Covalent dimerization of interleukin-like epithelial-to-mesenchymal transition (EMT) inducer (ILEI) facilitates EMT, invasion, and late aspects of metastasis

Abstract

The interleukin-like epithelial-to-mesenchymal transition (EMT) inducer(ILEI)/FAM3C is a member of the highly homologous FAM3 family andis essential for EMT and metastasis formation. It is upregulated in severalcancers, and its altered subcellular localization strongly correlates withpoor survival. However, the mechanism of ILEI action, including the struc-tural requirements for ILEI activity, remains elusive. Here, we show thatILEI formed both monomers and covalent dimers in cancer cell lines andin tumors. Using mutational analysis and pulse-chase experiments, wefound that the four ILEI cysteines, conserved throughout the FAM3 fam-ily and involved in disulfide bond formation were essential for extracellularILEI accumulation in cultured cells. Modification of a fifth cysteine(C185), unique for ILEI, did not alter protein secretion, but completelyinhibited ILEI dimerization. Wild-type ILEI monomers, but not C185Amutants, could be converted into covalent dimers extracellularly uponoverexpression by intramolecular-to-intermolecular disulfide bond isomer-ization. Incubation of purified ILEI with cell culture medium showed thatdimerization was triggered by bovine serum in a dose- and time-dependentmanner. Purified ILEI dimers induced EMT and trans-well invasion ofcancer cellsin vitro. In contrast, ILEI monomers and the dimerization-defective C185A mutant affected only cell motility as detected by scratchassays and cell tracking via time-lapse microscopy. Importantly, tumorcells overexpressing wild-type ILEI caused large tumors and lung metas-tases in nude mice, while cells overexpressing the dimerization-defectiveC185A mutant behaved similar to control cells. These data show that cova-lent ILEI self-assembly is essential for EMT induction, elevated tumorgrowth, and metastasis.