Rusnita, Dewi
(2024)
Investigating the role of Cut Like Homeobox 1 CUX1 in colorectal cancer progression.
PhD thesis, University of Nottingham.
Abstract
CUX1, also known as Cut-like homeobox 1, is a gene that has been identified as a transcriptional factor capable of regulating the expression of other genes involved in various cellular processes. It has emerged as a gene of interest due to its potential role in the progression of colorectal cancer (CRC), as indicated by analysis of cases from The Cancer Genome Atlas (TCGA). However, the precise function of CUX1 in colorectal cancer progression is not yet fully understood, and there is limited research examining its expression and role in this type of cancer, particularly with regards to its impact on cancer progression. Therefore, this study aimed to investigate the role of CUX1 in colorectal cancer progression using immunohistochemistry to assess CUX1 staining, in vitro functional studies, and RNA sequencing of CRC cell lines.
The expression of CUX1 in CRC tissues was evaluated using immunohistochemistry on tissue microarray slides, and the results showed that higher expression levels of CUX1 were associated with higher stage, tumour grade, infiltrative growth pattern, nodal involvement and metastasis in both Cohort and MMR-proficient CRC cases. Kaplan Meir and Cox’s regression analysis indicated that higher CUX1 expression was associated with lower overall survival and potentially served as an independent prognostic factor.
Functional studies were conducted on CRC cell lines using siRNA to transiently knockdown CUX1. The PrestoBlue assay revealed that knocking down CUX1 decreased cell proliferation. Furthermore, clonogenic assay showed that cell survival was also decreased following CUX1 knockdown.
To assess cell migration and invasion, wound healing assay, transwell migration and invasion assay were conducted, showing decreased cell migration and invasion. Additionally, F-actin staining experiment showed a reduction of lamellipodia and filopodia structures on knockdown. Flowcytometry results indicated an increase in cell apoptosis and a G1/S phase cell cycle arrest, and the cells became more sensitive to irinotecan treatment after CUX1 knockdown.
RNA sequencing was performed on CRC cell lines to investigate the downstream genes and pathways affected by CUX1 knockdown. The GSEA (gene set enrichment analysis) tool indicated that DNA repair was the most downregulated hallmark in HCT116, while KRAS signalling was significantly upregulated in DLD1 and SW480 cell lines. Gene ontology analysis revealed that mitochondria, endoplasmic reticulum, and golgi apparatus were the most affected organelles. The top 4 downregulated biological processes were 'organonitrogen compound biosynthetic process', 'organophosphate metabolic process', 'small molecule catabolic process', and 'lipid metabolic process', as indicated by gene ontology analysis. The PPI network analysis revealed that CUX1 interacts with multiple proteins to regulate cellular structure, processes, and pathways such as the golgi apparatus and intracellular membrane-bounded organelle, transport within the golgi network, actin cytoskeleton regulation, apoptosis, carbohydrate metabolism, and cellular signal transduction.
To conclude, altered CUX1 expression in CRC may disrupt various cellular processes and molecular pathways, leading to a breakdown of cellular homeostasis, which is essential for providing cancer cells with the necessary resources to sustain cancer growth and proliferation. Impacting these pathways perhaps translates into a survival advantage for CUX1 overexpressing colorectal cancers. The findings suggest that CUX1 may help prognostication and be a potential target for therapy.
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