Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3′,4′-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser⁴²⁸. This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser¹⁵) and Chk1 (Ser²⁹⁶) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo.     

Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase

Engagement of the TCR (CD3-Ti) by Ag/MHC, CD3 mAb, or lectin mitogen stimulates the very early tyrosine phosphorylation of several cellular substrates including TCR-zeta. The T cell specific protein-tyrosine kinase (PTK), p56lck, has been implicated in the tyrosine phosphorylation of TCR-zeta. However, the significance of this event with regard to CD3-Ti signal transduction remains unclear. Herein, we have investigated the effect of the selective PTK inhibitor genistein (4',5,7-trihydroxyisoflavone) on cellular events associated with activation via CD3-Ti triggering. Genistein inhibited the T cell PTK, p56lck, in a dose-dependent fashion with an ID50 = 40 microM. Genistein also inhibited CD3 mAb or PHA-induced TCR-zeta chain phosphorylation in intact peripheral blood T cells. Genistein blocked the expression of IL-2 and IL-2R (CD25) in T cells stimulated with PHA/PMA or CD3 mAb/PMA, but did not inhibit the de novo expression of the CD69 early activation Ag, which is induced primarily by a PKC-dependent pathway. IL-2 and CD25 expression induced by calcium ionophore A23187 and PMA was largely refractory to inhibition by genistein, suggesting an effect of the drug on calcium-dependent pathways stimulated via CD3-Ti triggering. In this last regard, genistein partially inhibited the CD3 mAb-induced rise in [Ca2+]i but did not inhibit PHA- or CD3 mAb-induced phosphatidylinositol hydrolysis. Consequently, protein-tyrosine phosphorylation does not appear to be a prerequisite for CD3-Ti-mediated activation of phosphatidylinositol-specific phospholipase C activity and PIP2 hydrolysis. An alternative role for PTK in CD3-Ti signal transduction is suggested.     

Genistein blocks breast cancer cells in the G(2)M phase of the cell cycle

Genistein, a natural isoflavone phytoestrogen present in soybeans, caused a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 microM genistein showed a dose-dependent accumulation in the G(2)M phase of the cell cycle. At the highest tested concentration, there was a sevenfold increase in the percentage of cells in G(2)M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate fourfold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G(2)M arrest was coupled with a parallel depletion of the G(0)/G(1) phase. To understand the mechanism of action underlying the block in G(2)M induced by genistein, we investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G(2)-->M transition. As expected, p34(cdc-2) expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34(cdc-2) catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a fourfold decrease in kinase activity evaluated using the histone H1 as substrate. Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 microM genistein, cyclin B(1) (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells. We therefore conclude that genistein causes a G(2)M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34(cdc-2)/cyclin B(1) complex only in all of the studied cell lines.     

Effects of the tyrosine-kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells

We have examined the effects of the tyrosine kinase inhibitor genistein on hormone dependent cell proliferation and intracellular signalling in mouse 10T1/2 fibroblasts and rat liver T51B epithelial cells. Genistein inhibits both PDGF and EGF induced mitogenesis with an IC50 of 40 uM and 10 uM respectively. Genistein also inhibits inositol phosphate generation and calcium signalling in response to PDGF, and 1,2-diacylglycerol generation and calpactin II translocation in response to EGF. By contrast genistein does not inhibit inositol phosphate production, Ca2+ signalling or 1,2-diacylglycerol generation in response to ATP or angiotensin II. These data demonstrate that genistein selectively inhibits tyrosine kinase dependent processes without effecting similar responses obtained to hormones which are not dependent upon tyrosine kinase activation.     

Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line

The expression of GPR41 and 43, which have recently been identified as G-protein-coupled cell-surface receptors for short-chain fatty acids (SCFAs), was detected in a human breast cancer cell line (MCF-7) by RT-PCR. Acetate, propionate and butyrate induced an increase in intracellular Ca2+ in these cells that was not blocked by treatment with pertussis toxin (PTX). SCFAs significantly reduced forskolin-induced cAMP levels in these cells. The phosphorylation of mitogen-activated protein kinase (MAPK) p38 was selectively increased by SCFAs. The downstream substrate heat shock protein 27 (HSP27) was also phosphorylated by SCFAs at Ser-78 and-82, but not-15. Propionate induced elevations in intracellular Ca2+ and the phosphorylation of p38 were inhibited by the silencing of GPR43 using a specific siRNA. These results suggest that GPR41 and 43 mediate SCFA signaling in mammary epithelial cells and thereby play an important role in their stress management.     

Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2.

Neu differentiation factor (NDF)-induced signaling involves the activation of members of the ErbB family of receptor tyrosine kinases. Although ectopic expression of recombinant ErbB receptors has yielded valuable insight into their signaling properties, the biological function and in vivo interplay of these receptors are still poorly understood. We addressed this issue by studying NDF signaling in various human cell lines expressing moderate levels of all known ErbB receptors. NDF-induced phosphorylation of ErbB-2 and ErbB-3 was found in the breast epithelial cell line MCF10A, the breast tumor cell lines T47D and MCF7, and the ovarian tumor cell line OVCAR3. Despite similar expression levels, NDF-induced phosphorylation of ErbB-4 was cell specific and only detected in T47D and OVCAR3 cells. Blocking cell surface expression of ErbB-2 by intracellular expression of a single-chain antibody revealed that in these two cell lines, ErbB-2 significantly enhanced phosphorylation of ErbB-4. Efficient NDF-induced phosphorylation of ErbB-3 was strictly ErbB-2 dependent in the breast tumor cell lines T47D and MCF7, while it was largely ErbB-2 independent in MCF10A and OVCAR3 cells. Consequently, NDF-stimulated intracellular signaling and induction of a biological response displayed a cell-specific requirement for ErbB-2. Thus, while ErbB-2 cooperates with NDF receptors in the breast tumor cell lines, ErbB-2 independent mechanisms seem to prevail in other cellular contexts.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.     

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.     

Genistein induces Ca2+ -mediated, calpain/caspase-12-dependent apoptosis in breast cancer cells

Genistein, a soy-derived isoflavone, has been suggested for breast cancer prevention; however, use of soy products for this purpose remains controversial. Genistein has been reported to regulate growth of tumor cells, although the involved molecular mechanisms are not defined. Here we report that genistein induces apoptosis in breast cancer cells via activation of the Ca2+ -dependent proapoptotic proteases, mu-calpain, and caspase-12. The treatment of MCF-7 breast cancer cells with genistein induced a sustained increase in concentration of intracellular Ca2+ resulting from depletion of the endoplasmic reticulum Ca2+ stores. This increase in Ca2+ was associated with activation of mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 substrates and antibodies to active (cleaved) forms of the enzymes. Selective inhibition of Ca2+ binding sites of mu-calpain, forced increase of the cytosolic Ca2+ buffering capacity, and caspase inhibition decreased apoptotic indices in the genistein-treated cells. Our results suggest that Ca2+ -dependent proteases are potential targets for genistein in breast cancer cells and that the cellular Ca2+ regulatory activity of genistein underlies its apoptotic mechanism.     

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.     

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.     

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.     

Positive and negative regulation of insulin action by genistein in the endothelium

Genistein is an isoflavone phytoestrogen with biological activities in management of metabolic disorders. This study aims to evaluate the regulation of insulin action by genistein in the endothelium. Genistein inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and attenuated downstream Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, leading to a decreased nitric oxide (NO) production in endothelial cells. These results demonstrated its negative regulation of insulin action in the endothelium. Palmitate (PA) stimulation evoked inflammation and induced insulin resistance in endothelial cells. Genistein inhibited IKKβ and nuclear factor-кB (NF-кB) activation with down-regulation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production and expression. Genistein inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-mediated tyrosine phosphorylation. Genistein restored insulin-mediated Akt and eNOS phosphorylation, and then led to an increased NO production from endothelial cells, well demonstrating its positive regulation of insulin action under insulin-resistant conditions. Meanwhile, genistein effectively inhibited inflammation-enhanced mitogenic actions of insulin by down-regulation of endothelin-1 and vascular cell adhesion protein-1 overexpression. PA stimulation impaired insulin-mediated vessel dilation in rat aorta, while genistein effectively restored the lost vasodilation in a concentration-dependent manner (0.1, 1 and 10 μM). These results suggested that genistein inhibited inflammation and ameliorated endothelial dysfunction implicated in insulin resistance. Better understanding of genistein action in regulation of insulin sensitivity in the endothelium could be beneficial for its possible applications in controlling endothelial dysfunction associated with diabetes and insulin resistance.     

PBK/TOPK在乳腺癌细胞中介导MEK非依赖性ERK激活中作用机制分析

该文探讨了乳腺癌细胞中表皮生长因子(EGF)介导的MEK非依赖性ERK激活通路。Western blot检测EGF刺激下,siRNA抑制MEK1/2后的T47D细胞的p-ERK水平,以验证T47D细胞中存在EGF介导的MEK非依赖性ERK激活的通路。接着使用可能参与MEK非依赖性ERK激活的激酶的小分子抑制剂抑制相关激酶(AC、PKC、Src、PI3K、PDK1和Akt)活性后,检测T47D细胞EGF介导ERK的磷酸化水平。siRNA抑制MEK1/2表达后,T47D细胞在EGF刺激后的仍保留部分p-ERK,即在T47D细胞中,存在EGF介导的MEK非依赖性的ERK磷酸化通路。小分子抑制剂抑制AC、PKC、Src对MEK非依赖性ERK激活途径影响不大。而使用小分子抑制剂抑制PI3K、PDK1和Akt后,ERK的磷酸化水平显著降低,提示PI3K/Akt通路下游的激酶参与T47D中EGF介导的MEK非依赖性ERK激活途径。siRNA干扰PI3K/Akt通路下游PBK/TOPK后并使用U0126抑制MEK功能后,几乎检测不到p-ERK,提示PBK/TOPK参与T47D细胞中EGF介导的MEK非依赖性ERK激活途径。乳腺癌抗雌激素药物耐药株T47D细胞存在EGF介导的MEK非依赖性ERK激活途径,且该途径受PI3K/Akt下游的PBK/TOPK调控。     

Extracellular matrix enhances heregulin-dependent BRCA1 phosphorylation and suppresses BRCA1 expression through its C terminus

Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [(3)H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-L-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through alpha(6) integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.     

Impact of protein tyrosine kinase 6 (PTK6) on human epidermal growth factor receptor (HER) signalling in breast cancer

PTK6, also known as Brk, is highly expressed in over 80% of breast cancers. In the last decade several substrates and interaction partners were identified localising PTK6 downstream of HER receptors. PTK6 seems to be involved in progression of breast tumours, in particular in HER receptor signalling. Here, we show the down-regulation effects of PTK6 in the T47D, BT474 and JIMT-1 breast cancer cell lines. PTK6 knockdown leads to a decreased phosphorylation of HER2, PTEN, MAPK (ERK), p38 MAPK, STAT3 and to a reduced expression of cyclin E. Our findings show that silencing PTK6 impairs the downstream targets of HER receptors and consequently the activation of signalling molecules. Furthermore, lower levels of PTK6 result in reduced migration of T47D and JIMT-1 breast cancer cells. Due to decreased migration, the PTK6 RNA interference might contribute to reduced metastasis and malignant potential of breast cancer cells. Since PTK6 plays an important role in HER receptor signal transduction, its down-regulation might be suitable for future therapy approaches in breast cancer.