02. Master's Thesis

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Browsing 02. Master's Thesis by Subject "AXL"

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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.
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    STUDY OF AXL ISOFORMS IN BLADDER CANCER CELLS USING CRISPR/CAS9 TECHNOLOGY
    (Nazarbayev University School of Medicine, 2025-04-25) Ospanova, Sabina
    One of the three receptors of the TAM family, AXL, is associated with the survival of cancer cells, making them resistant to chemotherapeutic drugs (Graham et al., 2014). AXL binding to its ligand Gas6 is important for activation of pathways involved in tumor cell invasion, metastasis, and survival. Therefore, developing AXL/Gas6-targeted anticancer drugs is of great interest (Tanaka & Siemann, 2021). The extracellular fragment of AXL can be proteolytically cleaved by ADAM10 and ADAM17. This results in soluble AXL (sAXL) that can inhibit AXL by binding to its ligand. High sAXL concentration in patient plasma is a tumor biomarker of adverse outcome (Miller et al., 2016). In humans there are only two well established isoforms; isoform 1 containing exon 10 and isoform 2 lacking exon 10. Significantly, the ADAM10/17 cleavage site for sAXL is found at exon 10 of isoform 1. However, sAXL formation leads to activation of signalling pathways that promote cancer metastasis and survival (Shen et al., 2020). The exact mechanism is not known, but it is thought that after receptor shedding, AXL may form heterodimers with non-TAM family receptors, which induces non-canonical pathways in cancer (Malikova et al., 2025). CRISPR/Cas9 technology will be used to study the differences between the two isoforms (such as the lack of cleavage site in isoform 2 and sAXL formation) and the effect of these isoforms on cancer cells. In a bladder cancer cell line it is used to express only isoform 2 by deleting exon 10. Cells expressing sAXL will be observed in the conditioned media. With this study, we will be able to understand how isoforms impact AXL function and how much they impact bladder cancer survival.