Endocrine Abstracts (2015) 38 P148 | DOI: 10.1530/endoabs.38.P148

PTTG is phosphorylated at residue T60 and regulates p53 stability, in conjunction with PBF, in head and neck cancer

Bhavika Modasia1, Martin L Read1, Vicki E Smith1, Andy S Turnell2, Peter C Rae3, Rachel J Watkins3, Waraporn Imruetaicharoenchoke1, Vikki L Poole1, Hisham Mehanna3 & Chris J McCabe1


1Centre for Endocrinology, Diabetes and Metabolism, Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; 2School of Cancer Sciences, University of Birmingham, Birmingham, UK; 3Institute of Head and Neck Studies and Education, University of Birmingham, Birmingham, UK.


PTTG is a multifunctional proto-oncogene, overexpressed in thyroid, pituitary and other endocrine cancers. PTTG is also implicated in the pathogenesis of head and neck cancer, where high PTTG expression independently correlates with advanced tumour stage and reduced disease-free survival. Recently, abrogation of residue threonine-60 (T60) has been associated with altered PTTG half-life, chromosomal instability and cell invasion. We therefore generated a phospho-specific antibody against T60 PTTG. Transient transfection of wild-type PTTG resulted in a significant increase in T60-phosphorylated PTTG protein expression in HeLa cells (3-fold, p=0.001), which was blocked by a phospho-peptide. Detection was also lost with a PTTG-T60 mutant (T60A). Antibody specificity was further confirmed by immunoprecipitation assays. Paraffin-embedded formalin-fixed tumour sections were obtained for immunohistochemical analysis from patients with primary oropharyngeal squamous cell carcinoma. Abundant total PTTG protein expression was evident both in the cytoplasm and nucleus. In contrast, expression of T60-phosphorylated PTTG was predominantly nuclear. As interaction with its binding partner PBF facilitates PTTG nuclear localisation, and both proto-oncogenes alter p53 stability and function, we assessed the relative contributions of PTTG and PBF to p53 stability. Preliminary experiments demonstrated that transfection of wild-type PBF or PTTG into HPV-positive 93-VU-147T HNSCC cells decreased p53 protein levels compared to controls. Further, half-life studies demonstrated reduced p53 stability in 93-VU-147T cells transfected with either PBF or PTTG. Interestingly, transfection with a PBF mutant incapable of PTTG interaction, or a PTTG mutant unable to bind PBF, resulted in an initial decrease in p53 stability followed by subsequent stabilisation. These data indicate a potential role for both PTTG and PBF in modulation of p53 stability in head and neck cancers. Furthermore, PTTG is phosphorylated at residue T60 in head and neck tumours, which may alter its well described mitotic regulatory function.