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Background: Keratinocyte growth factor (KGF) acts at the KGF receptor (KGFR) to produce a rapid stimulation of breast cancer cell proliferation and motility which is mediated via the Erk signaling pathway. Enhancement of KGF/KGFR signal transduction may be an early step in the metastatic progression of breast cancer. Receptor modeling of KGFR was used to identify selective KGFR tyrosine kinase (TK) inhibitor molecules that have the potential to bind selectively to the KGFR. The present study evaluated the biological activity of 57 of these KGFR TK inhibitor compounds on breast cancer cells., Materials and Methods: These compounds were tested for their ability to inhibit KGF-mediated breast cancer cell proliferation in MCF-7 breast cancer cells. Furthermore, the effects of the most effective proliferation inhibitors were examined on Erk signaling and on the relative density of cell membrane KGFR., Results: It was observed that 27 of the 57 compounds tested produced a 20% or greater reduction in KGF-mediated proliferation; while five compounds produced greater than 50% inhibition. In addition, the most potent inhibitors also reduced Erk signaling and cell membrane density of the KGFR., Conclusion: The compounds examined appear to be selective KGFR inhibitors which inhibit KGF-mediated activity and reduce the expression of KGFR on cancer cells. These results may lead to the development of a novel class of anticancer agents for the prevention of metastatic cancer progression.

Background: Keratinocyte growth factor (KGF) produces a rapid increase in the proliferation and motility of estrogen receptor (ER)-positive breast cancer cells which is abolished by estrogen deprivation and/or anti-estrogen treatment. The present study examined the hypothesis that ER-alpha is involved in the KGF proliferation in MCF-7 cancer cells using small interfering RNA (siRNA) to selectively inhibit ER-alpha expression., Materials and Methods: At 48 hours following ER-alpha siRNA transfection, the MCF-7 cells were treated with KGF (50 ng/ml) or vehicle for 24 hours. Cell proliferation was measured using a MTT assay. ER-alpha protein levels were quantified by Western blotting., Results: ER-alpha siRNA transfection significantly reduced ER-alpha expression and MCF-7 cell proliferation. KGF-mediated enhancement of cell proliferation and motile cell morphology were reduced or absent in the siRNA transfected MCF-7 cells., Conclusion: ER-alpha expression is associated with KGF-induced proliferation of breast cancer cells.

Background: Keratinocyte growth factor (KGF) has been shown to induce breast cancer metastasis in animal models. cDNA microarrays have revealed that KGF increased Wilms tumor 1 (WT1) and focal adhesion kinase (FAK) expression in breast cancer cells. The role of WT1 and FAK in KGF signaling was investigated., Materials and Methods: A cell culture wounding model was used to study the effects of WT1 and FAK down-regulation on KGF-induced proliferation and motility in breast cancer cells., Results: WT1 down-regulation inhibited KGF-mediated proliferation and motility of breast cancer cells, while FAK down-regulation inhibited proliferation, but had no significant effect on cell motility. WT1 down-regulation, but not FAK down-regulation, led to Erk1,2 inactivation., Conclusion: KGF-mediated signaling employs WT1 and FAK to regulate breast cancer cell proliferation and motility and may represent therapeutic targets for the prevention of breast cancer progression.

Background: The mammary glands of adult female animals are remarkably sensitive to keratinocyte growth factor (KGF). KGF acts at the KGF receptor (KGFR) to produce a rapid and profound stimulation of breast cancer cell proliferation and motility. Further, KGF-induced motility in breast cancer cells is mediated via the Erk1/2 signaling pathway. Thus, enhancement of KGF/KGFR signal transduction may be an early step in the metastatic progression of breast cancer. Receptor modeling of KGFR was used to identify selective KGFR tyrosine kinase inhibitor (TKI) molecules with high receptor affinity. The present study describes the synthesis and biological activity of three of the KGFR TKI compounds., Materials and Methods: Computer modeling of the KGFR was used to create a virtual library of compounds that have the potential to bind with high affinity to the KGFR. Three of these compounds were synthesized and tested in this study. The compounds were tested for their ability to inhibit KGF-mediated breast cancer cell proliferation and motility using a culture wounding assay. In addition, the effect of the most potent KGFR TKI compound on the relative density of cell membrane KGFR was measured using immunocytochemistry., Results: It was observed that the KGFR TKIs decreased KGF-mediated activity as predicted by computer modeling. In addition, the most potent inhibitor also reduced the density of the KGFR on the membrane of the cancer cells., Conclusion: The novel inhibitors identified in this project are selective KGFR inhibitors which appear to reduce the expression of KGFR on cancer cells. These results may lead to the development of a novel class of anticancer agents for the chemoprevention of metastatic cancer development and provide a new approach in the treatment of breast cancer.

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.

Background: We previously reported that a novel fusion protein (consisting of an amino-terminal fragment of urokinase which binds to the urokinase receptor, and L-methioninase which depletes methionine and arrests the growth of methionine-dependent tumors) inhibited MCF-7 breast cancer cells in vitro., Materials and Methods: We produced this fusion protein, L-methioninase, and a mutated fusion protein without L-methioninase activity by recombinant methods. MCF-7 cell proliferation and mobility were measured in vitro in a culture wounding assay. Protein binding to MCF-7 cells was measured by immunocytochemical localization. MCF-7 tumor xenograft growth was measured in nude mice., Results: The fusion protein was significantly more effective than L-methioninase in either the in vitro or in vivo assays. The binding assay showed that the unmutated and mutated fusion protein bound to the cells, but L-methioninase did not., Conclusion: Our results suggest that this fusion protein has potential as a therapeutic agent for cancer treatment.

Background: In a previous study, we reported that keratinocyte growth factor (KGF) produced a rapid increase in the motility of ER-positive breast cancer cells. Others have demonstrated that KGF treatment in rodent species produces rapid mammary ductal hyperplasia. Epithelial cells do not produce KGF; thus, in the present study, MCF-7 cells were stably transfected with a KGF-expressing vector and the motility and morphology of the transfected, non-transfected and empty vector cell lines compared., Materials and Methods: A mammalian expression vector containing a KGF cDNA was transfected into MCF-7/beta cells, and two stable clones (MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9) were identified. Western blotting of conditioned medium from these clones was used to confirm the expression of KGF. The motility of wild-type and KGF-transfected MCF-7 cells was compared using time-lapse videomicroscopy and a cell culture wounding model which examined cell migration over a period of 1-3 days., Results: The Western blots demonstrated that the expression of KGF in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines was higher than the wild-type and MCF-7/beta cell lines. The cell proliferation and migration distance was significantly greater for both KGF-transfected MCF-7 cell lines than the wild-type and MCF-7/beta cell lines under the same experimental conditions. Further, changes in motile morphology were observed in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines. In addition, the MCF-7/beta/KGF-T8 clone was found to produce much larger tumors than both the MCF-7/beta/KGF-T9 and EV clones in mouse xenografts. These results indicated that autocrine production of KGF in the KGF-transfected MCF-7 cell lines enhanced cell migration, migration-related morphology and xenograft tumor growth., Conclusion: KGF-transfected MCF-7 cells displayed a much greater motility than non-transfected cells, confirming the KGF motility enhancement effect which we previously reported. The use of KGF-transfected breast cancer cells in the xenograft model may help to study the mechanism of KGF-mediated cell motility and to identify specific KGF antagonists that may be used to prevent or impede KGF-mediated metastatic progression.

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.

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 of Grb2 mRNA by 2 3-fold. Since Grb2 plays an important role in tyrosine kinase signaling, we examined 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.

Background: The metastasis of breast cancer is known to be directly associated with the motility of breast cancer cells. We have previously shown that keratinocyte growth factor (KGF) enhances the motility of estrogen receptor (ER)-positive breast cancer cells and that this motility response is associated with cellular levels of the KGF receptor (KGFR). Further, we observed that KGF treatment enhanced KGFR gene expression in MCF-7 cells. The objective of the present study was to examine the influence of antisense KGFR oligonucleotide treatment on the KGF-induced motility response in breast cancer cells., Materials and Methods: Both time-lapse video microscopy (TLVM) and culture wounding experiments were used to quantify cell motility. KGFR antisense effects on the expression of KGFR were determined by Western blotting, real time PCR and immunocytochemistry., Results: Antisense KGFR treatment significantly reduced both KGFR mRNA and protein expression. In addition, the antisense KGFR abolished the KGF-mediated cell motility response as early as 2 h following KGF treatment as observed by TLVM and lasting for up 48 h as observed by culture wounding. CONCLUSIOS: The results of this study indicate that KGFR activation and intracellular signaling mediates the KGF motility effect and suggests that KGFR may be an important new therapeutic target for the treatment or prevention of metastatic progression in breast cancer.

It has been shown that methionine depletion inhibits tumor cell growth and reduces tumor cell survival. A novel fusion protein targeted specifically to tumor cells was developed. The fusion protein contained two components: the amino terminal fragment of human urokinase (amino acids 1-49) that binds to the urokinase receptor protein expressed on the surface of invasive cancer cells, and the enzyme L-methioninase (containing 398 amino acids) which depletes methionine and arrests the growth of methionine-dependent tumors. The influence of the fusion protein on the growth and motility of human breast cancer cells was examined using a culture wounding assay. It was determined that MCF-7 breast cancer cells, used in this study, were methionine-dependent and that the fusion protein bound specifically to urokinase receptors of the surface of the cancer cells. Further treatment of the cancer cells with fusion protein over the concentration range 10(-8) to 10(-6) M produced a dose-dependent inhibition of both the migration and proliferation index of MCF-7 cells in the culture wounding assay over a period of 1 to 3 days. The results of this study suggest that this novel fusion protein may serve as a prototype for specific targeting of methioninase and perhaps other cytotoxic agents to cancer cells.

Background: Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is a mesenchymally derived mediator of epithelial cell proliferation and migration. In a previous study, we reported that KGF enhanced the motility of human breast cancer cells. The objective of the present study was to examine the influence of specific and non-specific KGF inhibitors on KGF-induced motility and proliferation in ER-positive MCF-7 cells., Materials and Methods: In the present study three KGF inhibitors were employed [Heparin, Innohep, a low molecular weight heparin (LMWH) and KGFR2 beta (IIIb)/Fc, a chimeric KGFR fragment]. Heparin and LMWH bind to low affinity sites on KGF and produce non-specific inhibition, while KGFR2 beta (IIIb)/Fc, a soluble chimera of an extracellular KGFR fragment, is a more specific KGF inhibitor. Cellular motility was measured using two methods: culture wounding over a period of 48 hours; and secondly, time-lapse videomicroscopy (TLVM)., Results: In these experiments KGF was found to produce a dose-dependent enhancement of MCF-7 cell motility over a dosage range of 5 to 500 ng/ml. In the TLVM experiments, Heparin (30 ng/ml), LMWH (30 ng/ml) and KGFR2 beta (IIIb)/Fc (50 micrograms/ml) completely inhibited KGF-induced motility of MCF-7 cells during the initial 2-hour observation period. In the culture wounding assay, LMWH produced a greater reduction in KGF-induced motility than heparin at 48 hours post-treatment., Conclusion: The results of this study indicate that KGF-mediated enhancement of breast cancer cells motility and proliferation is inhibited by both specific and non-specific KGF inhibitors. LMWH appears to produce an inhibition of KGF with a much longer duration of action than Heparin. Our results suggest that KGF inhibition may be a potential new therapeutic approach for the treatment of metastatic breast cancer.

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.

Background: In a previous study we compared the influence of several growth factors on cancer cells in culture and observed that transforming growth factor-beta (TGF-beta) enhanced the invasiveness of several breast cancer cell lines. The objective of the present study was to determine the influence of three unique antiestrogens on TGF-beta-mediated chemotaxis of human breast cancer cells., Materials and Methods: The chemotactic activity of TGF-beta was assayed in vitro using collagen IV-coated transwell chambers with either MCF-7 or MDA-MB-231 human breast cancer cells. The cells were pretreated with antiestrogen for 4 days, harvested and placed in the upper transwell chamber. TGF-beta was added to the lower chamber and cell migration and collagenase release were determined following a 6-hour incubation., Results: In this study TGF-beta enhanced the chemotaxis of both cell types with a much greater effect on the MDA-MB-231 cells. However, TGF-beta-mediated chemotaxis of the MCF-7 cells was inhibited by antiestrogen pretreatment while TGF-beta-mediated chemotaxis of the MDA-MB-231 cells was not altered. Further, neither TGF-beta nor antiestrogen treatment altered collagenase release from either cell line., Conclusion: The data demonstrate that pure antiestrogens are capable of inhibiting TGF-beta-mediated chemotaxis in estrogen receptor (ER)-positive breast cancer cells by a mechanism which is independent of collagenase release. Finally, the results of this study suggest a coupling of the TGF-beta and ER signaling pathways and indicate that TGF-beta may be an important therapeutic target for the treatment or inhibition of breast cancer metastasis.