title: Translational Regulation Of Target Gene Expression By G3BPs In Breast Cancer Cells.
Creators: name: Alam, Umber
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Gene expression
subject: Breast cancer
subject: G3BP1
subject: G3BP2
subject: Carcinogenesis.
subject: Binding proteins

description: RNA binding proteins (RBPs) play key roles in the post-transcriptional regulation of RNAs, which along with transcriptional regulation, is a major pathway that controls patterns of gene expression for development and proper cell signaling. Post-transcriptional control can occur at many different steps in RNA metabolism including; splicing, polyadenylation, mRNA stability, mRNA localization and translation. The over-expression of various RBPs in several different cancers leads to the notion that disrupted RNA metabolism has a role in carcinogenesis. Nevertheless, it is exceptionally challenging to discover the mechanisms behind RBP functions due to the difficulty in identifying the RNA targets of RBPs. This problem is compounded by the finding that RBPs frequently have multiple RNA targets which could be bound and regulated under different cellular contexts. Ras-GTPase-activating protein SH3-domain-binding proteins (G3BPs) are members of a highly conserved family of multi-functional RNA binding proteins, which appear to co-ordinate signal transduction and post-transcriptional gene regulation. Both G3BP1 and G3BP2 proteins are over-expressed in cancer, and G3BP1 promotes cell proliferation and survival. Aberrant expression of G3BP proteins is common in cancer, and their over-expression influence tumorigenesis. Furthermore, there is growing evidence that G3BPs are implicated in the aetiology of cancer metastasis. G3BP1 is involved in breast cancer epithelial to mesenchymal (EMT) metastasis via the Smad signalling pathway, whereas G3BP2 suppresses EMT metastasis by interacting with TWIST1 and localising it in the cytoplasm. The TWIST1-G3BP2 mechanotransduction pathway responds to biomechanical signals from the tumour environment and promotes EMT metastasis through the release of TWIST1 from G3BP2. G3BP2 has also been implicated in breast tumour initiation by stabilizing Squamous cell carcinoma antigen recognised by T cells 3 (SART3) transcripts which is responsible for the expression of pluripotent transcription factors Octamer-binding protein 4 (Oct-4) and Nanog Homeobox (Nanog). In addition, G3BPs possess antiviral activities and are targeted by various viruses, such as Polio virus, Chikungunya virus and Semliki Forest virus, to promote infection. Moreover, G3BPs, along with Caprin 1, have been reported to be responsible for the accumulation of interferon stimulated genes (ISGs) by facilitating their translation. Therefore, a detailed examination of G3BPs’ RNA transcripts may provide insights into the post-transcriptional mechanisms underlying tumorigenesis and viral infections. G3BPs are likely to be involved in the regulation of multiple transcript targets and the identification of more, or all, RNA targets of G3BPs will be an important step in a comprehensive understanding of molecular and cellular significances of G3BP’s activity by analyzing gene transcription, mRNA stability and translation, in different cellular contexts. Identification of different transcript targets of G3BPs will aid in the understanding of how G3BPs exert coordinated control of different cellular functions in a concerted fashion through their RNA targets. This research project was conceived from previous studies suggesting that G3BPs support translation of ISGs. The involvement of G3BPs in the translation of ISGs implies that G3BPs are involved in the regulation of the interferon system in response to viral infections and/or cellular stress, regulating the cellular immune response. Therefore, their antiviral property, or involvement in cancer metastasis could, in part, be due to the regulation of various ISGs which inhibit viral infections and promote cancer metastasis. The recent literature shows that interferon induced transmembrane (IFITM) proteins (IFITM1, IFITM2 and IFITM3) are antiviral proteins, involved in the restriction of various viruses and are also emerging to have a role in cancer progression and metastasis. Therefore, this gene family was selected as potential transcript targets of G3BPs. The main aim of this research study was to identify the individual roles of G3BP1 and G3BP2 in the regulation of IFITM1, IFITM2 and IFITM3 (IFITM1-3) proteins in breast cancer cell lines. G3BPs were hypothesised to interact with the 3´-UTRs of the IFITM1-3 transcripts and regulate their translation. IFITM1-3 proteins are type I ISGs and are not expressed in all cell lines, therefore, the interferon (IFN) sensitive breast cancer line, MCF7, was selected to induce the expression of these proteins and to analyse the role of G3BPs in their regulation. Although IFITM1-3 are ISGs and their expression can be induced by type I IFN, the study design required a cell line which constitutively express these proteins as this would be beneficial to characterize the pathways regulating their translation. The interferon system is dysregulated in drug resistant cell lines in response to DNA damage, therefore, the breast cancer multidrug resistant (MDR) cell line, MDR.MCF7 (developed in the host laboratory) was chosen as a cell line which may constitutively express the IFITM1-3 proteins due to the dysregulation of the IFN system. Chapter 3 describes the induction and optimisation of IFITM1-3 proteins expression at both transcriptional and translational levels in MCF7 cells. The expression of IFITM1-3 proteins were also assessed in MDR.MCF7 cells. Chapter 4 and 5 were designed to study the individual role of G3BP1 and G3BP2, by performing siRNA-mediated knockdown of G3BPs in these cell lines and analysing the effects of their downregulation on the regulation of IFITM1-3 endogenous transcripts and proteins. The initial knockdown studies confirmed that both G3BP1 and G3BP2 are essential for the accumulation of IFITM1-3 proteins, without affecting their transcript levels. The research was extended to study the role of G3BPs in the regulation of IFITM1-3 through an interaction with their 3´-UTRs by performing luciferase reporter assays and RNase assisted RNA chromatography (RARC) assay. These assays confirmed that both G3BP1 and G3BP2 interact with the 3´-UTRs of IFITM1-3 and regulate their translation, supporting the hypothesis made at the start of this study. G3BPs have been reported to have a role in regulating the phosphorylation of the MEK/ERK pathway which is subsequently implicated in the translational regulation of ISGs. Based on these findings and other reports which show that one of the downstream effectors of the MEK pathway, eIF4E, is involved in the export of a certain subset of mRNAs, the role of this pathway was analysed in the regulation of IFITM1-3 proteins. Results have shown that knockdown of G3BPs in MDR.MCF7 cells led to a decrease in the phosphorylation status of MEK, ERK and eIF4E, supporting the idea that G3BPs could have role in the regulation of IFITM1-3 through this pathway as well. Preliminary studies were performed to further analyse this notion, by inhibiting the phosphorylation of MEK by using U0126, a well-known inhibitor of MEK, which acts by downstream inhibition of the phosphorylation of ERK and eIF4E. The mRNA and protein expression levels of IFITM1-3 were then analysed in the U0126 treated MDR.MCF7 cells by qRT-PCR and immunoblotting. The data analysis supports a role of G3BPs in the regulation of IFITM1-3 proteins via MEK/ERK pathway, although further experimental studies are required to confirm this role. Overall, this research study shows that both G3BP1 and G3BP2 are essential for the accumulation of IFITM1-3 proteins by interacting with the 3´-UTRs of their transcripts and also suggests an involvement of the MEK/ERK pathway in the translational regulation of IFITM1-3 via G3BPs. The data suggests that G3BPs intersect twice in the regulation of IFITM1-3 expression, firstly through MEK/ERK pathway and then through an interaction with the 3´-UTRs of IFITM1-3. However, the experiments performed here cannot resolve whether the two apparent functions are part of a single control mechanism or the two functions are mutually exclusive. Considering the relevance of these findings in the aetiology of cancer, further research is required to determine if these pathways can be targeted for future anti-cancer therapies.