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

  • Tiannan Guo
    Affiliations
    §School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551,

    ¶Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610,
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  • Yi Zhu
    Affiliations
    §School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551,
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  • Chee Sian Gan
    Affiliations
    §School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551,
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  • Sze Sing Lee
    Affiliations
    ¶Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610,
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  • Jiang Zhu
    Affiliations
    ‖Center for Stem Cell Research and Application, Union Hospital, Huazhong University of Science and Technology, Wuhan, P.R. China 430022,
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  • Haixia Wang
    Affiliations
    ‖Center for Stem Cell Research and Application, Union Hospital, Huazhong University of Science and Technology, Wuhan, P.R. China 430022,
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  • Xin Li
    Affiliations
    §School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551,
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  • James Christensen
    Affiliations
    **Translational Pharmacology, Pfizer Global Research and Development, La Jolla Laboratories, San Diego, California, 92121
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  • Shiang Huang
    Affiliations
    ‖Center for Stem Cell Research and Application, Union Hospital, Huazhong University of Science and Technology, Wuhan, P.R. China 430022,
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  • Oi Lian Kon
    Correspondence
    To whom correspondence should be addressed:Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 Siu Kwan Sze: Tel:65-6514-1006. Fax:65-6791-3856; Oi Lian Kon: Tel:65-6436-8307; Fax:65-6372-0161;
    Affiliations
    ¶Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610,
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  • Siu Kwan Sze
    Correspondence
    To whom correspondence should be addressed:Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 Siu Kwan Sze: Tel:65-6514-1006. Fax:65-6791-3856; Oi Lian Kon: Tel:65-6436-8307; Fax:65-6372-0161;
    Affiliations
    §School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551,
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  • Author Footnotes
    * This work is supported by grants from the Ministry of Education (ARC: T206B3211 to SKS) and the Agency for Science, Technology and Research (BMRC: 08/1/22/19/575 to SKS) of Singapore. This work was also supported by the National Cancer Centre of Singapore Research Fund. We declare no conflicts of interest.
    This article contains supplemental Figs. 1–4 and Table 1 and 2.
    1 The abbreviations used are:LCliquid chromatographyIPIInternational Protein IndexFDRfalse discovery rateETCelectron transfer chainmPTPmitochondrial permeability transition pore.
      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.
      Recent improvements in survival of some malignancies owe much to advances in uncovering aberrantly active molecular pathways, against which molecularly targeted agents have been developed as new strategies to control cancers (
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      ). However, molecular mechanisms underlying the curious dependence of some cancer cells, which contain multiple genomic, genetic, and epigenetic abnormalities, on a single oncogenic molecule (the phenomenon of “oncogene addiction”) are incompletely understood (
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      Receptor tyrosine kinases are the most extensively studied oncogenic targets and receptor tyrosine kinase inhibitors have proven anticancer therapeutic efficacy. A receptor tyrosine kinase, MET, whose ligand is hepatocyte growth factor (HGF), is frequently amplified and overexpressed (
      • Kuniyasu H.
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      ,
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      ). Human gastric cancer cell lines harboring MET amplicons and overexpressing MET are readily induced to apoptosis by selective inhibitors of MET (
      • Smolen G.A.
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      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
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      • Christensen J.G.
      • Settleman J.
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      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ,
      • Zou H.Y.
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      ), several of which are under active development for clinical use (
      • Comoglio P.M.
      • Giordano S.
      • Trusolino L.
      Drug development of MET inhibitors: targeting oncogene addiction and expedience.
      ). One of the selective small molecular inhibitors, PHA-665752, designed chemically as (3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one (molecule weight of 641.61), specifically suppresses tyrosine phosphorylation of MET. PHA-665752 has >50-fold higher selectivity for MET than for other tyrosine and serine/threonine kinases (
      • Christensen J.G.
      • Schreck R.
      • Burrows J.
      • Kuruganti P.
      • Chan E.
      • Le P.
      • Chen J.
      • Wang X.
      • Ruslim L.
      • Blake R.
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      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ). The inhibition of MET kinase function by PHA-665752 on cancer cells had been confirmed with siRNA knockdown of MET, and a number of downstream effectors of MET signaling pathways were confirmed to be effectively abrogated by this compound (
      • Smolen G.A.
      • Sordella R.
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      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ,
      • Christensen J.G.
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      • Kuruganti P.
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      • Blake R.
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      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ). PHA-665752 has been widely used as a potent and selective tool for the evaluation of MET-dependent cellular functions and signal transduction (
      • Smolen G.A.
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      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ,

      Okamoto, W., Okamoto, I., Yoshida, T., Okamoto, K., Takezawa, K., Hatashita, E., Yamada, Y., Kuwata, K., Arao, T., Yanagihara, K., Fukuoka, M., Nishio, K., Nakagawa, K., Identification of c-Src as a potential therapeutic target for gastric cancer and of MET activation as a cause of resistance to c-Src inhibition. Mol. Cancer Ther. 9, 1188–1197,

      ,
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      ,
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      ,
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      ).
      The fact that only a subset of cancers is sensitive to killing by MET-directed therapeutics (hereafter referred to as sensitive cells) (
      • Comoglio P.M.
      • Giordano S.
      • Trusolino L.
      Drug development of MET inhibitors: targeting oncogene addiction and expedience.
      ), raises an unexplained paradox. MET-overexpressing cancer cells could reasonably be expected to be more tolerant of MET kinase inhibition compared with cancer cells that do not overexpress MET. In reality, the opposite occurs. The underlying molecular mechanisms are incompletely understood.
      To investigate this paradox we undertook a systematic exploration of responses of a MET-overexpressing gastric cancer cell line, SNU5, to sublethal MET inhibition using the iTRAQ-based quantitative proteomics approach. Our results unexpectedly showed a predominant perturbation of mitochondrial proteins in response to MET inhibition. Next, we found that MET inhibition was rapidly associated with altered mitochondrial functions. These observations raised the possibility that mitochondria might be a direct target of MET inhibition. Both protein immunoblotting and confocal microscopy showed the presence of highly activated MET in the mitochondria of sensitive cancer cells. Furthermore, we observed that activating phosphorylation of tyrosine residues of mitochondrial MET was critically modulated by sublethal PHA-665752 treatment.

      EXPERIMENTAL PROCEDURES

       Chemicals

      All chemicals were purchased from Sigma-Aldrich unless otherwise stated. A selective MET inhibitor PHA-665752 (
      • Christensen J.G.
      • Schreck R.
      • Burrows J.
      • Kuruganti P.
      • Chan E.
      • Le P.
      • Chen J.
      • Wang X.
      • Ruslim L.
      • Blake R.
      • Lipson K.E.
      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ) was from Pfizer Global Research and Development (La Jolla Laboratories, San Diego, CA). Stock solutions of this compound were prepared in DMSO, stored in −80 °C and diluted with fresh medium before use. In all experiments, the final concentration of DMSO was <0.1%.

       Cell culture

      Gastric cancer cell lines AGS, Kato III, SNU1, SNU5, SNU16, NCIN87, and Hs746T, and a human fibroblast cell line, Hs68, were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured as recommended. MKN7, and IM95 cells were from Japan Health Science Research Resource Bank and were cultured as recommended. YCC cells were a gift from Dr. Sun Young Rha (Yonsei Cancer Center, Seoul, Korea) and were grown in MEM supplemented with 10% fetal bovine serum (Hyclone, Thermo Fisher Scientific, Waltham, MA), 100 U penicillin, and 100 μg streptomycin per ml (Invitrogen).

       Gene expression profiling

      Total RNA was extracted from cell lines using the RNeasy Mini kit (Qiagen, Valencia, CA) and profiled using Affymetrix HG-U133 and HG-U133 Plus 2.0 GeneChip® (Affymetrix, Santa Clara, CA). Each RNA sample was amplified, labeled, and hybridized according to the manufacturer's protocols. Normal gastric tissue RNA samples from two commercial sources were employed as controls. FirstChoice Human Stomach Total RNA (Ambion, Austin, TX) was RNA from a single individual. MVP Total RNA, Human Stomach (Stratagene, La Jolla, CA) was pooled RNA from two individuals. Four probe sets (203510_at, 211599_x_at, 213807_x_at and 213816_s_at) for MET were printed on the arrays.

       MTT assay

      Cell viability based on redox enzyme activity was quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the MTT assay as described (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ).

       iTRAQ protein sample preparation

      Four experimental groups of SNU5 cells were prepared in the absence or presence of PHA-665752. Three groups were exposed to 50 nm PHA-665752 for three time periods i.e. 4 h, 24 h, and 72 h. A parallel group without treatment served as the control. After treatment, proteins were extracted and three independent biological replicate flasks for each experimental condition were pooled and quantified by bicinchoninic acid protein assay kit as described previously (
      • Guo T.
      • Gan C.S.
      • Zhang H.
      • Zhu Y.
      • Kon O.L.
      • Sze S.K.
      Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation.
      ).

       Isobaric labeling

      Two-hundred micrograms of protein from each experimental condition were tryptically digested and labeled with 4-plex iTRAQ reagents (Applied Biosystems, Foster City, CA) as follows: control, 114; 4 h, 115; 24 h, 116; 72 h, 117. The labeled samples were pooled and resolved into 20 fractions using strong cation exchange (SCX)(
      • Guo T.
      • Gan C.S.
      • Zhang H.
      • Zhu Y.
      • Kon O.L.
      • Sze S.K.
      Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation.
      ). Eluted fractions were vacuum dried and desalted using SEP-PAK C18 cartridges (Waters, Milford, MA). Dried peptides were stored at −80 °C before MS analysis.

       Liquid chromatography (LC)
      The abbreviations used are:
      LC
      liquid chromatography
      IPI
      International Protein Index
      FDR
      false discovery rate
      ETC
      electron transfer chain
      mPTP
      mitochondrial permeability transition pore.
      -MS/MS analysis

      The LC-MS/MS analysis was performed as previously described (
      • Guo T.
      • Gan C.S.
      • Zhang H.
      • Zhu Y.
      • Kon O.L.
      • Sze S.K.
      Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation.
      ,

      Datta, A., Park, J. E., Li, X., Zhang, H., Ho, Z. S., Heese, K., Lim, S. K., Tam, J. P., Sze, S. K., Phenotyping of an in vitro model of ischemic penumbra by iTRAQ-based shotgun quantitative proteomics. J. Proteome Res. 9, 472–484,

      ) with some modifications. Briefly, dried iTRAQ-labeled peptide samples were dissolved in HPLC grade water (Mallinckrodt Baker) acidified with 0.1% formic acid, and sequentially injected and separated in a home-packed nanobored C18 column with a picofrit nanospray tip (75 μm ID × 15 cm, 5-μm particles) (New Objectives, Woburn, MA) on a TempoTM nano-MDLC system coupled with a QSTAR® Elite Hybrid LC-MS/MS system (Applied Biosystems). Each sample was divided into two equal aliquots and independently analyzed by the LC-MS/MS over a gradient of 120 min. The flow rate of LC system was set constantly at 300 nl/min. Data acquisition in QSTAR Elite was set to positive ion mode using Analyst® QS 2.0 software (Applied Biosystems). Precursors with a mass range of 300–2000 m/z and a calculated charge of +2 to +4 were selected for fragmentation. For each MS spectrum, a maximum of three most abundant peptides above a five-count threshold were selected for MS/MS. Each selected precursor ion was dynamically excluded for 30 s with a mass tolerance of 0.03 Da. Smart information-dependent acquisition was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 s.

       MS spectrum analysis

      Spectra acquired in LC-MS/MS system from the two independent runs were submitted in a batch to ProteinPilot (v2.0.1, Applied Biosystems) for peak-list generation, as well as protein identification and quantification against the International Protein Index (IPI) human database (version 3.34; 67758 sequences) supplemented with porcine trypsin. The Paragon algorithm in ProteinPilot software was configured as previously described (

      Datta, A., Park, J. E., Li, X., Zhang, H., Ho, Z. S., Heese, K., Lim, S. K., Tam, J. P., Sze, S. K., Phenotyping of an in vitro model of ischemic penumbra by iTRAQ-based shotgun quantitative proteomics. J. Proteome Res. 9, 472–484,

      ) with some modifications. Briefly, default parameters including fixed and variable modifications for tryptically digested samples labeled with 4-plex iTRAQ reagents (peptide labeled) were employed. The search was done thoroughly where all cleavage variants were considered. The confidence threshold for both peptide and protein identification was set to 70%. Default precursors and the fragments mass tolerance for QSTAR ESI MS instrument were adopted by the software. A concatenated target-decoy database search strategy was also employed to estimate the false discovery rate (FDR) (
      • Elias J.E.
      • Gygi S.P.
      Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.
      ). FDR was calculated as twofold of the percentage of decoy matches divided by the total matches. After stringent filtering as described in Results, FDR of the reported iTRAQ data set was <1%. ProteinPilot software employed the peak area of iTRAQ reporters for quantification. Details of the quantification algorithm can be found in the supplier's manual. Isoform-specific strategy was adopted to deal with quantification of isoforms. Quality control of the data set is addressed in Results.

       Bioinformatics

      Gene IDs of the proteins of interest were searched in a batch using PANTHER classification system (
      • Thomas P.D.
      • Campbell M.J.
      • Kejariwal A.
      • Mi H.
      • Karlak B.
      • Daverman R.
      • Diemer K.
      • Muruganujan A.
      • Narechania A.
      PANTHER: a library of protein families and subfamilies indexed by function.
      ) against NCBI (H. sapiens) dataset and the results were presented as genes. Most protein groups had more than one molecular function hit. Cellular localization information of the 50 proteins of interest was checked manually in Gene Ontology (
      • Ashburner M.
      • Ball C.A.
      • Blake J.A.
      • Botstein D.
      • Butler H.
      • Cherry J.M.
      • Davis A.P.
      • Dolinski K.
      • Dwight S.S.
      • Eppig J.T.
      • Harris M.A.
      • Hill D.P.
      • Issel-Tarver L.
      • Kasarskis A.
      • Lewis S.
      • Matese J.C.
      • Richardson J.E.
      • Ringwald M.
      • Rubin G.M.
      • Sherlock G.
      Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.
      ).

       Western blotting

      Western blotting was performed using primary antibodies at the dilutions indicated: 1:500 SDHB (clone 21A11), 1:500 NDUFS3 (clone 17D95), 1:1000 VDAC1 (clone 20B12), 1:1000 MET (clone C-12), 1:1000 phospho-MET (Y1234/1235), 1:1000 phospho-MET (Y1349), 1:1000 E-cadherin (G-10), 1:2500 actin (Clone C4), 1:2000 α-tubulin (clone B-7). Phospho-MET antibodies were from Cell Signaling (Danvers, MA), actin antibody was from Millipore (Billerica, MA), whereas the other primary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against integrin αL (1:500), MHM23, was kindly supplied by Dr Alex Law (School of Biological Sciences, Nanyang Technological University, Singapore).

       Mitochondrial membrane potential analysis

      Cells with or without PHA-665752 treatment were washed with ice-cold PBS and incubated with 5 μg/ml rhodamine 123 for 1 h, followed by flow cytometric analysis on FACS Calibur and CellQuest Pro software (Becton Dickinson, Franklin Lakes, NJ).

       Mitochondrial permeability transition pore analysis

      The activity of mitochondrial transition pore was evaluated by the MitoProbe™ Transition Pore Assay Kit (Becton Dickinson) following the manufacturer's instruction. Briefly, cells were washed twice with ice-cold Hanks' balanced salt solution containing 1.3 mm calcium (Invitrogen) before incubation in the presence or absence of cobalt chloride at 37 °C for 15 min, followed by flow cytometry analysis as described earlier.

       Confocal microscopy

      SNU5 cells were washed with HEPES twice, before incubating with 500 nm Mito Tracker Red CMXRos (Invitrogen) for 15 min. Cells were then fixed in 3% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 2 min. After blocking nonspecific antibody binding sites with 1% BSA for 1 h at 37 °C, cells were probed with primary antibodies (1:500) overnight at 37 °C and Alexa 488- conjugated goat-anti-rabbit secondary antibodies (Invitrogen) for 1 h at 37 °C. Finally the cells were washed with PBS and counterstained with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were captured with a Zeiss LSM 710 confocal microscope.

       Mitochondria isolation

      Mitochondria isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was employed to isolate mitochondria following the manufacturer's protocol. Briefly, 5 × 107 SNU5 cells with or without treatment were washed twice with PBS, and lysed in 2 ml of the provided lysis buffer supplemented with Complete Protease Inhibitor Mixture Tablets and phosSTOP (Roche, Basel, Switzerland). The crude cell lysate was incubated with anti-TOM22 MicroBeads for 1 h at 4 °C with gentle shaking. Subsequently, the suspension was loaded onto a pre-equilibrated MACS column, washed three times with separation buffer before removing the column from the magnetic field and eluting the mitochondria.

      RESULTS

       MET expression and susceptibility of gastric cancer cells to PHA-665752

      As PHA-665752 is differentially cytotoxic in cancer cells depending on MET expression levels (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ), we first evaluated MET expression data of a panel of 16 gastric cancer cell lines, (AGS, Kato III, SNU1, SNU5, SNU16, NCIN87, Hs746T, MKN7, IM95, YCC1, YCC2, YCC3, YCC6, YCC9, YCC11, and YCC16) in order to focus on a model cell line for systematic proteomics exploration. Our transcriptome data showed that SNU5 cells had markedly elevated levels of MET transcription (>40-fold compared with normal human stomach tissues), while MET expression of SNU1 cells was comparable to the controls (supplemental Fig. 1). MET protein expression levels of these two cell lines were compared by immunoblotting (supplemental Fig. 2). SNU5 and SNU1 cells showed high and low expression of MET, respectively, in agreement with our transcriptome data as well as a previous study (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ). We determined cytotoxic responses of the two gastric cancer cell lines to PHA-665752 using MTT assay (supplemental Fig. 3). The mean IC50 of PHA-665752 in SNU5 cells was ∼77 nm, whereas SNU1 cells were relatively resistant to the compound (IC50 > 500 nm). SNU5 was selected as the model cell line in subsequent temporal quantitative proteomics analyses because it was highly sensitive to PHA-665752. Conversely, SNU1 was chosen as being representative of gastric cancer cells resistant to MET inhibition in functional studies.

       Temporal quantitative proteomics analysis

      We treated SNU5 cells with PHA-665752 and analyzed the temporal dynamics of the proteome. First, we sought to determine an appropriate concentration of PHA-665752 for quantitative proteomic investigation of SNU5 cells, in order to trace early cellular responses of the cells to MET inhibition. The ideal treatment conditions with PHA-665752 should suppress phosphorylation of MET without causing substantial cell death. SNU5 cells were exposed for varying durations to two concentrations of PHA-665752 around its IC50 (determined at 72 h) and tested for viability using MTT assays. Our results showed that 50 nm PHA-665752 did not significantly impair cell viability, whereas 150 nm was rapidly cytotoxic (supplemental Fig. 4). As such, we regarded 50 nm as a sublethal concentration for SNU5 cells, and adopted these conditions for the subsequent proteomics study.
      It is worth noting that it remains a daunting challenge that some small-molecule kinase inhibitors exhibit off-target effects that cannot be ignored (
      • Zhang J.
      • Yang P.L.
      • Gray N.S.
      Targeting cancer with small molecule kinase inhibitors.
      ). To minimize off-target effects in this study, we selected one of the most potent and specific MET inhibitors PHA-665752, which is >50 times more selective for MET than other protein kinases (
      • Christensen J.G.
      • Schreck R.
      • Burrows J.
      • Kuruganti P.
      • Chan E.
      • Le P.
      • Chen J.
      • Wang X.
      • Ruslim L.
      • Blake R.
      • Lipson K.E.
      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ). Moreover, we applied it to a cell line SNU5 that overexpresses MET at unusually high levels i.e. >40 times higher than normal stomach tissue. In addition, we intentionally employed a low concentration of PHA-665752, i.e. 50 nm, which is sublethal to SNU5 cells but sufficient to inhibit MET activity. This further refined the data as arising from specific inhibition of MET because most off-target effects happen when inhibitors are used at high concentrations, such as >1 μm. We believe, in this scenario, the probability of inhibiting other proteins with even comparable or higher affinity than MET is low or negligible. Furthermore, previous work has documented that the differential effects of PHA-665752 are truly attributed to its effect on MET using small interfering (si) RNA targeting the MET receptor transcript in SNU5 cells (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ). Finally, growth factor effectors in the downstream of MET signaling pathway, including ERK1/2, AKT, STAT3, and FAK, were effectively abrogated by 50 nm PHA-665752 (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ). Thus, MET could reasonably be considered the main target of 50 nm PHA-665752 in SNU5 cells in this study.
      We used iTRAQ reagents to label the tryptically digested proteome, coupled with shotgun multidimensional liquid chromatography and tandem mass spectrometry (
      • Ross P.L.
      • Huang Y.N.
      • Marchese J.N.
      • Williamson B.
      • Parker K.
      • Hattan S.
      • Khainovski N.
      • Pillai S.
      • Dey S.
      • Daniels S.
      • Purkayastha S.
      • Juhasz P.
      • Martin S.
      • Bartlet-Jones M.
      • He F.
      • Jacobson A.
      • Pappin D.J.
      Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
      ) to profile the temporal proteome responses (Fig. 1A). This approach allowed simultaneous comparison of the proteomes at four time points (0, 4 h, 24 h, and 72 h) after PHA-665752 treatment, to capture both the early and late responses of the SNU5 proteome.
      Figure thumbnail gr1
      Fig. 1Quantitative proteomic analysis shows dominant effects on mitochondrial proteins. A, Schematic diagram of the iTRAQ-LC-MS/MS experimental workflow. B, Distribution of the relative expression levels of temporal proteomes. Ratios were calculated in log space before converting into linear space. C, Subcellular classification of the differentially expressed proteins based on gene ontology. Details of the proteins are shown in . Note that one protein may have >1 subcellular localization hits. D, Schematic display of the functions and localizations of PHA-665752-perturbed expression of mitochondrial proteins. *, mitochondrial proteins whose exact localization within the mitochondrion is uncertain; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; mPTP, mitochondrial permeability transition pore; ETC, electron transport chain.

       Quality control of quantitative MS data set

      To ensure the reliability of the quantitative datasets, three independent biological replicates of SNU5 cells were pooled for the proteomics study (Fig. 1A). Moreover, the iTRAQ-labeled samples were analyzed twice by LC-MS/MS to minimize technical variations. The ProteinPilot database search in a concatenated target and decoy strategy returned 26276 target matches and only one decoy match. A total of 1908 target proteins were identified and quantified with estimated FDR of <1%. We next employed stringent inclusion criteria to filter the data set. A total of 806 proteins quantified with high confidence i.e. quantified from at least five peptides, of which there are at least two unique peptides, and having error factors <1.5, were advanced to the next phase of analysis (supplemental Table 1).

       Estimation of cutoff for confidently defining perturbed proteins

      The cutoff for defining perturbed and unperturbed protein expression in iTRAQ experiments depends on the characteristics of biological samples as well as MS instruments. To avoid setting the cutoffs arbitrarily, we examined the distribution of the expression levels of the 806 proteins (Fig. 1B). The three relative expression levels of these proteins i.e. 4 h/control, 24 h/control, and 72 h/control, were all normally distributed indicating that sublethal treatment of this compound only modulated a small percentage of the SNU5 proteome. Thus, we focused on the top 5% proteins whose expression was most perturbed by PHA-665752 treatment. With this criterion, protein ratios <0.774 were regarded as underexpressed, whereas ratios >1.181 were considered overexpressed, thereby narrowing the reliable differentially expressed proteins to a small number of 50 (supplemental Table 2), which reflected significant effects of MET inhibition in SNU5 cells.

       Western blotting validation of iTRAQ ratios

      To further evaluate the accuracy of iTRAQ ratios for the shortlisted 50 proteins, we examined the expression of three representative proteins, NDUFS3, SDHB, and VDAC1, by semiquantitative Western blot analysis. NDUFS3 and SDHB, proteins of the mitochondrial ETC, were quantified by MS with unique peptide numbers of six and three, respectively, whereas VDAC1, a component of mPTP, was quantified with 10 unique peptides. As shown in Fig. 2 and Table I, Western blot data showed similar trends corresponding iTRAQ ratios.
      Figure thumbnail gr2
      Fig. 2Western blotting validation of selected proteins in the iTRAQ data set. A, DNUFS3 from ETC Complex I; B, SDHB from ETC Complex II; and C, VDAC1 were validated using Western blot analysis. Matched results from Western blots and iTRAQ (bar chart) are shown together for comparison.
      Table IDifferentially expressed mitochondrial proteins in MET-inhibited SNU5 cells as quantified by iTRAQ. Proteins reliably identified from the iTRAQ data set and documented to be localized in mitochondria are listed. *, proteins validated using western blotting
      Mitochondrial functionAccessionsProtein descriptionGene symbol% of sequence coverage# of unique peptide4h/ctrl24hr/ctrl72hr/ctrl
      RatioNRatioNRatioN
      ETC Complex IIPI00025239.2NADH dehydrogenase iron-sulfur protein 2SLC25A1326.130.838 ± 0.11660.613 ± 0.05860.772 ± 0.0596
      ETC Complex IIPI00025796.3NADH dehydrogenase iron-sulfur protein 3NDUFS3*41.760.813 ± 0.07050.712 ± 0.05550.922 ± 0.0545
      ETC Complex IIIPI00294911.1Succinate dehydrogenase iron-sulfur subunitSDHB*29.630.921 ± 0.04550.701 ± 0.04150.922 ± 0.0625
      ETC Complex IIIIPI00013847.4Ubiquinol-cytochrome-c reductase complex core protein 1UQCRC145.480.921 ± 0.045160.612 ± 0.053140.811 ± 0.04015
      ETC Complex IIIIPI00305383.1Ubiquinol-cytochrome-c reductase complex core protein 2UQCRC242.840.863 ± 0.06680.551 ± 0.03280.802 ± 0.0628
      ETC Complex IVIPI00017510.3Cytochrome c oxidase subunit 2COX223.330.691 ± 0.034130.431 ± 0.029130.772 ± 0.05213
      ETC Complex IVIPI00025086.3Cytochrome c oxidase subunit 5ACOX5A64.730.784 ± 0.07480.615 ± 0.08080.894 ± 0.0858
      ETC electron transporterIPI00029264.3Cytochrome c1 heme proteinCYC144.340.761 ± 0.04460.603 ± 0.05760.856 ± 0.1046
      mPTPIPI00216308.5Voltage-dependent anion-selective channel protein 1VDAC1*40.3100.713 ± 0.068140.532 ± 0.046140.874 ± 0.08314
      mPTPIPI00007188.5Adenine nucleotide translocator 2ANT272.1100.818 ± 0.11460.563 ± 0.05960.814 ± 0.0856
      OthersIPI00604707.4Dihydrolipoamide S-acetyltransferaseDLAT40.061.621 ± 0.53680.896 ± 0.16951.083 ± 0.0838
      OthersIPI00073772.5Fructose-1,6-bisphosphatase 1FBP150.690.942 ± 0.055271.163 ± 0.079331.483 ± 0.10033
      OthersIPI00022793.4Trifunctional enzyme β subunitHADHB45.950.784 ± 0.07470.722 ± 0.05560.926 ± 0.1047
      OthersIPI00007242.1Galectin-2LGALS259.161.037 ± 0.117150.986 ± 0.111151.963 ± 0.22115
      OthersIPI00017334.1ProhibitinPHB58.160.844 ± 0.08080.523 ± 0.05480.766 ± 0.1569
      OthersIPI00027252.6Prohibitin-2PHB263.260.862 ± 0.058110.502 ± 0.048110.642 ± 0.04911
      OthersIPI00183695.9Protein S100-A10S100A1070.140.661 ± 0.038280.712 ± 0.048281.062 ± 0.06229
      OthersIPI00007084.2Mitochondrial aspartate-glutamate carrier proteinSLC25A1329.170.856 ± 0.10480.662 ± 0.05790.903 ± 0.0698
      OthersIPI00790115.1CDNA FLJ90278 fis, mitochondrial precursorSLC25A339.620.681 ± 0.03380.581 ± 0.03980.892 ± 0.0528

       Quantitative proteomics dataset reveals perturbed cellular responses after sublethal PHA-665752 treatment

      The iTRAQ dataset provided identification and quantification of 806 proteins with various molecular functions as classified by PANTHER (Fig. 3), including 50 proteins (supplemental Table 2) whose expression had been perturbed by PHA-665752-induced MET inhibition. These proteins represent various cellular responses as shown in Fig. 3. Consistent with the specificity of MET inhibition, although proteins involved in nucleic acid binding and the cytoskeleton were the two most abundant groups, very few (<3%) were perturbed in expression by PHA-665752. Remarkably, sublethal concentration of this compound mainly affected the expression of several other groups of proteins, including oxidoreductases, calcium-binding proteins, transfer/carrier proteins involved in transport of specific substances, and signaling proteins.
      Figure thumbnail gr3
      Fig. 3Classification of molecular functions of proteins quantified by iTRAQ. Gene IDs of the 806 proteins were batch searched using PANTHER classification system against NCBI (H. sapiens). 728 unique genes were mapped to a total of 891 hits of molecular functions. Fifty perturbed proteins mapped to 49 unique genes and a total of 59 hits of molecular functions. The numbers of nonperturbed and perturbed hits are shown.
      Our data showed that 7 (29%) of the 24 molecular function hits were associated with calcium-binding proteins, including annexins (i.e. annexin A2 (IPI00455315.4), annexin A4 (IPI00793199.1), annexin A5 (IPI00329801.12)), calmodulin-related proteins comprising hippocalcin-like protein 1 (IPI00219344.4), protein S100-A4 (IPI00032313.1), protein S100-A10 (IPI00183695.9), and mitochondrial aspartate-glutamate carrier protein (IPI00007084.2). Modulation of calcium-binding proteins by PHA-665752 treatment suggested that calcium signaling might be a downstream response to inhibition of MET kinase activity. Calcium signaling has been extensively documented to be tightly modulated e.g. by G protein-coupled receptors and tyrosine kinase receptors (
      • Clapham D.E.
      Calcium signaling.
      ). It is worth noting that the link between calcium and MET signaling is supported by the recent observation that MET, when stimulated by hepatocyte growth factor, regulated calcium signals in human liver tumor cells (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ).
      Of the 29 proteins classified as having transfer/carrier function, six proteins (21%) responded to PHA-665752 at nanomolar concentration. Aside from the three annexin proteins and mitochondrial aspartate-glutamate carrier protein that were also classified in the calcium-binding protein category, another two phosphate carrier proteins i.e. adenine nucleotide translocator 2 (IPI00007188.5) and mitochondrial phosphate carrier protein (IPI00790115.1) were modulated by this compound.
      Five of 17 (29%) proteins classified as signaling molecules, cytokine macrophage migration inhibitory factor (IPI00790382.1), myristoylated alanine-rich protein kinase C substrate (IPI00219301.7), COP9 constitutive photomorphogenic homolog subunit 8 (IPI00009480.1), galectin 2 (IPI00007242.1), and Rho GDP dissociation inhibitor α(IPI00794402.1), showed perturbed expression (Fig. 3), as a probable consequence of altered signal transduction induced by PHA-665752.
      As shown in Table I, most of the mitochondrial proteins showed a similar trend of response to the compound. They were down-regulated at 4 h, further down-regulated at 24 h, and partially recovered at 72 h. This typical pattern of perturbed mitochondrial protein expression indicated a gradual but reversible effect of PHA-665752 on the SNU5 proteome.

       Dominant roles of mitochondrial proteins in PHA-665752-induced MET inhibition

      Interestingly, we found that many of the perturbed proteins in this dataset were associated with mitochondria. Hence, we performed a bioinformatics classification based on subcellular localization information from Gene Ontology (
      • Ashburner M.
      • Ball C.A.
      • Blake J.A.
      • Botstein D.
      • Butler H.
      • Cherry J.M.
      • Davis A.P.
      • Dolinski K.
      • Dwight S.S.
      • Eppig J.T.
      • Harris M.A.
      • Hill D.P.
      • Issel-Tarver L.
      • Kasarskis A.
      • Lewis S.
      • Matese J.C.
      • Richardson J.E.
      • Ringwald M.
      • Rubin G.M.
      • Sherlock G.
      Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.
      ) to determine how many perturbed proteins were mitochondrial. Not surprisingly, a significant number of the perturbed proteins were cytosolic. However, it was noteworthy that 19 proteins, i.e. 38% of the 50 proteins whose expressions were altered by PHA-665752, were localized in mitochondria (Fig. 1C), indicating a disproportionately dominant role of mitochondria in cellular responses to PHA-665752 treatment. Specifically, proteins from the two pivotal mitochondrial complexes i.e. electron transfer chain (ETC) and mitochondrial permeability transition pore (mPTP), were significantly perturbed by PHA-665752. Additionally, mitochondrial proteins involved in metabolism, signal transduction, survival, and apoptosis were also affected by PHA-665752 treatment (Fig. 1D).

       Mitochondrial ETC is perturbed by PHA-665752

      Eight (42%) of the 19 dysregulated mitochondrial proteins were components of the ETC in the inner mitochondrial membrane (IMM) (Fig. 1D, Table I). ETC is comprised of complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex), and complex IV (cytochrome c oxidase). Proteins from all four ETC complexes had decreased by 4 h after PHA-665752 treatment, were further inhibited at 24 h and had partially recovered at 72 h. These findings showed that PHA-665752 at a sublethal concentration rapidly modulated the expression of multiple ETC component proteins.
      The ETC operates mainly through its constituent oxidoreductase enzyme activities. As the MTT assay measures cellular oxidoreductase enzyme activities, mainly of the ETC, we asked if oxidoreductase activity of the ETC was diminished by PHA-665752. By cross-referencing MTT assay data (supplemental Fig. 4) with iTRAQ data (Table I) at 24 h of treatment with 50 nm PHA-665752, significant decreases in ETC protein expression levels were associated with only a small decrease of ETC oxidoreductase enzyme activity at the same time point, suggesting that despite inhibition of ETC protein expression, mitochondria retained substantial oxidoreductase enzyme activities required for cell survival.
      One of the most important functions of ETC is to maintain the MMP (
      • Chen L.B.
      Mitochondrial membrane potential in living cells.
      ). The energy released by electron transport pumps protons across the IMM, generating the electrochemical and pH gradients. We examined the MMP using rhodamine 123 staining and flow cytometry (
      • Foster K.A.
      • Galeffi F.
      • Gerich F.J.
      • Turner D.A.
      • Müller M.
      Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration.
      ). Rhodamine 123, a cationic dye, has a strong emission at 529 nm that is quenched as it accumulates within mitochondrial intermembrane space, and then dequenched (with increased fluorescence) when released into the cytosol. Our data showed that rhodamine 123 fluorescence of SNU5 cells did not change upon treatment with nanomolar concentration of PHA-665752 until exposure was prolonged beyond 24 h (Fig. 4). The decrease in rhodamine 123 fluorescence at 48 h and 72 h was the evidence of an increase in MMP and hyperpolarization of the mitochondria (
      • Foster K.A.
      • Galeffi F.
      • Gerich F.J.
      • Turner D.A.
      • Müller M.
      Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration.
      ). However, MMP of the PHA-665752-resistent cell line, SNU1, did not change with the same treatment (Fig. 4) and even at a higher concentration (300 nm, data not shown).
      Figure thumbnail gr4
      Fig. 4PHA-665752 effects on MMP of SNU5 and SNU1 cells. Mitochondrial membrane potentials were analyzed in SNU5 (left panel) and SNU1 (right panel) cells treated with 50 nm (bold solid line) PHA-665752 for 0.5, 2, 24, 48 and 72 h. Control untreated cells were analyzed in parallel (dotted line). Flow cytometry data are presented as histograms of the FL1 channel.

       mPTP responses to PHA-665752 treatment

      We observed inhibited expression of two core components of mPTP i.e. VDAC1 and ANT2 (
      • Bernardi P.
      • Krauskopf A.
      • Basso E.
      • Petronilli V.
      • Blalchy-Dyson E.
      • Di Lisa F.
      • Forte M.A.
      The mitochondrial permeability transition from in vitro artifact to disease target.
      ,
      • Bernardi P.
      Mitochondrial transport of cations: channels, exchangers, and permeability transition.
      ), from the quantitative proteomics datasets (Table I, Fig. 1). The effect on VDAC1 was confirmed by Western blot analysis (Fig. 2). mPTP is generally regarded as a crucial channel that permits the exchange of metabolites and ions (
      • Lemasters J.J.
      • Holmuhamedov E.
      Voltage-dependent anion channel (VDAC) as mitochondrial governator–thinking outside the box.
      ). Exchange of molecules is controlled by the flickering of mPTP between open and closed states (
      • Bernardi P.
      Mitochondrial transport of cations: channels, exchangers, and permeability transition.
      ). Channel opening or an increase in the frequency of mPTP flickering is extensively documented as an event tightly associated with both necrotic and apoptotic cell death (
      • Halestrap A.P.
      What is the mitochondrial permeability transition pore?.
      ). Therefore, we next asked whether mPTP was functionally associated with MET inhibition by PHA-665752. Flickering of mPTP was evaluated using the calcein AM/CoCl2 method (
      • Petronilli V.
      • Miotto G.
      • Canton M.
      • Colonna R.
      • Bernardi P.
      • Di Lisa F.
      Imaging the mitochondrial permeability transition pore in intact cells.
      ). Strikingly, mPTP was rapidly and effectively inhibited by 50 nm PHA-665752 in SNU5 cells within 30 min (Fig. 5). It is worth noting that inhibition of the mPTP was sustained for at least 72 h. The resistant gastric cancer line SNU1 did not show inhibition of mPTP, but only rapid and transient activation of mPTP (Fig. 5).
      Figure thumbnail gr5
      Fig. 5mPTP of SNU5 and SNU1 cells in response to PHA-665752 treatment. mPTP of SNU5 (left panel) and SNU1 (right panel) cells was determined after treatment with 50 nm PHA-665752 (bold solid line) for 0.5, 2, 24, 48, and 72 h. Control untreated cells were analyzed in parallel (dotted line). Flow cytometry data are presented as histograms of the FL1 channel.

       MET is present in mitochondria of SNU5 cells

      The rapid effects of PHA-665752 on mitochondrial proteins and functions of SNU5 cells raised the possibility that this MET-selective inhibitor may act directly on mitochondria. Two reasons led us to hypothesize that MET might be present in mitochondria where it would be a target for PHA-665752. First, as PHA-665752 has high specificity for MET kinase (
      • Christensen J.G.
      • Schreck R.
      • Burrows J.
      • Kuruganti P.
      • Chan E.
      • Le P.
      • Chen J.
      • Wang X.
      • Ruslim L.
      • Blake R.
      • Lipson K.E.
      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ), the rapid mitochondrial responses could well be mediated by MET. Second, cells in which MET gene is highly amplified overexpress MET proteins that may be constitutively activated. High intracellular levels of activated MET could facilitate its translocation or localization to intracellular organelles such as mitochondria. Indeed EGFR, another oncogenic receptor tyrosine kinase, was found to translocate to the mitochondria and nucleus when activated (
      • Boerner J.L.
      • Demory M.L.
      • Silva C.
      • Parsons S.J.
      Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol. Cell.
      ,
      • Lin S.Y.
      • Makino K.
      • Xia W.
      • Matin A.
      • Wen Y.
      • Kwong K.Y.
      • Bourguignon L.
      • Hung M.C.
      Nuclear localization of EGF receptor and its potential new role as a transcription factor.
      ). Moreover, MET has been reported to be translocate to the nucleus when stimulated by HGF (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Matteucci E.
      • Bendinelli P.
      • Desiderio M.A.
      Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells.
      ). However, no publication to date has reported the presence of MET in the mitochondria.
      We employed two approaches to investigate whether MET is present in mitochondria of sensitive cells. Immunoblotting analysis could demonstrate the presence of MET in the isolated mitochondria fraction in a semiquantitative manner, although it may suffer from contamination of proteins from other organelles because no current technique is capable of enriching mitochondria to 100% purity. Confocal microscopic analysis provided additional complementary evidence visually.
      Immunoblotting was used to probe MET and phospho-MET in SNU5 whole-cell lysate and isolated mitochondrial lysate. In order to maximize mitochondria enrichment efficiency and minimize contaminations from other organelles, we employed a newly developed method for mitochondria isolation based on superparamagnetic microbeads conjugated to anti-TOM22 antibody (
      • Hornig-Do H.T.
      • Günther G.
      • Bust M.
      • Lehnartz P.
      • Bosio A.
      • Wiesner R.J.
      Isolation of functional pure mitochondria by superparamagnetic microbeads.
      ). The protocol is fast, reproducible, and standardized, resulting in mitochondria of high purity, with minimal contamination from cytoskeleton, cytosol, Golgi apparatus, endosome, endoplasmic reticulum, and nucleus (
      • Hornig-Do H.T.
      • Günther G.
      • Bust M.
      • Lehnartz P.
      • Bosio A.
      • Wiesner R.J.
      Isolation of functional pure mitochondria by superparamagnetic microbeads.
      ). We employed several controls to further confirm the purity of isolated mitochondria in this study. Known plasma membrane proteins including E-calcium-dependent adhesion molecules (E-cadherin)/CD324 and integrin αL/CD18 were present in whole-cell lysate, but were almost absent in mitochondria fractions, indicating minimal plasma membrane contamination (Fig. 6A). The cytoskeleton protein, actin, displayed a similar pattern, whereas mitochondrial protein, SDHB, exhibited the opposite distribution, further proving the purity of isolated mitochondria (Fig. 6A). Equal loading of proteins for whole-cell lysate and mitochondria fraction was confirmed by Ponceau S staining (
      • Zhang J.
      • Liem D.A.
      • Mueller M.
      • Wang Y.
      • Zong C.
      • Deng N.
      • Vondriska T.M.
      • Korge P.
      • Drews O.
      • Maclellan W.R.
      • Honda H.
      • Weiss J.N.
      • Apweiler R.
      • Ping P.
      Altered proteome biology of cardiac mitochondria under stress conditions.
      ).
      Figure thumbnail gr6
      Fig. 6Expression of MET and phospho-MET in mitochondria. A, Whole cell lysate and isolated mitochondrial lysate from untreated SNU5 and SNU1 were immunoblotted using antibodies as indicated. B, Confocal images of SNU5 cells loaded with MitoTracker Red CMCRos, DAPI, and Alexa 488-conjugated antibodies against MET, phospho-MET (Y1234/1235), and phospho-MET (Y1349). Channels for DAPI, MET or pMET, MitoTracker, and merge are shown.
      In striking contrast to E-cadherin, integrin αL and actin that were present in whole-cell lysate but not in the mitochondrial fraction, MET was expressed at a high level in mitochondria of SNU5 cells (Fig. 6A). Remarkably, phosphorylated MET appeared to be enriched in mitochondria. Signals of pMET (Y1234/1235) and pMET (Y1349) were higher in mitochondria than in whole-cell lysate, indicating that mitochondrial MET was highly activated. SNU1 cells demonstrated minimal expression of MET compared with SNU5 cells.
      To further confirm the presence of MET in mitochondria of SNU5 cells visually, we employed confocal microscopy to determine if MET and mitochondria were colocalized. Fig. 7B shows SNU5 cells fluorescently labeled with DAPI (blue channel), Mito Tracker Red CMXRos (red channel), and Alexa 488-conjugated antibodies against MET, pMET (Y1234/1235), and pMET (Y1349) (green channel). As expected, both MET and phosphorylated MET were found in high abundance in the plasma membrane of SNU5 cells. Remarkably, MET and phosphorylated MET also colocalized with mitochondria, as indicated by yellow colored areas in merged images. These results were consistent with immunoblotting experiments (Fig. 6A). In contrast, SNU1 displayed very weak fluorescence for MET and phospho-MET using the same staining protocol (data not shown).
      Figure thumbnail gr7
      Fig. 7Mitochondrial MET is inhibited by MET inhibitor. Whole cell lysate and isolated mitochondrial lysate were prepared from control untreated SNU5 cells and SNU5 cells treated with 50 nm PHA-665752 for 0.5 h and 2 h. Antibodies against MET, phospho-MET (Y1234/1235), phospho-MET (Y1349), and actin were used in immunoblotting.
      Taken together, these data provided convincing experimental evidence for the presence of MET in mitochondria of SNU5 cells.

       PHA-665752 inhibits phosphorylation of mitochondrial MET in SNU5 cells

      Next we asked whether mitochondrial MET that was highly phosphorylated would respond to treatment with PHA-665752 for 0.5 h and 2 h. As shown in Fig. 7, immunoblotting demonstrated that MET expression level in whole-cell lysates was not altered by the compound at both time points but phosphorylation of MET was inhibited, consistent with previous reports (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ,
      • Christensen J.G.
      • Schreck R.
      • Burrows J.
      • Kuruganti P.
      • Chan E.
      • Le P.
      • Chen J.
      • Wang X.
      • Ruslim L.
      • Blake R.
      • Lipson K.E.
      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ). Importantly, phosphorylation of mitochondrial MET was inhibited in isolated mitochondrial lysates. Phosphorylation of MET at Y1234/Y1235 was suppressed by 50 nm PHA-665752 as early as 0.5 h after treatment. Phosphorylation of Y1234/1235 was further inhibited when the treatment was prolonged for 2 h. Phosphorylation of Y1349 in mitochondrial MET was inhibited by PHA-665752 at 2 h.

      DISCUSSION

      A curious observation in cancer research is that some cancers appear to be highly dependent for survival and progression on a single oncogene that is usually overexpressed (
      • Sharma S.V.
      • Settleman J.
      Oncogene addiction: setting the stage for molecularly targeted cancer therapy.
      ,
      • Weinstein I.B.
      • Joe A.
      Oncogene addiction.
      ). MET has essential functions in both normal and malignant cells (
      • Birchmeier C.
      • Birchmeier W.
      • Gherardi E.
      • Vande Woude G.F.
      Met, metastasis, motility and more.
      ). It is not entirely clear why gastric cancer cells that overexpress MET are paradoxically much more sensitive to MET inhibition than cells whose MET expression is comparable to normal stomach (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ).
      In an effort to dissect the underlying molecular mechanisms, we have established a model to inhibit MET activity using a potent MET inhibitor PHA-665752 in a sensitive gastric cancer cell line SNU5. A resistant line SNU1 was used as control. A sublethal concentration of PHA-665752, sufficient to inhibit MET activation, was intentionally used to minimize off-target effects, and to investigate early cellular responses. Previously it has been demonstrated that effects of PHA-665752 are truly attributed to its effect on MET in SNU5 cells (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ). iTRAQ-based proteomics experiments allowed us to identify and quantify >1900 proteins with a FDR<1%. After stringent quality control of the data set, we narrowed down our focus to >800 proteins with at least five peptides quantified, and 50 proteins that were significantly deregulated upon MET inhibition. Our study revealed several cellular responses to PHA-665752 treatment in SNU5 cells, including modulation of oxidoreductases, calcium-binding proteins, transfer/carrier proteins involved in transport of specific substances, and signaling proteins. Remarkably, we observed deregulation of mitochondrial proteins from ETC, mPTP, among others, formed a dominant protein group among the 50 deregulated proteins.
      The observations prompted us to ask whether perturbation of mitochondrial functions is one of the early responses of PHA-665752 treated SNU5 cells. It is worth noting that, in the current literature, aberrant mitochondrial functions are mainly documented when cells are induced to apoptosis or other forms of cell death by lethal interventions as downstream consequences of cytotoxic factors (
      • Vander Heiden M.G.
      • Chandel N.S.
      • Li X.X.
      • Schumacker P.T.
      • Colombini M.
      • Thompson C.B.
      Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival.
      ,
      • Vander Heiden M.G.
      • Li X.X.
      • Gottleib E.
      • Hill R.B.
      • Thompson C.B.
      • Colombini M.
      Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane.
      ,
      • Nowak G.
      Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells.
      ). Here we speculated that mitochondria might be a driving determinant of cellular death-survival decisions. Our cytometric data demonstrated that mitochondria of sensitive SNU5 cells rapidly responded to sublethal concentration of PHA-665752. In contrast, the resistant cell SNU1 displayed different patterns. These data indicated that mitochondrial deregulation appeared to be early response during MET inhibition.
      To further understand the mechanisms, we hypothesized that MET might be present in mitochondria of sensitive cells as a direct target of PHA-665752. Currently, MET is known to be present in the plasma membrane, and MET inhibitors, including small molecular inhibitors and monoclonal antibodies, are considered to act through plasma membrane MET. However, it is possible that MET might reside in other organelles. Both MET and EGFR, another receptor tyrosine kinase that was once considered be in the plasma membrane only, are capable of translocating to the nucleus (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Lin S.Y.
      • Makino K.
      • Xia W.
      • Matin A.
      • Wen Y.
      • Kwong K.Y.
      • Bourguignon L.
      • Hung M.C.
      Nuclear localization of EGF receptor and its potential new role as a transcription factor.
      ). Activated EGFR translocates to mitochondria where it actively modulates mitochondrial proteins (
      • Boerner J.L.
      • Demory M.L.
      • Silva C.
      • Parsons S.J.
      Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol. Cell.
      ). Our immunoblotting and confocal data both demonstrated that MET is present in mitochondria. Importantly, mitochondrial MET is hyperphosphorylated, indicating it is functionally highly active. In contrast, SNU1 cells display minimal expression of MET and phosphorylated MET. Furthermore, we demonstrated that PHA-665752 could effectively suppress the phosphorylation of mitochondrial MET. These data suggest that mitochondrial MET is a direct target of PHA-665752.
      Accumulating evidence suggests that many mitochondrial proteins, including ETC and mPTP components, that maintain the functions of this organelle, are tightly modulated by protein kinases including protein kinase A (PKA), PI3K/Akt/PKB, Raf-MEK-ERK, and MAPK (
      • Salvi M.
      • Brunati A.M.
      • Toninello A.
      Tyrosine phosphorylation in mitochondria: a new frontier in mitochondrial signaling.
      ,
      • Horbinski C.
      • Chu C.T.
      Kinase signaling cascades in the mitochondrion: a matter of life or death.
      ). Phosphorylated mitochondrial MET may play active roles in modulating phosphorylation status of various mitochondrial proteins involved in mitochondrial functions. Deregulation of mitochondrial proteins and dysfunctional mitochondrial processes are likely consequences of inhibition of mitochondrial MET. Our findings also plausibly link receptor tyrosine kinase-targeted therapeutics with the Warburg effect (
      • Warburg O.
      On respiratory impairment in cancer cells.
      ). Mitochondria are key regulators of glycolysis. We have found expression of several glycolysis-associated mitochondrial proteins, for example, VDAC1 and ANT2, was concurrently regulated rapidly in response to PHA-665752 treatment in SNU5 cells (supplemental Table 2).
      Our observations have uncovered novel mechanisms through which a kinase inhibitor directly acts on mitochondrial targets and influences mitochondrial functions in sensitive cells. This may explain the rapidly lethal effects of some targeted therapies and advance understanding of how these anticancer agents work. As localization of MET to mitochondria was not found in resistant cells, this may be a hallmark of sensitive cells and have potential implications in personalized cancer therapeutics.
      Our findings do not contradict but rather enhance the conventional paradigm that MET inhibitors including small molecule inhibitors and monoclonal antibodies act on plasma membrane MET (
      • Comoglio P.M.
      • Giordano S.
      • Trusolino L.
      Drug development of MET inhibitors: targeting oncogene addiction and expedience.
      ). As shown in Fig. 7, phosphorylation of MET in whole-cell lysate was rapidly inhibited by 50 nm PHA-665752, consistent with global suppression of MET molecules by the inhibitor as others have reported (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ,
      • Christensen J.G.
      • Schreck R.
      • Burrows J.
      • Kuruganti P.
      • Chan E.
      • Le P.
      • Chen J.
      • Wang X.
      • Ruslim L.
      • Blake R.
      • Lipson K.E.
      • Ramphal J.
      • Do S.
      • Cui J.J.
      • Cherrington J.M.
      • Mendel D.B.
      A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
      ). As reported previously, baseline phosphorylation of downstream signaling effectors, such as ERK1/2, AKT, STAT3, and FAK, were effectively abrogated by 50 nm PHA-665752, confirming that canonical MET signaling pathway was inhibited by this compound (
      • Smolen G.A.
      • Sordella R.
      • Muir B.
      • Mohapatra G.
      • Barmettler A.
      • Archibald H.
      • Kim W.J.
      • Okimoto R.A.
      • Bell D.W.
      • Sgroi D.C.
      • Christensen J.G.
      • Settleman J.
      • Haber D.A.
      Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752.
      ). However, this study provides evidence for an additional and novel locus of MET inhibition i.e. mitochondrial MET, that may be critical in contributing to the efficacy of MET inhibition in sensitive cells.
      We are still unclear about the origin of the mitochondrial MET. One possibility is translocation of autophosphorylated plasma membrane MET to mitochondria. It is also possible that a fraction of synthesized MET is directly localized to mitochondria because of certain signal peptide sequence or posttranslational modification. Localization of MET to mitochondria may correlate with the high genetic expression of MET in sensitive cells. The substrates of mitochondrial MET remain to be determined. Future work is also needed to demonstrate whether the failure of MET to localize to mitochondria affects the behavior and response of sensitive cells.

      CONCLUSION

      In an effort to understand molecular mechanisms of the curious sensitivity of MET-overexpressing cancer cells to MET inhibition, we have uncovered novel mechanisms of MET inhibition in sensitive cells. In response to low concentration of MET inhibitor, sensitive cells displayed substantial deregulation of mitochondrial proteins and functions. This study is the first to show the presence of activated MET in mitochondria of sensitive cancer cells that might be a direct target of MET inhibitor.

      Acknowledgments

      We thank Yiting You, Siyan Lu, Dr Xu Jian, Dr Jung Eun Park, Elynn Hui Qun Phang, Myat Thiri Maw, Siew Hong Leong, Jaichandran Sivalingam, Nelson Chen, Cheryl Lee, and Frank Kyaw Myo Lwin for helpful discussions and assistance.

      Supplementary Material

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