title: Inhibition of nestin suppresses stem cell phenotype of glioblastomas through the alteration of post-translational modification of heat shock protein HSPA8/HSC71.
abstract: Nestin, a class VI intermediate filament, was first described as a neuronal stem/progenitor cell marker. We previously reported that knockdown of nestin expression in human glioblastoma cells suppresses cell proliferation, migration, and invasion. In the present study, we examined the effect of nestin on stemness, and identified molecules involved in modulating nestin function in glioblastoma cells. Nestin expression was shown to be higher in high-grade gliomas than in low-grade gliomas. Furthermore, compared with control cells, nestin short hairpin RNA (shRNA)-transfected glioblastoma cells exhibited reduced sphere formation, decreased expression of NANOG, N-cadherin, CD133, and Oct-4, and decreased tumor size in vivo. To examine the proteins regulated by nestin in glioblastomas, we carried out two-dimensional electrophoresis using nestin shRNA-transfected glioblastoma cells. As a result, nestin shRNA-transfected glioblastoma cells exhibited a decrease in the level of phosphorylation of heat shock cognate 71 kDa protein (HSC71; gene HSPA8). From immunoprecipitation experiments, we demonstrated the direct binding of nestin, HSC71, and cyclin D1 in vitro. Overexpression of nestin in glioblastoma cells increased cell growth, sphere formation, and cell invasion. Transfection with HSC71 siRNA restored nestin expression and cell behavior; therefore, HSC71 knockdown will interfere with enhanced tumorigenic properties of glioblastoma cells that ectopically overexpress nestin. We have demonstrated that HSC71 and nestin regulate each other's expression levels or patterns, and that cyclin D1 is located downstream of nestin and HSC71. In conclusion, nestin regulates stemness, cell growth, and invasion in glioblastoma cells through the alteration of HSC71. Inhibition of nestin and HSC71 may thus be a useful molecular target in the treatment of glioblastomas.
authors.affiliation: Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. Electronic address: yoko_matsuda@tmghig.jp.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Matsuda
authors.affiliation: Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ishiwata
authors.affiliation: Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan; Division of Physiological Pathology, Department of Applied Science, School of Veterinary Nursing and Technology, Nippon Veterinary and Life Science University, 1-7-1, Kyonan-cho, Musashino-shi, Tokyo 180-8602.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Yoshimura
authors.affiliation: Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Hagio
authors.affiliation: Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, 35-2, Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
authors.forename: Tomio
authors.initials: T
authors.lastname: Arai
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D001921:Brain / Q000378:metabolism / Q000473:pathology; D015820:Cadherins / Q000235:genetics; D063646:Carcinogenesis / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006023:Glycoproteins / Q000235:genetics; D050883:HSC70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D013058:Mass Spectrometry; D016688:Mice, Inbred NOD; D000071317:Nanog Homeobox Protein; D064231:Nestin / Q000235:genetics / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D010455:Peptides / Q000235:genetics; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism*; D014183:Transplantation, Heterologous
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D015820:Cadherins; D006023:Glycoproteins; D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D064231:Nestin; D050814:Octamer Transcription Factor-3; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse
keywords: Glioblastoma; Heat shock cognate 71 kDa protein; Invasion; Nestin; Stem cells