Assessing the effect of intrinsic and extrinsic stressors on myosin VI distribution

Overexpression of the unconventional motor protein myosin VI (MYOVI) has been seen in aggressive cancer phenotypes. Its ability to interact with numerous intracellular pathways influences downstream targets and promotes cell growth. Recently MYOVI has been found to positively impact the ER pathway directly through the estrogen receptor. Activation of this receptor results in downstream signaling via estrogen responsive elements (EREs) to regulate transcription of a vast plethora of genes including P53. Furthermore interactions with Dab2, which binds to the WWY motif located in MYOVI's cargo binding domain, hinder MYOVI's functionality in transcription factories within the nucleus, hence negatively impacting ER-driven gene expression. To assess, in the context of cancer, what is regulating MYOVI distribution within cells we performed experiments to compare the effect of intrinsic protein interactions, using the ESR1-DAB2 fusion gene isolated from an endocrine therapy resistant metastatic breast cancer (MBC) patient treated with tamoxifen, to extrinsic environmental changes, in this case hypoxia. While the majority of breast cancers in patients are ER-positive (ER+), and hence respond to endocrine therapy, acquired resistance to therapy is a big problem. Determining the localization and interactions in vitro of ESR1-DAB2 and elucidating its association with MYOVI could highlight novel therapeutic targets in MBC. Hypoxia characteristics are found in many tumours and are associated with a poor prognosis. Low oxygen levels alter the transcriptome of cells drastically, typically favouring pathways that upregulate vascular endothelial growth factor (VEGF) encouraging angiogenesis to relieve the hypoxic stress. Microscopy revealed that the effect of ESR1-DAB2 varies between MCF7 and HeLa cell lines and MYOVI distribution can be compared, with the plasmid exhibiting higher toxicity in HeLa potentially due to the loss of ER in this cell line. While hypoxia did not result in increased expression levels of MYOVI, the distribution was altered depending on the duration of hypoxia. The data presented in this thesis is not conclusive but lays a foundation to build upon in future work and contributes to the growing collection of roles MYOVI has been found to play.