ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185ErbB2). In addition to p185ErbB2, truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2+ breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2+ breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2+ breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs. Mol Cancer Ther; 10(8); 1367–74. ©2011 AACR.

ErbB2, a 185-kDa transmembrane receptor tyrosine kinase (p185ErbB2), is deregulated in 25% of all breast cancers, where it predicts for a poor clinical outcome (1). ErbB2 activation requires autophosphorylation of tyrosine (Y) residues within the cytoplasmic domain of the receptor (e.g., Y1248; ref. 2). These phosphotyrosine residues serve as docking sites for adaptor proteins that link ErbB2 to downstream mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) signaling networks that promote the growth and survival of breast cancer cells (2–6). In addition to p185ErbB2, truncated forms of ErbB2 lacking all or most of the N-terminus extracellular domain (ECD) exist in ErbB2+ breast cancer cell lines and clinical tumors (7–10). The most extensively studied truncated forms retain the transmembrane region and are expressed at the cell surface. Historically referred to as p95, truncated forms of ErbB2 expressed at the cell surface form heterodimers with other ErbB receptors (11) and interact with the p85 subunit of PI3K (12), thereby activating downstream signal transduction cascades in a manner similar to p185ErbB2. The generation of p95 has been shown to be dependent upon metalloproteinase activity (7). p95-positive breast cancers exhibit an aggressive clinical phenotype characterized by an increased incidence of lymph node involvement at the time of initial diagnosis (13, 14) and are more resistant to trastuzumab, as they lack the ECD (15, 16).

Lapatinib is a highly selective small molecule inhibitor of the ErbB2 and EGFR tyrosine kinases. Inhibition of ErbB2 tyrosine autophosphorylation by lapatinib leads to the inactivation of downstream cell growth and survival signals (17–19). Although a significant advancement in the treatment of breast cancer, the clinical efficacy of lapatinib has been limited by the development of acquired therapeutic resistance (20, 21). To address this problem, we generated clinically relevant models of acquired resistance to lapatinib using human ErbB2+ breast cancer cell lines (22, 23).

We now show that treatment with ErbB2 tyrosine kinase inhibitors (TKI) increased the expression of a tyrosine phosphorylated, truncated form of ErbB2 that was expressed in the nuclei of ErbB2+ breast cancer cells, which will herein be referred to as p95L (lapatinib-induced p95). In contrast to truncated forms of ErbB2 expressed at the cell surface, the phosphorylation of p95L, and similar truncated forms that were also expressed in tumor cell nuclei, was resistant to ErbB2 TKI. The data supporting the activation and nuclear localization of p95L in response to ErbB2 TKI, and the role of nuclear, truncated forms of ErbB2 in the development of therapeutic resistance to ErbB2 TKIs, will be discussed.

Cell culture and reagents

BT474, SKBR3, Au565, MCF7, and T47D breast cancer cell lines were obtained from the American Type Culture Collection. Lapatinib-resistant breast cancer cells were generated as previously described (22). All cells were cultured as previously described (11, 17, 22). No independent authentication of these cells was done by the authors. Anti-phosphotyrosine (p-tyr) antibody, GW2974, and calpain inhibitor 1 were purchased from Sigma-Aldrich. Anti-c-ErbB2 (Ab-11) monoclonal antibody was from Neo Markers. Anti-ErbB2 (AA1243-1255) and anti-phospho-ErbB2 (Y1248) antibodies were from Upstate Biotechnology. MG132, γ-secretase inhibitor, and lactacystin were from Calbiochem. BB94 (batimastat) was from Kimia Corp. Protein G agarose was purchased from Boehringer Ingelheim. IRDye800 conjugated affinity purified anti-rabbit IgG and anti-mouse IgG were from Rockland. Alexa Fluor 680 goat anti-rabbit IgG was obtained from Molecular Probes. Lapatinib (GW572016) and N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (Fig. 1A) was purchased from LC Laboratories. Lapatinib for cell culture work was dissolved in dimethyl sulfoxide (DMSO; 0.01%).

Isolation of nuclear extracts, SDS-PAGE, and Western blot analysis

Details of cell fractionation, immunoprecipitation, SDS-PAGE, and Western blot analysis were previously described (22). Membranes were probed with specific antibodies recognizing target proteins and visualized using the Odyssey Infrared Imaging System (LI-COR, Inc.). Membranes were incubated with fluorescent-labeled secondary antibody at a 1:10,000 dilution with 3% bovine serum albumin in PBS for 60 minutes protected from light. After washing in PBS + 0.1% Tween-20, the membranes were scanned using an Odyssey imaging system.

Human tumor xenografts, animal treatment, and human tumor biopsies

NOD.CB17-Prkdcscid/J (NOD/SCID) mice were purchased from Jackson Laboratory and bred in the Duke Comprehensive Cancer Center Isolation Facility. BT474 and rBT474 cells were suspended in HBSS and mixed with Matrigel (BD Biosciences) at 1:1 ratio to make final concentrations of 1 × 104 cells per 50 μL. A 50-μL tumor cell suspension was inoculated into bilateral mammary fat pads of female NOD/SCID mice (5 ∼ 6 weeks old, 4 mice per group). Animals were treated with lapatinib (75 mg/kg/d) by oral gavage until they were sacrificed. Tumor dimensions were measured serially, and tumor volumes calculated using the following formula: long axis × (short axis)2 × 0.52. The mice were euthanized with CO2 inhalation and tumor xenografts excised 59 days after implantation of tumor cells. All animal studies were conducted in compliance with Duke animal care regulations. Human biopsies were collected from breast cancer skin metastasis after informed written consent was obtained as part of an Institutional Review Board–approved tissue collection protocol. Tumor specimens were flash frozen in liquid nitrogen and stored at −80°C. Tissue extracts were prepared for Western blot analysis by homogenization in radioimmunoprecipitation assay buffer at 4°C.

Expression of truncated forms of ErbB2 in human breast cancer cell lines

C-terminal fragments (CTF; c-611, c-676, and c-678) were generated on the basis of ErbB2 open reading frames from LTR-2/ErbB2 (8) and subcloned into the pcDNA 3.1 (+). c-611, c-676, and c-678 were subcloned into the pcDNA3.1 vector (Invitrogen) with forward primers: 5′-ACAAGCTT ACCATGCCCATCTGGAAG-3′, 5′-ACAAGCTTACCATGAAGCGACGGCAGCA-3′ and 5′-ACAAGCTT ACCATGCGGAGACTGCTG-3′, and reverse primer: 5′-AACTCGAG TCACACTGGCACGTCCAG-3′. MCF7 and T47D breast cancer cells were transfected with empty vector alone (controls) or the same vector containing p185ErbB2 or the various CTFs using the Lipofectamine 2000 Reagent from Invitrogen according to the manufacturer's protocol. Stably transfected cells were selected using G418 (400 μg/mL), and the expression levels of CTFs were confirmed by Western blot analysis.

Immunofluorescence microscopy

Cells were cultured in 6-well plates with or without the indicated treatments. After washing with PBS, cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.2% Triton X-100 for 20 minutes, and blocked with 2% bovine serum albumin in PBS at room temperature followed by washing with PBS and incubated with anti-ErbB2 or anti-phosphotyrosine–specific antibodies overnight at 4°C. After extensive washings, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit or rabbit anti-mouse antibodies followed by counterstaining with 1.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) from Vector Labs. An Olympus L Fluoview FV1000 was used for all photographs.

Proliferation and apoptosis assay

The proliferation assay was carried out in a 96-well plate format in a final volume of 100 μL per well cell culture medium with the cell proliferation reagent WST-1 from Roche Diagnostics. Details of the WST-1 proliferation and Annexin V/nexin 7-AAD apoptosis assays were previously published (17, 22).

Statistical analysis

Data were expressed as means with standard error bars included. Student's t test was used to determine statistical significance between 2 groups. P < 0.05 was considered a statistically significant difference.

ErbB2 TKIs increase the expression of phospho-p95L in tumor cell nuclei

The effects of ErbB2 TKI on ErbB2 tyrosine phosphorylation were determined in BT474 cells, a human ErbB2+ breast cancer cell line, using immunofluorescence microscopy. Total ErbB2 protein and phosphotyrosine expression were determined using an ErbB2-specific antibody and a phosphotyrosine (p-tyr) antibody, respectively. ErbB2 and p-tyr signals were visualized using a secondary FITC-conjugated antibody (green). Total ErbB2 expression was unchanged in response to GW2974, an ErbB2 TKI (Fig. 1B). The p-tyr signal primarily localized to the cell surface and cytoplasm in vehicle-treated controls (−). Relatively little p-tyr signal was seen in the nuclei (blue/DAPI) of control cells (Merge). Whereas cell surface and cytoplasmic p-tyr were markedly reduced in response to GW2974, nuclear p-tyr persisted (Fig. 1B, Merge). We treated another ErbB2+ breast cancer cell line, Au565, with lapatinib and examined phospho-ErbB2 (p-ErbB2) expression using an ErbB2 phosphotyrosine–specific antibody and an FITC-conjugated secondary antibody (green). Similar to BT474 cells, p-ErbB2 at the cell surface, but not in the nuclei of some ErbB2 cells, was markedly reduced by lapatinib (Fig. 1C).

We next isolated nuclear extracts from BT474 and Au565 cells treated with vehicle alone (controls) or lapatinib. The purity of nuclear extracts was confirmed using Oct 1, IκB, and E-cadherin, which represent nuclear, cytoplasmic, and cell membrane proteins, respectively. Steady-state levels of total p95L protein increased in lapatinib-treated cells without an appreciable change in p185ErbB2 (Fig. 1D). Additional molecular weight bands above 98 kDa, which have been seen previously in ErbB2 blots, were observed.

In Fig. 1E, total ErbB2 protein was immunoprecipitated (IP) from nuclear extracts isolated from Au565 cells treated with vehicle alone (control) or GW2974. Steady-state levels of total (green) and phosphorylated (red) p185ErbB2 and p95L were determined by Western blot analysis. Although both p185ErbB2 and p95L were expressed in a phosphorylated state, GW2974 inhibited phosphorylation of p185ErbB2 but not p95L. Similar results were seen in other ErbB2+ breast cancer cell lines (data not shown).

Increased expression of p95L in lapatinib-resistant breast cancer cell lines and tumor xenografts

P95L protein levels were increased in models of acquired resistance to lapatinib (e.g., rBT474; rAu565; ref. 22) compared with their lapatinib-sensitive cell counterparts (Fig. 2A). In Fig. 2B, the growth of tumor xenografts established from resistant cells (rBT474) was significantly increased compared with tumors derived from parental cells (BT474), in animals treated with lapatinib (P < 0.05). Steady-state p95L protein levels were increased in rBT474 compared with BT474 tumor xenografts (data not shown).

To determine whether a truncated form(s) of ErbB2 similar to p95L could be detected in clinical tumors, we analyzed steady-state ErbB2 protein levels in biopsies from metastatic breast cancer sites that had developed while patients were on lapatinib therapy. A truncated form of ErbB2, similar in molecular weight to p95L in rBT474 cells, was seen in both clinical samples (Fig. 2C, MCB1 and MCB2).

Comparison of p95L with CTFs of ErbB2 generated by alternate initiation of translation

CTFs of ErbB2 generated by alternate initiation of translation (e.g., c-611, c-676, c-687) have been reported (8). c-611 lacks most of the ECD, whereas c-676 and c-687 lack the ECD and transmembrane regions. We synthesized CTFs by alternate initiation of translation from methionines 611 (c-611) and 676 (c-676) and used the pcDNA3.1 vector to express them in non–ErbB2-overexpressing MCF7 and T47D breast cancer cells. In Fig. 3A, the subcellular localization of p185ErbB2, c-611, c-676, and c-687 expressed in MCF7 transfected cells was determined by immunofluorescence microscopy using an ErbB2-specific primary and FITC-conjugated secondary antibody (green). While c-611 localized to the cell membrane and cytoplasm, c-676 was seen primarily in tumor cell nuclei (blue). In Fig. 3B, the effects of GW2974 on the phosphorylation of c-676 expressed in MCF7 transfected cells were examined by immunofluorescence microscopy using a phosphotyrosine (p-tyr) antibody and FITC-conjugated secondary antibody (green). Phosphorylation of nuclear c-676 was not inhibited by GW2974. The effect of GW2974 on steady-state phosphoprotein levels of the indicated CTFs was next determined by Western blot analysis using an ErbB2 phosphotyrosine–specific antibody in whole cell extracts from T47D cells transfected with c-611, c-676, c-687, or vector alone (Fig. 3C). GW2974 inhibited tyrosine phosphorylation of c-611 but not c-676 or c-687. Expression of p95L in BT474 cells treated with GW2974 was included as a reference. Similar results were observed in MCF7 transfected cells (data not shown).

Proteasome inhibitors block p95L induction by ErbB2 TKI

We examined the effects of protease inhibitors on p95L expression in lapatinib-treated Au565 cells. Cells were treated as indicated in Fig. 4A. Briefly, cells were treated with lapatinib alone, the indicated protease inhibitors alone, or (24) a combination of lapatinib plus protease inhibitor. Included among the protease inhibitors were BB-94, a metalloproteinase inhibitor that blocked phorbol ester–induced p95 expression (7), and a γ-secretase inhibitor that reduced ErbB4 truncation (25). The BB-94 and the γ-secretase inhibitor had little effect on the induction of p95L by lapatinib (Fig. 4A). However, inhibitors of the 20S proteasomal subunit (lactacystin, MG132, calpain I inhibitor) blocked the induction of p95L in lapatinib-treated Au565 cells (Fig. 4A). Cells treated with vehicle alone served as controls.

Treatment with lactacystin alone, at the same concentration that blocked induction of p95L, had relatively little antitumor activity in Au565 cells (Fig. 4B). However, there was enhanced antitumor activity when lactacystin was combined with a sublethal concentration of lapatinib (0.1 μmol/L) that was otherwise sufficient to induce p95L.

Expression of truncated ErbB2 reduces the antitumor activity of lapatinib

To determine the impact of nuclear, truncated forms of ErbB2 on the antitumor activity of lapatinib, we expressed c-676 in BT474 cells (Fig. 5A). We chose c-676 because of its similarities to p95L, for example, molecular weight, nuclear localization, and resistant to ErbB2 TKI. Using an ErbB2 phosphotyrosine–specific antibody in Western blot analysis, we found that lapatinib increased steady-state p95L phosphoprotein levels in cells transfected with vector alone (Fig. 5A). In contrast, the phosphorylation of c-676 and p95L was unaffected by lapatinib. Cells transfected with vector alone served as controls.

Importantly, BT474 cells, which are normally highly sensitive to the antitumor effects of lapatinib, became significantly more resistant to lapatinib after c-676 was expressed in the nuclei of BT474 cells (P = 0.015; Fig. 5B). Cells transfected with vector alone served as controls.

The development of acquired therapeutic resistance represents a significant barrier limiting the clinical efficacy of lapatinib. Acquired resistance to lapatinib does not appear to be related to loss of target sensitivity as tyrosine phosphorylation of p185ErbB2 remains inhibited in ErbB2+ breast cancer cells that have developed resistance to lapatinib (22, 26). We now show that p95L is expressed in the nuclei of ErbB2+ breast cancer cells, in a tyrosine phosphorylated, presumably activated state. In contrast to truncated forms of ErbB2 expressed at the cell surface, the phosphorylation of truncated ErbB2 in the nucleus was resistant to ErbB2 TKIs. Importantly, expression of a truncated form of ErbB2 (c-676) in the nuclei of ErbB2+ breast cancer cells rendered cells resistant to the antitumor effects of lapatinib (Fig. 5).

Distinct forms of ErbB2 differing in their (i) subcellular localization, (ii) regulation of expression, and (iii) sensitivity to ErbB2 targeted therapies exist in breast cancer cells. Expressed at the cell surface, p95 and c-611 mediate resistance to trastuzumab but not ErbB2 TKIs (11, 15). In contrast, truncated forms of ErbB2 expressed in tumor cell nuclei in a tyrosine phosphorylated state are resistant to inhibition by ErbB2 TKIs. Whereas some nuclear, truncated forms of ErbB2 are generated through alternate initiation of translation (8), p95L appears to be mediated by the activation of the proteasome, as proteasome inhibitors block its induction (Fig. 4A). As summarized in Fig. 6, cleavage at a putative proteasome recognition site located within the intracellular domain (see *) would generate a truncated form of ErbB2 with a predicted molecular weight similar to p95L. This truncated form contains tyrosine autophosphorylation sites (e.g., Y1248) and the nuclear localization signal (NLS). In the nucleus, tyrosine phosphorylation (Y1248) of p95L is resistant to lapatinib (Fig. 6B). In contrast, phosphorylation of p185ErbB2, which is also expressed in the nucleus, is inhibited by lapatinib. The role of the proteasome in the induction of p95L is consistent with recent findings from our laboratory showing evidence of proteasome activation in lapatinib-treated ErbB2+ breast cancer cells (27). It is tempting to speculate that deregulation of intracellular calcium, which occurs in lapatinib-treated ErbB2+ cells (28), leads to the accumulation of unfolded proteins, which in turn activates the proteasome.

It is not clear why the phosphorylation of truncated ErbB2 in the nucleus is resistant to ErbB2 TKI. One potential explanation is that the structural conformation of p95L and c-676 differs from p185ErbB2, preventing ErbB2 TKIs to gain access to their active sites. Studies to elucidate the structural conformation of nuclear, truncated forms of ErbB2 should help answer this question.

Although the function of nuclear, truncated forms of ErbB2 is unknown, insight from studies of p185ErbB2 may be informative. When expressed at the cell surface, p185ErbB2 promotes tumor growth and survival by activating downstream cell signaling cascades (2–6). In contrast, nuclear p185ErbB2 directly regulates gene transcription (29–31). Nuclear p185ErbB2 has been shown to activate thymidylate synthase gene transcription, which is blocked by lapatinib (29). This is consistent with our observation that phosphorylation of nuclear p185ErbB2 is inhibited by lapatinib, presumably abrogating its transcriptional activity. It is tempting to speculate that phosphorylated forms of truncated ErbB2 expressed in tumor cell nuclei are also involved in regulating gene transcription, especially in light of the proteasome-dependent regulation of p95L. Although generally associated with complete proteolysis of proteins, proteasomal processing has been shown to generate biologically active proteins, particularly those involved in regulating gene transcription (32). Studies to elucidate the function(s) of nuclear, truncated forms of ErbB2 are warranted.

Patients take lapatinib on a chronic, daily basis. Our model would have predicted that this schedule would lead to the accumulation of p95L in breast cancer cells, thereby contributing to the development of acquired resistance. Results in models of acquired lapatinib resistance confirm this prediction (Fig. 2A). In addition, lower molecular weight forms of ErbB2, similar to p95L, were expressed in clinical biopsies from ErbB2+ breast cancers that had progressed on lapatinib therapy (Fig. 2C). Although intriguing, these findings will require confirmation in larger studies.

Although the exact function of nuclear, truncated forms of ErbB2 remains unknown, we have provided evidence supporting their role in the development of therapeutic resistance to lapatinib and GW2974 (Fig. 5B). Strategies to enhance the clinical efficacy of ErbB2 TKIs may now include therapies that prevent induction of p95L and/or inactivate other truncated forms of ErbB2 that are expressed in tumor cell nuclei.

No potential conflicts of interest were disclosed.

This work was supported by Department of Defense Breast Cancer Research Program (34 W81WXH-09-0065), Sisko Foundation, and Balderacchi Gift (N.L. Spector).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Slamon
DJ
,
Clark
GM
,
Wong
SG
,
Levin
WJ
,
Ullrich
A
,
McGuire
WL
. 
Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene
.
Science
1987
;
235
:
177
82
.
2.
Dankort
D
,
Jeyabalan
N
,
Jones
N
,
Dumont
DJ
,
Muller
WJ
. 
Multiple ErbB-2/neu phosphorylation sites mediate transformation through distinct effector proteins
.
J Biol Chem
2001
;
276
:
38921
8
.
3.
Dougall
WC
,
Qian
X
,
Peterson
NC
,
Miller
MJ
,
Samanta
A
,
Greene
MI
. 
The neu- oncogene: signal transduction pathways, transformation mechanisms and evolving therapies
.
Oncogene
1994
;
9
:
2109
23
.
4.
Olayioye
MA
,
Graus-Porta
D
,
Beerli
RR
,
Rohrer
J
,
Gay
B
,
Hynes
NE
. 
ErbB-1 and ErbB-2 acquire distinct signaling properties dependent upon their dimerization partner
.
Mol Cell Biol
1998
;
18
:
5042
51
.
5.
Olayioye
MA
,
Neve
RM
,
Lane
HA
,
Hynes
NE
. 
The ErbB signaling network: receptor heterodimerization in development and cancer
.
EMBO J
2000
;
19
:
3159
67
.
6.
Yarden
Y
,
Sliwkowski
MX
. 
Untangling the ErbB signalling network
.
Nat Rev Mol Cell Biol
2001
;
2
:
127
37
.
7.
Codony-Servat
J
,
Albanell
J
,
Lopez-Talavera
JC
,
Arribas
J
,
Baselga
J
. 
Cleavage of the HER2 ectodomain is a pervanadate-activable process that is inhibited by the tissue inhibitor of metalloproteases-1 in breast cancer cells
.
Cancer Res
1999
;
59
:
1196
201
.
8.
Anido
J
,
Scaltriti
M
,
Bech Serra
JJ
,
Santiago Josefat
B
,
Todo
FR
,
Baselga
J
, et al
Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation
.
EMBO J
2006
;
25
:
3234
44
.
9.
Lin
YZ
,
Clinton
GM
. 
A soluble protein related to the HER-2 proto-oncogene product is released from human breast carcinoma cells
.
Oncogene
1991
;
6
:
639
43
.
10.
Zabrecky
JR
,
Lam
T
,
McKenzie
SJ
,
Carney
W
. 
The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3
.
J Biol Chem
1991
;
266
:
1716
20
.
11.
Xia
W
,
Liu
LH
,
Ho
P
,
Spector
NL
. 
Truncated ErbB2 receptor (p95ErbB2) is regulated by heregulin through heterodimer formation with ErbB3 yet remains sensitive to the dual EGFR/ErbB2 kinase inhibitor GW572016
.
Oncogene
2004
;
23
:
646
53
.
12.
Chandarlapaty
S
,
Scaltriti
M
,
Angelini
P
,
Ye
Q
,
Guzman
M
,
Hudis
CA
, et al
Inhibitors of HSP90 block p95-HER2 signaling in Trastuzumab-resistant tumors and suppress their growth
.
Oncogene
2010
;
29
:
325
34
.
13.
Saez
R
,
Molina
MA
,
Ramsey
EE
,
Rojo
F
,
Keenan
EJ
,
Albanell
J
, et al
p95HER-2 predicts worse outcome in patients with HER-2-positive breast cancer
.
Clin Cancer Res
2006
;
12
:
424
31
.
14.
Christianson
TA
,
Doherty
JK
,
Lin
YJ
,
Ramsey
EE
,
Holmes
R
,
Keenan
EJ
, et al
NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer
.
Cancer Res
1998
;
58
:
5123
9
.
15.
Scaltriti
M
,
Rojo
F
,
Ocana
A
,
Anido
J
,
Guzman
M
,
Cortes
J
, et al
Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer
.
J Natl Cancer Inst
2007
;
99
:
628
38
.
16.
Pedersen
K
,
Angelini
PD
,
Laos
S
,
Bach-Faig
A
,
Cunningham
MP
,
Ferrer-Ramon
C
, et al
A naturally occurring HER2 carboxy-terminal fragment promotes mammary tumor growth and metastasis
.
Mol Cell Biol
2009
;
29
:
3319
31
.
17.
Xia
W
,
Mullin
RJ
,
Keith
BR
,
Liu
LH
,
Ma
H
,
Rusnak
DW
, et al
Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways
.
Oncogene
2002
;
21
:
6255
63
.
18.
Rusnak
DW
,
Lackey
K
,
Affleck
K
,
Wood
ER
,
Alligood
KJ
,
Rhodes
N
, et al
The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo
.
Mol Cancer Ther
2001
;
1
:
85
94
.
19.
Konecny
GE
,
Pegram
MD
,
Venkatesan
N
,
Finn
R
,
Yang
G
,
Rahmeh
M
, et al
Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2- overexpressing and trastuzumab-treated breast cancer cells
.
Cancer Res
2006
;
66
:
1630
9
.
20.
Geyer
CE
,
Forster
J
,
Lindquist
D
,
Chan
S
,
Romieu
CG
,
Pienkowski
T
, et al
Lapatinib plus capecitabine for HER2-positive advanced breast cancer
.
N Engl J Med
2006
;
355
:
2733
43
.
21.
Johnston
S
,
Trudeau
M
,
Kaufman
B
,
Boussen
H
,
Blackwell
K
,
LoRusso
P
, et al
Phase II study of predictive biomarker profiles for response targeting human epidermal growth factor receptor 2 (HER-2) in advanced inflammatory breast cancer with lapatinib monotherapy
.
J Clin Oncol
2008
;
26
:
1066
72
.
22.
Xia
W
,
Bacus
S
,
Hegde
P
,
Husain
I
,
Strum
J
,
Liu
L
, et al
A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer
.
Proc Natl Acad Sci U S A
2006
;
103
:
7795
800
.
23.
Xia
W
,
Bacus
S
,
Husain
I
,
Liu
L
,
Zhao
S
,
Liu
Z
, et al
Resistance to ErbB2 tyrosine kinase inhibitors in breast cancer is mediated by calcium-dependent activation of RelA
.
Mol Cancer Ther
2010
;
9
:
292
9
.
24.
Ni
CY
,
Murphy
MP
,
Golde
TE
,
Carpenter
G
. 
gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase
.
Science
2001
;
294
:
2179
81
.
25.
Spector
NL
,
Xia
W
,
Burris
H
 III
,
Hurwitz
H
,
Dees
EC
,
Dowlati
A
, et al
Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies
.
J Clin Oncol
2005
;
23
:
2502
12
.
26.
Xia
W
,
Bisi
J
,
Strum
J
,
Liu
L
,
Carrick
K
,
Graham
KM
, et al
Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers
.
Cancer Res
2006
;
66
:
1640
7
.
27.
Spector
NL
,
Yarden
Y
,
Smith
B
,
Lyass
L
,
Trusk
P
,
Pry
K
, et al
Activation of AMP- activated protein kinase by human EGF receptor 2/EGF receptor tyrosine kinase inhibitor protects cardiac cells
.
Proc Natl Acad Sci U S A
2007
;
104
:
10607
12
.
28.
Kim
HP
,
Yoon
YK
,
Kim
JW
,
Han
SW
,
Hur
HS
,
Park
J
, et al
Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, downregulates thymidylate synthase by inhibiting the nuclear translocation of EGFR and HER2
.
PLoS One
2009
;
4
:
e5933
.
29.
Wang
SC
,
Lien
HC
,
Xia
W
,
Chen
IF
,
Lo
HW
,
Wang
Z
, et al
Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2
.
Cancer Cell
2004
;
6
:
251
61
.
30.
Xie
Y
,
Hung
MC
. 
Nuclear localization of p185neu tyrosine kinase and its association with transcriptional transactivation
.
Biochem Biophys Res Commun
1994
;
203
:
1589
98
.
31.
Hervas-Aguilar
A
,
Rodriguez
JM
,
Tilburn
J
,
Arst
HN
 Jr.
,
Penalva
MA
. 
Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC
.
J Biol Chem
2007
;
282
:
34735
47
.
32.
Tian
L
,
Holmgren
RA
,
Matouschek
A
. 
A conserved processing mechanism regulates the activity of transcription factors Cubitus interruptus and NF-kappaB
.
Nat Struct Mol Biol
2005
;
12
:
1045
53
.