Epithelial to mesenchymal transition (EMT) of human stem cell-derived retinal pigment epithelium shares commonalities with malignancy-associated EMT: a proteomic analysis

Abstract Stress and injury to the retinal pigment epithelium (RPE) often lead to de-differentiation and epithelial to mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), and age-related macular degeneration (AMD). Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human induced pluripotent stem cell (iPSC)-based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag (TMT) labeling followed by high-resolution tandem mass spectrometry for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantifed 7,937 protein groups in our TMT-based mass spectrometry analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Further, we integrated our proteomic data with prior transcriptomic (RNA-seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label free single shot data-independent acquisition (DIA) mass spectrometry study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple AMD-associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease.