Tie2/TEK modulates the interaction of glioma and brain tumor stem cells with endothelial cells and promotes an invasive phenotype

Malignant gliomas are the prototype of highly infiltrative tumors and this characteristic is the main factor for the inevitable tumor recurrence and short survival after most aggressive therapies. The aberrant communication between glioma cells and tumor microenvironment represents one of the major factors regulating brain tumor dispersal. Our group has previously reported that the tyrosine kinase receptor Tie2/TEK is expressed in glioma cells and brain tumor stem cells and is associated with the malignant progression of these tumors. In this study, we sought to determine whether the angiopoietin 1 (Ang1)/Tie2 axis regulates crosstalk between glioma cells and endothelial cells. We found that Ang1 enhanced the adhesion of Tie2-expressing glioma and brain tumor stem cells to endothelial cells. Conversely, specific small interfering RNA (siRNA) knockdown of Tie2 expression inhibited the adhesion capability of glioma cells. Tie2 activation induced integrin β1 and N-cadherin upregulation, and neutralizing antibodies against these molecules inhibited the adhesion of Tie2-positive glioma cells to endothelial cells. In 2D and 3D cultures, we observed that Ang1/Tie2 axis activation was related to increased glioma cell invasion, which was inhibited by using Tie2 siRNA. Importantly, intracranial co-implantation of Tie2-positive glioma cells and endothelial cells in a mouse model resulted in diffusely invasive tumors with cell clusters surrounding glomeruloid vessels mimicking a tumoral niche distribution. Collectively, our results provide new information about the Tie2 signaling in glioma cells that regulates the cross-talk between glioma cells and tumor microenvironment, envisioning Tie2 as a multi-compartmental target for glioma therapy.     

Macrophage-derived SHP-2 inhibits the metastasis of colorectal cancer via Tie2-PI3K signals

This research aimed to explore the influence of Src homology-2 containing protein tyrosine phosphatase (SHP- 2) on the functions of tyrosine kinase receptors with immunoglobulin and EGF homology domains 2 (Tie2)-expressing monocyte/macrophages (TEMs) and the influence of the angiopoietin(Ang)/Tie2-phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) (Ang/Tie2-PI3K/Akt/mTOR) signaling pathway on the tumor microvascular remodeling in an immunosuppressive microenvironment. In vivo, SHP-2- deficient mice were used to construct colorectal cancer (CRC) liver metastasis models. SHP-2-deficient mice had significantly more metastatic cancer and inhibited nodules on the liver surface than wild-type mice, and the high-level expression of p-Tie2 was found in the liver tissue of the macrophages’ specific SHP-2-deficient mice (SHP-2MACKO) + planted tumor mice. Compared with the SHP-2 wild type mice (SHP-2WT) + planted tumor group, the SHP-2MAC-KO + planted tumor group experienced increased expression of p-Tie2, p-PI3K, p-Akt, p-mTOR, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), matrix metalloproteinase 2 (MMP2), and MMP9 in the liver tissue. TEMs selected by in vitro experiments were co-cultured with remodeling endothelial cells and tumor cells as carriers. It was found that when Angpt1/2 was used for stimulation, the SHP-2MAC-KO + Angpt1/2 group displayed evident increases in the expression of the Ang/Tie2-PI3K/Akt/mTOR pathway. The number of cells passing through the lower chamber and the basement membrane and the number of blood vessels formed by cells compared with the SHP-2WT + Angpt1/2 group, while these indexes were subjected to no changes under the simultaneous stimulation of Angpt1/2 + Neamine. To sum up, the conditional knockout of SHP-2 can activate the Ang/Tie2-PI3K/Akt/mTOR pathway in TEMs, thereby strengthening tumor micro angiogenesis in the microenvironment and facilitating CRC liver metastasis.     

Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2 expression through the alphavbeta1 integrin and focal adhesion kinase signaling pathway

Accumulating evidence reveals a significant correlation between angiopoietin 2 (Ang2) expression and tumor invasion and metastasis in various human cancers, but the major focus of recent studies has been on the angiogenic effects of Ang2. We recently reported that Ang2-stimulated glioma cell invasion results from the up-regulation and activation of matrix metalloprotease 2 (MMP-2) in tumor cells. In this study, we identify a novel mechanism by which Ang2 stimulates MMP-2 expression leading to glioma cell invasion. We show that Ang2 interacts with alpha(v)beta(1) integrin in Tie2-deficient human glioma cells, activating focal adhesion kinase (FAK), p130(Cas), extracellular signal-regulated protein kinase (ERK) 1/2, and c-jun NH(2)-terminal kinase (JNK) and substantially enhancing MMP-2 expression and secretion. The Ang2/alpha(v)beta(1) integrin signaling pathway was attenuated by functional inhibition of beta(1) and alpha(v) integrins, FAK, p130(Cas), ERK1/2, and JNK. Furthermore, expression of a negative regulator of FAK, FAK-related nonkinase, by U87MG/Ang2-expressing glioma xenografts suppressed Ang2-induced MMP-2 expression and glioma cell infiltration in the murine brain. These data establish a functional link between Ang2 interaction with alpha(v)beta(1) integrin and glioma cell invasion through the FAK/p130(Cas)/ERK1/2 and JNK-mediated signaling pathway.     

Angiopoietin-2 stimulates breast cancer metastasis through the alpha(5)beta(1) integrin-mediated pathway

Acquisition of a metastatic phenotype by breast cancer cells includes alternations of multigenic programs that permit tumor cells to metastasize to distant organs. Here, we report that angiopoietin-2 (Ang2), a known growth factor, is capable of promoting breast cancer cell invasion leading to metastasis. Analysis of 185 primary human breast cancer specimens that include 97 tumors showing lymph node and/or distant metastasis reveals a significant correlation between the expression of Ang2 and E-cadherin, Snail, metastatic potential, tumor grade, and lymph-vascular invasion during breast cancer progression. Using a xenograft model, we show that overexpression of Ang2 in poorly metastatic MCF-7 breast cancer cells suppresses expression of E-cadherin and induces Snail expression and phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) promoting metastasis to the lymph nodes and lung. In cell culture, Ang2 promotes cell migration and invasion in Tie2-deficient breast cancer cells through the alpha(5)beta(1) integrin/integrin-linked kinase (ILK)/Akt, GSK-3beta/Snail/E-cadherin signaling pathway. Inhibition of ILK and the alpha(5)beta(1) integrin abrogates Ang2 modulation of Akt, GSK-3beta, Snail, and E-cadherin and Ang2-stimulated breast cancer cell migration and invasion. Together, these results underscore the significant contribution of Ang2 in cancer progression, not only by stimulating angiogenesis but also by promoting metastasis, and provide a mechanism by which breast cancer cells acquire an enhanced invasive phenotype contributing to metastasis.     

Ser-10 phosphorylation of histone H3 and immediate early gene expression in oncogene-transformed mouse fibroblasts.

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) pathway by growth factors, phorbol esters, and oncoproteins results in the phosphorylation of histone H3. Rsk-2 and MSK1 have been reported to be H3 kinases activated by the Ras-MAPK signal transduction pathway. In this study, we used inhibitors of Rsk-2 and MSK1 to decide which of these kinases was responsible for the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of H3 in 10T(1/2) and Ciras-3 (H-ras-transformed 10T(1/2)) mouse fibroblasts. These studies demonstrated that MSK1, but not Rsk-2, was the H3 kinase activated in these cells. Furthermore, assays with Rsk-2 showed that this kinase phosphorylates H2B but not H3 in vitro. H89, a potent MSK1 inhibitor, prevented TPA induction of H3 phosphorylation and diminished the TPA-induced expression of the c-fos and urokinase plasminogen activator genes. We propose that persistent activation of the Ras-MAPK pathway and MSK1 resulting in the elevation of phosphorylated H3 levels may contribute to the aberrant gene expression observed in the oncogene-transformed cells.     

促血管生成素１、２及Ｔｉｅ２受体ｍＲＮＡ在卵巢上皮性浆液性腺癌中的表达

目的：探讨卵巢上皮性浆液性腺癌中促血管生成素（Ａｎｇ）１、Ａｎｇ２及Ｔｉｅ２受体ｍＲＮＡ的表达及其临床意义。方法：采用逆转录聚合酶链反应（ＲＴ ＰＣＲ）分析４３例卵巢上皮性浆液性腺癌及１０例正常卵巢组织中Ａｎｇ１、Ａｎｇ２及Ｔｉｅ２受体的表达。结果：Ａｎｇ２ｍＲＮＡ在卵巢上皮性浆液性腺癌中表达明显上调（Ｐ＜０.００１）,并随着病情的进展其表达水平进一步提高（Ｐ＜０.００１）;卵巢上皮性浆液性腺癌中Ａｎｇ１ｍＲＮＡ的表达低于正常组织（Ｐ＜０.０５）,且与临床分期密切相关（Ｐ＜０.０５）;Ｔｉｅ２ｍＲＮＡ的表达量在腺癌与正常组织之间以及在腺癌的不同临床分期中差异无显著性（Ｐ＞０.０５）。结论：Ａｎｇ２的表达水平可能与卵巢上皮性浆液性腺癌的血管生成增强以及早期转移有关。     

Cellular response and activation of apoptosis by mithramycin SK in p21-deficient HCT116 human colon carcinoma cells

HCT116 (p21^−/−) human colon carcinoma cells treated with mithramycin SK (MSK), a novel analog of the antitumor antibiotic mithramycin A (MTA), were transiently arrested in G2/M, with some cells entering a faulty mitotic cycle without cytokinesis that resulted in G1-like cell arrest, which consisted of post-mitotic aneuploid G1 cells. Some of these cells synthesized DNA and elicited an apoptotic response. The absence of p21^WAF1 made HCT116 cells more sensitive to MSK than to the related MTA. MSK also showed higher antiproliferative activity than MTA on HCT116 cells with different genetic backgrounds, including those lacking the p53 gene. Apoptosis in MSK-treated p21^−/− cells involved caspase 2 rather than caspase 3. Untreated HCT116 (p21^−/−) cells presented a little caspase 3 activity, which increased slightly after treatment with MSK. The apoptotic response in p21^−/− cells comprised caspase 2 acting as an executor caspase together with a loss of mitochondrial membrane potential that may be initiated by caspase 2. In contrast, caspase 3 was activated in wild-type HCT116 after treatment with MSK.     

Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway

The angiopoietin-Tie2 system in endothelial cells is an important regulator of vasculogenesis and vascular integrity. High levels of angiopoietin-2 (Ang2) mRNA are observed in vascular activation during tumorigenesis. Although Ang2 is known to be a naturally occurring antagonist of angiopoietin-1 (Ang1) in vivo, the exact function of Ang2 itself is not known. Here, we found that a high concentration of Ang2 (800 ng/ml) acts as an apoptosis survival factor for endothelial cells during serum deprivation apoptosis. The survival effect of high concentration Ang2 was blocked by pre-treatment with soluble Tie2 receptor and the PI 3'-kinase-specific inhibitors, wortmannin and LY294002. Accordingly, 800 ng/ml of Ang2 induced phosphorylation of Tie2, the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase), and serine-threonine kinase Akt at Ser473 in the human umbilical vein endothelial cells; lower concentrations of Ang2 (50 - 400 ng/ml) did not produce notable effects. These findings indicate that at high concentrations, Ang2, like Ang1, can be an apoptosis survival factor for endothelial cells through the activation of the Tie2 receptor, PI 3'-kinase and Akt, and thus may be a positive regulator of tumor angiogenesis. Oncogene (2000) 19, 4549 - 4552.     

Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice.

Angiopoietin-1 (Ang1) is an angiogenic growth factor that functions through activation of its endothelium-specific tyrosine kinase receptor Tie2; it mediates the interaction between endothelial and surrounding cells to promote the remodeling, maturation and stabilization of blood vessels. Although Ang1 is expressed constitutively in many adult tissues, its role in tumor growth and metastasis is not clear. Here we describe experiments in which Ang1 expression was inhibited in HeLa cells by an antisense RNA approach. The modified HeLa cells produced significantly less Ang1 protein both in cultured cells and in tumors formed when these cells were injected into immunodeficient mice. The Ang1 antisense tumors grew much more slowly, with significantly reduced tumor angiogenesis compared with control tumors. Furthermore, they also had substantially increased tumor cell apoptosis and decreased tumor necrosis. Our results indicate that the perturbation of Ang1 expression in tumors could be an effective method to control tumor growth by inhibiting tumor angiogenesis and that antisense RNA is an efficient way to inhibit Ang1 protein production in tumor cells.     

Expression and hypoxic regulation of angiopoietins in human astrocytomas

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and edema in human astrocytomas by its interaction with cognate endothelial-specific receptors (VEGFR1/R2). Tie1 and Tie2/Tek are more recently identified endothelial-specific receptors, with angiopoietins being ligands for the latter. These angiogenic factors and receptors are crucial for the maturation of the vascular system, but their role in tumor angiogenesis, particularly in astrocytomas, is unknown. In this study, we demonstrate that the angiopoietin family member Ang1 is expressed by some of the astrocytoma cell lines. In contrast to VEGF, Ang1 is down regulated by hypoxia. Ang2 was not overexpressed. Expression profiles of low-grade astrocytoma specimens were similar to those of normal brain, with low levels of Ang1, Ang2, and VEGF expression. Glioblastoma multiforme expressed higher levels of Angl, but not to the same degree as pseudopalisading astrocytoma cells around necrotic and hypoxic zones expressed VEGF, as shown in previous studies. Ang2 expression in the highly proliferative tumor vascular endothelium was also increased, as was phosphorylated Tie2/Tek. The expression profile of these angiogenic factors and their endothelial cell receptors in human glioblastomas multiforme was similar to that in a transgenic mouse model of glioblastoma multiforme. These data suggest that both VEGF and angiopoietins are involved in regulating tumor angiogenesis in human astrocytomas.     

Hydrogen peroxide produced by angiopoietin-1 mediates angiogenesis

Angiopoietin-1 (Ang1) mediates angiogenesis by enhancing endothelial cell survival and migration. It is also known that Ang1 activates Tie2, an endothelial-specific tyrosine kinase receptor, but the molecular mechanism of this process is not clear. In this study, we investigated whether reactive oxygen species (ROS) production plays a role in Ang1-mediated angiogenesis. We found that human umbilical vein endothelial cells treated with Ang1 produce ROS transiently, which was suppressed by NADPH oxidase inhibitor, diphenylene-iodonium chloride, and rotenone. The Ang1-induced ROS was identified as hydrogen peroxide (H2O2) using adenovirus-catalase infection. Removal of H2O2 by adenovirus-catalase significantly suppressed Ang1-induced in vitro endothelial cell migration, in vivo tubule formation and angiogenesis, and activation of p44/42 mitogen-activated protein kinase (MAPK), involved in cell migration, and delayed the deactivation of Akt phosphorylation involved in cell survival. Supporting to in vitro data, Ang1-induced vascular remodeling in catalase (-/-) mice was more prominent than in catalase (+/+) mice: Ang1-induced increases of the diameter of terminal arterioles and the postcapillary venules in catalase (-/-) mice were significant compared with catalase (+/+) mice. These results show that Ang1-induced H2O2 plays an important role in Ang1-mediated angiogenesis by modulating p44/42 MAPK activity.     

Enrichment of N-Cadherin and Tie2-bearing CD34/CD38/CD123 leukemic stem cells by chemotherapy-resistance

Acute myeloid leukemia (AML) arises from genetic changes at the level of stem cell, various mutations have been elucidated, including AML1–ETO fusion gene has been shown as the representative target of cellular transformation for LSCs originating from hematopoietic stem cells (HSCs) compartment. LSCs resemble HSCs with respect to self-renewal capacity and chemotherapy-resistance. However, LSCs possess specific cell-surface markers, they are proposed to reside within the CD34⁺/CD38⁻/CD123⁺ compartment. And the interaction mediated by adhesion molecules between LSCs and niche played a role in chemoresistance of LSCs. Therefore, study on the LSCs surface makers related to niche is helpful for the potential target therapy in the future. In this study, the proportions of CD34⁺/CD38⁻/CD123⁺ LSCs compartment co-expressing the three adhesion molecules, N-Cadherin, Tie2 and CD44, respectively, from AML patients before and after chemotherapy were analyzed. We demonstrated N-Cadherin and Tie2 positive CD34⁺/CD38⁻/CD123⁺ LSCs populations could be enriched by chemotherapy. Furthermore, AML1/ETO fusion signals and MDR1 expression were detected on the CD34⁺/CD38⁻/CD123⁺ LSCs populations expressing N-Cadherin and Tie2. Therefore, N-Cadherin and Tie2 are probably the potential markers for identification of LSCs.     

Mitogen- and stress-activated kinase 1-mediated histone H3 phosphorylation is crucial for cell transformation

Mitogen- and stress-activated kinase 1 (MSK1) belongs to a family of dual protein kinases that are activated by either extracellular signal-regulated kinase or p38 mitogen-activated protein kinases in response to stress or mitogenic extracellular stimuli. The physiologic role of MSK1 in malignant transformation and cancer development is not well understood. Here, we report that MSK1 is involved in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced neoplastic transformation of JB6 Cl41 cells. H89, a potent inhibitor of MSK1, strongly suppressed TPA-induced or EGF-induced cell transformation. When cells overexpressing wild-type MSK1 were treated with TPA or EGF, colony formation increased substantially compared with untreated cells or cells that did not overexpress MSK1. In contrast, MSK1 COOH terminal or NH(2) terminal dead dominant negative mutants dramatically suppressed cell transformation. Introduction of small interfering RNA-MSK1 into JB6 Cl41 cells resulted in suppressed TPA-induced or EGF-induced cell transformation. In addition, cell proliferation was inhibited in MSK1 knockdown cells compared with MSK1 wild-type cells. In wild-type MSK1-overexpressing cells, activator protein (AP-1) activation increased after TPA or EGF stimulation, whereas AP-1 activation decreased in both MSK1 dominant-negative mutants and in MSK1 knockdown cells. Moreover, TPA-induced or EGF-induced phosphorylation of histone H3 at Ser(10) was increased in wild-type cells but the induced phosphorylation was abolished in MSK1 dominant-negative mutant or MSK1 knockdown cells. Thus, MSK1 is required for tumor promoter-induced cell transformation through its phosphorylation of histone H3 at Ser(10) and AP-1 activation.     

Angiopoietin‐1 alters tumor growth by stabilizing blood vessels or by promoting angiogenesis

The maturation of blood vessels requires mural cell adhesion to endothelial cells. Angiopoietin‐1 (Ang1), a ligand for Tie2 receptor expressed on endothelial cells, plays a critical role in cell adhesion between mural cells and endothelial cells and in endothelial cell sprouting from preexisting vessels in the absence of mural cells. Much information has been amassed on the Tie2–Ang1 system in physiological blood vessel formation during embryogenesis; however, the role of Ang1 in the tumor environment and its interaction with mural cells has not been well documented. Here we studied how Ang1 regulates maturation of blood vessels using the human colon cancer cell line HT29 and the human prostate cancer cell line PC3, and studied how Ang1 affects tumor growth. In a xenograft tumor model using female nude mice, we found that Ang1 enhanced angiogenesis and resulted in tumor growth in the case of PC3 tumors but suppressed tumor growth in the case of HT29 tumors. In PC3 tumors, the number of mural cells adhering to endothelial cells was less than that in HT29 tumors. Ang1 induced sprouting angiogenesis in PC3 tumors although there was little maturation of blood vessels. On the other hand, there was abundant mural cell adhesion to endothelial cells in HT29 tumors and Ang1 did not induce angiogenesis. These results suggest that Ang1 alters tumor growth in a manner that is dependent on the adhesion of mural cells and their localization in the tumor environment. (Cancer Sci 2008; 99: 2373–2379)