Mutations in the ERBB2 gene were recently found in approximately 2% of primary non-small cell lung cancer (NSCLC) specimens; however, little is known about the functional consequences and the relevance to responsiveness to targeted drugs for most of these mutations. Here, we show that the major lung cancer-derived ERBB2 mutants, including the most frequent mutation, A775insYVMA, lead to oncogenic transformation in a cellular assay. Murine cells transformed with these mutants were relatively resistant to the reversible epidermal growth factor receptor (EGFR) inhibitor erlotinib, resembling the resistant phenotype found in cells carrying the homologous mutations in exon 20 of EGFR. However, the same cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor HKI-272, as were those overexpressing wild-type ERBB2. Finally, the NSCLC cell line, Calu-3, overexpressing wild-type ERBB2 owing to a high-level amplification of the ERBB2 gene were highly sensitive to HKI-272. These results provide a rationale for treatment of patients with ERBB2-mutant or ERBB2-amplified lung tumors with HKI-272.
The epidermal growth factor receptor (EGFR) has emerged as a major therapeutic target in non-small cell lung cancer (NSCLC): the tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib lead to responses in approximately 10% of Caucasian patients and in up to 40% of East Asian patients (Fukuoka et al., 2003; Kris et al., 2003; Perez-Soler et al., 2004). Moreover, erlotinib improved overall survival of NSCLC patients in a phase-III trial (Shepherd et al., 2005).
Systematic re-sequencing efforts revealed mutations in the kinase domain of EGFR to be correlated with response to EGFR TKIs (Lynch et al., 2004; Paez et al., 2004; Pao et al., 2004). In in vitro experiments, cells transformed with these mutants were exquisitely sensitive to EGFR TKIs, thus recapitulating the clinical observations (Sordella et al., 2004; Greulich et al., 2005). In these experiments, cells carrying the exon 20 insertion mutants of EGFR were however resistant to erlotinib and gefitinib, yet, they retained sensitivity to an irreversible EGFR TKI, CL-387,785 (Greulich et al., 2005). Another irreversible EGFR inhibitor with dual specificity against EGFR and ERBB2, HKI-272, was recently found to be active against the T790M resistance mutation (Kwak et al., 2005) found in exon 20 of EGFR in patients relapsing after an initial response to gefitinib or erlotinib (Kwak et al., 2005; Pao et al., 2005; Kobayashi et al., 2005b). These findings suggest that irreversible EGFR inhibitors may be beneficial to patients with tumors harboring mutations in exon 20 of EGFR.
Somatic mutations in the kinase domain of ERBB2 were recently discovered in approximately 2–4% of NSCLC (Stephens et al., 2004; Shigematsu et al., 2005). The mutations target residues that are highly conserved in the Erbb family, and are homologous to the exon 20 insertion mutations of EGFR. Furthermore, although erlotinib has high activity against purified EGFR (IC50=2 nM) but low activity against purified ERBB2 (IC50=350 nM), HKI-272 exhibits low IC50 values for both purified EGFR (IC50=92 nM) and ERBB2 (IC50=59 nM) (Rabindran, 2005). We therefore speculated that ERBB2-mutant lung tumors might be amenable to targeted treatment using the irreversible, dual-specificity EGFR/ERBB2 TKI, HKI-272.
We have previously shown that the NSCLC cell line H-1781 carrying the G776insV_G/C mutation of ERBB2 as well as Ba/F3 cells stably expressing this mutant are sensitive to HKI-272 (Shimamura et al., 2006). We now set out to characterize additional NSCLC-derived ERBB2 mutants, including the most prevalent one, the insertion A775insYVMA.
We ectopically expressed the three mutants G776insV_G/L, P780insGSP and A775insYVMA, as well as wild-type ERBB2, in murine Ba/F3 cells using retroviruses. Ba/F3 cells rely on continuous supplementation of recombinant interleukin-3 (IL-3) for their growth; introduction of dominant oncogenes renders these cells IL-3 independent (Azam et al., 2003; Jiang et al., 2004). Ba/F3 cells therefore represent an ideal model to assay for oncogenicity and to analyse different oncogene mutants for their sensitivity to targeted drugs. Note that the A775insYVMA and the M774insAYVM lead to identical amino-acid changes; the A775insYVMA mutant analysed here is therefore representative of both mutations. Thus, with the recently published G776insV_G/C, these mutations represent about 85% of all mutation instances reported for ERBB2 in the COSMIC database to date (www.sanger.ac.uk/genetics/CGP/cosmic).
Upon selection with hygromycin, Ba/F3 cells carrying the different ERBB2 mutants as well as the ones harboring wild-type ERBB2 became growth factor independent and proliferated rapidly in the absence of exogenously added IL-3 (data not shown). Analysis of expression of the transgene revealed different levels of expression in the various mutants (Figure 1a). Consistent with reports demonstrating that oncogenic transformation by wild-type ERBB2 is dependent on high levels of ERBB2 expression (Di Fiore et al., 1987; Pierce et al., 1991), Ba/F3 cells transformed by wild-type ERBB2 showed several-fold higher transgene expression compared to the various mutants (Figure 1a). Ectopic expression of mutant ERBB2 as well as massive overexpression of wild-type ERBB2 led to constitutive kinase activity as revealed by autophosphorylation of the ERBB2 protein in the absence of exogenously added EGF (data not shown).
Treatment of Ba/F3 cells transformed with NSCLC-derived ERBB2 mutants as well as cells carrying wild-type ERBB2 with the reversible EGFR inhibitor erlotinib revealed that all of these cells were significantly more resistant to this treatment (Figure 1b) than Ba/F3 cells harboring the L858R mutation of EGFR (Jiang et al., 2005; Kobayashi et al., 2005b) known to be sensitive to erlotinib (Paez et al., 2004; Greulich et al., 2005), with an IC50 (drug concentration at which growth is inhibited by 50%) of <40 nM (Figure 1b). In contrast, the T790M resistance mutation of EGFR (Pao et al., 2005; Kobayashi et al., 2005a) led to complete resistance to erlotinib when co-introduced with L858R (Figure 1b), thus validating the overall approach. We noted some differences in the sensitivity of the different ERBB2 mutants to erlotinib that seemed to correlate to some extent to expression levels of ERBB2 (Figure 1a and b). In cells, erlotinib has activity against ERBB2 in the low micromolar range. Since Ba/F3 cells do not express endogenous ERBB family members, we speculate that the mutants with low levels of transgene expression are affected by erlotinib owing to low-level inhibition of ERBB2.
When treated with the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor, HKI-272, all ERBB2 mutants as well as the cells overexpressing wild-type ERBB2 displayed a high degree of sensitivity (Figure 2a). Most of the mutants as well as the cells expressing wild-type ERBB2 were comparable in their sensitivity (IC50<2 nM) to the L858R mutant of EGFR previously reported to be highly sensitive to HKI-272 (Figure 2a) (Kwak et al., 2005). All mutants were significantly more sensitive than the L858R/T790M double mutant of EGFR, which was recently reported to be sensitive to HKI-272 (Kwak et al., 2005). Biochemical analyses of response recapitulated the strong cytotoxic activity of HKI-272 against Ba/F3 cells expressing the different NSCLC-derived ERBB2 mutants as well as wild-type ERBB2 in that autophosphorylation of ERBB2 was readily inhibited following a 12-h treatment with low nanomolar concentrations of HKI-272 (Figure 2b). Thus, although the reversible EGFR inhibitor erlotinib was only marginally effective in inhibiting the growth of Ba/F3 cells carrying the major NSCLC-derived mutants of ERBB2 as well as Ba/F3 cells overexpressing wild-type ERBB2, these cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor, HKI-272.
The high degree of sensitivity of the Ba/F3 cells overexpressing wild-type ERBB2 to HKI-272 prompted us to analyse the responsiveness of the NSCLC cell line Calu-3, which we found to harbor a high-level amplification of the ERBB2 gene by single-nucleotide polymorphism (SNP) arrays (Figure 3a). Calu-3 cells do not, however, harbor an EGFR or ERBB2 mutation as revealed by re-sequencing of the exons encoding the EGFR and ERBB2 kinase domain (data not shown). Furthermore, Calu-3 cells do not display amplification of the EGFR locus as evidenced by SNP array analysis (data not shown). Expression levels of the ERBB family members EGFR, ERBB2 and ERBB3 are shown in Supplementary Figure 1 online. Treatment of Calu-3 cells with HKI-272 revealed that these naturally occurring NSCLC cells overexpressing ERBB2 were sensitive (IC50<0.1 μ M, Figure 3b) to a degree comparable to the ERBB2-mutant H-1781 cell line previously described by us to be sensitive to HKI-272 (Shimamura et al., 2006), whereas the KRAS-mutant cell line A549 was resistant to HKI-272. In concordance with published results, Calu-3 cells were less resistant to erlotinib treatment than H-1781 cells (Figure 3c) (Engelman et al., 2005); the IC50 for erlotinib was however about 10-fold higher than that for HKI-272. Biochemical pharmacodynamic analyses revealed that treatment of both H-1781 and Calu-3 cells with HKI-272 led to inhibition of receptor phosphorylation and inhibition of the phosphoinositide 3 (PI3)-kinase and mitogen-activated protein (MAP)-kinase pathways at nanomolar concentrations (Figure 4).
In a direct comparison of biochemical response between EGFR and ERBB2-mutant Ba/F3 cells with Calu-3 and H-1781 cells, we found that the absolute IC50 values found in growth inhibition assays (Figures 2a and 3b) reflected the drug's ability to act on the target (Supplementary Figure 2a and b online). The difference in absolute IC50 values between genetically engineered Ba/F3 mutants and naturally occurring cancer cell lines might be attributable to changes in intracellular drug metabolism as the biochemical IC50 values were similar to the values determined in growth-inhibition assays. These differences could also be observed in H-3255 cells, carrying the L858R mutation of EGFR when their responsiveness to the EGFR inhibitor erlotinib was compared to that of L858R-mutant Ba/F3 cells (Supplementary Figure 2c online). Importantly, both H-3255 and L858R-mutant Ba/F3 cells are well-established models of EGFR-mutant and erlotinib-sensitive lung tumors (Paez et al., 2004; Jiang et al., 2005; Kobayashi et al., 2005b; Sharma et al., 2006). Thus, although differences in absolute IC50 values can be observed in naturally occurring cancer cell lines and Ba/F3 cells expressing the identical genetic lesion, they typically display similar phenotypes in drug assays.
In summary, we have shown that the most prevalent NSCLC-derived ERBB2 mutants as well as overexpression of wild-type ERBB2 induce oncogenic transformation in a murine cellular model of transformation. Transformation was accompanied by constitutive autophosphorylation of transgenic mutant as well as wild-type ERBB2. Finally, although the resulting cells were relatively resistant to the reversible EGFR TKI erlotinib, they exhibited exquisite sensitivity to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor, HKI-272. In addition to the somatic ERBB2 mutations, amplification of the ERBB2 gene has recently been reported to occur in a fraction of NSCLC (Stephens et al., 2004; Zhao et al., 2005). In accordance with these observations, a preliminary analysis of gene copy number using SNP arrays revealed amplification of ERBB2 in one out of 95 primary NSCLC tumors (our unpublished results). Taken together, our results may provide a rationale for the deployment of irreversible dual EGFR/ERBB2 inhibitors, such as HKI-272, in patients with ERBB2-mutant or ERBB2-amplified lung tumors.
Materials and methods
Generation of ERBB2-mutant Ba/F3 cells
ERBB2 cDNA was subcloned into pBabe-hygro. The most prevalent NSCLC-derived mutants (http://www.sanger.ac.uk/genetics/CGP/cosmic/) were introduced into the retroviral construct using site-directed mutagenesis (Quick-Change Mutagenesis XL kit; Stratagene, La Jolla, CA, USA) and viral supernatant was produced as described (Greulich et al., 2005). Murine Ba/F3 cells were stably transduced with the retroviruses and IL-3 was withdrawn.
Cells were seeded into 96-well plates and treated with different doses of erlotinib or HKI-272 for 72 h. Viability was determined colorimetrically by the WST assay (Roche Applied Science, Indianapolis, IN, USA) following the recommendations of the manufacturer. Viability was calculated as a percentage of the untreated control following subtraction of the background values (media only).
Cells were either cultured in the presence or absence of varying doses of drugs, harvested and lysed as described (Greulich et al., 2005). Whole cell lysates were assayed for protein levels using antibodies recognizing EGFR, ERBB2, Akt, Erk or the phosphorylation status of these proteins by immunoblotting using standard procedures (Greulich et al., 2005). Blots were probed for actin or tubulin as loading controls.
Single-nucleotide polymorphism arrays
SNP arrays (250 K; Sty1 arrays, Affymetrix, Santa Clara, CA, USA) were hybridized with 250 ng of genomic DNA of Calu-3 cells overexpressing ERBB2 (not shown) and compared to HCC-1954 breast cancer cells with high-level amplification of ERBB2 (Gazdar et al., 1998; data not shown). Arrays were scanned, the data were processed and raw copy numbers were obtained using dCHIP (http://biosun1.harvard.edu/complab/dchip) as described (Zhao et al., 2005).
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RK Thomas is a Fellow of the International Association for the Study of Lung Cancer (IASLC). This work was supported in part by the Deutsche Krebshilfe through a Mildred-Scheel Fellowship to RK Thomas. T Shimamura holds a Career Development Award, as part of the Dana-Farber/Harvard Cancer Center Specialized Program of Research Excellence in Lung Cancer, NIH grant P20 CA90578.
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Cite this article
Minami, Y., Shimamura, T., Shah, K. et al. The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI-272. Oncogene 26, 5023–5027 (2007). https://doi.org/10.1038/sj.onc.1210292
- lung cancer
- targeted therapy
- oncogene mutation
- kinase inhibitors
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