Modulation of alternative splicing regulators during epithelial-mesenchymal transition in cancer progression

Abstract

The epithelial-mesenchymal transition (EMT), one of the hallmarks of cancer, is a set of biochemical changes that allow epithelial cells to lose their polarization to transform into mobile mesenchymal cells. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and 2) are RNA-binding proteins that act as splice factors and regulate a large number of transcripts that promote epithelial characteristics in the cell. Among the genes regulated by ESRPs, fibroblast growth factor receptor 2 (FGFR2) is well-known that has two mutually exclusive exons switching in the EMT. ESRPs were identified as key regulators of EMT during cancer progress. The aim of my project is to reveal ESRPs’ role in prostate cancer.

To explore ESRPs’ functions in tumours, I constructed stable PC3 cell lines overexpressing either ESRP1, or ESRP2, or both ESRP1 and 2. These cells were injected in nude mice subcutaneously to grow xenografts, and tumour sizes were measured by calliper. Functional properties of these cells such as cell growth, migration rate, and EMT properties were also measured.

As ESRPs regulate splicing of FGFR2 in EMT, I have used a biochromatic reporter whose fluorescent output is dependent on the inclusion/exclusion of FGFR2 exon IIIc. Compounds from the LOPAC library were screened using HEK293 cells with the reporter to find hits that promote exclusion of exon IIIc in FGFR2 splicing and possibly block EMT. Several assays were carried out including cell growth assay, EMT markers staining, cell migration and proliferation assays in vitro. While in vivo, xenografts were developed by injecting PC3 cells in nude mice subcutaneously, and then treated with one of the hit compounds to explore whether it could affect tumour growth.

Three chemicals were selected that switch splicing of FGFR2, verified as modulators of EMT by increasing E-cadherin expression and junctional localization, showed different activities in cell functional assays in vitro, and one of them significantly decreased tumour growth in vivo in prostate cancer xenografts mouse models. I have also found that ESRPs overexpression may suppress tumour growth in vivo in prostate cancer xenografts mouse models.

Date of Award	24 Mar 2020
Original language	English
Awarding Institution	The University of Bristol
Supervisor	Kevin Gaston (Supervisor), Sebastian Oltean (Supervisor) & Zuner A Bortolotto (Supervisor)