Theses and Dissertations

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Browsing Theses and Dissertations by Subject "alternative splicing"

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    Genetic manipulations with the AXL gene in bladder cancer cells using CRISPR-Cas9 system
    (Nazarbayev University School of Medicine, 2024-04-29) Syzdykova, Aizhan
    The TAM family of receptor tyrosine kinases (RTKs), composed of AXL, TYRO3, and MER, substantially influences various biological processes during tissue homeostasis (Lemke, 2013). A growing focus within cancer research is centred around the AXL RTK and its ligand GAS6, as abnormal activations and overexpression of the former appear to be linked to cancer progression, poor prognosis, metastasis, and lesser sensitivity to anti-cancer therapies (Lemke, 2013; Wieman et al., 2005). Mediated in a concentration-dependent manner, GAS6 is a ligand not only for AXL but for TYRO3 and MER as well, although its binding affinity is 3- to 10-fold stronger for AXL specifically (Weinger et al., 2009; Wu et al., 2014). AXL is critical in conferring resistance to conventional and targeted cancer treatment (Auyez et al., 2021). It accomplishes this by activating multiple downstream intracellular signalling routes, including AKT, MEK/ERK, and NF-κB, when it binds to GAS6 (Antony & Huang, 2017; Ekman et al., 2010). These pathways collectively create an anti-apoptotic environment, enhancing cellular survival and tumour invasiveness. Additionally, AXL has been implicated in the epithelial-to-mesenchymal transition (EMT), a process essential for cancer metastasis and progression (Antony & Huang, 2017). AXL undergoes a series of post-translational modifications involving proteolytic enzymes like ADAM10 and ADAM17 (Miller et al., 2016; Lu et al., 2017). These enzymes cleave AXL to create its soluble form (sAXL), which can dampen AXL activation by interacting with GAS6 and an intracellular domain (Lu et al., 2017). This mechanism also presents how cancer cells evade therapies targeting the BRAF/MAPK pathway (Rankin & Giaccia, 2016). Elevated sAXL levels in plasma have been correlated with cancer progression to advanced stages in different tumour types, suggesting its potential utility as a biomarker (Martínez- Bosch et al., 2022; Flem-Karlsen et al., 2020). However, a significant gap exists in our understanding, particularly concerning the effect of the inactivation of AXL on its downstream effectors in urinary bladder cancer cell lines . This thesis aims to fill this gap using CRISPR/Cas9 gene editing technology, a novel approach in this field, and inactivating the AXL gene. This will allow us to generate bladder cancer cell lines without AXL, providing a unique opportunity to study its role. This study will also explore the influence of AXL expression on mesenchymal cells. We plan to quantify the expression levels of sAXL in conditioned media obtained from our genetically engineered bladder cancer cell lines. Subsequent analyses will assess the influence of the deactivation on the expression of AXL's nuclear and soluble forms and further AXL's phosphorylation through Western blotting techniques. This thesis aims to shed new light on the complexity of AXL signalling in urinary bladder cancer by employing cutting-edge genome editing technologies. The experience and knowledge gained from this could significantly improve our understanding of cancer biology and potentially guide the development of more effective therapeutic strategies.