Breast Cancer Research and Treatment

, Volume 122, Issue 1, pp 111–124

Transactivation of ErbB-2 induced by tumor necrosis factor α promotes NF-κB activation and breast cancer cell proliferation

Authors

  • Martín A. Rivas
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
  • Mercedes Tkach
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
  • Wendy Beguelin
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
  • Cecilia J. Proietti
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
  • Cinthia Rosemblit
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
  • Eduardo H. Charreau
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
  • Patricia V. Elizalde
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
    • Laboratory of Molecular Mechanisms of CarcinogenesisInstituto de Biología y Medicina Experimental (IBYME)
Preclinical study

DOI: 10.1007/s10549-009-0546-3

Cite this article as:
Rivas, M.A., Tkach, M., Beguelin, W. et al. Breast Cancer Res Treat (2010) 122: 111. doi:10.1007/s10549-009-0546-3

Abstract

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine which, acting locally, induces tumor growth. Accumulating evidence, including our findings, showed that TNFα is mitogenic in breast cancer cells in vitro and in vivo. In the present study, we explored TNFα involvement on highly aggressive ErbB-2-overexpressing breast cancer cells. We found that TNFα 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 TNFα-induced c-Src activation. Moreover, TNFα promoted ErbB-2/ErbB-3 heterocomplex formation, Akt activation and NF-κB 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 TNFα-induced NF-κB activation and proliferation. However, the humanized monoclonal antibody anti-ErbB-2 Herceptin could not inhibit TNFα ability to promote breast cancer growth. Interestingly, our work disclosed that TNFα is able to transactivate ErbB-2 and use it as an obligatory downstream signaling molecule in the generation of mitogenic signals. As TNFα 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-2TNFαHerceptinc-Src

Introduction

Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine originally characterized to cause hemorrhagic necrosis of tumors at high doses [1]. However, it is now widely accepted that TNFα, acting locally, induces the growth of certain tumor types such as ovary and breast [25]. In particular, TNFα 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 TNFα 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 TNFα 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-terminal kinase (JNK), Akt and NF-κB transcriptional activation [5]. However, the exact mechanism by which TNFα enhances 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-κB 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 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. Herceptin™ (Trastuzumab) is a monoclonal 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-targeted 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 TNFα through the activation of matrix metalloproteinases (MMPs) which are able to release EGFR ligands from the cell membrane [1820]. In contrast, there are no reports demonstrating TNFα ability to transactivate ErbB-2 in breast cancer.

In the present work, we explore the effect of TNFα on breast cancer cells that overexpress ErbB-2 and found that TNFα 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 TNFα is a rapid event that does not involve either ligand release or MMPs activation, and leads to cell proliferation even in the presence of Herceptin. Interestingly, we showed that TNFα regulates ErbB-2 phosphorylation as a requisite to activate NF-κB and cell proliferation in human and murine breast cancer cells. This is the first demonstration of ErbB-2 transactivation by TNFα 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 Biología 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 [2123].

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 IκBα (Ser32/36), IκBα, 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 Calbiochem (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 mammary tumor C4HD, growing in medroxyprogesterone acetate (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 TNFα (mTNFα, hTNFα, respectively) (Cell Sciences, Canton, MA, USA) or with 20 ng/ml of recombinant human β1 heregulin (HRG, Upstate, Millipore, Bedford, MA, USA). The following inhibitors were added to cells 60 min before incubation with TNFα: 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 indazolyamino quinazoline family and Bay 11-7082, inhibitor of IκB phosphorylation (both from Sigma). To perform certain experiments, cells were pre-incubated for 16 h with 10 μg/ml of the anti-ErbB-2 antibody Herceptin™ (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 incubated with TNFα for 48 h. 1 μCi [3H]-thymidine (NEN, Dupont, Boston, MA, USA; specific activity 20 Ci/mmol) was added at hour 24 of said culture. Cells were then trypsinized 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 μM AG825, it was of 67.6 ± 4.7%, for BT-474 cells cultured with 100 μM AG825, 10 μg/ml Herceptin, 1 μM GW2974, 0.5 μM Dasatinib and 1 μM 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 μM AG825 and 10 μg/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 coverslips were treated with TNFα 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 hTNFα 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. Trotter, 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 μg/ml) staining for 30 min at room temperature in the dark. Cell cycle analysis was performed using a FACScalibur 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 experiments as previously described [24]. Briefly, 500 μg of protein lysates was incubated with 2 μg of rabbit anti-EGFR, ErbB-2 or ErbB-3 antibody and the immunocomplexes 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 TNFα for 5 min or preincubated before TNFα stimulation for 60 min with PP2 (10 μM). 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 μg protein extracts using an anti-v-Src antibody. ErbB-2 was immunoprecipitated from 500 μg 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 previously described [25]. Proteins were separated by electrophoresis; gels were transferred onto nitrocellulose and immunoblotted with anti-Tyr877/927 ErbB-2 and anti-total c-Src antibodies and ErbB-2.

Transient transfections

κB-Luc vector (κB 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 were transiently co-transfected with 1 μg of κB-Luc or 1 μg 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 κB. Transfected cells were lysed and luciferase assays carried out using the Dual-Luciferase Reporter Assay System (Promega).

siRNAs targeting mouse ErbB-2 mRNAs were synthesized by Dharmacon Inc. (Lafayette, CO, USA) (ErbB-2 siRNA#02: antisense, 5′-GAUGUCCUCCGUAAGAAUA-3′, ErbB-2 siRNA#03 antisense 5′-GAUGGUGCUUACUCAUUGA-3′, ErbB-2 siRNA#04 antisense 5′-GGAAUCCUAAUCAAACGAA-3′). The non-silencing siRNA oligonucleotide from Dharmacon, which does not target any known mammalian gene, was used as negative control. Transfection of siRNA plus 1 μg of empty vector (pcDNA3.1) or 1 μg of human ErbB-2 expression vector and 1 μg of κB-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

TNFα-induced breast cancer cell proliferation requires ErbB-2 phosphorylation

We have already shown that TNFα 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 signaling in the growth stimulatory effects of TNFα. 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 TNFα was inhibited by addition of 100 μM AG825 (Fig. 1a). As expected, AG825 completely 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 TNFα induced 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 TNFα-induced p42/p44 MAPK nor JNK phosphorylation in C4HD cells (Supplemental Fig. 2). However, addition of AG825 completely blocked HRG-induced p42/p44 MAPK activation (Supplemental Fig. 2). These contrasting results suggest that p42/p44MAPK activation has a different up-stream signaling pathway that is dependent on ErbB-2 phosphorylation when cells are stimulated with HRG, and is ErbB-2 phosphorylation-independent when the mitogen used is TNFα.
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Fig. 1

TNFα requires ErbB-2 phosphorylation for inducing breast cancer cell proliferation. a, e Cells were preincubated with 100 μM AG825 for 60 min and then treated or not with 20 ng/ml of TNFα 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 TNFα-induced proliferation. b C4HD cells were treated with TNFα for the times shown and subjected to ErbB-2 immunoprecipitation. Immunoprecipitates were blotted with anti-phosphotyrosine antibody (top panel) and membranes were stripped and blotted with anti-ErbB-2 antibody (bottom panel). 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 TNFα for the times shown. Whole cell lysates were subjected to Western blot analysis for ErbB-2 phosphorylation 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 μM AG825 for 60 min and SK-BR-3 and BT-474 cells with 100 μM AG825 and then treated with TNFα for the times shown. ErbB-2 phosphorylation at Tyr1222/1272 residue was performed as described in (c). This is a representative experiment out of a total of three. g ErbB-2 expression by flow cytometry in cells treated with TNFα. 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 TNFα for 30 min are shown. The delta mean fluorescence intensity of control and TNFα-treated SK-BR3 cells was of 4.4 and 2.9, respectively (P <  0.01) and of control and TNFα-treated BT-474 cells it was of 22.2 and 10.0, respectively (P < 0.001). Kolmorgorov–Smirnov statistical test was used. h Localization of ErbB-2 by immunofluorescence and confocal microscopy in TNFα-treated cells. Each image is representative of at least 10 (scale bars 10 μm). 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 4′,6-diamidino-2-phenylindole (DAPI). hTNFα human TNFα, mTNFα murine TNFα

We examined ErbB-2 phosphorylation levels after TNFα treatment. Figure 1b shows that TNFα 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 TNFα treatment in C4HD cells, with maximum phosphorylation at 5 min (Fig. 1c). Addition of AG825 blocked TNFα-induced Tyr1272 phosphorylation in a concentration-dependent manner (Fig. 1d). Since our goal was to determine whether TNFα-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 TNFα induced SK-BR-3 and BT-474 cell proliferation, addition of AG825 blocked TNFα mitogenic effect (Fig. 1e). Similar effects were obtained with HRG-treated cells. In addition, TNFα induced phosphorylation of the Tyr1222 residue after 2 min stimulation, with a peak at 5 min (Fig. 1f) which was inhibited by addition of AG825. Comprehensively, our present findings indicate that TNFα is able to transactivate ErbB-2 in breast cancer cells.

To examine the effect of TNFα 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 TNFα treatment in both cell lines. To confirm these data, we performed confocal microscopy studies. ErbB-2 was localized primarily to the plasma membrane in unstimulated cells. TNFα 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 content in whole cell extracts. Comprehensively, these results show for the first time that TNFα is able to activate ErbB-2 inducing its phosphorylation and internalization into the cytoplasm of breast cancer cells.

The finding that TNFα is able to induce ErbB-2 transactivation, prompted us to explore the mechanism underlying this phosphorylation. It is known that TNFα can induce EGFR transactivation through MMP-dependent EGFR ligand release. Treatment of C4HD and SK-BR-3 cells with 10 μM GM6001, a broad MMPs inhibitor, did not modify TNFα-induced ErbB-2 phosphorylation (Supplemental Fig. 3a). In addition, the use of blocking antibodies to ErbB-3 and ErbB-4 to impede ligand binding did not modify TNFα-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 TNFα does not activate ErbB-2 through ligand release in these breast cancer cells.

TNFα-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 activation loop of the kinase domain, is important for its intrinsic kinase activity [28]. Interestingly, we found that TNFα induced an increase on Tyr877/927 ErbB-2 phosphorylation 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 TNFα 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, dramatically decreased TNFα-induced ErbB-2 phosphorylation at Tyr877 in SK-BR-3 and BT-474 (Fig. 2b).
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Fig. 2

TNFα phosphorylates ErbB-2 through c-Src activation. a Cells were treated with TNFα for the times shown. Whole cell lysates were subjected to Western blot analysis for ErbB-2 phosphorylation 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 representative of a total of five with same results. b C4HD and SK-BR-3 cells were preincubated with 10 μM PP2 for 60 min and BT-474 cells with 10 μM PP2 or 0.5 μM Dasatinib and then treated or not with TNFα. 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 performed 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 TNFα 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 μg protein extract from C4HD cells treated 5 min with TNFα were immunoprecipitated with 2 μg 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 preincubated with 10 μM GF109203X for 60 min and then treated with TNFα. c-Src phosphorylation and ErbB-2 Tyr927 phosphorylation were performed as described in (b)

To further explore whether activated c-Src can phosphorylate Tyr877 ErbB-2 in vitro, we performed a cold phosphorylation assay. For this purpose, we immunoprecipitated c-Src from C4HD cells treated or not with TNFα for 5 min, and from C4HD cells treated with TNFα 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 TNFα 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 TNFα binding to its receptors, protein kinase C (PKC) is involved in c-Src phosphorylation [30]. Addition of 10 μM GF109203X, a PKC inhibitor, effectively abolished TNFα 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 TNFα induces c-Src activation which phosphorylates ErbB-2 at Tyr877/927 residue.

TNFα 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 molecular mechanisms triggered by TNFα-induced ErbB-2 transactivation, we monitored ErbB-3 and EGFR phosphorylation and ErbB-2 heterodimerization with ErbB-3. TNFα 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 TNFα-induced ErbB-3 phosphorylation in C4HD cells (Fig. 3a). By co-immunoprecipitation experiments in C4HD cells, we showed that TNFα induced ErbB-2 and ErbB-3 heterodimerization, which was reduced by addition of PP2 (Fig. 3b). The reverse experiment, in which we immunoprecipitated 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. TNFα 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 TNFα was significantly inhibited by AG825 in SK-BR-3 and C4HD cells (Fig. 3c). Blockage of ErbB-2 expression by siRNA also leads to inhibition of TNFα-induced Akt activation in C4HD cells (Fig. 3c). Notably, in the absence of c-Src activation by preincubation with PP2, TNFα capacity to activate Akt was blocked (Fig. 3c).
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Fig. 3

TNFα induces ErbB-2/ErbB-3 heterodimerization and PI3-K/Akt activation. a Cells were treated with TNFα for the times shown and subjected to ErbB-3 or EGFR immunoprecipitation. Immunoprecipitates 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 mTNFα treatment. NRS normal rabbit serum, T total cell lysates, IP immunoprecipitation. 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 TNFα. In ErbB-2 immunoprecipitation, cells were preincubated with PP2 for 60 min and then treated or not with TNFα. 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 TNFα or 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 μM AG825 or 10 μM PP2 for 60 min and then treated or not with TNFα for the indicated times. C4HD cells were transiently transfected with 25 nM mouse ErbB-2 siRNA #03 or control siRNA before mTNFα treatment. Akt activation in cell lysates was determined by Western blot using anti-phospho-specific antibodies and total kinases are shown as loading control

ErbB-2 tyrosine kinase inhibitor but not Herceptin blocks TNFα-induced NF-κB transcriptional activation, cyclin D1 expression and cell proliferation

Recently we have demonstrated that NF-κB activation is a key factor in TNFα-induced C4HD cell proliferation [5]. Using the IκB pharmacological inhibitor Bay 11-7082, NF-κB activation is also shown to be a requisite in TNFα-induced BT-474 cell proliferation (Supplemental Fig. 5). Therefore, we examined whether ErbB-2 participates in NF-κB activation by TNFα. SK-BR-3 and BT-474 cells were transiently transfected with a κB-luciferase reporter construct. Treatment with TNFα induced a six- or twofold increase in κB 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 μg/ml Herceptin had no effect on TNFα-induced activation of NF-κB (Fig. 4a). On the other hand, AG825 blocked TNFα-induced NF-κB transcriptional activation (Fig. 4a). A similar result was observed using AG825 in C4HD cells (not shown). HRG induced a threefold induction in κB 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-κB transcriptional activation through the canonical pathway, cytoplasmic inhibitor IκBα must be phosphorylated, ubiquitinated and degraded. Treatment of SK-BR-3 and BT-474 cell lines with Herceptin did not modify TNFα-induced phosphorylation of IκB (Fig. 4a). On the other hand, addition of AG825 completely inhibited TNFα-induced phosphorylation of IκB, confirming the transcriptional activity results (Fig. 4a).
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Fig. 4

ErbB-2 mediates TNFα-induced NF-κB trancriptional activation. a kB luciferase-transfected cells were preincubated with 100 μM AG825 or control DMSO or with 10 μg/ml Herceptin for 60 min and then treated or not with 20 ng/ml hTNFα or with 20 ng/ml HRG for 18 h. Cells were harvested for NF-κB transcriptional activation as described in “Materials and methods” (*P < 0.01, **P < 0.001 vs. control). Right panel shows Western blot analysis of phospho IκBα from cells preincubated with AG825 or Herceptin and treated or not with hTNFα or HRG for 10 min. As loading control, membranes were stripped and hybridized with an anti-IκBα 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 κB-luciferase construct. Cell treatment and reporter activity were performed as described in (a). SK-BR-3 cells transfected with 2 μg ErbB-2-Y877F vector or pCDNA3.1 and treated with TNFα 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 μg 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 κB-luciferase construct before mTNFα treatment. Similar results were obtained with siRNA #02 and #04. In reconstitution experiments, 1 μg/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 IκBα phosphorylation, as described in (a), from C4HD cells transiently transfected with 25 nM mouse ErbB-2 siRNA #03 or control siRNA before mTNFα treatment

To further explore the role of ErbB-2 on NF-κB transcriptional 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 plasmid in which tyrosine 877 was point mutated to phenylalanine (ErbB-2-Y877F), together with a κB-Luc reporter vector. The presence of the mutated ErbB-2 constructs inhibited endogenous 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 TNFα ability to induce NF-κB transcriptional activation (Fig. 4b), suggesting that phosphorylation at Tyr877 residue and the ErbB-2 autophosphorylation are indispensable events for TNFα-induced NF-κB transcriptional activation. Silencing ErbB-2 expression with ErbB-2 siRNAs completely inhibited TNFα-stimulated NF-κB transcriptional activation and IκBα phosphorylation in C4HD cells (Fig. 4c). Co-transfection with a human ErbB-2 expression vector completely recovered TNFα-induced NF-κB 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-κB biding sites [5, 32]. TNFα treatment of SK-BR-3 cells transfected with a cyclin D1 promoter-luciferase reporter vector stimulated luc activity threefold (Fig. 5a). The presence of Herceptin did not modify TNFα-induced cyclin D1 promoter expression in SK-BR-3 cells. On the other hand, AG825 blocked TNFα-induced cyclin D1 promoter activation. Moreover, in BT-474 cells, Herceptin could not block TNFα-induced up-regulation of cyclin D1 expression, while AG825 and GW2974, structurally related to the ErbB-2 inhibitor clinically used Lapatinib, completely inhibited TNFα-induced expression of cyclin D1 (Fig. 5b). Interestingly, addition of Herceptin to BT-474 cells did not affect TNFα-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 TNFα-induced BT-474 breast cancer cell proliferation. These results show that ErbB-2 transactivation is essential for TNFα-induced NF-κB transcriptional activation and proliferation in breast cancer cells.
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Fig. 5

TNFα-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 μM AG825, 1 μM GW2974 or 10 μg/ml Herceptin for 60 min and treated or not with 20 ng/ml hTNFα 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 μM AG825 or 10 μg/ml Herceptin for 60 min and treated or not with 20 ng/ml hTNFα. 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 TNFα vs. control; G2/M phase P < 0.05 TNFα vs. control; G0/G1 phase P < 0.05 TNFα + Herceptin vs. Herceptin; G2/M phase P < 0.05 TNFα + 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 μg/ml Herceptin for 6 h and treated or not with 20 ng/ml hTNFα. c-Src phosphorylation was performed as described in Fig. 2b. e BT-474 cells were preincubated with 0.5 μM Dasatinib for 60 min and then treated or not with 20 ng/ml of TNFα for 48 h. Proliferation was performed by [3H]-thymidine incorporation assay as described in Fig. 1 (*P < 0.01 vs. control)

According to our results in which c-Src acts as an upstream activator of ErbB-2, we reasoned out that Herceptin should not inhibit c-Src activation by TNFα. Indeed in the presence of Herceptin, TNFα 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 TNFα. We performed a proliferation assay that showed that Dasatinib completely blocked TNFα ability to induce BT-474 proliferation (Fig. 5e). These results disclose that c-Src is a key player on TNFα-induced signaling cascade leading to breast cancer cell growth.

Discussion

In the present study we disclosed, for the first time, that TNFα transactivates ErbB-2 in breast cancer cells, thus becoming another player in the scenario of ErbB-2 oncogenic activity. Our results provided a time course of events triggered by TNFα leading to breast cancer proliferation.We unraveled that in ErbB-2-overexpressing cells, TNFα 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 autophosphorylation (5–15 min) and ErbB-2/ErbB-3 heterodimer formation, leading to the activation of PI3-K/Akt and finally transcriptional activation of NF-κB. 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 TNFα treatment (data not shown) continuing high 48 h later.
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Fig. 6

Model of TNFα transactivation of ErbB-2 and proliferation induction in breast cancer cells. TNFα binds to TNFα receptor and activates PKC which in turn activates c-Src which is able to phosphorylate ErbB-2 at Tyr877 residue. ErbB-2 becomes autophosphorylated at Tyr1222 residue inducing ErbB-2/ErbB-3 heterodimerization, ErbB-3 phosphorylation and the concomitant recruitment of p85 PI3-K. This event activates Akt and leads to NF-κB transcriptional activation and the consequent expression of cyclin D1 and proliferation

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 phosphorylation. 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 TNFα 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 TNFα induces Tyr416 phosphorylation of c-Src, and that this activated kinase phosphorylates ErbB-2 Tyr877/927 residue in vitro. Moreover, we also demonstrated that blockage of c-Src with the clinically used inhibitor, Dasatinib, inhibits TNFα-induced BT-474 cell proliferation. This piece of information would be useful in future evaluations of breast cancer specimens with the expression of TNFα and activated c-Src, of patients undergoing anti-ErbB-2 therapy. It was recently demonstrated that TNFR1 is constitutively associated with c-Src, and that TNFα 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 TNFα-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 TNFα-induced proliferation of breast cancer cells overexpressing ErbB-2, and that TNFα 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 TNFα and HRG stimulate p42/p44 MAPK activation, blockage of ErbB-2 by AG825 did not inhibit the activation of p42/p44 MAPK induced by TNFα. 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-4 ligand (HRG) or a “non-classic” activator (TNFα). On the other hand, transactivation of EGFR by TNFα is a well-documented mechanism that involves MMPs stimulation; in particular, of the TNFα converting enzyme, which releases EGFR ligands [1820]. However, in this work we could not detect any evidence supporting the fact that ErbB-2 transactivation by TNFα was induced by cleavage of membrane-tethered ErbB ligands, since pretreatment with GM6001, a broad spectrum inhibitor of MMPs, did not modify TNFα-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 TNFα utilizes ErbB-2 as a downstream signaling partner in the generation of mitogenic signals. Our data supporting the fact that TNFα-induced proliferation of C4HD cells is dependent on ErbB-2 kinase activity was also confirmed in SK-BR-3 cells, where TNFα has already been reported as mitogenic [8], and in BT-474 cells. Our findings suggest that NF-κB activation induced by TNFα 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 TNFα capacity to activate NF-κB and (d) the fact that co-transfection with siRNA to mouse ErbB-2 and reconstitution of ErbB-2 expression with a vector encoding human ErbB-2, restored TNFα-induced NF-κB activation. However, in NIH 3T3 cells, which have low levels of ErbB-2, transiently transfected with ErbB-2 plasmid, we observed no difference of TNFα effect on NF-κB activation as compared to the cells transfected with the empty vector (data not shown). These data suggest that ErbB-2-overexpressing cells have other/s signaling molecule/s that allow/s the cross talk between TNFα receptors and ErbB-2. We observed that either inhibition of ErbB-2 with AG825 or silencing ErbB-2 by siRNA treatment inhibited TNFα-induced activation of PI3-K/Akt and IκBα phosphorylation. These results are in line with our previous report showing that Akt behaves as an upstream molecule in the NF-κB 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 TNFα-induced cytotoxicity [37], the mechanisms that promote this effect have scarcely been characterized. ErbB-2 overexpression has been found to constitutively activate Akt/NF-κB anti-apoptotic pathway, conferring resistance to TNFα in cancer cell lines [10]. Yamaoka et al. [36] determined that loss of EGFR expression suppressed TNFα activation of Akt and induce apoptosis in YAMC cells, although they did not observe any modulation of IκB. On the other hand, Biswas et al. [38] demonstrated that NF-κB 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-κB activation in this subclass of breast cancer is essential for tumorigenesis and tumor progression [39]. In the present work, we illustrate that TNFα activates ErbB-2 and uses it as an intermediate to promote NF-κB activation, adding yet another layer of complexity in ErbB-2 overexpressing 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 mechanism 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 TNFα as a new ErbB-2 transactivating factor unexplored before, and demonstrate that TNFα-induced ErbB-2 transactivation leading to NF-κB 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 TNFα-induced NF-κB activity and IκBα 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 explanation for the resistance to Herceptin as observed in clinical practice, and hypothesize that TNFα signaling may have augmented in patients unresponsive to monoclonal antibody therapy. Even when TNFα has been shown to be expressed in a significant proportion of human infiltrating breast cancers [6], the eventual worth of TNFα 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. Molinolo (NIH, Bethesda, MD) for his constant help and support. We thank Dr C. Lanari for providing the MPA-induced mammary tumor model.

Supplementary material

10549_2009_546_MOESM1_ESM.pdf (209 kb)
Supplementary material 1 (PDF 209 kb)

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© Springer Science+Business Media, LLC. 2009