Several lines of evidence exist that lend support to T790M as a putative susceptibility allele. T790M has been detected somatically in tumors that have not been treated with gefitinib or erlotinib, which may suggest that this mutant may have arisen during drug-free cancer progression (15
). Furthermore, the NCI-H1975 BAC cell line, which has never undergone tyrosine kinase inhibitor treatment, has both activating L858R and resistant T790M mutations, suggestive that T790M may be growth promoting (5
). Bell et al. (8
) also have observed that the T790M mutation seems to occur in cis
with the activating mutations. Perhaps more persuasive is that the analogous resistance mutation in CML patients, BCR-ABL-T315I, displays increased in vitro
kinase activity (16
). Similarly, the analogous mutations in Src (T341M) and FGFR1 (V561M) also result in increased phosphorylation and activation (10
). Why T790M in EGFR does not reportedly function in a similar manner is unclear.
Our results suggest that the T790M mutation may in fact provide a proliferative advantage in normal cells by increasing kinase activity and downstream signaling. Our data suggest that EGFR-T790M does in fact exhibit higher kinase activity than WT molecule in HEK293T and COS-7 cell lines. Overexpression in our HBEC3 cell line showed increased tyrosine phosphorylation at Y992 and Y1068 and increased colony formation. Increased proliferation is most evident in the absence of EGF in the medium, suggesting that the activating T790M EGFR mutation is involved in the proliferative effects observed. EGFR kinase activity is essential for oncogenic transformation. EGFR extracellular domain deletion mutants and overexpression of EGFR are commonly found in human cancers (17
). Our data also show that phosphorylation levels between WT and T790M are indistinguishable at higher expression levels (), yet differences are more apparent at lower expression levels. We believe this may be an explanation why others have seen no difference between EGFR and T790M.
EGFR signaling activates many pathways that lead to proliferative advantages. The Ras/MAPK pathway is activated via ligation of Grb2 to an activated EGFR molecule. In our 293T overexpression system, we observed that MAPK activity is increased by T790M versus WT EGFR. However, a significant effect of T790M on these downstream kinases was not evident in our HBEC3 system, which might suggest that other signaling/transcriptional events are responsible for the proliferative changes. Recent work has revealed transcriptional changes in cyclin D1, may be a key response to gefitinib resistance by T790M and susceptibility to the irreversible inhibitor CL-387,785 (14
). We did not observe changes in cyclin D1 levels in the HBEC3 system and believe other, yet unidentified, mechanisms exist to account for the proliferative advantages caused by mutant EGFR.
A recent study, using a mutant-enriched PCR system, revealed the presence of the T790M mutation as a minor clone in non–small cell lung cancer tumors (19
). It is suggested that these clones are selected for during gefitinib treatment and are enriched in the resistant tumor but do not provide the main proliferative function for oncogenesis. Our observations suggest that T790M as a germ-line mutation (i.e., in all cells) may provide the mild proliferative push for lung cancer development.
Many lines of evidence suggest the existence of a limited number of genetic factors that control susceptibility to lung cancer (20
). However, due to a high-case fatality rate (5-year survival rate of 15%), obtaining biospecimen samples for DNA analysis is particularly difficult (23
). A collaborative effort of GELCC has accrued DNA from families with lung, throat, and laryngeal cancers since the early 1990s. At present, 771 families with three or more first-degree relatives affected with lung cancer have been collected, of which 11% have sufficient family-wide biospecimen availability for any future studies. Subsequent sequencing of 237 families with predisposition to lung cancer, 45 BAC tumors and 60 fresh-frozen resected tumors, did not reveal any mutations, suggesting that T790M is likely a rare mutation. Nevertheless, our data provide a basis for EGFR-T790M
as a rare susceptibility allele in human lung cancer.