A transcriptomic and molecular approach uncovering ASCL2 as a novel tumourigenic gene in breast cancer
Creators: name: Faramarzi, Nicola
Publisher: name: University of Westminster
Year: 2019

description: Breast cancer is highly heterogeneous and is considered a collection of molecularly distinct tumour subtypes. Substantial efforts have been made to explore the gene expression profiles underlying the subtypes, and to elucidate possible markers associated with clinical outcomes. However, research in this area has been met with significant challenges and despite ongoing advancements in diagnostics and targeted therapeutics, incidence and mortality continues to rise. Thus, there is a need for greater molecular characterisation of breast tumours, to further understand the mechanistic roles of genes within their respective signalling pathways. With the advent of high-throughput technologies in transcriptomics, as well as the use of open databases and bioinformatics analysis tools, it is now possible to examine thousands of genes in parallel, generating an unprecedented amount of information. This provides a means for researchers to identify novel genes and targets from large volumes of gene expression data. However, the task of extracting clinically relevant results, is a prominent challenge. Therefore, the aim of this study was to use a streamlined in silico pipeline, integrated with in vitro methods to identify and functionally investigate a novel genetic marker demonstrating a key role in breast carcinogenesis. Gene expression profiles from breast cancer cell lines were obtained from public databases (Array Express and Gene Expression Omnibus). Data was filtered and subjected to an extreme variation analysis to generate a list of differentially expressed genes. Subsequently, multiple pathway analysis tools were used to identify a novel candidate gene for further investigation. Achaete-scute complex homolog 2 (ASCL2) is a transcription factor and Wnt-target gene, recognised as a regulator of stem cell identity and embryogenesis. Gene expression was validated in vitro by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR), and to assess the tumourigenic potential of ASCL2, siRNA knockdown was performed; assays were employed to measure proliferation, wound-healing and apoptosis. Data mining of patient tumours obtained from the METABRIC study was also undertaken to ascertain the potential of ASCL2 as a prognostic indicator. This work utilised a systematic pipeline used by the wider scientific community for the identification of candidate genes from transcriptomic data. Differential expression of ASCL2 was observed across multiple breast cancer cell lines, with largest the expression seen in MCF7 cells. Although evidence did not support the usage of ASCL2 as a prognostic indicator in patient tumours, data integrated from multiple lines of investigation suggested that this gene may influence the migratory capacity of breast tumour cells, whilst exercising its tumourigeneic function via the Wnt signalling pathway in breast cancer. Thus, this potential novel role of ASCL2 in breast tumourigenesis highlights a prominent area for further exploration.