Shi Yuan Cheng

Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment.

Platelet-derived growth factor (PDGF)-B and its receptor (PDGF-R) beta are overexpressed in human gliomas and responsible for recruiting peri-endothelial cells to vessels. To establish the role of PDGF-B in glioma angiogenesis, we overexpressed PDGF-B in U87MG glioma cells. Although PDGF-B stimulated tyrosine phosphorylation of PDGF-Rbeta in U87MG cells, treatment with recombinant PDGF-B or overexpression of PDGF-B in U87MG cells had no effect on their proliferation. However, an increase of secreted PDGF-B in conditioned media of U87MG/PDGF-B cells promoted migration of endothelial cells expressing PDGF-R beta, whereas conditioned media from U87MG cells did not increase the cell migration. In mice, overexpression of PDGF-B in U87MG cells enhanced intracranial glioma formation by stimulating vascular endothelial growth factor (VEGF) expression in neovessels and by attracting vessel-associated pericytes. When PDGF-B and VEGF were overexpressed simultaneously by U87MG tumors, there was a marked increase of capillary-associated pericytes as seen in U87MG/VEGF(165)/PDGF-B gliomas. As a result of pericyte recruitment, vessels induced by VEGF in tumor vicinity migrated into the central regions of these tumors. These data suggest that PDGF-B is a paracrine factor in U87MG gliomas, and that PDGF-B enhances glioma angiogenesis, at least in part, by stimulating VEGF expression in tumor endothelia and by recruiting pericytes to neovessels.

Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3

Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However, molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further, Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway, comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3, were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover, inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last, radiation treatment of PN GSCs up-regulated Mes-associated markers and down-regulated PN-associated markers, whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together, our data suggest that two subtypes of GSCs, harboring distinct metabolic signaling pathways, represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3-mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature.

Angiopoietin-2 Stimulates Breast Cancer Metastasis through the α5β1 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.

Neuropilin-1 promotes human glioma progression through potentiating the activity of the HGF/SF autocrine pathway.

Neuropilin-1 (NRP1) functions as a coreceptor through interaction with plexin A1 or vascular endothelial growth factor (VEGF) receptor during neuronal development and angiogenesis. NRP1 potentiates the signaling pathways stimulated by semaphorin 3A and VEGF-A in neuronal and endothelial cells, respectively. In this study, we investigate the role of tumor cell-expressed NRP1 in glioma progression. Analyses of human glioma specimens (WHO grade I–IV tumors) revealed a significant correlation of NRP1 expression with glioma progression. In tumor xenografts, overexpression of NRP1 by U87MG gliomas strongly promoted tumor growth and angiogenesis. Overexpression of NRP1 by U87MG cells stimulated cell survival through the enhancement of autocrine hepatocyte growth factor/scatter factor (HGF/SF)/c-Met signaling. NRP1 not only potentiated the activity of endogenous HGF/SF on glioma cell survival but also enhanced HGF/SF-promoted cell proliferation. Inhibition of HGF/SF, c-Met and NRP1 abrogated NRP1-potentiated autocrine HGF/SF stimulation. Furthermore, increased phosphorylation of c-Met correlated with glioma progression in human glioma biopsies in which NRP1 is upregulated and in U87MG NRP1-overexpressing tumors. Together, these data suggest that tumor cell-expressed NRP1 promotes glioma progression through potentiating the activity of the HGF/SF autocrine c-Met signaling pathway, in addition to enhancing angiogenesis, suggesting a novel mechanism of NRP1 in promoting human glioma progression.

Angiopoietin 2 Induces Glioma Cell Invasion by Stimulating Matrix Metalloprotease 2 Expression through the αvβ1 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 induces human glioma invasion through the activation of matrix metalloprotease-2.

A hallmark of highly malignant human gliomas is their infiltration of the brain. We analyzed a large number of primary human glioma biopsies and found high levels of expression of an angiogenic regulator, angiopoietin-2 (Ang2), in the invasive areas, but not in the central regions, of those tumors. In the invasive regions where Ang2 was overexpressed, increased levels of matrix metalloprotease-2 (MMP-2) were also apparent. Consonant with these features, intracranial xenografts of glioma cells engineered to express Ang2 were highly invasive into adjacent brain parenchyma compared with isogenic control tumors. In regions of the Ang2-expressing tumors that were actively invading the brain, high levels of expression of MMP-2 and increased angiogenesis were also evident. A link between these two features was apparent, because stable expression of Ang2 by U87MG cells or treatment of several glioma cell lines with recombinant Ang2 in vitro caused activation of MMP-2 and acquisition of increased invasiveness. Conversely, MMP inhibitors suppressed Ang2-stimulated activation of MMP-2 and Ang2-induced cell invasion. These results suggest that Ang2 plays a critical role in inducing tumor cell infiltration, and that this invasive phenotype is caused by activation of MMP-2.

Vascular Endothelial Growth Factor Contributes to the Prostate Cancer-Induced Osteoblast Differentiation Mediated by Bone Morphogenetic Protein

Human prostate cancer has a high predisposition to metastasize to bone, resulting in the formation of osteoblastic metastases. The mechanism through which prostate cancer cells promote osteoblastic lesions is undefined. Vascular endothelial growth factor (VEGF) has been implicated as a mediator of osteoblast activity. In the present study, we examined if prostate cancer cells promote osteoblastic activity through VEGF. We found that LNCaP and C4-2B prostate cancer cell lines and primary tumor and metastatic prostate cancer tissues from patients expressed VEGF. Bone morphogenetic proteins (BMPs), which are normally present in the bone environment, induced VEGF protein and mRNA expression in C4-2B cells. Furthermore, BMP-7 activated the VEGF promoter. Noggin, a BMP inhibitor, diminished VEGF protein expression and promoter activity in C4-2B cells. Conditioned media (CM) from C4-2B cells induced pro-osteoblastic activity (increased alkaline phosphatase, osteocalcin, and mineralization) in osteoblast cells. Both noggin alone and anti-VEGF antibody alone diminished C4-2B CM-induced pro-osteoblastic activity. Transfection of C4-2B cells with VEGF partially rescued the C4-2B CM-induced pro-osteoblastic activity from noggin inhibition. These observations indicate that BMPs promote osteosclerosis through VEGF in prostate cancer metastases. These results suggest a novel function for VEGF in skeletal metastases. Specifically, VEGF promotes osteoblastic lesion formation at prostate cancer bone metastatic sites.

miR-182 as a Prognostic Marker for Glioma Progression and Patient Survival

Accumulating evidence indicates that miRNA expression can be used as a diagnostic and prognostic marker for human cancers. We report that the expression level of miR-182 was markedly up-regulated in glioma cell lines and in human primary glioma specimens. Quantitative PCR analysis showed that miR-182 was significantly increased by up to 32-fold in glioma tumors compared with the adjacent nontumor brain tissues obtained from the same patient. Elevated expression of miR-182 was further identified by in situ hybridization in 248 of 253 (98%) archived human glioma biopsies tested. Statistical analysis revealed a significant correlation between miR-182 expression and World Health Organization glioma grading (P < 0.001). The cumulative 5-year survival rate of glioma patients was 51.54% (95% confidence interval, 0.435 to 0.596) in the low miR-182-expression group, whereas it was only 7.23% (95% confidence interval, 0.027 to 0.118) in the high miR-182-expression group (P = 0.001), and multivariate Cox regression analysis indicated that miR-182 expression was an independent prognostic indicator for the survival of glioma patients. Moreover, the correlations of miR-182 level with the clinical features of glioma suggested in the in situ hybridization analysis were further verified by the real-time RT-PCR analysis. Taken together, our results suggest that miR-182 could be a valuable marker of glioma progression and that high miR-182 expression is associated with poor overall survival in patients with malignant glioma.

Up-Regulation of Angiopoietin-2, Matrix Metalloprotease-2, Membrane Type 1 Metalloprotease, and Laminin 5 γ 2 Correlates with the Invasiveness of Human Glioma

Diffuse infiltration of malignant human glioma cells into surrounding brain structures occurs through the activation of multigenic programs. We recently showed that angiopoietin-2 (Ang2) induces glioma invasion through the activation of matrix metalloprotease-2 (MMP-2). Here, we report that up-regulation of Ang2, MMP-2, membrane type 1-MMP (MT1-MMP), and laminin 5 gamma 2 (LN 5 gamma 2) in tumor cells correlates with glioma invasion. Analyses of 57 clinical human glioma biopsies of World Health Organization grade I to IV tumors displaying a distinct invasive edge and 39 glioma specimens that only contain the central region of the tumor showed that Ang2, MMP-2, MT1-MMP, and LN 5 gamma 2 were co-overexpressed in invasive areas but not in the central regions of the glioma tissues. Statistical analyses revealed a significant link between the preferential expression of these molecules and invasiveness. Protein analyses of microdissected primary glioma tissue showed up-regulation and activation of MT1-MMP and LN 5 gamma 2 at the invasive edge of the tumors, supporting this observation. Concordantly, in human U87MG glioma xenografts engineered to express Ang2, increased expression of MT1-MMP and LN 5 gamma 2, along with MMP-2 up-regulation, in actively invading glioma cells was also evident. In cell culture, stimulation of glioma cells by overexpressing Ang2 or exposure to exogenous Ang2 promoted the expression and activation of MMP-2, MT1-MMP, and LN 5 gamma 2. These results suggest that up-regulation of Ang2, MMP-2, MT1-MMP, and LN 5 gamma 2 is associated with the invasiveness displayed by human gliomas and that induction of these molecules by Ang2 may be essential for glioma invasion.

ELMO1 and Dock180, a Bipartite Rac1 Guanine Nucleotide Exchange Factor, Promote Human Glioma Cell Invasion

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.