Dissecting the evolution of resistance to microtubule hyper-stabilization

Macaluso, Francesca
When microtubules are hyper-stabilized, they do not provide the proper tension at the kinetochore of the metaphase chromosomes. Low tension leads to the activation of the spindle assembly checkpoint which stops cells in mitosis. Eventually cells can bypass the checkpoint and resume proliferation with defective microtubules; some cells die in this process, but others missegregate chromosomes and eventually become resistant to the microtubule hyper-stabilizing stress. For example, cancer cells develop resistance to chemotherapeutic drugs that hyper-stabilize microtubules such as taxanes. Here, we aimed to investigate the evolution of resistance to microtubule hyper-stabilizing conditions. We characterized in yeast the tub2- 150 mutation which hyper-stabilizes microtubules similarly to taxanes and shows a growth defect compared to WT cells. We evolved in the laboratory haploid yeast cells carrying the tub2-150 mutation for over ~190 generations. We then investigated by Next Generation Sequencing the genetic alterations in cells that had resumed cell proliferation. We found that tub2-150 mutants were able to evolve and recover the initial growth defect by two temporally distinct solutions. The first evolutionary solution was the selection of recurrent aneuploidies, in particular the disomy of chromosome XII. We found that overexpression of Stu2, a regulator of microtubule-kinetochore attachments, was responsible for the disomy. We confirmed that STU2 duplication was able to partially rescue the tub2-150 growth defect by lowering the mis-segregation rate of the mutant. In the long-term, aneuploidies were replaced by point mutations of the TUB genes. Some representative chosen TUB mutations, when re-introduced in the mutant ancestor before evolution, largely rescued the tub2-150 growth defect by destabilizing microtubules to different extents. Interestingly, several compensatory TUB mutations that emerged during the evolution experiment had been previously found in mammalian cell lines resistant to taxane-like treatments or in breast cancer patients. These results provide new insights on the evolutionary dynamics underlying resistance to microtubules hyper stabilization and potentially lead to the development of new strategies in order to stop or delay the emergence of resistance to drugs which stabilize microtubules.
NUI Galway
Publisher DOI
Attribution-NonCommercial-NoDerivs 3.0 Ireland