Osteopontin selectively regulates p70S6K/mTOR phosphorylation leading to NF-κB dependent AP-1-mediated ICAM-1 expression in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Breast cancer is one of the most frequently diagnosed cancer and accounts for over 400,000 deaths each year worldwide. It causes premature death in women, despite progress in early detection, treatment, and advances in understanding the molecular basis of the disease. Therefore, it is important to understand the in depth mechanism of tumor progression and develop new strategies for the treatment of breast cancer. Thus, this study is aimed at gaining an insight into the molecular mechanism by which osteopontin (OPN), a member of SIBLING (Small Integrin Binding LIgand N-linked Glycoprotein) family of protein regulates tumor progression through activation of various transcription factors and expression of their downstream effector gene(s) in breast cancer.</p> <p>Results</p> <p>In this study, we report that purified native OPN induces ICAM-1 expression in breast cancer cells. The data revealed that OPN induces NF-κB activation and NF-κB dependent ICAM-1 expression. We also observed that OPN-induced NF-κB further controls AP-1 transactivation, suggesting that there is cross talk between NF-κB and AP-1 which is unidirectional towards AP-1 that in turn regulates ICAM-1 expression in these cells. We also delineated the role of mTOR and p70S6 kinase in OPN-induced ICAM-1 expression. The study suggests that inhibition of mTOR by rapamycin augments whereas overexpression of mTOR/p70S6 kinase inhibits OPN-induced ICAM-1 expression. Moreover, overexpression of mTOR inhibits OPN-induced NF-κB and AP-1-DNA binding and transcriptional activity. However, rapamycin further enhanced these OPN-induced effects. We also report that OPN induces p70S6 kinase phosphorylation at Thr-421/Ser-424, but not at Thr-389 or Ser-371 and mTOR phosphorylation at Ser-2448. Overexpression of mTOR has no effect in regulation of OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424. Inhibition of mTOR by rapamycin attenuates Ser-371 phosphorylation but does not have any effect on Thr-389 and Thr-421/Ser-424 phosphorylation of p70S6 kinase. However, OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424 is being controlled by MEK/ERK pathway.</p> <p>Conclusion</p> <p>These results suggest that blocking of OPN-induced ICAM-1 expression through mTOR/p70S6 kinase signaling pathway may be an important therapeutic strategy for the treatment of breast cancer.</p

Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor κB-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-Kinase activity in breast cancer cells

Tumor growth and metastasis are multifaceted processes that mainly involve cell adhesion, proteolytic degradation of the extracellular matrix, and cell migration. Syk is a member of a tyrosine kinase family that is expressed mostly in hematopoietic cells. Syk is expressed in cell lines of epithelial origin, but its function in these cells remains unknown. Here we report that Syk is expressed in MCF-7 cells but not in MDA-MB-231 cells. The overexpression of wild type Syk kinase but not kinase-negative Syk suppressed cell motility and inhibited the activation of phosphatidylinositol (PI) 3'-kinase in MDA-MB-231 cells. In contrast, when Syk-specific antisense S-oligonucleotide but not the sense S-oligonucleotide was transfected to MCF-7 cells the level of PI 3'-kinase activity as well as cell motility were increased. The MDA-MB-231 cells transfected with wild type Syk cDNA followed by treatment with piceatannol, a Syk inhibitor, enhanced cell motility and PI 3'-kinase activity. Pervanadate, a phosphotyrosine phosphatase inhibitor, induced PI 3'-kinase activity and stimulated the interaction between the inhibitor of nuclear factor κBα (IκBα) and the p85α domain of PI 3'-kinase through tyrosine phosphorylation of the IκBα, which ultimately resulted in nuclear factor κB (NFκB) activation. Pervanadate had no effect on the activation of Syk in these cells. However, Syk suppressed the NFκB transcriptional activation and interaction between IκBα and PI 3'-kinase by inhibiting the tyrosine phosphorylation of IκBα. Syk, PI 3'-kinase inhibitors, and NFκB inhibitory peptide inhibited urokinase type plasminogen activator (uPA) secretion and cell motility in these cells. To our knowledge, this is the first report that Syk suppresses the cell motility and inhibits the PI 3'-kinase activity and uPA secretion by blocking NFκB activity through tyrosine phosphorylation of IκBα. These data further demonstrate a functional molecular link between Syk-regulated PI 3'-kinase activity and NFκB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells

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

We have recently demonstrated that osteopontin (OPN) induces nuclear factor &#954;B (NF&#954;B)-mediated promatrix metalloproteinase-2 activation through I&#954;B&#945;/I&#954;B&#945; 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 &#945;v&#946;3 integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKK&#945;/&#946; 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&#954;B activation through ERK/IKK&#945;/&#946; -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, IKKa/&#223;, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKa/&#946; (dn IKK&#945;/&#946;), and dn ERK1/2 suppressed the OPN-induced NF&#954;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&#954;B activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKK&#945; /&#946;-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth

The crucial role of cyclooxygenase-2 in osteopontin-induced protein kinase C α/c-Src/IκB Kinase α/β-dependent prostate tumor progression and angiogenesis

The regulation of tumor progression towards its malignancy needs the interplay among several cytokines, growth factors, and enzymes, which are controlled in the tumor microenvironment. Here, we report that osteopontin, a small integrin-binding ligand N-linked glycoprotein family of calcified extracellular matrix-associated protein, regulates prostate tumor growth by regulating the expression of cyclooxygenase-2 (COX-2). We have shown that osteopontin stimulates the activation of protein kinase C a/nuclear factor-inducing kinase/nuclear factor-&#954;B-dependent signaling cascades that induces COX-2 expression, which in turn regulates the prostaglandin E2 production, matrix metalloproteinase-2 activation, and tumor progression and angiogenesis. We have revealed that suppression of osteopontin-induced COX-2 expression by the nonsteroidal anti-inflammatory drug celecoxib or blocking the EP2 receptor by its blocking antibody resulted in significant inhibition of cell motility and tumor growth and angiogenesis. The data also showed that osteopontin-induced mice PC-3 xenograft exhibits higher tumor load, increased tumor cell infiltration, nuclear polymorphism, and neovascularization. Interestingly, use of celecoxib or anti-EP2 blocking antibody drastically suppressed osteopontin-induced tumor growth that further indicated that suppression of COX-2 or its metabolites could significantly inhibit osteopontin-induced tumor growth. Human clinical prostate cancer specimen analysis also supports our in vitro and animal model studies. Our findings suggest that blockage of osteopontin and/or COX-2 is a promising therapeutic approach for the inhibition of prostate tumor progression and angiogenesis

Transcriptional regulation of human osteopontin promoter by histone deacetylase inhibitor, trichostatin A in cervical cancer cells

<p>Abstract</p> <p>Background</p> <p>Trichostatin A (TSA), a potent inhibitor of histone deacetylases exhibits strong anti-tumor and growth inhibitory activities, but its mechanism(s) of action is not completely understood. Osteopontin (OPN) is a secreted glycoprotein which has long been associated with tumor metastasis. Elevated OPN expression in various metastatic cancer cells and the surrounding stromal cells often correlates with enhanced tumor formation and metastasis. To investigate the effects of TSA on OPN transcription, we analyzed a proximal segment of OPN promoter in cervical carcinoma cells.</p> <p>Results</p> <p>In this paper, we for the first time report that TSA suppresses PMA-induced OPN gene expression in human cervical carcinoma cells and previously unidentified AP-1 transcription factor is involved in this event. Deletion and mutagenesis analyses of OPN promoter led to the characterization of a proximal sequence (-127 to -70) that contain AP-1 binding site. This was further confirmed by gel shift and chromatin immunoprecipitation (ChIP) assays. Western blot and reverse transcription-PCR analyses revealed that TSA suppresses c-jun recruitment to the OPN promoter by inhibiting c-jun levels while c-fos expression was unaffected. Silencing HDAC1 followed by stimulation with PMA resulted in significant decrease in OPN promoter activity suggesting that HDAC1 but not HDAC3 or HDAC4 was required for AP-1-mediated OPN transcription. TSA reduces the PMA-induced hyperacetylation of histones H3 and H4 and recruitment of RNA pol II and TFIIB, components of preinitiation complex to the OPN promoter. The PMA-induced expression of other AP-1 regulated genes like cyclin D1 and uPA was also altered by TSA. Interestingly, PMA promoted cervical tumor growth in mice xenograft model was significantly suppressed by TSA.</p> <p>Conclusions</p> <p>In conclusion, these findings provide new insights into mechanisms underlying anticancer activity of TSA and blocking OPN expression at transcriptional level by TSA may act as novel therapeutic strategy for the management of cervical cancer.</p

Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth and angiogenesis via autocrine and paracrine mechanisms

Angiogenesis is the hallmark of cancer, and development of aggressiveness of primary tumor depends on de novo angiogenesis. Here, using multiple in vitro and in vivo models, we report that osteopontin (OPN) triggers vascular endothelial growth factor (VEGF)-dependent tumor progression and angiogenesis by activating breast tumor kinase (Brk)/nuclear factor-inducing kinase/nuclear factor-κB (NF-κB)/activating transcription factor-4 (ATF-4) signaling cascades through autocrine and paracrine mechanisms in breast cancer system. Our results revealed that both exogenous and tumor-derived OPN play significant roles in VEGF-dependent tumor angiogenesis. Clinical specimen analysis showed that OPN and VEGF expressions correlate with levels of neuropilin-1, Brk, NF-κB, and ATF-4 in different grades of breast cancer. Consequently, OPN plays essential role in two key aspects of tumor progression: VEGF expression by tumor cells and VEGF-stimulated neovascularization. Thus, targeting OPN and its regulated signaling network could be a novel strategy to block tumor angiogenesis and may develop an effective therapeutic approach for the management of breast cancer

Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-κB-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-&#954;B (NF-&#954;B)) into the nucleus. However, when the super-repressor form of I&#954;B&#945; (inhibitor of NF-&#954;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-&#954;B-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with &#945; v&#946;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 antisenseS-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-&#954;B-mediated induction of MT1-MMP

Tyrosine kinase, p56<SUP>lck</SUP>-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways

We have recently reported that tyrosine kinase, p56lck regulates cell motility and nuclear factor &#954;B-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of I&#954;B&#945; following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56lck and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56lck in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells

Hypoxia regulates cross-talk between Syk and Lck leading to breast cancer progression and angiogenesis

Hypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer

Prostaglandin E<SUB>2</SUB> regulates tumor angiogenesis in prostate cancer

In cancer management, the cyclooxygenase (COX)-targeted approach has shown great promise in anticancer therapeutics. However, the use of COX-2 inhibitors has side effects and health hazards; thus, targeting its major metabolite prostaglandin E2 (PGE2)-mediated signaling pathway might be a rational approach for the next generation of cancer management. Recent studies on several in vitro and in vivo models have revealed that elevated expression of COX-2 correlates with prostate tumor growth and angiogenesis. In this study, we have shown the in-depth molecular mechanism and the PGE2 activation of the epidermal growth factor receptor and &#946;3 integrin through E prostanoid 2 (EP2)-mediated and EP4-mediated pathways, which lead to activator protein-1 (AP-1) activation. Moreover, PGE2 also induces activating transcription factor-4 (ATF-4) activation and stimulates cross-talk between ATF-4 and AP-1, which is unidirectional toward AP-1, which leads to the increased expressions of urokinase-type plasminogen activator and vascular endothelial growth factor and, eventually, regulates prostate tumor cell motility. In vivo Matrigel angiogenesis assay data revealed that PGE2 induces angiogenesis through EP2 and EP4. Human prostate cancer specimen analysis also supported our in vitro and in vivo studies. Our data suggest that targeting PGE2 signaling pathway (i.e., blocking EP2 and EP4 receptors) might be a rational therapeutic approach for overcoming the side effects of COX-2 inhibitors and that this might be a novel strategy for the next generation of prostate cancer management