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.

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24534号 / 医科博第148号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 寺田 智祐, 教授 川上 浩司, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le mélanome est un cancer très agressif en raison de sa capacité rapide à former des métastases et de développer une résistance aux traitements existants.
 MITF (Micropthalmia-associated Transcription Factor) est un facteur de transcription clé à toutes les étapes de développement du lignage mélanocytaire et dans la physiopathologie du mélanome. Afin de comprendre les mécanismes impliqués dans la régulation de l’activité et de la stabilité de MITF, nous avons identifié ses partenaires protéiques parmi lesquels figurent de nombreuses sous-unités des complexes de remodelage de la chromatine ATP-dépendant PBAF et NURF. Ce travail caractérise le rôle et l’étendue de la coopération entre BRG1/PBAF et plusieurs facteurs de transcription clés tels que MITF et SOX10 dans le fonctionnement des cellules de mélanome, qui recrutent activement de BRG1 à la chromatine et contribuent ainsi à la mise en place de la signature épigénétique caractéristique des cellules de mélanome prolifératives. Par ailleurs, l’utilisation de différents modèles murins a permis de révéler in vivo la contribution fonctionnelle distincte mais complémentaire de ces deux complexes de remodelage associé à MITF aux cours de trois stades majeurs du lignage mélanocytaire : le développement embryonnaire des mélanocytes, leur différentiation ainsi que lors de l’initiation et la progression du mélanome. Ce travail contribue ainsi à une meilleure compréhension du fonctionnement biologique des mélanocytes, du mélanome et du remodelage de la chromatine chez les eucaryotes. / Malignant melanoma is the most deadly form of skin cancer due to its quick metastatic spread and the development of resistance to available treatments.
MITF (Micropthalmia-associated Transcription Factor) is a transcription factor and master regulator of melanocyte lineage development and melanoma physiopathology. In order to investigate the mechanisms involved in the regulation of MITF activity and stability, we identified its numerous partners by tandem affinity purification coupled to mass spectrometry, which include several subunits of the PBAF and NURF ATP-dependant chromatin remodelling complexes. The present work characterizes the role and extent of cooperation between BRG1/PBAF and several key transcription factors including MITF and SOX10 in melanoma cell function, that actively recruit BRG1 to chromatin to establish the epigenetic landscape of proliferative melanoma cells. Furthermore, using different mouse models we revealed the distinct but complementary functional contribution of these two MITF-associated chromatin remodelers in vivo at three majors stages of melanocyte lineage development: embryonic development of melanocytes, their differentiation and during melanomagenesis. Thus, this work contributes to a better understanding of processes regulating the biological function of melanocytes, melanoma and more widely chromatin remodelling events in eukaryotes.