Alternative Splicing (AS) and Epithelial-to-Mesenchymal Transition (EMT) programming in cellular model of breast cancer

It is increasingly evident that alternative pre- mRNA splicing events (AS) play a profound role in tumor progression and metastasis, frequently via the activation of cellular processes that can trigger epithelial-to-mesenchymal transition (EMT) program in the cells. Alterations in mRNA splicing pattern can arise from altered expression of splicing factors as well as interactions with cancer-related signal transductions. This study investigated the potential role of key hormones, estrogen and CRH in the regulation of pre-mRNA splicing mechanism, and the molecular pathways mediating CRH actions in promoting EMT in ER (+) MCF7 and ER (-) SKBR3 breast cancer cell line. Estrogen signaling has been reported to be involved in the alternative splicing of specific set of genes, and this study found that estrogen can induce the phosphorylation and nuclear translocation of a key splicing kinase, SRPK1 via Akt dependent pathway, and thus triggers the production of CD44s splice variant. Additionally, CRH regulates numerous protein kinases including Akt, therefore the effects of CRH on SRPK1 status was also investigated. In this study, CRH was shown to modulate SRPK1 activity via similar pathway as E2 did in MCF7 cells, resulted in the accumulation of CD44v6 mRNA isoform. Apparently, while this effect of CRH on SRPK1 was not seen in SKBR3 cells, increased expression of CD44v6 mRNA was detected, suggesting that CRH exerted its effects via different pathways in SKBR3 cells. Furthermore, increased level of CD44 splice variant, particularly CD44v6 in the stimulated cells positively correlated with EMT gene expression Twist/Snail as well as the migration and invasion properties of the cells. Additionally, EMT gene profiling analysis revealed that in addition to Twist and Snail, CRH was able to drive the transcription of several other prominent EMT markers such as VIM, TGFB1/2, NOTCH1, MMP9 and Wnt5A when either Akt or SRPK1 kinase was inhibited in the cells, thus signifying the potential convergence of CRH signaling pathway in the MCF7 cells. Given that the role and the mechanism of E2 and CRH actions in the progression of breast cancer are still not fully elucidated, the link between these two key hormones with alternative splicing events and the activation of EMT in ER+ cells demonstrated in this project may contribute considerably to the current understanding of breast cancer development and progression