Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity

Abstract

In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.alternative splicing ͉ mesenchymal-epithelial transitions ͉ tumor plasticity R egulation of alternative splicing is essential for normal gene expression (1), and alterations of this regulation are linked to disease (2), as illustrated by the association between cancer and splicing defects (3-7). An elegant example of this is provided by the splicing of transcripts encoding fibroblast growth factor receptor 2 (FGFR2). The status of FGFR2 alternative splicing depends on the interplay between several cis-acting elements in the FGFR2 premRNA and transacting factors, some of which are cell-type-specific (8). In mesenchymal cells, exon IIIb is silenced by the action of an exonic splicing silencer (9) and two flanking intronic splicing silencers (10-12). This silencing is mediated by Polypyrimidine tract-binding protein (PTB), hnRNP A1, and heretofore unknown factors. In epithelial cells, exon IIIb silencing is countered by several intronic elements. The best characterized are the intronic activating sequence 2, the intronic splicing activator and repressor (ISAR, also known as IAS3), and several GCAUG repeats (13-18). These elements have a dual function in epithelial cells, because they are also involved in silencing exon IIIc (14-18).FGFR2 splicing has been studied in tumors derived from the R-3327 Dunning rat prostate tumor, which arose spontaneously from the dorsal lobe of the prostate in a Copenhagen rat (19). Some R-3327-derived tumors (DT or DT3) express FGFR2(IIIb), which is consistent with their epithelial phenotype (20), whereas AT tumors (e.g., AT3), which have lost epithelial markers and display many mesenchymal indicators (21), express FGFR2(IIIc) (20). The significance of these alternative decisions for tumor behavior is underscored by the fact that forced expression of FGFR2(IIIb) suppresses tumor progression of AT3 tumors (22). Most importantly, however, the differential splicing of FGFR2 transcripts in these two cell types highlights broad differences in gene expression programs. Arguably, monitoring alternative sp...

Epithelial—mesenchymal and mesenchymal—epithelial transitions in carcinoma progression

“…This is consistent with findings in clinical material, in which membranous E-cadherin was detected in hepatic metastasis using immunohistochemistry, and vimentin was absent in the tumor cells. In the Dunning prostate cancer model, mapping of FGF receptor-2 splice variant expression demonstrated the occurrence of METs (indicated by co-expression of E-cadherin and FGF receptor-2(IIIb)) in primary tumors, typically where the tumor cells were in contact with the stroma (Oltean et al, 2006). In lung micrometastases, tumor cells in contact with the lung parenchyma and adjacent to blood vessels also frequently underwent MET.…”

Mechanism of the Mesenchymal–Epithelial Transition and Its Relationship with Metastatic Tumor Formation

“…Several transcription factors, including zinc finger proteins of the Snail and Twist families (e.g., dEF1/ZEB1/ TCF8 and SIP1/ZEB2/ZFHX1B) and the basic helix-loophelix factor E12/E47, which have been shown to be associated with the repression of E-cadherin (22), are also depressed during MET. As described above, downregulation of ZEB1, Slug, Snail, SMA, or Twist is usually induced during MET (49,61,64).…”

“…By using the bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo via systemic seeding, Chaffer and colleagues (21) showed the importance of the epithelial phenotype in the formation of secondary tumors, as epithelial characteristics were dramatically associated with increased bone and soft-tissue colonization after intracardiac or intratibial injection. Furthermore, Oltean and colleagues (22,23) observed an unexpected MET among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels by visualizing the fibroblast growth factor receptor-2 (FGFR2) exon IIIc in a prostate cancer model. In addition, non-EMT hamster cheek pouch carcinoma-1 cells, but not EMT cells, were found to be able to form overt lung metastases after being inoculated into the bloodstream by tail-vein injection (24), suggesting that EMT is not conducive to the formation of metastases and that EMT cells in vivo might possibly be converted into the non-EMT cells via MET at the site of metastases.…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…To examine FGFR2 splicing in vivo, Garcia-Blanco and colleagues (Oltean et al 2006) developed a minigene reporter system designed to express RFP in cells in which exon IIIc is not included. They injected mesenchymal AT3 prostate tumor cells expressing the minigene into mice and found that, after invading the lungs, the tumor cells frequently exhibited IIIc skipping, indicative of a mesenchymal-to-epithelial transition (MET), which was confirmed by the presence of the epithelial marker E-cadherin.…”