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.

Lung cancer is the leading cause of cancer death, accounting for over 150,000 deaths annually in the United States alone (1). Current treatment options are thus inadequate for the majority of patients and additional therapies are needed. Mutationally activated oncogenes that promote tumorigenesis represent potential drug targets due to frequent dependency of tumor cells on such oncogenes (2, 3), and somatically altered receptor tyrosine kinases in particular have been successfully exploited as therapeutic targets in several cancers.

The prototypical therapy targeted to a somatically activated tyrosine kinase oncogene is imatinib mesylate, which targets the BCR-ABL fusion protein in chronic myelogenous leukemia (4). Targeted therapies developed for lung cancer include gefitinib and erlotinib, small-molecule inhibitors of mutationally activated EGFR in lung adenocarcinoma (5–8), and crizotinib, a small-molecule inhibitor of the EML4-ALK translocation product in lung adenocarcinoma (9). Trastuzumab, a monoclonal antibody inhibitor targeting ERBB2, and the small-molecule EGFR/ERBB2 inhibitor lapatinib are effective in ERBB2-amplified patients with breast cancer (10, 11).

The advent of next-generation sequencing technologies has enabled compilation of large somatic mutation datasets from cancer sequencing studies. Statistical methods that examine differences in gene mutation frequency can reveal evidence of positive selection; however, demonstration of the contribution of a mutated gene to tumorigenesis additionally requires functional validation. To identify new lung cancer oncogenes, we systematically assessed somatic alleles of significantly mutated receptor tyrosine kinase genes reported in patients with lung adenocarcinoma (12) for activity in cellular transformation assays. Although most receptor tyrosine kinase mutations tested failed to score, novel extracellular domain mutations of ERBB2 were oncogenic. Our results indicate a unique therapeutic opportunity for patients with lung and breast cancer who harbor extracellular domain mutations of ERBB2.

Results

Extracellular Domain Mutations of ERBB2 Found in Cancer are Oncogenic.

In the most comprehensive lung adenocarcinoma targeted sequencing experiment thus far, 623 genes were sequenced in 188 lung adenocarcinomas, identifying 1,013 nonsynonymous somatic mutations and 26 significantly mutated genes (12). In addition to mutated genes already well characterized in lung adenocarcinoma (13), the significant genes included known tumor suppressors and several receptor tyrosine kinases, putative but unproven oncogenes. In an effort to determine whether these uncharacterized receptor tyrosine kinase mutations are oncogenic, we analyzed the four most significantly mutated receptor tyrosine kinase genes identified by multiple statistical methods, EPHA3, ERBB4, FGFR4, and NTRK3, and two genes that failed to achieve statistical significance, NTRK2 and ERBB2, due to a cluster of mutations in the kinase domain of NTRK2 and an extracellular domain mutation of unknown significance in ERBB2 (Fig. S1). We expressed the mutant alleles in NIH 3T3 cells and examined oncogenic activity in soft agar assays.

None of the somatic alleles of EPHA3, ERBB4, FGFR4, NTRK2, or NTRK3 were found to support anchorage-independent proliferation in soft agar assays (Figs. S1 and S2A). In contrast, ectopic expression of FGFR4 V550E, recurrent in rhabdomyosarcoma and oncogenic in NIH 3T3 cells (14), and FGFR4 K645E, modeled after the activating FGFR3 K650E mutation found in multiple cancers (15), resulted in soft agar colony formation (Fig. S2A). Moreover, we could not detect EPHA3 protein expression in three lung cancer cell lines harboring EPHA3 mutations (Fig. S2B). Somatic mutations of EPHA3, ERBB4, FGFR4, NTRK2, and NTRK3 reported in lung adenocarcinoma thus do not confer phenotypes expected of receptor tyrosine kinase oncogenes.

Of the four mutations reported in ERBB2, S310F and A775_G776insYVMA (“insYVMA”) are predicted to encode the full-length protein (Fig. S1). Whereas the insYVMA mutation of the kinase domain of ERBB2 is already well characterized (16, 17), mutations of the extracellular domain have not been functionally analyzed. We therefore focused on the S310F mutation in exon 8 of ERBB2, found in 1/188 lung adenocarcinoma samples (12). Additional reports of extracellular domain mutations of ERBB2 included G309E in 1/183 breast cancer samples and S310Y in 1/63 squamous lung cancer samples (18), S310F in 2/112 breast cancers (19), 1 S310F and 1 S310Y in 258 lung adenocarcinomas sequenced by the Cancer Genome Atlas Network (Fig. S3 A and B), S310F in 1/65 breast cancers (20), and S310F in 1/316 ovarian cancers (21). An S310F mutation was also found in a bladder cancer cell line, 5637 (22).

We examined genomic data for samples with extracellular domain mutations of ERBB2. One breast cancer sample harbored an additional kinase domain mutation of ERBB2, L755S, and one lung cancer sample harbored a mutation of KRAS, G12F (Fig. S3C); none had mutations of EGFR. One breast cancer sample exhibited high-level amplification of ERBB2 in the genome, whereas the other samples did not (Fig. S3C). Two of four patients with lung cancer were former smokers. However, given the small number of samples analyzed, we lack power to determine whether there are any systematic associations of ERBB2 extracellular domain mutations with the presence or absence of other known driver mutations, ERBB2 amplification, or smoking status.

NIH 3T3 cells overexpressing wild-type ERBB2 exhibited a weak anchorage-independent phenotype (Fig. 1 A and B), consistent with previous reports (23). In contrast, the G309E, S310F, and S310Y mutants supported robust colony formation in soft agar (Fig. 1 A and B), similar to an ERBB2 kinase domain insertion mutant (16, 24–26). A kinase-inactive mutant, D845A, failed to form any colonies. AALE human lung epithelial cells were similarly transformed to anchorage independence by the extracellular mutants of ERBB2 (Fig. 1 C and D). We have thus identified oncogenic somatic mutations of the extracellular domain of ERBB2 in lung and breast cancer, occurring at a rate of about 1%, approximately half that of the ERBB2 kinase domain mutations previously described in lung adenocarcinoma (12, 24, 26).

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.

ERBB2 was reported to be a significantly mutated gene in glioblastoma (27). Paradoxically, only three of the reported mutations, C311R, E321G, and C334S, were transforming (Fig. 1 E and F). Upon closer inspection, it became apparent that all 7 glioblastoma samples harboring ERBB2 mutations were from a single TCGA sample batch. Because the reported data were derived from 91 samples in four sample batches, it was unlikely that all 7 samples would be in the same sample batch by chance (Fisher’s exact P = 0.000002). Nor could prior patient treatment account for this cluster of ERBB2-mutated samples. This analysis raised the possibility that the reported mutations were artifacts of whole-genome amplification, as the mutations in the TCGA study were not validated in unamplified DNA.

Sequenom genotyping of the original unamplified glioblastoma DNA samples, corresponding to the whole-genome amplified material in which the ERBB2 mutations were discovered, failed to detect the reported ERBB2 mutations (Fig. S4A), whereas most mutations reported in other significantly mutated genes in this sample batch were present in the unamplified DNA (Fig. S4B). Consistent with the possibility that these ERBB2 mutations are artifacts, no mutations of ERBB2 were reported in a parallel study (28). Because of these inconsistencies, we checked unamplified tumor DNA for the three mutations in lung and breast cancer originally detected in whole-genome amplified material. All three mutations were confirmed in native DNA (Figs. S3C and S4 C–F).

ERBB2 Extracellular Domain Mutations Activate the Receptor by Two Distinct Mechanisms.

The oncogenic mutations of the ERBB2 extracellular domain cluster in subdomain II, a region characterized by 11 disulfide bonds (29). Because two ERBB2 mutants with in vitro transforming activity, C311R and C334S, affect cysteine residues, we examined the crystal structure of ERBB2 (29) to ask whether these changes affect disulfide bonds. Both C311 and C334 are involved in disulfide bond formation, with C299 and C338, respectively (Fig. 2A). These intramolecular disulfide bonds are presumably disrupted in the C311R and C334S mutants.

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.

We hypothesized that disruption of intramolecular disulfide bonds might result in formation of intermolecular disulfide bonds by the remaining unpaired cysteines. We tested this hypothesis by running lysates on nonreducing and reducing gels in parallel. Whereas wild-type ERBB2 showed no evidence of dimerization under these conditions, C311R and C334S formed high-molecular-weight species consistent with ERBB2 dimers on nonreducing SDS/PAGE gels (Fig. 2B). E321G did as well, possibly due to disruption of salt bridges that E321 forms with K369 and R434 that stabilize the structure of the disulfide-bonded loops (Fig. 2 A and B). In contrast, there was no evidence of dimerization by the transforming insYVMA kinase domain mutant (Fig. 2B).

We then examined the mechanism of activation of the mutants found in breast and lung cancer. The S310F mutant protein was hyperphosphorylated, similar to the kinase domain mutant insYVMA (Fig. 3A). However, the C-terminal tail of the G309E mutant was not hyperphosphorylated (Fig. 3A), like that of other mutants of ERBB2 that dimerized by intermolecular disulfide bonding (Figs. 1F and 3A). We therefore investigated the dimerization capacity of ERBB2 G309E and found that this mutant did indeed form reduction-sensitive dimers (Fig. 3B).

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.

There are six cysteine residues involved in three intramolecular disulfide bonds in the region below the dimerization arm of ERBB2; replacement of any of these six cysteines with serine conferred the ability to form reduction-sensitive dimers and transform NIH 3T3 cells (Fig. S5 A and B). A decrease in C-terminal phosphorylation was also observed on the ERBB2 cysteine mutants despite a robust soft agar phenotype (Fig. S5C).

ERBB2 Extracellular Domain Mutants Effect Transformation Using Common Downstream Machinery.

We have thus defined two distinct mechanisms of activation of extracellular domain mutants of the ERBB2 receptor tyrosine kinase: elevation of C-terminal phosphorylation and formation of disulfide-linked dimers. In order to determine whether these two classes of ERBB2 mutants use similar pathways to effect oncogenic transformation, we used stable isotope labeling by amino acids in cell culture (SILAC) combined with immunoaffinity enrichment of tyrosine-phosphorylated peptides to compare differences in global protein tyrosine phosphorylation using quantitative mass spectrometry.

Whereas only a slight increase in phosphopeptide ratios was seen in the ERBB2 G309E-expressing cells over wild type, the cells expressing ERBB2 S310F exhibited a more substantial increase in peptide phosphorylation (Fig. S6), correlating with the greater oncogenic activity of S310F. Forty-four of 47 endogenous proteins with peptides phosphorylated twofold or higher in the ERBB2 S310F-expressing cells (Table 1) were also hyperphosphorylated in the G309E-expressing cells (Dataset S1). Furthermore, the 92 individual peptides phosphorylated twofold or higher in the ERBB2 S310F-expressing cells compared with ERBB2 wild-type–expressing cells exhibit a fold change distribution that is skewed toward the top of the list of hyperphosphorylated peptides in the G309E-expressing cells in a statistically significant manner, with a rank-test P value of 2.2 × 10⁻¹⁶ (SI Experimental Procedures). These data indicate that despite activation by distinct mechanisms, the two ERBB2 mutants use similar downstream effector pathways to transform cells.

Table 1.

Proteins containing peptides phosphorylated twofold or higher in cells expressing ERBB2 S310F than in cells expressing wild-type ERBB2 implicate events that impact cell motility

Gene	S310F/WT*	No. phosphopeptides†
CSNK1A1	83.5	1
CRK	19.3	1
DLG3	17.1	1
AHNAK	14.8	2
DLG1	11.1	1
ERBB2	10.2	11
CCDC88A	9.3	2
IQGAP1	9.3	1
ERRFI1	9.1	3
SEMA4C	8.0	1
PEAK1	7.0	3
STXBP3	7.0	1
CAV1	6.4	3
PTPN11	6.2	1
ERBB2IP	5.6	4
ANXA1	5.4	2
TLN1	5.3	2
ANXA2	5.1	9
DOK1	5.1	5
G6PD	4.9	1
TNK2	4.8	1
SH2B3	4.6	1
LAYN	4.4	2
CFL1	4.3	2
SHC1	4.2	2
ACTB	3.5	1
VIM	3.5	3
AXL	3.5	3
EPS8	3.0	1
PLCG1	2.9	1
PABPC3	2.8	1
CALM1	2.7	2
LDLR	2.7	1
CTNND1	2.6	1
GAB1	2.6	1
EEF1A2	2.5	1
CDK2	2.4	1
VASP	2.2	1
RIN1	2.2	1
STAT3	2.2	2
EEF1A1	2.2	1
PIK3R2	2.2	1
CDK1	2.2	1
MAPK1	2.1	1
ABI1	2.1	1
RPS27	2.1	2
RPS10	2.1	1
MYO9B	2.0	1

*Mean fold increase in phosphorylation of the most phosphorylated peptide for each protein.

^†Total number of distinct phosphopeptides detected for each protein.

ERBB2 itself was hyperphosphorylated in S310F-expressing cells but not G309E-expressing cells, consistent with immunoblot data (Table 1, Dataset S1, and Fig. 3A). Interestingly, the EGFR/ERBB2 inhibitor MIG6, encoded in human DNA by ERRFI1, was hyperphosphorylated in both the G309E- and S310F-expressing cells (Table 1 and Dataset S1), correlating with the previously described dependence of association with ERBB2 on ERBB2 activity but not C-terminal autophosphorylation (30).

A number of proteins regulating cytoskeletal dynamics and cell motility were found to be prominently hyperphosphorylated in the ERBB2 S310F cells (Table 1), including the murine homologs of CRK, DLG1, CCD88A, IQGAP, and PEAK1, as well as components of the cytoskeleton (Table 1). Altered cell motility may thus be closely linked to the transformed phenotype measured by the soft agar assay. PTPN11, a phosphatase involved in activation of Erk proteins in response to growth factor stimulation and intriguingly required for growth and metastasis of HER2-positive breast cancer cells (31, 32), was also prominently phosphorylated in the ERBB2 S310F cells. Of note, there was considerable overlap between the proteins phosphorylated in response to ERBB2 S310F expression and proteins reported to be phosphorylated in human mammary epithelial cells in response to knockdown of PTPN12, a negative regulator of EGFR and ERBB2 (33).

Oncogenic Activity of ERBB2 Extracellular Domain Mutants Is Sensitive to Treatment with ERBB2 Inhibitors.

Introduction of a kinase-inactivating D845A mutation into cDNAs harboring ERBB2 extracellular domain mutations prevented soft agar colony formation by transduced NIH 3T3 cells (Fig. S5B, Bottom Right). To facilitate inhibitor testing, we expressed the ERBB2 mutants in murine Ba/F3 cells and derived IL-3 independent lines. Expression of the extracellular mutations of ERBB2 conferred IL-3 independence more efficiently than wild-type ERBB2, whereas the vector control and kinase-inactive form of ERBB2 were not able to support IL-3–independent growth (Fig. 4A).

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.

Ba/F3 cells transformed with the ERBB2 extracellular domain mutants were treated with the irreversible ERBB2 inhibitors neratinib and afatinib, resulting in effective abrogation of cell survival, with IC50s in the low nanomolar range (Fig. 4 B and C). Cells expressing the extracellular domain mutants exhibited increased sensitivity to these inhibitors relative to cells expressing the wild-type ERBB2 or the kinase domain mutant, insYVMA. Importantly, the 95% confidence intervals of the IC50s for extracellular domain mutants S310F, S310Y, and E321G in response to treatment with small-molecule inhibitors were generally lower than the corresponding limits for wild-type ERBB2 or insYVMA (Fig. S7A). Inhibitor efficacy furthermore correlated with inhibition of ERBB2 phosphorylation (Fig. S7 B–E).

The reversible inhibitor lapatinib was 5- to 10-fold less effective than neratinib and afatinib (Fig. 4D), perhaps due to the more efficient recovery of receptor activity, evidenced by increases in phospho-ERBB2 and phospho-Akt following inhibitor washout in lapatinib-treated cells but not in neratinib-treated cells (Fig. S8). However, cells expressing the extracellular domain mutants were significantly more sensitive to lapatinib than cells expressing insYVMA (Fig. 4D). Trastuzumab treatment effectively inhibited survival of Ba/F3 cells expressing mutants of G309 and S310, but curiously had less of an effect on cells transformed by the other mutants (Fig. 4E). Although the cancer-derived mutations are located in the same region of the receptor as the epitope bound by trastuzumab, these results indicate that mutations of G309 or S310 do not inhibit trastuzumab binding.

We have previously shown that the lung cancer cell line NCI-H1781, harboring an ERBB2 kinase domain mutation, is sensitive to treatment with the irreversible inhibitor afatinib (34). In contrast, the endometrial cancer cell line AN3CA is characterized by FGFR2 mutation but not by ERBB2 mutation (35). Using these two cancer cell lines as controls, we tested whether ERBB2 inhibition affected survival of a bladder cancer cell line, 5637, harboring an ERBB2 S310F mutation (22). Whereas ERBB2 inhibition alone was effective against the NCI-H1781 cells, a combination of ERBB2 inhibition and Mek inhibition was necessary for abrogation of 5637 cell survival with an IC₅₀ comparable to that for the NCI-H1781 cells (Fig. 4F). Neither inhibitor had a significant effect on the AN3CA cells alone or in combination. These results suggest a possible treatment option for patients with lung and breast cancer harboring these mutations.

Discussion

We functionally analyzed mutated receptor tyrosine kinase genes found in lung adenocarcinomas. None of the somatic alleles of EPHA3, ERBB4, FGFR4, NTRK3, or NTRK2 were found to be oncogenic in NIH 3T3 cells. There are three possible explanations for the lack of oncogenic transformation by these mutant receptor tyrosine kinase genes. The reported significantly mutated genes may in fact be tumor suppressor genes, contributing to tumorigenesis but not scoring in a transformation assay designed to detect dominant gain-of-function oncogenes. The absence of nonsense and frameshifting mutations of EPHA3, ERBB4, NTRK2, and NTRK3 argues against a role in tumor suppression, as all known significantly mutated tumor suppressor genes found in the lung adenocarcinoma study harbored mutations resulting in premature termination.

A second explanation for the absence of oncogenic transformation is that the tested somatic alleles are in fact gain-of-function and oncogenic, and we simply used the wrong assay to uncover an oncogenic phenotype. This argument is difficult to refute; however, the ability of FGFR4 V550E and K645E and an ETV6-NTRK3 gene fusion (34) to support anchorage-independent growth argues against such an explanation. Furthermore, we find it unlikely that three of three lung adenocarcinoma cell lines harboring EPHA3 mutations would fail to express detectable EPHA3 if such mutations were in fact oncogenic.

A third explanation for the absence of oncogenic transformation is that the reported mutations are passenger mutations, and more refined statistical methods are needed to detect evidence of positive selection. For example, as nonexpressed genes may exhibit higher mutation rates than expressed genes (37), incorporation of sample-specific gene expression data from parallel RNA sequencing may assist in a more accurate estimation of gene-specific background mutation rates. Moreover, the effect of replication timing on mutation of individual genes (38, 39) was not modeled into the background mutation rate calculation and may similarly confound the results of significance testing.

In contrast to the other receptor tyrosine kinase mutants tested, an extracellular domain mutation of ERBB2 transformed NIH 3T3 cells to anchorage independence. This result demonstrates that infrequently mutated but genuine oncogenes can be found in cancer sequencing data even if the genes fail to achieve statistical significance. ERBB2 was reported as significantly mutated in glioblastoma (27) but the observed mutations could not be confirmed in patient-matched unamplified tumor DNA, raising the possibility that the reported mutations were artifacts of whole-genome amplification. In response to these data, the TCGA consortium is no longer to our knowledge performing any sequence analysis of whole-genome–amplified DNA.

We have identified a unique mechanism of activation of ERBB2 in tumor cells, namely, formation of covalent dimers linked by intermolecular disulfide bonds in subdomain II. It is straightforward to envision how the cysteine substitution mutants of the ERBB2 extracellular domain may lead to intermolecular disulfide bonding. G309 is located in close proximity to the C299-C311 disulfide bond; replacement of this compact residue with a bulkier residue such as glutamate may prevent formation of this intracellular disulfide bond, leaving unpaired cysteine residues available for intermolecular disulfide bond formation. We furthermore speculate that the S310F and S310Y mutations may result in hydrophobic interactions between the aromatic rings of the newly introduced 310F or 310Y with Y274 and F279 of the neighboring molecule, promoting noncovalent dimerization and kinase activation.

There is precedent in the literature for activation of receptor tyrosine kinases by disulfide-mediated dimerization. Somatic and germ-line mutations of the extracellular domain of the receptor tyrosine kinase FGFR3 that introduce unpaired cysteine residues have been described in bladder cancer and thanatophoric dysplasia, respectively (15). These mutations caused reduction-sensitive dimer formation, activated FGFR3 kinase activity, and supported anchorage-independent proliferation of NIH 3T3 cells (40, 41). The rat neu oncogene, an ERBB2 ortholog identified as the transforming agent in nitrosoethylurea-induced rat neuroblastomas (42), harbors a mutation corresponding to V659E in the transmembrane domain of human ERBB2 (43). Mutant neu but not wild-type neu formed covalent high-molecular-weight species under nonreducing conditions, consistent with disulfide bond-mediated receptor dimerization (44). Mice expressing a mouse mammary tumor virus encoding wild-type neu developed mammary tumors from which spontaneously mutated forms of neu, characterized by in-frame deletions in domain IV of the extracellular domain, could be isolated (45). These spontaneous deletion mutants, which typically removed a single cysteine residue, were oncogenic and migrated as dimers on nonreducing gels (46).

The availability of inhibitors effective against the extracellular ERBB2 mutants present in lung, breast, ovarian, and bladder cancer raises therapeutic possibilities. The efficacy of a combination of ERBB2 and Mek inhibition on a bladder cancer cell line harboring an ERBB2 S310F mutation further indicates the clinical utility of this approach; however, it is not yet mechanistically clear why both inhibitors are necessary for this effect, requiring further study. More broadly, our results suggest that a clinical trial of ERBB2 inhibition, alone or in combination with other agents, in patients with cancer harboring extracellular domain mutations of ERBB2 across tumor types is warranted.

Experimental Procedures

Cell Culture.

NIH 3T3 cells (ATCC) were maintained in DMEM (Cellgro) supplemented with 10% (vol/vol) calf serum (Invitrogen). Ba/F3 cells were maintained in RPMI 1640 (Cellgro) with 10% FCS (Gemini Bioproducts) and 10 ng/mL interleukin-3 (BD Biosciences). AALE cells were grown in SAGM media (Lonza). NCI-H1781 and 5637 cells were grown in RPMI 1640 with 10% FCS, and AN3CA cells were grown in MEM (Cellgro) with 10% FCS.

Retroviral Transduction.

cDNAs were ectopically expressed in NIH 3T3, AALE, and Ba/F3 using a Gateway-modified pBabe puro vector. Details can be found in SI Experimental Procedures.

Soft Agar Assay.

Soft agar assays were performed as described in ref. 47. Briefly, 5 × 10³ to 5 × 10⁴ cells were suspended in media containing 0.33% Select agar (Invitrogen) and plated on a bottom layer of media containing 0.5% Select agar in a six-well plate. Plates were incubated at 37 °C 2 wk before imaging. AALE colonies were photographed at 5× magnification and one field was counted 2–3 wk after plating.

Inhibitor Assays.

Totals of 2,000–4,000 Ba/F3 or 3,000 NCI-H1781, 5637, and AN3CA cells were seeded in 96-well plates, incubated with the indicated concentrations of inhibitor for 3 d, and assessed for cell survival with the WST-1 reagent (Roche). PD184352 (CI-1040) was obtained from Sigma. Afatinib, neratinib, and trastuzumab were purchased commercially, and lapatinib was purified from patient-discarded tablets by James Bradner. Survival data were analyzed using the Prism GraphPad software.

Dimerization Assay.

The dimerization assay was performed as described in ref. 46. Briefly, cells were washed twice with cold PBS containing 10 mM iodoacetamide (Sigma) and lysed with 500 μL TGP buffer (50 mM Tris, pH 7.4, 1% Triton, 10% glycerol, 10 mM iodoacetamide) containing protease inhibitors (Roche) and phosphatase inhibitors (Calbiochem). One aliquot was boiled in NuPage LDS 4× sample buffer (Invitrogen) containing 100 mM DTT (final concentration of 20 mM DTT) and one aliquot was boiled in sample buffer without reducing agent. Samples were run on 4–12% gradient polyacrylamide gels (Invitrogen).

SILAC Experiments.

Experiments were performed as described in ref. 47. Full experimental details and statistical methods can be found in SI Experimental Procedures.