Novel Roles of E3 Ubiquitin Ligase in Cancer Pathogenesis

The ubiquitin-proteasome system plays a crucial role in maintaining cellular ‎homeostasis by regulating ‎‎protein degradation and turnover. Central to this system are ‎E3 ubiquitin ligases, responsible for transferring ‎‎ubiquitin molecules onto target ‎proteins, marking them for degradation by the proteasome. Mounting ‎‎evidence suggests ‎that the deregulation of E3 ubiquitin ligases is a critical factor in the development and ‎‎‎progression of human tumours. Any mutation or aberrant expression in E3 ligases can ‎lead to oncogene ‎‎overexpression or tumour suppressor gene downregulation, ‎contributing to cancer development. ‎‎Understanding the molecular targets and activities ‎of E3 ligases provides a foundation for developing novel ‎‎cancer therapeutics.‎ ‎ This PhD thesis focuses on investigating the potential of E3 ubiquitin ligases as ‎cancer targets, with a ‎‎specific focus on three uncharacterized E3 ligases: FBXO24, ‎DCAF12L2, and RNF32. These ligases exhibit a ‎‎high mutation frequency or ‎overexpression in various human tumours, making them potential targets for ‎‎therapeutic ‎intervention.‎ To thoroughly examine the landscape of E3 ubiquitin ligase genes, we conducted a ‎‎comprehensive bioinformatic analysis that encompasses over 600 E3 ligase genes utilizing ‎the Catalogue of Somatic Mutations in ‎Cancer (COSMIC) database. Our investigation ‎led to the identification of two E3 ligases, namely FBXO24 and DCAF12L2, exhibiting ‎notable hypermutation. These hypermutated E3 ligases displayed a cancer-type-specific ‎pattern, with clustered mutations consistently observed across multiple patients ‎diagnosed with specific cancer types. This finding highlights the potential role of these ‎ligases in driving tumorigenesis and underscores their significance as cancer-associated ‎genetic alterations. The mutations affected critical functional domains, such as the F-box ‎motif in FBXO24 and the ‎‎WD40 repeats in DCAF12L2. We revealed that these ‎substitutions, found in cancer genomic databases, ‎‎are sufficient to disrupt the binding of ‎the E3 ligases to their interactors. In the case of FBXO24, the ‎‎T65P mutation abolished ‎the interaction with Skp1 and disrupted the SCF complex formation. For DCAF12L2, ‎‎‎the WD40 domain mutations affected the interaction and ubiquitination of substrates.‎ ‎ Affinity purification, combined with mass spectrometry analysis, unveiled potential ‎substrates for FBXO24 and DCAF12L2. FBXO24 demonstrated a strong interaction ‎with ZNF24. We demonstrated a decreased turnover of ZNF24 upon FBXO24 ‎overexpression but not with FBXO24T65P, suggesting that FBXO24 mediates the ‎ubiquitination and degradation of ZNF24. ZNF24 is a pleiotropic factor associated with ‎various cancer-related processes, such as proliferation, differentiation, migration, and ‎invasion. Additionally, the deubiquitinating enzyme USP34 was identified as a candidate ‎involved in the SCF-FBXO24 ubiquitination machinery. For DCAF12L2, FAM91A1 and ‎MEKK4 were identified as potential targets for ubiquitination by CRL4-DCAF12L2. ‎The accumulation of MEKK4 through the p38/JNK signaling pathway and the ‎accumulation of FAM91A1 through stabilization of the WDR11 complex have been ‎implicated in cancer progression. Remarkably, a C-terminal diglutamic (EE) region was ‎discovered to function as a specific degron in these substrates, facilitating recognition and ‎interaction with DCAF12L2.‎ Analyzing the TCGA dataset using cBioportal, we identified RNF32 as an E3 ‎ligase upregulated in various ‎‎cancers. We attempt to characterize the RNF32 structure, ‎consisting of two zinc finger RING domains known ‎as the N-RING and C-RING. Our ‎investigation specifically ‎highlighted the functional significance of the C-‎RING domain ‎in autoubiquitination. Moreover, our study revealed a unique regulation mechanism ‎involving ‎ Ca2+-Calmodulin ‎. RNF32 interacts with Calmodulin through its IQ domain, ‎influencing its ‎conformation ‎and homodimerization. Furthermore, using AP/MS we ‎identified the IKK-complex as ‎a potential target regulated by RNF32. The IKK complex ‎is a key regulator of the NF-κB ‎signaling pathway. ‎Dysregulation of the NF-κB pathway ‎has been linked to cancer pathogenesis, as aberrant ‎activation of NF-κB ‎promotes cell ‎survival, angiogenesis, and metastasis. We believe that the upregulation ‎of RNF32 may ‎‎disrupt the balance of NF-κB signaling, potentially contributing to cancer development ‎.‎ In conclusion, this research elucidates novel roles of E3 ubiquitin ligases in human ‎cancer. The study highlights the disruptive effects of clustered mutations on substrate ‎ubiquitination. Moreover, our research suggests that the upregulation ‎‎of RNF32 results ‎in deregulation of NF-κB signaling, ‎thereby contributing to the development of cancer. ‎‎The findings from this project hold promise for the development of new treatments, ‎drugs, and gene therapy ‎‎targets for cancer therapy. ‎