An Investigation of the role of the MDM2-NME interaction in cancer cells

Chang, Shie
An Investigation of the role of the MDM2-NME interaction in cancer cells. PhD thesis, University of Liverpool.

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MDM2 is a proto-oncogene well known for its role as a negative regulator of the p53 tumour suppressor. It has also been demonstrated that co-upregulation of MDM2 and wild-type p53 is linked with reduced disease specific survival in renal cell carcinoma (RCC). Moreover, MDM2 expression has been shown to promote cell motility and invasiveness in several cancer cell types including RCC cells in a p53- and RING-finger- independent manner, suggesting a role for protein-protein interactions in mediating this effect. The work presented in this thesis has aimed to study the role of MDM2 interaction with other proteins, in particular with members of the NME (Non-metastatic protein) family of metastasis suppressing genes, in order to decipher the mechanisms of action by which MDM2 may contribute to a more aggressive phenotype in cancer cells. To accomplish this we have used a range of methodologies including investigating the consequences of MDM2 expression using exon array analysis in an attempt to identify changes in gene expression induced by wild type MDM2 (Clone 9) and an MDM2 RINGmutant (RFM9) expressed stably in clonal derivatives of H1299 cells. In parallel studies in our research group, an interaction was identified between MDM2 and NME2. Since NME2 is a member of a family of genes that has been implicated in metastasis suppression, and since metastatic spread is a primary determinant of patient survival, we also focused on an analysis of this interaction. Thus the primary aim of this work became an examination of the mechanistic details and consequences of MDM2-NME interaction in cancer cells. The two most highly expressed forms of NME genes in most cell types are the highly related NME1 and NME2 genes which both encode nucleoside diphosphate kinase (NDPK) activity, essential for maintaining cellular pools of nucleoside triphosphates. They are also metastasis suppressor genes with an ability to suppress cellular motility. Since MDM2 possesses an E3 ubiquitin ligase activity, we have investigated whether MDM2 has an ability to modify NME proteins by ubiquitinating them and also whether MDM2 modulates the NDPK activity of NME proteins and/or their motility suppressing activity. Our results suggest that MDM2 may abrogate motility suppressive effects of NME2 through down-regulation of NME2 (and also of NME1) protein levels with potential consequences for suppression of NDPK activity. More interestingly, our studies have identified NM23-LV, which is derived from a specific read-through transcription and alternative splicing event of the adjacent NME1 and NME2 genes, as an MDM2-interacting protein. In addition, we have identified NM23-LV as a novel substrate for MDM2-mediated ubiquitination that occurs in an MDM2 E3 ligase activity-dependent manner. Ubiquitination of NM23-LV by MDM2 seems to be highly specific, since neither NME1 nor NME2 are substrates for MDM2-mediated ubiquitination. Furthermore, our results have also shown that NM23-LV can substantially promote MDM2 stabilisation and enhance MDM2-dependent p53 inactivation, adding another layer of complexity to the interactions between MDM2 and NME family members. Functional studies of NM23-LV have suggested that NM23-LV plays a role in promoting cell motility, which contrasts with the canonical role of NME1 and NME2 as metastasis suppressors. Taken together, studies presented in this thesis have identified a novel relationship between MDM2 and NM23-LV, which could provide valuable insights into the underlying mechanisms leading to increased cell motility promoted by MDM2 and suggests that new studies examining how MDM2-NME interactions may regulate cell motility and cancer metastasis are needed. Given the role of MDM2 in promoting more aggressive RCCs, insights gained from these studies may ultimately identify opportunities for therapeutic interventions for RCC, as well as for other human cancers.

Item Type: Thesis (PhD)
Additional Information: Date: 2014-04 (completed)
Depositing User: Symplectic Admin
Date Deposited: 27 Aug 2015 11:16
Last Modified: 17 Dec 2022 01:33
DOI: 10.17638/02003899