Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors

BMC Clinical Pathology

Jerome B Rattner¹  Marvin J Fritzler¹  Joanna J Moser¹ 

[1]Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB, Canada

关键词: MGMT promoter methylation;    IDH1/2 mutation;    EGFR amplification;    Glioblastoma multiforme;    Distal appendages;    Basal body;    Centriole;    Cilium-pit;    Ciliogenesis;    Primary cilia;   

Others  :  1084476
DOI  :  10.1186/1472-6890-14-40

 received in 2013-12-30, accepted in 2014-09-09,  发布年份 2014

【 摘 要 】

Background

Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors.

Methods

Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy.

Results

We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium.

Conclusions

Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.

【 授权许可】

   
2014 Moser et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Moser JJ, Fritzler MJ, Ou Y, Rattner JB: The PCMbasal body/primary cilium coalition. Semin Cell Dev Biol 2010, 21:148-155.
• [2]Hassounah NB, Bunch TA, McDermott KM: Molecular pathways: the role of primary cilia in cancer progression and therapeutics with a focus on hedgehog signaling. Clin Cancer Res 2012, 18:2429-2435.
• [3]Davenport JR, Watts AJ, Roper VC, Croyle MJ, van Groen T, Wyss JM, Nagy TR, Kesterson RA, Yoder BK: Disruption of intraflagellar transport in adult mice leads to obesity and slow-onset cystic kidney disease. Curr Biol 2007, 17:1586-1594.
• [4]Satir P: Cilia biology: stop overeating now! Curr Biol 2007, 17:R963-R965.
• [5]Ingham PW: Transducing hedgehog: the story so far. EMBO J 1998, 17:3505-3511.
• [6]Dahmane N, Altaba A: Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 1999, 126:3089-3100.
• [7]Wallace VA: Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr Biol 1999, 9:445-448.
• [8]Ou YY, Mack GJ, Zhang M, Rattner JB: CEP110 and ninein are located in a specific domain of the centrosome associated with centrosome maturation. J Cell Sci 2002, 115:1825-1835.
• [9]Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DYR, Reiter JF: Vertebrate Smoothened functions at the primary cilium. Nature 2005, 437:1018-1021.
• [10]Haycraft C, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ, Yoder BK: Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet 2005, 1:e53.
• [11]Rohatgi R, Milenkovic L, Scott MP: Patched1 regulates hedgehog signaling at the primary cilium. Science 2007, 317:372-376.
• [12]Breunig JJ, Sarkisian MR, Arellano JI, Morozov YM, Ayoub AE, Sojitra S, Wang B, Flavell RA, Rakic P, Town T: Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc Natl Acad Sci 2008, 105:13127-13132.
• [13]Danilov AI, Gomes-Leal W, Ahlenius H, Kokaia Z, Carlemalm E, Lindvall O: Ultrastructural and antigenic properties of neural stem cells and their progeny in adult rat subventricular zone. Glia 2009, 57:136-152.
• [14]Badano JL, Mitsuma N, Beales PL, Katsanis N: The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet 2006, 7:125-148.
• [15]Wong SY, Seol AD, So PL, Ermilov AN, Bichakjian CK, Epstein EH, Dlugosz AA, Reiter JF: Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis. Nat Med 2009, 15:1055-1061.
• [16]Han YG, Kim HJ, Dlugosz AA, Ellison DW, Gilbertson RJ, Alvarez-Buylla A: Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med 2009, 15:1062-1065.
• [17]Moser JJ, Fritzler MJ, Rattner JB: Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells. BMC Cancer 2009, 9:448. BioMed Central Full Text
• [18]Louis D, Ohgaki H, Wiestler O, Cavenee W, Burger P, Jouvet A, Scheithauer B, Kleihues P: The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 2007, 114:547.
• [19]Ostrom QT, Gittleman H, Farah P, Ondracek A, Chen Y, Wolinsky Y, Stroup NE, Kruchko C, Barnholtz-Sloan JS: CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2006-2010. Neuro Oncol 2013, 15(suppl 2):ii1-ii56.
• [20]Wen PY, Kesari S: Malignant gliomas in adults. N Engl J Med 2008, 359:492-507.
• [21]Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C, Chin L, DePinho RA, Cavanee WK: Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 2007, 21:2683-2710.
• [22]Nobusawa S, Watanabe T, Kleihues P, Ohgaki H: IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas. Clin Cancer Res 2009, 15:6002-6007.
• [23]Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD: IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009, 360:765-773.
• [24]Zhang K, Wang XQ, Zhou B, Zhang L: The prognostic value of MGMT promoter methylation in Glioblastoma multiforme: a meta-analysis. Fam Cancer 2013, 12:449-458.
• [25]Perizzolo M, Winkfein B, Hui S, Krulicki W, Chan JA, Demetrick DJ: IDH mutation detection in formalin-fixed paraffin-embedded gliomas using multiplex PCR and single-base extension. Brain Pathol 2012, 22:619-624.
• [26]Williams NE, Frankel J: Regulation of microtubules in tetrahymena : I. electron microscopy of oral replacement. J Cell Biol 1973, 56:441-457.
• [27]Sorokin S: Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol 1962, 15:363-377.
• [28]Hagiwara H, Ohwada N, Aoki T, Takata K: Ciliogenesis and ciliary abnormalities. Med Electron Microsc 2000, 33:109-114.
• [29]Hagiwara H, Ohwada N, Takata K: Cell Biology of normal and abnormal ciliogenesis in the ciliated epithelium. In International Review of Cytology. Volume 234. Academic Press: Kwang WJ; 2004::101-141. ISBN 9780123646385
• [30]Tukachinsky H, Lopez LV, Salic A: A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFuGli protein complexes. J Cell Biol 2010, 191:415-428.
• [31]Schraml P, Frew IJ, Thoma CR, Boysen G, Struckmann K, Krek W, Moch H: Sporadic clear cell renal cell carcinoma but not the papillary type is characterized by severely reduced frequency of primary cilia. Mod Pathol 2008, 22:31-36.
• [32]Seeley ES, Carrière C, Goetze T, Longnecker DS, Korc M: Pancreatic cancer and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia. Cancer Res 2009, 69:422-430.
• [33]Yang Y, Roine N, Makela TP: CCRK depletion inhibits glioblastoma cell proliferation in a cilium-dependent manner. EMBO Rep 2013, 14:741-747.
• [34]Guarguaglini G, Duncan PI, Stierhof YD, Holmstrom T, Duensing S, Nigg EA: The Forkhead-associated domain protein Cep170 Interacts with Polo-like Kinase 1 and serves as a marker for mature centrioles. Mol Biol Cell 2005, 16:1095-1107.
• [35]Mogensen MM, Malik A, Piel M, Bouckson-Castaing V, Bornens M: Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein. J Cell Sci 2000, 113:3013-3023.
• [36]Chang P, Giddings TH, Winey M, Stearns T: epsilon-Tubulin is required for centriole duplication and microtubule organization. Nat Cell Biol 2003, 5:71-76.
• [37]Ishikawa H, Kubo A, Tsukita S, Tsukita S: Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia. Nat Cell Biol 2005, 7:517-524.
• [38]Soung NK, Park JE, Yu LR, Lee KH, Lee JM, Bang JK, Veenstra TD, Rhee K, Lee KS: Plk1-dependent and -independent roles of an ODF2 splice variant, hCenexin1, at the centrosome of somatic cells. Dev Cell 2009, 16:539-550.
• [39]Si Y, Egerer J, Fuchs E, Haas AK, Barr FA: Functional dissection of Rab GTPases involved in primary cilium formation. J Cell Biol 2007, 178:363-369.
• [40]Gromley A, Jurczyk A, Sillibourne J, Halilovic E, Mogensen M, Groisman I, Blomberg M, Doxsey S: A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase. J Cell Biol 2003, 161:535-545.
• [41]Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le Clech M, Nigg EA: Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol 2007, 179:321-330.
• [42]Sillibourne JE, Hurbain I, Grand-Perret T, Goud B, Tran P, Bornens M: Primary ciliogenesis requires the distal appendage component Cep123. Biol Open 2013.
• [43]Town T, Breunig JJ, Sarkisian MR, Spilianakis C, Ayoub AE, Liu X, Ferrandino AF, Gallagher AR, Li MO, Rakic P, Flavell RA: The stumpy gene is required for mammalian ciliogenesis. Proc Natl Acad Sci 2008, 105:2853-2858.
• [44]Garcia-Gonzalo FR, Corbit KC, Sirerol-Piquer MS, Ramaswami G, Otto EA, Noriega TR, Seol AD, Robinson JF, Bennett CL, Josifova DJ, García-Verdugo JM, Katsanis N, Hildebrandt F, Reiter JF: A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet 2011, 43:776-784.
• [45]Williams CL, Li C, Kida K, Inglis PN, Mohan S, Semenec L, Bialas NJ, Stupay RM, Chen N, Blacque OE, Yoder BK, Leroux MR: MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol 2011, 192:1023-1041.
• [46]Chih B, Liu P, Chinn Y, Chalouni C, Komuves LG, Hass PE, Sandoval W, Peterson AS: A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat Cell Biol 2012, 14:61-72.
• [47]Zhang D, Aravind L: Identification of novel families and classification of the C2 domain superfamily elucidate the origin and evolution of membrane targeting activities in eukaryotes. Gene 2010, 469:18-30.
• [48]Dowdle W, Robinson J, Kneist A, Sirerol-Piquer M, Frints S, Corbit K, Zaghloul N, van Lijnschoten G, Mulders L, Verver D, et al.: Disruption of a Ciliary B9 protein complex causes Meckel syndrome. Am J Hum Genet 2011, 89:94-110.
• [49]Gate D, Danielpour M, Levy R, Breunig J, Town T: Basic biology and mechanisms of neural ciliogenesis and the B9 family. Mol Neurobiol 2012, 45:564-570.