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Background: In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes in pre-mRNA processing occurring as fibroblasts enter the proliferative cell cycle are also important for promoting their migration., Results: RNA sequencing of fibroblasts induced into quiescence by contact inhibition reveals downregulation of genes involved in mRNA processing, including splicing and cleavage and polyadenylation factors. These genes also show differential exon use, especially increased intron retention in quiescent fibroblasts compared to proliferating fibroblasts. Mapping the 3' ends of transcripts reveals that longer transcripts from distal polyadenylation sites are more prevalent in quiescent fibroblasts and are associated with increased expression and transcript stabilization based on genome-wide transcript decay analysis. Analysis of dermal excisional wounds in mice reveals that proliferating cells adjacent to wounds express higher levels of cleavage and polyadenylation factors than quiescent fibroblasts in unwounded skin. Quiescent fibroblasts contain reduced levels of the cleavage and polyadenylation factor CstF-64. CstF-64 knockdown recapitulates changes in isoform selection and gene expression associated with quiescence, and results in slower migration., Conclusions: Our findings support cleavage and polyadenylation factors as a link between cellular proliferation state and migration.

Cell migration is a highly conserved process involving cytoskeletal reorganization and restructuring of the surrounding extracellular matrix. Although there are many studies describing mechanisms underlying cell motility, little has been reported about the contribution of alternative isoform use toward cell migration. Here, we investigated whether alternative isoform use can affect cell migration focusing on reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), an established inhibitor of cell migration. We found that a shorter isoform of RECK is more highly expressed in proliferating fibroblasts, in TGF-β-treated fibroblasts, and in tumors compared with differentiated tissue. Knockdown of this short RECK isoform reduces fibroblast migration through Matrigel. Thus, this short isoform of RECK generated by a combination of alternative splicing and alternative polyadenylation plays an opposing role to the canonical RECK isoform, as knockdown of canonical RECK results in faster cell migration through Matrigel. We show that the short RECK protein competes with matrix metalloprotease 9 (MMP9) for binding to the Kazal motifs of canonical RECK, thus liberating MMP9 from an inactivating interaction with canonical RECK. Our studies provide a new paradigm and a detailed mechanism for how alternative isoform use can regulate cell migration by producing two proteins with opposing effects from the same genetic locus.

Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network.