Genome-wide analysis in human colorectal cancer cells reveals ischemia-mediated expression of motility genes via DNA hypomethylation

DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes. © 2014 Skowronski et al

Lack of Efficacy of Combined Antiangiogenic Therapies in Xenografted Human Melanoma

Antiangiogenic therapy is theoretically a promising anticancer approach but does not always produce significant tumor control. Combinations of antiangiogenic therapies are therefore being investigated as strategies to enhance clinical benefit. Common targets for angiogenic blockade include endothelial specific receptors, such as Tie2/Tek, which signal blood vessel stabilization via recruitment and maturation of pericytes. Here, we report on the effects of targeted Tie2 antiangiogenic therapy (TekdeltaFc) in combination with nontargeted metronomic cyclophosphamide (LDM CTX) (reported to also act in antiangiogenic fashion) in xenografted human melanoma. Individually, these therapies showed transient antitumor activity, but, in combination, there was no significant reduction in tumor growth. In addition, while TekdeltaFc caused the expected increased pericyte coverage in treated blood vessels, LDM CTX alone or in combination with TekdeltaFc resulted in increased levels of VEGF production. Collectively, our data highlight the complexity of molecular interactions that may take place when antiangiogenic regimens are combined

Differential response of lymphatic, venous and arterial endothelial cells to angiopoietin-1 and angiopoietin-2

BACKGROUND: The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Angiopoietins 1 and 2 (Ang1 and Ang2) are regulators of both angiogenesis and lymphangiogenesis through the Tek/Tie-2 receptor tyrosine kinase. The response of endothelial cells to stimulation with either Ang1 or Ang2 is thought to be dependent upon the origin of the endothelial cells. In this study, we examined the effects of the angiopoietins on lymphatic, venous and arterial primary endothelial cells (bmLEC, bmVEC and bmAEC, respectively), which were isolated and cultured from bovine mesenteric vessels. RESULTS: BmLEC, bmVEC and bmAEC cell populations all express Tie-2 and were shown to express the appropriate cellular markers Prox-1, VEGFR3, and Neuropilin-1 that define the particular origin of each preparation. We showed that while bmLECs responded slightly more readily to angiopoietin-2 (Ang2) stimulation, bmVECs and bmAECs were more sensitive to Ang1 stimulation. Furthermore, exposure of bmLECs to Ang2 induced marginally higher levels of proliferation and survival than did exposure to Ang1. However, exposure to Ang1 resulted in higher levels of migration in bmLECs than did to Ang2. CONCLUSION: Our results suggest that although both Ang1 and Ang2 can activate the Tie-2 receptor in bmLECs, Ang1 and Ang2 may have distinct roles in mesenteric lymphatic endothelial cells

Differential expression of Tie2 receptor and VEGFR2 by endothelial clones derived from isolated bovine mononuclear cells.

The purpose of these experiments was to evaluate the expression of endothelial markers, such as Tie2 and VEGFR2 in endothelial cells derived from blood mononuclear endothelial progenitor cells. Bovine mononuclear cells were isolated using separation by centrifugation and were grown in endothelial specific media supplemented with growth factors. Isolation of the whole cell population of mononuclear cells (MNC) from bovine peripheral blood gave rise to progenitor-like cells (CD45(-)) that, although morphologically similar, have different phenotypes revealed by expression of endothelial specific markers Tie2 and VEGFR2. Plating of MNCs on collagen and fibronectin gave rise to more colonies than non-coated dishes. Occasional colonies from MNC isolations had a mural cell phenotype, negative for Tie2 and VEGFR2 but positive for smooth muscle actin and PDGFRβ. Although cells expressing high levels of VEGFR2 and low levels of Tie2, and vice versa were both able to form cords on Matrigel, cells with higher expression of Tie2 migrate faster in a scratch assay than ones with lower expression of Tie2. When these different clones of cells were introduced in mice through tail vein injections, they retained an ability to home to angiogenesis occurring in a subcutaneous Matrigel plug, regardless of their Tie2/VEGFR2 receptor expression patterns, but cells with high VEGFR2/low Tie2 were more likely to be CD31 positive. Therefore, we suggest that active sites of angiogenesis (such as wounds, tumors, etc.) can attract a variety of endothelial cell precursors that may differentially express Tie2 and VEGFR2 receptors, and thus affect our interpretation of EPCs as biomarkers or therapies for vascular disease

<i>In vitro</i> angiogenic capacity of clones with differential expression of Tie2/VEGFR2 receptors.

<p>(<b>A</b>) Representative images showing that clones with higher expression of either Tie2 or VEGFR2 are able to form cord-like structures on Matrigel with equal efficiency. <i>Scale bar  = 100 µm</i> (<b>B</b>) Scratch wound assay reveals that clones with higher Tie2 expression migrate and close the wound faster than cells with low Tie2 expression, although both clones have high levels of VEGFR2 expression. BAECs were used as control. <i>Scale bar  = 100 µm.</i></p

Phenotype of clones with a differential expression of Tie2/VEGFR2 receptors.

<p>(<b>A–C</b>) Clones with high or low Tie2 and/or high and low VEGFR2 do not reveal any obvious differences in monolayer morphology between one another and compared to BAECs, by phase contrast microscopy. <i>Scale bar  = 100 µm</i> (<b>D</b>) Western blot analysis of representative clones isolated from MNCs grown on either collagen (C) or fibronectin (FN) matrices or non-coated plates (NC) revealed highly differential expression of VEGFR2 and Tie2; proteins were normalized to α-tubulin. Representative clones are marked with boxes – clone 9 has high VEGFR2 and low Tie2 expression and clone 13 has high Tie2 and VEGFR2 expression, relative to one another and to BAECs. (<b>E</b>) Random clones from several isolations were stained for the expression of monocyte surface antigen CD45, revealing that all the clones are negative for this marker. Lanes 9 and 10 depict positive controls for CD45: BJAB (human T-cell lymphoma cells) and lysed freshly isolated buffy-coat mononuclear cells obtained from bovine peripheral blood, respectively. Note that these mononuclear cells are negative for endothelial cell markers CD31, Tie2 and VEGFR2.</p

Western blot analysis of isolated bovine clones grown on different extracellular matrices.

<p>(<b>A</b>) Composite blots of 5 different clones grown on non-coated plates (control conditions) shows clear expression of endothelial markers. Blots also demonstrate that some of the clones express N-cadherin. (<b>B</b>) A composite blot of 12 different clones from the same bovine mononuclear cell isolation as in (<b>A</b>) but grown on fibronectin (FN) or collagen (C) coated plates. (<b>C</b>) Composite blot of a different bovine mononuclear cell isolation showing phenotype of clones obtained from collagen (C) and fibronectin (FN) coated plates.</p