Chaiwatanasirikul, K.-A.; (2012) Clusterin (CLU) and its interacting proteins in cell signalling and neuroblastoma. Doctoral thesis , UCL (University College London).

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Abstract

Neuroblastoma is the most common extracranial solid tumour in children, with a high mortality rate among patients with aggressive disease. In a previous study we showed that Clusterin (CLU) inhibits the transcription factor NF-ϰB in a neuroblastoma cell line by stabilizing the NF-ϰB inhibitors (IϰBs). Moreover, suppression of CLU could elicit NF-ϰB activation and increased the expression of markers for the epithelial-to-mesenchymal transition (EMT), a developmental process utilized by aggressive cancer cells for invasion, in a mouse neuroblastoma model. The expression of CLU is also negatively regulated by the proto-oncogene MYCN, which is associated with aggressive stages of neuroblastoma tumours. Thus, we hypothesised that CLU is a tumour suppressor gene, which negatively regulates NF-ϰB and metastasis. In this study, we investigated the role of the different isoforms of CLU in the regulation of signalling pathways. We also aimed at identifying the precise mechanisms by which CLU regulates NF-ϰB. The results show that intracellular, but not secreted CLU, inhibits NF-ϰB activity. Interestingly, extracellular CLU (secreted CLU) positively regulates AKT and the Phosphoinositide-3 Kinase (PI3K) pathway. Mass-spectrometry analysis and co-immunoprecipitation experiments demonstrated that a chaperone protein named Heat Shock Protein 60 (HSP60) is bound to the N-terminal region of intracellular CLU in neuroblastoma cells. Suppression of HSP60 by shRNA knock down experiments caused decreased neuroblastoma cell proliferation and increased cell death. Our results suggest that HSP60 exert its oncogenic activity by inhibiting the function of CLU and promoting NF-κB activity. In summary, in this report we demonstrate that a direct interaction between intracellular CLU and HSP60 could play an important role in the regulation of NF-κB activity and neuroblastoma development.

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