Abstract
Chordoma, a rare neoplasm derived from intraosseous notochordal remnants, is unresponsive to conventional chemotherapy and radiotherapy. Sonic Hedgehog (Shh) is a crucial fetal notochord–secreted morphogen that directs notochordal development. The aim of this study was to determine the functional roles and therapeutic potential of Shh-Gli1 signaling in chordomas. Tissue samples and clinical profiles were collected from 42 patients with chordoma. The chordoma cell lines U-CH1 and MUG-Chor1 were used for functional experiments. Shh-Gli1 signaling pathway genetic alterations were screened, and the functions of the identified novel variants were analyzed using in silico analyses, real-time quantitative PCR, and minigene assays. Ligand-dependent Shh-Gli1 signaling activation was assessed using single- and dual-label immunostaining, western blot analysis, and a Shh-responsive Gli-luciferase reporter assay. The small-molecule inhibitor vismodegib was used to target Shh-Gli1 signaling in vitro and in vivo. Overall, 44 genetic alterations were identified, including four novel variants (c.67_69dupCTG in SMO, c.-6_-4dupGGC and c.3306 + 83_3306 + 84insG in PTCH1, and c.183-67_183-66delinsA in SUFU). Shh, PTCH1, SMO, SUFU, and Gli1 were extensively expressed in chordomas, and higher Gli1 expression correlated with poorer prognosis. A luciferase reporter assay and dual-label immunostaining indicated the occurrence of juxtacrine ligand-dependent Shh-Gli1 signaling activation. Vismodegib significantly inhibited cell proliferation and induced apoptosis and G1/S cell cycle arrest. In vivo investigation demonstrated that vismodegib effectively inhibited chordoma xenograft growth. This current preclinical evidence elucidates the therapeutic potential of Shh-Gli1 signaling pathway targeting for chordoma treatment. Vismodegib may be a promising targeted agent, and further clinical trials are warranted.
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References
Lee IJ, Lee RJ, Fahim DK. Prognostic factors and survival outcome in patients with chordoma in the United States: a population-based analysis. World Neurosurg. 2017;104:346–55.
Chen H, Garbutt CC, Spentzos D, Choy E, Hornicek FJ, Duan Z. Expression and Therapeutic Potential of SOX9 in Chordoma. Clin Cancer Res. 2017;23:5176–86.
Ahmed AT, Abdel-Rahman O, Morsy M, Mustafa K, Testini P, Aleem IS, et al. Management of sacrococcygeal chordoma: a systematic review and meta-analysis of observational studies. Spine (Philos Pa 1976). 2018;43:E1157–E1169.
Otani R, Mukasa A, Shin M, Omata M, Takayanagi S, Tanaka S, et al. Brachyury gene copy number gain and activation of the PI3K/Akt pathway: association with upregulation of oncogenic Brachyury expression in skull base chordoma. J Neurosurg. 2018;128:1428–37.
Kitamura Y, Sasaki H, Yoshida K. Genetic aberrations and molecular biology of skull base chordoma and chondrosarcoma. Brain Tumor Pathol. 2017;34:78–90.
Yakkioui Y, van Overbeeke JJ, Santegoeds R, van Engeland M, Temel Y. Chordoma: the entity. Biochim Biophys Acta. 2014;1846:655–69.
Williams S, Alkhatib B, Serra R. Development of the axial skeleton and intervertebral disc. Curr Top Dev Biol. 2019;133:49–90.
Zhu J, Kwan KM, Mackem S. Putative oncogene Brachyury (T) is essential to specify cell fate but dispensable for notochord progenitor proliferation and EMT. Proc Natl Acad Sci. 2016;113:3820–5.
Gotz W, Kasper M, Fischer G, Herken R. Intermediate filament typing of the human embryonic and fetal notochord. Cell Tissue Res. 1995;280:455–62.
Sun X, Hornicek F, Schwab JH. Chordoma: an update on the pathophysiology and molecular mechanisms. Curr Rev Musculoskelet Med. 2015;8:344–52.
Choi KS, Cohn MJ, Harfe BD. Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation. Dev Dyn. 2008;237:3953–8.
Cortes JE, Gutzmer R, Kieran MW, Solomon JA. Hedgehog signaling inhibitors in solid and hematological cancers. Cancer Treat Rev. 2019;76:41–50.
Cates JM, Itani DM, Coffin CM, Harfe BD. The sonic hedgehog pathway in chordoid tumours. Histopathology. 2010;56:978–9.
Akhavan-Sigari R, Schulz-Schaeffer W, Angelika Harcej A, Rohde V. The importance of the hedgehog signaling pathway in tumorigenesis of spinal and cranial chordoma. J Clin Med. 2019;8:248.
Scheil-Bertram S, Kappler R, von Baer A, Hartwig E, Sarkar M, Serra M, et al. Molecular profiling of chordoma. Int J Oncol. 2014;44:1041–55.
Wu F, Zhang Y, Sun B, McMahon AP, Wang Y. Hedgehog signaling: from basic biology to cancer therapy. Cell Chem Biol. 2017;24:252–80.
Sari IN, Phi LTH, Jun N, Wijaya YT, Lee S, Kwon HY. Hedgehog signaling in cancer: a prospective therapeutic target for eradicating cancer stem cells. Cells. 2018;7:208.
Begnini A, Tessari G, Turco A, Malerba G, Naldi L, Gotti E, et al. PTCH1 gene haplotype association with basal cell carcinoma after transplantation. Br J Dermatol. 2010;163:364–70.
Wang Y, Wang J, Pan W, Zhou Y, Xiao Y, Zhou K, et al. Common genetic variations in Patched1 (PTCH1) gene and risk of hirschsprung disease in the Han Chinese population. PLoS One. 2013;8:e75407.
Bari R, Hartford C, Chan WK, Vong Q, Li Y, Gan K, et al. Genome-wide single-nucleotide polymorphism analysis revealed SUFU suppression of acute graft-versus-host disease through downregulation of HLA-DR expression in recipient dendritic cells. Sci Rep.2015;5:11098.
Hancock DB, Eijgelsheim M, Wilk JB, Gharib SA, Loehr LR, Marciante KD, et al. Meta-analyses of genome-wide association studies identify multiple loci associated with pulmonary function. Nat Genet. 2010;42:45–52.
Magic M, Zeljic K, Jovandic S, Stepic J, Pejovic M, Colic S, et al. Hedgehog signaling pathway and vitamin D receptor gene variants as potential risk factors in odontogenic cystic lesions. Clin Oral Investig. 2019;23:2675–84.
Szkandera J, Pichler M, Absenger G, Stotz M, Weissmueller M, Samonigg H, et al. A functional germline variant in GLI1 implicates hedgehog signaling in clinical outcome of stage II and III colon carcinoma patients. Clin Cancer Res. 2014;20:1687–97.
Armas-Lopez L, Zuniga J, Arrieta O, Avila-Moreno F. The Hedgehog-GLI pathway in embryonic development and cancer: implications for pulmonary oncology therapy. Oncotarget. 2017;8:60684–703.
Regl G, Neill GW, Eichberger T, Kasper M, Ikram MS, Koller J, et al. Human GLI2 and GLI1 are part of a positive feedback mechanism in Basal Cell Carcinoma. Oncogene. 2002;21:5529–39.
Incardona JP, Roelink H. The role of cholesterol in Shh signaling and teratogen-induced holoprosencephaly. Cell Mol Life Sci. 2000;57:1709–19.
Skoda AM, Simovic D, Karin V, Kardum V, Vranic S, Serman L. The role of the Hedgehog signaling pathway in cancer: A comprehensive review. Bosn J Basic Med Sci. 2018;18:8–20.
Reifenberger J, Wolter M, Knobbe CB, Kohler B, Schonicke A, Scharwachter C, et al. Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas. Br J Dermatol. 2005;152:43–51.
Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW, Bonifas JM, et al. Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science. 1996;272:1668–71.
Raffel C, Jenkins RB, Frederick L, Hebrink D, Alderete B, Fults DW, et al. Sporadic medulloblastomas contain PTCH mutations. Cancer Res. 1997;57:842–5.
Taylor MD, Liu L, Raffel C, Hui CC, Mainprize TG, Zhang X, et al. Mutations in SUFU predispose to medulloblastoma. Nat Genet. 2002;31:306–10.
Berman DM, Karhadkar SS, Maitra A, Montes De Oca R, Gerstenblith MR, Briggs K, et al. Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature. 2003;425:846–51.
Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H, Nomura M, et al. Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Res. 2004;64:6071–4.
Theunissen JW, de Sauvage FJ. Paracrine Hedgehog signaling in cancer. Cancer Res. 2009;69:6007–10.
Tian H, Callahan CA, DuPree KJ, Darbonne WC, Ahn CP, Scales SJ, et al. Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis. Proc Natl Acad Sci USA. 2009;106:4254–9.
Sekulic A, Migden MR, Oro AE, Dirix L, Lewis KD, Hainsworth JD, et al. Efficacy and safety of vismodegib in advanced basal-cell carcinoma. N. Engl J Med. 2012;366:2171–9.
Duman-Scheel M, Weng L, Xin S, Du W. Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E. Nature. 2002;417:299–304.
Bigelow RL, Chari NS, Unden AB, Spurgers KB, Lee S, Roop DR, et al. Transcriptional regulation of bcl-2 mediated by the sonic hedgehog signaling pathway through gli-1. J Biol Chem. 2004;279:1197–205.
Amini P, Ettlin J, Opitz L, Clementi E, Malbon A, Markkanen E. An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing. BMC Mol Biol. 2017;18:22.
Sinicrope FA, Ruan SB, Cleary KR, Stephens LC, Lee JJ, Levin B. bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis. Cancer Res. 1995;55:237–41.
Vinall RL, Hwa K, Ghosh P, Pan CX, Lara PN Jr, de Vere White RW. Combination treatment of prostate cancer cell lines with bioactive soy isoflavones and perifosine causes increased growth arrest and/or apoptosis. Clin Cancer Res. 2007;13:6204–16.
Sebaugh JL. Guidelines for accurate EC50/IC50 estimation. Pharm Stat. 2011;10:128–34.
Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep. 1966;50:219–44.
Charan J, Kantharia ND. How to calculate sample size in animal studies? J Pharm Pharmacother. 2013;4:303–6.
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This work was supported by the National Natural Science Foundation of China (81641103 to XL; 81901202 to CY) and the Postdoctoral Science Foundation of China (2018M630047 to CY).
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Yang, C., Yong, L., Liang, C. et al. Genetic landscape and ligand-dependent activation of sonic hedgehog-Gli1 signaling in chordomas: a novel therapeutic target. Oncogene 39, 4711–4727 (2020). https://doi.org/10.1038/s41388-020-1324-2
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DOI: https://doi.org/10.1038/s41388-020-1324-2
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