Biochemical and Biological Significance of a S100A4 Dimer

Abstract

S100A4, a member of the Ca2+-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high expression levels of S100A4 correlate with poor patient survival in several cancers. Biophysical and structural studies show that S100A4 is a non-covalent homodimer. Although studies have addressed the molecular determinants necessary for S100A4 dimerization, it is unknown whether a bivalent S100A4 dimer is required for regulating the activity of its protein targets and promoting cell invasion. To address this question, we developed covalently linked S100A4 dimers (sc-S100A4), which, exhibit comparable binding affinities for calcium and myosin-IIA as the wild-type dimer, and promote the disassembly of myosin-IIA filaments. Furthermore, expression of sc-S100A4 proteins in HCT116 S100A4 -/- colon carcinoma cells restores tumor cell invasion in a comparable manner as re-expression of wild-type S100A4. To investigate the requirement for a bivalent S100A4 dimer, the Ca2+-binding motifs in one or both sc-S100A4 monomers were mutated. These mutants exhibited reduced binding affinities for myosin-IIA and did not promote myosin-IIA depolymerization. Moreover, these mutants were unable to restore HCT116 S100A4-/- cell invasion indicating that two functional monomeric subunits are necessary for S100A4 function in vitro and in vivo. In addition to these biochemical studies, we have begun examining the specific signaling pathways that regulate S100A4 activation in colon carcinoma cells, and which mediate paracrine interactions with macrophages to promote tumor cell invasion. Altogether these studies have provided a biochemical and molecular foundation for how S100A4 mediates colon carcinoma cell invasion.