Characterisation of Newcastle disease virus (NDV)-mediated oncolysis of putative bladder cancer stem cells
Background: Cancer stem cells (CSC) are intricately linked to the high recurrence and therapeutic resistance risks in bladder cancer. Newcastle disease virus (NDV) possesses a strong and selective oncolytic effect against various cancer types. However, some cancer cells resist NDV-mediated oncolysis...
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| Format: | Thesis |
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2022
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| Online Access: | http://eprints.sunway.edu.my/2391/ |
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| Summary: | Background: Cancer stem cells (CSC) are intricately linked to the high recurrence and therapeutic resistance risks in bladder cancer. Newcastle disease virus (NDV) possesses a strong and selective oncolytic effect against various cancer types. However, some cancer cells resist NDV-mediated oncolysis and develop persistency of infection. Thus, this study aimed at characterising the oncolytic effect of NDV-AF2240 against putative bladder CSC and the genes and signalling pathways associated with NDV-mediated oncolysis. Methods: Putative bladder CSC were selectively grown in the form of 3D-spheroids. The stemness property of the spheroids and the oncolytic effect of NDV against the spheroids were assessed. Finally, the next generation sequencing (NGS)-generated transcriptomic profiles of the NDV-sensitive and –resistant spheroids were analysed using various bioinformatics tools. Results: Spheroids were successfully established from UMUC1, UMUC10, J82, TCCSUP, RT112 and EJ28 bladder cell lines. These spheroids expressed significantly high levels of CSC genes (SOX2, OCT4 and NANOG) expression; indicative of their stemness property. However, immunophenotyping analysis of the CSC markers (CD44, CD49f, ALDH1, and CD133) identified heterogeneity in expression in both the spheroid and monolayer cells across the six different bladder cancer cell lines. Interestingly, different spheroids displayed varying degrees of sensitivity towards NDV-mediated oncolysis. By confirming the presence of NDV viral genes (HN, NP and F), persistency of infection (PI) was detected in bladder CSC spheroids that were resistant towards NDV-mediated oncolysis. The NDV-resistant bladder CSC spheroids were also resistant to superinfection and expressed interferon-beta (IFN-β). Nevertheless, the spheroid forming ability of established NDV PI bladder cancer cell lines, EJ28-PI and TCCSUP-PI were shown to be impaired. Both EJ28-PI and TCCSUP-PI cells, expressed significantly high levels of EN2, the knockdown of which, resulted in reduced cellular viability. The NGS analysis of NDV-resistant versus NDV-sensitive spheroids revealed cohesive dysregulation of the cancer cell transcriptome. Key DEGs that are involved in specific pathways were identified. Pathway enrichment analysis revealed pathways such as viral carcinogenesis and NF-kappa B signalling that were significantly upregulated in NDV-resistant spheroids. Protein-protein interaction (PPI) network analysis was performed and the top upregulated hub genes: UBB, UBC, NFKB1 and RELA were identified. The NGS analysis of NDV-sensitive spheroids cultured in different media identified that various metabolic pathways and proteoglycans in cancer were significantly upregulated in these spheroids. Top hub genes such as SRC and EGFR1 were downregulated in NDV-sensitive spheroids. Signalling pathways regulating the pluripotency of stem cells were significantly downregulated in NDV PI spheroids. Top hub genes such as TP53 and RPS27A were upregulated, while UBC and SRC were downregulated in NDV PI spheroids. Cross-validation of gene expression patterns using RT-qPCR supports the robustness of the NGS experiments. Conclusion: NDV resistance and persistency of infection are associated with the cohesive dysregulation of the bladder CSC transcriptome, involving key genes, and signalling pathways in bladder tumorigenesis. Potential combinatorial strategies such as the inhibition of NF-KB1 signalling to overcome resistance to NDV-mediated oncolysis may accelerate the bench to bedside translation of NDV. In addition, further functional characterisation of key genes such as EN2 in bladder CSC and NDV sensitivity will be critical towards improving our understanding of bladder tumorigenesis. |
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