PRECLINICAL STUDY

Transactivation of ErbB-2 induced by tumor necrosis factora promotes NF-jB activation and breast cancer cell proliferation

Martın A. Rivas Æ Mercedes Tkach Æ Wendy Beguelin ÆCecilia J. Proietti Æ Cinthia Rosemblit Æ Eduardo H. Charreau ÆPatricia V. Elizalde Æ Roxana Schillaci

Received: 3 June 2009 / Accepted: 3 September 2009

� Springer Science+Business Media, LLC. 2009

Abstract Tumor necrosis factor alpha (TNFa) is a

pleiotropic cytokine which, acting locally, induces tumor

growth. Accumulating evidence, including our findings,

showed that TNFa is mitogenic in breast cancer cells in

vitro and in vivo. In the present study, we explored TNFainvolvement on highly aggressive ErbB-2-overexpressing

breast cancer cells. We found that TNFa induces ErbB-2

phosphorylation in mouse breast cancer C4HD cells and in

the human breast cancer cell lines SK-BR-3 and BT-474.

ErbB-2 phosphorylation at Tyr877 residue was mediated by

TNFa-induced c-Src activation. Moreover, TNFa promoted

ErbB-2/ErbB-3 heterocomplex formation, Akt activation

and NF-jB transcriptional activation. Inhibition of ErbB-2

by addition of AG825, an epidermal growth factor receptor/

ErbB-2-tyrosine kinase inhibitor, or knockdown of ErbB-2

by RNA interference strategy, blocked TNFa-induced NF-

jB activation and proliferation. However, the humanized

monoclonal antibody anti-ErbB-2 Herceptin could not

inhibit TNFa ability to promote breast cancer growth.

Interestingly, our work disclosed that TNFa is able to

transactivate ErbB-2 and use it as an obligatory downstream

signaling molecule in the generation of mitogenic signals.

As TNFa has been shown to be present in the tumor

microenvironment of a significant proportion of human

infiltrating breast cancers, our findings would have clinical

implication in ErbB-2-positive breast cancer treatment.

Keywords ErbB-2 � TNFa � Herceptin � c-Src

Introduction

Tumor necrosis factor alpha (TNFa) is a pleiotropic cyto-

kine originally characterized to cause hemorrhagic necrosis

of tumors at high doses [1]. However, it is now widely

accepted that TNFa, acting locally, induces the growth of

certain tumor types such as ovary and breast [2–5]. In

particular, TNFa has been shown to be produced by

malignant or host cells in the tumor microenvironment of

human infiltrating breast cancer and to be associated with

increasing malignancy [6]. Treatment of cancer cells with

TNFa enhances cell proliferation in vitro [7, 8] and, as we

have recently demonstrated, also supports murine breast

cancer growth in vivo [5]. In addition, we have shown that

TNFa induces in vitro proliferation of breast cancer cells

through a mechanism which requires activation of p42/p44

mitogen-activated protein kinase (MAPK), c-jun NH2-ter-

minal kinase (JNK), Akt and NF-jB transcriptional acti-

vation [5]. However, the exact mechanism by which TNFaenhances tumor growth and its involvement in different

breast cancer subtypes remains elusive.

ErbB-2, a transmembrane tyrosine kinase receptor, is

overexpressed in nearly 30% of human breast cancer and

has been associated with enhanced tumor aggressiveness

and poor clinical outcome [9]. These tumors also display

NF-jB activation [10]. ErbB-2 is an orphan receptor

belonging to the family of type I tyrosine kinase receptors,

which signals by forming heterodimers with epidermal

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10549-009-0546-3) contains supplementarymaterial, which is available to authorized users.

M. A. Rivas � M. Tkach � W. Beguelin �C. J. Proietti � C. Rosemblit � E. H. Charreau �P. V. Elizalde � R. Schillaci (&)

Laboratory of Molecular Mechanisms of Carcinogenesis,

Instituto de Biologıa y Medicina Experimental (IBYME),

CONICET, Vuelta de Obligado 2490,

Buenos Aires C1428ADN, Argentina

e-mail: rschilla@dna.uba.ar

123

Breast Cancer Res Treat

DOI 10.1007/s10549-009-0546-3

growth factor receptor (EGFR), ErbB-3 and ErbB-4 [11] in

response to ligands including heregulins [12]. After ligand

binding, all ErbB receptors are phosphorylated, serving as

docking sites for the recruitment of cytoplasmic adaptor

proteins, initiating signaling cascades that control multiple

cellular processes. HerceptinTM (Trastuzumab) is a mono-

clonal antibody which binds to the extracellular domain of

the receptor [13] and is administrated to breast cancer

patients whose tumors overexpress ErbB-2. However, the

clinical efficacy of Herceptin is limited to 30% of these

patients. An important reason for this is that other tumor-

cell alterations may influence the response to ErbB-2-tar-

geted inhibitors [14]. Thus, understanding the mechanisms

by which ErbB-2 can be activated through non-classical

receptors and ligands is relevant in order to design a new

therapeutic approach and to predict patients’ response.

Ligands of G protein-coupled receptors which act through

c-Src kinase activation [15], as well as hormones such as

prolactin, acting through Janus kinase 2 activation [16],

and cytokines such as interleukin-6 [17] have all been

demonstrated to transactivate ErbB-2. Several groups have

so far shown transactivation of EGFR by TNFa through the

activation of matrix metalloproteinases (MMPs) which are

able to release EGFR ligands from the cell membrane [18–

20]. In contrast, there are no reports demonstrating TNFaability to transactivate ErbB-2 in breast cancer.

In the present work, we explore the effect of TNFa on

breast cancer cells that overexpress ErbB-2 and found that

TNFa induces not only ErbB-2 autophosphorylation, but

also phosphorylation of its Tyr877 residue through the

activation of the tyrosine kinase c-Src. We further found

that it promotes association between ErbB-2 and ErbB-3.

ErbB-2 transactivation by TNFa is a rapid event that does

not involve either ligand release or MMPs activation, and

leads to cell proliferation even in the presence of Hercep-

tin. Interestingly, we showed that TNFa regulates ErbB-2

phosphorylation as a requisite to activate NF-jB and cell

proliferation in human and murine breast cancer cells. This

is the first demonstration of ErbB-2 transactivation by

TNFa in breast cancer cells, which may be one of the

mechanisms by which ErbB-2-overexpressing tumors show

resistance to anti-ErbB-2 monoclonal antibodies therapy.

Materials and methods

Animals and tumors

Experiments were carried out in virgin female Balb/c mice,

raised at the Instituto de Biologıa y Medicina Experimental

of Buenos Aires. All animal studies were conducted as

described [5]. C4HD mouse mammary tumor expresses

progesterone and estrogen receptors, lacks EGFR

expression, overexpresses ErbB-2 and exhibits high levels

of ErbB-3 and low expression of ErbB-4 [21–23].

Antibodies

Antibodies to the following proteins were used: Neu/ErbB-

2 (C-18), ErbB-3 (C-17), phosphotyrosine (PY99), p85

phosphatidyl inositol 3-kinase (PI3-K), p42/p44 MAPK (C-

14), phospho-p42/p44 MAPK (E-4), JNK (N-18) and

phospho JNK (G-7) all from Santa Cruz Biotechnology

(Santa Cruz, CA, USA), Akt, phospho Akt (Ser 473),

phospho IjBa (Ser32/36), IjBa, phosphotyrosine c-Src

(Tyr416), c-Src (36D10), phospho-ErbB-2 (Tyr1221/

1222), phospho-ErbB-2 (Tyr877) and EGFR from Cell

Signaling (Beverly, MA, USA), v-Src (ab-1) from Cal-

biochem (La Jolla, CA, USA) and actin (Clone ACTN05)

and cyclin D1 from Neomarkers (Fremont, CA, USA).

Cell culture and treatments

Primary cultures of epithelial cells from the mouse mam-

mary tumor C4HD, growing in medroxyprogesterone ace-

tate (MPA)-treated mice, were performed as previously

described [5, 24]. As C4HD cells are sensitive to progestin,

all the experiments were performed in Dulbecco’s Modified

Eagle’s Medium/F12 without phenol red (Sigma, St. Louis,

MO, USA) (DMEM) ? 0.1% charcoal-stripped fetal calf

serum (ChFCS). Human breast cancer cell lines SK-BR-3

and BT-474 were obtained from the American Type Culture

Collection and maintained in Mc Coy’s 5A ? 10% FCS

(Gen SA, Buenos Aires, Argentina) and RPMI 1640 ? 10%

FCS, respectively. Experiments were performed in their

respective medium ? 1% FCS. Cells were treated for

the indicated times with 20 ng/ml of murine or human TNFa(mTNFa, hTNFa, respectively) (Cell Sciences, Canton,

MA, USA) or with 20 ng/ml of recombinant human b1

heregulin (HRG, Upstate, Millipore, Bedford, MA, USA).

The following inhibitors were added to cells 60 min before

incubation with TNFa: AG825, EGFR/ErbB-2 tyrosine

kinase inhibitor from the benzylidene malononitrile family;

PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,

4-d]pyrimidine, c-Src inhibitor; GF109203X, PKC inhibitor;

GM6001, a broad MMPs inhibitor, and its corresponding

negative control (all from Calbiochem); Dasatinib, c-Src

inhibitor (LC Laboratories, Woburn, MA, USA); GW2974,

EGFR/ErbB-2 tyrosine kinase inhibitor from the indazol-

yamino quinazoline family and Bay 11-7082, inhibitor of

IjB phosphorylation (both from Sigma). To perform certain

experiments, cells were pre-incubated for 16 h with 10 lg/ml

of the anti-ErbB-2 antibody HerceptinTM (Hoffmann-La

Roche Ltd, Basel, Switzerland) for Western blot analysis, and

1 h for proliferation and reporter assays. Cell proliferation was

evaluated by [3H]-thymidine incorporation assay. Cells were

Breast Cancer Res Treat

123

incubated with TNFa for 48 h. 1 lCi [3H]-thymidine (NEN,

Dupont, Boston, MA, USA; specific activity 20 Ci/mmol)

was added at hour 24 of said culture. Cells were then tryp-

sinized and harvested and radioactivity was counted using

standard scintillation procedures [5]. Assays were performed

in octuplicate. In earlier experiments we demonstrated that

[3H]-thymidine uptake correlated with the number of cells/

well [5]. Cell viability was performed by triplicate by Trypan

blue exclusion at 48 h of treatment with the corresponding

inhibitors. For C4HD cells, cultured with 100 lM AG825, it

was of 67.6 ± 4.7%, for BT-474 cells cultured with 100 lM

AG825, 10 lg/ml Herceptin, 1 lM GW2974, 0.5 lM Da-

satinib and 1 lM Bay 11-7082, it was of 62.3 ± 7.2,

70.9 ± 7.6, 73.7 ± 10.3, 77.0 ± 1.3 and 73 ± 8.3%,

respectively, and for SK-BR-3 cells cultured with 100 lM

AG825 and 10 lg/ml Herceptin it was of 55.8 ± 9.3 and

65.6 ± 1.3%, respectively.

Immunofluorescence staining and confocal microscopy

SK-BR-3, BT-474 and C4HD cells grown on glass cover-

slips were treated with TNFa 20 ng/ml for 30 min. Cells

were fixed and permeabilized in ice-cold methanol and

ErbB-2 was localized using ErbB-2 9G-6 (Santa Cruz

Biotechnology) followed by incubation with a rhodamine

conjugated anti-mouse secondary antibody (The Jackson

Laboratory, Bar Harbor, ME, USA). Stained cells were

analyzed using a Nikon C1 confocal laser scanning

microscope.

Flow cytometry analysis

SK-BR-3 and BT-474 cells treated with hTNFa for the

indicated times were harvested with PBS ? EDTA 1% and

incubated with anti-ErbB-2 9G-6 antibody followed by

incubation with anti-mouse phycoerythrin (PE)-conjugated

antibody (Santa Cruz Biotechnology). A total of 104 cells/

sample was analyzed using FACSAria cytometer (Becton–

Dickinson, La Jolla, CA, USA). Background staining was

evaluated in cells incubated with an isotype control IgG

followed by anti-mouse PE-conjugated antibody. Data

analysis was performed using WinMDI software (J. Trot-

ter, Scripps Research Institute, San Diego, CA, USA).

Delta mean fluorescence intensity (MFI) values were

obtained by subtracting the MFI of the cells incubated with

control isotype antibody from the MFI of cells incubated

with ErbB-2 antibody.

For cell cycle analysis, BT-474 cells were subjected to

the different treatments, harvested at 36 and 48 h, and fixed

in 70% ethanol for 24 h at 4�C, as we previously described

[5]. They were washed twice with PBS, followed by RNA

digestion (RNAse A 50 U/ml) and propidium iodide

(20 lg/ml) staining for 30 min at room temperature in the

dark. Cell cycle analysis was performed using a FAC-

Scalibur flow cytometer (Becton–Dickinson) and Modfit

LT software.

Western blot and immunoprecipitation

Lysates were prepared from cells subjected to the different

treatments described in each experiment, as previously

detailed, and proteins were subjected to SDS-PAGE [5].

Association among ErbB-2, ErbB-3 and p85 PI3-K was

studied by performing co-immunoprecipitation experi-

ments as previously described [24]. Briefly, 500 lg of

protein lysates was incubated with 2 lg of rabbit anti-

EGFR, ErbB-2 or ErbB-3 antibody and the immunocom-

plexes were captured by adding protein A-agarose (Santa

Cruz Biotechnology). Beads were washed, boiled in sample

buffer, and proteins were electroblotted as described above.

As negative control, normal rabbit serum was used.

In vitro cold phosphorylation assay

C4HD cells were treated with TNFa for 5 min or prein-

cubated before TNFa stimulation for 60 min with PP2

(10 lM). Cells were lysed in kinase lysis buffer (20 mM

HEPES pH 7.5, 10 mM EGTA, 1% NP-40, 2.5 mM

MgCl2) and Src was immunoprecipitated from 500 lg

protein extracts using an anti-v-Src antibody. ErbB-2 was

immunoprecipitated from 500 lg protein extract from

unstimulated C4HD cells. The immunoprecipitated ErbB-2

was then subjected to an in vitro phosphorylation assay

with Src immunoprecipitated from each treatment as pre-

viously described [25]. Proteins were separated by elec-

trophoresis; gels were transferred onto nitrocellulose and

immunoblotted with anti-Tyr877/927 ErbB-2 and anti-total

c-Src antibodies and ErbB-2.

Transient transfections

jB-Luc vector (jB sites from HIV promoter) and cyclin

D1 promoter-Luc vector were kindly provided by Dr M.

Bell (Mayo Clinic, Rochester, MN, USA) and Dr R. Pestell

(Northwestern University Medical School, Chicago, IL,

USA), respectively. Human ErbB-2 wild type, kinase-

negative (KN) ErbB-2 and ErbB-2-Y877F were kindly

provided by Dr T. Akiyama (Gumma University, Gumma,

Japan), Dr A. Gertler (Protein Laboratories Rehovot,

Israel) and Dr O. Segatto (Centro Recerca Sperimentale,

Rome, Italy). C4HD cells were transfected for 24 h in

DMEM supplemented with 10 nM MPA and 2.5% ChFCS,

and cell lines in the corresponding growth medium without

antibiotics. FuGENE 6 transfection reagent technique

(Roche Biochemicals, Indianapolis, IN, USA) was used in

accordance with the manufacturer’s instructions. Cells

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123

were transiently co-transfected with 1 lg of jB-Luc or

1 lg cyclin D1-luc construct plus 10 ng Renilla luciferase

expression vector CMV-pRL (Promega, Madison, WI,

USA) used to correct variations in transfection efficiency.

As control, cells were transfected with a pGL3-basic

reporter lacking jB. Transfected cells were lysed and

luciferase assays carried out using the Dual-Luciferase

Reporter Assay System (Promega).

siRNAs targeting mouse ErbB-2 mRNAs were synthe-

sized by Dharmacon Inc. (Lafayette, CO, USA) (ErbB-2

siRNA#02: antisense, 50-GAUGUCCUCCGUAAGAAUA-

30, ErbB-2 siRNA#03 antisense 50-GAUGGUGCUUACU

CAUUGA-30, ErbB-2 siRNA#04 antisense 50-GGAAUC

CUAAUCAAACGAA-30). The non-silencing siRNA oli-

gonucleotide from Dharmacon, which does not target any

known mammalian gene, was used as negative control.

Transfection of siRNA plus 1 lg of empty vector

(pcDNA3.1) or 1 lg of human ErbB-2 expression vector

and 1 lg of jB-Luc vector plus 10 ng Renilla luciferase

expression vector was performed using the DharmaFECT

Duo transfection reagent following the manufacturer’s

directions, using 25 nM of siRNA for 3 days. Cell treatment

and reporter activity were measured as described above. In

some experiments, siRNA at a final concentration of 25 nM

was transfected with Dharmafect 1 reagent following the

manufacturer’s directions.

Statistical analysis

The differences between control and experimental groups

were analyzed by ANOVA followed by Tukey t test among

groups. Kolmorgorov–Smirnov test was used for flow

cytometric studies.

Results

TNFa-induced breast cancer cell proliferation

requires ErbB-2 phosphorylation

We have already shown that TNFa induces proliferation of

the murine mammary adenocarcinoma C4HD, both in vitro

and in vivo [5]. As C4HD cells overexpress ErbB-2 and

since ErbB-2 is a key player in C4HD cell proliferation [21,

22, 24], we investigated the potential role of ErbB-2 sig-

naling in the growth stimulatory effects of TNFa. For that

purpose, cell proliferation assays were conducted in the

presence of the selective dual EGFR/ErbB-2 tyrosine kinase

inhibitor tyrphostin AG825. Previous studies have shown

that C4HD cells do not express EGFR [21, 23, 24], therefore

the results obtained with AG825 will be attributable entirely

to ErbB-2 blockage. Interestingly, proliferation of C4HD

cells induced by 20 ng/ml TNFa was inhibited by addition

of 100 lM AG825 (Fig. 1a). As expected, AG825 com-

pletely suppressed HRG-induced growth (Fig. 1a) and

ErbB-2 phosphorylation at Tyr1272, one of the main

C-terminal tail autophosphorylation sites (Supplemental

Fig. 1). In a previous work, we demonstrated that TNFainduced p42/p44 MAPK and JNK activation in breast

cancer cells [5]. We now wanted to explore the involvement

of ErbB-2 on the activation of these signaling pathways.

Addition of AG825 did not affect TNFa-induced p42/p44

MAPK nor JNK phosphorylation in C4HD cells (Supple-

mental Fig. 2). However, addition of AG825 completely

blocked HRG-induced p42/p44 MAPK activation (Sup-

plemental Fig. 2). These contrasting results suggest that

p42/p44MAPK activation has a different up-stream sig-

naling pathway that is dependent on ErbB-2 phosphoryla-

tion when cells are stimulated with HRG, and is ErbB-2

phosphorylation-independent when the mitogen used is

TNFa.

Fig. 1 TNFa requires ErbB-2 phosphorylation for inducing breast

cancer cell proliferation. a, e Cells were preincubated with 100 lM

AG825 for 60 min and then treated or not with 20 ng/ml of TNFa or

20 ng/ml heregulin (HRG) for 48 h. Proliferation was performed by

[3H]-thymidine incorporation assay. Data are presented as

mean ± SE of octuplicate samples (*P \ 0.05, **P \ 0.001 vs.

control). The experiments shown are representative of a total of four.

Controls were performed in order to verify that dimethyl sulfoxide

(DMSO) (1:2,000) did not modify TNFa-induced proliferation.

b C4HD cells were treated with TNFa for the times shown and

subjected to ErbB-2 immunoprecipitation. Immunoprecipitates were

blotted with anti-phosphotyrosine antibody (top panel) and mem-

branes were stripped and blotted with anti-ErbB-2 antibody (bottompanel). IP immunoprecipitation, NRS normal rabbit serum. Bands

were quantified using Image J with untreated cell samples (first lines)

set as 1.0. This is a representative experiment out of a total of three.

c C4HD cells were treated with TNFa for the times shown. Whole cell

lysates were subjected to Western blot analysis for ErbB-2 phos-

phorylation at Tyr1222/1272 residue. ErbB-2 is shown as loading

control and was used for quantification as described in (b). d, f C4HD

cells were preincubated with 50, 80 or 100 lM AG825 for 60 min

and SK-BR-3 and BT-474 cells with 100 lM AG825 and then treated

with TNFa for the times shown. ErbB-2 phosphorylation at Tyr1222/

1272 residue was performed as described in (c). This is a represen-

tative experiment out of a total of three. g ErbB-2 expression by flow

cytometry in cells treated with TNFa. ErbB-2 expression was

revealed by incubation with anti-ErbB-2 antibody followed by a

secondary phycoerythrin (PE)-conjugated antibody (ErbB-2 PE).

Representative histograms of ErbB-2 expression in SK-BR-3 and BT-

474 cells treated or not with TNFa for 30 min are shown. The delta

mean fluorescence intensity of control and TNFa-treated SK-BR3

cells was of 4.4 and 2.9, respectively (P \ 0.01) and of control and

TNFa-treated BT-474 cells it was of 22.2 and 10.0, respectively

(P \ 0.001). Kolmorgorov–Smirnov statistical test was used. hLocalization of ErbB-2 by immunofluorescence and confocal micros-

copy in TNFa-treated cells. Each image is representative of at least 10

(scale bars 10 lm). Control experiments demonstrated no detectable

staining with secondary antibody incubation only or with anti-ErbB-2

antibody preincubated with the specific blocking peptide. Nuclei were

stained with 40,6-diamidino-2-phenylindole (DAPI). hTNFa human

TNFa, mTNFa murine TNFa

c

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We examined ErbB-2 phosphorylation levels after TNFatreatment. Figure 1b shows that TNFa augmented total

levels of ErbB-2 tyrosine phosphorylation from 2 to 15 min

of treatment and decline thereafter. Then, we addressed the

specific mouse ErbB-2 tyrosine phosphorylation at Tyr1272,

analogous to human Tyr1222 ErbB-2. There was an increase

in Tyr1272 phosphorylation 2 min after TNFa treatment in

C4HD cells, with maximum phosphorylation at 5 min

(Fig. 1c). Addition of AG825 blocked TNFa-induced

Tyr1272 phosphorylation in a concentration-dependent

manner (Fig. 1d). Since our goal was to determine whether

TNFa-induced ErbB-2 transactivation was taking place also

in human breast cancer, we then used SK-BR-3 [26] and BT-

474 cell lines [27] that are widely used models of ErbB-2-

overexpression. While TNFa induced SK-BR-3 and BT-474

cell proliferation, addition of AG825 blocked TNFa mito-

genic effect (Fig. 1e). Similar effects were obtained with

HRG-treated cells. In addition, TNFa induced phosphory-

lation of the Tyr1222 residue after 2 min stimulation, with a

peak at 5 min (Fig. 1f) which was inhibited by addition of

A

B

0 2 105 15 6030 mTNFα, min

pTyr

ErbB-2

C4HD

IP: ErbB2 NRS

5

pTyr-1272-ErbB-2

ErbB-2

-

- -

+ + + + mTNFαAG825

-- -

C4HD

pTyr-1272-ErbB-2

ErbB-2

C0 2 105 15 6030 mTNFα, min

C4HD

F

0

10

20

- + - - + -mTNFα- - -HRG + - +

AG825 - - ++ +-

[3 H]-

Th

ymid

ine

inco

rpo

rati

on

(C

PM

x 1

03)

*

**C4HD

[3 H]-

Th

ymid

ine

inco

rpo

rati

on

(C

PM

x 1

03)

0

125

250

- + - - + -hTNFα- - -HRG + - +

AG825 - - ++ +-

**

*

SK-BR-3

0

15

30

45

- + - - + -

- - -+ - +

- - ++ +-

* *

BT-474E

ErbB2 DAPI

hTN

Fα

30 m

inC

ontr

ol

SK-BR-3

pTyr-1222-ErbB-2

ErbB-2

SK-BR-3

+ +------

0 2 105 15 30 0 10

AG825

hTNFα, min

BT-474

+ +------

0 2 105 15 30 0 10

AG825

hTNFα, min

pTyr-1222-ErbB-2

ErbB-2

G

BT-474

ErbB2 DAPIErbB2 DAPI

C4HDH

mT

NF

α 30

min

Con

trol

D

1 1.6 1.51.6 1.5 0.90.8

1 1.5 1.71.7 1.6 0.41.8 0.4 1 1.6 2.62.4 2.6 0.62.0 0.6

1 3.3 1.53.7 1.0 0.91.0

Isotype control

Control

hTNFα 30 min

64

0100 101 102

ErbB-2-PE

SK-BR-3 64

100 101 102

ErbB-2-PE

0

BT-474

3232

Breast Cancer Res Treat

123

AG825. Comprehensively, our present findings indicate that

TNFa is able to transactivate ErbB-2 in breast cancer cells.

To examine the effect of TNFa stimulation on ErbB-2

expression on the surface of SK-BR-3 and BT-474 cells,

we performed immunofluorescence and flow cytometry

analysis. ErbB-2 plasma membrane expression was

observed to decrease, reaching its minimum at 30 min

(Fig. 1g) and staying low for at least 1 h of TNFa treatment

in both cell lines. To confirm these data, we performed

confocal microscopy studies. ErbB-2 was localized pri-

marily to the plasma membrane in unstimulated cells.

TNFa treatment for 30 min led to a significant increase in

ErbB-2 localization in the cytoplasm of C4HD, SK-BR-3

and BT-474 cells (Fig. 1h) without affecting ErbB-2 con-

tent in whole cell extracts. Comprehensively, these results

show for the first time that TNFa is able to activate ErbB-2

inducing its phosphorylation and internalization into the

cytoplasm of breast cancer cells.

The finding that TNFa is able to induce ErbB-2 trans-

activation, prompted us to explore the mechanism under-

lying this phosphorylation. It is known that TNFa can

induce EGFR transactivation through MMP-dependent

EGFR ligand release. Treatment of C4HD and SK-BR-3

cells with 10 lM GM6001, a broad MMPs inhibitor, did

not modify TNFa-induced ErbB-2 phosphorylation (Sup-

plemental Fig. 3a). In addition, the use of blocking anti-

bodies to ErbB-3 and ErbB-4 to impede ligand binding did

not modify TNFa-induced cell growth (Supplemental

Fig. 3b) although both antibodies were able to inhibit HRG

proliferative effect in C4HD cells [22]. Thus, these data

suggest that TNFa does not activate ErbB-2 through ligand

release in these breast cancer cells.

TNFa-induced c-Src kinase activation mediates

ErbB-2 phosphorylation

It has been shown recently that ErbB-2 phosphorylation at

the residue Tyr877 (Tyr927 in mice), located in the acti-

vation loop of the kinase domain, is important for its

intrinsic kinase activity [28]. Interestingly, we found that

TNFa induced an increase on Tyr877/927 ErbB-2 phos-

phorylation in C4HD, SK-BR-3 and BT-474 cells (Fig. 2a).

Since tyrosine kinase c-Src directly phosphorylates ErbB-2

at Tyr877 residue [29], we hypothesized that c-Src could be

involved in this event. We observed that TNFa induced

c-Src phosphorylation at Tyr416 in C4HD cells, with a

clear activation at 2–5 min of treatment (Fig. 2b). Addition

of PP2 or Dasatinib, a clinically used c-Src inhibitor, dra-

matically decreased TNFa-induced ErbB-2 phosphoryla-

tion at Tyr877 in SK-BR-3 and BT-474 (Fig. 2b).

To further explore whether activated c-Src can phos-

phorylate Tyr877 ErbB-2 in vitro, we performed a cold

phosphorylation assay. For this purpose, we immunopre-

cipitated c-Src from C4HD cells treated or not with TNFa for

5 min, and from C4HD cells treated with TNFa and PP2. We

also immunoprecipitated ErbB-2 from unstimulated cells

and used it as a source of unphosphorylated ErbB-2 in the

assay. As shown in Fig. 2c, c-Src activated by TNFa was

able to phosphorylate ErbB-2 at Tyr927 residue. Neither

c-Src obtained from control cells nor c-Src inactivated by

PP2 increased ErbB-2 phosphorylation (Fig. 2c). Moreover,

it is known that after TNFa binding to its receptors, protein

kinase C (PKC) is involved in c-Src phosphorylation [30].

Addition of 10 lM GF109203X, a PKC inhibitor, effectively

abolished TNFa ability to induce c-Src phosphorylation and

subsequently ErbB-2 phosphorylation at Tyr927 in C4HD

cells (Fig. 2d). Taken together, these results strongly suggest

that TNFa induces c-Src activation which phosphorylates

ErbB-2 at Tyr877/927 residue.

TNFa induces ErbB-2/ErbB-3 heterodimerization

and PI3-K/Akt pathway activation

In the last years, it has been established that ErbB-2 and

ErbB-3 function as an oncogenic unit responsible for

driving breast tumor-cell proliferation by activating PI3-K/

Akt pathway [31]. To gain further insight into the molec-

ular mechanisms triggered by TNFa-induced ErbB-2

transactivation, we monitored ErbB-3 and EGFR phos-

phorylation and ErbB-2 heterodimerization with ErbB-3.

TNFa caused ErbB-3 phosphorylation in C4HD and

SK-BR-3 cells but did not substantially affect EGFR

tyrosine phosphorylation in SK-BR-3 cells (Fig. 3a).

Silencing ErbB-2 expression with 25 nM ErbB-2 siRNAs

(Supplemental Fig. 4) completely inhibited TNFa-induced

ErbB-3 phosphorylation in C4HD cells (Fig. 3a). By co-

immunoprecipitation experiments in C4HD cells, we

showed that TNFa induced ErbB-2 and ErbB-3 heterodi-

merization, which was reduced by addition of PP2

(Fig. 3b). The reverse experiment, in which we immuno-

precipitated ErbB-3 and sought for ErbB-2 presence, is

shown in Fig. 3b with similar results. HRG-induced ErbB-

3/ErbB-2 association was included as control. Among the

members of the ErbB family, ErbB-3 has the unique ability

of binding p85 subunit of PI3-K. TNFa also induced a

rapid p85 PI3-K association with ErbB-3 similar to that

observed upon HRG treatment (Fig. 3b). The activation of

the downstream kinase of PI3-K, Akt, by TNFa was sig-

nificantly inhibited by AG825 in SK-BR-3 and C4HD cells

(Fig. 3c). Blockage of ErbB-2 expression by siRNA also

leads to inhibition of TNFa-induced Akt activation in

C4HD cells (Fig. 3c). Notably, in the absence of c-Src

activation by preincubation with PP2, TNFa capacity to

activate Akt was blocked (Fig. 3c).

Breast Cancer Res Treat

123

ErbB-2 tyrosine kinase inhibitor but not Herceptin

blocks TNFa-induced NF-jB transcriptional activation,

cyclin D1 expression and cell proliferation

Recently we have demonstrated that NF-jB activation is a

key factor in TNFa-induced C4HD cell proliferation [5].

Using the IjB pharmacological inhibitor Bay 11-7082, NF-

jB activation is also shown to be a requisite in TNFa-

induced BT-474 cell proliferation (Supplemental Fig. 5).

Therefore, we examined whether ErbB-2 participates in

NF-jB activation by TNFa. SK-BR-3 and BT-474 cells

were transiently transfected with a jB-luciferase reporter

construct. Treatment with TNFa induced a six- or twofold

increase in jB transcriptional activation, respectively

(Fig. 4a). We next examined the effect of Herceptin, the

humanized anti-ErbB-2 antibody widely used in ErbB-2

positive breast cancer patients [13]. Addition of 10 lg/ml

Herceptin had no effect on TNFa-induced activation of

NF-jB (Fig. 4a). On the other hand, AG825 blocked

TNFa-induced NF-jB transcriptional activation (Fig. 4a).

A similar result was observed using AG825 in C4HD cells

(not shown). HRG induced a threefold induction in jB

transcriptional activation that was blocked by Herceptin

and AG825 treatment (Fig. 4a). Herceptin effectiveness

was proved through its ability to block Akt phosphorylation

in SK-BR-3 and BT-474 cells (Supplemental Fig. 6). To

accomplish NF-jB transcriptional activation through the

canonical pathway, cytoplasmic inhibitor IjBa must be

phosphorylated, ubiquitinated and degraded. Treatment of

SK-BR-3 and BT-474 cell lines with Herceptin did not

modify TNFa-induced phosphorylation of IjB (Fig. 4a).

On the other hand, addition of AG825 completely inhibited

A

B

D

-- -+ + mTNFα

GF109203X- ++

pTyr-927-ErbB-2

ErbB-2

Src

pTyr-416-Src

C4HDC

pTyr-927-ErbB-2

ErbB-2

Src

IP Src Control

IP Src mTNFα + PP2IP Src mTNFα

+-

---

-+---+-

IP ErbB2 Control++++C4HD

C4HD

0 2 105 15 30 60

SK-BR-3

0 2 105 15 30

pTyr-877/927-ErbB-2

ErbB-2

TNFα, min0 2 105 15 30

BT-474

C4HD

0 2 5 mTNFα, min0 5

pTyr-416-Src

Src

pTyr-927-ErbB-2

ErbB-2

- PP2- ++- -- -+ +

SK-BR-3

- ++

hTNFαPP2

pTyr-877-ErbB-2

ErbB-2

-- -+ +

BT-474

- ++

1 1.2 0.82.7 1.0 1.11.0 1 1.3 1.71.6 2.0 1.6 1 1.9 2.82.7 3.6 3.3

1 1.4 1.01.4 0.9

1 1.9 0.41.4 0.5

1 2.0 0.40.41 1.4 0.40.4

1 2.4 1.41.3

1 5.0 1.31.4

hTNFαDasatinibpTyr-877-ErbB-2

ErbB-2

-- -+ +

BT-474

- ++

1 1.7 0.30.3

Src

pTyr-416-Src

1 2.4 0.60.5

Fig. 2 TNFa phosphorylates ErbB-2 through c-Src activation.

a Cells were treated with TNFa for the times shown. Whole cell

lysates were subjected to Western blot analysis for ErbB-2 phos-

phorylation at Tyr877/927 residue. ErbB-2 is shown as loading

control. Bands were quantified using Image J with untreated cell

samples (first lines) set as 1.0. The experiment shown is represen-

tative of a total of five with same results. b C4HD and SK-BR-3 cells

were preincubated with 10 lM PP2 for 60 min and BT-474 cells with

10 lM PP2 or 0.5 lM Dasatinib and then treated or not with TNFa.

c-Src activity in cell lysates was determined by Western blot using

anti-phospho Tyr416 Src antibody and total c-Src is shown as loading

control. ErbB-2 phosphorylation at Tyr877/927 residue was per-

formed as described in (a). These experiments were repeated four

times with same results. c Cold in vitro phosphorylation assay was

performed with C4HD cells preincubated or not with PP2 and then

treated with TNFa for 5 min. c-Src was immunoprecipitated from

each treatment and ErbB-2 immunoprecipitated from unstimulated

C4HD cells was used as substrate. A cold in vitro phosphorylation

assay was performed as described in ‘‘Materials and methods’’. As

specificity control 500 lg protein extract from C4HD cells treated

5 min with TNFa were immunoprecipitated with 2 lg of normal

mouse serum and subjected to the same in vitro phosphorylation

protocol. Western blots of c-Src, anti-phospho Tyr927 ErbB-2 and

ErbB-2 are shown. These experiments were repeated four times with

same results. IP immunoprecipitation. d C4HD cells were preincu-

bated with 10 lM GF109203X for 60 min and then treated with

TNFa. c-Src phosphorylation and ErbB-2 Tyr927 phosphorylation

were performed as described in (b)

Breast Cancer Res Treat

123

TNFa-induced phosphorylation of IjB, confirming the

transcriptional activity results (Fig. 4a).

To further explore the role of ErbB-2 on NF-jB transcrip-

tional activation, we used different human ErbB-2 mutants.

SK-BR-3 cells were co-transfected with a kinase-negative

(KN)-ErbB-2 expression vector or an ErbB-2 expression plas-

mid in which tyrosine 877 was point mutated to phenylalanine

(ErbB-2-Y877F), together with a jB-Luc reporter vector. The

presence of the mutated ErbB-2 constructs inhibited endoge-

nous ErbB-2 phosphorylation, revealing a dominant negative

activity exerted on the wild-type ErbB-2 molecules (Fig. 4b).

Our results showed that KN-ErbB-2 or ErbB-2-Y877F blocked

TNFa ability to induce NF-jB transcriptional activation

(Fig. 4b), suggesting that phosphorylation at Tyr877 residue

and the ErbB-2 autophosphorylation are indispensable events

for TNFa-induced NF-jB transcriptional activation. Silencing

ErbB-2 expression with ErbB-2 siRNAs completely inhibited

TNFa-stimulated NF-jB transcriptional activation and IjBaphosphorylation in C4HD cells (Fig. 4c). Co-transfection with

a human ErbB-2 expression vector completely recovered

TNFa-induced NF-jB transcriptional activation that had been

lost by siRNA to mouse ErbB-2 (Fig. 4c; Supplemental Fig. 4).

We next explored cyclin D1 promoter activation, key

cell cycle regulator protein whose promoter has NF-jB

biding sites [5, 32]. TNFa treatment of SK-BR-3 cells

transfected with a cyclin D1 promoter-luciferase reporter

CC4HD

++-----+ - -+--- ++--

-- + - -+--

SK-BR-3

AG825PP2

0 5 10 TNFα, min0 5 0 5

p-Ser 473-Akt

Akt

0 10 0 10 0 10

A

- + + mTNFαPP2+- -

NRSTIP: ErbB-2

IP: ErbB-3

- + - mTNFαHRG+- -

NRST

ErbB-3

ErbB-2

ErbB-2

ErbB-3

p85 PI3-K

C4HDB

+-

+-

+-

+-

IP: ErbB-3TNRS

0 10

C4HD

10 10 mTNFα

siRNA Control- -++siRNA ErbB-2+ +--

pTyr

ErbB-3

0 100 10

siRNA Control- -++siRNA ErbB-2+ +--

p-Ser 473-Akt

Akt

TNFα, min0 10 0 10

0 5 10 hTNFα, min

pTyr

EGFR

IP: EGFRTNRS

IP: ErbB-3TNRS

0 10

SK-BR-3

10 10 10 10

pTyr

ErbB-3

AG825PP2

TNFα, min

Fig. 3 TNFa induces ErbB-2/ErbB-3 heterodimerization and PI3-K/

Akt activation. a Cells were treated with TNFa for the times shown

and subjected to ErbB-3 or EGFR immunoprecipitation. Immunopre-

cipitates were blotted with anti-phosphotyrosine antibody (top panel)and membranes were stripped and blotted with anti-ErbB-3 or EGFR

antibody (bottom panel). C4HD cells were transiently transfected

with 25 nM mouse ErbB-2 siRNA #03 or control siRNA before

mTNFa treatment. NRS normal rabbit serum, T total cell lysates, IPimmunoprecipitation. These are representative experiments out of a

total of three. b Association of ErbB-2 with ErbB-3 was performed by

immunoprecipitation of C4HD cells treated for 10 min with TNFa. In

ErbB-2 immunoprecipitation, cells were preincubated with PP2 for

60 min and then treated or not with TNFa. ErbB-3 was analyzed by

Western blot and the membrane was stripped and ErbB-2 was

detected to verify that nearly equal amounts of immunoprecipitated

proteins were loaded. The inverse immunoprecipitation using anti-

ErbB-3 antibodies was also performed. Cells were treated with TNFaor HRG for 10 min. ErbB-2 was analyzed by Western blot and

membrane was stripped and ErbB-3 was detected to verify that nearly

equal amounts of immunoprecipitated proteins were loaded. In the

lower part of the membrane, p85 PI3-K was detected. All these results

are representative of three performed experiments. c Cells were

preincubated with 100 lM AG825 or 10 lM PP2 for 60 min and then

treated or not with TNFa for the indicated times. C4HD cells were

transiently transfected with 25 nM mouse ErbB-2 siRNA #03 or

control siRNA before mTNFa treatment. Akt activation in cell lysates

was determined by Western blot using anti-phospho-specific antibod-

ies and total kinases are shown as loading control

Breast Cancer Res Treat

123

vector stimulated luc activity threefold (Fig. 5a). The

presence of Herceptin did not modify TNFa-induced cyclin

D1 promoter expression in SK-BR-3 cells. On the other

hand, AG825 blocked TNFa-induced cyclin D1 promoter

activation. Moreover, in BT-474 cells, Herceptin could not

block TNFa-induced up-regulation of cyclin D1 expres-

sion, while AG825 and GW2974, structurally related to the

ErbB-2 inhibitor clinically used Lapatinib, completely

inhibited TNFa-induced expression of cyclin D1 (Fig. 5b).

Interestingly, addition of Herceptin to BT-474 cells did not

affect TNFa-induced proliferation measured either by cell

count (96 h), propidium iodide staining and flow cytometry

analysis (48 h) or by thymidine incorporation (48 h) as

shown in Fig. 5c. Treatment with Herceptin diminished

BT-474 cell count and induced G0/G1 arrest, although

inhibition of thymidine incorporation was very slight

(Fig. 5c). In line with our previous data on cyclin D1

promoter and protein expression, the presence of AG825

blocked TNFa-induced BT-474 breast cancer cell prolif-

eration. These results show that ErbB-2 transactivation is

essential for TNFa-induced NF-jB transcriptional activa-

tion and proliferation in breast cancer cells.

According to our results in which c-Src acts as an

upstream activator of ErbB-2, we reasoned out that

A

0

3

6

9

HerceptinAG825

κB-l

uc

/ ren

illa

(fo

ld in

du

ctio

n)

- + - - + -- - -+ - +

+- -- - +

++ +- - - - - -++ +- - -- - -

hTNFαHRG

**

*

**

SK-BR-3

0

1,5

3

4,5

- + - +- - - -

+-- -

+ +- - - -+ +- -- -

BT-474

**

**

-

- -

+ +-

+ +

SK-BR-3

-

- -

+ - -

- -

+ -

- -

- +- - +-

- - - + + +

hTNFαAG825

phospho-IκBα

IκBα

HRGHerceptin

-

- -

+ - +

- -

- -- -

- - + +

BT-474

-

- -

+ +-

+ +

1 4.1 1.11.3 1 2.0 1.31.5 1.22.3 1 4.7 0.70.4 1 4.7 101.1

B

0

2

4

6

8

mTNFα

siRNA control

siRNA ErbB-2

- + - +

+ +

- - + +

- -

- +

+ +

- -

κB-l

uc

/ ren

illa

(fo

ld in

du

ctio

n)

pCDNA3.1 hErbB-2wt

*

*

C

0

0,5

1

1,5

2

2,5

κB-l

uc

/ ren

illa

(fo

ld in

du

ctio

n)

- + - + - +

*

pCDNA3.1 ErbB-2-Y877F KN-ErbB-2

hTNFα

SK-BR-3 C4HD

siRNA Control- -++siRNA ErbB-2+ +--TNFα, min0 10 0 10phospho-IκBα

IκBα

- -+ +pCDNA3.1 KN-ErbB-2

- -+ + hTNFαpCDNA3.1 ErbB-2-Y877F

hTNFα

pTyr-877-ErbB-2

pTyr-1222-ErbB-2

ErbB-2

ErbB-2

Fig. 4 ErbB-2 mediates TNFa-induced NF-jB trancriptional activa-

tion. a kB luciferase-transfected cells were preincubated with 100 lM

AG825 or control DMSO or with 10 lg/ml Herceptin for 60 min and

then treated or not with 20 ng/ml hTNFa or with 20 ng/ml HRG for

18 h. Cells were harvested for NF-jB transcriptional activation as

described in ‘‘Materials and methods’’ (*P \ 0.01, **P \ 0.001 vs.

control). Right panel shows Western blot analysis of phospho IjBafrom cells preincubated with AG825 or Herceptin and treated or not

with hTNFa or HRG for 10 min. As loading control, membranes were

stripped and hybridized with an anti-IjBa antibody. Bands were

quantified using Image J with untreated cell samples (first lines) set as

1.0. b SK-BR-3 cells were transiently co-transfected with the

indicated vectors and jB-luciferase construct. Cell treatment and

reporter activity were performed as described in (a). SK-BR-3 cells

transfected with 2 lg ErbB-2-Y877F vector or pCDNA3.1 and treated

with TNFa for 10 min were subjected to Western blot analysis for

ErbB-2 phosphorylation at Tyr877/927 residue as described in Fig. 2a

(right panel). Cells transfected with 2 lg KN-ErbB-2 vector or

pCDNA3.1 were subjected to Western blot analysis for ErbB-2

phosphorylation at Tyr1222 residue as described in Fig. 1f. c C4HD

cells were transiently co-transfected with 25 nM mouse ErbB-2

siRNA #03 or control siRNA, and with jB-luciferase construct before

mTNFa treatment. Similar results were obtained with siRNA #02 and

#04. In reconstitution experiments, 1 lg/well of a human ErbB-2

expression vector was used. Cell treatment and reporter activity were

performed as described in (a) (*P \ 0.001 vs. control). Inset shows

Western Blot analysis of IjBa phosphorylation, as described in (a),

from C4HD cells transiently transfected with 25 nM mouse ErbB-2

siRNA #03 or control siRNA before mTNFa treatment

Breast Cancer Res Treat

123

BT-474

[3 H]-

Thy

mid

ine

inco

rpo

rati

on

(C

PM

x 1

03)

0

50

100

150

200

250

- + - +mTNFα

- - +Dasatinib +

*

* *

D

- + - + - +

AG825 - - ++ - -

Herceptin - - +- +-

Cyc

lin D

1-lu

c / r

enill

a(f

old

ind

uct

ion

)A

SK-BR-3

hTNFα

B

0

1

2

3 * *

0

- -

24 48

+ +- - - -- --+

hTNFα (h)

AG825

GW 2974

Herceptin

0 24 48 0 24 48 0 24 48

- - + +- - - -- -- +- - + +- - - -- -- +

Cyclin D1

Actin

BT-474

1 1.8 0.31.9 0.20.3 0.1 0.2 0.80.1 1.7 1.4

0

25

50

75

100

G0/G1 S G2/M

- + - + - +

- - ++ - -

- - +- +-

BT-474%

Cel

ls

SK-BR-3mTNFα, min

pTyr-416-Src

Src

- Herceptin- + +

0 5 50

- - + +

0 5 50

BT-474

E

- + - -+ +

- - ++ - -

- - +- +-[3

H]-

Thy

mid

ine

inco

rpo

rati

on

(C

PM

x 1

03)

0

5

10

15

20

25

30*

*

0

5

10

15

20

Cel

l nu

mb

er /

wel

l (10

4)

- + - + - +

AG825 - - ++ - -

Herceptin - - +- +-

hTNFα

*

*

†

* *

C

Fig. 5 TNFa-induced proliferation is blocked by pharmacological

inhibitors of ErbB-2 and c-Src but not by Herceptin in human breast

cancer cells. a SK-BR-3 cells were transiently transfected with cyclin

D1-luciferase promoter and then treated as described in Fig. 4a

(*P \ 0.001 vs. control). b BT-474 cells were preincubated with

100 lM AG825, 1 lM GW2974 or 10 lg/ml Herceptin for 60 min

and treated or not with 20 ng/ml hTNFa for 24 or 48 h. Whole cell

lysates were subjected to Western blot analysis for cyclin D1

expression. Actin is shown as loading control and was used for

quantification. c BT-474 cells were preincubated with 100 lM

AG825 or 10 lg/ml Herceptin for 60 min and treated or not with

20 ng/ml hTNFa. Proliferation assay was performed by cell count

with Trypan blue at 96 h of culture (*P \ 0.05 vs. control, �P \ 0.05

vs. Herceptin), left panel, by cell cycle analysis with propidium iodide

staining and flow cytometry analysis at 48 h of culture (G0/G1 phase

P \ 0.02 TNFa vs. control; G2/M phase P \ 0.05 TNFa vs. control;

G0/G1 phase P \ 0.05 TNFa ? Herceptin vs. Herceptin; G2/M phase

P \ 0.05 TNFa ? Herceptin vs. Herceptin), central panel, and by

[3H]-thymidine incorporation assay, as described in Fig. 1a

(*P \ 0.01 vs. control) right panel. d BT-474 and SK-BR-3 cells

were preincubated with 10 lg/ml Herceptin for 6 h and treated or not

with 20 ng/ml hTNFa. c-Src phosphorylation was performed as

described in Fig. 2b. e BT-474 cells were preincubated with 0.5 lM

Dasatinib for 60 min and then treated or not with 20 ng/ml of TNFafor 48 h. Proliferation was performed by [3H]-thymidine incorpora-

tion assay as described in Fig. 1 (*P \ 0.01 vs. control)

Breast Cancer Res Treat

123

Herceptin should not inhibit c-Src activation by TNFa.

Indeed in the presence of Herceptin, TNFa induced c-Src

activity to the same degree as in control cells (Fig. 5d).

These results led us to assess whether c-Src was involved in

the mitogenic effect of TNFa. We performed a proliferation

assay that showed that Dasatinib completely blocked TNFaability to induce BT-474 proliferation (Fig. 5e). These

results disclose that c-Src is a key player on TNFa-induced

signaling cascade leading to breast cancer cell growth.

Discussion

In the present study we disclosed, for the first time, that

TNFa transactivates ErbB-2 in breast cancer cells, thus

becoming another player in the scenario of ErbB-2 onco-

genic activity. Our results provided a time course of events

triggered by TNFa leading to breast cancer prolifera-

tion.We unraveled that in ErbB-2-overexpressing cells,

TNFa initiates ErbB-2 signal transduction by the activation

of c-Src (2–5 min), the kinase involved in Tyr877 ErbB-2

phosphorylation. These induce ErbB-2 autophosphoryla-

tion (5–15 min) and ErbB-2/ErbB-3 heterodimer forma-

tion, leading to the activation of PI3-K/Akt and finally

transcriptional activation of NF-jB. This transcription

factor in turn increases the expression of its target gene,

cyclin D1 which is a key protein involved in breast cancer

proliferation (Fig. 6). Cyclin D1 increase was detectable as

soon as 4 h of TNFa treatment (data not shown) continuing

high 48 h later.

Previous studies have shown that c-Src activation is

dependent on ErbB-2, but it has recently been reported that

c-Src itself plays a role upstream on ErbB-2 phosphoryla-

tion. c-Src interacts directly with the catalytic domain of

ErbB-2 [33, 34] and phosphorylates ErbB-2 at Tyr877/927

residue within the activation loop of the kinase domain,

stabilizing it in an open and extended conformation which

increases its intrinsic kinase activity [28]. Here, we found

that TNFa induces Tyr877/927 ErbB-2 phosphorylation

through c-Src activation, in a PKC-dependent manner, in

murine C4HD and human SK-BR-3 and BT-474 cells. We

observed that TNFa induces Tyr416 phosphorylation of

c-Src, and that this activated kinase phosphorylates ErbB-2

Tyr877/927 residue in vitro. Moreover, we also demon-

strated that blockage of c-Src with the clinically used

inhibitor, Dasatinib, inhibits TNFa-induced BT-474 cell

proliferation. This piece of information would be useful in

future evaluations of breast cancer specimens with the

expression of TNFa and activated c-Src, of patients

undergoing anti-ErbB-2 therapy. It was recently demon-

strated that TNFR1 is constitutively associated with c-Src,

and that TNFa induces recruitment of additional c-Src and

increases its activity in HEK293 and MCF-7 cell lines [35].

During the preparation of this manuscript, Yamaoka et al.

[36] demonstrated that Src kinase activity as well as

transactivation of EGFR and ErbB-2 is required for TNFa-

induced survival of young adult mouse colon (YAMC)

epithelial cells. In the present study, we also confirmed that

c-Src is the kinase responsible for ErbB-2 transactivation,

and we proved that c-Src mediates the phosphorylation on

ErbB-2 Tyr877 residue. Moreover, we demonstrated that

ErbB-2 transactivation is responsible for TNFa-induced

proliferation of breast cancer cells overexpressing ErbB-2,

and that TNFa elicits heterodimerization of ErbB-2 with

ErbB-3. However, we could not detect any participation of

EGFR. These differences might be attributed to tissue-

specific signaling. Another interesting finding is that,

although both TNFa and HRG stimulate p42/p44 MAPK

activation, blockage of ErbB-2 by AG825 did not inhibit the

activation of p42/p44 MAPK induced by TNFa. In contrast,

AG825 blocked HRG-induced p42/p44 MAPK activation.

These results reveal different signaling pathways induced

by ErbB-2 transactivation caused by a classic ErbB-3/ErbB-

TNFR

PKC

c-Src

TNFα

ErbB2ErbB3

Akt

PI3-K

P

P P

IκB

α

> Cyclin D1p65p50

877

1222

Fig. 6 Model of TNFa transactivation of ErbB-2 and proliferation

induction in breast cancer cells. TNFa binds to TNFa receptor and

activates PKC which in turn activates c-Src which is able to

phosphorylate ErbB-2 at Tyr877 residue. ErbB-2 becomes autophos-

phorylated at Tyr1222 residue inducing ErbB-2/ErbB-3 heterodimer-

ization, ErbB-3 phosphorylation and the concomitant recruitment of

p85 PI3-K. This event activates Akt and leads to NF-jB transcrip-

tional activation and the consequent expression of cyclin D1 and

proliferation

Breast Cancer Res Treat

123

4 ligand (HRG) or a ‘‘non-classic’’ activator (TNFa). On the

other hand, transactivation of EGFR by TNFa is a well-

documented mechanism that involves MMPs stimulation;

in particular, of the TNFa converting enzyme, which

releases EGFR ligands [18–20]. However, in this work we

could not detect any evidence supporting the fact that ErbB-

2 transactivation by TNFa was induced by cleavage of

membrane-tethered ErbB ligands, since pretreatment with

GM6001, a broad spectrum inhibitor of MMPs, did not

modify TNFa-induced ErbB-2 phosphorylation either in

C4HD cells or in SK-BR-3 cells.

An exciting novel finding of this study has been the

demonstration that TNFa utilizes ErbB-2 as a downstream

signaling partner in the generation of mitogenic signals.

Our data supporting the fact that TNFa-induced prolifera-

tion of C4HD cells is dependent on ErbB-2 kinase activity

was also confirmed in SK-BR-3 cells, where TNFa has

already been reported as mitogenic [8], and in BT-474

cells. Our findings suggest that NF-jB activation induced

by TNFa requires a functional ErbB-2 because (a) the

inhibition of ErbB-2 by AG825, (b) the protein knockdown

of ErbB-2 by siRNA strategy, (c) the fact that transfection

with KN-ErbB-2 or ErbB-2-Y877F plasmids, impaired

TNFa capacity to activate NF-jB and (d) the fact that co-

transfection with siRNA to mouse ErbB-2 and reconstitu-

tion of ErbB-2 expression with a vector encoding human

ErbB-2, restored TNFa-induced NF-jB activation. How-

ever, in NIH 3T3 cells, which have low levels of ErbB-2,

transiently transfected with ErbB-2 plasmid, we observed

no difference of TNFa effect on NF-jB activation as

compared to the cells transfected with the empty vector

(data not shown). These data suggest that ErbB-2-over-

expressing cells have other/s signaling molecule/s that

allow/s the cross talk between TNFa receptors and ErbB-2.

We observed that either inhibition of ErbB-2 with AG825

or silencing ErbB-2 by siRNA treatment inhibited TNFa-

induced activation of PI3-K/Akt and IjBa phosphoryla-

tion. These results are in line with our previous report

showing that Akt behaves as an upstream molecule in the

NF-jB activation cascade and this activation proceeds

through the canonical pathway [5]. Although it has long

been known that amplified expression of ErbB-2 induces

breast cancer resistance to TNFa-induced cytotoxicity [37],

the mechanisms that promote this effect have scarcely been

characterized. ErbB-2 overexpression has been found to

constitutively activate Akt/NF-jB anti-apoptotic pathway,

conferring resistance to TNFa in cancer cell lines [10].

Yamaoka et al. [36] determined that loss of EGFR

expression suppressed TNFa activation of Akt and induce

apoptosis in YAMC cells, although they did not observe

any modulation of IjB. On the other hand, Biswas et al.

[38] demonstrated that NF-jB activation is predominantly

found in the estrogen receptor (ER)-negative/ErbB-2

positive subclass in comparison with ER-positive human

breast cancer tumors. Recently, this group demonstrated

that NF-jB activation in this subclass of breast cancer is

essential for tumorigenesis and tumor progression [39]. In

the present work, we illustrate that TNFa activates ErbB-2

and uses it as an intermediate to promote NF-jB activation,

adding yet another layer of complexity in ErbB-2 over-

expressing tumor signaling.

An extensive body of evidence has established ErbB-2

as a key mediator of tumor cell growth and survival, since

ErbB-2 overexpression in nearly 30% of human breast

cancers predicts an aggressive course of disease and poor

prognosis [9]. The first anti-ErbB-2 agent used in clinical

practice is the humanized monoclonal antibody Herceptin

[13]. Given alone as first line treatment to metastatic breast

cancer patients, Herceptin shows an overall response rate

of 38% [40]. It has been suggested that one likely mech-

anism of acquired resistance to Herceptin can be the

amplification of ligand-induced activation of the ErbBs

receptor [41]. Consequently, small-molecule inhibitors of

EGFR/ErbB-2 have progressed to clinical trials [42]. Here,

we report TNFa as a new ErbB-2 transactivating factor

unexplored before, and demonstrate that TNFa-induced

ErbB-2 transactivation leading to NF-jB activation and

cell proliferation can be blocked at the level of ErbB-2

phosphorylation through AG825 or GW2974, specific

EGFR/ErbB-2 tyrosine kinase inhibitors, but not at the

level of antibody blockage of the receptor. Interestingly,

Herceptin increased TNFa-induced NF-jB activity and

IjBa phosphorylation in BT-474 but not in SK-BR-3 cells

(Fig. 4a). Since BT-474 cell line expresses estrogen and

progesterone receptors (PR) and as we have demonstrated

that PR is involved in ErbB-2 phosphorylation [43], further

studies have to be carried out to shed light on the above-

mentioned finding.

In this work, we provide a suitable molecular explana-

tion for the resistance to Herceptin as observed in clinical

practice, and hypothesize that TNFa signaling may have

augmented in patients unresponsive to monoclonal anti-

body therapy. Even when TNFa has been shown to be

expressed in a significant proportion of human infiltrating

breast cancers [6], the eventual worth of TNFa signaling as

a prognostic factor in anti-ErbB-2 therapy is yet to be

determined.

Acknowledgments This work was supported by grants IDB 1728/

OC-AR PICT 2006 0211 and PICT 2004 05-25301, both from the

National Agency of Scientific Promotion of Argentina, PIP 5391 from

the Argentine National Council of Scientific Research (CONICET)

and by Oncomed-Reno CONICET 1819/03, from the Henry Moore

Institute of Argentina and by grant KG090250 from the Susan G.

Komen for the Cure. The authors wish to thank Dr Alfredo A. Mo-

linolo (NIH, Bethesda, MD) for his constant help and support. We

thank Dr C. Lanari for providing the MPA-induced mammary tumor

model.

Breast Cancer Res Treat

123

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