Genetic studies have shown that Eph receptor tyrosine kinases have both kinase-dependent and kinase-independent functions through incompletely understood mechanisms. We report here that ephrin-B1 stimulation of endogenous EphB kinases in LS174T colorectal epithelial cells inhibited integrin-mediated adhesion and HGF/SF-induced directional cell migration. Using 293 cells stably transfected with wild type (WT)- or kinase-deficient (KD-EphB3), we found that inhibition of integrin-mediated cell adhesion and induction of cell rounding was kinase-dependent. Unexpectedly, in two independent assays, both KD- and WT-EphB3 significantly inhibited directional cell migration. Upon ephrin-B1 stimulation, the activities of Rac1 and Cdc42 were reduced in both WT- and KD-EphB3-expressing cells that were induced to migrate. Pharmacological evidence demonstrates that a relative increase in RhoA signaling as a result of decreased Rac1/Cdc42 activities contributes to the inhibitory effects. Furthermore, EphB3-mediated inhibitory effect on cell adhesion but not migration was abolished by the integrin activating antibodies, suggesting that the inhibition of cell migration is not because of down-regulation of integrin function. These results uncover a differential requirement for EphB3 catalytic activity in the regulation of cell adhesion and migration, and suggest that while catalytic activity of EphB3 is required for inhibition of integrin-mediated cell adhesion, a distinct signaling pathway to Rho GTPases shared by WT- and KD-EphB3 receptor mediates inhibition of directional cell migration.

The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant epidermal growth factor receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors are minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met, which begins at 8 h and continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of mitogen-activated protein kinase (MAPK) or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of hepatocyte growth factor (HGF) or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pretreatment with pan-Src family kinase inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared with control, further confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft antitumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.

Hybridoma growth factor (HGF) is a 20-25 kD protein, supporting the growth of hybridoma cells in vitro and capable of replacing feeder cells. It was shown to be produced by human monocytes and a number of cultured cell lines. Recently, HGF was found to be identical to interferon-beta 2 or 26 kD protein and BSF-2, and was renamed interleukin 6 (IL-6). Using a sensitive bio-assay we were able to measure IL-6 activity in the serum and urine of healthy volunteers and renal transplant recipients. Low levels of IL-6 were present in the serum but not in the urine of healthy individuals. In contrast, both serum and urine of renal transplant recipients contained high levels of IL-6 directly after transplantation and during acute rejection episodes. On the basis of kinetic studies of the IL-6 response, it is concluded that serial measurement of IL-6, especially in urine, may be of value in monitoring renal transplant recipients. Moreover, the sensitivity of the bioassay will allow for detailed studies as to the biological significance of IL-6 in health and disease.

OBJECTIVE

Resistance to current cytotoxic therapies limits the treatment of most solid malignancies. This results, in part, from the overactivation of receptor tyrosine kinases and their downstream pathways in tumor cells and their associated vasculature. In this report, we ask if targeting the multifunctional mitogenic, cytoprotective, and angiogenic scatter factor/hepatocyte growth factor (SF/HGF)/c-Met pathway potentiates antitumor responses to gamma-radiation.

METHODS

Endogenous expression of SF/HGF and c-Met was targeted in U87 MG human malignant glioma cells and xenografts using chimeric U1/ribozymes. The effects of U1/ribozymes +/- gamma-radiation on glioma cell proliferation, apoptosis, xenograft growth, and animal survival were examined.

RESULTS

U1/ribozymes knocked down SF/HGF and c-Met mRNA and protein levels, sensitized cells to gamma-radiation (P < 0.005), and enhanced radiation-induced caspase-dependent cytotoxicity in vitro (P < 0.005). Intravenous U1/ribozyme therapy as liposome/DNA complexes or radiation alone modestly and transiently inhibited the growth of s.c. U87 xenografts. Combining the therapies caused tumor regression and a 40% tumor cure rate. In animals bearing intracranial xenografts, long-term survival was 0% in response to radiation, 20% in response to intratumoral adenoviral-based U1/ribozyme delivery, and 80% (P < 0.0005) in response to combining U1/ribozymes with radiation. This apparent synergistic antitumor response was associated with a approximately 70% decrease in cell proliferation (P < 0.001) and a approximately 14- to 40-fold increase in apoptosis (P < 0.0001) within xenografts.

CONCLUSIONS

Targeting the SF/HGF/c-Met pathway markedly potentiates the anti-glioma response to gamma-radiation. Clinical trials using novel SF/HGF/c-Met pathway inhibitors in glioma and other malignancies associated with c-Met activation should ultimate include concurrent radiation and potentially other cytotoxic therapeutics.

BACKGROUND

Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS1435/1449) and penta-acylated (LPS1690) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.

RESULTS

This study systematically investigated the effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS1435/1449 and LPS1690 differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS1690 but not by LPS1435/1449. In addition, LPS1690 induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS1690 while down-regulated by LPS1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS1690-induced expression of IL-6 and IL-8 in HGFs.

CONCLUSIONS

The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

The bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is found at high concentrations in the bone marrow of patients with multiple myeloma. Here we show that HGF inhibited bone morphogenetic protein (BMP)-induced in vitro osteoblastogenesis. Thus, HGF inhibited BMP-induced expression of alkaline phosphatase in human mesenchymal stem cells (hMSCs) and the murine myoid cell line C2C12, as well as mineralization by hMSCs. Furthermore, the expression of the osteoblast-specific transcription factors Runx2 and Osterix was reduced by HGF treatment. HGF promoted proliferation of hMSCs, and the BMP-induced halt in proliferation was overridden by HGF, keeping the cells in a proliferative, undifferentiating state. BMP-induced nuclear translocation of receptor-activated Smads was inhibited by HGF, providing a possible explanation of how HGF inhibits BMP signaling. The in vitro data were supported by the observation of a negative correlation between HGF and a marker of osteoblast activity, bone-specific alkaline phosphatase (rho = -0.45, P = .008), in sera from 34 patients with myeloma. These observations suggest that HGF inhibits bone formation in multiple myeloma.

OBJECTIVE

It remains largely unknown whether tumor-associated macrophages (TAMs) are involved in invasion and metastasis of human lung cancer. The aim of our study was to obtain an accurate overview of the broad range of changes occurring in monocytes that develop into TAMs, and the roles of TAMs during the progression of non-small cell lung cancer.

METHODS

TAM was isolated from 98 primary lung cancer tissues by short term cultivation in serum-free medium. The mRNA expression levels of 9 genes, including EGF, Cathepsin K, Cathepsin S, COX-2, MMP-9, PDGF, uPA, VEGFA, HGF, were evaluated by real-time PCR in 98 NSCLC. The relationships between those gene expression levels and clinicopathological features were investigated. The effects of conditioned medium from TAMs on the invasive properties of different lung cancer cell lines were measured using Transwell chambers.

RESULTS

We successfully achieved up to 95% purity of TAM, derived from 98 primary lung cancer tissues. TAM expressed high levels of Cathepsin K, COX-2, MMP-9, PDGF-B, uPA, VEGFA, and HGF. Phenotypic expression on TAMs, like MMP9, was shown to be correlated with disease progression by analyzing lung cancer tissues. Conditioned medium from TAM significantly increased cell migration and invasion in SPC-A1 cells, H460 cells and A549 cells. Anti-uPA and anti-MMP-9, but not anti-VEGF monoclonal antibodies, can inhibit TAM-induced invasion. The increase of invasiveness in the lung cancer cell lines was also correlated with their gelatinase activity, through MMP9.

CONCLUSIONS

Short-term culture in serum free medium is an effective way to isolate TAM in NSCLC. The results of this study also demonstrated that those up-regulated genes in TAMs contributed to suitable microenvironments for lung cancer invasion and metastasis. These findings may be useful in developing novel therapeutic strategies to prevent lung cancer progression.

Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from chemical transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined and functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics. 1MEA cells express and secrete more HGF than BNL cells. There was significantly decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1, vimentin, snail and slug in 1MEA cells. There was also increased expression of cyclooxygenase-2 (COX-2), Akt and phosphorylated Akt (pAkt) in 1MEA cells. Moreover, 1MEA cells had increased migratory capacity inhibited by inhibition of COX-2 and Akt but not extracellular signal regulated kinase (ERK). Molecular mesenchymal characteristics of 1MEA cells were reversed by inhibition of COX-2, Akt and ERK. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, snail, slug, COX-2, Akt, pAkt and increased migration, invasiveness and clonogenicity. We conclude that development of HCC is associated with upregulation of HGF which promotes EMT and carcinogenesis via upregulation of COX-2 and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

Fimbriae of Porphyromonas gingivalis, a periodontopathogen, play an important role in its adhesion to and invasion of host cells. The fimA genes encoding fimbrillin (FimA), a subunit protein of fimbriae, have been classified into five types, types I to V, based on nucleotide sequences. We previously reported that P. gingivalis with type II fimA was strongly associated with adult periodontitis. In the present study, we compared the abilities of recombinant FimA (rFimA) types I to V to adhere to and invade human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells) by using rFimA-conjugated microspheres (rFimA-MS). There were no significant differences in the abilities of the rFimA-MS to adhere to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than those of other types of rFimA-MS. We also observed that type II rFimA-MS invaded epithelial cells and accumulated around the nuclei. These adhesion and invasion characteristics were eliminated by the addition of antibodies to type II rFimA and alpha5beta1-integrin. In contrast, Arg-Gly-Asp-Ser peptide and a synthetic peptide of proline-rich protein C had negligible inhibitory effects. Furthermore, P. gingivalis strain HW24D1 with type II fimA adhered to cells and invaded them more than strains with other fimA genotypes. These results suggest that type II FimA can bind to epithelial cells most efficiently through specific host receptors.

We previously reported the Wnt receptor low-density lipoprotein receptor-related protein 5 (LRP5) was frequently expressed in osteosarcoma (OS) tissue and correlated with metastasis and a lower disease-free survival. Subsequent in vitro analysis revealed that dominant-negative, soluble LRP5 (sLRP5) can reduce in vitro cellular invasion. In the current study, we examined the molecular mechanisms of blocking canonical Wnt signaling by sLRP5 in Saos-2 osteosarcoma cells. Transfection of sLRP5 caused a marked up-regulation of E-cadherin in this cell line. This increase in E-cadherin, seen primarily at the cell-cell contact borders, was associated with down-regulation of Slug and Twist, transcriptional repressors which mediate cancer invasion and metastasis. In contrast, N-cadherin, a mesenchymal marker, was reduced by sLRP5. In addition, blocking Wnt signaling by sLRP5 modulated other epithelial and mesenchymal markers (keratin 8 and 18, fibronectin), suggesting a reversal of epithelial-mesenchymal transition (EMT) seen during cancer progression. SLRP5 also reduced the expression of matrix metalloproteinase (MMP) 2 and 14, consistent with a decrease in invasive capacity. SLRP5 transfection decreased both Met expression and hepatocyte growth factor (HGF)-induced cell motility. Taken together, these results support a role for Wnt/LRP5 signaling in invasiveness of a subset of OS cells.

OBJECTIVE

Hepatocyte growth factor (HGF/SF) and its receptor c-Met have previously been shown to be up-regulated in multiple human cancers, including glioblastoma multiforme. To better understand if AMG 102, a fully human, anti-HGF/SF-neutralizing antibody, could be incorporated into current clinical practice, AMG 102 was tested preclinically in combination with temozolomide or docetaxel to determine if enhanced efficacy was observed compared with AMG 102 alone.

METHODS

The effects of AMG 102 were tested for antiproliferative activity in combination with temozolomide or docetaxel on U-87 MG cells in vitro and for antitumor activity in a U-87 MG xenograft model in vivo. Apoptotic activity was also measured for AMG 102 and docetaxel combined in vitro.

RESULTS

Treatment with temozolomide combined with AMG 102 resulted in increased inhibition of cell growth in vitro compared with treatment with either single agent alone. In U-87 MG xenografts in vivo, AMG 102 combined with temozolomide or docetaxel significantly increased the inhibitory effect on tumor growth when compared with treatment with either agent alone (P < 0.0001 and P < 0.015, respectively). In vitro, docetaxel alone induced both caspase-3/7 activity as well as poly(ADP)ribose polymerase and caspase-7 cleavage in U-87 MG cells; these events were enhanced when used in combination with AMG 102. Importantly, there was no evidence of interference between AMG 102 and either temozolomide or docetaxel in vitro or in vivo.

CONCLUSIONS

These studies support testing of AMG 102 in combination with temozolomide or docetaxel. Such combinations may represent promising, novel clinical therapeutic strategies for cancers that are dependent on the HGF/SF/SF:c-Met pathway in the oncology setting.

Mammary epithelial cells comprise the functional component of the normal gland and are the major target for carcinogenesis in mammary cancer. However, the stromal compartment of the normal gland and of tumors plays an important role in directing proliferative and functional changes in the epithelium. In vivo and in vitro studies of the murine mammary gland have provided insights into novel stroma-dependent mechanisms by which estrogen and progesterone action in the epithelium can be modulated by hepatocyte growth factor (HGF) and the extracellular matrix proteins, collagen type I, fibronectin and laminin. In vitro and in vivo studies of estrogen receptor positive, estrogen-responsive human breast cancer cells have also demonstrated that estrogen responsiveness of tumor cells can also be modulated by extracellular matrix proteins, collagen type I and laminin.

BACKGROUND

Mesenchymal stem cells (MSC) are pluripotent cells, present in the bone marrow and other tissues that can differentiate into cells of all germ layers and may be involved in tissue maintenance and repair in adult organisms. Because of their plasticity and accessibility these cells are also prime candidates for regenerative medicine. The contribution of stem cell aging to organismal aging is under debate and one theory is that reparative processes deteriorate as a consequence of stem cell aging and/or decrease in number. Age has been linked with changes in osteogenic and adipogenic potential of MSCs.

RESULTS

Here we report on changes in global gene expression of cultured MSCs isolated from the bone marrow of mice at ages 2, 8, and 26-months. Microarray analyses revealed significant changes in the expression of more than 8000 genes with stage-specific changes of multiple differentiation, cell cycle and growth factor genes. Key markers of adipogenesis including lipoprotein lipase, FABP4, and Itm2a displayed age-dependent declines. Expression of the master cell cycle regulators p53 and p21 and growth factors HGF and VEGF also declined significantly at 26 months. These changes were evident despite multiple cell divisions in vitro after bone marrow isolation.

CONCLUSIONS

The results suggest that MSCs are subject to molecular genetic changes during aging that are conserved during passage in culture. These changes may affect the physiological functions and the potential of autologous MSCs for stem cell therapy.

BACKGROUND

To investigate whether copy number gain of MET or hepatocyte growth factor (HGF) affect trastuzumab sensitivity in HER2-positive metastatic breast cancer (MBC).

METHODS

We analysed 130 HER2-positive MBC treated with trastuzumab-based therapy. MET and HGF gene copy numbers (GCN) were assessed by fluorescence in situ hybridisation (FISH) in primary breast cancer samples. Receiver operating characteristic analysis was applied to find the best cutoff point for both MET and HGF GCN.

RESULTS

MET FISH-positive cases (N=36, mean 3.72) had a significantly higher trastuzumab failure rate (44.4% vs 16.0%; P=0.001) and a significantly shorter time to progression (5.7 vs 9.9 months; HR 1.74; P=0.006) than MET FISH-negative cases (N=94, mean <3.72). Hepatocyte growth factor GCN was evaluated in 84 cases (64.6%). Receiver operating characteristic analysis identified 33 HGF FISH-positive patients (mean HGF GCN 3.01). HGF FISH-positive status was significantly associated with higher risk of failure (30.3% vs 7.8%; P=0.007) as compared with HGF FISH-negative cases (N=51, mean <3.01). MET and HGF FISH-positive status was highly correlated (P<0.001) and combination of both biomarkers did not increase predictive value of either considered separately.

CONCLUSIONS

High GCNs of MET and HGF associate with an increased risk of trastuzumab-based therapy failure in HER2-positive MBC.

Multiple myeloma (MM) was the first haematological malignancy in which a prognostic relevance of bone marrow microvessel density (MVD) was shown. Myeloma-induced angiogenesis involves either the direct production of angiogenic molecules by myeloma cells or their induction in bone marrow stromal cells or endothelial cells (EC). Recent data demonstrate an increased angiogenic potential and a paracrine stimulatory effect of bone marrow EC on plasma cells (PC) in MM. Soluble angiogenic factors are elevated in bone marrow (BM) and in peripheral blood samples from myeloma patients. Furthermore, correlation with disease stage and prognosis was shown for serum levels of the angiogenic factors basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). In this review we summarize recent data which give strong evidence for an increased angiogenic activity in bone marrow microenvironment and support the hypothesis that angiogenesis is not only an epiphenomenon of tumour growth but may also promote PC growth in MM.

Omentum metastasis is a common occurrence in epithelial ovarian cancer (EOC), which is often accompanied by ascites that facilitates the spread of EOC cells. A subpopulation of fibroblasts-the cancer-associated fibroblasts (CAFs) are important promoters of tumor progression. We have shown previously that CAFs exist not only in omentum with EOC metastasis but also in omentum without metastasis. In the present study, using primary human fibroblasts isolated from normal omentum (NFs) and omentum with ovarian cancer metastasis (CAFs), we established in vitro coculture models and a 3D culture model mimicking human omentum structure for investigation of interactions between fibroblasts and cancer cells. We demonstrate that EOC cells activate NFs and promote their proliferation via transforming growth factor-β1 (TGF-β1) signaling, and the activated fibroblasts contribute to the invasion and adhesion of EOC cells. Moreover, EOC cells and NFs coculture led to overexpression of hepatocyte growth factor (HGF) and matrix metalloproteinase-2 (MMP-2) and adhesion and invasion of EOC cells could be partially suppressed by blocking the function of HGF or MMP-2. Additionally, mouse peritoneal dissemination models of EOC confirmed the activation of fibroblasts by cancer cells and the tumor growth- and metastasis-promoting effects of activated fibroblasts in vivo. Our findings indicate that activated fibroblasts in omentum form a congenial environment to promote EOC cells implantation. It is an intriguing concept that targeting the activation of omentum fibroblast through the inhibition of TGF-β1 signaling can be used as a new therapeutic strategy against ovarian cancer omentum metastases, which needs further study.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Scatter factor (SF) was first identified as a fibroblast-derived protein which disperses (i.e., "scatters") cohesive colonies of epithelium. SF-like proteins were found in human smooth muscle cell conditioned medium, amniotic fluid, and placental tissue. SFs markedly stimulate migration of epithelial, carcinoma, and vascular endothelial cell types at picomolar concentrations. Hepatocyte growth factors (HGFs) were originally described as platelet- and serum-derived proteins which stimulate hepatocyte DNA synthesis. Partial amino acid sequence data for mouse and human SFs indicate significant homology with HGFs. We used biological, biochemical, and immunological assays to evaluate and compare the activities, properties, and mechanisms of action of mouse SF, human SF (fibroblast or placenta derived), and recombinant human HGF (hrHGF). We report the following findings: (a) mouse SF exhibits species-related differences in biological activities relative to the human factors; (b) human SF and hrHGF show significant overlap in biological activities (i.e., hrHGF stimulates motility of multiple normal and carcinoma cell types, whereas human SF stimulates DNA synthesis in several normal cell types); (c) the three factors contain common antigenic determinants; and (d) all three proteins stimulate rapid phosphorylation of tyrosine residues on the c-met protooncogene protein product (the putative receptor for HGF) and on another protein with Mr 110,000. A few biological and immunological differences between human SFs and hrHGF were observed. These may reflect minor variations in amino acid sequence or posttranslational modification related to the sources of the factors. Taken as a whole, our findings suggest that by structural, functional, immunological, and mechanistic criteria, human SF and human HGF are essentially identical.

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Cell migration and invasion requires reorganisation of the actin cytoskeleton; processes mediated by the Rho family GTPases. p21 activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is activated downstream of HGF. We report here the novel finding that in prostate cancer cells PAK4 binds to and phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner. We show for the first time that variations in the level of PAK4 expression change the level of cofilin phosphorylation in cells, a change we correlate with LIMK1 activity, cell morphology and migratory behaviour. We identify for the first time a direct and localised interaction between PAK4 and LIMK1 within cells using FRET: FLIM. Moreover we show here that HGF mediates this interaction which is concentrated in small foci at the cell periphery. PAK4 and LIMK1 act synergistically to increase cell migration speed, whilst a reduction in PAK4 expression decreases cell speed. It is well established that unphosphorylated (active) cofilin is a required to drive cell migration. Our results support a model whereby HGF-stimulated cell migration also requires a cofilin phosphorylation step that is mediated by PAK4.

Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.

Progesterone is the hormone of pregnancy and unequivocally required in all mammals for maternal support of conceptus (embryo/fetus and associated membranes) survival and development. The actions of progesterone are mediated by the progesterone receptor (PR). However, the endometrial lumenal (LE) and glandular epithelia (GE) of a number of species exhibit a loss of PR expression prior to the stages of uterine receptivity and implantation. In sheep, PR expression becomes undetectable in the endometrial LE after Day 11 and then in the GE after Day 13. Loss of PR in the GE appears to be required for onset of differentiated functions in terms of production of secretory proteins, such as uterine milk proteins (UTMP) and osteopontin (OPN). Therefore, the actions of progesterone on endometrial epithelia during most of gestation appear to be mediated by the endometrial stroma that remains PR-positive throughout pregnancy. Stromal cells produce several growth factors, such as hepatocyte growth factor (HGF) and fibroblast growth factors-7 and -10 (FGF-7, FGF-10), that have receptors expressed specifically in the endometrial epithelia. These factors may be progesterone-responsive and mediate epithelial-mesenchymal interactions that are crucial for support of pregnancy. Studies of the uterine gland knockout (UGKO) ewe indicate that uterine glands and, by default, their secretions are required for peri-implantation conceptus survival and growth. A complex servomechanism, involving hormones from the ovary and conceptus as well as endogenous betaretroviruses expressed in the endometrial LE and GE, is proposed to regulate endometrial gland differentiation and function during gestation. At estrus, estrogen increases PR expression in the endometrial epithelia. High levels of endogenous Jaagsiekte sheep retroviruses (enJSRVs) are expressed in the PR-positive endometrial LE and GE in response to increasing progesterone and are hypothesized to stimulate trophoblast proliferation and production of interferon (IFN) tau. IFN tau, the pregnancy recognition hormone produced by the trophoblast from Days 10 to 21, acts in a paracrine manner on the PR-negative endometrial LE and superficial GE to inhibit transcription of estrogen receptor alpha (ER) and oxytocin receptor (OTR) genes. These actions of IFN tau maintain progesterone production from the corpus luteum by abrogating release of luteolytic pulses of prostaglandin F2 alpha (PGF) from the endometrial epithelium. The antiluteolytic effects of IFN tau are dependent on progesterone. Progesterone stimulation over 8-10 days suppresses expression of the PR gene in the LE and then GE. Loss of the PR in the LE is concomitant with decreases in mucin glycoprotein one (MUC-1), an inhibitor of blastocyst implantation. As the conceptus begins implantation on Day 15, the binucleate trophectodermal cells then differentiate and produce placental lactogen (PL), a member of the prolactin (PRL) and growth hormone (GH) family. PL stimulates GE proliferation and production of secretory proteins, such as UTMP and OPN. Interestingly, the effects of PL on the GE appear to require the absence of PR and prior exposure to IFN tau. During mid-pregnancy, the mononuclear trophectodermal cells produce GH that can also act on a progestinized uterus to stimulate GE hypertrophy and secretory function. The actions of this servomechanism are proposed to stimulate GE hyperplasia from Days 20 to 50 and then GE hypertrophy and maximal differentiated function after Day 50 when the majority of fetal growth and development occurs during gestation.