Investigating the effects of varying levels of EB2 expression on migration in cancer cells

Render, Jasmine (2022) Investigating the effects of varying levels of EB2 expression on migration in cancer cells. Masters thesis, University of East Anglia.

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Abstract

Metastasis is the process whereby cancer cells migrate from the primary tumour to other tissues and organs where they colonise and form new tumours and metastasis is responsible for the majority of cancer associated deaths. End-binding protein 2 (EB2) is a microtubule (MT) associated protein that associates with the plus-end of MTs. EB2 has been shown to play a role in MT organisation and contributes to efficient cell migration, however its full functional role is currently unknown. EB2 is overexpressed in a variety of cancer types and therefore it is essential that the effects of varying EB2 expression be investigated. Additionally, the mechanism by which EB2 expression is regulated has not yet been identified and it has been suggested that fibroblast growth factor receptor 3 (FGFR3) promotes MT associated protein 2 (MAPRE2) expression. Therefore, the objective of this investigation was to further investigate the functional role of EB2 in cell migration and determine whether FGFR3 effects EB2 expression. Two cell lines were used in this study, the pancreatic cancer cell line PANC1 and the breast cancer cell line MDA-MB-231. PANC1 cells were subcloned for increased and decreased EB2 expression (PANC1EB2Hi and PANC1EB2Low cells) and MDAMB-231 cells with a partial EB2 knockout (MDAEB2KO) in addition to wildtype MDA-MB-231 cells (MDAWT) were used. To determine whether EB2 expression is affected by FGFR3, PANC1EB2Hi and PANC1EB2Low cells were treated with an FGFR inhibitor. Fluorescence intensity analysis of EB2 determined that there was no significant difference in EB2 fluorescence following FGFR inhibition in PANC1EB2Low cells and a significant decrease in EB2 fluorescence in FGFR inhibited PANC1EB2Hi cells. However, as the significant decrease in intensity in PANC1EB2Hi cells was only slight and there was no change in PANC1EB2Low cells, it was concluded that FGFRs do not affect the expression of EB2 in PANC1 cells, suggesting that EB2 expression must be regulated through a different mechanism. Immunofluorescence labelling for MTs in PANC1EB2Hi and PANC1EB2Low cells demonstrated that PANC1EB2Hi cells have a more polarised MT organisation and thus a more migratory phenotype. This is supported by time-lapse imaging of MDAWT and MDAEB2KO cells that showed that MDAWT cells had a higher migration velocity. Furthermore, immunolabelling for proteins involved in cell migration (GEFH1, pMLC, mDia1, dynamin2) as well as the cytoskeleton (MTs and actin) determined the effects of varying EB2 expression on cell migration. The organisation and localisation of these proteins in migrating PANC1EB2Hi cells suggests that these proteins are essential for cell migration and that EB2 may have an effect on these proteins. It was found that dynamin2 localises along the leading edge of migrating cells and strongly appears to co-localise with EB2, which builds off of previous work in the Mogensen lab to suggest that these two proteins interact. While the function of this co-localisation is unknown, it appears from the immunofluorescence labelling that these proteins may interact at focal adhesions. Additionally, migrating PANC1EB2Hi and PANC1EB2Low cells were treated with the ROCK inhibitor, Y27632, and the fluorescence intensity of GEF-H1 significantly increased. Therefore, in PANC1EB2Hi cells, when ROCK is inhibited, it appears that the concentration of GEF-H1 increases, suggesting that ROCK inhibition leads to an increase in MT dynamics. In conclusion, this study contributes to the growing literature on the role of EB2, particularly in cell migration, and highlights several avenues for further investigation.

(Due to limitations of the form textbox super- and subscript are not included in the abstract)

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Kitty Laine
Date Deposited:	27 Jun 2023 12:46
Last Modified:	20 Feb 2024 10:40
URI:	https://ueaeprints.uea.ac.uk/id/eprint/92515
DOI:

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