Functional analysis of receptor tyrosine kinase
mutations in lung cancer identifies oncogenic
extracellular domain mutations of ERBB2
Heidi Greulicha,b,c,d,1, Bethany Kaplana,d, Philipp Mertinsd, Tzu-Hsiu Chend, Kumiko E. Tanakaa,d, Cai-Hong Yune,
Xiaohong Zhanga, Se-Hoon Leea, Jeonghee Choa, Lauren Ambrogiod, Rachel Liaoa,d, Marcin Imielinskia,d,
Shantanu Banerjia,d, Alice H. Bergera,d, Michael S. Lawrenced, Jinghui Zhangf, Nam H. Phoa,d, Sarah R. Walkera,
Wendy Wincklerd, Gad Getzd, David Franka, William C. Hahna,b,d,g, Michael J. Eckh, D. R. Manid, Jacob D. Jaffed,
Steven A. Carrd, Kwok-Kin Wonga,b,c, and Matthew Meyersona,d,g,i,j
aDepartment of Medical Oncology,gCenter for Cancer Genome Discovery, andhCancer Biology, Dana–Farber Cancer Institute, Boston, MA 02115;
Departments ofbMedicine andiPathology, Brigham and Women’s Hospital, Boston, MA 02115; Department ofcMedicine andjPathology, Harvard Medical
School, Boston, MA 02115;dBroad Institute of Harvard and MIT, Cambridge, MA 02142;eDepartment of Biophysics, Peking University Health Science Center,
Beijing 100191, China; andfDepartments of Biotechnology and Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105
Edited by William Pao, Vanderbilt–Ingram Cancer Center, Nashville, TN, and accepted by the Editorial Board July 24, 2012 (received for review February
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 extra-
cellular domain mutants were activated by two distinct mecha-
nisms, 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.
HER2|breast cancer|bladder cancer
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 ac-
tivated oncogenes that promote tumorigenesis represent poten-
tial 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 thera-
peutic 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 system-
atically assessed somatic alleles of significantly mutated receptor
tyrosine kinase genes reported in patients with lung adenocar-
cinoma (12) for activity in cellular transformation assays.
ung cancer is the leading cause of cancer death, accounting
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.
Extracellular Domain Mutations of ERBB2 Found in Cancer are
Oncogenic. In the most comprehensive lung adenocarcinoma tar-
geted sequencing experiment thus far, 623 genes were sequenced in
188 lung adenocarcinomas, identifying 1,013 nonsynonymous so-
to mutated genes already well characterized in lung adenocarci-
noma (13), the significant genesincluded known tumor suppressors
and several receptor tyrosine kinases, putative but unproven
receptor tyrosine kinase mutations are oncogenic, we analyzed the
four most significantly mutated receptor tyrosine kinase genes
identified by multiplestatistical methods, EPHA3, ERBB4, FGFR4,
and NTRK3, and two genes that failed to achieve statistical signif-
icance, 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 pro-
liferation in soft agar assays (Figs. S1 and S2A). In contrast, ectopic
expression of FGFR4 V550E, recurrent in rhabdomyosarcoma and
the activating FGFR3 K650E mutation found in multiple can-
cers (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 NTRK3reported inlung adenocarcinoma thus donot confer
phenotypes expected of receptor tyrosine kinase oncogenes.
Author contributions: H.G., P.M., S.R.W., W.W., D.F., J.D.J., S.A.C., and K.-K.W. designed
research; H.G., B.K., P.M., T.-H.C., K.E.T., S.-H.L., L.A., R.L., and W.W. performed research;
M.S.L. and G.G. contributed new reagents/analytic tools; H.G., P.M., C.-H.Y., X.Z., J.C., M.I.,
S.B., A.H.B., M.S.L., J.Z., N.H.P., W.W., G.G., D.F., W.C.H., M.J.E., D.M., K.-K.W., and M.M.
analyzed data; and H.G. and M.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. W.P. is a guest editor invited by the Editorial
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 4, 2012
| vol. 109
| no. 36www.pnas.org/cgi/doi/10.1073/pnas.1203201109
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 mu-
tation in exon 8 of ERBB2, found in 1/188 lung adenocarcinoma
samples (12). Additional reports of extracellular domain muta-
tions 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 Net-
work (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 do-
main 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), con-
sistent 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 simi-
larly 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).
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 glioblas-
toma DNA samples, corresponding to the whole-genome am-
plified 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 ex-
tracellular 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.
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
Number of Colonies
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_G776-
insYVMA; insV, ERBB2 A775_G776insV/G776C; WT, wild-type ERBB2.
Extracellular domain mutations of ERBB2 found in lung and breast
Greulich et al.PNAS
| September 4, 2012
| vol. 109
| no. 36
mutants of ERBB2 that dimerized by intermolecular disulfide
bonding (Figs. 1F and 3A). We therefore investigated the di-
merization capacity of ERBB2 G309E and found that this mutant
did indeed form reduction-sensitive dimers (Fig. 3B).
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 trans-
form 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-phosphor-
ylated 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 in-
crease 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 hyper-
phosphorylated in the G309E-expressing cells (Dataset S1). Fur-
thermore, 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 distri-
bution that is skewed toward the top of the list of hyper-
phosphorylated 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 de-
spite activation by distinct mechanisms, the two ERBB2 mutants
use similar downstream effector pathways to transform cells.
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 homo-
logs 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-inacti-
vating D845A mutation into cDNAs harboring ERBB2 extracel-
lular 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
glioblastoma cause disulfide bond remodeling. (A) Model of the ERBB2 di-
mer 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.
Oncogenic extracellular domain mutations of ERBB2 reported in
sensitive dimers that exhibit diminished C-terminal tail phosphorylation. (A)
Immunoblot analysis of ERBB2 protein and tyrosine 1221/1222 phosphory-
lation state on lysates of NIH 3T3 expressing ERBB2 extracellular domain
mutations. (B) Immunoblot analysis of ERBB2 dimers trapped by non-
reducing SDS/PAGE. pBp, pBabe puro vector; WT, wild-type ERBB2.
ERBB2 mutants found in lung and breast cancer form reduction-
| www.pnas.org/cgi/doi/10.1073/pnas.1203201109Greulich et al.
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).
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 in-
creased 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 ex-
tracellular 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 in-
hibition 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 inhibi-
ted 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 character-
ized 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.
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 explan-
ations for the lack of oncogenic transformation by these mutant
receptor tyrosine kinase genes. The reported significantly mu-
tated 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 muta-
tions resulting in premature termination.
A second explanation for the absence of oncogenic trans-
formation 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 har-
boring EPHA3 mutations would fail to express detectable
EPHA3 if such mutations were in fact oncogenic.
A third explanation for the absence of oncogenic trans-
formation is that the reported mutations are passenger muta-
tions, 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
or higher in cells expressing ERBB2 S310F than in cells expressing
wild-type ERBB2 implicate events that impact cell motility
Proteins containing peptides phosphorylated twofold
*Mean fold increase in phosphorylation of the most phosphorylated peptide
for each protein.
†Total number of distinct phosphopeptides detected for each protein.
Greulich et al. PNAS
| September 4, 2012
| vol. 109
| no. 36
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 demon-
strates 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 mu-
tated 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 se-
quence 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 di-
sulfide bond, leaving unpaired cysteine residues available for in-
termolecular 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 di-
mer 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 neuroblasto-
mas (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 mam-
mary tumors from which spontaneously mutated forms of neu,
characterized by in-frame deletions in domain IV of the extra-
cellular domain, could be isolated (45). These spontaneous de-
letion 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 combi-
nation 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 mechanis-
tically 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.
Cell Culture. NIH 3T3 cells (ATCC) were maintained in DMEM (Cellgro) sup-
plemented 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.
H1781+PD AN3CA+PD 5637+PD
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 to IL-3 independence
| www.pnas.org/cgi/doi/10.1073/pnas.1203201109Greulich et al.
Soft Agar Assay. Soft agar assays were performed as described in ref. 47. Download full-text
Briefly, 5 × 103to 5 × 104cells 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. Afati-
nib, 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 iodoace-
tamide (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
ACKNOWLEDGMENTS. We thank Dr. James Bradner for providing lapatinib,
Dr. Jesse Boehm for supplying receptor tyrosine kinase cDNAs, Dr. Somase-
kar Seshagiri for genotyping of ERBB2 mutations found in ref. 18 in native
DNA, Dr. Emanuele Pelscandolo and Ms. Christina Go of the Dana-Farber
Cancer Institute Center for Cancer Genome Discovery for genotyping the
ERBB2 mutation found in ref. 12 in native DNA, Drs. Angela Brooks and
Andrew Cherniack for assistance with genomic data, and Drs. Hideo Wata-
nabe and Rameen Beroukhim for helpful discussions. H.G. is supported by
a grant from Uniting Against Lung Cancer. This work was also supported in
part by National Cancer Institute Grants R01CA109038, R01CA116020, and
P20CA90578 (to M.M.); the American Lung Association; the Seaman Foun-
dation; and the Monopoli Foundation (M.M.).
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| September 4, 2012
| vol. 109
| no. 36