Characterising the role of trim16 in differentiation, proliferation and tumour development in neuroblastoma

Abstract

Neuroblastoma is the most common solid tumour in childhood and represents a significant 15% of all cancer deaths in children. Retinoids are used in the conventional treatment of several human cancers, including neuroblastoma. Retinoids are essential for normal differentiation processes, and can induce terminal differentiation of cancer cells with minimal side-effects. TRIM16, a member of the RING B-box coiled-coil (RBCC)/Tripartite Motif (TRIM) protein family, is a positive transcriptional regulator of the retinoid anti-cancer signal. This characteristic led to the hypothesis that TRIM16 regulates cell proliferation and differentiation in neuroblastoma. This thesis examines TRIM16 s role in neuroblastoma proliferation and tumour development, and also investigates the function of TRIM16 s putative protein domains. TRIM16 overexpression was shown to decrease neuroblastoma proliferation in vitro. Significantly, overexpression of TRIM16 in a neuroblastoma xenograft mouse model also decreased tumour growth. Additionally, overexpression of TRIM16 enhanced retinoid-induced differentiation in vitro. This indicated a role of TRIM16 in neuroblastoma cell differentiation. Immunohistochemical staining in a transgenic neuroblastoma mouse model revealed that tumour cells have weak cytoplasmic TRIM16 expression compared with strong nuclear TRIM16 staining in newly differentiated ganglia cells. Furthermore, in vitro, immunofluorescence studies showed that strong nuclear expression of TRIM16 is also seen during the G1 cell cycle phase of synchronised neuroblastoma cells. Significantly, knockdown of TRIM16 affected several G1 cell cycle phase regulatory proteins including downregulation of p27 and Cyclin D1. Furthermore, domain deletion mutants demonstrated that TRIM16 s B-boxes were required for in vivo ubiquitin binding and its coiled-coil domain conferred TRIM16 homodimerisation ability. Moreover, TRIM16 also heterodimerised with other TRIM proteins; MID1, PML and TRIM24. Most importantly, both TRIM16 s nuclear localisation and growth inhibition traits were attributed to a region linking the coiled-coil and B30.2 domains of TRIM16. Taken together, this research strongly indicates that nuclear localisation of the TRIM16 protein is required for its role in neuroblastoma proliferation and differentiation. Furthermore, this study provides compelling evidence that TRIM16 is a novel regulator of differentiation and proliferation in neuroblastoma and is a potential tumour suppressor.