The development of drug resistance during treatment is possibly the most important factor hampering the success of cancer therapy. In order to survive in the presence of chemotherapeutic drugs cells must quickly adapt to their altered environment. This may involve a collective stress response of interacting cells, whose mechanism is not yet clear. In the course of this work we interrogated the conceptual framework used to describe cancer and examined different aspects of drug resistance. While the main focus was on the role of ABC transporters in the rapid acquisition of drug resistance following a short period of drug treatment, the long-term adaptation to continuous drug treatment was also studied. As a tangent to this subject, the possible role of endocytosis in the process of adaptation to continuous presence of drug and subsequent resistance was also assessed. Cancer cell populations inexorably develop resistance to therapeutic treatment. In addition to selection of genetic variants, resistance may arise through two possible non-genetic mechanisms, (1) Darwinian selection of cells occupying (non-genetic) resistant microstates, or (2) Lamarckian instruction, in which cells adopt a resistant (treatment) induced phenotype. To examine the relative contribution of these two mechanisms we studied the population dynamics of leukemic cells (HL60 cell line) following treatment with the mitotic inhibitor vincristine. Single-cell analysis and mathematical modelling of state transition kinetics demonstrated that the appearance of multi-drug resistance phenotype within 24h was overwhelmingly the result of instruction. Transcriptome dynamics pointed towards a genome-wide state transition into a stress response state. Resistance induction correlated with Wnt pathway upregulation and was suppressed by beta-catenin knockdown, revealing a new opportunity for early therapeutic intervention against the development of drug resistance. By addressing the adaptation of the cell culture to prolonged drug treatment we observed that the survivor cells mounted a cellular response that neutralised the cytotoxic stress. That response involved the stabilisation of a transcriptome state that confers drug resistance. Our results suggested that the positive correlation between Wnt signalling and ABC transporters expression is important not only for the short-term survival but also for the enduring MDR phenotype. As we explored population heterogeneity we realised that the dead cells might also help the rest of the population to survive. Thus, our results support the need for examining the role of each population fraction, and ultimately each individual cell, in the overall story of cancer adaptation towards multidrug resistance. Subsequently we examined the differential endocytic behaviour between drug-sensitive and drug-resistant cells. By combining confocal time-lapse microscopy with flow cytometry we demonstrated that fluid-phase endocytosis was reduced in the resistant cells. The differences in the endocytic pathway only became noticeable after MDR1 expression has become constitutive, suggesting another protective role of the ABC transporters. All the results obtained support the idea that acquired drug resistance is not simply the passive selection of pre-existing mutants but can be accelerated by active adaptation. Cancer treatment is a double-edge sword: while the weakest cells die, the survivors cope cell-autonomously with the therapeutic perturbation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603169 |
| Date | January 2014 |
| Creators | Oliveira Pisco, Angela |
| Contributors | Jackson, Dean; Pedrosa Mendes, Pedro |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/drug-resistance-mechanisms-in-cancer-heterogeneous-populations(a5f2d318-3fd2-4491-84a5-fd2d69ac1b40).html |
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