Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2

Abstract

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.

Fig. 1.

Extracellular domain mutations of ERBB2 found in lung and breast cancer are oncogenic. (A) NIH 3T3 cells expressing ERBB2 extracellular mutants were assessed for colony formation in soft agar. (B) Anti-ERBB2 immunoblot on NIH 3T3 lysates. (C) AALE human airway epithelial cells expressing ERBB2 extracellular mutants also exhibited an increase in soft agar colony formation. (D) Anti-ERBB2 immunoblot on AALE lysates. (E) NIH 3T3 cells expressing ERBB2 mutants reported in glioblastoma were assessed for colony formation in soft agar. (F) Immunoblot analysis of ERBB2 protein and phosphorylation state on lysates of NIH 3T3 expressing ERBB2 mutations reported in glioblastoma. pBp, pBabe puro vector; insYVMA, A775_G776insYVMA; insV, ERBB2 A775_G776insV/G776C; WT, wild-type ERBB2.

Fig. 2.

Oncogenic extracellular domain mutations of ERBB2 reported in glioblastoma cause disulfide bond remodeling. (A) Model of the ERBB2 dimer made by superimposing the human [Protein Data Bank (PDB) ID code 2A91] and rat (PDB ID code 1N8Y) ERBB2 extracellular domain crystal structures onto an EGF-bound EGFR extracellular domain dimer crystal structure (PDB ID code 1IVO). Intramolecular disulfide bonds are indicated in green. (B) Immunoblot analysis of ERBB2 extracellular mutants reported in glioblastoma reveals formation of covalent dimers on nonreducing gels. pBp, pBabe puro vector; WT, wild-type ERBB2.

Fig. 3.

ERBB2 mutants found in lung and breast cancer form reduction-sensitive dimers that exhibit diminished C-terminal tail phosphorylation. (A) Immunoblot analysis of ERBB2 protein and tyrosine 1221/1222 phosphorylation state on lysates of NIH 3T3 expressing ERBB2 extracellular domain mutations. (B) Immunoblot analysis of ERBB2 dimers trapped by nonreducing SDS/PAGE. pBp, pBabe puro vector; WT, wild-type ERBB2.

Fig. 4.

Ba/F3 cells transformed to IL-3 independence with the ERBB2 extracellular domain mutants are sensitive to ERBB2 inhibition. (A) Proliferation of Ba/F3 cells expressing mutant forms of ERBB2 upon IL-3 withdrawal. (B) Survival of ERBB2-transformed Ba/F3 cells in response to neratinib. (C) Survival of ERBB2-transformed Ba/F3 cells in response to afatinib. (D) Survival of ERBB2-transformed Ba/F3 cells in response to lapatinib. (E) Survival of ERBB2-transformed Ba/F3 cells in response to trastuzumab. (F) Response of cancer cell lines NCI-H1781, AN3CA, and 5637 to a combination of Mek and ERBB2 inhibition. The concentration of Mek inhibitor PD184352, 1 μM, was chosen for lack of an effect alone on survival of these cell lines.