HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tagliabue, E. Articles by Ménard, S. Search for Related Content PubMed PubMed Citation Articles by Tagliabue, E. Articles by Ménard, S. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 8, 5388-5394, February 25, 2000

Nerve Growth Factor Cooperates with p185^HER2 in Activating Growth of Human Breast Carcinoma Cells^*

Elda Tagliabue, Fabio Castiglioni, Cristina Ghirelli, Michele Modugno, Laura Asnaghi, Giulia Somenzi, Cecilia Melani^§, and Sylvie Ménard^¶

From the  Molecular Targeting Unit and ^§ Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nerve growth factor (NGF) is known to exert a mitogenic effect on human breast cancer cells through proto-TrkA activation. Reverse transcriptase-PCR analysis of proto-TrkA expression in human breast carcinoma specimens and cell lines revealed trkA transcript in 12 of 14 human breast carcinoma specimens and in all of four cell lines tested. While cytofluorimetric and Western blot analysis indicated proto-TrkA expression in three of the four cell lines, NGF stimulated growth in only two of the three positive cell lines. Inhibition of NGF-induced MAPK activation by an antibody directed against the extracellular domain of TrkA but not by an inhibitor of TrkA phosphorylation demonstrated the requirement of NGF binding but not of proto-TrkA kinase activity for MAPK activation, suggesting the recruitment of another kinase for transmission of the mitogenic signaling. Indeed, NGF induced tyrosine phosphorylation and stimulated kinase activity of p185^HER2, a kinase receptor of the HER family. A TrkA phosphorylation inhibitor did not affect this activation. Moreover, the two receptors were coprecipitated by antibodies directed against proto-TrkA and p185^HER2. Down-modulation of p185^HER2 expression in a breast carcinoma line transfected with a construct containing an anti-p185^HER2 antibody sequence and expressing proto-TrkA impaired NGF-induced MAPK activation and proliferation. Together these data show that in cells expressing low levels of TrkA such as breast carcinoma cells, NGF must recruit other overexpressed receptors such as p185^HER2 in order to generate a biological signal that can induce breast cancer cell growth.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The development and progression of breast cancer is a multifactorial process that includes the activation of oncogenes and loss of tumor suppressor genes and other genetic disruptions (1). There is evidence that breast cancer cells can be stimulated by various growth factors (2). Recently, nerve growth factor (NGF),¹ the archetypal neurotrophic factor, was shown to act as a mitogen for human breast cancer cell lines (3) through activation of proto-TrkA (4, 5), a transmembrane 140-kDa glycoprotein (gp140^trk) with tyrosine kinase activity that functions as high affinity NGF receptor.

NGF has previously been implicated in the growth modulation of human tumor cells with high affinity binding sites for this molecule, such as neuroblastoma (6, 7), glioblastoma (8), and pancreatic carcinoid (9) cell lines, and medullary thyroid carcinoma (10, 11), prostate (12), and lung (8) carcinomas.

A percentage of breast tumors overexpress growth factor receptors that confer a selective growth advantage over those that do not express these receptors. One of the most relevant receptors described for breast cancer progression is p185^HER2, the transmembrane glycoprotein with intrinsic tyrosine kinase activity that is encoded by the c-erbB2 proto-oncogene (13) and overexpressed in 30% of human primary breast carcinomas (14). A direct association between tumor aggressiveness and p185^HER2 overexpression has been reported (15). Like proto-TrkA, p185^HER2 induces an intracellular signaling pathway that leads to stimulation of breast cancer cell growth through activation of the MAPK pathway (16, 17).

In the present study, we analyzed the expression of the high affinity NGF receptor in breast carcinoma cell lines and in human breast carcinoma specimens to determine the molecules involved in NGF-induced signaling, in particular those that cooperate with other tyrosine kinase receptors at the cell surface, such as p185^HER2, in signal transduction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Monoclonal and Polyclonal Antibodies-- The following mouse monoclonal antibodies (mAb) were used: MGR12, directed against the extracellular domain of proto-TrkA (18); MGR2, directed against the extracellular domain of p185^HER2 (19); MGR1, directed against the extracellular domain of EGF receptor (20); c-neu Ab3, directed against the carboxyl-terminal peptide of p185^HER2 (Oncogene Science, Inc. Manhasset, NY); EGF receptor Ab7, directed against EGF receptor (NeoMarkers, Freemont, CA); and 4G10, directed against phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY). Polyclonal sera were directed against human proto-TrkA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p44/42 MAPK, or phospho-p44/42 MAPK (New England Biolabs, Inc., Beverly, MA).

Cell Lines and Culture Conditions-- Human breast carcinoma cell lines SKBr3, MDAMB453, MDAMB361, and DU4475 were provided by ATCC (Manassas, VA). Cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (FCS), L-glutamine, and antibiotics. Experiments involving treatment with NGF, the TrkA inhibitor K252a (21, 22) and mAb MGR12 were performed using recombinant NGF (Roche Molecular Biochemicals) at 100 ng/ml and mAb MGR12 at 20 µg/ml. K252a (Calbiochem) was used at 200 nM, the concentration at which proto-TrkA phosphorylation was inhibited after stimulation with NGF in PC12 cells (23) and NIH3T3 cells overexpressing proto-TrkA (E25427) (kindly provided by M. Barbacid, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) (data not shown). Human breast carcinoma cell lines MDAMB453-5R and MDAMB453-LBSN were maintained in Dulbecco's modified Eagle's medium (Sigma), supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM Hepes, antibiotics, and 1 mg/ml Geneticin (G418) (Life Technologies, Inc., Paisley, Scotland).

Human Tumor Samples-- 14 cases of fresh surgical tissues of pathologically confirmed primary breast carcinoma were used in this study. All patients were enrolled at the National Cancer Institute of Milan for primary breast carcinoma and underwent breast surgery with complete axillary dissection. Mean tumor size at pathological examination was 33.7 mm (range 15-80 mm). All patients had infiltrating ductal carcinoma. 10 patients had axillary lymph node involvement at histologic examination. All human tissues were collected within 5 min of surgical resection, snap frozen in liquid nitrogen, and stored at 80 °C until use.

Intracellular Expression of p185^HER2-specific Single Chain Antibody-- cDNA encoding the p185^HER2-specific single chain antibody 5R (scFv-5R), cloned in the retroviral vector pBabe-Puro (a generous gift from Nancy Hynes, Friedrich Miescher-Institue, Basel), was excised and cloned in the LXSN retroviral vector. Ecotropic virus encoding scFv-5R as well as a control virus encoding the LXSN vector carrying the E. coli lacZ gene (LBSN) were used to infect the amphotropic packaging cell line gp + AM12 in the presence of 8 µg/ml Polybrene. 2 days after infection, cells were selected in G418 (800 µg/ml) (24-26). Virus-containing conditioned medium was collected from pools of resistant gp + AM12 and used to infect the human mammary carcinoma cell lines SKBr3 and MDAMB453. To improve infection efficacy, the same infection cycle was repeated three times. Individual colonies as well as bulk cultures of G418-selected (1 mg/ml) cells were tested for p185^HER2 surface expression by FACScan analysis. G418-resistant colonies recovered from cells infected with the LBSN vector were used as controls.

Determination of Proto-trkA Transcript-- Total RNA was isolated using RNAzol (Paesel and Lorei, Frankfurt, Germany) according to the manufacturer's instructions. RNA was quantitated spectrophotometrically. Reverse transcription was carried out at 37 °C for 60 min using 2 µg of total RNA, 75 pmol of random primer, a 0.5 mM concentration of each deoxynucleotide triphosphate, and 50 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). PCR was performed in a total volume of 50 µl using primer pairs of proto-trkA extracellular domain previously described (27), composed of the (+)-primer 5'-CCATCGTGAAGAGTGGTCTC-3' and the ()-primer 5'-GGTGACATTGGCCAGGGTCA-3' with an expected amplicon length of 476 base pairs. After an initial denaturation at 95 °C for 4 min, 35 cycles at 95 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min were carried out followed by a final extension at 72 °C for 10 min.

PCR products (25 µl) were electrophoresed in a 2% agarose gel, transferred to nylon Hybond-N membranes (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom), and cross-linked by UV irradiation. Prehybridization was carried out at 42 °C for at least 5-6 h with 50% (v/v) formamide, 5× Denhardt's solution (1× Denhardt's solution: 0.02% each of bovin serum albumin, Ficoll, and polyvinylpyrrolidone), 1% SDS, and 10 µg/ml salmon sperm DNA. Hybridization with ³²P-random-primed labeled proto-trkA full-length probe was carried out at 42 °C for 18 h in the same solution used for prehybridization. Unbound probe was washed from the membranes by treating twice at room temperature for 30 min with 2× SSC (1× SSC: 0.15 M sodium chloride, 0.015 M sodium citrate) plus 0.1% SDS. E25427 cells were used as a positive control. mRNA load, integrity, and retrotranscription success were demonstrated by human -actin gene amplification using the two primers (+) 5'-CATCGTGGGCCGCTCTAGGCA-3' and () 5'-CCGGCCAGCCAAGTCCAGACG-3'. An amplicon length of 482 base pairs was expected using the following conditions. After an initial denaturation at 95 °C for 4 min, 30 cycles at 95 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min were carried out, followed by a final extension at 72 °C for 10 min. Amplified DNA was visualized under a UV lamp after separation in 2% agarose gel.

Flow Cytometric Analysis-- Indirect immunofluorescence assay was performed on live cells using purified mAb (10 µg/ml) or ascitic fluid (diluted 1:100 in PBS, 1% bovine serum albumin). Cells were incubated with 100 µl of antibody for 30 min at 37 °C, washed twice, and incubated with FITC-labeled goat anti-mouse Ig (1:100) (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) for 30 min at 0 °C. After a final wash, cells were suspended in PBS. Fluorescence was evaluated by a FACScan using LYSYS II software (Becton Dickinson, Mountain View, CA).

[³H]Thymidine Incorporation-- Cells were grown to 60% confluency in 96-well plates and maintained in serum-free medium for 24 h. Cells were treated with medium containing 100 ng/ml NGF or 10% FCS for 24 h and labeled with 1 µCi/well of [methyl-³H]thymidine (Amersham Pharmacia Biotech) during the last 4 h. Cell monolayers were washed twice with ice-cold PBS, precipitated with 10% trichloroacetic acid, and solubilized in 100 µl of 0.2 N NaOH, 1% SDS. Lysates were neutralized with 100 µl of 0.2 N HCl, and incorporated radioactivity was quantitated by scintillation counting.

Immunoprecipitation and Western Blot Analysis-- Cells were incubated with K252a or mAb MGR12 for 1 h and with 100 ng/ml NGF or with PBS alone for 5 min at 37 °C. Cells were then washed with cold PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton, 10% glycerol, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). The crude lysate was centrifuged at 13,000 rpm for 10 min at 4 °C. For immunoprecipitation, 2 mg of precleared lysate was reacted with mAb MGR12, MGR2, or MGR1, all previously conjugated to protein A/G-Sepharose for 3 h at 4 °C. Immunoprecipitates were washed three times with 1 ml of washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton, 10% glycerol, and the same protease and phosphatase inhibitors as in the lysis buffer) and eluted with Laemmli buffer. Samples were then boiled for 5 min at 95 °C.

Total cell lysates (50 µg/lane) or immunoprecipitates were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE), and proteins were blotted to nitrocellulose membranes (Amersham Pharmacia Biotech). After blocking with Blotto solution (5% low fat dry milk in PBS), filters were probed with antibodies, and proteins were visualized with peroxidase-coupled secondary antibody using the ECL detection system (Amersham Pharmacia Biotech). Filters were stripped in buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol for 30 min at 65 °C, washed three times in PBS, blocked, and reprobed with the indicated antibodies.

Kinase Assay-- Immunoprecipitates with mAb MGR2 were washed three times with washing buffer and once in kinase buffer (25 mM Hepes, 12.5 mM MgCl₂, 1.25 mM dithiothreitol, 1 mM sodium orthovanadate, and 1.25 mM EGTA). Each pellet was incubated with 50 µl of kinase buffer containing 20 µM ATP and 5 µCi of [-³²P]ATP (Amersham Pharmacia Biotech) for 15 min at room temperature. The reaction was terminated by the addition of 50 µl of 2× Laemmli sample buffer. Samples were boiled, electrophoresed in an 8% polyacrylamide gel, and autoradiographed. p185^HER2 was quantified by Western blot with c-neu mAb Ab3.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Proto-TrkA in Breast Carcinomas-- Breast carcinoma surgical specimens and cell lines were analyzed for the presence of proto-trkA transcript via amplification of the corresponding cDNA using a primer pair of the extracellular domain of this high affinity NGF receptor. Specificity of the transcripts was tested by Southern blot of PCR products with the full-length probe for proto-trkA. Load and integrity of mRNA was controlled by PCR amplification of the human -actin gene. All of the four cell lines tested and 12 of 14 human breast carcinoma specimens (Fig. 1a) expressed the proto-trkA transcript. Immunofluorescence assay with mAb MGR12 on live cells from breast carcinoma lines revealed low level proto-TrkA expression on the cell surface of three of the four lines tested (SKBr3, MDAMB453, and DU4475) (Fig. 1b). Immunoprecipitation of total cell lysates from cell lines using mAb MGR12, followed by Western blot with anti-proto-TrkA polyclonal antibody confirmed the presence of gp140^trk in the three cell lines positive in immunofluorescence (Fig. 1c, lanes 1, 2, and 4) and the complete negativity of MDAMB361 line (Fig. 1c, lane 3).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Detection of proto-TrkA on breast carcinoma cells. a, total RNA (2 µg) from SKBr3 (lane 2), MDAMB453 (lane 3), MDAMB361 (lane 4), and DU4475 (lane 5) breast cancer cell lines and 14 different breast carcinoma specimens (lanes 6-19) was reverse-transcribed and analyzed for the presence of proto-trkA mRNA by amplification of the corresponding cDNA using a primer pair of the receptor extracellular domain. PCR products were electrophoresed, transferred to nylon Hybond-N membrane, and probed with proto-trkA full-length probe. Lane 1 shows transcript present in E25427 positive control cells. 5 µl of each cDNA, except murine E25427 cells, was used to amplified human -actin gene to justify the comparability of amplification results of the proto-trkA-specific target region and the load and integrity of mRNAs. b, SKBr3, MDAMB453, MDAMB361, and DU4475 breast carcinoma cell lines were analyzed by indirect immunofluorescence using mAb MGR12 and FITC-conjugated goat anti-mouse Ig. Light lines indicate background values. Panel c, extracts of SKBr3 (lane 1), MDAMB453 (lane 2), MDAMB361 (lane 3), and DU4475 (lane 4) were immunoprecipitated (IP) with mAb MGR12. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis was performed using an anti-proto-TrkA polyclonal antibody. bp, base pairs.

Effect of NGF on Cell Proliferation-- [³H]Thymidine uptake (Fig. 2a) using cell lines treated with NGF for 24 h revealed significant growth stimulation of SKBr3 and MDAMB453 cell lines, which express both proto-TrkA and p185^HER2, another tyrosine kinase receptor. On the contrary, no significant cell growth increase was observed in DU4475 cells expressing proto-TrkA but not p185^HER2 and in the MDAMB361 cell line, which vice versa presents a large amount of p185^HER2 but is negative for proto-TrkA expression (Fig. 1b and 2b). NGF-induced cell proliferation resulted that was lower than that of cells treated with medium containing 10% FCS, indicating that other factors together with NGF contribute to breast carcinoma cell growth.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of NGF on cell proliferation. a, SKBr3, MDAMB453, MDAMB361, and DU4475 cells maintained in serum-free medium for 24 h were treated with medium containing 100 ng/ml NGF (filled bars) or 10% FCS (black bars) or not treated (open bars) for 24 h and labeled with [methyl-3H]thymidine (1 µCi/well) during the last 4 h. Cell monolayers were treated with 10% trichloroacetic acid, and incorporated radioactivity in lysates was quantitated by scintillation counting. Results are expressed as the mean ± S.D. of three separate experiments. *, statistically significant (p < 0.05) as determined using Student's t test. b, SKBr3, MDAMB453, MDAMB361, and DU4475 breast carcinoma cell lines were analyzed by indirect immunofluorescence using mAb MGR2 and FITC-conjugated goat anti-mouse Ig. Light lines indicate background values.

MAPK Activation and Role of Different Proto-TrkA Regions in This Activation-- Western blot analysis of MAPK phosphorylation in SKBr3, MDAMB361, and DU4475 revealed an increase in MAPK activity upon NGF treatment only in cell lines expressing proto-TrkA (SKBr3 and DU4475), whereas in MDAMB361 cells, no MAPK activation was detected. K252a, an inhibitor of proto-TrkA kinase activity, in DU4475 cells inhibited this activation (Fig. 3a). In SKBr3 cells, which showed the highest NGF-induced proliferation, MAPK activation was blocked by pretreament of cells with an excess of mAb MGR12 directed against the extracellular domain of gp140^trk (Fig. 3b, lane 4). This activation was not inhibited by K252a (Fig. 3a and 3b, lane 3), suggesting the involvement of other membrane kinases in the activation of MAPK.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Effect of NGF treatment on MAPK activation. a, SKBr3, MDAMB361, and DU4475 cells treated with K252a or not treated were stimulated for 5 min with 100 ng/ml NGF or with 10% FCS or not stimulated. Total lysates were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis was performed using anti-phospho-p44/42 MAPK polyclonal antibody. The filter was stripped and reprobed with anti-p44/42 MAPK polyclonal antibody. b, SKBr3 cells treated with K252a (lane 3) or mAb MGR12 (lane 4) or not treated (lanes 1 and 2) were stimulated with 100 ng/ml of NGF for 5 min (lanes 2-4) or not stimulated (lane 1). Total lysates were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis was performed using anti-phospho-p44/42 MAPK polyclonal antibody. The filter was stripped and reprobed with anti-p44/42 MAPK polyclonal antibody.

Effect of NGF on Proto-TrkA and p185^HER2 Receptor Activation-- Because SKBr3 cells overexpress p185^HER2, the status of tyrosine phosphorylation was determined for both gp140^trk and p185^HER2 receptors in these cells after treatment with NGF. As detected by Western blot analysis of anti-proto-TrkA immunoprecipitates with anti-Tyr(P) antibody, tyrosine phosphorylation of NGF receptor was increased after treatment with NGF (Fig. 4a, lane 2). K252a was found to inhibit gp140^trk phosphorylation (Fig. 4a, lane 3). A significant increase in p185^HER2 tyrosine phosphorylation was also observed as shown by anti-Tyr(P) Western blotting of anti-p185^HER2 immunoprecipitates (Fig. 4b, lane 2), which, on the contrary, was not blocked by pretreatment of the cells with K252a (Fig. 4b, lane 3). Analysis to estimate intrinsic p185^HER2 kinase activity using anti-p185^HER2 immunoprecipitates incubated with [-³²P]ATP indicated that NGF stimulated p185^HER2 kinase activity (Fig. 4c, lane 2) and that K252a treatment did not affect this activation (Fig. 4c, lane 3).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of NGF on activation of proto-TrkA and p185HER2. a, extracts of SKBr3 cells treated with K252a (lane 3) or not treated (lanes 1 and 2) and stimulated with 100 ng/ml NGF for 5 min at 37 °C (lanes 2 and 3) or not stimulated (lane 1) were immunoprecipitated (IP) with mAb MGR12. Immunoprecipitates were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed using anti-Tyr(P) mAb. The filter was stripped and reprobed with anti-TrkA polyclonal antibody. b, extracts of SKBr3 cells treated with K252a (lane 3) or not treated (lanes 1 and 2) and stimulated with 100 ng/ml of NGF for 5 min at 37 °C (lanes 2 and 3) or not stimulated (lane 1) were immunoprecipitated with mAb MGR2. Immunoprecipitates were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed using anti-Tyr(P) mAb. The filter was stripped and reprobed with c-neu mAb Ab3. c, extracts of SKBr3 cells treated with K252a (lane 3) or not treated (lanes 1 and 2) and stimulated with 100 ng/ml of NGF for 5 min at 37 °C (lanes 2 and 3) or not stimulated (lane 1) were immunoprecipitated with mAb MGR2. Immunoprecipitates were incubated in the presence of 5 µCi of [-32P]ATP at RT for 15 min. Immunocomplexes were electrophoresed in an 8% polyacrylamide gel and autoradiographed.

Coprecipitation of p185^HER2 with Proto-TrkA-- Immunoprecipitation of soluble extracts obtained from SKBr3 cells, either treated with NGF or untreated, was performed with mAb to proto-TrkA, p185^HER2, and EGF receptor. Western blot analysis of immunoprecipitated proteins using anti c-erbB2 mAb revealed p185^HER2 in the material immunoprecipitated with anti-trkA mAb (Fig. 5a, lane 1). The amount of p185^HER2 found in the TrkA immunoprecipitate did not increase after NGF treatment (Fig. 5a, lane 2). Western blot analysis of the immunoprecipitated proteins using anti-TrkA polyclonal antibody revealed gp140^trk in the material immunoprecipitated with anti-p185^HER2 mAb (Fig. 5b, lane 1), whereas no co-immunoprecipitation of gp140^trk was found with mAb directed to EGF receptor (Fig. 5c, lanes 1 and 2). Proto-TrkA was not detectable in p185^HER2 immunoprecipitate from soluble extracts of cells treated with NGF (Fig. 5b, lane 2). It is important to note that p185^HER2 in anti-TrkA immunoprecipitates was detectable after a few minutes of filter exposure using the ECL detection system, whereas gp140^trk in anti p185^HER2 immunoprecipitates was detectable only after 40 min of filter exposure. This may depend on a low number of gp140^trk molecules compared with highly overexpressed p185^HER2 receptors.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Co-precipitation of p185HER2 with proto-TrkA. Extracts of SKBr3 cells treated with 100 ng/ml of NGF for 5 min at 37 °C (lane 2) or not treated (lane 1) were immunoprecipitated (IP) with mAb MGR12 (a), mAb MGR2 (b), or mAb MGR1 (c). Immunoprecipitates were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed using c-neu mAb Ab3 (a) or anti-trkA polyclonal antibody (b and c). The filters were stripped and reprobed with anti-TrkA polyclonal antibody (a), c-neu mAb Ab3 (b), or EGF receptor mAb Ab7 (c).

p185^HER2-dependent NGF Activation-- To examine the role of p185^HER2 in the activation of TrkA induced by NGF, a gene construct that encodes a single chain antibody directed against the p185^HER2 extracellular domain was expressed intracellularly in SKBr3 and MDAMB453 breast carcinoma cells, which express both proto-TrkA and p185^HER2. p185^HER2 expression was down-modulated in a MDAMB453-transfected subline (MDAMB453-5R) as compared with the mock-transfected cells (MDAMB453 LBSN) (Fig. 6a). On the contrary, for the SKBr3 cell line, which highly overexpresses p185^HER2, no subline showing significant inhibition of oncoprotein expression was obtained (data not shown). Treatment of the p185^HER2-expressing MDAMB453 LBSN cells with NGF induced an increase in MAPK activation (Fig. 6b, lane 2), whereas no increase was detectable in the transfected cells in which p185^HER2 was down-modulated (Fig. 6b, lane 4). Cell proliferation analysis by [³H]thymidine uptake revealed that the stimulation of MDAMB453 LBSN proliferation upon NGF treatment was not observed in the subline showing p185^HER2 down-modulation (Fig. 6c).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   p185HER2 down-modulation and NGF-induced signal. a, MDAMB453 LBSN and MDAMB453-5R cells were analyzed by indirect immunofluorescence using mAb MGR12 (dark line) or MGR2 (gray line) and FITC-conjugated goat anti-mouse Ig. Light lines indicate background values. b, MDAMB453 LBSN (lanes 1 and 2) and MDAMB453-5R (lanes 3 and 4) cells were stimulated with 100 ng/ml of NGF for 5 min (lanes 2 and 4) or not stimulated (lanes 1 and 3). Total lysates were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using anti-phospho-p44/42 MAPK polyclonal antibody. The filter was stripped and reprobed with anti-p44/42 MAPK polyclonal antibody. c, MDAMB453 LBSN and MDAMB453-5R cells maintained in serum-free medium for 24 h were treated with 100 ng/ml of NGF (filled bars) or not treated (open bars) for 24 h and labeled with [methyl-3H]thymidine (1 µCi/well) during the last 4 h. Cell monolayers were treated with 10% trichloroacetic acid, and incorporated radioactivity in lysates was quantitated by scintillation counting. Results are expressed as the mean ± S.D. of three separate experiments. *, statistically significant (p < 0.05) as determined using Student's t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study demonstrates that NGF-induced activation of breast carcinoma cells growth requires co-operation between the proto-TrkA receptor and p185^HER2 in assembling the signal transduction machinery. In keeping with a previous study (3), we detected proto-trkA mRNA expression in all four breast cancer cell lines and 86% of surgical specimens from primary breast carcinomas, raising the possibility that NGF plays a role in breast cancer progression. The absence of detectable gp140^trk in a cell line showing proto-trkA transcript (MDAMB361) is probably due to a rapid degradation of the receptor or a mechanism that inhibits mRNA translation in these breast carcinoma cells. However, preliminary analysis of protein expression so far carried out in 3 of 14 breast carcinoma specimens revealed the presence of gp140^trk in all of the tested cases, indicating the presence in vivo of the membrane receptor. Consistent with the role of NGF in breast carcinoma progression and according with the data reported by Descamps et al. (3) is our finding that NGF is mitogenic for human breast cancer cell lines through MAPK activation. However, NGF stimulated growth in only two out of three breast carcinoma cell lines expressing similar levels of proto-TrkA. In one of these cell lines (SKBr3), which express p185^HER2, a membrane tyrosine kinase receptor of a different family (28-30), NGF treatment activated both proto-TrkA and p185^HER2 receptor phosphorylation, stimulating also the intrinsic kinase activity of c-erbB2-encoded receptor. It is possible that NGF binding to its receptor induces receptor clustering at the plasma membrane and consequent activation of p185^HER2 overexpressed by these tumor cells. Indeed, p185^HER2 coprecipitated with gp140^trk, indicating that these molecules are structurally associated on the cell membrane with the possibility of trans-activation. The large amount of p185^HER2 already associated with gp140^trk in cells not treated with NGF, which does not increase upon NGF treatment, suggests a basal co-activation between the two receptors. Such a conclusion is further proved by detection of gp140^trk in p185^HER2 immunoprecipitate. The low detectability of gp140^trk associated with p185^HER2 is probably due to the low level of gp140^trk in comparison with the overexpressed p185^HER2. The disappearance of coprecipitated gp140^trk upon NGF treatment is consistent with the processes of internalization of the NGF receptor after interaction with its own growth factor. The cooperation between the two receptors also explains why K252a, an inhibitor of TrkA tyrosine kinase activity, does not significantly inhibit NGF-induced cell activation, measured as MAPK activation. In fact, the signaling pathways of proto-TrkA and of p185^HER2 are both mediated through MAPK. The lack of K252a inhibition of MAPK and p185^HER2 activation indicates that gp140^trk kinase activity is not required for NGF-mediated p185^HER2 activation. On the contrary, inhibition of MAPK activation by anti-proto-TrkA mAb, which was also found to impair NGF-induced p185^HER2 phosphorylation (data not shown), indicates that proto-TrkA provides the initial signal, since NGF must bind to the extracellular domain of gp140^trk to trigger p185^HER2 activation. This evidence suggests that the two receptors, even if belonging to different families, work together by dimerizing. Consistent with this conclusion are the results of the kinase assay that indicates NGF stimulates the kinase activity of p185^HER2 also upon cell treatment with K252a. However, the possibility remains that interaction between the two receptors occurs through other still unknown molecules. The lack of co-precipitation of gp140^trk with EGF receptor seems to exclude the possibility that NGF, upon interaction with TrkA, induces an aspecific receptor clustering at the plasma membrane and consequently, the heterodimerization and activation of p185^HER2 with receptors of the HER family. The recruitment of p185^HER2, which is highly overexpressed in some breast cancer cells, leads to amplification of the NGF-induced signal. Apparently, only after this amplification does the signal become relevant for tumor cell proliferation and detectable through analysis of MAPK activation.

Together, our results show that overexpression of the p185^HER2 receptor is required in generating NGF-induced biological signals relevant for the growth of cells expressing low levels of TrkA, such as breast cancer cells. The observation that down-modulation of p185^HER2 overexpression impairs NGF-induced signaling is consistent with this conclusion.

Cross-talk between receptors of the same family has been widely documented (31-33). However, our data indicate an interaction between receptors of different families, leading to amplification of a growth factor-mediated response. It remains unknown whether this cross-talk represents a physiological interaction or instead occurs only in cells that pathologically overexpress a receptor.

The simultaneous expression of p185^HER2 and the NGF receptor, detected in 4 of 12 proto-TrkA-positive breast carcinoma specimens, might confer a proliferative advantage to breast carcinoma cells in the presence of NGF in tissue surrounding the tumor. Although little is known about NGF expression in human breast tissues, our data indicate that this growth factor, produced by targets of sympathetic innervation and found in circulating blood (34) might activate p185^HER2, one of the most important receptors involved in the growth regulation of breast carcinoma cells.

	ACKNOWLEDGEMENTS

We thank Dr. Marco Pierotti and Dr. Elena Ardini for critical comments, Piera Aiello for excellent technical assistance, and Laura Mameli for manuscript preparation.

	FOOTNOTES

^* This work was supported in part by grants from FIDIA and Associazione Italiana per la Ricerca sul Cancro.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Molecular Targeting Unit, Dept. of Experimental Oncology, Istituto Nazionale Tumori, via Venezian 1, 20133 Milano, Italy. Tel.: 39-02-2390571; Fax: 39-02-2362692; E-mail: menard@istitutotumori.mi.it.

	ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; EGF, epidermal growth factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				E. Adriaenssens, E. Vanhecke, P. Saule, A. Mougel, A. Page, R. Romon, V. Nurcombe, X. Le Bourhis, and H. Hondermarck Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer Cancer Res., January 15, 2008; 68(2): 346 - 351. [Abstract] [Full Text] [PDF]

				E. Com, C. Lagadec, A. Page, I. El Yazidi-Belkoura, C. Slomianny, A. Spencer, D. Hammache, B. B. Rudkin, and H. Hondermarck Nerve Growth Factor Receptor TrkA Signaling in Breast Cancer Cells Involves Ku70 to Prevent Apoptosis Mol. Cell. Proteomics, November 1, 2007; 6(11): 1842 - 1854. [Abstract] [Full Text] [PDF]

				B. Davidson, P. Lazarovici, A. Ezersky, J. M. Nesland, A. Berner, B. Risberg, C. G. Trope, G. B. Kristensen, M. Goscinski, G. van de Putte, et al. Expression Levels of the Nerve Growth Factor Receptors TrkA and p75 in Effusions and Solid Tumors of Serous Ovarian Carcinoma Patients Clin. Cancer Res., November 1, 2001; 7(11): 3457 - 3464. [Abstract] [Full Text] [PDF]

				S. Descamps, V. Pawlowski, F. Revillion, L. Hornez, M. Hebbar, B. Boilly, H. Hondermarck, and J.-P. Peyrat Expression of Nerve Growth Factor Receptors and Their Prognostic Value in Human Breast Cancer Cancer Res., June 1, 2001; 61(11): 4337 - 4340. [Abstract] [Full Text] [PDF]

				O. Tikhomirov and G. Carpenter Geldanamycin Induces ErbB-2 Degradation by Proteolytic Fragmentation J. Biol. Chem., August 18, 2000; 275(34): 26625 - 26631. [Abstract] [Full Text] [PDF]

				S. Descamps, R.-A. Toillon, E. Adriaenssens, V. Pawlowski, S. M. Cool, V. Nurcombe, X. Le Bourhis, B. Boilly, J.-P. Peyrat, and H. Hondermarck Nerve Growth Factor Stimulates Proliferation and Survival of Human Breast Cancer Cells through Two Distinct Signaling Pathways J. Biol. Chem., May 18, 2001; 276(21): 17864 - 17870. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tagliabue, E. Articles by Ménard, S. Search for Related Content PubMed PubMed Citation Articles by Tagliabue, E. Articles by Ménard, S. Social Bookmarking                      What's this?