Dab2 is pivotal for endothelial cell migration by mediating VEGF expression in cancer cells

Although angiogenesis is crucial for tumor growth and metastasis, the molecular mechanisms controlling this process are not clearly understood. Here, we explore the role of Dab2 in tumor angiogenesis. We found that Dab2 is expressed in several cancer cells, including A549 lung cancer cells, but it is hardly detectable in SW480 colon cancer cells. Migration and Erk phosphorylation were enhanced in human umbilical vein endothelial cells (HUVECs) treated with the conditioned medium obtained from Dab2-overexpressing SW480 stable cells. In addition, vascular endothelial growth factor (VEGF) protein was strongly detected in conditioned medium derived from Dab2-overexpressing SW480 cells, and Erk phosphorylation enhanced by Dab2(+) CM was restored by VEGF inhibition. Moreover, Dab2 depletion in A549 cells led to a decrease in HUVEC migration and Erk phosphorylation. Furthermore, we show that Dab2 is required for the TGFβ-induced gene expression of angiogenic factors such as VEGF and FGF2. Taken together, these results suggest that Dab2, which is expressed in cancer cells, is pivotal for endothelial cell migration by affecting VEGF expression.     

Matrix metalloproteinase 2 promotes cell growth and invasion in colorectal cancer

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the western world. In this study, we evaluated the expression of matrix metalloproteinase 2 gene (MMP2) in CRC and analyzed its correlation with clinicopathological features. We found that the expression of MMP2 was significantly higher in CRC tissues than in the colorectal tissues. In addition, high levels of MMP2 protein were positively correlated with the status of tumor size, lymph node metastasis, distant metastasis, Dukes' stage, and tumor invasion. Moreover, patients with higher MMP2 levels had markedly shorter overall survivals than those with low MMP2 levels. Multivariate analysis results suggested that the level of MMP2 expression is an independent prognostic indicator for the survival of patients with CRC. Silencing MMP2 expression in CRC cell lines with lentiviral-mediated shRNA markedly suppressed cell proliferation, colony formation, and invasion. Furthermore, we observed that vascular endothelial growth factor (VEGF) and membrane type 1 (MT1)-MMP protein levels were decreased in MMP2-down-regulated colorectal cells. Therefore, our study demonstrated that MMP2 is an important factor related to carcinogenesis and metastasis of CRC, and MMP2 promotes CRC cell growth and invasion by up-regulating VEGF and MT1-MMP expression, which makes this pathway a potential target for cancer treatment.     

(−)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells

(?)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1α, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3 h, EGCG caused a decrease in the expression of HIF-1α protein and VEGF, HIF-1α, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6 h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1α and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.     

MicroRNA-22 regulates hypoxia signaling in colon cancer cells

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.     

Downregulation of developmentally regulated endothelial cell locus-1 inhibits the growth of colon cancer

Developmentally regulated endothelial cell locus-1 (Del1) is an embryonic angiogenic factor expressed in early embryonic endothelial cells, but recently has been found to be expressed in some forms of cancers including colon and breast cancers, and melanoma, and human cancer cell lines. Overexpression of Del1 accelerates tumor growth by enhancing vascular formation, implying Del1 may be a potential target for anti-angiogenic cancer therapy. The study aims to investigate whether downregulation of Del1 could inhibit the growth of tumors established in nude Balb/c mice by subcutaneous implantation of human LS-174T colon cancer cells. The shRNA expression vectors targeting human Del1, and vascular endothelial growth factor (VEGF) were constructed. Gene transfection of Del1-shRNA downregulated expression of Del1 in LS-174T cells in vivo and in vitro, but did not alter the proliferative or survival properties of cells in vitro. Gene transfection of VEGF-shRNA downregulated expression of both VEGF and Del1 in LS-174T cells in vivo and in vitro. Both Del1-shRNA and VEGF-shRNA gene therapies exhibited anti-tumor activities and they also showed a synergistic effect in suppressing growth of colon tumors by anti-angiogenesis and anti-proliferation. Although further investigation to clarify the mechanisms explaining the role of Del1 in tumor growth, and the interaction between VEGF and Del1, is required, the results indicate that downregulation of Del1 presents a potent therapeutic strategy to combat colon cancer.     

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.     

NADPH oxidase subunit p22phox-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22^phox expression are greatly increased in human prostate cancer tissues, and knockdown of p22^phox by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22^phox in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22^phox resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22^phox in tumor angiogenesis and tumor growth, and suggest that p22^phox is a potential novel target for prostate cancer treatment.     

Regulation of bone morphogenetic protein-4 by matrix GLA protein in vascular endothelial cells involves activin-like kinase receptor 1

Matrix GLA protein (MGP) has previously been shown to enhance expression of vascular endothelial growth factor (VEGF) through the activin-like kinase receptor 1 (ALK1) in bovine aortic endothelial cells. MGP has also been identified as an inhibitor of bone morphogenetic protein-2 (BMP-2). This study showed that the effect of MGP on ALK1 signaling and VEGF expression in bovine aortic endothelial cells was dose-dependent, that a progressive increase of MGP levels ceased to be stimulatory and instead turned inhibitory. We identified a new regulatory pathway involving BMP that may explain this response. BMP-2 and BMP-4 induced expression of ALK1 in a dose-dependent fashion as determined by real-time PCR and immunoblotting. Activation of ALK1 signaling induced expression of MGP in addition to that of VEGF, allowing for negative feedback regulation of BMP by MGP. MGP inhibited BMP-4 activity similarly to that of BMP-2 and interacted with BMP-4 on a protein level as determined by co-immunoprecipitation. The dose-dependent effect on ALK1 expression and the stimulation of MGP and VEGF expression were dependent on signaling by transforming growth factor-beta (TGF-beta) and ALK1. Inhibition of TGF-beta by neutralizing antibodies abolished the inhibitory effect of high BMP-4 levels on ALK1 expression and the induction of MGP and VEGF. Depletion of ALK1 by small interfering RNA abolished the induction of MGP and VEGF. MGP promoter activity was also stimulated by BMP-4 in a TGF-beta-dependent fashion. The results suggest that the effects of BMP on endothelial cells occur in part through induction of ALK1, an effect that may be limited by ALK1-induced MGP.     

CXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP-9 activation

Colorectal cancer (CRC) is characterized by a distinct metastatic pattern resembling chemokine-induced leukocyte trafficking. This prompted us to investigate expression, signal transduction and specific functions of the chemokine receptor CXCR4 in CRC cells and metastases. Using RT-PCR analysis and Western blotting, we demonstrated CXCR4 and CXCL12 expression in CRC and CRC metastases. Cell differentiation increases CXCL12 mRNA levels. Moreover, CXCR4 and its ligand are inversely expressed in CRC cell lines with high CXCR4 and low or not detectable CXCL12 expression. CXCL12 activates ERK-1/2, SAPK/JNK kinases, Akt and matrix metalloproteinase-9. These CXCL12-induced signals mediate reorganization of the actin cytoskeleton resulting in increased cancer cell migration and invasion. Moreover, CXCL12 increases vascular endothelial growth factor (VEGF) expression and cell proliferation but has no effect on CRC apoptosis. Therefore, the CXCL12/CXCR4 system is an important mediator of invasion and metastasis of CXCR4 expressing CRC cells.     

Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression,which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells

Stimulation of human colon cancer cells with insulin-like growth factor 1 (IGF-1) induces expression of the VEGF gene, encoding vascular endothelial growth factor. In this article we demonstrate that exposure of HCT116 human colon carcinoma cells to IGF-1 induces the expression of HIF-1 alpha, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. In contrast to hypoxia, which induces HIF-1 alpha expression by inhibiting its ubiquitination and degradation, IGF-1 did not inhibit these processes, indicating an effect on HIF-1 alpha protein synthesis. IGF-1 stimulation of HIF-1 alpha protein and VEGF mRNA expression was inhibited by treating cells with inhibitors of phosphatidylinositol 3-kinase and MAP kinase signaling pathways. These inhibitors also blocked the IGF-1-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1 alpha protein synthesis. Forced expression of a constitutively active form of the MAP kinase kinase, MEK2, was sufficient to induce HIF-1 alpha protein and VEGF mRNA expression. Involvement of the MAP kinase pathway represents a novel mechanism for the induction of HIF-1 alpha protein expression in human cancer cells.     

PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E₂ (PGE₂) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE₂ directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE₂-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE₂ increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE₂ EP₄ receptor was responsible for transducing the signal to mTORC1. Moreover, PGE₂ increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE₂-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE₂ increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE₂ mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.     

Effect of arginine on angiogenesis induced by human colon cancer: in vitro and in vivo studies

This study investigated the effect of arginine (Arg) supplementation on angiogenesis in human colon cancer. The in vitro study investigated the effects of different Arg levels and inducible nitric oxide (iNO) synthase inhibitor on angiogenic protein expressions stimulated by SW480 cells. The results showed that the production of vascular endothelial growth factor (VEGF), basic fibroblast growth factor with 100 and 1000 μmol/L Arg and matrix metalloproteinase (MMP)-2 with 1000 μmol/L Arg was lower than that with 0 and 50 μmol/L Arg. Inhibition of iNO resulted in higher angiogenic protein expressions comparable with groups with low Arg administration, indicating that Arg administration at levels similar to or higher than physiological concentrations reduced the progression of colon cancer, and iNO may partly play a role in reducing angiogenesis. The in vivo study used a human colon cancer xenograft model in nude mice. Mice were inoculated with 1×10⁷ SW480 cells and assigned to two groups. The control group was fed a semipurified diet, while the experimental group was supplied an Arg-supplemented diet. After 5 weeks, tumors were harvested and spleens were excised for further analysis. Results showed that the MMP-2, MMP-9 and VEGF receptor levels in tumors were significantly lower, whereas tumor NO levels and spleen natural killer (NK) cell activities were higher in the Arg group than in the control group. These results were consistent with the in vitro study that dietary Arg supplementation inhibits the progression of colon cancer possibly by increasing NO secretion and consequently enhancing NK cell activity.     

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Gene Expression of Neuropilin-1 and Its Receptors,VEGF/Semaphorin 3a,in Normal and Cancer Cells

Extracellular domains of the transmembrane glycoprotein, neuropilin-1 (Np1), specifically bind an array of factors and co-receptors including class-3 semaphorins (Sema3a), vascular endothelial growth factor (VEGF), hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-β 1 (TGF-β1), and fibroblast growth factor2 (FGF2). Np1 may have a role in immune response, tumor cell growth, and angiogenesis, but its relative expression in comparison to its co-primary receptors, VEGF and Sema3a, is not known. In this study we determined the mRNA expression of Np1 and its co-receptors, VEGF and Sema3a, and the ratio of VEGF/Sema3a in different human and rodent cell lines. Expression of Np1, VEGF and Sema3a is very low in cells derived from normal tissues, but these proteins are highly expressed in tumor-derived cells. Furthermore, the ratio of VEGF/Sema3a is highly variable in different tumor cells. The elevated mRNA expression of Np1 and its putative receptors in tumor cells suggests a role for these proteins in tumor cell migration and angiogenesis. As different tumor cells exhibit varying VEGF/Sema3a ratios, it appears that cancer cells show differential response to angiogenic factors. These results bring to light the individual variation among the cancer-related genes, Np1, VEGF, and Sema3a, and provide an important impetus for the possible personalized therapeutic approaches for cancer patients.