The Effect of Epithelial-Mesenchymal Transition on Ribosomal Protein Heterogeneity in Circulating Tumor Cells
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CitationLi, Selena. 2020. The Effect of Epithelial-Mesenchymal Transition on Ribosomal Protein Heterogeneity in Circulating Tumor Cells. Doctoral dissertation, Harvard Medical School.
AbstractMetastasis is the primary cause of morbidity and mortality in cancer patients. During the metastatic progression, a cancer cell must detach from the primary site and intravasate into a blood vessel, survive in circulation, extravasate from the bloodstream, survive and proliferate in a distant location. Recent evidence suggests that cancer cells that successfully metastasize exhibit features of epithelial plasticity that parallels the developmental processes of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). EMT was first described in embryonic development as precursor cells migrate to populate distant differentiated tissues; however, in cancer, epithelial tumor cells co-opt some of these same pathways for metastatic dissemination, acquiring invasive properties including increased motility and loss of cellular attachments and polarity. Our lab has previously demonstrated that breast circulating tumor cells (CTCs) isolated from the peripheral blood of patients with breast cancer exhibit both epithelial and mesenchymal cellular phenotypes. The role of these heterogeneous cellular states and the epithelial plasticity of CTCs in their survival, persistence and potential for colonization of a distant metastatic site is unknown.
This dramatic shift in cellular phenotype requires significant changes in protein expression. Much of the prior work in this field has focused on the transcriptional programs that regulate EMT and MET. However, translational regulation of protein synthesis is an underappreciated mechanism by which cells can rapidly respond to various stimuli and alter their cellular phenotype. Importantly, single cell RNA-sequencing and clustering of patient-derived CTCs demonstrated two distinct subpopulations of CTCs defined by either high or low ribosomal protein (RP) expression. Of the 81 core ribosomal proteins, 53 were identified as among the most differentially regulated genes. Remarkably, this heterogeneity in ribosome protein expression strongly correlates with epithelial or mesenchymal markers with CTCs exhibiting high RP expression also expressing epithelial markers and CTCs with low RP expression expressing mesenchymal markers. We sought to investigate the connection between EMT and the translational machinery in a well-defined model of EMT. By stimulating EMT in a breast epithelial cell line using transforming growth factor beta (TGF-beta), I observed that as a cell shifts towards a mesenchymal phenotype, ribosomal protein expression decreases, along with rRNA and global protein synthesis paralleling our observations in patient-derived CTCs.
To investigate the dynamics of ribosomal protein regulation, I fused three ribosomal protein gene promoters (RPS6, RPL10a, RPL12) to a green fluorescent protein (GFP) gene, allowing quantification of GFP fluorescence to serve as a dynamic readout of RP expression. I stably infected both a CTC line and breast epithelial cell line that undergoes a well-characterized EMT, in order to study temporal changes to ribosomal protein expression at both a single cell and bulk population level. Using these reporter cell lines, I observed reduced RP promoter activity after 24 hours but an effect on rRNA levels as early as 4 hours after TGF-beta treatment. Ultimately, this study aimed to characterize the regulation of translational machinery and its relation to EMT, expanding our understanding of the beginning stages of metastasis and potential targets for inhibition. We demonstrate that cellular plasticity in ribosomal protein regulation leads to increased circulating tumor cell aggression. In light of this, future efforts to modulate translational machinery may prevent the occurrence of distant metastases.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37364785