Prostaglandin E2 enhances pancreatic cancer invasiveness through an Ets-1-dependent induction of matrix metalloproteinase-2

Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2) in the pathogenesis of a wide range of malignancies. Here we tested the hypothesis that the COX-2 product prostaglandin E(2) (PGE(2)) increases cellular invasive potential by inducing matrix metalloproteinase-2 (MMP-2) expression and activity through an extracellular signal-regulated kinase (ERK)/Ets-1-dependent mechanism in pancreatic cancer. PANC-1 and MIAPaCa-2 pancreatic cancer cells were treated with PGE(2) or rofecoxib, a selective COX-2 inhibitor. MMP-2 expression and activity were assayed using Western blot analysis and zymography, respectively. MMP-2 promoter activity was analyzed with a luciferase-based assay. Ets-1 activity was analyzed using gel shift assay. Ets-1 expression was specifically silenced using RNA interference. Cellular invasive and migratory potentials were determined using a Boyden chamber assay with or without Matrigel, respectively. Exogenous PGE(2) induced MMP-2 expression and activity and increased ERK1/2 phosphorylation, Ets-1 binding activity, and MMP-2 promoter activity. PGE(2) also increased cellular migratory and invasive potentials. The mitogen-activated protein kinase kinase inhibitor PD98059 and Ets-1 silencing each abolished PGE(2)-induced increases in MMP-2 expression. PD98059 and Ets-1 silencing each abrogated the effect of PGE(2) on cellular invasive potential but not on cellular migratory potential. Rofecoxib suppressed MMP-2 expression and activity, Ets-1 binding activity, MMP-2 promoter activity, and cellular migratory and invasive potentials. These results suggest that PGE(2) mediates pancreatic cancer cellular invasiveness through an ERK/Ets-1-dependent induction of MMP-2 expression and activity. They also suggest that COX-2 inhibition may represent a strategy to inhibit invasive potential in pancreatic cancer.     

Gli1 contributes to the invasiveness of pancreatic cancer through matrix metalloproteinase-9 activation

The hedgehog (Hh) signaling pathway has been reported to be associated with the growth of pancreatic cancer, but its role in the invasive phenotype is poorly understood. Therefore, we investigated the role of the Hh pathway in pancreatic cancer cell invasiveness using a Matrigel invasion assay. Blockade of the Hh pathway by cyclopamine inhibited pancreatic cancer cell invasion in association with a decreased expression of matrix metalloproteinase (MMP)-9. By contrast, activation of the Hh pathway by the addition of exogenous Sonic hedgehog increased cell invasion and MMP-9 expression. Stable transfection of pancreatic cancer cells with Gli1 increased their invasiveness, which was associated with activation of MMP-9. We also showed that inhibition of MMP-9 by small interfering RNA blocked the increased invasiveness of Gli1-transfected cells. Furthermore, inhibition of Gli1 by small interfering RNA suppressed the invasiveness and MMP-9 expression of pancreatic cancer cells. Taken together, these findings suggest that members of the Hh pathway, especially Gli1, play an important role in the invasiveness of pancreatic cancer cells through the regulation of MMP-9 expression. ( Cancer Sci 2008; 99: 1377–1384)     

Involvement of matrix metalloproteinase-7 in invasion-metastasis through induction of cell dissociation in pancreatic cancer

Epidermal growth factor receptor (EGFR) mediated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was isolated as invasion-metastasis related factor in pancreatic cancer in our previous studies. Matrix metalloproteinase-7 (MMP-7) and tight junction (TJ) proteins are indicated to be involved in cancer invasion-metastasis. To clarify the underlying mechanism of involvement of MMP-7 in cancer invasion, western blotting, invasion assay and immunohistochemistry were performed in dissociated (PC-1.0 and AsPC-1) and non-dissociated (PC-1 and Capan-2) pancreatic cancer cells, as well as pancreatic cancer tissues. Intracellular MMP-7 protein presented as pre-proenzyme and its expression was decreased by AG1478 (EGFR inhibitor) or U0126 (MEK inhibitor) treatment in pancreatic cancer cells. Activated MMP-7 protein was only detected in the medium of PC-1.0 and AsPC-1 cells, but not detected in the medium of PC-1 and Capan-2 cells. Moreover, MMP-7 treatment significant induced the dissociation of cell colonies in PC-1 and Capan-2 cells. Synchronously, TJ structure was apparently disrupted and translocation of TJ proteins to cytoplasm or extracellular medium was induced in PC-1 and Capan-2 cells. Furthermore, MMP-7 treatment markedly increased the in vitro invasion of PC-1 and Capan-2 cells. In addition, MMP-7 expression at the invasive front was obviously stronger than that at the center of pancreatic cancer tissues. Activation of MMP-7 protein is closely involved in disruption of TJ structure and consequent induction of cell dissociation as well as invasion in pancreatic cancer. EGFR mediated MEK/ERK signaling pathway is implied to be involved in regulation of MMP-7 expression in pancreatic cancer cells.     

Mullerian-inhibiting substance induces Gro-beta expression in breast cancer cells through a nuclear factor-kappaB-dependent and Smad1-dependent mechanism

Mullerian-inhibiting substance (MIS), a transforming growth factor-beta family member, activates the nuclear factor-kappaB (NF-kappaB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-beta. Inhibiting NF-kappaB activation with a phosphorylation-deficient IkappaBalpha mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-beta promoter-driven reporter expression and Gro-beta mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-beta mRNA, suggesting that regulation of Gro-beta expression by MIS was dependent on activation of NF-kappaB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of kappaB-binding sequences within Gro-beta, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-kappaB complex bound to the kappaB site of the Gro-beta promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-beta promoter in vivo, whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-kappaB signaling in which enhancement of NF-kappaB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the kappaB consensus site within the promoter.     

TGF-beta-induced invasiveness of pancreatic cancer cells is mediated by matrix metalloproteinase-2 and the urokinase plasminogen activator system.

TGF-beta strongly promotes local tumor progression in advanced epithelial tumors, though the underlying mechanisms are poorly understood. In the present study, we demonstrate the potential of TGF-beta to increase the invasiveness of the pancreatic cancer cell lines PANC-1 and IMIM-PC1. TGF-beta-induced tumor cell invasion occurred in a time-dependent manner, started after 12 hr and continued to increase even 48 hr after a single application of the growth factor. Blocking of secreted TGF-beta1 by application of neutralizing antibodies 24 hr after TGF-beta treatment completely prevented the sustained effects of TGF-beta on tumor cell invasion. Together with our previous observation that TGF-beta1 up-regulates its own expression in both cell lines, our data suggest that TGF-beta1 acts in an autocrine manner to maintain tumor cell invasion. As measured by Northern blot hybridization and zymography, TGF-beta treatment of PANC-1 and IMIM-PC1 cells resulted in strong up-regulation of expression and activity of both matrix metalloproteinase-2 (MMP-2) and the urokinase plasminogen activator (uPA) system. Treatment with MMP inhibitors or inhibitors of the uPA system caused significant reduction of TGF-beta-induced invasiveness in both cell lines. In contrast, expression and activity of MMP-2 and uPA as well as tumor cell invasiveness remained unaffected in cell lines with defects of the TGF-beta type II receptor (MiaPaca2) or the Smad4 gene (IMIM-PC2 and CAPAN-1). In these cell lines, TGF-beta also failed to auto-induce its own expression. In conclusion, our results suggest that TGF-beta1 is a strong promotor of pancreatic cancer progression. TGF-beta thereby acts in an autocrine manner to induce tumor cell invasion, which is mediated by MMP-2 and the uPA system.     

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.     

Aspirin inhibits tumor cell invasiveness induced by Epstein-Barr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression

Matrix metalloproteinases (MMPs) are thought to play crucial roles in tumor invasion and metastasis. Because we have shown that EBV latent membrane protein 1 (LMP1) enhances MMP-9 expression by activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 (T. Yoshizaki, et al., Proc. Natl. Acad. Sci. USA, 95: 3621-3626, 1998), we therefore tested whether up-regulation of MMP-9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP-9 expression in tumors grown in nude mice were also tested. C33A cells stably expressing LMP1 had increased expression of MMP-9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells (P < 0.02). Treatment with aspirin or sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells (P < 0.03) and suppressed both the LMP1-induced MMP-9 expression in zymographic analyses and LMP1-induced MMP-9 promoter activity in CAT reporter assays (P < 0.01). Endogenous MMP-2 levels were unaffected by either drug. Both drugs repressed the CAT activity of the truncated MMP-9 promoter construct, which only contained a binding site for AP-1, to the basal level (P < 0.05). Moreover, EMSA indicated that the effects of the salicylates were through the inhibition of not only NF-kappaB but also AP-1 binding activity. Inhibitory effect of salicylates could be reversed by p50/p65 subunits of NF-kappaB or c-Jun overexpression. The inhibitory effect of aspirin on NF-kappaB activity was attributable to the inhibition of IkappaB kinase activity. Finally, tumors derived from C33A cells stably expressing LMP1 grown in nude mice showed enhanced MMP-9 levels compared with tumors derived from vector-transfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9.     

Dexamethasone inhibits invasiveness of a human pancreatic cancer cell line

Recent studies have shown that dexamethasone inhibits gelatinases. We investigated the effect of dexamethasone on the invasiveness of a human pancreatic cancer cell line. Tumor cells were treated with dexamethasone at various concentrations for 24 h. Cells were examined in an invasion assay. The expression and activity of MMP-9 in culture media were quantitated by Western blotting and gelatin zymogram. The invasiveness of cancer cells and expression of MMP-9 were decreased by dexamethasone in a dose-dependent manner. Dexamethasone may be a useful therapeutic agent in the treatment of pancreatic cancer.     

Chemopreventive allylthiopyridazine derivatives induce apoptosis in SK-Hep-1 hepatocarcinoma cells through a caspase-3-dependent mechanism

Dietary organosulphur compounds including diallylsulphide, a component of garlic oil, were shown to inhibit the proliferation of tumour cells. Since hepatocellular carcinoma is one of the most lethal malignancies and there is no effective preventive measure to date, we wished to pursue the chemopreventive potential of the synthetic allylthiopyridazine derivatives (K compounds) on hepatocarcinoma cells. Here, we report that the K compounds efficiently inhibited SK-Hep-1 cell proliferation through induction of apoptosis. Increased chain length at the 3-position of allylthiopyridazine ring improved the potency of growth inhibition. K compounds downregulated Bcl-2, while Bax remained unchanged, reducing the ratio of Bcl-2 to Bax. We also provide evidence that the K compound-induced apoptosis involves cytochrome c release and caspase-3 activation. These results suggest that the allythiopyridazine derivatives, especially 3-propoxy-6-allylthiopyridazine, induce apoptosis in SK-Hep-1 cells through a caspase-3-dependent mechanism, which may contribute to the chemopreventive function for hepatocellular carcinoma.     

RETRACTED ARTICLE: MiR-429 regulates gastric cancer cell invasiveness through ZEB proteins

Tumor Biology - MicroRNAs (miRNAs) play an essential role in the tumorigenesis of gastric carcinoma (GC). MiR-429 has been recently reported to inhibit GC growth; however, whether miR-429 may also...     

Integrin fibronectin receptors in matrix metalloproteinase-1-dependent invasion by breast cancer and mammary epithelial cells

Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of alpha(5)beta(1) with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of alpha(5)beta(1) and alpha(4)beta(1) fibronectin receptors in regulating matrix metalloproteinase (MMP)-1-dependent invasion by human breast cancer and mammary epithelial cells. We found that a peptide consisting of fibronectin PHSRN sequence, Ac-PHSRN-NH(2), induces alpha(5)beta(1)-mediated invasion of basement membranes in vitro by human breast cancer and mammary epithelial cells. PHSRN-induced invasion requires interstitial collagenase MMP-1 activity and is suppressed by an equimolar concentration of a peptide consisting of the LDV sequence of the fibronectin connecting segment, Ac-LHGPEILDVPST-NH(2), in mammary epithelial cells, but not in breast cancer cells. This sequence interacts with alpha(4)beta(1), an integrin that is often down-regulated in breast cancer cells. Immunoblotting shows that the PHSRN peptide stimulates MMP-1 production by serum-free human breast cancer and mammary epithelial cells and that the LDV peptide represses PHSRN-stimulated MMP-1 production only in mammary epithelial cells. Furthermore, PHSRN stimulates MMP-1 activity in breast cancer cells and mammary epithelial cells with a time course that closely parallels invasion induction. Thus, down-regulation of surface alpha(4)beta(1) during oncogenic transformation may be crucial for establishment of the alpha(5)beta(1)-induced, MMP-1-dependent invasive phenotype of breast cancer cells.     

Bone morphogenetic protein 2 stimulation of tumor growth involves the activation of Smad-1/5

Morphogenetic protein 2 (BMP-2) is normally expressed in the embryo promoting the development of several organs. Aberrant expression of BMP-2 occurs in approximately 98% of lung carcinomas, however, its role in regulating tumor growth is poorly understood. We show that BMP-2 induces Id-1 expression in lung cancer cell lines through its activation of Smad-1/5, which is dependent on cell culture conditions. A549 cells in DMEM/5% FCS BMP-2 activated Smad-1/5 and caused a transient increase in proliferation. In serum-free medium, BMP-2 induced significantly less Smad-1/5 activation and Id-1 expression, and produced significant growth inhibition. The affect of BMP-2 on tumor growth in vivo was substantially more significant. Recombinant BMP-2 coinjected with A549 cells, into nude mice increased proliferation and produced an increase in Id-1 expression. Forced expression of BMP-2 in A549 cells significantly enhanced tumor growth in the lungs following intravenous injection but not of subcutaneous tumors. Tumors in the lung were found to have an activated Smad-1/5 and expressed Id-1. Subcutaneous tumors expressed less activated Smad-1/5 and Id-1 than that of controls. Human lung carcinomas were also found to express an activated Smad-1/5 and Id-1. We provide evidence that BMP-2 promotes tumor growth. This paper highlights that cell culture experiments may not reveal the full biological affects of BMP-2, and its activity varies depending of the local environment.     

Deleted in liver cancer 1 controls cell migration through a Dia1-dependent signaling pathway

Deleted in liver cancer (DLC) 1 and 2 are Rho GTPase-activating proteins that are frequently down-regulated in various types of cancer. Ectopic expression in carcinoma cell lines lacking these proteins has been shown to inhibit cell migration and invasion. However, whether the loss of DLC1 or DLC2 is the cause of aberrant Rho signaling in transformed cells has not been investigated. Here, we have down-regulated DLC1 and DLC2 expression in breast cancer cells using a RNA interference approach. Silencing of DLC1 led to the stabilization of stress fibers and focal adhesions and enhanced cell motility in wound-healing as well as chemotactic Transwell assays. We provide evidence that enhanced migration of cells lacking DLC1 is dependent on the Rho effector protein Dia1 but does not require the activity of Rho kinase. By contrast, DLC2 knockdown failed to affect the migratory behavior of cells, suggesting that the two proteins have distinct functions. This is most likely due to their differential subcellular localizations, with DLC1 found in focal adhesions and DLC2 being mainly cytosolic. Collectively, our data show that DLC1 is critically involved in the control of Rho signaling and actin cytoskeleton remodeling and that its cellular loss is sufficient for the acquisition of a more migratory phenotype of breast cancer cells.