Identification of a pivotal endocytosis motif in c-Met and selective modulation of HGF-dependent aggressiveness of cancer using the 16-mer endocytic peptide

Abstract

Since c-Met has an important role in the development of cancer, it is considered as an attractive target for cancer therapy. Although molecular mechanisms for oncogenic property of c-Met have been actively investigated, regulatory elements for c-Met endocytosis and its effect on c-Met signaling remain unclear. In this study, we identified a pivotal endocytic motif in c-Met and tested it for selective modulation of HGF-induced c-Met response. Using various chimeric constructs with the cytoplasmic tail of c-Met, we were able to demonstrate that a dileucine motif located in the C-terminus of c-Met acts to regulate its endocytosis. Synthetic peptide Ant-3S, consisting of antennapedia-derived protein transduction domain (designated as Ant) and c-Met-derived 16 amino-acids (designated as 3S, spanning amino-acids 1378 to 1393), rapidly moved into cancer cells and disrupted c-Met trafficking. Importantly, an extension of c-Met retention time on the membrane by Ant-3S peptide significantly decreased phosphorylation-dependent c-Met signal transduction. Additionally, the peptide effectively inhibited HGF-induced cell growth, scattering and migration. The underlying molecular mechanism for these observations has been investigated and revealed that the dileucine motif interacts with endocytic machinery, including adaptin β and caveolin-1, for sustained and enhanced signal transduction. Finally, Ant-3S peptide specifically blocked internalization of interleukin-2 receptor α-subunit/3S chimeric protein, but not the other receptors, including Glut4, Glut8 and transferrin receptor. Such results indicate the presence of a selective endocytic assembly for c-Met. It also suggests a potential for c-Met-specific anti-cancer therapy using the identified endocytic motif in this study.

Reciprocal Activating Crosstalk between c-Met and Caveolin 1 Promotes Invasive Phenotype in Hepatocellular Carcinoma

“…HGF induced c-Met/CAV1 complex formation and enhanced co-localization of phosphorylated-c-Met with phosphorylated-CAV1, predominantly accumulated in the perinuclear region of cells. This data is consistent with the data reported by Cho et al [2012] that HGF induces the internalization of c-Met, which is co-localized with endocytic machinery members including CAV1 in 239E human embryonic kidney cell line [40]. They demonstrated that, CAV1 has a mediator function for c-Met trafficking to the perinuclear region towards signal promotion and inhibition of the association between endocytic machinery and c-Met inhibits HGF induced phosphorylation of MAPK and consequently reduces scattering and migration [40].…”

“…This data is consistent with the data reported by Cho et al [2012] that HGF induces the internalization of c-Met, which is co-localized with endocytic machinery members including CAV1 in 239E human embryonic kidney cell line [40]. They demonstrated that, CAV1 has a mediator function for c-Met trafficking to the perinuclear region towards signal promotion and inhibition of the association between endocytic machinery and c-Met inhibits HGF induced phosphorylation of MAPK and consequently reduces scattering and migration [40]. Accumulation of CAV1 and activated c-Met in the perinuclear region suggested ligand-mediated receptor internalization.…”

The Chemokine Receptor CXCR4 and c-MET Cooperatively Promote Epithelial-Mesenchymal Transition in Gastric Cancer Cells

“…Recent work indicated that Cav-1 is a molecular hub, integrating the transduction of multiple signals and acting as a stress-related oncotarget for drug resistance and metastasis [17] , [32] . Studies have demonstrated that Cav-1 can bind c-MET and regulate its endocytosis, and consequently its effects on downstream signaling [33] . Reciprocal activating cross talk between c-MET and Cav-1 was shown previously to promote migration, invasion, and branching morphogenesis in hepatocellular carcinoma [34] .…”

HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer

“…As such, this activation mechanism was insensitive to the potent, active site-directed MET inhibitor PHA-665752. In fact, a fourth category of inhibitor, involving a peptide that interferes with the C-terminal docking site of MET and which has been shown to display greater inhibitory effect and toxicity tolerance than the MET inhibitor PHA-665752 (Cantelmo et al 2010;Cho et al 2013), becomes a more attractive strategy in this context. Considering the data presented here, it is possible that combinations of agents that block both liganddependent and ligand-independent activation of MET may be the most effective strategies for therapeutic intervention.…”