EXPRESSION OFMet PROTEIN IN THYROID TUMOURS

Ruco, Luigi; Ranalli, Teresa; Marzullo, A; Bianco, Paolo; Prat, María; Comoglio, Paolo M.; Baroni, Carlo D.

doi:10.1002/(sici)1096-9896(199611)180:3<266::aid-path658>3.0.co;2-q

Cited by 85 publications

(84 citation statements)

References 25 publications

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“…Immunoprecipitation and Western blotting were performed as described (19). In particular, the Met protein was immunoprecipitated with the monoclonal antibody DQ13 (20) and detected by Western blotting with DL21 monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Tyrosine phosphorylation was detected with the monoclonal antibody clone 4G10 (Upstate Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

Genetic targeting of the kinase activity of the Met receptor in cancer cells

Arena

Pisacane²,

Mazzone

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The development of kinase inhibitors is revolutionizing cancer treatment. Assessing the oncogenic potential of individual kinase activities and ensuring that a drug of interest acts by direct inhibition of its putative target kinase are clear priorities. We developed a genetic strategy to selectively inactivate the catalytic activity of kinases. This approach generates isogenic cells in which a given kinase gene is expressed but is devoid of enzymatic activity. As a model to test this approach, we chose the MET receptor, which is involved in multiple cancers and is the focus of several therapeutic efforts. The exon encoding the ATP-binding site of MET was deleted from the genome of colorectal, bladder, and endometrial cancer cells. The derivative isogenic cells expressed a kinase-inactive Met (MET-KD) and were completely unresponsive to its ligand hepatocyte growth factor (HGF), indicating the exclusivity of this ligand-receptor axis. The in vivo tumorigenic potential of MET-KD cells was reduced but could be partially restored by HGF, suggesting that concomitant targeting of the receptor and its ligand should be therapeutically exploited. A reportedly selective Met-kinase inhibitor (SU-11274) markedly affected the growth of MET-KD cancer cells, indicating this compound exerts its effects not only through the intended target. The genetic strategy presented here is not limited to kinase genes but could be broadly applicable to any drug/protein combination in which the target enzymatic domain is known.knockout ͉ somatic knockout ͉ targeted therapy ͉ kinase inhibitor ͉ invasive growth

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Section: Methodsmentioning

confidence: 99%

Genetic targeting of the kinase activity of the Met receptor in cancer cells

Arena

Pisacane²,

Mazzone

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…WT and MET exon 16 KO DLD-1 cells were incubated for 24 hours in 0.5% FBS in the presence of 1,000 nmol/L tivantinib or JNJ-38877605 and then stimulated with 50 ng/mL recombinant human HGF (R&D Systems) for 10 minutes. Cell lysates were immunoprecipitated as described previously (24) using the DQ-13 anti-MET antibody (25) and resolved by electrophoresis. The levels of phosphorylated and total MET was determined by Western blotting using the appropriate antibodies as described above.…”

Section: Met Expression and Activationmentioning

confidence: 99%

Tivantinib (ARQ197) Displays Cytotoxic Activity That Is Independent of Its Ability to Bind MET

Basilico¹,

Pennacchietti²,

Vigna³

et al. 2013

Clinical Cancer Research

161

127

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Purpose: MET, the high-affinity receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Tivantinib (ARQ197; Arqule), a staurosporine derivative that binds to the dephosphorylated MET kinase in vitro, is being tested clinically as a highly selective MET inhibitor. However, the mechanism of action of tivantinib is still unclear.Experimental Design: The activity of tivantinib was analyzed in multiple cellular models, including: cells displaying c-MET gene amplification, strictly 'addicted' to MET signaling; cells with normal c-MET gene copy number, not dependent on MET for growth; cells not expressing MET; somatic knockout cells in which the ATP-binding cleft of MET, where tivantinib binds, was deleted by homologous recombination; and a cell system 'poisoned' by MET kinase hyperactivation, where cells die unless cultured in the presence of a specific MET inhibitor.Results: Tivantinib displayed cytotoxic activity independently of c-MET gene copy number and regardless of the presence or absence of MET. In both wild-type and isogenic knockout cells, tivantinib perturbed microtubule dynamics, induced G 2 /M arrest, and promoted apoptosis. Tivantinib did not rescue survival of cells 'poisoned' by MET kinase hyperactivation, but further incremented cell death. In all cell models analyzed, tivantinib did not inhibit HGF-dependent or -independent MET tyrosine autophosphorylation.Conclusions: We conclude that tivantinib displays cytotoxic activity via molecular mechanisms that are independent from its ability to bind MET. This notion has a relevant impact on the interpretation of clinical results, on the design of future clinical trials, and on the selection of patients receiving tivantinib treatment.

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“…MKN-45 and H1993 cell lysates were also analyzed by Western blotting using anti-GAB1 antibodies and anti-phospho-GAB1 antibodies (Cell Signaling). For assessment of MET-associated phospho-proteins, cell lysates were immunoprecipitated as described (28) using the DQ13 anti-MET antibody (29) followed by Western blotting with antiphospho-HER3 antibodies (Cell Signaling), anti-phospho-GAB1 antibodies (Cell Signaling), and anti-PI3K antibodies (Millipore). For the assessment of PI3K-associated phospho-proteins, cell lysates were immunoprecipitated as above using anti-PI3K antibodies (Millipore) followed by Western blotting with anti-phospho-HER3 antibodies (Cell Signaling), anti-phospho-GAB1 antibodies (Cell Signaling), and anti-PI3K antibodies (Millipore).…”

Section: Signal Transduction Analysismentioning

confidence: 99%

Microenvironment-Derived HGF Overcomes Genetically Determined Sensitivity to Anti-MET Drugs

et al. 2014

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Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are "addicted" to MET signaling and therefore are very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, hepatocyte growth factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of six MET-addicted human tumor cells to three MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and one antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma-tumor coculture system, and by combining anti-MET drugs with an HGF neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET-amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic GAB1-mediated PI3K activation that compensated for loss of aberrant HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in coculture systems and overcame resistance to JNJ-38877605, crizotinib, and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET-amplified tumor cells-which normally exhibit ligand-independent, constitutive MET activation-become dependent on HGF for survival upon pharmacologic MET inhibition. Because HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof of principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial and opens new perspectives for the treatment of MET-dependent tumors. Cancer Res; 74(22); 6598-609. Ó2014 AACR.

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EXPRESSION OFMet PROTEIN IN THYROID TUMOURS

Cited by 85 publications

References 25 publications

Genetic targeting of the kinase activity of the Met receptor in cancer cells

Genetic targeting of the kinase activity of the Met receptor in cancer cells

Tivantinib (ARQ197) Displays Cytotoxic Activity That Is Independent of Its Ability to Bind MET

Microenvironment-Derived HGF Overcomes Genetically Determined Sensitivity to Anti-MET Drugs

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