Original Article

Oncogene (2014) 33, 2040–2052; doi:10.1038/onc.2013.173; published online 27 May 2013

Phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis

X Xie1,2, L Piao1,2, G S Cavey3,4, M Old1,2, T N Teknos1,2, A K Mapp5 and Q Pan1,2

  1. 1Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA
  2. 2Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA
  3. 3Van Andel Research Institute, Grand Rapids, MI, USA
  4. 4Southwest Michigan Innovation Center, Kalamazoo, MI, USA
  5. 5Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

Correspondence: Professor Q Pan, Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Wexner Medical Center, 442 Tzagournis Medical Research, 420 West 12th Avenue, Columbus, OH 43210, USA. E-mail: Quintin.Pan@osumc.edu

Received 3 May 2012; Revised 19 March 2013; Accepted 28 March 2013
Advance online publication 27 May 2013

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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 Cε 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.

Keywords:

Head and neck cancer; protein kinase C; p300; cancer stem cells; cancer-initiating cells.