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

Abstract

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.

Fig. 1.

Targeted deletion of the ATP-binding site of the MET receptor in cancer cells. (A) The schematic structure of the Met genomic locus, the targeting vector, and the effects of the deletion on the MET protein are represented. (+/−), heterozygous; (−/−), homozygous. PC, post-CRE recombinase treatment. (B) Electrophoresis analysis of PCR products generated by using the cDNA obtained from WT and targeted (−/−) DLD1 cells using primers flanking exon 16 of the MET gene. The PCR strategy and the position of the primers are represented. (C) Sequence analysis of the MET transcript in WT and MET-KD cells. Upper and Lower represent the sequence of the MET cDNA obtained from WT and targeted cells, respectively. The arrow indicates the location of the boundary between exons.

Fig. 2.

MET-KD is expressed on the surface of cancer cells but is catalytically and signaling inactive. (A) The Met protein was immunoprecipitated from lysates of the indicated cells, untreated or stimulated with HGF, and detected by immonoblotting with anti-MET and antiphosphotyrosine antibodies. (B) The Met protein was immunoprecipitated as in A from the indicated cells, and its ability to autophosphorylate on tyrosine residues was assessed. (C) The surface proteins of the indicated cells were labeled with biotin, the Met receptor was immunoprecipitated by using streptavidin (StAv) and revealed by Western blotting analysis. MDA cells were used as positive control. (D) The multidocking protein Gab1, which acts as the main MET phosphorylation substrate, was immunoprecipitated from the indicated cells in the presence and absence of HGF. The ability of the indicated Met proteins to bind and phosphorylate Gab1 was evaluated by Western blotting by using anti-MET and antiphosphotyrosine antibodies. (+/−), heterozygous; (−/−), homozygous; PC, post-CRE recombinase treatment; MDA, MDA 435S cancer cells; ATP, AdenosinTriPhosphate.

Fig. 3.

Biological and tumorigenic properties of cancer cells expressing a catalytically inactive MET receptor. (A) Proliferation of cancer cells lacking Met kinase activity. The indicated cells were seeded at the same concentration on day 0, and cell density was measured for 5 consecutive days. Each point represents the mean of triplicates; error bars, SD. (B) Anchorage-independent growth of cancer cells lacking Met kinase activity. The indicated cells were seeded in soft agar, and the colonies appearing after 15 days of incubation were counted. Each column represents the mean of triplicates; error bars, SD. (C) Measurement of the “invasive growth” potential of cancer cells lacking the Met kinase activity. The indicated cells were seeded in collagen and examined for their ability to form branched tubules in response to HGF stimulation. Representative pictures are presented. (D) Tumorigenic potential of cancer cells lacking the Met tyrosine activity. Cells were transduced with a control vector or with a lentivirus expressing HGF and were injected s.c. into the right posterior flank of 6-week-old immunodeficient nude mice. Tumor growth was measured for 30 days. Statistical significance was determined by two-tailed Student's t test. Mean ± SEM, n = 4.

Fig. 4.

Biological and biochemical effects of the MET inhibitor SU11274 in cancer cells lacking the MET kinase activity. (A) The ability of SU11274 to inhibit the kinase activity of the MET receptor was evaluated on WT DLD1 cells. Cells were treated for 16 h with 2.5-μM SU11274 and then lysed. The Met protein was immunoprecipitated and analyzed by Western blotting by using the specified antibodies. (B) Cell lines were treated as indicated, and the cell number was evaluated by using an ATP-based assay. Each column represents the mean of triplicates; error bars, SD. RLU, relative light units. (C) Modulation of intracellular signaling pathways by the MET enzymatic activity and by SU11274. The indicated cells were incubated for 16 h in the absence and presence of 2.5 μM SU11274. Phosphorylation of intracellular signaling molecules was assessed by using the Human Phospho-MAPK Array Kit. The columns represent the result of the densitometric analysis of the dot images corresponding to the phosphorylation status of individual proteins. The numbers are referred to the untreated DLD1 WT cells that were given an arbitrary value of 1. Statistical significance was determined by two-tailed Student's t test by using WT untreated cells as reference.