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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-B (NF-B)) into the nucleus. However, when the super-repressor form of IB␣ (inhibitor of NF-B) 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-B-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with ␣ v ␤ 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-B-mediated induction of MT1-MMP.Cell migration and extracellular matrix invasion are some of the major steps in embryonic development (1, 2) and wound healing and cancer cell metastasis (3, 4). However, the exact molecular mechanisms that regulate these processes are not well understood. In the past, several investigators have shown that matrix metalloproteinases (MMPs) 1 and the tissue inhibitor of matrix metalloproteinase (TIMP) play a major role in the regulation of cancer cell migration, extracellular matrix (ECM) invasion, and metastasis by degrading the ECM proteins (3-5). Current investigations have focused on the understanding of molecular mechanism(s) by which osteopontin (OPN), an ECM protein, regulates MMP expression both in vitro and in vivo and controls invasiveness and tumor growth in B16F10 cells.OPN is a noncollagenous, sialic acid-rich, and glycosylated phosphoprotein (6, 7). It has an N-terminal signal sequence, a highly acidic region consisting of nine consecutive aspartic acid residues, and a GRGDS cell adhesion sequence predicted to be flanked by the ␤-sheet structure (8). This protein has a functional thrombin cleavage site and is a substrate for tissue transglutaminase (7). OPN binds with type I collagen (9), fibronectin (...

We have recently demonstrated that osteopontin (OPN) induces nuclear factor B (NF B)-mediated promatrix metalloproteinase-2 activation through I B␣/ I B␣ 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 ␣ v ␤ 3 integrinmediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKK␣/␤ in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NF B activation through ERK/ IKK␣/␤-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, IKK␣/␤, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKK␣/␤ (dn IKK␣/␤), and dn ERK1/2 suppressed the OPN-induced NF B 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 NF B activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKK␣/␤-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.

This review focuses on new possibilities to exploit OPN as a tumor and stroma-derived therapeutic target to combat cancer.

SummaryCancer 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 kB (NFkB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkBa/IKK (IkBa 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 NFkB 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 (Dp85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkBa kinase (IKK) and phosphorylated Akt. OPN also induces NFkB activation through phosphorylation and degradation of IkBa by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECMinvasion. Furthermore, cells transfected with Dp85 or superrepressor form of IkBa suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkB 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 NFkB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells. IUBMB Life, 57: 441 -447, 2005

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-kappaB 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 IkappaBalpha phosphorylation and NF-kappaB activation.

A better understanding of the signalling mechanism by which osteopontin promotes tumourigenesis may be useful in crafting novel osteopontin -based anticancer therapy. The role of osteopontin in promoting cancer progression is the subject of in depth investigation and thus targeting osteopontin might be a suitable therapeutic approach for the treatment of cancer.

Background Cancer stem cells (CSCs) play crucial role in tumor progression, drug resistance and relapse in various cancers. CSC niche is comprised of various stromal cell types including Tumor-associated macrophages (TAMs). Extrinsic ques derived from these cells help in maintenance of CSC phenotype. TAMs have versatile roles in tumor progression however their function in enrichment of CSC is poorly explored. Methods Mouse macrophages (RAW264.7) cells were activated by interaction with conditioned media (CM) of murine breast cancer cells (4T1) into TAMs and the effect of activated macrophage (TAM) derived factors was examined on enrichment of cancer stem cells (CSCs) and tumor growth using in vitro and in vivo models. Results In this study, we report that macrophages upon interaction with breast cancer cells activate tumor promoting function and exhibit differential expression of various proteins as shown by secretome analysis using proteomics studies. Based on secretome data, we found that Interleukin-6 (IL-6) is one of the up-regulated genes expressed in activated macrophages. Further, we confirm that TAMs produce high levels of IL-6 and breast cancer cell derived factors induce IL-6 production in activated macrophages via p38-MAPK pathway. Furthermore, we demonstrate that tumor activated macrophages induce enrichment of CSCs and expression of CSC specific transcription factors such as Sox-2, Oct-3/4 and Nanog in breast cancer cells. We further prove that TAM derived IL-6 plays a key role in TAM mediated CSC enrichment through activation of Signal transducer and activator of transcription 3 (STAT-3) signaling. TAM derived IL-6 influences breast cancer cell migration and angiogenesis. Moreover, our in vivo findings indicated that TAM derived IL-6 induces CSC population and resulting tumor growth in breast cancer. Conclusion These finding provide evidence that TAM derived IL-6 plays a major role in CSC enrichment and tumor progression in breast cancer and IL-6 and its regulated signalling network may act as potential therapeutic target for management of breast cancer.