A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM) have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicating CD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possess various genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we compared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressions typical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patients produced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed more proliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast to previous reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cells also possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells for therapeutic targeting. A recent concept in brain tumor biology is that brain tumors arise from cancer stem cells (CSCs) that are CD133 positive (CD133 ( þ ) ). It has been reported that a small number of CD133 ( þ ) glioblastoma multiforme (GBM) cells are able to recapitulate the original tumor in vivo, whereas millions of CD133-negative (CD133 (À) ) cells could not produce brain tumor masses. 1-6 However, accumulating evidence suggests that CD133 (À) GBM cells can also regenerate heterogenous tumors in vivo, 7,8 and generation of the huge and rapidly growing tumors by only CD133 ( þ ) CSCs would be difficult because more than 50% of GBM patients have few CD133 ( þ ) cells. 9 As a majority of neurogenic astrocytes in the adult brain are not recognized by a CD133 antibody, 8 it is likely that CD133 might be newly expressed in GBM CSCs that are derived from CD133 (À) adult neural stem cells (NSCs) or terminally differentiated brain cells, such as astrocytes, neurons, and oligodendrocytes. Given that the gene expression profile is changed when GBM recurs after treatments, 10 it is plausible that new CD133 expression may occur if the characteristics of CSCs are changed or if some CSCs are selected by treatment. Furthermore, the wide-range variation in CD133 ( þ ) cell ratio (0.1-50% in GBM patients) 1-6 also suggests the existence of other GBM CSCs that do not express CD133.Therefore, we hypothesize that there are several kinds of CSCs in the tumor mass of GMB, and these diverse CSCs

Glioblastomas are highly vascularized tumors and anti-angiogenic strategy is one of the most promising therapeutic approaches to treat brain tumors. Interferon • (IFN-•) as a single agent or combined with standard chemotherapy has been shown to inhibit various tumors, but the effect of combination anti-angiogenic therapy on brain tumors has not been well studied. We determined the optimal dose and schedule of pegylated IFN-• (PEG-IFN-•) against U-87MG human glioblastoma cells growing orthotopically in nude mice, since several clinical trials reported that PEG-IFN-• administered at higher or lower doses was less effective. The group treated two times per week with injections of 10 KU of PEG-IFN-• for 4 weeks showed significant decreases in cell proliferation and angiogenesis. Moreover, the optimal dose and schedule of PEG-IFN-• determined in this study and combined with paclitaxel treatment potently inhibited tumor growth in vivo. The mechanisms of the significant therapeutic effects were most likely caused by directly inhibiting cell proliferation and angiogenesis, and rendering apoptosis increased. Specifically PEG-IFN-•/paclitaxel combination induced apoptosis of tumor-associated endothelial cells more than that of tumor cells. These results suggest that optimal biological dosage and scheduling of PEG-IFN-• and paclitaxel combination is a potent strategy for glioblastoma patients as a new synergistic anti-endothelial treatment.

Abstract.The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the antiangiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo.

The Notch signaling pathway plays a central role in the development of various organisms. However, dysregulation of microRNAs (miRNAs), which are crucial regulators of gene expression, can disrupt signaling pathways at all stages of development. Although Notch signaling is involved in wing development in Drosophila, the mechanism underlying miRNA‐based regulation of the Notch signaling pathway is unclear. Here, we report that loss of Drosophila miR‐252 increases the size of adult wings, whereas the overexpression of miR‐252 in specific compartments of larval wing discs leads to patterning defects in the adult wings. The miR‐252 overexpression‐induced wing phenotypes were caused by aberrant Notch signaling with intracellular accumulation of the full‐length Notch receptor during development, which could be due to defects in intracellular Notch trafficking associated with its recycling to the plasma membrane and autophagy‐mediated degradation. Moreover, we identified Rab6 as a direct target of miR‐252‐5p; Rab6 encodes a small Ras‐like GTPase that regulates endosomal trafficking pathways. Consistent with this finding, RNAi‐mediated downregulation of Rab6 led to similar defects in both wing patterning and Notch signaling. Notably, co‐overexpression of Rab6 completely rescued the wing phenotype associated with miR‐252 overexpression, further supporting that Rab6 is a biologically relevant target of miR‐252‐5p in the context of wing development. Thus, our data indicate that the miR‐252‐5p‐Rab6 regulatory axis is involved in Drosophila wing development by controlling the Notch signaling pathway.