(Received for publication, October 5, 1995; and in revised form, January 17, 1996)
From the
Overexpression of Neu (ErbB-2/HER2) is found in 20% of
breast tumors. Activation of Neu by a point mutation (Neu
)
causes constitutive tyrosine kinase activity of this transmembrane
receptor and transforming activity in fibroblasts. To identify
downstream targets of Neu, we have analyzed the ability of Neu to
activate gene expression. Expression of Neu
, but not normal
Neu, caused transcriptional activation of Ets, AP-1, or
NF-
B-dependent reporter genes. Dominant inhibitory Ras or Raf
mutants blocked the Neu-mediated transcriptional activation, confirming
that Ras signaling pathways were required for this activation. Analysis
with Ets2 mutants indicated that activation of Ets2 transcriptional
activity mediated by Neu
or oncogenic Ras required
phosphorylation of the same Ets2 residue, threonine 72. Cotransfection
of dominant inhibitory Ets2 mutants specifically blocked
Neu
-mediated activation of Ets-dependent reporter genes.
Furthermore, in focus formation assays using NIH 3T3 cells, the
transforming activity of Neu
was inhibited 5-fold when
Neu
was cotransfected with a dominant negative Ets2 mutant.
However, parallel colony formation assays showed that the Ets2 dominant
negative mutant did not inhibit the growth of normal cells. Together,
these data show that Neu
activates a variety of
transcription factor families via the Ras signaling pathway and that
Ets activation is required for Neu
-mediated cellular
transformation. Thus, downstream targets of Neu, including Ets
transcription factors, may be useful points for therapeutic
intervention in Neu/ErbB-2-associated cancers.
The c-neu oncogene product (also called ErbB-2 or HER2) is a 185-kDa transmembrane receptor tyrosine kinase that belongs to the epidermal growth factor family(1, 2, 3) . Overexpression of Neu/ErbB-2 is found in 20-30% of human breast cancers and is also seen in ovarian, lung, and gastric adenocarcinomas(4, 5, 6, 7, 8) . A number of potential activating ligands for Neu/c-ErbB-2 have been studied(8) , and this receptor can be constitutively activated by a point mutation in the transmembrane domain (9) or by deletions in the extracellular domain(10, 11) . Constitutive activation by mutation causes Neu to exhibit strong transforming activity, both in cultured cells and in transgenic mice(9, 12) . Expression of very high levels of normal Neu can transform fibroblasts in vitro(13) . Overexpression of normal Neu in transgenic mice also leads to tumor formation, but because these tumors are infrequent and focal, it appears that a second event is required for tumor formation(14, 15) . Cellular transformation is associated with alterations in the expression of multiple genes, many of which are likely regulated by the abundance or activity of specific transcription factors. Although downstream targets of Neu have been identified, like other receptor tyrosine kinases, the details of the pathways for cellular transformation remain unclear(16, 17) .
Neu that has been activated by a
point mutation in the transmembrane domain (called Neu) has
enhanced tyrosine kinase activity (9) and has been found to
activate a variety of signaling pathway components(18) .
Neu
-activated signaling components include phospholipase
C
and phosphatidylinositol 3`-kinase(19, 20) ,
Src(21, 22) , Shc, and
Grb2/SOS(23, 24, 25) . More downstream
signaling components activated by Neu
also include Ras,
mitogen-activated protein kinases, and AP-1
activity(24, 26, 27) . Therefore, although
mutations in ras are rarely found in breast cancer, the
Neu-mediated activation of Ras signaling pathway components suggests
that this pathway may nonetheless play an important role in breast
cancers(28) .
We have focused on transcription factor
activation by oncogenes(29, 30) , because there is a
close correlation between the ability of non-nuclear oncogenes to
activate gene expression and to transform
cells(31, 32) . We previously examined the
requirements for promoter DNA binding sites that could function as Ras
responsive elements, and found that at the right spacing and
orientation, two or more binding sites for members of either the Ets,
AP-1 or NF-B transcription factor families are sufficient to
confer Ras responsiveness(29) . The importance of
oncogene-mediated transcription factor activation is revealed by the
findings that inhibition of either AP-1 or Ets activation blocks
Ras-mediated cellular
transformation(33, 34, 35, 36) . In
the present study, we have determined that Neu
increases
the activities of several different families of transcription factors,
including Ets, AP-1, and NF-
B, and that activation of these
downstream targets is mediated by the Ras signaling pathway. We further
show evidence that Neu
and oncogenic Ras share a common
molecular target for activating transcription, by mediating the
phosphorylation of Ets2 threonine 72, leading to increased Ets2
transactivation activity. Finally, to examine the biological
significance of Ets activation, we have used dominant negative Ets
mutants to show that Ets activation is required for
Neu
-mediated cellular transformation.
The FNpcDNA3 expression vector was constructed
using the cytomegalovirus promoter-driven expression vector pcDNA3
(Invitrogen, San Diego, CA), and inserting between the HindIII
and BamHI sites a peptide leader sequence encoding
MDYKDDDDKPKKKRKVGS, preceded by a consensus translational start site
(GCCACC). This leader sequence contains the FLAG epitope tag and the
SV40 nuclear localization signal. The Ets2 coding sequence was modified
to insert into this vector by standard polymerase chain reaction
mutagenesis, by addition of an in-frame BamHI site (GGA TCC)
at the 5` end and a SmaI site just 3` of the termination
codon. The BamHI-SmaI cut Ets2 coding sequence was
inserted into the BamHI-EcoRV cut FNpcDNA3 vector, to
create FN-Ets2. The Ets2 coding sequence inserted directly into pcDNA3
gave the same results as the epitope tagged FN-Ets2 construct. ()Mutant FN-Ets2 constructs with altered residue 72 were
constructed using the Chameleon site-directed mutagenesis kit
(Stratagene, La Jolla, CA) as recommended by the manufacturer. The Ets
dominant negative mutants were constructed using the FNpcDNA vector in
the same manner as full-length Ets2. E2TAD contains Ets2 residues
1-331, and E2DBD contains Ets2 residues 332-468. Similarly,
E1TAD contains Ets1 residues 1-305, and E1DBD contains residues
Ets1 residues 306-466.
The results of cotransfection experiments (Fig. 1) showed
that expression of Neu activated the transcription of the
reporter genes containing synthetic binding sites for Ets (E.18), AP-1
(6AP-1), or NF-
B (NF-
B), and a reporter gene containing
single overlapping Ets and AP-1 binding sites (Py). Expression of
Neu
did not significantly activate reporter gene expression
(data not shown). The transcriptional activation for each reporter gene
was measured by determining the ratio of the CAT activity from cells
transiently cotransfected with Neu expression construct relative to
cells cotransfected with empty pSV2 expression plasmid. The
transcription of the minimal promoter-containing CAT reporter
construct,
56FosdE, was not significantly activated by Neu
(Fig. 1). However, the transcriptional activation by
Neu
of reporter genes containing Ets, AP-1, or NF-
B
binding sites ranged from 8.6-fold for the E.18 reporter to 20.6-fold
for the NF-
B reporter (Fig. 1). The difference in
transactivation activity between the expression plasmids for Neu
and Neu
, which differ by only a single nucleotide,
parallels the difference in their transformation activity, because
pSV2neu
, but not pSV2neu
, can
transform NIH 3T3 cells(9) . Under the conditions of this
assay, oncogenic Ras activated the transcription of these reporter
genes to an even higher level than Neu
(data not shown). To
demonstrate the specificity of Neu
-mediated activation, we
tested whether the expression of a reporter gene containing 2 copies of
the somatostatin CRE was activated by Neu
. This cAMP
response element has been previously shown to confer
cAMP-responsiveness, but not Ras-responsiveness(42) . The 2CRE
reporter gene was not transactivated by Neu
(Fig. 1)
or by oncogenic Ras, but was transactivated approximately 10-fold by
treatment of the cells with forskolin and isobutylmethylxanthine to
induce cAMP levels (data not shown). Similar to the results we
previously found for oncogenic Ras (29) , reporter genes
containing the minimal fos promoter with only a single added
binding site for Ets, AP-1, or NF-
B family members were not
significantly transactivated by expression of Neu
(data not
shown), indicating that the presence of a single binding site for any
of these transcription factors was not sufficient to create an Neu
responsive promoter element.
Figure 1:
Neu-induced transcription
activation of Ets, AP-1, and NF-
B-dependent reporter genes.
Reporter plasmid DNA (3 µg of
56FosdE or 1 µg or the other
reporters) and either pSV2neuT or pSV2 (4 µg) were
cotransfected in to NIH 3T3 cells, and the resulting CAT activity was
determined. The fold activation is the ratio of the CAT activity from
each reporter gene in the presence of pSV2neuT, relative to
the CAT activity from the same reporter gene with empty pSV2 expression
vector. The bar graph results display the average of at least three
separate experiments, and the standard deviation is shown by error
bars.
Figure 2:
Neu-induced transcription
activation is Ras- and Raf-dependent. The CAT cotransfection assays and
determination of fold activation by Neu
were the same as
for Fig. 1, except that 3 µg of an additional expression
plasmid was cotransfected with the reporter and Neu expression
plasmids. The added plasmids were either pZIP vector, (solid
bars), pZIPrasH(17N) (striped bars), or Raf-N3 (open bars).
Figure 3:
Neu-mediated enhancement of
Ets2 activity targets Ets2 residue 72. A, CAT cotransfection
assays were performed using the E.18 reporter gene (1.5 µg) and 100
ng of the indicated Ets2 expression plasmid, along with 4 µg of
empty expression vector (pSV2, open bars) or Neu
expression vector (pSV2neuT, closed bars). The
fold activation and error bars were determined as described in Fig. 1. B, immunoprecipitation analysis of Ets2 protein
levels. Cells were cotransfected with the Ets2 expression construct
indicated beneath each lane or with no expression vector denoted by
``(-),'' and either pSV2 or pSV2neuT.
The cells were treated as in Fig. 3A, except that they
were metabolically labeled with [
S]methionine
for 4 h prior to harvest. Subsequently, the cells were lysed and the
transiently expressed epitope tagged Ets2 proteins were
immunoprecipitated, separated by SDS-polyacrylamide gel
electrophoresis, and visualized by autoradiography (see
``Experimental Procedures''). The specific Ets2 signal is
indicated with an arrow.
Figure 4: Dominant negative Ets mutants block E.18 transactivation by Neu or Ras. A, schematic representation of the Ets2 protein, with the potential helix-loop-helix region denoted by HLH. The regions present in the truncated E2TAD and E2DBD mutants are indicated. B, CAT cotransfection assays were performed with 1 µg of E.18 reporter plasmid (solid bars) or of 6AP-1 reporter (striped bars) the indicated amounts of Ets dominant negative construct and 4 µg of either pSV2 or pSV2neuT expression construct. Fold activation was determined as in Fig. 1. C, CAT cotransfection assays as described in B, except that the E.18 reporter gene was cotransfected with the indicated amount of Ets2 dominant negative expression construct and either 2 µg of pZIP or pZIPrasH(L61).
The other type of Ets2 inhibitory mutant we tested consisted of the
Ets2 sequences lacking the DNA binding domain (Fig. 4). This
portion of Ets2 contains the transactivation domains of Ets2 (44, 45) including a predicted helix-loop-helix
domain(46) , and we called this construct E2TAD. The only known
ETS family member to contain extensive identity to Ets2 in this region
is Ets1, making this putative dominant negative mutant potentially more
specific. We also tested the corresponding Ets1 transactivation domains
construct, E1TAD. High level expression of either E2TAD or E1TAD (4
µg of cotransfected plasmid) also strongly inhibited E.18 reporter
gene activation by either Neu (Fig. 4B) or
oncogenic Ras (Fig. 4C) and of the Py reporter gene
(data not shown). Plasmid titration studies revealed that inhibition of
Neu
or Ras-mediated transactivation by the E2TAD expression
construct required 30-100 fold more expression plasmid DNA than
was required with E2DBD, and Western blot analysis revealed that this
difference was reflected in protein levels, as substantially more TAD
protein than DBD protein was required for dominant negative function
(data not shown). However, the observed inhibition of both types of Ets
dominant negative mutants was specific, and furthermore, not due to
inhibition of Neu expression, because control experiments showed that
Neu-mediated activation of the AP-1 dependent reporter gene, 6AP-1, was
not inhibited by either of the Ets dominant negative constructs (Fig. 4B). Similar specificity was seen with
Ras-mediated activation, as Ets-dependent, but not AP-1-dependent
reporter gene activation was strongly inhibited by Ets dominant
negative mutants (data not shown).
Figure 5:
Dominant negative Ets mutants inhibit
Neu-mediated cellular transformation. Results of focus
formation assay using 6-cm dishes of NIH 3T3 cells transfected with 250
ng of the indicated oncogene expression construct. Where indicated
below, 250 ng of pSV2neuT was cotransfected with 50 ng of
empty expression vector (FNpcDNA3) or dominant inhibitory Ets plasmids
(E1DBD or E2DBD). Each bar graph shows the average of six
plates from two separate experiments, and the standard deviation is
indicated by error bars.
The neu oncogene product has been reported to
activate an array of signaling molecules, the most downstream of which
is AP-1 transactivation activity, which results in increased
AP-1-dependent gene expression(27) . The purpose of the current
study was to identify other downstream targets of Neu by
determining whether Neu
mediates activation of other
transcription factor families, and to assess the biological
significance of Neu
-mediated transactivation. Therefore, we
used cotransfection analysis with Neu
expression plasmids
and reporter genes that we had shown previously to be activated by
oncogenic Ras(29) . These reporter genes contained synthetic
transcription factor binding sites placed in front of a minimal
promoter-CAT reporter gene and allow analysis of the activation of
specific transcription factor families by oncogenes(29) . This
analysis showed that the transactivation activity of Ets and NF-
B
transcription factors, as well as AP-1, is activated by
Neu
, but not Neu
. These results form the basis
for more detailed future studies to determine which individual members
of these large transcription factor families are targets of
Neu
.
As an example of such analysis, we have shown here,
using cotransfection studies with wild-type and mutant Ets2 proteins,
that Ets2 is a downstream target of Neu. The observed
Neu
-mediated transcriptional activation was not simply a
consequence of Neu
increasing cell growth or generally
stimulating transcription, because the minimal promoter-CAT reporter
gene alone (
56FosdE) or reporter plasmids containing nonfunctional
oncogene response elements had clearly measurable basal levels of
expression but were not transactivated by Neu
. Furthermore,
a reporter gene inducible by signals distinct from the Ras pathway, the
cAMP-inducible reporter gene 2CRE, was also not transcriptionally
activated by Neu
. The finding that dominant negative Ras or
Raf blocked transcription factor activation indicated that an essential
component of Neu
signaling is the Ras signaling pathway.
However, because transcriptional activation was measured after 2 days
of coexpression, this inhibition by dominant negative Ras and Raf does
not necessarily mean that there is a direct linear pathway from Neu
through Ras and Raf. For example, it is possible that Neu behaves
similar to Raf, which stimulates the production of autocrine factors
which in turn activate other components of the Ras signaling
pathway(47) . The fact that Ets2 is a downstream target of Neu
is intriguing, given the finding that there is a binding site for an
unidentified ETS protein in the promoter of the HER/neu gene
that influences its expression(48) . Although we found that the
three families of transcription factors examined were induced in
parallel by Neu
and oncogenic Ras in NIH 3T3 cells, the
transcription signaling pathways of these oncogenes do not appear to be
identical. Two examples of differential gene regulation by Ras and Neu
that are found in Ras transformed cells, but not in the same cell lines
transformed with Neu
, are the elevation of the
transcription of the parathyroid hormone-related peptide gene (49) and decreased expression of the NF-1/CTF
gene(50) .
A question that arises from our transactivation
results, is why overexpression of normal Neu, which is the defect in
Neu/ErbB-2 associated with human cancers, did not significantly
activate transcription factor activity in our assays. One possible
explanation is that normal Neu requires its ligand to stimulate
transcription, and this ligand is not present in the mouse fibroblasts.
In fact, studies where the normal Neu/ErbB-2 receptor was stimulated
with antibodies or by using a hybrid epidermal growth factor-Neu
receptor and epidermal growth factor or with heregulin and
cotransfected ErbB-3, the same activation of signal transduction
(including AP-1 activation), is seen as with
Neu(51, 52, 53, 54, 55) .
A second partial explanation may be that while previous studies of
human breast tumors found no activating Neu/ErbB-2
mutations(56, 57) , many activated Neu mutants may not
have been detected. Recent work with transgenic mice overexpressing
normal Neu has revealed that in 65% of the tumors examined, the neu gene contained an activating mutation, but outside of the
previously examined sequences encoding the transmembrane domain (11) . Another potential reason that Neu
was not
active in the transactivation assay is that the pSV2neuN
expression construct does not sufficiently overexpress Neu to achieve
the amplified levels of Neu/ErbB-2 found in human tumors. In support of
this idea is the finding that transfection of pSV2neuN does
not transform cells ( (9) and see Fig. 5), whereas
transfection of an expression plasmid that causes much higher levels of
normal Neu expression can transform NIH 3T3 cells(13) .
Because oncogene expression can have widespread effects on cells, it
was important to establish whether Neu-mediated activation
of Ets transcription factors is an necessary component of
Neu
-mediated cellular transformation. For this analysis, we
made the dominant inhibitory Ets mutants. The truncated Ets mutants
that expressed only the Ets1 or Ets2 DNA binding domains (DBD) were
potent inhibitors of Ets-dependent transcription activation by
Neu
or Ras. Presumably these mutant proteins act by binding
to Ets binding sites, and blocking the function of endogenous Ets
proteins. These mutant proteins may effectively compete with the
endogenous protein due to the loss of the postulated intramolecular
inhibition of DNA binding present in full-length Ets1 and
Ets2(58, 59) . It is likely that E2DBD or E1DBD
inhibit the activity of most ETS family members, due to the similarity
of ETS family DNA binding sites (60) . The potential for
cross-inhibition of ETS family members was illustrated by a recent
study in which overexpression of either of two of the most divergent
ETS domains, Ets1 or PU.1, has similar inhibitory effects on
Ras-mediated transactivation and transformation(36) . We found
that overexpression of the transactivation domains (TAD) of Ets1 or
Ets2 also blocked Neu
-mediated activation of Ets-dependent
transcription, but much less efficiently than the DBD mutants. We
postulate that the TAD mutants act by titrating out some limiting Ets
interaction partner. However, the inhibition of transactivation by TAD
is not a generalized squelching of all transcription or by inhibiting
Neu
expression, because neither the TAD nor the DBD mutants
efficiently blocked the Neu
or Ras-mediated activation of
an AP-1-dependent reporter gene.
The potential biological importance
of the activation of Ets-dependent transcription by Neu was
revealed by the observation that the Ets dominant negative mutant
Ets2DBD specifically inhibited Neu
-mediated focus formation
over 5-fold in an NIH 3T3 cell cotransfection assay (Fig. 5).
Thus, Ets mutants that blocks Neu
-mediated transcriptional
activation of an Ets-dependent reporter gene also block cellular
transformation. We do not yet understand why the Ets1DBD mutant
inhibited Neu
-mediated focus formation less efficiently
than Ets2DBD, but this inhibition was still significant. We have
further found that both Ets1DBD and Ets2DBD inhibit focus formation by
oncogenic Ras and that the Ets2DBD mutant also inhibits this focus
formation more efficiently than Ets1DBD. (
)The results of
the colony formation assay, in which equivalent numbers and size of
G418-resistant colonies were obtained with empty expression vector or
EtsDBD mutants indicated that these dominant negative mutants did not
block focus formation by growth inhibition or toxicity. Thus, with the
appropriate expression of an Ets dominant negative protein, it appears
that Neu
-mediated cellular transformation can be blocked
without interfering with normal cell growth. It has previously been
found that Ras transformed cells can actually be reverted to normal
morphology and growth characteristics upon expression of dominant
negative Ets(35, 36) , and it will now be of great
interest to determine whether introduction of dominant negative Ets
mutants can revert Neu
transformed cell lines or human
breast cancer cell lines back to nontransformed cells. Previous
experimental approaches to inhibiting Neu/ErbB-2-mediated cellular
transformation and tumor growth have largely been based on directly
interfering with Neu/ErbB-2 function, either with dominant negative
ErbB-2 mutants, antibodies against ErbB-2, or ErbB-2 antisense
oligonucleotides (61, 62, 63, 64, 65) . The
results described here suggest that inhibiting targets well downstream
of Neu/ErbB-2, including activation of transcription factors, may
complement other approaches for therapy in tumors associated with
Neu/ErbB-2.