We found that many NSCLC cell lines and bronchial epithelial cells expressed total MET protein. However, a more restricted subset of MET-expressing NSCLC cell lines also expressed the phosphorylated forms. Phospho-MET expression was significantly related to increased MET
copy number and was significantly more frequent in EGFR
mutant cell lines. As additional evidence for the relation between EGFR
mutation and MET activation, recent reports suggested that other tyrosine kinases were also activated in cells with EGFR activation by EGFR
supporting our results. Indeed, the results of siRNA experiments confirmed effect of activated EGFR to MET status. Three EGFR
mutant cell lines harboring phospho-MET expression without MET
amplification lost or decreased phospho-MET expression whereas MET
amplified cell lines with or without EGFR
mutations retained phospho-MET expression after knockdown of EGFR, indicating that EGFR
mutation or MET
amplification activate MET protein.
Regarding clinical samples, we found increased MET
copy number in 2 of 100 previously untreated primary resected lung cancers. For clinical samples, contamination with non-malignant cells is invariably present (estimated average per tumor = 50% tumor cells, 50% non-malignant cells). Thus cut-off value of amplification may be reduced. One of these 2 MET
amplified tumors also had an activating EGFR
mutation (L858R), but lacked the resistance associated with T790M mutation. This patient had an advanced tumor and died within 20 days after the start of gefitinib treatment. While he would appear to have been refractory to gefitinib therapy, the short survival duration renders a firm conclusion difficult. We did not examine phospho-MET protein status in primary tumors because formalin fixed materials were unsuitable for such studies.33
We neither had enough frozen clinical samples for Western blot analysis. It is mandatory to confirm the relation among phospho-MET expression, MET
copy number and EGFR
mutations in primary tumors.
We studied Japanese patients and found the expected high frequency of EGFR
mutations in NSCLC arising in patients of East Asian ethnicity.7
The frequency of MET
amplification in untreated lung cancers was relatively low. These results were similar with the previous report.14
On the other hand, Beau-Faller et al
. recently reported relatively higher frequency (21%) of MET
amplification in 106 non-Asian NSCLC.34
Their definition of “amplification” is normalized ratio over mean plus 2 standard deviations of 30 normal lung DNA samples. Even if we use this definition for our sample set, that is, normalized ratio over mean plus 2 standard deviations of 100 normal lung DNA samples, we only have 8 samples with MET
amplification out of 100 tumors. The discrepancy of frequency of MET
amplification between them may be explained by ethnic difference.
We also examined the IC50
of gefitinib to investigate the impact of phospho-MET on NSCLC cell lines, but the effect of gefitinib did not depend on MET status but depended on EGFR
mutation or PTEN status. Our results suggested that although biological significance of MET activation as a result of MET
amplification by gefitinib exposure caused acquired resistance to gefitnib,13
MET activation was not solo factor for inherent resistance to gefitinib.
The clinical course of our 2 cases with MET
amplification suggests that tumors with MET
amplification are very aggressive in nature, that is, MET
amplification may have the potential to be involved with development, progression or metastasis of lung cancer, consistent with the known relation of MET expression and tumor invasion and metastasis.35
Lutterbach et al
. reported that lung cancer cell lines with MET
amplification were dependent on MET for growth and survival.36
Although phospho-MET expression was not analyzed in our clinical samples because of technical issues, further investigation for phospho-MET expression status in primary tumors is important to understand biological significance of MET expression in lung cancer.
In conclusion, MET amplification is present in a subset of untreated lung cancers and cell lines with or without EGFR mutations, and EGFR mutation or MET amplification lead to activation of MET. Further studies clarifying biological significance of MET activation may shed light on the pathogenesis of lung cancer.