Ethnic differences and functional analysis of MET mutations in lung cancer

Abstract

Purpose: African Americans have higher incidence and poorer response to lung cancer treatment compared with Caucasians. However, the underlying molecular mechanisms for the significant ethnic difference are not known. The present study examines the ethnic differences in the type and frequency of MET proto-oncogene (MET) mutation in lung cancer and correlated them with other frequently mutated genes such as epidermal growth factor receptor (EGFR), KRAS2, and TP53.

Experimental design: Using tumor tissue genomic DNA from 141 Asian, 76 Caucasian, and 66 African American lung cancer patients, exons coding for MET and EGFR were PCR amplified, and mutations were detected by sequencing. Mutation carriers were further screened for KRAS2 and TP53 mutations. Functional implications of important MET mutations were explored by molecular modeling and hepatocyte growth factor binding studies.

Results: Unlike the frequently encountered somatic mutations in EGFR, MET mutations in lung tumors were germline. MET-N375S, the most frequent mutation of MET, occurred in 13% of East Asians compared with none in African Americans. The frequency of MET mutations was highest among male smokers and squamous cell carcinoma. The MET-N375S mutation seems to confer resistance to MET inhibition based on hepatocyte growth factor ligand binding, molecular modeling, and apoptotic susceptibility to MET inhibitor studies.

Conclusions: MET in lung cancer tissues contained nonsynonymous mutations in the semaphorin and juxtamembrane domains but not in the tyrosine kinase domain. All the MET mutations were germline. East Asians, African-Americans, and Caucasians had different MET genotypes and haplotypes. MET mutations in the semaphorin domain affected ligand binding.

Fig. 1

MET mutations in lung cancer. A, frequency of MET mutations based on histologic subtypes of lung cancer and ethnic groups. The percentage frequencies of the occurrence of nonsynonymous mutations are given in parenthesis (blue). Adeno, adenocarcinoma. B, occurrence of MET-N375S mutation among histologic subtypes in different ethnic groups. Carriers of MET-N375S mutation are given as a percentage of total samples. C, frequency distribution of MET-N375S mutation in lung cancer patients of East Asian origin based on gender (i) and smoking history (ii). D, the pie chart shows tumors with nonsynonymous mutations in MET alone, MET and EGFR, and MET and KRAS2 as a percentage of total number of MET mutation carriers.

Fig. 2

MET mutations: type, frequency, and distribution. Mutations of MET identified in lung tumor tissue samples from different ethnic groups. A, various mutations are illustrated schematically in the context of functional domains of MET. The numbers denote amino acid residues at indicated position in the molecule. Sema, semaphorin domain; PSI, plexins, semaphorins, and integrins domain; IPT1-4, found in immunoglobulin-like regions, plexins, and transcriptional factors; TM, transmembrane region; JM, juxtamembrane domain; TK, catalytic tyrosine kinase domain. Genotype frequencies are represented as a percentage of total number of tumor samples tested in each group. The altered nucleotides as well as the corresponding amino acids (top row) are numbered according to full-length MET (Ensembl transcript ID: ENST00000318493). B, effect of MET mutations on the ligand binding pocket in MET. Structural changes in MET semaphorin domain due to nonsynonymous mutations and their effect on interaction with HGF β-chain are shown by homology modeling: yellow, HGF β-chain; purple, the semaphorin domain of MET as ribbon models. Altered residues are colored red and labeled by residue number; residues 168 and 229 can be seen in direct contact with HGF. C, Van der Waals or space-filling spheres representation of positively selected residues P169, S170, and P210 with high probabilities of ω > 1 are spatially close to mutation E168D, indicating that residue 168 is in the ligand binding region. D, stereo magnification of the mutation N375S; N375 (left) has two potential hydrogen bonds (green dash line), whereas S375 modeling structure (right) shows loss of one hydrogen bond, and therefore likely to weaken ligand binding.

Fig. 3

The functional significance of MET-N375S mutation. A, relative purity of MET-WT-Fc and MET-N375S-Fc fusion proteins. B, relative binding of HGF to MET-WT-Fc and MET-N375S-Fc. C, susceptibility of COS-7 cells expressing MET-WT or MET-N375S to SU11274 induced apoptosis.