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The estrogen-regulated protein, TFF1, stimulates migration of human breast cancer cells¹

Department of Pathology, School of Clinical and Laboratory Sciences, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK

²Correspondence: Department of Pathology, School of Clinical and Laboratory Sciences, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK. E-mail: B.R.Westley{at}ncl.ac.uk

SPECIFIC AIMS

TFF1 is a small cysteine-rich secreted protein that is normally expressed at highest levels in the mucosa of the gastrointestinal tract. It is, however, also widely expressed at high levels in malignant breast epithelial cells where its expression is regulated by estrogen. The objective of this study was to identify a biological role for the TFF1 protein in breast cancer.

PRINCIPAL FINDINGS

1. Estrogen receptor positive but not negative breast cancer cells secrete high concentrations of TFF1
Concentrations of TFF1 were measured by Western transfer analysis using known amounts of recombinant TFF1 as standards in samples of medium conditioned by five estrogen-responsive cell lines: MCF-7, T47D, ZR 75, EFM-19, and EFF3; and by the estrogen-receptor negative cell line MDA MB231. TFF1 was present at highest levels in medium from MCF-7 and ZR 75 cells (3.2±0.3 µg/ml and 2.4±0.2 µg/ml, respectively), at lower levels in medium from EFM-19 (0.4±0.05 µg/ml) and EFF3 cells, and was not detected in medium from T47D cells or MDA MB231 cells.

Single domain trefoil proteins have a conserved cysteine residue at the carboxyl terminus outside of the trefoil domain. Recombinant TFF1 forms homodimers of 13.3 kDa by an intermolecular disulfide bond via the extratrefoil domain residue Cys⁵⁸. Recombinant TFF1 does not form dimers and is a monomer of 6.7 kDa.

The molecular forms of TFF1 secreted by the estrogen-responsive breast cancer cells were analyzed using nondenaturing PAGE with Western transfer analysis. Three molecular forms (TFF1 monomer, TFF1 homodimer, and a TFF1 heterodimer) were identified. The latter two forms result from the ability of the extratrefoil domain cysteine to form intermolecular disulfide bonds; the identity of the protein forming the heterodimer is not known. The intensities of the bands relative to the standards suggest that each cell line secretes a TFF1 monomer and TFF1 dimer in approximately equal amounts into the medium.

2. TFF1 stimulates breast cancer cell movement
Monolayer cultures of MCF-7 breast cancer cells, which secrete TFF1, were wounded and cultured in the absence and presence of recombinant TFF1 dimer using a concentration range that encompassed the concentration of TFF1 found in medium conditioned by MCF-7 cells: 1, 10, and 20 µg/ml TFF1 all significantly increased cell movement (P<0.01) (Fig. 1 A).

View larger version (40K): [in this window] [in a new window]   Figure 1. Stimulation of the movement of breast cancer cell monolayers in an in vitro wounding and cell migration assay. Confluent monolayers of MCF-7 (A) or MDA MB231 cells (B) were cultured in DMEM and BSA in the absence and presence of different concentrations of TFF1 for 24 h after ‘wounding’ of the cell layer with a pipette tip. The distance the wounded edge of cells had moved was measured. Means ± SE of at least 7 measurements from each of 3 experiments are shown. C–E) Cells were trypsinized, allowed to recover for 1 h, then 1.5 x 104 cells were placed in the upper wells of a micro-chemotaxis chamber in medium containing 0.01% BSA. They were left to migrate for 5 h on polyvinylpyrrolidone membranes with 8 µm pores coated with 20 µg/ml collagen IV. The lower wells contained medium and 0.01% BSA and varying concentrations of TFF1 dimer. The chamber was dismantled; the cells were fixed on the membrane with methanol, stained with Harris’s hematoxylin, and attached to the underside of the membrane. The cells in 5 high-powered fields (40x) were counted in triplicate wells for each TFF concentration. Means ± SE from a representative experiment for MCF-7 cells (C) and MDA MB231 cells (D) are shown. E) The lower wells contained phenol red-free DMEM, 0.01% BSA, and varying concentrations of TFF1 Ser58 monomer (filled square) or TFF1 Cys58 dimer (filled circle).

Recombinant TFF1 at concentrations of 1, 10, and 20 µg/ml also stimulated movement of MDA MB 231 cells, but 10 µg/ml stimulated more movement than 1 µg/ml (P<0.02) (Fig. 1B ). This shows that TFF1 can stimulate movement of breast cancer cells that do not secrete trefoil peptides, but a higher concentration is required for a maximum response.

The effect of TFF1 on cell movement was measured using a modified Boyden chamber assay. The dose response curve was bell shaped. Migration was increased significantly by 0.5, 1, 10, and 20 µg/ml, with a fourfold maximum stimulation (Fig. 1C ). For MDA MB 231, markedly higher concentrations were required to stimulate cell migration. Migration was not stimulated by TFF1 at 1 µg/ml but was stimulated significantly at 20, 50, and 100 µg/ml TFF1, with maximum stimulation at 50 µg/ml (P<0.0001) (Fig. 1D ).

3. Dimeric TFF1 is more potent in stimulating cell migration than TFF1 monomer
As breast cancer cells produce significant amounts of TFF1 monomer and TFF1 dimer, the relative potency of these two molecular forms were compared. Figure 1E shows dose response curves for the stimulation of MCF-7 cell migration by recombinant TFF1 Ser⁵⁸ monomer and TFF1 Cys⁵⁸ dimer. TFF1 monomer stimulated migration of MCF-7 cells, but a higher concentration was required than for TFF1 dimer. Half-maximal induction of MCF-7 cell migration was induced by 5 µg/ml TFF1 monomer whereas 0.6 µg/ml of TFF1 dimer was required. The dose response curves for both proteins were bell shaped; migration was maximal at 20 µg/ml for TFF1 monomer and 1 µg/ml for TFF1 dimer.

A similar displacement of the dose response curve was observed for TFF1-stimulated migration of MDA MB231 by recombinant TFF1 monomer and dimer; higher concentrations (100 µg/ml TFF1 monomer) were required to stimulate maximum migration.

4. TFF1 stimulates cell migration principally by chemotaxis
Motogens can stimulate random cell movement, chemokinesis, or directional movement (also known as chemotaxis) along a concentration gradient of the motogen. Checkerboard analysis was used to determine whether TFF1 is chemotactic or chemokinetic for breast cancer cells (summarized in Fig. 2 ).

View larger version (56K): [in this window] [in a new window]   Figure 2. The motile response of MCF-7 cells to varying concentrations of TFF1 in the upper and lower wells of a microchemotaxis chamber were measured as in Fig. 1 . Significant increases in cell migration above control wells are indicated: **P < 0.001;*P < 0.05.

In all cases, cell migration was increased with a higher concentration of TFF1 monomer or dimer in the lower well for MCF-7 and MDA MB 231 cell lines, demonstrating that TFF1 is strongly chemotactic for breast cancer cells. For instance, MCF-7 cells migrated toward 50 µg/ml of TFF1 monomer in the bottom well when the cell suspension in the upper well contained 0, 0.5, or 5 µg/ml of TFF1 monomer (P<0.001), but migration was not stimulated when there was no difference in concentration between the top and bottom wells. Although most cell migration appeared to be chemotactic, TFF1 may also stimulate cell migration by chemokinesis. This was observed for MCF-7 cells at 1 µg/ml TFF1 dimer and for MDA MB231 cells at 50 µg/ml of TFF1 dimer (Fig. 2) .

CONCLUSIONS AND SIGNIFICANCE

We have shown that TFF1 is a potent chemoattractant for breast cancer cells, and this is the first direct evidence for a biological function of TFF1 in breast cancer. Early studies focused on its possible role as a mitogen, but failed to demonstrate biological activity. The majority of the work described in this study uses a modified Boyden chamber migration assay to measure cell movement. This assay is highly relevant to breast tumor biology as tumor cells have to move across an extracellular matrix protein, change shape, and move through membrane pores, thereby recapitulating some processes involved in tumor cell dissemination in vivo.

Estrogen increases the expression and secretion of TFF1 into the medium of estrogen-responsive breast cancer cells in culture. Three different molecular forms have been identified and are shown schematically in Fig. 3 A. The nature of the partner in the TFF1 heterodimer is unknown. The biological activities of TFF1 monomer and dimer were measured.

View larger version (30K): [in this window] [in a new window]   Figure 3. Effects of TFF1 on breast cancer cell migration. A) Estrogen-responsive breast cancer cells contain estrogen receptor (ER) in the cell nucleus. Estrogen induces the transcription of the TFF1 gene resulting in secretion of three molecular forms of the TFF1 protein. The TFF1 molecule is shown as a surface rendered model, with areas of red indicating the strong positive charge in the carboxyl terminus of the molecule. The TFF1 homodimer is formed via 2 cysteine residues at the carboxyl terminus of the protein. The protein partner forming the heterodimer is not known and appears as a gray ovoid. The putative cell surface TFF1 receptor is shown in blue. B) Model for TFF1 acting as an estrogen-induced motogen. Cells have little motility in the absence of TFF1. Addition of exogenous recombinant TFF1 or estrogen will stimulate cell movement. C) That dimeric TFF1 is more potent than monomeric TFF1 suggests that binding of TFF1 to its receptor involves a bivalent interaction.

Our current model (illustrated in Fig. 3B ) suggests that in the absence of estrogen, there is little cell movement. Cell migration can be stimulated by the addition of exogenous TFF1 or estrogen. The concentrations at which TFF1 stimulates breast cancer cell migration are similar to the those detected in medium conditioned by estrogen-treated breast cancer cells in culture. As TFF1 expression is induced by estrogen, this suggests that TFF1 acts as an estrogen-regulated autocrine motogen in breast tumor cells (Fig. 3B ). The concentrations of TFF1 dimer that stimulate MCF-7 cell migration are similar to the active concentrations of growth factors that stimulate cell proliferation of breast cancer cells such as insulin-like growth factors. This suggests that TFF1 may stimulate cell migration by interacting with a high-affinity cell surface receptor. Our data show that though the TFF1 dimer and monomer both stimulate the migration of breast cancer cells, the dimer is more potent. Several peptide growth factors and hormones, (growth hormone, insulin, and insulin-like growth factors) bind to and activate their receptors by cross-linking receptor monomers; our data are consistent with TFF1 stimulating the migration of breast cancer cells via a bivalent interaction with a dimeric receptor, as illustrated in Fig. 3C .

It is 15 years since it was shown that TFF1 is induced dramatically by estrogens in breast cancer cells and expressed by the majority of estrogen receptor-positive breast tumors and 10 years since it was shown that TFF1 can be expressed in a broad spectrum of human epithelial tumors. Nevertheless, the biological function of TFF1 in these tumors has never been identified. The present study provides direct evidence for the hypothesis that TFF1 acts as a motogen for breast tumor cells and, in so doing, promotes their dissemination. This implies that TFF1 confers a selective advantage on tumor cells, facilitates invasion of surrounding tissue, and may be involved in establishing distant metastatic deposits. Such a selective advantage could explain the increased expression of TFF1 in tumor vs. normal cells. The chemotactic response of breast cancer cells to TFF1 may in part determine the pattern of metastatic spread. Tumor cells may be attracted to normal tissues that express the highest concentration of trefoil proteins such as the uterus, gastrointestinal tract, and lung.

TFF1 is a marker of endocrine response in breast cancer patients. This raises the possibility that the beneficial effects of anti-estrogen therapy may be due in part to a reduction in the expression of TFF1. The demonstration that TFF1 stimulates breast cancer cell motility suggests that anti-estrogens may inhibit tumor cell dissemination.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0498fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0498fje

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