Gene Regulatory Networks are a Mechanism for Drug Resistance

Abstract

Multidrug resistance has become a major issue in the treatment of both microbial infections and cancers. While genetically encoded drug resistance is fairly well understood, it cannot explain all observed cases of resistance, namely the ability of a subset of disease cells to persist in an otherwise susceptible population. This non-genetic resistance requires the heterogeneous expression of a drug resistance phenotype, which can be produced by certain gene regulatory network architectures. Two particular network motifs, the coherent feedforward loop (CFFL) and the positive feedback loop (PFL), have functional properties that implicate them in the development of non-genetic heterogeneity and response to changing conditions. Motivated by the observation that CFFL and PFL motifs are involved in the transcriptional regulation of multiple pleiotropic drug resistance (PDR) genes in yeast, it has been hypothesized that CFFLs and PFLs could contribute to the development of drug resistance. This hypothesis was based on model simulations and has not been tested experimentally. In this thesis, it is demonstrated experimentally that the PDR5 gene is indeed expressed heterogeneously within an isogenic population of yeast cells, and that this cell-to-cell variability enables a subset of cells to persist drug treatment. While these observations agree with model predictions, it is also observed that the resistant phenotype occurs within a subset of cells that are morphologically distinct. This subpopulation has previously been linked to abnormal mitochondrial function, which cannot be ruled out as a likely cause of the observed drug resistance. To validate the hypothesis that CFFLs and PFLs contribute to drug resistance, the expression of the PDR5 gene was placed under the control of synthetic gene regulatory networks constructed to contain different combinations of direct activation, indirect activation, and positive feedback. These networks are used to show that direct activation can provide a selective advantage enabling rapid responses, while indirect activation and positive feedback can provide a selective advantage by maintaining favourable gene expression states. These results demonstrate that a gene regulatory network combining CFFLs and PFLs can contribute to the development of drug resistance, and highlight plausible means by which cells can exploit certain network features to gain a fitness advantage.