An array of polypeptide growth factors contribute to the development of breast cancer, the most common tumor-related cause of death in women of Western countries. Therefore, breast cancer therapy should be aimed at inhibition of growth factor-dependent breast cancerous cell proliferation. However, the relative contribution of each individual factor in the development and maintenance of the transformed phenotype is largely unknown. Here we report for the first time that the proliferative effects of nerve growth factor, (NGF) a typical neurotrophin, are similar to those of epidermal growth factor (EGF) and insulin-like growth factor II, and are enhanced by 17β-estradiol in the human breast cancer cell line MCF-7. The effect of NGF appeared to be mediated by its trkA receptors (trkANGFR), as suggested by the potent inhibition of both MCF-7 cell proliferation and trkANGFR phosphorylation occurring upon treatment of cultures with the selective trkANGFR inhibitor K252a. Surprisingly, the antiestrogen drug tamoxifen (TAM) inhibited NGF-induced MCF-7 cell proliferation and trkANGFR phosphorylation in a concentration-related fashion. The effect of TAM seemed to be estrogen receptor-independent, because the pure estrogen receptor antagonist ICI 182.780 was unable to block NGF-induced trkANGFR phosphorylation. Our data underline the new emerging role of trkANGFR in breast tumor growth, and suggest a related novel therapeutic use of TAM in breast cancer.

Breast cancer is the most common cause of tumor-related death among women in the Western world (1). Transformation of normal breast cells, initiation, and maintenance of breast cancer growth all depend upon the interplay of a number of inhibitory and stimulatory factors, including estrogens, and an array of members of the cytokine/growth factor superfamily. The dedifferentiating and proliferative effects of each single factor are mediated by specific receptors (2). In addition, it is likely that breast cancer development and growth possibly require the concerted intervention of two or more of these growth factors (3). Indeed, it has been reported that estrogens may enhance the effects of EGF4 or of IGF-I and -II (4, 5).

NGF, a member of the neurotrophin family of growth factors, is a candidate pleiotropic agent that participates in a number of relevant biological processes other than neuronal differentiation and growth (6) including the proliferation of cancerous cells (7, 8).

The major direct evidence that the trkANGFR induces cell proliferation was achieved upon transfection of a variety of neuronal and non-neuronal cells, resulting in strong proliferation upon NGF treatment (9).

The working concept today is that trkANGFR serves as an autocrine loop to regulate cell proliferation (10). For instance, trkANGFR, along with the other NGF receptor, p75NTR, have been proposed to participate in the paracrine cross-talk between stromal and epithelial cells in the human prostate (11, 12), suggesting that NGF contributes to the genesis of androgen-dependent prostatic cancer. Indeed, it appears that the trkANGFR plays the prominent role in mediating the effects of NGF on prostatic cancerous cell proliferation (13). In this line, the trkANGFR inhibitors of the indol-carbazole series, such as K252a and CEP 7511, inhibit the effects of NGF (14), including NGF-stimulated tyrosin kinase activity (9), as well as prostate tumor growth in vivo in the rat (15). Recently, a trkANGFR-mediated mitogenic effect of NGF has also been described on different lines of breast cancer cells (16).

Therefore, we found it of interest to investigate the role of NGF in stimulating the proliferation of the human breast cancer cell line MCF-7, focusing on (a) the compared effects of NGF, EGF, and IGF-II on MCF-7 breast cancer cell proliferation, and the role of trkANGFR; and (b) the possibility of pharmacological inhibition of NGF-induced MCF-7 cell proliferation by antiestrogens.

In fact TAM, an antiestrogen used in the treatment of ER+ breast cancer (17), has also been successfully used as an agent in various estrogen-independent tumors (18, 19). Clinical data are supported by in vitro studies showing the effectiveness of TAM in inhibiting the proliferation of ER breast cancer cells (20). In addition, TAM has been shown to interfere with tyrosine phosphorylation promoted by IGF-I in breast cancer cell lines (21).

Thus, we finally decided to evaluate further the effects of TAM on NGF-induced MCF-7 human breast cancer cell proliferation and trkANGFR tyrosine residue phosphorylation.

Materials.

Tissue culture medium, FCS, and other supplies were purchased from Life Technologies Italia (Milan, Italy). E2, IGF-I, EGF, aprotinin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, glycerol, Triton X-100, BSA, and TAM were purchased from Sigma Chemicals (Milan, Italy). NGF and the tyrosine kinase inhibitor K252a were obtained from Calbiochem, Novabiochem (San Diego, CA). The pure ER antagonist ICI 182.780 was a kind gift from Prof. Alvin M. Kaye, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Normal rabbit serum, biotinylated goat antirabbit IgG, avidin-biotin complex, and 3,3′-diaminobenzidine kit were obtained from Vector (Burlingame, CA). IgG-agarose beads were purchased from Pharmacia Biotech AB (Uppsala, Sweden); second antibodies conjugated to horseradish peroxidase and ECL reagent were purchased from Amersham Life Science (Buckinghamshire, United Kingdom). The anti-pan-trkANGFR 203 antibody was the kind gift of Prof. David Kaplan, McGill University, Montreal Neurological Institute, Montreal, Canada (10). The monoclonal anti-phosphotyrosine antibody 4G10 (UBI, New York,) was a kind gift from Dr. Oreste Segatto, Istituto Regina Elena, Rome, Italy. Moloney murine leukemia virus-reverse transcriptase and Taq polymerase were purchased from Life Technologies Italia.

Cell Culture.

PC12 cells, originally from NIH, were cultured as described previously (9) in DMEM without sodium piruvate and with 7.5% horse serum and 7.5% FCS, penicillin (100 units/ml), and streptomycin (100 μg/ml). The human breast cancer MCF-7 cells were provided by Dr. Daniela Callari, Department of General Pathology, University of Catania, Italy. The cells were cultured in DMEM (with sodium pyruvate), supplemented with 10% FCS, penicillin and streptomycin. MCF-10 noncancerous mammary cells (from Dr. Antonino Belfiore, Institute of Internal Medicine and Endocrine and Metabolic Diseases, University of Catania School of Medicine, Catania, Italy), were cultured in DMEM/F12 medium supplemented with 5% horse serum, penicillin (100 units/ml), streptomycin (100 μg/ml), hydrocortisone (0.5 μg/ml), cholera toxin and (0.1 μg/ml) to stimulate cAMP formation, insulin (10 μg/ml), and EGF (0.02 μg/ml).

Cells were grown in 25-cm2 or 75-cm2 flasks with 10% FCS-DMEM, containing antibiotics, in a water-jacketed incubator at 37°C in a 5% CO2 atmosphere.

Proliferation Assay.

Cells were plated at a density of 5 × 104 in 35-mm plastic Petri dishes in a volume of 5 ml/dish. After 12 h, the medium was replaced with 3 ml of fresh DMEM (1% FCS), containing graded concentrations of E2 (10 pm to 100 nm), IGF-II (5, 10, 15, 20, and 25 ng/ml), EGF (5, 10, 15, 20, and 25 ng/ml), NGF (5, 10, 15, 20, and 25 ng/ml), or TAM (60 nm). In other experiments, TAM was used alone or in combination with E2 (1 nm), EGF (25 ng/ml), or NGF (25 ng/ml). The pure ER antagonist ICI 182.780 was used at a concentration of 60 nm. In the experiments for trkANGFR inhibition, the selective trkANGFR inhibitor K252a was used at a concentration of 200 nm(14). Cells were grown for 7 days and counted daily. Adherent cells were detached by rapid trypsinization. An adequate volume of medium containing trypan blue was added. Then cells were counted in a Hausser chamber. Experiments were performed in triplicate, and each experiment was repeated at least twice.

Immunohistochemistry.

Immunohistochemical analysis was performed on untreated, subconfluent cells cultured in 35-mm Petri dishes. Cells were fixed with 2% glutaraldehyde in Hanks’ balanced salt solution (pH 7.3) diluted 1:1 with culture medium. After 5 min, the mixture was removed and replaced with undiluted 2% glutaraldehyde solution. After 10 min, cells were washed twice for 3 min with PBS. Then a blocking buffer (PBS containing 5% normal goat serum) was added to the dishes for 15 min at room temperature. Cells were incubated with a rabbit polyclonal anti-trkANGFR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:100 in blocking buffer at room temperature, and shaken gently for 60 min. In control experiments, primary antiserum was replaced by normal rabbit serum. Cells were washed twice for 3 min with PBS and then incubated with biotinylated goat antirabbit IgG (1:200 in blocking buffer) and shaken slowly for 60 min at room temperature. After two 3-min washes with PBS, fresh avidin-biotin complex solution (Vector) was added for 30 min. Finally, cells were washed again with PBS, and immunoreactivity was visualized using the diaminobenzidine method according to the manufacturer’s instructions (Vector).

Immunoprecipitation Assay.

For signal transduction 2experiments, MCF-7 cells were grown for 3 days in 60-mm Petri dishes in 1% FCS-DMEM containing EGF and NGF alone or in combination with TAM. Upon termination of the experiments, cell proteins were extracted in lysis buffer [150 mm NaCl, 50 mm Tris-HCl (pH 7.5), 5 mm EDTA, 1 mm Na3VO4, 30 mm NaPPi, 50 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin, 10% glycerol, and 0.2% Triton X-100]. Cell lysates were stored at −80°C until used for immunoprecipitation assay.

Cell extracts were centrifuged for 15 min at 15,000 g at 4°C. The supernatants were incubated overnight at 4°C with anti-pan-trkANGFR 203 and rabbit polyclonal anti-p75NTR (sc-8317; Santa Cruz Biotechnology, Inc.). The complexes were precipitated with antimouse IgG-agarose beads for 1 h at 4°C. After 3 washes with 500 μl of lysis buffer, the precipitated material was eluted from the beads by boiling in 20 μl of elution buffer [20% glycerol, 10% 2-mercaptoethanol, 4.6% SDS, 0.125 m Tris (pH 6.8)], subjected to 10% SDS-PAGE under reducing conditions, and then transferred onto a Hybond ECL nitrocellulose membrane (Amersham Life Science) for 45 min at a fixed current of 150 mA. Membranes were blocked in 200 mm NaCl, 50 mm Tris (pH 7.5), and 1% BSA overnight at 4°C.

Immunoblot Analysis.

After blocking procedure, the membranes were incubated for 2 h at room temperature with a specific primary antibody in antibody buffer [50 mm Tris (pH 7.6), 200 mm NaCl, 0.05% Tween 20, and 1% BSA]. Then the blots were washed 3 times for 10 min in washing buffer [50 mm Tris (pH 7.6), 200 mm NaCl, 0.05% Tween 20] before incubation for 45 min with the specific secondary antibody conjugated to horseradish peroxidase diluted 1:4000 in blocking buffer at room temperature. After three 10-min washes, the blots were incubated with ECL reagent for 1 min before exposure to Kodak X-Omat AR film (10–60 min).

To analyze trkANGFR tyrosine phosphorylation, two aliquots of the same sample were processed as follows. The first aliquot was immunoprecipitated with anti-trkANGFR rabbit polyclonal antibody (sc118; Santa Cruz Biotechnology); the second aliquot was immunoprecipitated with the monoclonal anti-phosphotyrosine (PY) antibody. After SDS-PAGE and electroblotting, the membranes were blocked with 1% BSA and incubated with anti-trkANGFR antibody for 2 h at 4°C, rinsed four times, and incubated with horseradish peroxidase-conjugated antirabbit IgG (second antibody) for 45 min at room temperature. The membranes were again washed four times for 15 min and exposed to ECL.

Reverse Transcriptase-PCR.

Total RNA from cells grown to confluence was isolated after solubilization in guanidinium thiocyanate by phenol-chloroform extraction and precipitation (22). For first-strand cDNA synthesis, 5 μg of total RNA was reverse-transcribed using 25 μg/ml oligo(dT)12–18 primer, in a final volume of 20 μl, in the presence or absence of 200 units of Moloney murine leukemia virus reverse transcriptase. After first heating at 70°C for 15 min, the reaction mixture was carried out at 42°C for 1 h and subsequently heated for an additional 5 min at 95°C. PCR was performed in a total volume of 25 μl containing 1 μl of the cDNA reaction mixture, 5 pmol of each upstream and downstream primer, and 1.2 units of Taq polymerase. The cycle program for each pair of primers consisted of 40 runs of denaturation at 94°C for 45 s, annealing at 62°C for 1 min, and elongation at 72°C for 1 min. The cycle program was preceded by an initial denaturation at 94°C for 3 min before a final extension at 72°C for 10 min. PCR products were analyzed by 1.5% agarose gel electrophoresis and visualized with ethidium bromide. The following RNA transcripts were detected via amplification of the corresponding cDNAs: (a) glyceraldehyde-3-phosphate dehydrogenase using the primer set composed of the sense 5′-TAGACAAGATGGTGAAGG and the antisense primer 5′-TCCTTGGAGGCCATGTAG, yielding an amplicon of 1006 bp; (b) the p75NTR using a primer pair, already described (23), composed of the sense primer 5′-AGCCACCAGACCGTGTGTG and the antisense primer 5′-TTGCAGCTGTTCCACCTCTT, yielding a 663-bp PCR product; and (c) trkANGFR using a primer pair described previously (24), composed of the sense primer 5′-CCATCGTGAAGAGTGGTCTC and the antisense primer 5′-GGTGACATTGGCCAGGGTCA, with an expected amplicon length of 476 bp. Amplification of glyceraldehyde-3-phosphate dehydrogenase was used to judge DNA contamination in the RNA samples examined and as a reference for estimating the level of NGF receptor mRNAs in the different cell lines.

Statistical Analysis of Results.

Results were analyzed by one-way ANOVA, followed by Fisher’s least significant difference test. P < 0.05 was considered significant.

NGF Proliferative Effects on MCF-7 and MCF-10 Cells.

NGF stimulated proliferation of MCF-7 cells in a concentration-dependent manner, with an EC50 value of 7.1 ng/ml (Table 1). We also assessed the relative potencies of other growth factors, compared with that of E2. IGF-II, EGF, and E2 stimulated MCF-7 cell proliferation in a concentration-dependent fashion, but with different potencies. The relative EC50s were 5.0, 10.8, and 0,027 ng/ml, respectively, for IGF-II, EGF, and E2 (Table 1). Coincubation of MCF-7 cells with both EGF and NGF resulted in an additive stimulatory effect on MCF-7 cell proliferation (EC50, 4.4 ng/ml; P < 0.05).

NGF-stimulated MCF-7 cell proliferation was inhibited by treatment with the specific trkANGFR inhibitor K252a (Ref. 14; Fig. 1), suggesting that the NGF high-affinity receptor trkANGFR mediates the NGF proliferative effect. On the other hand, treatment with an equal concentration of K252a did not affect both EGF- and E2-stimulated MCF-7 cell proliferation (data not shown).

In addition, NGF failed to stimulate proliferation when added to MCF-10 human noncancerous breast cells incubated with low, nonproliferative concentrations of EGF (2 ng/ml; Fig. 1).

Analysis of NGF Receptors Protein and mRNA Expression in MCF-7 and MCF-10 Cells.

Western blot analysis showed a band of 75 kDa corresponding to the p75NTR protein in the positive control-transfected PC12 cells (Fig. 2,A, Lane 3), a cell line known to express both NGF receptors (10) as well as in MCF-7 cells (Fig. 2,A, Lane 2). However p75NTR protein expression was undetectable in MCF-10 noncancerous human breast cells (Fig. 2 A, Lane 1).

A 145 kDa molecular mass protein, corresponding to the trkANGFR protein was also present both in MCF-7 cells (Fig. 2,A, Lane 5) and in the positive control PC12 cells (Fig. 2,A, Lane 6). Densitometric analysis revealed a 8- to 9-fold increase in trkANGFR expression in MCF-7 human breast cancerous cells in comparison with the noncancerous human breast epithelial cell line MCF-10 (Fig. 2 A, Lane 4).

Immunohistochemical analysis also revealed the presence of the high-affinity trkANGFR-like immunoreactivity in MCF-7 cells (Fig. 2 B).

Total mRNA was analyzed in PC12, MCF-10, and MCF-7 cells by reverse transcriptase-PCR for the presence of p75NTR and trkANGFR transcripts. The p75NTR transcript was present in either PC12 (Fig. 2,C, Lane 3) or MCF-7 (Fig. 2,C, Lane 2) but not in MCF-10 cells (Fig. 2,C, Lane 1). In addition, all cell lines contained the trkANGFR transcript (Fig. 2 C, Lanes 4, 5 and 6).

Autophosphorylation of the NGF trkANGFR receptors in MCF-7 cells is shown in Fig. 2 D. Indeed, NGF induced tyrosine phosphorylation of these receptors within 10 min, a selective effect that was completely inhibited by K252a at 200 nm. This concentration has been reported previously to be specific to the trkANGFR receptor (14, 25), a property shared with other compounds of the same family (15).

TAM Inhibits Estradiol-, EGF-, and NGF-stimulated MCF-7 Cell Proliferation.

Treatment with the estrogen receptor antagonist TAM resulted in significant inhibition of MCF-7 cell proliferation stimulated by E2 (Fig. 3,A1), NGF (Fig. 3,A2) and EGF (Fig. 3A3 ). The combination of NGF and E2 stimulated MCF-7 cell proliferation additively. Proliferation induced by combined treatment with NGF and E2 was inhibited by pretreatment with TAM (Fig. 3,B). The effect of TAM was compared with that of the pure ER antagonist ICI 182.780; both TAM and, to a lesser extent, ICI were able to inhibit NGF-stimulated MCF-7 cell proliferation (Fig. 3 C). The concentration of TAM and ICI 182.780 used (60 nm) is known not to affect protein synthesis (26).

Effect of TAM and ICI 182.780 on NGF-stimulated trkANGFR Tyrosine Phosphorylation.

To study the possible mechanism of action of TAM and ICI 182.780 as inhibitors of NGF-stimulated MCF-7 cell proliferation, we assessed their effects upon NGF-induced tyrosine phosphorylation of trkANGFR. MCF-7 cells were incubated respectively for 1 and 48 h with TAM and ICI182.780 and then stimulated for 10 min with NGF. No effect on trkANGFR phosphorylation was observable after 1 h of treatment with both drugs (Fig. 4,A). On the other hand, 48 h treatment with TAM, but not ICI182.780, resulted in significant inhibition (90%) of tyrosine phosphorylation of trkANGFR (Fig. 4,B). The expression of unphosphorylated trkANGFR in MCF-7 cells was comparable in all experiments (Fig. 4; A, top, and B, top), suggesting that TAM is effective on receptor activity rather than on its level of expression.

The expression of the NGF p75NTR receptor was not affected by 48 h of treatment with TAM or ICI182.780 (Fig. 4 C).

In additional experiments, we assessed the concentration-dependence of the effect of TAM on trkANGFR phosphorylation, by performing concentration-response experiments. Indeed, TAM inhibited trkANGFR phosphorylation in a concentration-dependent manner. The inhibitory effect of TAM appeared at 60 nm and reached its maximum at 120 nm (Fig. 5).

Here we provide the first evidence that the antiestrogen TAM is able to inhibit NGF-induced MCF-7 human breast cancer cell proliferation in a fashion similar to other breast cancer growth factors, such as EGF and IGF-II (27). Our results support the proposal of Descamps et al.(16) that NGF is a promotion mitogen for breast cancer cells. In addition, we found that the proliferative efficacy of NGF on MCF-7 cells was comparable with those of EGF and IGF-II. Moreover, our experiments indicate that the effect of NGF is additive to that of EGF, supporting the prevalent hypothesis that more than one factor is required at the same time for a breast tumor to reach and maintain its ideal rate of growth (2). The enhancing effect of NGF on MCF-7 breast cancer cell proliferation appears to be mediated by its tyrosine kinase trkANGFR(28). These findings are strongly supported by the ability of the specific trkANGFR inhibitor K252a (14, 25) to selectively block the proliferative effect of NGF on these cells at a concentration that completely inhibited its autophosphorylation at tyrosine residues after NGF stimulation.

The trkANGFR, which we have detected by immunohistochemistry on the surface of MCF-7 cells, has also been regarded among mediators of androgen-dependent cancer growth (e.g., prostate cancer; Ref. 29), perhaps indicating a prominent role for NGF in these tumors. It is of interest to note that NGF produced by normal prostatic stromal cells (11) has been suggested to act as a paracrine growth factor once normal prostatic epithelial cells have turned cancerous (30). In the light of the above reports, it seems plausible to speculate that, whereas androgens in the prostate and, similarly, estrogens in the breast, probably promote and/or support normal cell transformation and proliferation, NGF could act in a subsequent secondary phase to enhance the progression of cancer cell growth. Conceivably, NGF could function as a synchronizing promotion factor, inducing cells in the G0 phase to enter the cell cycle (31).

The notion that NGF is a mitogenic factor for cancerous breast cells might find additional support in the increased expression of trkANGFR that we observed in the cancerous MCF-7 cell line, as compared with the MCF-10 noncancerous human breast cells. On this basis, it seems reasonable that such significantly different expression of the trkANGFR in MCF-7 and MCF-10 could be one of the reasons underlying the lack of proliferative effects of NGF on the latter cell line, and adds convincing evidence to the important role of a high number of trkANGFRs in breast cancer growth.

It has been suggested that p75NTR increases the affinity of the trkANGFR in the presence of low concentrations of NGF in PC12 cells (32). Considered the lack of p75NTR expression in MCF-10 cells, the previous finding could well explain the higher activity of trkANGFR in MCF-7.

We found that the mixed ER agonist/antagonist TAM inhibited estradiol-, EGF-, and NGF-induced MCF-7 human breast cancerous cell proliferation.

Estrogens profoundly affect the organization and the activity of the nervous system (33), and tight relationships have been described between the expression of trkANGFR and ERs (33). In addition, signal transduction cross-talks have been proposed between estrogen and growth factor receptors (34, 35). With this in mind, it was not surprising to observe the inhibitory effect of TAM when used in combination with either estradiol, EGF, or NGF. In fact, clinical work provides evidence that TAM is quite efficient in the control of both estrogen (36), and non-estrogen-dependent breast tumor cell growth (17, 37), a hypothesis that has been verified experimentally in various human malignant cell lines (38), as well as clinically, in protocols for treatment of melanoma (39) and leukemias (40).

In fact, tyrosine residue phosphorylation of the IGF-I receptor in MCF-7 cells is inhibited by TAM (21), a result which goes along with the TAM-dependent inhibition of EGF receptor phosphorylation observed in MCF-7 cells,5 in accordance to Freiss et al.(41), describing that the TAM metabolite 4-hydroxy-TAM decreases autophosphorylation of the EGF receptor in vitro.

Although the bulk of clinical evidence, supported by meta-analysis (42, 43) and chemoprevention (44) data, strongly suggests that TAM is most effective in the treatment of ER+ breast cancer, growing experimental evidence describes an alternate estrogen-independent pharmacological mechanism of action of this antiestrogen. In fact, Kanter-Lewensohn et al.(45) reported that inhibition of proliferation of malignant, ER human melanoma cells by TAM is probably attributable to a direct effect of the antiestrogen with the IGF-I receptor phosphorylation, a concept affirmed previously by Guvakova and Surmacz (21), suggesting inhibition of the IGF-I receptor phosphorylation by TAM in MCF-7 cells. It appears that TAM is also able to inhibit tumor angiogenesis induced by an ER fibrosarcoma in vivo in the rat (46), and estrogen-independent effects of TAM have been shown in several ER+ and ER tumor cell lines, in synergism with IFN-γ (38). In addition, few clinical data support the pharmacological efficacy of TAM in ER-poor patients (47). Thus, our data, taken together with these findings, corroborate the concept that TAM may function in both an estrogen-dependent and an estrogen-independent fashion, leading to the hypothesis of its possible use in ER breast cancer.

It appears that the complete antagonistic effect of TAM requires the presence of estradiol, because the drug acts as a full agonist in the absence of the latter (48). In fact, it has been shown that, despite the initial inhibition of proliferation, prolonged treatment with TAM enhanced tumor growth in nude mice transplanted with wild-type nonconditioned MCF-7 cells (49). The latter effect appears estrogen-dependent, because ICI 164.384, a pure estrogen antagonist, appears to block the effects of TAM (49). These data could also explain, at least in part, the initial proliferation (days 1–3) of MCF-7 breast cancer cells observed in our experiments subsequent to treatment with TAM.

To corroborate the hypothesized estrogen receptor-independent antiproliferative mechanism of TAM on MCF-7 cells, we have shown in additional experiments that the pure ER antagonist ICI 182.780 was significantly less effective than TAM in inhibiting the effect of NGF on breast cancer cell proliferation. At any rate, such an inhibitory effect of ICI 182.780 appeared unrelated to NGF-stimulated trkANGFR tyrosine phosphorylation.

In conclusion, NGF most probably acts as a progression mitogen in conjunction with other growth factors, inducing breast cancer cell proliferation, as suggested by its additive effect in combination with EGF and E2. The proliferative effect of NGF on MCF-7 cells seems to be mediated by its trkANGFR. NGF-induced phosphorylation of tyrosine residues of the trkANGFR can be reduced by TAM, a drug routinely used in breast cancer treatment. Because TAM inhibited IGF-I- (21), EGF- (41),5 and NGF-stimulated receptor autophosphorylation, it could use a general mechanism not selective for a particular tyrosine kinase receptor. The signal transduction mechanism affected by TAM in modulating trkANGFR autophosphorylation requires additional investigation and should enhance our understanding of the antagonism between TAM and mitogens on breast cancer cells.

TAM might find its way into novel protocols of breast cancer chemotherapy as an adjuvant agent for the growth inhibition of tumors expressing elevated levels of trkANGFR.

Fig. 1.

Effects of NGF on proliferation of noncancerous and cancerous human breast cell lines. Time-related proliferation of MCF-7 human breast cancer cells stimulated by NGF (○), and in the presence of the trkANGFR selective inhibitor K252a (▵). ♦ curve represents the effect of NGF on the human noncancerous breast epithelial cells MCF-10. ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancerous cells. Symbols are the means (P < 0.05; ANOVA, and then Fisher’s test); bars, SE.

Fig. 1.

Effects of NGF on proliferation of noncancerous and cancerous human breast cell lines. Time-related proliferation of MCF-7 human breast cancer cells stimulated by NGF (○), and in the presence of the trkANGFR selective inhibitor K252a (▵). ♦ curve represents the effect of NGF on the human noncancerous breast epithelial cells MCF-10. ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancerous cells. Symbols are the means (P < 0.05; ANOVA, and then Fisher’s test); bars, SE.

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Fig. 2.

Expression of NGF receptors in noncancerous and cancerous human breast cell lines. A, Western blots of p75NTR and trkANGFR proteins in noncancerous (MCF-10; Lanes 1 and 4, respectively), and cancerous (MCF-7; Lanes 2 and 5, respectively) human breast cells, and PC12 cells (Lanes 3 and 6, respectively). B, immunostaining for trkANGFR in MCF-7 human breast cancer cell cultures; B1, samples treated with a specific anti-trkANGFR antibody; B2, nonspecific binding (samples incubated with normal rabbit serum). C, expression of p75NTR and trkANGFR mRNAs in noncancerous (MCF-10; Lanes 1 and 4, respectively), cancerous (MCF-7; Lanes 2 and 5, respectively) human breast cells, and PC12 cells (Lanes 3 and 6, respectively). Total RNA was analyzed by reverse transcriptase-PCR as described in “Material and Methods,” with primer sets designed to amplify fragments of the sequence encoding for p75NTR (663-bp PCR products) and trkANGFR (476-bp PCR products). Lane L, 100-bp marker. DNA ladder, with the 600 bp to provide internal orientation. D, effects of 15 min of treatment with the tyrosine kinase inhibitor K252a on trkANGFR expression (D1) and its tyrosine residue phosphorylation (D2) in MCF-7 cells incubated 10 min with NGF.

Fig. 2.

Expression of NGF receptors in noncancerous and cancerous human breast cell lines. A, Western blots of p75NTR and trkANGFR proteins in noncancerous (MCF-10; Lanes 1 and 4, respectively), and cancerous (MCF-7; Lanes 2 and 5, respectively) human breast cells, and PC12 cells (Lanes 3 and 6, respectively). B, immunostaining for trkANGFR in MCF-7 human breast cancer cell cultures; B1, samples treated with a specific anti-trkANGFR antibody; B2, nonspecific binding (samples incubated with normal rabbit serum). C, expression of p75NTR and trkANGFR mRNAs in noncancerous (MCF-10; Lanes 1 and 4, respectively), cancerous (MCF-7; Lanes 2 and 5, respectively) human breast cells, and PC12 cells (Lanes 3 and 6, respectively). Total RNA was analyzed by reverse transcriptase-PCR as described in “Material and Methods,” with primer sets designed to amplify fragments of the sequence encoding for p75NTR (663-bp PCR products) and trkANGFR (476-bp PCR products). Lane L, 100-bp marker. DNA ladder, with the 600 bp to provide internal orientation. D, effects of 15 min of treatment with the tyrosine kinase inhibitor K252a on trkANGFR expression (D1) and its tyrosine residue phosphorylation (D2) in MCF-7 cells incubated 10 min with NGF.

Close modal
Fig. 3.

The inhibitory effect of TAM on the proliferation of MCF-7 cells induced by different mitogens. A, time-dependent effects of: A1, TAM (□), E2 (•), or the combined treatment with TAM + E2 (▴); A2, TAM (□), NGF (•), or the combined treatment with TAM + NGF (▵); and A3, TAM (□), EGF (▴), or combined treatment with TAM + EGF (⋄) on MCF-7 human breast cancer cell proliferation. In all panels, ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancer cells. Symbols are the means (P < 0.05; ANOVA, and then Fisher’s test); bars, SE. B, time-dependent effects of TAM (□), E2 (•), NGF (○), combination E2 + NGF (♦), or coincubation with TAM, E2 + NGF (×) on MCF-7 human breast cancer cell proliferation. ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancer cells. Symbols are the means (P < 0.05; ANOVA, followed by Fisher’s test); bars, SE. C, time-dependent effects of TAM (▴) or the full estrogen receptor antagonist ICI 182.780 (▵) on NGF-stimulated (○) proliferation of MCF-7 human breast cancer cells. ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancerous cells. Symbols are the means (P < 0.05, ANOVA, followed by Fisher’s test); bars, SE.

Fig. 3.

The inhibitory effect of TAM on the proliferation of MCF-7 cells induced by different mitogens. A, time-dependent effects of: A1, TAM (□), E2 (•), or the combined treatment with TAM + E2 (▴); A2, TAM (□), NGF (•), or the combined treatment with TAM + NGF (▵); and A3, TAM (□), EGF (▴), or combined treatment with TAM + EGF (⋄) on MCF-7 human breast cancer cell proliferation. In all panels, ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancer cells. Symbols are the means (P < 0.05; ANOVA, and then Fisher’s test); bars, SE. B, time-dependent effects of TAM (□), E2 (•), NGF (○), combination E2 + NGF (♦), or coincubation with TAM, E2 + NGF (×) on MCF-7 human breast cancer cell proliferation. ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancer cells. Symbols are the means (P < 0.05; ANOVA, followed by Fisher’s test); bars, SE. C, time-dependent effects of TAM (▴) or the full estrogen receptor antagonist ICI 182.780 (▵) on NGF-stimulated (○) proliferation of MCF-7 human breast cancer cells. ▪ curve represents proliferation of nonstimulated, untreated MCF-7 human breast cancerous cells. Symbols are the means (P < 0.05, ANOVA, followed by Fisher’s test); bars, SE.

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Fig. 4.

Effect of treatment with TAM and ICI 182.780 on NGF-stimulated trkANGFR phosphorylation and p75NTR expression in MCF-7 cells. A, effect of 1 h of pretreatment with TAM or ICI 182.780 on trkANGFR expression and its tyrosine phosphorylation in MCF-7 cells incubated for 10 min with NGF. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells. B, effects of 48 h of pretreatment with TAM or ICI 182.780 on trkANGFR expression and its tyrosine phosphorylation in MCF-7 cells incubated for 10 min with NGF. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells. C, expression of the NGF low affinity receptor p75NTR after 48 h of treatment of MCF-7 human breast cancer cells incubated for 10 min with NGF with TAM or ICI 182.780. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells.

Fig. 4.

Effect of treatment with TAM and ICI 182.780 on NGF-stimulated trkANGFR phosphorylation and p75NTR expression in MCF-7 cells. A, effect of 1 h of pretreatment with TAM or ICI 182.780 on trkANGFR expression and its tyrosine phosphorylation in MCF-7 cells incubated for 10 min with NGF. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells. B, effects of 48 h of pretreatment with TAM or ICI 182.780 on trkANGFR expression and its tyrosine phosphorylation in MCF-7 cells incubated for 10 min with NGF. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells. C, expression of the NGF low affinity receptor p75NTR after 48 h of treatment of MCF-7 human breast cancer cells incubated for 10 min with NGF with TAM or ICI 182.780. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells.

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Fig. 5.

Concentration-dependent effect of TAM on NGF-stimulated trkANGFR phosphorylation. Concentration-dependent effect of 48 h of treatment of TAM (3, 30, 60, and 120 nm, and 3 μm) on trkANGFR expression (A) and its tyrosine phosphorylation (B) in MCF-7 cells incubated for 10 min with NGF. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells.

Fig. 5.

Concentration-dependent effect of TAM on NGF-stimulated trkANGFR phosphorylation. Concentration-dependent effect of 48 h of treatment of TAM (3, 30, 60, and 120 nm, and 3 μm) on trkANGFR expression (A) and its tyrosine phosphorylation (B) in MCF-7 cells incubated for 10 min with NGF. CTRL are nonstimulated, untreated MCF-7 human breast cancer cells.

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

1

Supported by the Associazione Italiana Ricerca sul Cancro and Ministero dell’Università e Ricerca Scientifica e Tecnologica (Cofin). Dr. Philip Lazarovici is a member of and supported in part by the David R. Bloom Center for Pharmacy at the Hebrew University of Jerusalem.

4

The abbreviations used are: EGF, epidermal growth factor; IGF, insulin-like growth factor; NGF, nerve growth factor; trkANGFR, trkA NGF receptor; TAM, tamoxifen; ER, estrogen receptor; E2,17β-estradiol; ECL, enhanced chemiluminescence.

5

R. Bernardini, unpublished data.

Table 1

Stimulation of MCF-7 human breast cancer cell proliferation by different mitogens

Proliferating stimulusEC50 (ng/ml)
EGF 10.8 
E2 0.027 
IGF-II 5.0 
NGF 7.12 
NGF + EGF 4.4 
NGF + IGF-II 2.84 
NGF + estradiol 3.44 
Proliferating stimulusEC50 (ng/ml)
EGF 10.8 
E2 0.027 
IGF-II 5.0 
NGF 7.12 
NGF + EGF 4.4 
NGF + IGF-II 2.84 
NGF + estradiol 3.44 

We thank Dr. Oreste Segatto, Istituto Regina Elena, Roma, Italy; and Prof. David Wallach, the Weizmann Institute of Science, Rehovot, Israel, for their helpful advice and suggestions. Dr. Bernardini thanks the Alex Grass Center for Drug Design and Novel Therapeutics at the School of Pharmacy, The Hebrew University, Jerusalem, Israel, for generous support.

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