A point mutation in the MET oncogene abrogates metastasis without affecting transformation

Abstract

The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 --> N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 --> H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive-metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.

Figure 1

Expression of Tpr-Met mutants in Fisher rat fibroblasts. The same amounts of Tpr-Met proteins (arrow) were immunoprecipitated by using Met-specific antibodies. Immunoprecipitated proteins were separated on SDS/10% PAGE, blotted, and probed with anti-Met antibodies.

Figure 2

(Left) Schematic representation of Tpr-Met and Tpr-Sea proteins. White boxes represent the Tpr moiety; gray boxes represent the Met tyrosine kinase domain; the black box represents the Sea tyrosine kinase domain. YVHV-YVNV indicates the sequence of the Met multifunctional docking site; YVNL-YVNL indicates the sequence of the Sea multifunctional docking site. The N, critical for Grb2 binding, is underlined. (Right) Transformation refers to ability to induce foci of transformation, expressed as relative transforming activity (percent of Tpr-Met wild type). Metastasis indicates percentage of mice dead because of lung metastases, 3 weeks after injection of 10⁶ cells into the tail vein.

Figure 3

Rescue of the invasive phenotype by reconstitution in trans of Met multifunctional docking site. Cells expressing the wild-type Tpr-Met invaded the in vitro reconstituted basement membrane and migrated to the Transwell lower chamber. Invasion was severely impaired in cells expressing Tpr-Met mutants affecting the multifunctional docking site (Grb2⁻ and 2× Grb2, see text). Coexpression of the two inactive mutants restored the ability to invade the basement membrane. Cells migrating into the lower chamber were fixed with glutaraldehyde and stained with crystal violet. (×4.)

Figure 4

Cells expressing TPR-vSEA invaded the “in vitro” reconstituted basement membrane and migrated into the Transwell lower chamber. Invasion was severely impaired in cells expressing TPR-cSEA. Cells migrating into the lower chamber were fixed with glutaraldehyde and stained with crystal violet. (×4.)