Global Signaling Profiling in a Human Model of Tumorigenic Progression Indicates a Role for Alternative RNA Splicing in Cellular Reprogramming

Abstract

Intracellular signaling is controlled to a large extent by the phosphorylation status of proteins. To determine how human breast cells can be reprogrammed during tumorigenic progression, we profiled cell lines in the MCF10A lineage by phosphoproteomic analyses. A large cluster of proteins involved in RNA splicing were hypophosphorylated as cells progressed to a hyperplastic state, and then hyperphosphorylated after progression to a fully metastatic phenotype. A comprehensive transcriptomic approach was used to determine whether alterations in splicing factor phosphorylation status would be reflected in changes in mRNA splicing. Results indicated that the degree of mRNA splicing trended with the degree of tumorigenicity of the 4 cell lines tested. That is, highly metastatic cell cultures had the greatest number of genes with splice variants, and these genes had greater fluctuations in expression intensities. Genes with high splicing indices were mapped against gene ontology terms to determine whether they have known roles in cancer. This group showed highly significant associations for angiogenesis, cytokine-mediated signaling, cell migration, programmed cell death and epithelial cell differentiation. In summary, data from global profiling of a human model of breast cancer development suggest that therapeutics should be developed which target signaling pathways that regulate RNA splicing.