Investigation of the regulatory role of Nek4 in the alternative splicing in non-small cell lung cancer

Author(s)

Lívia Alves dos Reis Moura

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Farmacêuticas
• Defense date: 2024-08-28

Examining board members

Jörg Kobarg; Leticia Fröhlich Archangelo; Ana Lucia Tasca Gois Ruiz

• Advisor: Jörg Kobarg

Abstract

Nek4 is a serine/threonine kinase that, among other functions, participates in the regulation of mRNA alternative splicing. It is still unclear how this participation occurs, but it is known that its two main isoforms interact with several splicing factors. This project aimed to assess whether Nek4 is involved in the regulation of splicing of genes important in the context of lung cancer, particularly in response to chemotherapeutics. Using RNA-seq, the effects of Nek4 silencing were analyzed in lung cancer cells (A549) to identify changes in gene expression profiles and alternative splicing events. Among the genes with differential expression after Nek4 silencing, those involved in epithelial-mesenchymal transition (EMT), such as TGFBR1, CDH1, CLDN2, FOXA2, and HMOX1, stood out. These genes are crucial for regulating phenotypic changes in cancer cells, facilitating invasion and metastasis, suggesting that Nek4 overexpression may favor EMT and contribute to tumor progression. Additionally, differential splicing events were identified in EMT-related genes, such as CTNNB1 and SOS2, although the impact of these specific events was not assessed. RNA-seq analysis also indicated the regulation of genes involved in the DNA damage response, with differential splicing events in the MDM2 and DIABLO genes, highlighting a potential role of Nek4 in the regulation of apoptosis through the modulation of proteins that control p53 stability and caspase activity. Experimentally, we demonstrated that Nek4 appears to influence caspase 9 splicing. Our results show that Nek4 silencing reduces the expression of the 9b isoform (anti-apoptotic), favoring the 9a isoform (pro-apoptotic), while Nek4 overexpression promotes the opposite, suggesting that Nek4 contributes to an anti-apoptotic phenotype. Furthermore, cell viability experiments indicated that Nek4 silencing sensitizes lung cancer cells to UVC-induced cell death, suggesting that Nek4 plays a protective role against DNA damage-mediated apoptosis. Interestingly, Nek4 silencing did not have the same effect following treatment with cisplatin or etoposide, chemotherapeutic agents that also induce DNA damage, indicating that Nek4 may influence cell survival differently depending on the type of genotoxic stress or experimental conditions. Although our results demonstrate Nek4's involvement in alternative splicing regulation, the underlying molecular mechanisms remain unknown. Future studies may explore in greater detail how Nek4 regulates splicing events—whether through direct interactions with splicing factors, alterations in cellular signaling pathways, or regulation of gene expression. The data obtained in this study suggest that Nek4 not only regulates alternative splicing but may also influence key processes in tumor progression, such as apoptosis, EMT, and hypoxia, through gene expression regulation (AU)

• FAPESP's process: 21/09439-1 - Investigation of the regulatory role of Nek4 in the alternative splicing in non-small cell lung cancer
• Grantee: Lívia Alves dos Reis Moura
• Support Opportunities: Scholarships in Brazil - Master