Alternative pre-messenger RNA splicing (AS) events are regulated when breast cancer cells acquire metastatic features through epithelial mesenchymal transition (EMT). ASE following EMT in the PMC42 human breast cancer cell lines were investigated by (i) comparisons between the more mesenchymal parental PMC42-ET (ET) cells and the more epithelial PMC42-LA (LA) subline, and (ii) in response to epidermal growth factor (EGF), which stimulates EMT-like changes at the mRNA and protein level in both PMC42 variants. We examined Epithelial Splicing Regulatory Protein (ESRP)-associated AS in the PMC42 system and other human breast cancer cell lines, using 2D and 3D cultures, as well as upon ZEB1-knockdown in ET cells. AS events in the PMC42 model showed an epithelial vs. mesenchymal splicing pattern that was directly correlated with expression of ESRP 1 and 2. ESRP1 was significantly higher (p<0.001) in LA than ET. ESRP1 levels were much higher than ESRP2, implicating ESRP1 rather than ESRP2 was the primary regulator of these AS events. The ESRP1-regulated epithelial-specific exon FGFR2-IIIb was higher in PMC42-LA than ET (p<0.001), and reduced by EGF in LA (p<0.01). ESRP regulation was also reflected in increased levels of 6 other ESRP-enhanced exons (ENAH, FLNB, RALGPS2, SLK, BAIAP2, FNIP1) and reduced levels of 5 other ESRP-silenced exons (OSBPL3, MAGI1, SCRIB, ARHGAP17, MBNL1) in PMC42-LA compared to PMC42-ET. CD44 standard isoform (CD44s) was elevated from Luminal to Basal B cells, and increased post EGF treatment in both PMC42 variants. CD44 variable regions (exon 5-6 and 8-10) exhibited higher levels in PMC42-LA than ET. ZEB1-knockdown in PMC42-ET cells caused increased levels of ESRP1 and the FGFR2-IIIb splice variant.
In 3D environments (Matrigel or collagen (Vitrogen)), ESRP1 and FGFR2-IIIb were significantly higher in PMC42-LA than PMC42-ET irrespective of EGF treatment. ENAH expression in Matrigel was similar in both PMC42 variants, irrespective of EGF, but increased by EGF in Vitrogen. CD44 was regulated more in Vitrogen than in Matrigel. These results suggest that 3D environment influence ESRP-related AS