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.
Novel Roles of E3 Ubiquitin Ligase in Cancer Pathogenesis
Mohsen Hajisadeghian
Investigation
2023-01-01
Abstract
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. 
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PhD.Thesis.Mohsen.Hajisadeghian.pdf
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