Paclitaxel (Taxol) is a microtubule-stabilizing drug widely used for the treatment of breast, ovarian and lung cancers. For many years, it was thought to kill cancer cells by inducing mitotic arrest, thus preventing proliferation. Recently it became apparent that clinically relevant concentrations of paclitaxel are insufficient for mitotic arrest and instead cause chromosomal instability due to multipolar cell divisions. Correct bipolar divisions need cells to round up before mitosis, which requires disassembly of interphase microtubules.
While paclitaxel is highly efficient in some patients, many eventually develop resistance. Cells round due to changes in the cortical actin cytoskeleton, which is induced by interphase microtubule disassembly. Since paclitaxel stabilises microtubules, it is likely to interfere with microtubule disassembly and therefore cell rounding. Thus, actin regulators are likely candidates for paclitaxel resistance. Additionally, paclitaxel resistance correlates with post-translational modifications of microtubules, such as acetylation and tyrosination, but it is unclear how these modifications contribute to the resistance.
Objectives:
1. Characterise effects of paclitaxel on microtubule disassembly, mitotic rounding and cell proliferation in vivo (Year 1).
2. Identify actin regulators which suppress paclitaxel effects (Year 2-3).
3. Determine how microtubule posttranslational modifications alter the effects of paclitaxel (Year 2-3).
4. Examine effects of identified actin regulators and microtubule modifications on paclitaxel-treated breast cancer cells in vitro (Year 3-3.5).
Experimental approach:
1. Wing imaginal discs of Drosophila larvae fed with paclitaxel will be analysed using immunohistochemistry to quantitate effects on proliferation, apoptosis and chromosome segregation.
2. Wing imaginal discs expressing GFP-tagged tubulin or E-cadherin will be analysed using live imaging in presence of paclitaxel to measure effects on microtubule disassembly and cell rounding.
3. For the objectives 2 and 3, larvae overexpressing interfering RNA or full-length variants of actin regulators (e.g. Pebble, RhoGEF2) and microtubule modifying enzymes (e.g. Atat, TTLL1A) will be generated using the UAS-GAL4 system.
4. MCF7 cells transfected with siRNAs against identified regulators will be cultured with paclitaxel to measure changes in cell rounding and chromosome segregation.
Novelty and timeliness:
Effects of cell rounding and paclitaxel on chromosomal segregation only recently came into light. The student will test the hypothesis that paclitaxel inhibits microtubule disassembly and cell rounding, leading to chromosome mis-segregation and ultimately cell death in vivo. Understanding how paclitaxel acts on the cellular level and how actin regulators and microtubule modifications modulate its effects will clarify why some patients develop resistance and reveal new routes for combating paclitaxel resistance.
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