The PTEN/Akt Pathway Dictates the Direct V 3-Dependent Growth-Inhibitory Action of an Active Fragment of Tumstatin in Glioma Cells In vitro and In vivo

Department of Neurological Surgery, University of California, San Francisco, San Francisco, California, United States
Cancer Research (Impact Factor: 9.33). 01/2007; 66(23):11331-40. DOI: 10.1158/0008-5472.CAN-06-1540
Source: PubMed


The collagen type IV cleavage fragment tumstatin and its active subfragments bind to integrin alpha(V)beta(3) and inhibit activation of focal adhesion kinase, phophoinositol-3 kinase, Akt, and mammalian target of rapamycin (mTOR) in what is thought to be an endothelial cell-specific manner. The resultant endothelial cell apoptosis accounts for the ability of tumstatin to function as an endogenous inhibitor of angiogenesis and an indirect suppressor of tumor growth. We hypothesized that the inability of tumstatin to directly suppress tumor cell growth might be the result of the constitutive activation of the Akt/mTOR pathway commonly seen in tumors. Consistent with this idea, several integrin alpha(V)beta(3)-expressing glioma cell lines with PTEN mutations and high levels of phospho-Akt (pAkt) were unaffected by exposure to an active fragment of tumstatin (T3), whereas alpha(V)beta(3)-expressing glioma cell lines with a functional PTEN/low levels of pAkt exhibited T3-induced growth suppression that could be bypassed by small interfering RNA-mediated suppression of PTEN, introduction of a constitutively expressed Akt, or introduction of the Akt and mTOR target eukaryotic translation initiation factor 4E. The direct tumor-suppressive actions of T3 were further shown in an alpha(V)beta(3)-deficient in vivo mouse model in which T3, while unable to alter the tumstatin-insensitive vasculature contributed by the alpha(V)beta(3)-deficient host, nonetheless suppressed the growth and proliferative index of i.c. implanted alpha(V)beta(3)-expressing PTEN-proficient glioma cells. These results show that tumstatin, previously considered to be only an endogenous inhibitor of angiogenesis, also directly inhibits the growth of tumors in a manner dependent on Akt/mTOR activation.

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    • "These observations suggest that up-regulation of PRLR in GBM tumors after ES + Tum treatment was not a secondary response to the anti-angiogenic treatment, but rather mediated through direct action of both integrin targeting factors on tumor cells. Although little is known about the effects of ES and Tum on glioma cells at the molecular level, an integrin-mediated auto-regulation of cell proliferation and apoptosis in glioma cells have been recently described by our group and others [17,34]. In addition, an integrin-PRLR cross-talk has recently been described in breast cancer cells [46]. "
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    ABSTRACT: Tumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways. Therefore, multiple angiogenic pathways should be targeted to achieve significant angiogenic blockade. In this study we investigated the effects of a combined application of the angiogenic inhibitors endostatin and tumstatin in a model of human glioblastoma multiforme. Inhibitors released by stably transfected porcine aortic endothelial cells (PAE) showed anti-angiogenic activity in proliferation and wound-healing assays with endothelial cells (EC). Interestingly, combination of endostatin and tumstatin (ES + Tum) also reduced proliferation of glioma cells and additionally induced morphological changes and apoptosis in vitro. Microencapsulated PAE-cells producing these inhibitors were applied for local therapy in a subcutaneous glioblastoma model. When endostatin or tumstatin were applied separately, in vivo tumor growth was inhibited by 58% and 50%, respectively. Combined application of ES + Tum, in comparison, resulted in a significantly more pronounced inhibition of tumor growth (83%). cDNA microarrays of tumors treated with ES + Tum revealed an up-regulation of prolactin receptor (PRLR). ES + Tum-induced up-regulation of PRLR in glioma cells was also found in in vitro. Moreover, exogenous PRLR overexpression in vitro led to up-regulation of its ligand prolactin and increased proliferation suggesting a functional autocrine growth loop in these cells. Our data indicate that integrin-targeting factors endostatin and tumstatin act additively by inhibiting glioblastoma growth via reduction of vessel density but also directly by affecting proliferation and viability of tumor cells. Treatment with the ES + Tum-combination activates the PRLR pro-proliferative pathway in glioblastoma. Future work will show whether the prolactin signaling pathway represents an additional target to improve therapeutic strategies in this entity.
    Molecular Cancer 11/2013; 12(1):144. DOI:10.1186/1476-4598-12-144 · 4.26 Impact Factor
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    • "Immunodeficient mice (nu/nu; Charles River) were injected intracranially with 4×105 luciferase-expressing U87-Scr-Luc (N = 5) or U87-shPK-M2-Luc (N = 5) cells as described [25]. Tumor growth was monitored weekly by treating mice with D-luciferin (150 mg/kg IP, Gold-Biotechnology) and measuring bioluminescence using a Xenogen IVIS Bioluminescence imaging station (Caliper). "
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    ABSTRACT: Normal tissues express the M1 isoform of pyruvate kinase (PK) that helps generate and funnel pyruvate into the mitochondria for ATP production. Tumors, in contrast, express the less active PKM2 isoform, which limits pyruvate production and spares glycolytic intermediates for the generation of macromolecules needed for proliferation. Although high PKM2 expression and low PK activity are considered defining features of tumors, very little is known about how PKM expression and PK activity change along the continuum from low grade to high grade tumors, and how these changes relate to tumor growth. To address this issue, we measured PKM isoform expression and PK activity in normal brain, neural progenitor cells, and in a series of over 100 astrocytomas ranging from benign grade I pilocytic astrocytomas to highly aggressive grade IV glioblastoma multiforme (GBM). All glioma exhibited comparably reduced levels of PKM1 expression and PK activity relative to normal brain. In contrast, while grade I-III gliomas all had modestly increased levels of PKM2 RNA and protein expression relative to normal brain, GBM, regardless of whether they arose or progressed from lower grade tumors, showed a 3-5 fold further increase in PKM2 RNA and protein expression. Low levels of PKM1 expression and PK activity were important for cell growth as PKM1 over-expression and the accompanying increases in PK activity slowed the growth of GBM cells. The increased expression of PKM2, however, was also important, because shRNA-mediated PKM2 knockdown decreased total PKM2 and the already low levels of PK activity, but paradoxically also limited cell growth and . These results show that pyruvate kinase M expression, but not pyruvate kinase activity, is regulated in a grade-specific manner in glioma, but that changes in both PK activity and PKM2 expression contribute to growth of GBM.
    PLoS ONE 02/2013; 8(2):e57610. DOI:10.1371/journal.pone.0057610 · 3.23 Impact Factor
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    • "FAK acts as a receptor-proximal protein that bridges the growth-factor-receptor and integrin signaling pathways [33]. A previous study has shown that in endometrial epithelial cells, EMP2 regulates αvβ3 integrin expression [6], and it is known that in epithelial cells β3 integrin activation directly leads to Src and FAK phosphorylation resulting in stable focal adhesions [34], [35]. Analysis of EMP2-β3 integrin-FAK association data suggest that in the endometrium EMP2 may act as a molecular adaptor for efficient integrin-mediated FAK activation. "
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    ABSTRACT: Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.
    PLoS ONE 05/2011; 6(5):e19945. DOI:10.1371/journal.pone.0019945 · 3.23 Impact Factor
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