Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments.

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in 167L and 211L do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a 167L to 167A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to 131ELVTDFPTDLPATE144 had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These results suggest that a shorter motif that is found in MMP-cleaved OPN, 162SVVYG166, is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the beta1 integrins, which have been previously characterized to bind the SVVYGLR sequence.     

Talin2 mediates secretion and trafficking of matrix metallopeptidase 9 during invadopodium formation

Talin2 plays an important role in transduction of mechanical signals between extracellular matrix and actin cytoskeleton. Recent studies showed that talin2 is localized to invadopodia and regulates their maturation, subsequently cancer cell invasion and metastasis. However, the molecular mechanism whereby talin2 mediates invadopodium maturation is unknown. Here we show that ablation of talin2 in MDA-MB-231 cells inhibited the secretion of matrix metallopeptidase 9 (MMP9), a proteinase involved in extracellular matrix degradation in invadopodium maturation and metastasis. Furthermore, re-expression of talin2^WT in talin2-KO cells rescued MMP9 secretion, but talin2^S339C, a mutant with reduced β-integrin binding, did not, indicating that the talin2-β-integrin interaction is involved in the MMP9 secretion. Moreover, ablation of talin2 caused an accumulation of enlarged MMP9 vesicles. These vesicles co-localized with enlarged early, late endosomes and autophagosomes, suggesting talin2 controls MMP9 trafficking process. Therefore, these data suggest that talin2 regulates extracellular matrix degradation and invadopodium maturation by mediating MMP9 secretion.     

Up-regulation of alphavbeta3 integrin expression is a novel molecular response to chemotherapy-induced cell damage in a heregulin-dependent manner

alpha(v)beta(3) integrin has a dual role in apoptosis. Whereas ligated alpha(v)beta(3) activates cell survival pathways and suppresses pro-apoptotic signals, unligated alpha(v)beta(3) or integrins bound to soluble ligands promote apoptosis. In this study, we assessed the role of alpha(v)beta(3) in chemosensitivity of breast cancer cells expressing different levels of heregulin (HRG). Expression levels of the RGD-binding integrins alpha(v)beta(3) were measured in MDA-MB-231 human breast cancer cells and its low HRG-expressing derivative (MDA-MB-231/AS31) treated with the microtubule-interfering agents (MIAs) paclitaxel and vincristine. Following treatment, only alpha(v)beta(3) levels were significantly increased in MDA-MB-231 cells. Interestingly, alpha(v)beta(3) expression was more significantly up-regulated in the MDA-MB-231/AS31 cells than in the parental cells. This MIA-induced increase of alpha(v)beta(3) expression was correlated with a decrease in cell viability and an increase in apoptosis in MDA-MB-231/AS31 cells, indicating that overexpression of alpha(v)beta(3) is linked to chemotherapy-induced cell death in low HRG-expressing breast cancer models. Moreover, a paclitaxel-induced increase of alpha(v)beta(3) was also observed in MCF-7 cells but not in an doxorubicin-resistant derivative that shows cross-resistance to paclitaxel, further providing evidence that the extent of alpha(v)beta(3) up-regulation is related to cell damage. These results indicate that alpha(v)beta(3) integrin is dramatically up-regulated in low HRG-expressing breast cancer models that are highly responsive to MIAs, thus providing a novel molecular marker of chemosensitivity influenced by HRG levels in breast cancer cells.     

Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells

We have recently reported that osteopontin (OPN) stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells (Das, R., Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 28593-28606). However, the role(s) of OPN on AP-1-mediated uPA secretion and cell motility and the involvement of c-Src/epidermal growth factor receptor (EGFR) in these processes in breast cancer cells are not well defined. In this study we report that OPN induces alpha(v)beta(3) integrin-mediated c-Src kinase activity in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Ligation of OPN with alpha(v)beta(3) integrin induces kinase activity and tyrosine phosphorylation of EGFR in MDA-MB-231 and wild type EGFR-transfected MCF-7 cells, and this was inhibited by the dominant negative form of c-Src (dn c-Src) indicating that c-Src kinase plays a crucial role in this process. OPN induces association between alpha(v)beta(3) integrin and EGFR on the cell membrane in a macromolecular form with c-Src. Furthermore, OPN induces alpha(v)beta(3) integrin/EGFR-mediated ERK1/2 phosphorylation and AP-1 activation. Moreover, dn c-Src also suppressed the OPN-induced phosphatidylinositol (PI) 3-kinase activity in these cells indicating that c-Src acts as master switch in regulating MEK/ERK1/2 and phosphatidylinositol 3-kinase/Akt signaling pathways. OPN-induced ERK phosphorylation, AP-1 activation, uPA secretion, and cell motility were suppressed when cells were transfected with dn c-Src or pretreated with alpha(v)beta(3) integrin antibody, c-Src kinase inhibitor (pp2), EGFR tyrosine kinase inhibitor (PD153035), and MEK-1 inhibitor (PD98059). To our knowledge, this is the first report that OPN induces alpha(v)beta(3) integrin-mediated AP-1 activity and uPA secretion by activating c-Src/EGFR/ERK signaling pathways and further demonstrates a functional molecular link between OPN-induced integrin/c-Src-dependent EGFR phosphorylation and ERK/AP-1-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

A regulated interaction between alpha5beta1 integrin and osteopontin

The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

CD44v4 is a major E-selectin ligand that mediates breast cancer cell transendothelial migration

Background

Endothelial E-selectin has been shown to play a pivotal role in mediating cell–cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown.

Methodology/Principal Findings

By assessing migration of various breast cancer cells across TNF-α pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLe^x) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a ∼170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLe^x moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA.

Conclusions/Significance

We demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin–dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis.     

骨唾液酸蛋白通过整合素αvβ3调控ILK信号通路

旨在探究骨唾液酸蛋白(BSP)是否通过整合素αvβ3对整合素连接激酶(ILK)信号通路进行调控。BSP基因沉默乳腺癌MDA-MB-231细胞,流式细胞仪在细胞水平检测BSP不同水平的细胞株中整合素αvβ3的表达量。Western blotting检测磷酸化ILK水平的变化,MTT法检测细胞增殖能力。与对照组231BO-Scrambled细胞相比,BSP基因沉默组231BO-BSP27细胞中整合素αvβ3的表达水平明显下调(61.32±1.94)%(P<0.01)。整合素αvβ3鼠抗单克隆抗体(LM609)处理前的BSP基因沉默组231BO-BSP27细胞与21BO-Scrambled细胞相比,ILK磷酸化水平下调明显(39.38±1.38)%(P<0.01);LM609处理后的231BO-BSP27细胞与21BO-Scrambled细胞相比,ILK磷酸化水平下调明显(33.78±1.51)%(P<0.01)。向乳腺癌细胞231BO-scrambled和231BO-BSP27中添加LM609,MTT试验结果显示两株乳腺癌细胞的增殖能力均有降低(P<0.05)。BSP通过整合素αvβ3对乳腺癌MDA-MB-231细胞ILK信号通路进行调控,并影响细胞增殖。     

Combination of integrin siRNA and irradiation for breast cancer therapy

Up-regulation of integrin alpha(v)beta(3) has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin alpha(v)beta(3) in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin alpha(v) siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin alpha(v)beta(3)-positive). Integrin alpha(v)beta(3)-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin alpha(v)beta(3) expression in MDA-MB-435 cells and integrin alpha(v) siRNA can effectively reduce both alpha(v) and alpha(v)beta(3) integrin expression, leading to increased radiosensitivity. Integrin alpha(v) siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin alpha(v)beta(3)-positive cancer types.     

New signaling pathways from cancer progression modulators to mRNA expression of matrix metalloproteinases in breast cancer cells

We observed previously that each of seven cancer progression inhibitors suppresses the mRNA expression of some matrix metalloproteinases (MMPs), but stimulates that of others, in breast cancer cells. In the present study we tested the effect of overexpressing other cancer modulators on MMP expression. The MMPs tested are MMP1, MMP2, MMP7, MMP13, MMP14, MMP16, MMP19, and MMP25. The proteins that were overexpressed are cancer inhibitors (NME, DRG1, IL10), enhancers (SOD2, FAK, IL17, and CREB), and proteins that suppress cancer progression in cells of some cancers and promote it in others (FUT1, integrin beta3, serpin E1, TIAM1, and claudin 4). Unexpectedly, all of them only lowered MMP mRNA expression, mainly of MMP16, MMP2, and MMP13, in breast cancer cells. Signaling from SOD2 uncoupled the accumulation of two MMP16 mRNA splice variants, suggesting signaling to a late step in MMP16 mRNA accumulation, such as MMP16 mRNA stabilization or late mRNA processing. Signaling that modulates MMP expression differed widely among the total population of MDA-MB-231 cells and single-cell progenies cloned from that population. It also differed substantially between cells of two metastatic breast basal adenocarcinomas, MDA-MB-231 and MDA-MB-468. The present study detected 37 new signaling pathways from cancer progression modulators located upstream of MMP mRNA expression in human breast cancer cells. Our siRNA-induced MMP knockdown data support the interpretation that signaling from MMP19, MMP1, MMP7, MMP12, MMP14, and MMP11 each stimulates the mRNA expression of other MMPs in breast cancer cells.     

Beta(3) integrin expression increases breast carcinoma cell responsiveness to the malignancy-enhancing effects of osteopontin

Osteopontin (OPN) is a secreted phosphoprotein that has been associated with malignancy of breast and other cancers. OPN binds to several cell surface integrins including alpha(v)beta(3), alpha(v)beta(5), and alpha(v)beta(1). Although the relative contribution of these integrins to breast cancer cell malignancy is uncertain, correlative studies suggest that alpha(v)beta(3) may be particularly associated with increased tumor aggressiveness. Previously, we reported that tumorigenic, nonmetastatic 21NT mammary carcinoma cells respond to OPN through alpha(v)beta(5) and alpha(v)beta(1) but not alpha(v)beta(3). Here, we determined that 21NT cells lack beta(3) expression, and we asked whether expression of alpha(v)beta(3) could enhance the ability of breast cancer cells to respond to the malignancy-promoting effects of OPN both in vitro and in vivo. 21NT cells stably transfected with beta(3) showed significantly increased adhesion, migration, and invasion to OPN in vitro compared with vector control. To determine if beta(3) could also enhance the response of breast epithelial cells to OPN in vivo, cells stably transfected with both beta(3) and OPN (NT/Obeta(3)) were injected into the mammary fat pad of female nude mice and primary tumor growth was assessed relative to controls. Mice injected with NT/Obeta(3) cells demonstrated a significantly increased primary tumor take (75% of mice) compared with controls (0-12.5% of mice) as well as a decreased tumor doubling time and a decreased tumor latency period. These results suggest that increased expression of the alpha(v)beta(3) integrin during breast cancer progression can make tumor cells more responsive to malignancy-promoting ligands such as OPN and result in increased tumor cell aggressiveness.     

Receptor-type protein tyrosine phosphatase alpha (PTPα) mediates MMP14 localization and facilitates triple-negative breast cancer cell invasion

The ability of cancer cells to invade surrounding tissues requires degradation of the extracellular matrix (ECM). Invasive structures, such as invadopodia, form on the plasma membranes of cancer cells and secrete ECM-degrading proteases that play crucial roles in cancer cell invasion. We have previously shown that the protein tyrosine phosphatase alpha (PTPα) regulates focal adhesion formation and migration of normal cells. Here we report a novel role for PTPα in promoting triple-negative breast cancer cell invasion in vitro and in vivo. We show that PTPα knockdown reduces ECM degradation and cellular invasion of MDA-MB-231 cells through Matrigel. PTPα is not a component of TKS5-positive structures resembling invadopodia; rather, PTPα localizes with endosomal structures positive for MMP14, caveolin-1, and early endosome antigen 1. Furthermore, PTPα regulates MMP14 localization to plasma membrane protrusions, suggesting a role for PTPα in intracellular trafficking of MMP14. Importantly, we show that orthotopic MDA-MB-231 tumors depleted in PTPα exhibit reduced invasion into the surrounding mammary fat pad. These findings suggest a novel role for PTPα in regulating the invasion of triple-negative breast cancer cells.     

Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.     

Toll-Like Receptor 4 Prompts Human Breast Cancer Cells Invasiveness via Lipopolysaccharide Stimulation and Is Overexpressed in Patients with Lymph Node Metastasis

Toll-like receptor (TLR)4-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study investigated the expression and biological role of TLR4 in human breast cancer metastasis. MCF-7 and MDA-MB-231 are human breast cancer cell lines with low and high metastatic potential, respectively. Using lipopolysaccharide (LPS) to stimulate MCF-7 and MDA-MB-231 cells, expression of TLR4 mRNA and protein increased compared with that in control cells. TLR4 activation notably up-regulated expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor(VEGF) mRNA and their secretion in the supernatants of both cell lines. LPS enhanced invasion of MDA-MB-231 cells by transwell assay and MCF-7 cells by wound healing assay. LPS triggered increased expression of TLR4 downstream signaling pathway protein myeloid differentiation factor 88(MyD88) and resulted in interleukin (IL)-6 and IL-10 higher production by human breast cancer cells. Stimulation of TLR4 with LPS promoted tumorigenesis and formed metastatic lesions in liver of nude mice. Moreover, expression of TLR4 and MyD88 as well as invasiveness and migration of the cells could be blocked by TLR4 antagonist. Combined with clinicopathological parameters, TLR4 was overexpressed in human breast cancer tissue and correlated with lymph node metastasis. These findings indicated that TLR4 may participate in the progression and metastasis of human breast cancer and provide a new therapeutic target.     

Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA

Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.     

Cancer cell-derived interleukin 1alpha contributes to autocrine and paracrine induction of pro-metastatic genes in breast cancer

Invasion and metastasis of cancer cells is a complex process requiring the activity of proteins that promote extracellular matrix degradation, motility of cancer cells, and angiogenesis. Although exclusively the cancer cells make several of these proteins, few key proteins are derived from stromal cells in response to cancer cell-stromal cell interaction. In this report, we show that the breast cancer cell-derived interleukin-1alpha (IL-1alpha) plays an important role in expression of pro-metastatic genes in cancer as well as in stromal cells. Neutralizing antibody against IL-1alpha inhibited IL-6, and IL-8 expression in IL-1alpha-expressing cancer cells. In addition, this antibody also prevented induction of IL-6, IL-8, and matrix metalloproteinase 3 (MMP3) but not vascular endothelial growth factor (VEGF) in fibroblasts by conditioned medium (CM) from IL-1alpha-expressing breast cancer cells. These results suggest that inhibition of IL-1alpha activity by either neutralizing antibody against IL-1alpha or chemical inhibitor of IL-1alpha processing may prevent invasion and metastasis of breast cancer.     

S-Allylcysteine reduces breast tumor cell adhesion and invasion

Previous studies show that aqueous garlic extract and its derivatives (e.g. S-allylcysteine [SAC]) prevent carcinogen-induced breast tumorigenesis. However, investigations testing the effect of SAC on later stages of breast tumorigenesis and/or metastasis have produced mixed results. Here we show that SAC significantly reduced anchorage-dependent and -independent growth of MDA-MB-231 breast tumor cells in a dose- and time-dependent fashion, and sub-lethal SAC-treatment altered mammary tumor cell adhesion and invasion through components of the extracellular matrix. We provide evidence to suggest increased expression of E-cadherin and reduced MMP-2 expression and activity are partially responsible for inhibition of mammary tumor cell invasion by SAC. Because E-cadherin and MMP-2 are important in cancer metastasis, these results suggest a link between SAC induction of E-cadherin and reduction of MMP2 activity with the inhibition of cell motility and invasion; thus providing evidence that events leading to breast cancer metastasis are repressed by sub-lethal SAC-treatment.     

Platycodin D inhibits migration,invasion, and growth of MDA-MB-231 human breast cancer cells via suppression of EGFR-mediated Akt and MAPK pathways

Platycodin D (PD), an active triterpenoid saponin from Platycodon grandiflorum, has been known to inhibit the proliferation of a variety of cancer cells, but the effect of PD on the invasiveness of cancer cells is largely unknown. In this study, we first determined the molecular mechanism by which PD inhibits the migratory and invasive abilities of the highly metastatic MDA-MB-231 breast cancer cell line. We demonstrated that a non-cytotoxic concentration of PD markedly suppressed wound healing migration, invasion through the matrigel, and adhesion to an ECM-coated substrate in a dose-dependent manner. Moreover, PD inhibited cell invasion by reducing matrix metalloproteinase (MMP)-9 enzyme activity and mRNA expression. Western blot analysis indicated that PD potently suppressed the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) as well as blocked the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling pathway. Furthermore, PD treatment inhibited the DNA binding activity of NF-κB, which is known to mediate the expression of epidermal growth factor receptor (EGFR), as observed by electrophoretic mobility shift assay. Specific mechanisms of action exerted by PD involved the downregulation of EGFR and the inhibition of EGF-induced activation of the EGFR, MAPK, and PI3K/Akt pathways. The in vivo studies showed that PD significantly inhibited the growth of MDA-MB-231 xenograft tumors in BALB/c nude mice. These results suggest that PD might be a potential therapeutic candidate for the treatment of breast cancer metastasis.     

Benzo-[a]-pyrene induces FAK activation and cell migration in MDA-MB-231 breast cancer cells

Benzo-[a]-pyrene (B[a]P) is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant. It is a mammary carcinogen in rodents and contributes to the development of human breast cancer. However, the signal transduction pathways induced by B[a]P and its role in breast cancer progression have not been studied in detail. Here, we demonstrate that B[a]P induces cell migration through a lipoxygenase- and Src-dependent pathway, as well as the activation of focal adhesion kinase, Src, and the extracellular signal-regulated kinase 2 in MDA-MB-231 breast cancer cells. However, B[a]P is not able to promote migration in the mammary nontumorigenic epithelial cells MCF12A. Moreover, B[a]P promotes an increase of αvβ3 integrin–cell surface levels and an increase of metalloproteinase (MMP)-2 and MMP-9 secretions. In summary, our findings demonstrate that B[a]P induces the activation of signal transduction pathways and biological processes involved in the invasion/metastasis process in MDA-MB-231 breast cancer cells.     

MicroRNA-224 targets RKIP to control cell invasion and expression of metastasis genes in human breast cancer cells

The Raf kinase inhibitor protein (RKIP) is a tumor suppressor that protects against metastasis and genomic instability. RKIP is downregulated in many types of tumors, although the mechanism for this remains unknown. MicroRNAs silence target genes via translational inhibition or target mRNA degradation, and are thus important regulators of gene expression. In the current study, we found that miR-224 expression is significantly upregulated in breast cancer cell lines, and especially in highly invasive MDA-MB-231 cells, compared to human normal breast epithelial cells. In addition, miR-224 inhibits RKIP gene expression by directly targeting its 3'-untranslated region (3'-UTR). Moreover, metastasis, as assayed by Transwell migration, 3D growth in Matrigel, and wound healing, was enhanced by ectopic expression of miR-224 and inhibited by miR-224 downregulation. Promotion of metastasis in response to miR-224 downregulation was associated with derepression of the stroma-associated RKIP target genes, CXCR4, MMP1, and OPN, which are involved in breast tumor metastasis to the bone. Taken together, our data indicate that miR-224 play an important role in metastasis of human breast cancer cells to the bone by directly suppressing the RKIP tumor suppressor.