Downregulation of cPLA2γ expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway

Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA₂γ in chemotaxis of human breast cancer cells. Inhibition of cPLA₂γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA₂γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA₂γ regulated migration. Knockdown of cPLA₂γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA₂γ also impaired the phosphorylation of integrin β1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA₂γ plays an important role in cancer cell chemotaxis.     

Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis

BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.     

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Protein Kinase C�� Supports Survival of MDA-MB-231 Breast Cancer Cells by Suppressing the ERK1/2 Pathway

Mechanisms that mediate apoptosis resistance are attractive therapeutic targets for cancer. Protein kinase Cδ (PKCδ) is considered a pro-apoptotic factor in many cell types. In breast cancer, however, it has shown both pro-survival and pro-apoptotic effects. Here, we report for the first time that down-regulation of PKCδ per se leads to apoptosis of MDA-MB-231 cells. Inhibition of MEK1/2 by either PD98059 or U0126 suppressed the induction of apoptosis of PKCδ-depleted MDA-MB-231 cells but did not support survival of MCF-7 or MDA-MB-468 cells. Basal ERK1/2 phosphorylation was substantially higher in MDA-MB-231 cells than in the other cell lines. PKCδ depletion led to even higher ERK1/2 phosphorylation levels and also to lower expression levels of the ERK1/2 phosphatase MKP3. Depletion of MKP3 led to apoptosis and higher levels of ERK1/2 phosphorylation, suggesting that this may be a mechanism mediating the effect of PKCδ down-regulation. However, PKCδ silencing also induced increased MEK1/2 phosphorylation, indicating that PKCδ regulates ERK1/2 phosphorylation both upstream and downstream. Moreover, PKCδ silencing led to increased levels of the E3 ubiquitin ligase Nedd4, which is a potential regulator of MKP3, because down-regulation led to increased MKP3 levels. Our results highlight PKCδ as a potential target for therapy of breast cancers with high activity of the ERK1/2 pathway.     

Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.     

Investigate the role of PTEN in chemotaxis of human breast cancer cells

Chemotaxis plays an important role in metastasis of cancer cells. In the current study, we investigated the role of PTEN, a tumor suppressor, in chemotaxis of human breast cancer cells. Over-expression of PTEN inhibited EGF-induced chemotaxis, probably due to an overall reduction of PIP(3) levels. Disruption of PTEN by siRNA caused a marked decrease in chemokinesis, cell adhesion, and membrane spreading, resulting in a severe defect in chemotaxis. In PTEN disrupted cells, PDK1, AKT, and PKCzeta exhibited elevated basal activities, which prevented EGF-induced further activation of these molecules. In the absence of EGF, active PDK1 was detected on multiple directions of the plasma membranes of PTEN disrupted cells, which competed against EGF-induced gradient sensing. To confirm the biological relevance of in vitro studies, both PTEN disrupted cells and its parental human breast cancer cells were injected into tail veins of SCID mice. Mice injected with PTEN disrupted cancer cells showed a marked decrease in lung metastasis. Taken together, our data show that PTEN plays a non-redundant role in EGF-induced chemotaxis of human breast cancer cells, and an optimal level of PTEN is required in these responses.     

Phosphorylated LIM Kinases Colocalize with Gamma-Tubulin in Centrosomes During Early Stages of Mitosis

LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with γ-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with γ-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that γ-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with γ-tubulin and play a role in mitotic spindle assembly.     

NUAK1增加乳腺癌细胞的趋化和粘附能力

本课题组先前已证明NUAK1/ARK5可通过影响F-actin的聚合从而促进乳腺癌细胞的侵袭和转移. 但是NUAK1是否还通过其它机制影响乳腺癌的侵袭和转移尚有待于探讨.本文证明NUAK1还可以影响乳腺癌细胞趋化、粘附能力从而在乳腺癌细胞侵袭转移中起重要作用. 应用化学合成的小RNA干扰质粒转染到乳腺癌细胞系MDA MB 231中,用免疫印迹技术检测NUAK1蛋白的表达情况. 结果显示,在敲除NUAK1的细胞（siNUAK1/MDA231）中, NUAK1蛋白表达水平明显降低;趋化运动实验结果显示, siNUAK1/MDA231细胞的趋化运动能力比未处理组（Scr/MDA231）细胞明显降低;细胞粘附实验结果显示, EGF刺激5 min、15 min后,siNUAK1/MDA231细胞比Scr/MDA231细胞粘附细胞数量均明显减少;免疫印迹技术检测integrin β1磷酸化验证NUAK1影响乳腺癌细胞粘附的机制. 结果显示,siNUAK1/MDA231细胞内integrin β1磷酸化比Scr/MDA231细胞不同程度降低. 上述结果表明, NUAK1通过磷酸化integrin β1促进乳腺癌细胞与纤维粘连蛋白的粘附,从而促进乳腺癌的侵袭和转移.     

NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移

本课题组先前研究证明NUAK1/ARK5参与乳腺癌、胶质瘤侵袭转移,但机制尚不清楚.本文证明,NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移.应用小RNA干扰技术敲除乳腺癌细胞系MDA-MB-231中的NUAK1,用G418进行稳定筛选,并用Western印迹进行蛋白质表达检测,结果显示,成功敲除MDA-MB-231细胞中的NUAK1;采用Transwell侵袭实验检测NUAK1在乳腺癌细胞侵袭转移中的作用,结果表明,NUAK1被干扰的SiNUAK1/MDA-231细胞的侵袭能力明显减弱;应用免疫荧光法对细胞的F-actin进行荧光染色,半定量F-actin聚合分析结果显示,IGF-1在转染空载的细胞组（Scr/MDA-231）能诱导肌动蛋白短暂的聚合反应,而在敲除NUAK1的细胞组（SiNUAK1/MDA-231）肌动蛋白的聚合显著减少;用细胞因子IGF-1刺激乳腺癌细胞观察cofilin磷酸化,结果显示,在敲除NUAK1的细胞组（SiNUAK1/MDA-231）,IGF-1诱导的cofilin的磷酸化明显受抑制.上述结果表明,NUAK1能通过促进F-actin的聚合从而影响乳腺癌细胞的侵袭转移.     

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.     

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.     

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.     

The involvement of lipid rafts in epidermal growth factor-induced chemotaxis of breast cancer cells

Metastasis is the major cause of morbidity and mortality in cancer. Recent studies reveal a role of chemotaxis in cancer cell metastasis. Epidermal growth factor receptors (EGFR) have potent chemotactic effects on human breast cancer cells. Lipid rafts, organized microdomain on plasma membranes, regulate the activation of many membrane receptors. In the current study, we investigated the role of lipid rafts in EGFR-mediated cancer cell chemotaxis. Our confocal microscopy results suggested that EGFR co-localized with GM1-positive rafts. Disrupting rafts with methyl-β-cyclodextrin (mβCD) inhibited EGF-induced chemotaxis of human breast cancer cells. Supplementation with cholesterol reversed the inhibitory effects. Pretreatment with mβCD also impaired directional migration of cells in an in vitro “wound healing” assay, EGF-induced cell adhesion, actin polymerization, Akt phosphorylation and protein kinase Cζ (PKCζ) translocation. Taken together, our study indicated that integrity of lipid rafts was critical in EGF-induced chemotaxis of human breast cancer cells.     

Integrin α2β1 Mediates Tyrosine Phosphorylation of Vascular Endothelial Cadherin Induced by Invasive Breast Cancer Cells

The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of β-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α₂β₁ integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α₂β₁ integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of β-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with β₁-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α₂β₁ integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.     

Modeling of Hypo/Hyperglycemia and Their Impact on Breast Cancer Progression Related Molecules

Breast cancer (BC) arises commonly in women with metabolic dysfunction. The underlying mechanism by which glycemic load can exert its action on tumor metastasis is under investigated. In this study we showed that glycemic microenvironment alters the expression of three classes of proteins, VEGF and its receptors, cell to cell, and cell to extracellular matrix (ECM) adhesion proteins in MDA-MB-231 parental cells and its two metastatic variants to the bone and brain (MDA-MB-231BO and MDA-MB-231BR, respectively). Using western blotting, we showed that VEGFR2 levels were higher in these variant cells and persisted in the cells under extreme hypoglycemia. Hypoglycemia did not alter VEGFR2 expression per se but rather suppressed its posttranslational glycosylation. This was reversed rapidly upon the restoration of glucose, and cyclohexamide (CHX) treatment demonstrated that this deglycosylated VEGFR2 was not a product of de-novo protein synthesis. VEGFR2 co-receptor Neuropilin-1 was up-regulated four-fold in all MDA-MB-231 cells (parental and two variants) compared to VEGFR2 expression, and was also susceptible to glycemic changes but resistant to CHX treatment for up to 72 hrs. Hypoglycemia also resulted in a significant decrease in specific catenin, cadherin, and integrin proteins, as well as cellular proliferation and colony forming ability. However, MDA-MB-231BR cells showed a unique sensitivity to hypo/hyperglycemia in terms of morphological changes, colony formation ability, integrin β3 expression and secreted VEGF levels. In conclusion, this study can be translated clinically to provide insight into breast cancer cell responses to glycemic levels relevant for our understanding of the interaction between diabetes and cancer.     

Arachidonic acid promotes migration and invasion through a PI3K/Akt-dependent pathway in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n−6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, however it might be present in the extracellular microenvironment. AA and its metabolites mediate FAK activation, adhesion and migration in MDA-MB-231 breast cancer cells. However, it remains to be investigated whether AA promotes invasion and the signal transduction pathways involved in migration and invasion. Here, we demonstrate that AA induces Akt2 activation and invasion in MDA-MB-231 cells. Akt2 activation requires the activity of Src, EGFR, and PIK3, whereas migration and invasion require Akt, PI3K, EGFR and metalloproteinases activity. Moreover, AA also induces NFκB-DNA binding activity through a PI3K and Akt-dependent pathway. Our findings demonstrate, for the first time, that Akt/PI3K and EGFR pathways mediate migration and invasion induced by AA in MDA-MB-231 breast cancer cells.     

The involvement of lipid rafts in epidermal growth factor-induced chemotaxis of breast cancer cells

Metastasis is the major cause of morbidity and mortality in cancer. Recent studies reveal a role of chemotaxis in cancer cell metastasis. Epidermal growth factor receptors (EGFR) have potent chemotactic effects on human breast cancer cells. Lipid rafts, organized microdomain on plasma membranes, regulate the activation of many membrane receptors. In the current study, we investigated the role of lipid rafts in EGFR-mediated cancer cell chemotaxis. Our confocal microscopy results suggested that EGFR co-localized with GM1-positive rafts. Disrupting rafts with methyl-beta-cyclodextrin (mbetaCD) inhibited EGF-induced chemotaxis of human breast cancer cells. Supplementation with cholesterol reversed the inhibitory effects. Pretreatment with mbetaCD also impaired directional migration of cells in an in vitro "wound healing" assay, EGF-induced cell adhesion, actin polymerization, Akt phosphorylation and protein kinase Czeta (PKCzeta) translocation. Taken together, our study indicated that integrity of lipid rafts was critical in EGF-induced chemotaxis of human breast cancer cells.