Microtubules and end-binding proteins in epithelial remodelling and breast cancer invasion

The cytoskeleton plays vital roles in several cellular functions and disruption to its normal organisation and dynamics could lead to cancer. Cell migration is important during wound healing and tissue repair, and it is also known to take place during tumour metastasis. Microtubule reorganisation is driven by their inherent dynamic properties and influenced by plus-end tracking proteins such as the end-binding protein (EB) family. A better understanding of cell migration mechanisms could lead to more efficient treatments for invasive cancers such as breast cancer. Random migration of MCF-7 and MDA-MB-231 cells were monitored using time-lapse microscopy. Analysis revealed that MDA-MB-231, that express relatively high levels of EB2, migrated faster compared with MCF-7 cells. Similar results were observed in EB2 overexpressing MDCKII compared with the Empty-vector MDCKII cells suggesting that elevated levels of EB2 expression is associated with increased rate of cell migration. Interestingly, immunolabelling revealed that many of these migratory cells showed EB2 accumulation at the leading edge. Resveratrol is a naturally occurring compound found in foods such as red wine and grapes and its potential in reducing the rate of cell migration in breast cancer was also investigated. Resveratrol was found to significantly reduce the migration speed of MDA-MB-231 and MCF-7 cells. In addition, resveratrol-treated cells had straighter and more radially organised microtubules with more rounded EB1 comets at the plus-ends. A dramatic redeployment of EB2 was also observed upon treatment with 50 and 75μM resveratrol. Rearrangement of the microtubule cytoskeleton and its regulators are known to be essential for polarity establishment. Therefore, the project also investigated the effect of EB2 overexpression on epithelial remodelling in order to understand the processes leading to the loss of normal tissue architecture and ultimately, an invasive breast cancer state. Upon 3D culturing of MDCKII cells in Matrigel, EB2 overexpression in MDCKII cysts showed an increase in multiple lumen formation. Mechanistically, this was found to be due to loss of normal spindle orientation and specifically defects in astral microtubule cortical contact. Spindle misorientation could be rescued by Taxol treatment. Additionally, EB2 overexpression caused multipolar spindles, which are associated with supernumerary centrosomes and chromosome instability, and may contribute to cancer progression. In conclusion, this suggests a possible role for EB2 as a prognostic bio-marker for breast biopsies