Xiao-Ping Zang

KGF-induced motility of breast cancer cells is dependent on Grb2 and Erk1,2

Breast cancer metastasis is directly associated with breast cancer cell motility. Using a cell culture wounding model, we have demonstrated that keratinocyte growth factor (KGF) enhanced the motility of estrogen receptor-positive breast cancer cells. However, the mechanisms by which KGF enhanced motility of breast cancer cells are not known. In the present study, we report that KGF-induced motility requires intact tyrosine kinase signaling since genistein, a tyrosine kinase inhibitor, led to decreased motility of breast cancer cells mediated by KGF. Using cDNA microarrays, we previously found that KGF increased the expression ofGrb2mRNA by 2-- the involvement of Grb2 in KGF-induced motility. Down-regulation of Grb2 protein expression inhibited KGF-induced motility. Since Grb2 is known to regulate Erk1,2 and Akt kinase activities we determined whether these downstream proteins may be vital to KGF-induced motility. Inhibiting the activation of Erk1,2 by PD98059 suppressed KGF-induced motility whereas inhibiting the activation of Akt by wortmannin did not affect KGF-induced motility. In conclusion, these results indicate that KGF mediated signal transduction employs Grb2 to transduce the tyrosine kinase signals resulting in the activation of Erk1,2 and breast cancer cell motility.

KERATINOCYTE GROWTH FACTOR STIMULATES THE MIGRATION AND PROLIFERATION OF BREAST CANCER CELLS IN A CULTURE WOUNDING MODEL

Growth factors are known to influence the progression, motility and invasiveness of tumor cells. In a previous study, we reported that conditioned media from NIH 3T3 cells (mouse fibroblast), which contains KGF, increased the motile morphology of estrogen receptor (ER)-positive breast cancer cells and produced no effect on ER-negative cells. The present study examined the influence of human KGF on two estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7 and T-47D) using a culture wounding model to evaluate cell proliferation and migration over a period of 48h. In the present study we observed that KGF enhanced the migration and proliferation of both MCF-7 and T-47D breast cancer cells. In both cell lines the response to KGF was found to be both dose- and time-dependent. However, the total migration and proliferation response of the MCF-7 cells to KGF was much greater than that observed in the T-47D cells. The results of this study demonstrate that human KGF enhances the migration and proliferation of human breast cancer cells. Further, these results support the concept that KGF may be an early signal in the progression of breast cancer to a more motile and metastatic phenotype.

Expression of prohibitin 3' untranslated region suppressor RNA alters morphology and inhibits motility of breast cancer cells.

The prohibitin 3′ untranslated region (3′UTR) belongs to a novel class of non-coding regulatory RNAs. It arrests cell cycle progression by blocking G1-S transition in breast and other cancers. Our previous studies comparing MCF7 derived clones constitutively expressing a common allelic form of prohibitin RNA (UTR/C) to various controls demonstrated that it functions as a tumor suppressor. Here, we further characterized the morphology and motility of these transgenic breast cancer cells when grown in cell culture and on nude mice. In contrast to empty vector (EV) cells, UTR/C cells were observed to grow in an organized manner with more cell-cell contact and differentiate into structures with a duct-like appearance. Computer assisted cytometry to evaluate differences in nuclear morphology was performed on UTR/C and EV tissues from nude mice. Receiver operator curve areas generated using a logistic regression model were 0.8, indicating the ability to quantitatively distinguish UTR/C from EV tissues. Keratinocyte growth factor-induced motility experiments showed that migration of UTR/C cells was significantly reduced (80–90%) compared to EV cells. Together, these data indicate that this novel 3′UTR influences not only the tumorigenic phenotype but also may play a role in differentiation and migration of breast cancer cells.

Expression of prohibitin 3? untranslated region suppressor RNA alters morphology and inhibits motility of breast cancer cells

The prohibitin 3′ untranslated region (3′UTR) belongs to a novel class of non-coding regulatory RNAs. It arrests cell cycle progression by blocking G1-S transition in breast and other cancers. Our previous studies comparing MCF7 derived clones constitutively expressing a common allelic form of prohibitin RNA (UTR/C) to various controls demonstrated that it functions as a tumor suppressor. Here, we further characterized the morphology and motility of these transgenic breast cancer cells when grown in cell culture and on nude mice. In contrast to empty vector (EV) cells, UTR/C cells were observed to grow in an organized manner with more cell-cell contact and differentiate into structures with a duct-like appearance. Computer assisted cytometry to evaluate differences in nuclear morphology was performed on UTR/C and EV tissues from nude mice. Receiver operator curve areas generated using a logistic regression model were 0.8, indicating the ability to quantitatively distinguish UTR/C from EV tissues. Keratinocyte growth factor-induced motility experiments showed that migration of UTR/C cells was significantly reduced (80–90%) compared to EV cells. Together, these data indicate that this novel 3′UTR influences not only the tumorigenic phenotype but also may play a role in differentiation and migration of breast cancer cells.

Effect of Antiestrogens on EGF-Mediated Movement of Human Breast Cancer Cells

In a previous study we compared the influence of several growth factors on breast cancer cells in culture and observed that epidermal growth factor (EGF) enhanced the invasiveness of estrogen receptor-positive breast cancer cells. The objective of the present study was to determine the influence of three unique antiestrogens on EGF-mediated movement of human breast cancer cells. The rate of movement of MCF-7 breast cancer cells was measured using time-lapse videomicroscopy (TLVM). The MCF-7 cells were pretreated with antiestrogen (either tamoxifen, ICI-182-780 (ICI) or 1,1-dichloro-cis-2,3-diarylcyclopropane (AII)) at 10–6 mol/l for 4 days, and then treated with EGF (10–10 mol/l) immediately prior to TLVM. EGF enhanced the motility of the MCF-7 cells at 30–90 min post-administration. However, EGF-mediated motility of the MCF-7 cells was inhibited by antiestrogen pretreatment, with TAM and ICI producing complete inhibition of EGF-induced motility. In conclusion, this study demonstrates that EGF enhances the rate of movement of MCF-7 breast cancer cells and that antiestrogen pretreatment inhibits EGF-mediated motility.

ANTIESTROGEN INHIBITION OF EGF-MEDIATED INVASIVENESS OF HUMAN BREAST CANCER CELLS

Dear Editor: The metastatic spread of tumor cells involves a complex cascade that includes cell proliferation, invasion, release of proteases, and establishment of a new microenvironment. It appears that peptide growth factors are associated with the metastatic process and have a profound effect on cancer cell motility (Gherardi, 1991). Epidermal growth factor (EGF) and the EGF receptor (EGFR) are known to be associated with tumor progression in highly malignant and invasive breast cancer (Klijn et al., 1992). Further, overexpression of EGFR in breast cancer is known to be associated with poor clinical prognosis (Sainsbury et al., 1987; Barker and Vinson, 1990). In a previous study, we compared the influence of EGF, insulin-like growth factor, and transforming growth factor (TGF-~) on the invasiveness of breast cancer ceils, using transwell membranes (Tong et al., 2000). We observed that both EGF and TGF-~ produced a chemoattractant effect in estrogen receptor (ER)-positive MCF-7 cells, although EGF was found to be the most active and produced a threefold increase in cell invasion. In ER-negative MDA-MB-231 ceils, EGF was found to produce very little invasive activity, whereas TGF-~ was an effective chemoattractant. Similarly, Matthay et al. (1993) observed transient effects of EGF on the motility of 184A1 immortalized cells, a nontumorigenic breast cell line. The results of these studies suggest that EGF may be an autocrine regulator of breast cancer cell invasion and metastasis. As EGF was found to be most effective in ER-positive cells, the present study was designed to examine the effects of three antiestrogens, (tamoxifen [TAM], ICI-182,780 [ICI] and 1,1-dichloro-cis-2,3-diarylcyclopropane [AII]), which differ in chemical structure and biological mechanism (Jain et al., 1997), on EGF-induced chemoattractant activity in MCF-7 cells. The cancer cells were obtained from American Type Culture Collection (Rockville, MD) and grown as monolayer cultures in serum-free Roswell Park Memorial Institute 1640 media as previously described (Jain et al., 1997). The chemoattractant activity of the growth factors was assayed in vitro using Matrigelcoated (100 ~g/well) transwell chambers (Costar, Corning, NY) as previously described (Tong et al., 2000). The cells were grown in T-25 flasks and pretreated with antiestrogen for 4 d at a concentration of 10 -6 M that was previously found to produce optimum inhibition of cell proliferation (Jain et al., 1997; Tong et al., 1999). A control group was pretreated with vehicle alone. MCF-7 cells (3 • 105) were then plated onto the upper surface of transwell membranes in media containing 10 -6 M antiestrogen. Human recombinant EGF (GIBCO, Gaithersburg, MD) at a concentration of 10 -l~ M, which was found to produce optimal chemoinvasion (Tong et al., 2000), was placed in the lower well of the transwell chambers. Cell movement through the Matrigelcoated membranes was quantified by counting the number of cells on the lower surface of the membrane at the end of a 48h incubation period. The cells were dyed with DiffQuick (Baxter Heahhcare Corp., Miami, FL), rinsed, and counted. Invasion data is reported as the percentage of cells that migrated through the membrane, relative to the control group that was treated with vehicle alone. Following incubation, supernatants were collected from upper wells and frozen for subsequent collagenase assays. The supernatants were assayed for collagenase activity using a radiometric assay in 96-well plates as previously described (Abidi et al., 1997). All experiments were performed in triplicate and the data are expressed as the percentage of collagenase activity, relative to the control group. In the present study, EGF treatment produced a threefold increase in MCF-7 cell chemoinvasion (Fig. 1), as previously reported (Tong et al., 2000). In addition, EGF produced a significant enhancement of collagenase release (Fig. 2). Pretreatment with each of the three antiestrogens inhibited the EGF-mediated chemoinvasion effect on these cells, with TAM producing the greatest inhibitory activity (P < 0.05) (Fig. 1). However, EGF-mediated collagenase release from the MCF-7 cells was not altered by antiestrogen pretreatment (Fig. 2). It has been shown that EGFR upregulation is inversely correlated with ER expression in breast cancer and predicts the failure of endocrine therapy (Klijn et al., 1992). It has been reported that antiestrogen pretreatment did not inhibit EGF-induced proliferation of MCF-7 cells (Cromier and Jordan, 1989). However, we and others have observed that antiestrogen treatment produces a direct inhibition of the mitogenic activity of EGF in MCF-7 cells (Chalbos et al., 1993; Tong et al., 1999). Yarden et al. (1996) demonstrated a rapid threefold increase in the expression of EGFR in MCF-7 cells, which was triggered by estradiol and inhibited by the steroidal antiestrogen ICI-164,385 (structurally similar to ICI-182,780). Fm~ ther, these investigators reported the presence of estrogen-response elements in the promoter region of the EGFR gene and suggested a direct transcriptional mechanism for estrogens in the regulation of EGFR expression. A link between ER and EGF-induced mitogenic activity in breast cancer has been established (Ignar-Trowbridge et al., 1992). Accordingly, antiestrogeus may reduce EGF effects on cancer cells by altering EGFR density or functionality because it has been shown that pretreatment with 4-OH-TAM (an active metabolite of TAM) leads to a large decrease in EGFR autophosphorylation in MCF-7 cells; although, 4-OH-TAM did not directly compete with EGF for EGFR binding in this study (Freiss et al., 1990). In conclusion, this study confirms the influence of EGF on the process of membrane invasion by MCF-7 breast cancer ceils. Further, our results demonstrated that antiestrogen pretreatment abolished EGF-induced cell invasion by a mechanism that is indepen-