Quantitative Proteomics Discloses MET Expression in Mitochondria as a Direct Target of MET Kinase Inhibitor in Cancer Cells

Abstract

Cancer cells with MET overexpression are paradoxically more sensitive to MET inhibition than cells with baseline MET expression. The underlying molecular mechanisms are incompletely understood. Here, we have traced early responses of SNU5, a MET-overexpressing gastric cancer cell line, exposed to sublethal concentration of PHA-665752, a selective MET inhibitor, using iTRAQ-based quantitative proteomics. More than 1900 proteins were quantified, of which >800 proteins were quantified with at least five peptides. Proteins whose expression was perturbed by PHA-665752 included oxidoreductases, transfer/carrier proteins, and signaling proteins. Strikingly, 38% of proteins whose expression was confidently assessed to be perturbed by MET inhibition were mitochondrial proteins. Upon MET inhibition by a sublethal concentration of PHA-665752, mitochondrial membrane potential increased and mitochondrial permeability transition pore was inhibited concomitant with widespread changes in mitochondrial protein expression. We also showed the presence of highly activated MET in mitochondria, and striking suppression of MET activation by 50 nM PHA-665752. Taken together, our data indicate that mitochondria are a direct target of MET kinase inhibition, in addition to plasma membrane MET. Effects on activated MET in the mitochondria of cancer cells that are sensitive to MET inhibition might constitute a novel and critical noncanonical mechanism for the efficacy of MET-targeted therapeutics. Molecular & Cellular Proteomics 9: 2629 -2641, 2010.

Discovery of biomarkers for gastric cancer: A proteomics approach

“…An iTRAQ-based quantitative proteomic approach was used to explore the paradox that MET-overexpressing cancer cells have a lower tolerance for the MET kinase inhibitor than cells with baseline expression. After exposure of a MET-overexpressing cancer cell line to a MET inhibitor, a number of proteins were identified as being altered, thus shedding light on the mechanism of MET inhibition [59]. Moreover, the analysis of gastric carcinoma cells (GTL16) treated with the MET inhibitor, PHA-665752, using a membrane-based protein array system revealed the differential expression of a number of cytokines and soluble molecules in response to MET inhibition [60].…”

Mitochondrial-Targeting MET Kinase Inhibitor Kills Erlotinib-Resistant Lung Cancer Cells

“…22 We have observed a hitherto unrecognized facet of MET kinase activity, namely, its localization to the mitochondria of cancer cells, which are high MET expressors 13 and the ensuing alteration of cellular functions that promote survival and hence resistance to TKIs. Our finding resonates with that of Ding et al, who showed that ERBB2 translocated to mitochondria of cancer cells and that localization was associated with greater resistance to cell killing by trastuzumab, a monoclonal antibody that blocks plasma membrane ERBB2 kinase activation.…”

“…12 We have previously shown that MET overexpression in cancer cells is associated with MET protein localization to the mitochondria while also remaining on the plasma membrane. 13 The presence of MET in the mitochondria shifted cellular metabolism to a state of heightened glycolysis, increased tricarboxylic acid (TCA) cycle activity, and oxidative phosphorylation. 13 Such metabolic remodeling would conceivably enhance the fitness of cancer cells for survival by increasing ATP production and providing abundant TCA cycle intermediates for biomass production.…”

“…13 The presence of MET in the mitochondria shifted cellular metabolism to a state of heightened glycolysis, increased tricarboxylic acid (TCA) cycle activity, and oxidative phosphorylation. 13 Such metabolic remodeling would conceivably enhance the fitness of cancer cells for survival by increasing ATP production and providing abundant TCA cycle intermediates for biomass production. Thus, we postulate that in these and other ways yet to be uncovered, mitochondrial MET increases the fitness of cancer cells for survival and may counteract anticancer therapies designed to achieve cell killing.…”

“…We were curious if the "extra" MET is localized to the mitochondria as this has been observed in other MET-amplified malignant cells. 13 Hence, we carried out an immunoblotting analysis of activated MET (MET phosphorylated at the catalytic tyrosine residues Y1234/1235) in whole cell lysates comprising equal numbers (10 5 ) of HCC827A or HCC827B cells and the subcellular fractions derived from this number of cells as they were fractionated to obtain purified mitochondria. The signal from each lane in Figure 1 reflects phosphorylated MET (p-MET) present in the same number of cells (10 5 cells) but not derived from identical total protein loading because the amounts of protein would progressively decline as purification progressed.…”