title: Phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.
abstract: Emerging evidence indicates that Nanog is intimately involved in tumorigenesis, in part, through regulation of the cancer-initiating cell (CIC) population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human protein kinase Cɛ at multiple residues, including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene-promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration and the CIC population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer-initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.
authors.affiliation: 1] Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA [2] Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: X
authors.initials: X
authors.lastname: Xie
authors.affiliation: 1] Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA [2] Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: L
authors.initials: L
authors.lastname: Piao
authors.affiliation: 1] Van Andel Research Institute, Grand Rapids, MI, USA [2] Southwest Michigan Innovation Center, Kalamazoo, MI, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Cavey
authors.affiliation: 1] Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA [2] Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: M
authors.initials: M
authors.lastname: Old
authors.affiliation: 1] Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA [2] Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: T N
authors.initials: TN
authors.lastname: Teknos
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Mapp
authors.affiliation: 1] Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA [2] Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Pan
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D000071317:Nanog Homeobox Protein; D010766:Phosphorylation; D063150:Polycomb Repressive Complex 1 / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014183:Transplantation, Heterologous
chemical_list: C069077:BMI1 protein, human; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D013912:Threonine; D063150:Polycomb Repressive Complex 1; D051744:Protein Kinase C-epsilon