Osteopontin: role in cell signaling and cancer progression

Cell migration and degradation of the extracellular matrix (ECM) are crucial steps in tumor progression. Several matrix-degrading proteases, including matrix metalloproteases, are highly regulated by growth factors, cytokines and ECM proteins. Osteopontin (OPN), a chemokine-like, calcified ECM-associated protein, plays a crucial role in determining the metastatic potential of various cancers. Since its first identification in bone, the multifaceted roles of OPN have been an area of intense investigation. Extensive research has elucidated the pivotal role of OPN in regulating the cell signaling that controls tumor progression and metastasis. This review focuses on recent advances in understanding the functional role of the OPN-induced signaling pathway in the regulation of cell migration and tumor progression and the implications for identifying novel targets for cancer therapy.     

Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappa B-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells

Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and play critical roles in tissue repair, tumor invasion, and metastasis. MMPs are regulated by different cytokines, ECM proteins, and other factors. However, the molecular mechanisms by which osteopontin (OPN), an ECM protein, regulates ECM invasion and tumor growth and modulates MMP activation in B16F10 cells are not well defined. We have purified OPN from human milk and shown that OPN induces pro-MMP-2 production and activation in these cells. Moreover, our data revealed that OPN-induced membrane type 1 (MT1) MMP expression correlates with translocation of p65 (nuclear factor-kappaB (NF-kappaB)) into the nucleus. However, when the super-repressor form of IkappaBalpha (inhibitor of NF-kappaB) was transfected into cells followed by treatment with OPN, no induction of MT1-MMP expression was observed, indicating that OPN activates pro-MMP-2 via an NF-kappaB-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with alpha(v)beta(3) integrin, but these effects were reduced drastically when the MMP-2-specific antisense S-oligonucleotide was used to suppress MMP-2 expression. Interestingly, when the OPN-treated cells were injected into nude mice, the mice developed larger tumors, and the MMP-2 levels in the tumors were significantly higher than in controls. The proliferation data indicate that OPN increases the growth rate in these cells. Both tumor size and MMP-2 expression were reduced dramatically when anti-MMP-2 antibody or antisense S-oligonucleotide-transfected cells were injected into the nude mice. To our knowledge, this is the first report that MMP-2 plays a direct role in OPN-induced cell migration, invasion, and tumor growth and that demonstrates that OPN-stimulated MMP-2 activation occurs through NF-kappaB-mediated induction of MT1-MMP.     

Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation

We have recently demonstrated that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IkappaBalpha kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced alpha(v)beta(3) integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKKalpha/beta in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NFkappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKKalpha/beta, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKalpha/beta (dn IKKalpha/beta), and dn ERK1/2 suppressed the OPN-induced NFkappaB activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NFkappaB activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKKalpha/beta-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.     

Matrix metalloproteinase-2: Mechanism and regulation of NF-κB-mediated activation and its role in cell motility and ECM-invasion

Matrix metalloproteinases belong to a family of enzymes that degrade the extracellular matrix (ECM) components and play an important role in tissue repair, tumor invasion, and metastasis. ECM proteins, cytokines, and certain other factors regulate MMP activity. OPN, an ECM protein, has been found to be overexpressed in various cancers, and it has been shown to correlate with the metastatic potential. Although such reports indicate that OPN plays an important role in the ability of tumor cells to survive and metastasize to secondary sites, the mechanism by which OPN regulates these processes is yet to be understood. In this study we report that native purified human OPN can induce cell migration and ECM invasion. Increased invasiveness and migration correlates with enhanced expression and activation of MMP-2. Our study provides evidence showing that OPN increases gelatinolytic activity by inducing MT1-MMP expression via activation of the NF-B pathway. Suppression of MMP-2 by ASMMP-2 reduces the OPN-induced cell migration and ECM invasion. Curcumin blocks OPN-induced MT1-MMP expression and pro-MMP-2 activation. Curcumin, a known anti-inflammatory and anticarcinogenic compound, suppresses OPN-induced cell migration, invasion and induces apoptotic morphology in OPN-treated cells. The mechanism by which curcumin suppresses the OPN-induced effects has also been delineated. Curcumin inhibits MT1-MMP gene expression by blocking signals leading to IKK activation. This in turn inhibits IB phosphorylation and NF-B activation. Published in 2004.     

Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met)

Osteopontin (OPN) is a secreted glycophosphoprotein which induces migration of mammary carcinoma cells, and has been implicated in the malignancy of breast carcinoma. Hepatocyte growth factor (HGF) induces cell migration of several mammary epithelial cell (MEC) lines, via activation of its cognate receptor (Met). This study examines the mechanism of OPN-induced MEC migration, in terms of the cell surface integrins involved and induction of the HGF/Met pathway. Three different MEC cell lines were used, representing different stages of tumor progression: 21PT, non-tumorigenic; 21NT, tumorigenic; non-metastatic; and MDA-MB-435, tumorigenic, highly metastatic. Human recombinant OPN was found to induce the migration of all three lines. OPN-induced migration of 21PT and 21NT cells was alphavbeta5 and beta1-integrin dependent, and alphavbeta3-independent, while that of MDA-MB-435 cells was alphavbeta3-dependent. HGF also induced migration of all three cell lines, and a synergistic response was seen to HGF and OPN together. The increased migration response to OPN was found to be associated with an initial increase in Met kinase activity (within 30 min), followed by an increase in Met mRNA and protein expression. OPN-induced cell migration is thus mediated by different cell surface integrins in MEC lines representing different stages of progression, and involves activation of the HGF receptor, Met.     

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.     

Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression

Cancer progression depends on an accumulation of metastasis supporting cell signaling molecules that target signal transduction pathways and ultimately gene expression. Osteopontin (OPN) is one such chemokine like metastasis gene which plays a key signaling event in regulating the oncogenic potential of various cancers by controlling cell motility, invasiveness and tumor growth. We have reported that OPN stimulates tumor growth and nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkappaBalpha/IKK (IkappaBalpha kinase) signaling pathway in melanoma cells. Urokinase type plasminogen activator (uPA), a widely acting serine protease degrades the ECM components and plays a pivotal role in cancer progression. However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and uPA secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt/PKB (protein kinase B) in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with dominant negative mutant of p85 domain of PI 3'-kinase (Deltap85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECM-invasion. Furthermore, cells transfected with Deltap85 or super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkappaB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN induced PI 3'-kinase dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells.     

Nuclear factor inducing kinase: A key regulator in osteopontin- induced MAPK/IκB kinase dependent NF-κB-mediated promatrix metalloproteinase-9 activation

Osteopontin (OPN) is a secreted, non-collagenous, sialic-acid rich, glycosylated adhesive phospho- protein. Several highly metastatic transformed cells synthesized a higher level of OPN compared with non-tumorigenic cells. We have recently reported that OPN induces nuclear factor-κB (NF-κB)-mediated promatrix metalloproteinase-2 activation through IκBα/IKK signaling pathways. However, the molecular mechanism(s) by which OPN regulates pro-matrix metalloproteinase-9 (pro-MMP-9) activation and involvement of upstream kinases in regulation of these processes that ultimately control cell motility and tumor growth in murine melanoma cells are not well defined. Here we report that OPN induces αvβ3 integrin-mediated phosphorylation and activation of nuclear factor inducing kinase (NIK) and enhances the interaction between phosphorylated NIK and IκBα kinase α/β (IKKα/β) in B16F10 cells. Moreover, NIK is involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induces NIK-dependent NF-κB activation through ERK/IKKα/β-mediated pathways. Furthermore, OPN enhances NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, and cell motility. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. Taken together, NIK acts as crucial regulator in OPN-induced MAPK/IKK-mediated NF-κB-dependent uPA secretion and MMP-9 activation thereby controlling melanoma cell motility and chemoinvasion. An erratum to this article is available at .     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

Mesenchymal stem cells require integrin ��1 for directed migration induced by osteopontin in vitro

Mesenchymal stem cells (MSCs) are characterized by their ability of self-renewal paired with the capacity to differentiate into multiple mesenchymal cell lineages. Numerous studies have reported beneficial effects of MSCs in tissue repair and regeneration. After in vivo administration, MSCs home to and engraft to injured tissues. However, the molecular mechanisms are not clear. Osteopontin (OPN) has been found to be elevated in response to injury and inflammation and its role on cell mobilization has been studied. Therefore, the facts imply that OPN may contribute to the recruitment of MSCs to the sites of injury. In this study, using transwell assay, we found that rat bone marrow-derived mesenchymal stem cells (rMSCs) migrated towards OPN in a concentration-dependent manner. To further examine the involved molecular mechanisms for OPN-induced rMSCs migration, RT-PCR, and Western blot were used to detect the expressions of integrin β1 and CD44v6, the two receptors of OPN. OPN promoted integrin β1 mRNA and protein expression while CD44v6 mRNA level was not altered. Blockade of integrin β1 also inhibited OPN-induced rMSCs migration, indicating the possible involvement of integrin β1 in OPN-induced migration in rMSCs. Our data have shown for the first time that OPN increases integrin β1 expression in rMSCs and promotes rMSCs migration through the ligation to integrin β1.     

CIZ/NMP4 is expressed in B16 melanoma and forms a positive feedback loop with RANKL to promote migration of the melanoma cells

Tumor metastasis to bone is a serious pathological situation that causes severe pain, and deterioration in locomoter function. However, the mechanisms underlying tumor metastasis is still incompletely understood. CIZ/NMP4 is a nucleocytoplasmic shuttling protein and its roles in tumor cells have not been known. We, therefore, hypothesized the role of CIZ/NMP4 in B16 melanoma cells that metastasize to bone. CIZ/NMP4 is expressed in B16 cells. The CIZ/NMP4 expression levels are correlated to the metastatic activity in divergent types of melanoma cells. Overexpression of CIZ/NMP4 increased B16 cell migration in Trans-well assay. Conversely, siRNA-based knockdown of CIZ/NMP4 suppressed migratory activity of these cells. As RANKL promotes metastasis of tumor cells in bone, we tested its effect on CIZ in melanoma cells. RANKL treatment enhanced CIZ/NMP4 expression. This increase of CIZ by RANKL promoted migration. Conversely, we identified CIZ/NMP4 binding site in the promoter of RANKL. Furthermore, luciferase assay indicated that CIZ/NMP4 overexpression enhanced RANKL promoter activities, revealing a positive feedback loop of CIZ/NMP4 and RANKL in melanoma. These observations indicate that CIZ/NMP4 is critical regulator of metastasis of melanoma cells.     

Osteopontin induction of hyaluronan synthase 2 expression promotes breast cancer malignancy

Osteopontin (OPN) is a tumor-associated, secreted phosphoprotein that has been implicated in breast cancer progression and metastasis. Research concerning how OPN functions in tumor progression has led to the identification of a limited number of genes that contribute functionally to OPN-induced cellular behaviors. Recent microarray analysis, comparing 21NT breast cancer cells transfected to constitutively overexpress OPN with control cells, revealed hyaluronan synthase 2 (HAS2) to be a gene highly up-regulated in OPN-overexpressing cells. In this study, we further examined the relationship between OPN and HAS2. We show that 21NT OPN-transfected cells express high levels of HAS2, which is associated with increased HA production and matrix retention and is necessary for tumor cell adhesion to bone marrow endothelial cells and anchorage-independent growth. Finally, stable transfection of antisense HAS2 into 21NT cells overexpressing OPN resulted in a reduction in HAS2 expression, HA production, and pericellular retention. Antisense-mediated down-regulation of HAS2 also resulted in a significant decrease in cellular proliferation and colony growth in soft agar. To our knowledge, this is the first report of the ability of OPN to regulate HAS2 expression and HA production in breast cancer cells and further illustrates a unique functional relationship by which enhanced HA production facilitates OPN-mediated cell behaviors.     

Overexpression of human osteopontin increases cell proliferation and migration in human embryo kidney-293 cells

Malignant tumors are characterized by dysregulated cell growth and the metastasis of secondary tumors. Numerous studies have documented that osteopontin (OPN) plays a key role in regulating tumor progression and metastasis. Here, we show that the overexpression of OPN in human embryo kidney-293 cells significantly increases both the level of cell proliferation, by provoking the G₁/S transition, and the level of cell migration in vitro. These findings suggest that augmented OPN contributes to cell growth and motility. Inhibiting OPN or the pathway it stimulates may therefore represent a novel approach for the treatment of primary tumors and associated metastases.     

Tumor-derived osteopontin is soluble, not matrix associated.

The secreted phosphoprotein osteopontin (OPN), when immobilized on a surface, supports cell adhesion, prevents apoptosis of endothelial cells, and is a ligand for the alpha(v)beta(3) integrin, which is important in endothelial cell biology and neovascularization. OPN synthesized by tumor cells stimulates tumor growth, but the mechanism by which the protein acts remains unclear. One possibility, therefore, is that OPN may exert its effects on tumor growth by enhancing angiogenesis. While OPN is found at high levels in bone, where it is a component of the mineralized matrix, we have asked here whether OPN present in tumors is similarly extracellular matrix associated. We have shown that OPN is detectable in tumor extracts and in serum of tumor-bearing mice, and that the protein in tumors and in serum can be synthesized by both tumor and the host cells. Biochemical fractionation of tumor tissue confirmed that there is little if any association of OPN with the insoluble fraction. Immunochemical analysis of murine mammary tumors shows no co-localization of OPN with the extracellular matrix, identified by laminin staining. Ras-transformed cells in culture produce abundant OPN, however, the protein was found to be associated with the cell fraction but not with the matrix fraction. An enzyme-linked immunosorbent assay was used to demonstrate that OPN in conditioned medium from these cells fails to associate with extracellular matrix components, including laminin and fibronectin, in vitro. Recombinant OPN (GST-OPN) when coated onto a plastic surface can support human umbilical vein endothelial cell adhesion, suppressing apoptosis and allowing cell cycle progression, at concentrations from 1 to 50 microg/ml. Soluble GST-OPN in the same concentration range has no effect on HUVECs held in suspension. Thus, we conclude that OPN associated with tumors is primarily soluble, and that soluble OPN can neither support endothelial cell proliferation nor prevent apoptosis of these cells in the absence of adhesion.     

骨桥蛋白对细胞生存的多重作用

骨桥蛋白(osteopontin, OPN)作为一种分泌性蛋白质广泛存在于多种组织细胞中,不仅调控肿瘤细胞的转移、侵袭和增殖,也在炎症反应、血管再生等生理过程中发挥作用。OPN通过与受体结合直接或间接地激活细胞内信号途径,介导细胞与细胞、细胞与细胞外基质之间的相互作用,从而参与调控细胞的生存。大量实验证实,OPN能够促进细胞的增殖,抑制细胞凋亡,尤其是对于肿瘤细胞。但在有些细胞中,OPN对细胞命运的影响却恰恰相反。本文综述了OPN在不同条件下对细胞存活、活化和增殖,细胞自噬和细胞凋亡的多重作用及其作用途径,为进一步研究OPN对不同细胞作用的受体及其信号网络机制提供重要理论基础。     

Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells

We have recently reported that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IKK signaling pathways and that curcumin (diferulolylmethane) down-regulates these pathways (Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487-14497). However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and urokinase type plasminogen activator (uPA) secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt in highly invasive MDA-MB-231 and low invasive MCF-7 cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (Deltap85) and enhanced when cells were transfected with an activated form of PI 3-kinase (p110CAAX), indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. However, both pharmacological (wortmannin and LY294002) and genetic (Deltap85) inhibitors of PI 3'-kinase inhibited OPN-induced Akt phosphorylation, IKK activity, and NFkappaB activation through phosphorylation and degradation of IkappaBalpha. OPN also enhances uPA secretion, cell motility, and extracellular matrix invasion. Furthermore, cells transfected with Deltap85 or the super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility, whereas cells transfected with p110CAAX enhanced these effects. Pretreatment of cells with PI 3-kinase inhibitors or NFkappaB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. To our knowledge, this is first report that OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN-induced PI 3'-kinase-dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-κB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-κB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.     

Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.     

Versican G3 promotes mouse mammary tumor cell growth, migration, and metastasis by influencing EGF receptor signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis.