Uncovering a role for RYK, a WNT-binding receptor tyrosine kinase, in cancer
AuthorRoy, James Philip
AffiliationSir Peter MacCallum Department of Oncology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-12-20.
© 2019 James Philip Roy
Cancer is the leading cause of disease burden in Australia. Targeted therapy utilises oncogenic mutations in tumours by modulating cancer-promoting proteins and signalling pathways. While these drugs deliver promising initial responses, cancers invariably develop resistance resulting in the recurrence of the cancer. A common theme of resistance mechanisms is the ability of cancer to hijack alternative signalling pathways to evade therapy. The identification and evaluation of these relatively understudied pathways could facilitate the development of novel therapies to overcome resistance development. One such understudied subset of signalling molecules, the WNT-binding RTKs, are at the interface of two protein families frequently dysregulated in cancer – receptor tyrosine kinases (RTKs) and WNTs. RTKs are targets of many drugs used clinically for cancer treatment, often through the inhibition of their kinase activity or their interactions with activating ligands. WNTs are key signalling morphogens in cancer but the complexity of their signalling has made them difficult drug targets. RYK is a WNT-binding RTK pivotal for embryonic development but with unusual biochemical properties that have meant it is understudied in a cancer context. This Thesis sought to uncover a role for RYK in cancer and evaluate its potential as a therapeutic target. This Thesis revealed the overexpression of RYK mRNA in lung squamous cell carcinoma and glioblastoma samples before identifying human tumour cell lines that readily express RYK mRNA and might be reliant on RYK signalling for its tumourigenic nature. Genetic perturbation of RYK through siRNA knockdown and CRISPR/Cas9 inactivation facilitated the discovery of human tumour cells from four different tumour types that were dependent on RYK for their viability. To assess the clinical expression of RYK protein, a novel chicken anti-RYKEC antiserum was generated, validated and utilised to uncover an upregulation of RYK in a subset of human NSCLC tumours. This antiserum also identified RYK expression in tumours from breast cancer patients and found an inverse correlation between RYK expression and the tumour grade of breast cancers. The establishment of a novel bioassay in pre-osteoblast cells dependent on WNT3A/RYK signalling is described. This assay is used to optimise the expression and purification of two RYK/WNT signalling inhibitors and confirm their RYK/WNT-inhibitory activity. One of these inhibitors, a neutralising anti-RYK antibody – RWD1 – then demonstrated an anti-cancer effect by inhibiting the viability of the RYK-dependent human tumour cells in vitro. RWD1 also reduced the growth of A549 NSCLC tumour xenografts, demonstrating the anti-cancer effect of targeting RYK signalling in vivo. RNA-sequencing analysis revealed an upregulation of epithelial to mesenchymal transition (EMT) genes in human tumour cells upon RWD1 treatment, implying RYK signalling supresses EMT. CRISPR/Cas9-mediated inactivation of RYK increased the metastatic potential of A549 tumour xenografts in vivo. This Thesis uncovered a dual role for RYK signalling in a cancer setting. RYK was found to promote the viability and growth of human tumour cells while also suppressing EMT and metastatic growth. The findings of these parallel pro- and anti-tumour functions of RYK signalling, suggest that RYK signalling could be exploited to deliver a therapeutic effect in cancer.
KeywordsRYK; WNT signalling; Receptor tyrosine kinase signalling; Cancer biology; WNT-binding receptor tyrosine kinase; Targeted therapy
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