Targeting ubiquitination signalling as a novel strategy against colorectal cancer

Background: Colorectal cancer (CRC) is a most common cancer and the 4th leading cause of cancer-related death worldwide. Australia has one of the highest incidence rates of CRC in the world. According to Australian statistics, the lifetime risk of developing CRC in the general populationis 1 in 13. Due to lack of effectivetargeted therapy, the 5-year overall survival rate of CRC is roughly 69%, lagging well behind other common cancers such as breast, melanoma and prostate (over 90 % survival rate). Thus, the development of novel targeted therapies is a most urgent needin the fight against colorectal cancer. The initiation and progression of CRC is mostly attributed to the deregulation of multiple oncogenic signalling pathways, particularly the Wnt/β-catenin signaling pathway. A majority of CRC arise from the intestinal polyps caused by genetic mutations in Wnt signalling components, such as Adenomatous polyposis coli (Apc), β-catenin or RNF43. This causes the constitutive activation of β-catenin and consequent malignant transformation of colon epithelial cells. However, directly targeting β-catenin as a transcription factor is challenging and may causes unintended adverse effects due to the central role of physiological β-catenin activity in various tissue homeostasis. Protein ubiquitination is a versatile post-translational modification (PTM) tightly involved in tumorigenesis. Ubiquitination modification is catalyzed by an enzymatic cascade composed of ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). This E1-E2-E3 cascade causes ubiquitin to be activated and specifically transferred to the lysine (K) residues of substrate proteins, leading to protein functional change or degradation. Wnt/β-catenin signaling transduction is closely regulated by the ubiquitination cascade, because β-catenin and its critical co-factors are mostly regulated by the ubiquitination-mediated degradation. In this project, we aim to identify critical regulators for oncogenic β-catenin and assess the potential of targeting these ubiquitination cascade as an alternative strategy to selectively abolish β-catenin oncogenic function. Methods: The present project plans to establish a reporter system of CSCs in colorectal cancer cell lines and organoids (a 3D in vitro tissue culture system), and screen the ubiquitination enzymes that are selectively required for oncogenic β-catein activity using the delicate β-catein reporter systems developed in our lab (Chunhua Wan et al. Science Advances. 2021). Proteomic studies will be conducted to identify the critical ubiquitination mechanisms leading β-catenin hyperactivation in the nucleus. The molecular mechanisms underpinning ubiquitination-mediated β-catenin hyperactivation may also be investigated using biochemical approaches in vitro. Overall Goals: 1. Clarify the ubiquitination mechanisms underlying constitutive activation of β-catenin; 2. Identify of druggable ubiquitination enzymes for blocking oncogenic β-catenin activity; 3. Identification of novel inhibitors of the ubiquitination cascades against β-catenin-driven CRC.