The Shc adaptor protein is critical for VEGF induction by Met/HGF and ErbB2 receptors and for early onset of tumor angiogenesis

Abstract

The etiology and progression of a variety of human malignancies are linked to the deregulation of receptor tyrosine kinases (RTKs). To define the role of RTK-dependent signals in various oncogenic processes, we have previously engineered RTK oncoproteins that recruit either the Shc or Grb2 adaptor proteins. Although these RTK oncoproteins transform cells with similar efficiencies, fibroblasts expressing the Shc-binding RTK oncoproteins induced tumors with short latency (approximately 7 days), whereas cells expressing the Grb2-binding RTK oncoproteins induced tumors with delayed latency (approximately 24 days). The early onset of tumor formation correlated with the ability of cells expressing the Shc-binding RTK oncoproteins to produce vascular endothelial growth factor (VEGF) in culture and an angiogenic response in vivo. Consistent with this, treatment with a VEGF inhibitor, VEGF-Trap, blocked the in vivo angiogenic and tumorigenic properties of these cells. The importance of Shc recruitment to RTKs for the induction of VEGF was further demonstrated by using mutants of the Neu/ErbB2 RTK, where the Shc, but not Grb2, binding mutant induced VEGF. Moreover, the use of fibroblasts derived from ShcA-deficient mouse embryos, demonstrated that Shc was essential for the induction of VEGF by the Met/hepatocyte growth factor RTK oncoprotein and by serum-derived growth factors. Together, our findings identify Shc as a critical angiogenic switch for VEGF production downstream from the Met and ErbB2 RTKs.

Fig. 1.

RTK oncoproteins coupling to Shc confer on fibroblasts the ability to form tumors in nude mice with short latency. (A) Diagram of the RTK oncoproteins specific for recruitment of Grb2 or Shc. The amino acid sequences substituted within the Tpr-Met Y482/489F cassette mutant and the inserted binding motifs are shown. The Grb2-binding site from the epidermal growth factor receptor was inserted to generate the Y-Grb2 RTK oncoprotein, whereas the Y-Shc-1 and Y-Shc-2 RTK oncoproteins contain, respectively, the Shc-binding sites from the TrkA or EGF receptors (3). (B) Expression and phosphorylation levels of RTK oncoproteins and control proteins in fibroblast cell lines. Lysates (500 μg) of cells expressing RTK oncoproteins or control proteins were subjected to immunoprecipitation with an antibody specific for Met (Ab 144) and immunoblotted with the same antibody or anti-pTyr. (C) The growth of tumor (mm³) over time (day) was measured after s.c. injection of 10⁵ cells (see Table 1). The results represent the mean tumor volume obtained from two independent experiments in which at least three mice were injected for each cell line.

Fig. 2.

Fibroblasts expressing Shc-binding RTK oncoproteins promote angiogenesis in vivo and enhance VEGF mRNA and protein. (A) Representative photographs of Matrigel plugs formed 10 days after injection of fibroblasts (10⁵ cells) expressing the Grb2 or Shc RTK oncoproteins, or the Tpr-Met Y482/489F control mixed with 250 μl of growth factor-depleted Matrigel solution. (B) Cells were seeded at a density of 10⁶ per 100-mm plate. The following day, medium was replaced with serum-free media, and CM was collected after 48 h. The level of VEGF was detected after enrichment with heparin by immunoblot analysis using a VEGF antibody. (C) The level of VEGF mRNA was detected by Northern blot analysis of total RNA (40 μg) isolated from serum-starved (24 h) cells expressing variant or control proteins.

Fig. 3.

VEGF-Trap abrogates the in vivo angiogenic response and tumor growth of cells expressing Shc-binding RTK oncoproteins. (A) Nude mice were treated with either vehicle or 25 mg/kg VEGF-Trap 2 days before the injection of fibroblasts expressing Shc-binding RTK oncoproteins mixed with Matrigel (10⁵ cells per 250 μl), and treatment was continued on a twice-weekly regimen for a period of 10 days. Representative photographs of the Matrigel plugs are shown. (B) Nude mice were treated twice a week with 25 mg/kg VEGF-Trap or vehicle after s.c. implantation of cells expressing Shc-binding RTK oncoproteins. Results represent the mean tumor volume ± SEM (cm³) over time (day) from two independent experiments, each conducted with three mice per treatment group.

Fig. 4.

A Neu/ErbB2 RTK mutant that recruits Shc but not Grb2 increases VEGF protein. (A) Diagram of the activated Neu/ErbB2 RTK add-back mutants that retain the Grb2- or Shc-binding site. (B) Expression and phosphorylation levels of RTK add-back mutant or control proteins in Rat-1 fibroblast cell lines. (C) Levels of VEGF produced in the culture media of cells expressing the Neu/ErbB2 RTK add-back mutant or control proteins were detected by immunoblot analysis using a VEGF antibody.

Fig. 5.

The binding of Shc but not of Grb2 to a ligand-activated transmembrane RTK induces VEGF. (A) Schematic representation of the design of RTKs specific for the binding of Grb2 or Shc (their binding specificities are shown in Fig. 8). (B) Expression levels of the Grb2 or Shc signaling specific RTK and control proteins in populations of Rat-1 fibroblast cells. (C) Levels of VEGF in CM of Rat-1 fibroblasts (10⁶ per 100-mm plate) after 48-h stimulation or not (CSF 100 ng/ml) was detected by immunoblot analysis using a VEGF antibody.

Fig. 6.

The induction of VEGF mediated by the Met RTK oncoprotein and serum-derived growth factors depends on Shc. (A) Expression and phosphorylation levels of the Tpr-Met and Shc proteins in wild-type (+/+) or ShcA-deficient (–/–) MEFs, or in ShcA-deficient MEF stably transfected with p52 ShcA cDNA that express or not the Tpr-Met oncoprotein. Lysates (250 μg) prepared from the different cell lines were subjected to immunoprecipitation with anti-Met and immunoblotted with Met or pTyr antibody. To detect the phosphorylation levels of Shc proteins, cell lysates (500 μg) were subjected to immunoprecipitation with an antibody specific for Shc and immunoblotted with anti-pTyr. The expression level of Shc proteins is shown by immunoblot analysis of whole-cell lysates with anti-Shc. (B) Levels of VEGF produced after 48 h in the CM of wild-type (+/+) or ShcA-deficient (–/–) MEFs, or in ShcA-deficient MEF stably transfected with p52 ShcA cDNA, which express or not Tpr-Met, was detected by immunoblot analysis. (C) The level of VEGF in the CM of wild-type (+/+) or ShcA-deficient (–/–) MEFs, or in ShcA-deficient MEF stably transfected with p52 ShcA cDNA after 48 h of serum stimulation.