Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells.

Acquired resistance to endocrine therapies presents a major obstacle to the successful treatment of breast cancer patients. Previously, we have shown that acquisition of resistance to tamoxifen in breast cancer cells is accompanied by an elevation in Src kinase activity which promotes an aggressive, invasive phenotype in vitro. Here, we have explored the potential therapeutic effects of combining Src inhibition with anti-oestrogen treatment on the development of endocrine insensitivity in breast cancer cells. Treatment of MCF7 and T47D cells with tamoxifen alone resulted in an initial growth inhibitory phase followed by the eventual development of tamoxifen resistance together with an elevation of Src kinase activity, which was central to their increased invasive capacity. Chronic exposure of both cell types to the Src inhibitor, AZD0530, as a monotherapy resulted in outgrowth of AZD0530-resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasive nature. Treatment of both MCF7 and T47D cells with AZD0530 in combination with tamoxifen resulted in a reduction of Src activity together with inhibition of focal adhesion kinase phosphorylation and a complete abrogation of their in vitro invasive behaviour. Furthermore, combination therapy significantly suppressed expression of cyclinD1 and c-myc and prevented cell proliferation and the subsequent emergence of a resistant phenotype, with total cell loss occurring by 12 weeks. These data demonstrate that pharmacological targeting of Src kinase, in conjunction with antihormone therapies, effectively prevents antihormone resistance in breast cancer cells in vitro and suggests a potential novel therapeutic benefit of Src kinase inhibitors clinically.

Growth factor signalling in endocrine and anti-growth factor resistant breast cancer.

Growth factors provide powerful mitogenic and survival signals to breast cancer cells and it is therefore not surprising that they are able to subvert inhibitory responses to anti-hormonal drugs. In this review we discuss several mechanisms by which this may be achieved and expand our observations to encompass recently emerging anti-growth factor treatments. The information presented is underpinned by inhibitor studies that show the targeting of such mechanisms in advance of anti-hormone or anti-growth factor resistance development is able to substantially delay this event, thus pointing the way forward to intelligent combination therapies relevant to the future management of breast cancer.

Deciphering antihormone-induced compensatory mechanisms in breast cancer and their therapeutic implications.

Breast cancer inhibition by antihormones is rarely complete, and our studies using responsive models reveal the remarkable flexibility of breast cancer cells in recruiting alternative signalling to limit maximal anti-tumour effects of oestrogen receptor alpha (ER) blockade. The recruited mechanism involves antihormone-induced expression of oestrogen-repressed signalling genes. For example, epidermal growth factor receptor gene (EGFR) is induced by antioestrogens and maintains residual kinase and ER phosphorylation, cell survival genes, and thereby allows incomplete antihormone response and emergence of resistance. Microarrays are revealing the breadth of antihormone-induced genes that may attenuate growth inhibition, including NFkappaB, Bag1, 14-3-3zeta and tyrosine kinases, such as HER2 and Lyn. Three concepts are emerging: first, some genes are induced exclusively by antioestrogens, while others extend to oestrogen deprivation; secondly, some are transiently induced, while others persist into resistance; finally, some confer additional adverse features when tumour cells are in an appropriate context. Among the latter is CD59 whose antioestrogen induction may permit evasion of immune surveillance in vivo. Also, induction of pro-invasive genes (including NFkappaB, RhoE and delta-catenin) may underlie our findings that antioestrogens can markedly stimulate migratory behaviour when tumour intercellular contacts are compromised. Based on our promising studies selectively inhibiting EGFR (gefitinib), NFkappaB (parthenolide) or CD59 (neutralising antibody) together with antioestrogens, we propose that co-targeting strategies could markedly improve anti-tumour activity (notably enhancing cell kill) during the antihormone-responsive phase. Furthermore, subverting those induced signalling genes that are retained into resistance (e.g. EGFR, NFkappaB, HER2) may prove valuable in this state. Alongside future deciphering and targeting of genes underlying antioestrogen-promoted invasiveness, embracing of intelligent combination strategies could significantly extend patient survival.

Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer.

De novo insensitivity and acquired resistance to the selective oestrogen receptor modulator tamoxifen and the pure anti-oestrogen fulvestrant (faslodex) severely limit their effectiveness in breast cancer patients. This is a major clinical problem, since each year upward of 1 million women are dispensed anti-oestrogenic drugs. In order to investigate the phenomenon of anti-oestrogen resistance and to rapidly screen drugs that target the resistance mechanism(s), we have previously established several in vitro breast cancer models that have acquired resistance to anti-hormones. Such cells commonly develop an ability to proliferate after approximately 3 months of exposure to 4-hydroxytamoxifen or fulvestrant, despite an initial endocrine-responsive (i.e. growth-suppressive) phase. The current paper explores the role that growth factor signalling plays in the transition of oestrogen receptor-positive endocrine-responsive breast cancer cells to anti-oestrogen resistance or insensitivity and how we might, in the future, most effectively use anti-growth factor therapies to treat or delay endocrine-resistant states.

Growth factor signalling networks in breast cancer and resistance to endocrine agents: new therapeutic strategies.

Recent evidence demonstrates that growth factor networks are highly interactive with the estrogen receptor (ER) in the control of breast cancer growth and development. As such, tumor responses to anti-hormones are likely to be a composite of the ER and growth factor inhibitory activity of these agents, with alterations/aberrations in growth factor signalling providing a mechanism for the development of anti-hormone resistance. In this light, the current article focuses on illustrating the relationship between growth factor signalling and anti-hormone failure in our in-house tumor models of breast cancer and describes how we are now beginning to successfully target their actions to improve the effects of anti-hormonal drugs and to block aggressive disease progression.

Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells.

De novo and acquired resistance to the anti-tumour drug gefitinib (ZD1839; Iressa), a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been reported. We have determined whether signalling through the IGF-I receptor (IGF-1R) pathway plays a role in the gefitinib-acquired resistance phenotype. Continuous exposure of EGFR-positive MCF-7-derived tamoxifen resistant breast cancer cells (TAM-R) to 1 microM gefitinib resulted in a sustained growth inhibition (90%) for 4 months before the surviving cells resumed proliferation. A stable gefitinib-resistant subline (TAM/TKI-R) was established after a further 2 months and this showed no detectable basal phosphorylated EGFR activity. Compared with the parental TAM-R cells, the TAM/ TKI-R cells demonstrated (a) elevated levels of activated IGF-1R, AKT and protein kinase C (PKC)delta, (b) an increased sensitivity to growth inhibition by the IGF-1R TKI AG1024 and (c) an increased migratory capacity that was reduced by AG1024 treatment. Similarly, the EGFR-positive androgen-independent human prostate cancer cell line DU145 was also continuously challenged with 1 microM gefitinib and, although substantial growth inhibition (60%) was seen initially, a gefitinib-resistant variant (DU145/TKI-R) developed after 3 months. Like their breast cancer counterparts, the DU145/TKI-R cells showed increases in the levels of components of the IGF-1R signalling pathway and an elevated sensitivity to growth inhibition by AG1024 compared with the parent DU145 cell line. Additionally, DU145/TKI-R cell migration was also decreased by this inhibitor. We have therefore concluded that in breast and prostate cancer cells acquired resistance to gefitinib is associated with increased signalling via the IGF-1R pathway, which also plays a role in the invasive capacity of the gefitinib-resistant phenotype.

Zinc transporter LIV-1: a link between cellular development and cancer progression.

Genes involved in normal developmental processes often have a role in tumour progression. Epithelial-mesenchymal transition (EMT) is a general phenomenon that is an essential event in embryonic development, tissue remodelling and wound repair. Genes that regulate EMT are attracting significant attention as mediators of tumour progression. Indeed, EMT is considered an important event during the evolution of malignant tumours and their progression to metastasis, with several reports of molecules involved in this process facilitating migration and invasion of epithelial tumour cells. Recent work linking the zinc transporter LIV-1 with the transcription factors STAT3 and Snail in zebrafish embryogenesis, throws new light on this transition and has important implications for the development of cancer.

Phenotypic variation in the production of bioactive hepatocyte growth factor/scatter factor by oral mucosal and skin fibroblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic growth factor produced principally by cells of mesenchymal origin. HGF/SF is an important mitogen, morphogen, and motogen and plays an important role in wound healing, tumorigenesis and particularly fetal development. Oral mucosal fibroblasts exhibit a fetal phenotype, including an increased extracellular matrix reorganizational ability, cellular migration and experimental wound repopulation in comparison to skin fibroblasts. In this study the expression, production, and bioactivity of HGF/SF by oral mucosal and skin fibroblasts was investigated. Although both oral mucosal and skin fibroblasts expressed HGF/SF, the oral mucosal fibroblasts produced significantly increased amounts of total HGF/SF (p < 0.01) as measured by enzyme-linked immunosorbent assay and bioactive HGF/SF as measured by cell scatter and cell-dissociation techniques (p < 0.01). The possible effect of increased HGF/SF in production mediating the previously described preferential responses of oral mucosal fibroblasts was studied in vitro. Reverse transcriptase-polymerase chain reaction-Western blotting and immunocytochemistry methods all showed that both oral mucosal and skin fibroblasts expressed and produced the c-Met receptor. Recombinant HGF (20-40 ng/mL) however, failed to affect fibroblast repopulation of monolayer wounds or cellular proliferation. In contrast, recombinant HGF significantly increased ECV304 wound repopulation. These studies provide direct evidence of another mechanism by which site-specific variations in fibroblast phenotype may contribute in a paracrine fashion to the rapid reepithelialization and revascularization of oral wounds.

Inhibition of HGF/SF-induced breast cancer cell motility and invasion by the HGF/SF variant, NK4.

NK4 is a variant form of HGF/SF, comprising the N-terminal and subsequent four kringle domains of mature HGF/SF. HGF/SF is a multifunctional cytokine that enhances the metastatic behaviour of tumour cells in vitro by stimulation of the c-met receptor tyrosine kinase and has been implicated in the development of tumour metastasis in vivo. The aims of this study were to further investigate the potential antagonistic effects of the recently described variant form of HGF/SF, NK4, on HGF/SF activity in breast cancer cells. All cell lines used expressed both the HGF/SF receptor gene and protein as shown by RT-PCR and Western blotting. NK4 inhibited HGF/SF-induced tumour cell invasion through an artificial basement membrane. Tumour cell motility and scattering induced by HGF/SF were also dramatically reduced by the inclusion of NK4. Immunoprecipitation studies revealed that NK4 inhibited the phosphorylation of the c-met receptor in response to HGF/SF. Treatment of these cells with NK4 alone did not have any significant effects on their metastatic behaviour. From this data we conclude that NK4 demonstrates significant antagonistic properties towards HGF/SF, inhibiting HGF/SF-stimulated breast tumour cell invasion, motility and migration. NK4 may therefore be of potential benefit in the development of anti-metastasis therapies.

Eryloside F, a novel penasterol disaccharide possessing potent thrombin receptor antagonist activity.

We report the discovery of Eryloside F, a novel disaccharide of the steroidal carboxylic acid penasterol, isolated from an extract of the marine sponge Erylus formosus. The compound is a potent thrombin receptor antagonist, and furthermore inhibits human platelet aggregation in vitro.

Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells.

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a heterodimeric molecule that plays a key role in the regulation of migration, invasion and angiogenesis in cancer, via activation of its receptor, c-met. HGF/SF is composed of an alpha-chain, containing the N-terminal hairpin domain and 4 kringle domains, plus the serine protease-like beta-chain. We have examined here the properties of NK4, an HGF variant containing the N-terminal hairpin plus the 4 kringle domains, on tumour cell proliferation, dissociation and invasion using human colorectal cancer cells (HT115). The expression of HGF/SF and its receptor was also examined by RT-PCR and Western blotting. Analysis revealed NK4 to be an HGF/SF antagonist that, at a wide range of concentrations, did not exert any biological effects of its own. HT115 cells were shown to express the HGF/SF receptor mRNA and protein. HGF/SF-induced receptor tyrosine phosphorylation was suppressed, in a dose-dependent manner, upon addition of NK4, whereas the addition of NK4 alone caused no phosphorylation. Tumour cell motility was induced by HGF and inhibited by NK4. Furthermore, HGF/SF induced the invasion of cells through Matrigel basement membrane components, and again this induced invasion was suppressed by NK4. Our results show that the ability of HGF/SF to stimulate tumour cell motility and invasion, properties required for metastatic spread, can be inhibited by NK4. Thus, NK4 may have an important role in the control of cancer metastasis.

Antagonistic effect of NK4, a novel hepatocyte growth factor variant, on in vitro angiogenesis of human vascular endothelial cells.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is known to act on cancer cells as well as endothelial cells and stimulate angiogenesis, thus playing an unwanted role in the development and progression of cancer. The current study examined the effects of a newly discovered HGF variant, NK4, on angiogenesis in vitro. Chemically generated NK4 (from recombinant human HGF/SF) was found to be able to inhibit HGF-induced activation (tyrosine phosphorylation) of the HGF/SF receptor cMET but was itself unable to activate cMET. Furthermore, NK4 was demonstrated to inhibit tubule formation from human umbilical vein endothelial cells that was induced by both HGF/SF and a HGF/SF-producing fibroblast (MRC5). Under the same settings, NK4 failed to increase tubular formation. NK4 had no effects on interleukin 8- and vascular endothelial growth factor-induced tubule formation. Using computer-assisted motion analysis, it was further shown that NK4 inhibited HGF-induced migration of human umbilical vein endothelial cells in a migration assay and in an endothelial wounding assay. These data show that NK4 is a complete antagonist to HGF. It inhibits HGF-induced endothelial movement and tubule formation. Thus, NK4 may have an important bearing on the control of cancer progression through its role in angiogenesis. Additional in vivo studies are warranted.

Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin.

Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. The function of these proteins is to link the plasma membrane to the actin cytoskeleton. Merlin is defective or absent in schwannomas and meningiomas and has been suggested to function as a tumour suppressor. In this study, we have examined the role of ezrin as a potential regulator of the adhesive and invasive behaviour of tumour cells. We have shown that following inhibition of ezrin expression in colo-rectal cancer cells using antisense oligonucleotides, these cells displayed a reduced cell-cell adhesiveness together with a gain in their motile and invasive behaviour. These cells also displayed increased spreading over matrix-coated surfaces. Immunofluorescence studies revealed that antisense-treated cells also displayed an increased staining of paxillin in areas representing focal adhesions. Furthermore, coprecipitation studies revealed an association of ezrin with E-cadherin and beta-catenin. Induction of the phosphorylation of ezrin by orthovanadate and hepatocyte growth factor/scatter factor resulted in changes similar to those seen with antisense treatment, together with a marked decrease in the association of ezrin with both beta-catenin and E-cadherin. It is concluded that ezrin regulates cell-cell and cell-matrix adhesion, by interacting with cell adhesion molecules E-cadherin and beta-catenin, and may thus play an important role in the control of adhesion and invasiveness of cancer cells.

Association of the HGF/SF receptor, c-met, with the cell-surface adhesion molecule, E-cadherin, and catenins in human tumor cells.

Tumour cell metastatic potential is significantly enhanced following treatment with HGF/SF, the ligand for the c-met receptor tyrosine kinase. Following c-met activation in tumour cells, phosphorylation of beta-catenin occurs, together with loss of intercellular adhesion and a gain in the motile and invasive nature of the cell. In this study we show that c-met is co-localised with beta-catenin and E-cadherin at regions of cell-cell contact in human colon cancer (HRT18 and HT115) and two breast cancer (MCF7 and MDA MB 231) cell lines. Immunoprecipitation studies demonstrated an association between c-met and members of the cadherin adhesion complex in these epithelial tumour cells, along with the membrane tyrosine protein phophatase, PTPmu. We conclude that the HGF/SF receptor, c-met, together with members of the cadherin/catenin cell-cell adhesion system and PTPmu, may form part of a protein complex in E-cadherin positive tumour cells that acts to regulate intercellular adhesion following HGF/SF stimulation.

Hepatocyte growth factor/scatter factor disrupts epithelial tumour cell-cell adhesion: involvement of beta-catenin.

Initial events in the metastatic spread of tumours involve loss of cell-cell adhesion within the primary tumour mass. The integrity and morphology of epithelial tumour cell colonies is maintained primarily by cell-cell adhesions mediated by E-cadherin and its associated intracellular catenin molecules. Hepatocyte growth factor/scatter factor (HGF/SF) is a potent promoter of the metastatic functions of tumour cells, including motility and invasion and also induces the dissociation of tumour cell colonies. In this study we report that HGF/SF promoted the scattering of an epithelial colorectal tumour cell line. Western blotting demonstrated that this was not due to a change in level of either E-cadherin or its associated catenin molecules. Immunoprecipitation studies revealed that HGF/SF elevated the level of tyrosine-phosphorylated beta-catenin within these cells together with reducing the amount of E-cadherin that was observed to co-precipitate with the beta-catenin. These results were confirmed with confocal scanning laser microscopy. We conclude that phosphorylation of beta-catenin by HGF/SF affects its association with E-cadherin at the cell surface and thus regulates E-cadherin function resulting in colony scattering phenomena.

Role of neuraminidase in influenza virus-induced apoptosis.

The virulent influenza virus clone 7a produced a greater level of apoptosis in MDCK cells compared with the attenuated strain A/Fiji. In both cases, apoptosis could be partially blocked by treatment with three anti-neuraminidase compounds [4-amino-(GR121158A) and 4-guanidino- (GG167; Zanamivir) 2,3-dehydro-N-acetylneuraminic acid and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA)] when they were given to cells during the virus attachment/entry phase, but not subsequent to this phase. In contrast, GG167, which does not enter cells, did not affect the numbers of infected cells and, in addition, acted late in the infection cycle to inhibit virus yields. Clone 7a neuraminidase was more active than A/Fiji neuraminidase when fetuin was used as the substrate. Similar differences in activity between the two viruses were seen when alpha-2,6 sialyl lactose was used as a substrate, but not with alpha-2,3 sialyl lactose. No sequence differences in the enzyme active site of the two neuraminidases were observed, indicating that differences in neuraminidase specificity and activity may be dictated by other residues. These results suggest that neuraminidase plays some role in the induction of apoptosis and that it acts prior to or during virus entry. However, apoptosis was considerably reduced when UV-irradiated virus, which retains >75% of its neuraminidase activity, was used. In addition, ammonium chloride, used to prevent virus entry, reduced virus-induced apoptosis. Amantadine, which inhibits virus uncoating, also inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2), but not the amantadine-resistant clone 7a. Hence, one or more intracellular processes are also involved in influenza virus-induced apoptosis.

Association of PTPmu with catenins in cancer cells: a possible role for E-cadherin.

Protein tyrosine phosphorylation and dephosphorylation is regulated by the action of protein tyrosine kinases (PTK) and phosphatases (PTP) respectively. The receptor type phosphatase, PTPmu, is located at the cell surface where it may function to regulate the phosphoryl status of members of the cadherin adhesion complex and thus cadherin function. We have investigated the association of PTPmu with E-cadherin and catenin molecules in human tumour cells and report that PTPmu; is associated with E-cadherin and alpha and beta-catenin in E-cadherin-positive cell lines. However, no association between PTPmu and catenin members could be detected in E-cadherin negative cells. These observations suggest that the association of PTPmu with catenin molecules may occur via E-cadherin rather than a direct interaction.

The effects of n-6 polyunsaturated fatty acids on the expression of nm-23 in human cancer cells.

This study examined the effect of n-6 polyunsaturated fatty acids (PUFAs) on the expression of nm-23, a metastasis-suppressor gene, in two highly invasive human cancer cell lines, HT115 and MDA MB 231. A range of n-6 and n-3 PUFAs were tested. We report that while linoleic acid and arachidonic acid reduced the expression of nm-23-H1, gamma linolenic acid (GLA) and its soluble lithium salt markedly increased the expression of the molecules. The stimulation of the expression of nm-23 by GLA was seen at both protein and mRNA levels. Up-regulation of nm-23 was also associated with a reduction of the in vitro invasiveness of these cells. It is concluded that gamma linolenic acid (GLA) enhances the expression of nm-23. This contributes to the inhibition of the in vitro invasion of tumour cells.

Regulation of desmosomal cell adhesion in human tumour cells by polyunsaturated fatty acids.

Desmosomes are key structures in cell-cell adhesion. In this study we examined the effect of n-6 essential fatty acids on the expression of desmoglein (Dsg), desmosomal cadherin and the formation of desmosomes in E-cadherin negative human breast, colon and lung cancer cells and melanoma cells. Electron microscopy revealed that cells cultured with gamma linolenic acid (GLA) showed increased cell-cell adhesion together with an increase in the formation of desmoglein-containing desmosomes. Western blotting studies of cellular proteins demonstrated that, following culture with fatty acids, Dsg expression was modified, with the greatest increase seen after GLA treatment. Other fatty acids increased Dsg expression, but to a lesser extent. It is concluded that GLA regulates desmosome-mediated cell-cell adhesion in human cancer cells, particularly in cells without E-cadherin.