RNA Methylation in Cancer Plasticity and Drug Resistance

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ ABSTRACT Cancer drug resistance occurs not only by selection of genetically resistant clones, but also through phenotypic plasticity via rapid induction of transcriptional programs that allow some cells to adapt and persist. In bladder cancer (BC), phenotypic plasticity is observed in a model established in our lab, in which two subpopulations of tumor cells reversibly and spontaneously transition from one to the other. Isolated and studied by Hoechst staining and flow cytometry, these subpopulations consist of a “side population” (SP) of highly tumorigenic, cisplatin-resistant, stem-like cells, and a “non-side population” (NSP) of cells lacking these properties. A potential contributing epigenetic factor to this plasticity is N6-methyladenosine (m6A) RNA modification. Deposited by m6A writers and removed by m6A erasers, m6A dynamically and reversibly regulates key cellular functions and essential features of cancer cells. Deregulation of m6A modifications and m6A effectors (writers, erasers, readers) has been implicated in drug resistance in various cancer types. To study the role of m6A modifications in BC, I set up and validated in our lab the gold standard epitranscriptomic assay, methyl-RNA-immunoprecipitation followed by high-throughput sequencing (MeRIP-seq). I used this assay to compare the SP and NSP subpopulations and identified differentially methylated candidate transcripts. I also found that pharmacological inhibition of a key m6A eraser, fat mass and obesity-associated protein (FTO), potentiates a shift to the SP state. Based on these preliminary data, I propose to test the hypothesis that m6A modifications regulate expression of transcripts that promote transition to a drug-resistant state in BC in vitro and in patient-derived samples. I will accomplish this with the following aims: Aim 1: I will use MeRIP-seq and RNA-seq to systematically identify differentially methylated and differentially expressed transcripts that drive the shift to and from a drug-resistant state. I will genetically modulate these targets, and measure the impact on cisplatin resistance, SP-NSP interconversion, colony formation, migration and invasiveness. Aim 2: I will define the function of FTO, a key m6A eraser that affects plasticity in our model by genetically and pharmacologically modulating FTO. I will measure the impact on cisplatin resistance, SP-NSP interconversion, colony formation, migration and invasiveness, and use MeRIP-seq and RNA-seq to identify transcripts that are both differentially methylated and differentially expressed. Aim 3: I will use RT-qPCR to test which candidate transcripts and m6A effectors are associated with clinical progression to cisplatin resistance using BC patient-derived solid and liquid biopsy samples. Characterizing this novel epitranscriptomic mechanism will provide strong evidence for new biomarkers and therapeutic targets aimed at short-circuiting BC drug resistance. The proposed research study will offer rigorous physician-scientist training in an outstanding environment offering top notch research facilities integrated with translational patient tissue resources and diverse mentoring expertise.