasion and Metastasis. Cancers (Basel) 2015; 7:1863-84. [PMID: 26389956 PMCID: PMC4586799 DOI: 10.3390/cancers7030866] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Revised: 09/09/2015] [Accepted: 09/10/2015] [Indexed: 12/18/2022] Open
Abstract
The epithelium of the lactiferous ducts in the breast is comprised of luminal epithelial cells and underlying basal myoepithelial cells. The regulation of cell fate and transit of cells between these two cell types remains poorly understood. This relationship becomes of greater importance when studying the subtypes of epithelial breast carcinoma, which are categorized according to their expression of luminal or basal markers. The epithelial mesenchymal transition (EMT) is a pivotal event in tumor invasion. It is important to understand mechanisms that regulate this process, which bears relation to the normal dynamic of epithelial/basal phenotype regulation in the mammary gland. Understanding this process could provide answers for the regulation of EMT in breast cancer, and thereby identify potential targets for therapy. Evidence points towards a role for hedgehog signaling in breast tissue homeostasis and also in mammary neoplasia. This review examines our current understanding of role of the hedgehog-signaling (Hh) pathway in breast epithelial cells both during breast development and homeostasis and to assess the potential misappropriation of Hh signals in breast neoplasia, cancer stem cells and tumor metastasis via EMT.
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Affiliation(s)
- Arwa Flemban
- Department of Biological, Biomedical and Analytical Sciences, Faculty of Health and Applied Sciences, University of West of England, Bristol BS16 1QY, UK.
- Department of Pathology, Faculty of Medicine, Umm Al-Qura University, Makkah 24382, Saudi Arabia.
| | - David Qualtrough
- Department of Biological, Biomedical and Analytical Sciences, Faculty of Health and Applied Sciences, University of West of England, Bristol BS16 1QY, UK.
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Schneider P, Miguel Bayo-Fina J, Singh R, Kumar Dhanyamraju P, Holz P, Baier A, Fendrich V, Ramaswamy A, Baumeister S, Martinez ED, Lauth M. Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1. Nat Commun 2015; 6:8023. [PMID: 26310823 PMCID: PMC4552080 DOI: 10.1038/ncomms9023] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2014] [Accepted: 07/09/2015] [Indexed: 12/12/2022] Open
Abstract
The Down syndrome-associated DYRK1A kinase has been reported as a stimulator of the developmentally important Hedgehog (Hh) pathway, but cells from Down syndrome patients paradoxically display reduced Hh signalling activity. Here we find that DYRK1A stimulates GLI transcription factor activity through phosphorylation of general nuclear localization clusters. In contrast, in vivo and in vitro experiments reveal that DYRK1A kinase can also function as an inhibitor of endogenous Hh signalling by negatively regulating ABLIM proteins, the actin cytoskeleton and the transcriptional co-activator MKL1 (MAL). As a final effector of the DYRK1A-ABLIM-actin-MKL1 sequence, we identify the MKL1 interactor Jumonji domain demethylase 1A (JMJD1A) as a novel Hh pathway component stabilizing the GLI1 protein in a demethylase-independent manner. Furthermore, a Jumonji-specific small-molecule antagonist represents a novel and powerful inhibitor of Hh signal transduction by inducing GLI1 protein degradation in vitro and in vivo.
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Affiliation(s)
- Philipp Schneider
- Department of Medicine, Philipps University, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology, Hans-Meerwein-Street 3, 35043 Marburg, Germany
| | - Juan Miguel Bayo-Fina
- Department of Pharmacology, UT Southwestern Medical Center, 6000 Harry Hines boulevard, Dallas, Texas 75390-8593, USA
| | - Rajeev Singh
- Department of Medicine, Philipps University, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology, Hans-Meerwein-Street 3, 35043 Marburg, Germany
| | - Pavan Kumar Dhanyamraju
- Department of Medicine, Philipps University, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology, Hans-Meerwein-Street 3, 35043 Marburg, Germany
| | - Philipp Holz
- Department of Medicine, Philipps University, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology, Hans-Meerwein-Street 3, 35043 Marburg, Germany
| | - Aninja Baier
- Department of Surgery, Philipps University, Baldingerstraße 1, 35033 Marburg, Germany
| | - Volker Fendrich
- Department of Surgery, Philipps University, Baldingerstraße 1, 35033 Marburg, Germany
| | - Annette Ramaswamy
- Department of Pathology, Philipps University, Baldingerstraße 1, 35033 Marburg, Germany
| | - Stefan Baumeister
- Department of Biology, Philipps University, Karl-von-Frisch-Straße 8, 35043 Marburg, Germany
| | - Elisabeth D. Martinez
- Department of Pharmacology, UT Southwestern Medical Center, 6000 Harry Hines boulevard, Dallas, Texas 75390-8593, USA
| | - Matthias Lauth
- Department of Medicine, Philipps University, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology, Hans-Meerwein-Street 3, 35043 Marburg, Germany
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Kumar RMR, Fuchs B. Hedgehog signaling inhibitors as anti-cancer agents in osteosarcoma. Cancers (Basel) 2015; 7:784-94. [PMID: 25985215 PMCID: PMC4491684 DOI: 10.3390/cancers7020784] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Revised: 04/30/2015] [Accepted: 05/07/2015] [Indexed: 12/31/2022] Open
Abstract
Osteosarcoma is a rare type of cancer associated with a poor clinical outcome. Even though the pathologic characteristics of OS are well established, much remains to be understood, particularly at the molecular signaling level. The molecular mechanisms of osteosarcoma progression and metastases have not yet been fully elucidated and several evolutionary signaling pathways have been found to be linked with osteosarcoma pathogenesis, especially the hedgehog signaling (Hh) pathway. The present review will outline the importance and targeting the hedgehog signaling (Hh) pathway in osteosarcoma tumor biology. Available data also suggest that aberrant Hh signaling has pro-migratory effects and leads to the development of osteoblastic osteosarcoma. Activation of Hh signaling has been observed in osteosarcoma cell lines and also in primary human osteosarcoma specimens. Emerging data suggests that interference with Hh signal transduction by inhibitors may reduce osteosarcoma cell proliferation and tumor growth thereby preventing osteosarcomagenesis. From this perspective, we outline the current state of Hh pathway inhibitors in osteosarcoma. In summary, targeting Hh signaling by inhibitors promise to increase the efficacy of osteosarcoma treatment and improve patient outcome.
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Affiliation(s)
- Ram Mohan Ram Kumar
- Laboratory for Orthopaedic Research, Balgrist University Hospital, Sarcoma Center-UZH University of Zurich, Zurich 8008, Switzerland.
| | - Bruno Fuchs
- Laboratory for Orthopaedic Research, Balgrist University Hospital, Sarcoma Center-UZH University of Zurich, Zurich 8008, Switzerland.
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Cherepanov SA, Baklaushev VP, Gabashvili AN, Shepeleva II, Chekhonin VP. [Hedgehog signaling in the pathogenesis of neuro-oncology diseases]. BIOMEDITSINSKAIA KHIMIIA 2015; 61:332-342. [PMID: 26215410 DOI: 10.18097/pbmc20156103332] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/07/2025]
Abstract
The review summarizes current knowledge on the Hedgehog signaling pathway, its role in normal embryogenesis and/or initiation and progression of neuro-oncological diseases, especially of high-grade gliomas, the most malignant neuroepithelial tumors. The main proteins forming the Hedgehog signaling pathway include Shh, PTCH1, SMO, HHIP, SUFU and GLI1 isoforms. Effects of other signaling pathways on the family of transcription factors GLI and other proteins are described. The review summarizes modern data about the impact of the Hedgehog signaling pathway on proliferation, migration activity and invasiveness, and also on tumor neoangiogenesis and tumor cell chemoresistance. The role of the Hedgehog signaling pathway in origin of cancer stem cells and epithelial-mesenchymal transition is also analyzed. Some prospects for new anticancer drugs acting on components of the Hedgehog signaling pathway inhibitors are demonstrated.
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Affiliation(s)
- S A Cherepanov
- Pirogov Russian National Research Medical University, Moscow, Russia
| | - V P Baklaushev
- Pirogov Russian National Research Medical University, Moscow, Russia
| | - A N Gabashvili
- Pirogov Russian National Research Medical University, Moscow, Russia
| | - I I Shepeleva
- Pirogov Russian National Research Medical University, Moscow, Russia
| | - V P Chekhonin
- Pirogov Russian National Research Medical University, Moscow, Russia
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Fan YH, Ding J, Nguyen S, Liu XJ, Xu G, Zhou HY, Duan NN, Yang SM, Zern MA, Wu J. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells. Oncogene 2015; 35:116-24. [DOI: 10.1038/onc.2015.67] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2014] [Accepted: 01/14/2015] [Indexed: 02/07/2023]
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Diao Y, Rahman MFU, Villegas VE, Wickström M, Johnsen JI, Zaphiropoulos PG. The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling. BMC Cancer 2014; 14:600. [PMID: 25134527 PMCID: PMC4152578 DOI: 10.1186/1471-2407-14-600] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Accepted: 08/11/2014] [Indexed: 01/20/2023] Open
Abstract
Background The crosstalk between Hedgehog (HH) signaling and other signal transduction cascades has been extensively studied in different cancers. In neuroblastoma, mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. Moreover, S6K1 kinase has been shown to phosphorylate GLI1, the effector of HH signaling, promoting GLI1 transcriptional activity and oncogenic function in esophageal adenocarcinoma. In this study, we examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma. Methods siRNA knockdowns were used to suppress S6K1 and GLI1 expression, and the siRNA effects were validated by real-time PCR and Western blotting. Cell proliferation analysis was performed with the EdU incorporation assay. Cytotoxic analysis with increasing concentrations of PI3K/mTOR and GLI inhibitors, individually and in combination, was used to determine drug response. Results Although knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. GLI1 overexpression can not rescue the reduced proliferation elicited by S6K1 knockdown. Moreover, inhibitors of PI3K/mTOR and GLI signaling reduced neuroblastoma cell growth, but no additional growth inhibitory effects were detected when the two classes of drugs were combined. Conclusion Our results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-600) contains supplementary material, which is available to authorized users.
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The Yes-associated protein controls the cell density regulation of Hedgehog signaling. Oncogenesis 2014; 3:e112. [PMID: 25111861 PMCID: PMC5189961 DOI: 10.1038/oncsis.2014.27] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2014] [Revised: 06/03/2014] [Accepted: 07/02/2014] [Indexed: 12/18/2022] Open
Abstract
The evolutionarily conserved Hedgehog (Hh) signaling pathway is essential for correct embryogenesis and is misregulated in several malignancies. In cell culture, Hh-sensitive cells display a striking dependence on cell density with active Hh signaling requiring cell-to-cell contact. As the Hippo/YAP system is tightly linked to cell density control and contact inhibition, we investigated the cross-talk between the two pathways. Our data reveal that the suppression of Hh signaling in the absence of cellular contacts is independent of primary cilia and is mediated by the YAP oncogene. Overexpression of YAP blocks Hh signaling whereas RNA interference-mediated knockdown of YAP enhances Hh/GLI activity. Despite this negative regulation, Hh signaling promotes YAP activity through post-transcriptional mechanisms, resulting in a negative feedback loop. In vivo, we found strong nuclear YAP immunoreactivity restricted to compartments with low Hh pathway activity in human and mouse pancreatic cancer. Finally, we identified protease-activated receptors (PARs) as molecules being able to override the inverse Hippo/Hh regulation, potentially giving tumors a mechanism to utilize both oncogenic pathways in parallel.
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Guo KT, Fu P, Juerchott K, Motaln H, Selbig J, Lah T, Tonn JC, Schichor C. The expression of Wnt-inhibitor DKK1 (Dickkopf 1) is determined by intercellular crosstalk and hypoxia in human malignant gliomas. J Cancer Res Clin Oncol 2014; 140:1261-70. [PMID: 24770633 DOI: 10.1007/s00432-014-1642-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2014] [Accepted: 03/07/2014] [Indexed: 11/30/2022]
Abstract
OBJECTIVE Wnt signalling pathways regulate proliferation, motility and survival in a variety of human cell types. Dickkopf 1 (DKK1) gene codes for a secreted Wnt inhibitory factor. It functions as tumour suppressor gene in breast cancer and as a pro-apoptotic factor in glioma cells. In this study, we aimed to demonstrate whether the different expression of DKK1 in human glioma-derived cells is dependent on microenvironmental factors like hypoxia and regulated by the intercellular crosstalk with bone-marrow-derived mesenchymal stem cells (bmMSCs). METHODS Glioma cell line U87-MG, three cell lines from human glioblastoma grade IV (glioma-derived mesenchymal stem cells) and three bmMSCs were selected for the experiment. The expression of DKK1 in cell lines under normoxic/hypoxic environment or co-culture condition was measured using real-time PCR and enzyme-linked immunoadsorbent assay. The effect of DKK1 on cell migration and proliferation was evaluated by in vitro wound healing assays and sulphorhodamine assays, respectively. RESULTS Glioma-derived cells U87-MG displayed lower DKK1 expression compared with bmMSCs. Hypoxia led to an overexpression of DKK1 in bmMSCs and U87-MG when compared to normoxic environment, whereas co-culture of U87-MG with bmMSCs induced the expression of DKK1 in both cell lines. Exogenous recombinant DKK1 inhibited cell migration on all cell lines, but did not have a significant effect on cell proliferation of bmMSCs and glioma cell lines. CONCLUSION In this study, we showed for the first time that the expression of DKK1 was hypoxia dependent in human malignant glioma cell lines. The induction of DKK1 by intracellular crosstalk or hypoxia stimuli sheds light on the intense adaption of glial tumour cells to environmental alterations.
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Affiliation(s)
- Ke-Tai Guo
- Tumour-Biological Laboratory, Department of Neurosurgery, Ludwig-Maximilians-University, Campus Grosshadern, Munich, Germany,
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Niu Y, Li F, Tang B, Shi Y, Hao Y, Yu P. Clinicopathological correlation and prognostic significance of sonic hedgehog protein overexpression in human gastric cancer. INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY 2014; 7:5144-5153. [PMID: 25197388 PMCID: PMC4152078] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Received: 06/04/2014] [Accepted: 07/23/2014] [Indexed: 06/03/2023]
Abstract
OBJECTIVES This study investigated the expression of Sonic Hedgehog (Shh) protein in gastric cancer, and correlated it with clinicopathological parameters. The prognostic significance of Shh protein was analyzed. METHODS Shh protein expression was evaluated in 113 cases of gastric cancer and 60 cases of normal gastric mucosa. The immunoreactivity was scored semi quantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely non-overexpression group with score 0 or 1, and overexpression group with score 2 or 3. The overexpression of Shh protein was correlated with clinicopathological parameters. Survival analysis was then performed to determine the Shh protein prognostic significance in gastric cancer. RESULTS In immunohistochemistry study, nineteen (31.7%) normal gastric mucosa revealed Shh protein overexpression, while eighty-one (71.7%) gastric cancer revealed overexpression. The expression of Shh protein were significantly higher in gastric cancer tissues than in normal gastric mucosa (P < 0.001), which was statistically correlated with age (P = 0.006), tumor differentiation (P < 0.001), depth of invasion (P = 0.042), pathologic staging (P = 0.017), and nodal metastasis (P = 0.019). We found no significant difference in both overall and disease free survival rates between Shh overexpression and non-expression groups P = 0.168 and 0.071). However, Shh overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 1.187, P = 0.041). CONCLUSIONS Shh protein expression is upregulated and is statistically correlated with age, tumor differentiation, depth of invasion, pathologic staging, and nodal metastasis. The Shh protein overexpression is a significant independent prognostic factor in multivariate Cox regression analysis in gastric cancer.
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Affiliation(s)
- Yanyang Niu
- General Surgery Center of People’s Liberation Army, Military General Surgery Center, Southwest Hospital, The Third Military Medical UniversityGaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China
| | - Fang Li
- Department of Stomatology, Southwest Hospital, The Third Military Medical UniversityGaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China
| | - Bo Tang
- General Surgery Center of People’s Liberation Army, Military General Surgery Center, Southwest Hospital, The Third Military Medical UniversityGaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China
| | - Yan Shi
- General Surgery Center of People’s Liberation Army, Military General Surgery Center, Southwest Hospital, The Third Military Medical UniversityGaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China
| | - Yingxue Hao
- General Surgery Center of People’s Liberation Army, Military General Surgery Center, Southwest Hospital, The Third Military Medical UniversityGaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China
| | - Peiwu Yu
- General Surgery Center of People’s Liberation Army, Military General Surgery Center, Southwest Hospital, The Third Military Medical UniversityGaotanyan Street, Shapingba District, Chongqing 400038, People’s Republic of China
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Villegas VE, Rahman MFU, Fernandez-Barrena MG, Diao Y, Liapi E, Sonkoly E, Ståhle M, Pivarcsi A, Annaratone L, Sapino A, Ramírez Clavijo S, Bürglin TR, Shimokawa T, Ramachandran S, Kapranov P, Fernandez-Zapico ME, Zaphiropoulos PG. Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1. Mol Oncol 2014; 8:912-26. [PMID: 24726458 PMCID: PMC4082767 DOI: 10.1016/j.molonc.2014.03.009] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2013] [Revised: 02/18/2014] [Accepted: 03/11/2014] [Indexed: 12/01/2022] Open
Abstract
Non‐coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non‐coding RNA located head‐to‐head with the gene encoding the Glioma‐associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer‐associated signaling pathways. The expression of this three‐exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up‐regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFβ and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up‐regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non‐coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non‐coding RNA‐based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor.
A novel negative feedback loop on Hedgehog signaling is demonstrated. The mechanism involves a non‐coding RNA antisense to the GLI1 gene, GLI1AS. GLI1AS is shown to be a target gene of the GLI1 transcription factor. GLI1AS represses gene expression at the GLI1/GLI1AS locus. GLI1AS acts as an epigenetic modifier eliciting repressive chromatin marks.
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Affiliation(s)
- Victoria E Villegas
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden; Faculty of Natural Sciences and Mathematics & Doctoral Program in Biomedical Sciences, Universidad del Rosario, Bogotá, Colombia
| | | | | | - Yumei Diao
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | - Eleni Liapi
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | - Enikö Sonkoly
- Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Solna, Sweden
| | - Mona Ståhle
- Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Solna, Sweden
| | - Andor Pivarcsi
- Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institutet, Solna, Sweden
| | - Laura Annaratone
- Department of Medical Sciences, University of Turin, Turin, Italy
| | - Anna Sapino
- Department of Medical Sciences, University of Turin, Turin, Italy
| | - Sandra Ramírez Clavijo
- Faculty of Natural Sciences and Mathematics & Doctoral Program in Biomedical Sciences, Universidad del Rosario, Bogotá, Colombia
| | - Thomas R Bürglin
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
| | - Takashi Shimokawa
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
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Mia MM, Boersema M, Bank RA. Interleukin-1β attenuates myofibroblast formation and extracellular matrix production in dermal and lung fibroblasts exposed to transforming growth factor-β1. PLoS One 2014; 9:e91559. [PMID: 24622053 PMCID: PMC3951452 DOI: 10.1371/journal.pone.0091559] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2013] [Accepted: 02/13/2014] [Indexed: 12/11/2022] Open
Abstract
One of the most potent pro-fibrotic cytokines is transforming growth factor (TGFβ). TGFβ is involved in the activation of fibroblasts into myofibroblasts, resulting in the hallmark of fibrosis: the pathological accumulation of collagen. Interleukin-1β (IL1β) can influence the severity of fibrosis, however much less is known about the direct effects on fibroblasts. Using lung and dermal fibroblasts, we have investigated the effects of IL1β, TGFβ1, and IL1β in combination with TGFβ1 on myofibroblast formation, collagen synthesis and collagen modification (including prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase), and matrix metalloproteinases (MMPs). We found that IL1β alone has no obvious pro-fibrotic effect on fibroblasts. However, IL1β is able to inhibit the TGFβ1-induced myofibroblast formation as well as collagen synthesis. Glioma-associated oncogene homolog 1 (GLI1), the Hedgehog transcription factor that is involved in the transformation of fibroblasts into myofibroblasts is upregulated by TGFβ1. The addition of IL1β reduced the expression of GLI1 and thereby also indirectly inhibits myofibroblast formation. Other potentially anti-fibrotic effects of IL1β that were observed are the increased levels of MMP1, −2, −9 and −14 produced by fibroblasts exposed to TGFβ1/IL1β in comparison with fibroblasts exposed to TGFβ1 alone. In addition, IL1β decreased the TGFβ1-induced upregulation of lysyl oxidase, an enzyme involved in collagen cross-linking. Furthermore, we found that lung and dermal fibroblasts do not always behave identically towards IL1β. Suppression of COL1A1 by IL1β in the presence of TGFβ1 is more pronounced in lung fibroblasts compared to dermal fibroblasts, whereas a higher upregulation of MMP1 is seen in dermal fibroblasts. The role of IL1β in fibrosis should be reconsidered, and the differences in phenotypical properties of fibroblasts derived from different organs should be taken into account in future anti-fibrotic treatment regimes.
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Affiliation(s)
- Masum M. Mia
- Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands,
| | - Miriam Boersema
- Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands,
| | - Ruud A. Bank
- Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands,
- * E-mail:
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Interleukin-1β attenuates myofibroblast formation and extracellular matrix production in dermal and lung fibroblasts exposed to transforming growth factor-β1. PLoS One 2014. [PMID: 24622053 DOI: 10.1371/journal.pone.0091559.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
One of the most potent pro-fibrotic cytokines is transforming growth factor (TGFβ). TGFβ is involved in the activation of fibroblasts into myofibroblasts, resulting in the hallmark of fibrosis: the pathological accumulation of collagen. Interleukin-1β (IL1β) can influence the severity of fibrosis, however much less is known about the direct effects on fibroblasts. Using lung and dermal fibroblasts, we have investigated the effects of IL1β, TGFβ1, and IL1β in combination with TGFβ1 on myofibroblast formation, collagen synthesis and collagen modification (including prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase), and matrix metalloproteinases (MMPs). We found that IL1β alone has no obvious pro-fibrotic effect on fibroblasts. However, IL1β is able to inhibit the TGFβ1-induced myofibroblast formation as well as collagen synthesis. Glioma-associated oncogene homolog 1 (GLI1), the Hedgehog transcription factor that is involved in the transformation of fibroblasts into myofibroblasts is upregulated by TGFβ1. The addition of IL1β reduced the expression of GLI1 and thereby also indirectly inhibits myofibroblast formation. Other potentially anti-fibrotic effects of IL1β that were observed are the increased levels of MMP1, -2, -9 and -14 produced by fibroblasts exposed to TGFβ1/IL1β in comparison with fibroblasts exposed to TGFβ1 alone. In addition, IL1β decreased the TGFβ1-induced upregulation of lysyl oxidase, an enzyme involved in collagen cross-linking. Furthermore, we found that lung and dermal fibroblasts do not always behave identically towards IL1β. Suppression of COL1A1 by IL1β in the presence of TGFβ1 is more pronounced in lung fibroblasts compared to dermal fibroblasts, whereas a higher upregulation of MMP1 is seen in dermal fibroblasts. The role of IL1β in fibrosis should be reconsidered, and the differences in phenotypical properties of fibroblasts derived from different organs should be taken into account in future anti-fibrotic treatment regimes.
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Koso H, Tsuhako A, Lyons E, Ward JM, Rust AG, Adams DJ, Jenkins NA, Copeland NG, Watanabe S. Identification of FoxR2 as an oncogene in medulloblastoma. Cancer Res 2014; 74:2351-61. [PMID: 24599127 DOI: 10.1158/0008-5472.can-13-1523] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Medulloblastoma is the most common pediatric brain tumor, and in ∼25% of cases, it is driven by aberrant activation of the Sonic Hedgehog (SHH) pathway in granule neuron precursor (GNP) cells. In this study, we identified novel medulloblastoma driver genes through a transposon mutagenesis screen in the developing brain of wild-type and Trp53 mutant mice. Twenty-six candidates were identified along with established driver genes such as Gli1 and Crebbp. The transcription factor FoxR2, the most frequent gene identified in the screen, is overexpressed in a small subset of human medulloblastoma of the SHH subtype. Tgif2 and Alx4, 2 new putative oncogenes identified in the screen, are strongly expressed in the SHH subtype of human medulloblastoma. Mutations in these two genes were mutually exclusive with mutations in Gli1 and tended to cooccur, consistent with involvement in the SHH pathway. Notably, Foxr2, Tgif2, and Alx4 activated Gli-binding sites in cooperation with Gli1, strengthening evidence that they function in SHH signaling. In support of an oncogenic function, Foxr2 overexpression transformed NIH3T3 cells and promoted proliferation of GNPs, the latter of which was also observed for Tgif2 and Alx4. These findings offer forward genetic and functional evidence associating Foxr2, Tgif2, and Alx4 with SHH subtype medulloblastoma.
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Affiliation(s)
- Hideto Koso
- Authors' Affiliations: Division of Molecular and Developmental Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Division of Genetics and Genomics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore; Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; and Cancer Research Program, The Methodist Hospital Research Institute, Houston, Texas
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Hou X, Chen X, Zhang P, Fan Y, Ma A, Pang T, Song Z, Jin Y, Hao W, Liu F, Wang W, Wang Y. Inhibition of hedgehog signaling by GANT58 induces apoptosis and shows synergistic antitumor activity with AKT inhibitor in acute T cell leukemia cells. Biochimie 2014; 101:50-9. [PMID: 24394624 DOI: 10.1016/j.biochi.2013.12.019] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2013] [Accepted: 12/19/2013] [Indexed: 12/11/2022]
Abstract
The hedgehog (Hh) signaling pathways have a crucial role in cell proliferation and survival, and the de-regulation of these pathways can lead to tumorigenesis. Here we investigated the expression and function of these pathways in acute T lymphocytic leukemia cells (T-ALL). Profiling of Hh pathway members revealed common expression of key Hh signaling effectors in all T-ALL cells. We found that T-ALL cells were insensitive to specific Smoothened (SMO) inhibition following the use of low concentrations of the SMO antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT58 reduced expression of the target gene Patched 1 as well as GLI family zinc finger 1 (GLI1) and preferentially decreased the viability of T-ALL cells. We also found perifosine, a novel AKT inhibitor, down-regulated GLI1 protein by dephosphorylation of AKT and GSK3β dose-dependently and that pre-treatment with PD98059, a MEK/ERK pathway inhibitor, enhanced this down-regulation by 20%-30%. Then we questioned whether use of both GANT58 and AKT inhibitor together could confer a synergistic effect to decrease T-ALL cell viability. By applying the Chou-Talalay method, low concentration of GANT58 induced T-ALL cell death in a synergism fashion with perifosine or GSK690693 when used simultaneously. These findings indicate that the combined use of GANT58 and AKT inhibitor could help treat a broad range of malignant tumors in conjunction with existing cancer treatments.
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Affiliation(s)
- Xiaoming Hou
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China.
| | - Xing Chen
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Ping Zhang
- Department of Neurosurgery, Qi Lu Hospital, Shandong University, Jinan, PR China
| | - Youfei Fan
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Aihua Ma
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Tingting Pang
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Zhao Song
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Youpeng Jin
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Wei Hao
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Fengqin Liu
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Wei Wang
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China
| | - Yulin Wang
- Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, PR China.
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MicroRNA218 inhibits glioma migration and invasion via inhibiting glioma-associated oncogene homolog 1 expression at N terminus. Tumour Biol 2013; 35:3831-7. [PMID: 24357514 DOI: 10.1007/s13277-013-1507-3] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2013] [Accepted: 12/03/2013] [Indexed: 12/18/2022] Open
Abstract
Glioma is characterized by high invasion, migration and proliferation abilities. However, the molecular mechanism that triggers the development and recurrence of this tumor is also elusive. This study aims to investigate the biological function and molecular mechanism of microRNA218 in glioma. Human glioma samples were obtained and employed to investigate the correlation between microRNA218 and glioma pathological grading. Glioma cell viability was detected by the cell-counting kit-8 (CCK-8) cell counting assay. Transwell assay and wound-healing assay were employed to examine the migration and invasion of the glioma cells. The mRNA transcription and protein expression of glioma-associated oncogene homolog 1 (GLI1) were analyzed by quantitative RT-PCR and Western blot analysis, respectively. Southwestern blot assay was utilized to explore the possible interaction site of GLI1 and microRNA218. The results indicated that microRNA218 is significantly down-regulated in glioma samples and negatively correlated with the pathological grading. The cell viability was significantly decreased, and migration and invasion were significantly inhibited in microRNA218 treated cells, compared with un-treated cells. GLI1 was discovered acting as a functional downstream target of microRNA218, by which microRNA218 inhibited glioma cell migration and invasion. Southwestern blot result showed that microRNA218 targeted directly the N terminus of GLI1 molecular, and repressed the GLI1 expression in U87MG cells. In conclusion, microRNA218 could reduce the invasion and migration, and inhibit proliferation of glioma cells by suppressing the expression of GLI1 protein at the interacting with the N terminus.
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Zhu H, Carpenter RL, Han W, Lo HW. The GLI1 splice variant TGLI1 promotes glioblastoma angiogenesis and growth. Cancer Lett 2013; 343:51-61. [PMID: 24045042 DOI: 10.1016/j.canlet.2013.09.014] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2013] [Revised: 09/10/2013] [Accepted: 09/11/2013] [Indexed: 01/27/2023]
Abstract
We investigated truncated glioma-associated oncogene homolog 1 (TGLI1) that behaves as gain-of-function GLI1 and promotes tumor cell migration and invasion. Herein, we report that TGLI1 had a higher propensity than GLI1 to enhance glioblastoma angiogenesis and growth, both in vivo and in vitro. TGLI1 has gained the ability to enhance expression of pro-angiogenic heparanase. In patient glioblastomas, TGLI1 levels are correlated with heparanase expression. Together, we report that TGLI1 is a novel mediator of glioblastoma angiogenesis and that heparanase is a novel transcriptional target of TGLI1, shedding new light on the molecular pathways that support tumor angiogenesis and aggressive growth.
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Affiliation(s)
- Hu Zhu
- Department of Surgery, Division of Surgical Sciences, Durham, NC 27710, USA
| | | | - Woody Han
- Department of Surgery, Division of Surgical Sciences, Durham, NC 27710, USA
| | - Hui-Wen Lo
- Department of Surgery, Division of Surgical Sciences, Durham, NC 27710, USA; Duke Center for RNA Biology, Durham, NC 27710, USA; Duke Cancer Institute, Duke University School of Medicine, Durham, NC 27710, USA.
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Laner-Plamberger S, Wolff F, Kaser-Eichberger A, Swierczynski S, Hauser-Kronberger C, Frischauf AM, Eichberger T. Hedgehog/GLI signaling activates suppressor of cytokine signaling 1 (SOCS1) in epidermal and neural tumor cells. PLoS One 2013; 8:e75317. [PMID: 24058673 PMCID: PMC3769249 DOI: 10.1371/journal.pone.0075317] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Accepted: 08/13/2013] [Indexed: 12/29/2022] Open
Abstract
Sustained hedgehog (Hh) signaling mediated by the GLI transcription factors is implicated in many types of cancer. Identification of Hh/GLI target genes modulating the activity of other pathways involved in tumor development promise to open new ways for better understanding of tumor development and maintenance. Here we show that SOCS1 is a direct target of Hh/GLI signaling in human keratinocytes and medulloblastoma cells. SOCS1 is a potent inhibitor of interferon gamma (IFN-y)/STAT1 signaling. IFN-у/STAT1 signaling can induce cell cycle arrest, apoptosis and anti-tumor immunity. The transcription factors GLI1 and GLI2 activate the SOCS1 promoter, which contains five putative GLI binding sites, and GLI2 binding to the promoter was shown by chromatin immunoprecipitation. Consistent with a role of GLI in SOCS1 regulation, STAT1 phosphorylation is reduced in cells with active Hh/GLI signaling and IFN-у/STAT1 target gene activation is decreased. Furthermore, IFN-у signaling is restored by shRNA mediated knock down of SOCS1. Here, we identify SOCS1 as a novel Hh/GLI target gene, indicating a negative role of Hh/GLI pathway in IFN-y/STAT1 signaling.
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Affiliation(s)
- Sandra Laner-Plamberger
- Department of Molecular Biology, University of Salzburg, Salzburg, Austria
- Department of Blood Group Serology and Transfusion Medicine, University Hospital of Salzburg, Paracelsus Medical University, Salzburg, Austria
- Spinal Cord Injury & Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Salzburg, Austria
- * E-mail:
| | - Florian Wolff
- Department of Molecular Biology, University of Salzburg, Salzburg, Austria
| | - Alexandra Kaser-Eichberger
- Department of Molecular Biology, University of Salzburg, Salzburg, Austria
- Department of Ophthalmology, University Hospital, Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Stefan Swierczynski
- Department of Pathology, University Hospital of Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Cornelia Hauser-Kronberger
- Department of Pathology, University Hospital of Salzburg, Paracelsus Medical University, Salzburg, Austria
| | | | - Thomas Eichberger
- Department of Molecular Biology, University of Salzburg, Salzburg, Austria
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Kelemen O, Convertini P, Zhang Z, Wen Y, Shen M, Falaleeva M, Stamm S. Function of alternative splicing. Gene 2013; 514:1-30. [PMID: 22909801 PMCID: PMC5632952 DOI: 10.1016/j.gene.2012.07.083] [Citation(s) in RCA: 548] [Impact Index Per Article: 45.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2012] [Revised: 07/21/2012] [Accepted: 07/30/2012] [Indexed: 12/15/2022]
Abstract
Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in 'splicing programs', which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.
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Affiliation(s)
- Olga Kelemen
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
| | - Paolo Convertini
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
| | - Zhaiyi Zhang
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
| | - Yuan Wen
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
| | - Manli Shen
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
| | - Marina Falaleeva
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
| | - Stefan Stamm
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, United States of America
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Shimokawa T, Rahman MFU, Tostar U, Sonkoly E, Ståhle M, Pivarcsi A, Palaniswamy R, Zaphiropoulos PG. RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling. RNA Biol 2013; 10:321-33. [PMID: 23324600 PMCID: PMC3594290 DOI: 10.4161/rna.23343] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.
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Affiliation(s)
- Takashi Shimokawa
- Department of Biosciences and Nutrition; Karolinska Institutet; Huddinge, Sweden
- Advanced Radiation Biology Research Program; Research Center for Charged Particle Therapy; National Institute of Radiological Sciences; Chiba-shi, Chiba, Japan
| | | | - Ulrica Tostar
- Department of Biosciences and Nutrition; Karolinska Institutet; Huddinge, Sweden
| | - Enikö Sonkoly
- Unit of Dermatology; Department of Medicine; Karolinska Institutet; Solna, Sweden
| | - Mona Ståhle
- Unit of Dermatology; Department of Medicine; Karolinska Institutet; Solna, Sweden
| | - Andor Pivarcsi
- Unit of Dermatology; Department of Medicine; Karolinska Institutet; Solna, Sweden
| | - Ramesh Palaniswamy
- Department of Biosciences and Nutrition; Karolinska Institutet; Huddinge, Sweden
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A feedback regulation between Kindlin-2 and GLI1 in prostate cancer cells. FEBS Lett 2013; 587:631-8. [PMID: 23337877 DOI: 10.1016/j.febslet.2012.12.028] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2012] [Revised: 12/05/2012] [Accepted: 12/21/2012] [Indexed: 01/15/2023]
Abstract
Kindlin-2 is engaged in tumor progression. However, the mechanism accounting for Kindlin-2 regulation in tumor cells remained largely unknown. Here, we report a regulatory loop between Kindlin-2 and GLI1, an effector of Hedgehog signaling pathway. We show that Kindlin-2 is transcriptionally downregulated via GLI1 occupancy on the Kindlin-2 promoter. Adversely, we found that Kindlin-2 promotes GLI1 expression through a mechanism involving GSK3β inactivation and is independent of Smoothened. Functionally, knockdown of Kindlin-2 cooperates with cyclopamine, a Smoothened antagonist, to decrease the viability of prostate cancer cells. Taken together, targeting the Kindlin-2-GLI1 feedback loop may facilitate the killing of prostate cancer cells.
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Wickström M, Dyberg C, Shimokawa T, Milosevic J, Baryawno N, Fuskevåg OM, Larsson R, Kogner P, Zaphiropoulos PG, Johnsen JI. Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo. Int J Cancer 2012; 132:1516-24. [PMID: 22949014 DOI: 10.1002/ijc.27820] [Citation(s) in RCA: 89] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2012] [Accepted: 08/17/2012] [Indexed: 12/15/2022]
Abstract
Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.
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Affiliation(s)
- Malin Wickström
- Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden.
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Alternative transcription and alternative splicing in cancer. Pharmacol Ther 2012; 136:283-94. [PMID: 22909788 DOI: 10.1016/j.pharmthera.2012.08.005] [Citation(s) in RCA: 94] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2012] [Accepted: 08/01/2012] [Indexed: 01/27/2023]
Abstract
In recent years, the notion of "one gene makes one protein that functions in one signaling pathway" in mammalian cells has been shown to be overly simplistic. Recent genome-wide studies suggest that at least half of the human genes, including many therapeutic target genes, produce multiple protein isoforms through alternative splicing and alternative usage of transcription initiation and/or termination. For example, alternative splicing of the vascular endothelial growth factor gene (VEGFA) produces multiple protein isoforms, which display either pro-angiogenic or anti-angiogenic activities. Similarly, for the majority of human genes, the inclusion or exclusion of exonic sequences enhances the generation of transcript variants and/or protein isoforms that can vary in structure and functional properties. Many of the isoforms produced in this manner are tightly regulated during normal development but are misregulated in cancer cells. Altered expression of transcript variants and protein isoforms for numerous genes is linked with disease and its prognosis, and cancer cells manipulate regulatory mechanisms to express specific isoforms that confer drug resistance and survival advantages. Emerging insights indicate that modulating the expression of transcript and protein isoforms of a gene may hold the key to impeding tumor growth and act as a model for efficient targeting of disease-associated genes at the isoform level. This review highlights the role and regulation of alternative transcription and splicing mechanisms in generating the transcriptome, and the misuse and diagnostic/prognostic potential of alternative transcription and splicing in cancer.
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