Anti-Angiogenic Therapy Induces Integrin-Linked Kinase 1 Up-Regulation in a Mouse Model of Glioblastoma

BACKGROUND: In order to improve our understanding of the molecular pathways that mediate tumor proliferation and angiogenesis, and to evaluate the biological response to anti-angiogenic therapy, we analyzed the changes in the protein profile of glioblastoma in response to treatment with recombinant human Platelet Factor 4-DLR mutated protein (PF4-DLR), an inhibitor of angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: U87-derived experimental glioblastomas were grown in the brain of xenografted nude mice, treated with PF4-DLR, and processed for proteomic analysis. More than fifty proteins were differentially expressed in response to PF4-DLR treatment. Among them, integrin-linked kinase 1 (ILK1) signaling pathway was first down-regulated but then up-regulated after treatment for prolonged period. The activity of PF4-DLR can be increased by simultaneously treating mice orthotopically implanted with glioblastomas, with ILK1-specific siRNA. As ILK1 is related to malignant progression and a poor prognosis in various types of tumors, we measured ILK1 expression in human glioblastomas, astrocytomas and oligodendrogliomas, and found that it varied widely; however, a high level of ILK1 expression was correlated to a poor prognosis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that identifying the molecular pathways induced by anti-angiogenic therapies may help the development of combinatorial treatment strategies that increase the therapeutic efficacy of angiogenesis inhibitors by association with specific agents that disrupt signaling in tumor cells

«La relation de limitation et d’exception dans le français d’aujourd’hui : excepté, sauf et hormis comme pivots d’une relation algébrique »

L’analyse des emplois prépositionnels et des emplois conjonctifs d’ “excepté”, de “sauf” et d’ “hormis” permet d’envisager les trois prépositions/conjonctions comme le pivot d’un binôme, comme la plaque tournante d’une structure bipolaire. Placées au milieu du binôme, ces prépositions sont forcées par leur sémantisme originaire dûment métaphorisé de jouer le rôle de marqueurs d’inconséquence systématique entre l’élément se trouvant à leur gauche et celui qui se trouve à leur droite. L’opposition qui surgit entre les deux éléments n’est donc pas une incompatibilité naturelle, intrinsèque, mais extrinsèque, induite. Dans la plupart des cas (emplois limitatifs), cette opposition prend la forme d’un rapport entre une « classe » et le « membre (soustrait) de la classe », ou bien entre un « tout » et une « partie » ; dans d’autres (emplois exceptifs), cette opposition se manifeste au contraire comme une attaque de front portée par un « tout » à un autre « tout ». De plus, l’inconséquence induite mise en place par la préposition/conjonction paraît, en principe, tout à fait insurmontable. Dans l’assertion « les écureuils vivent partout, sauf en Australie » (que l’on peut expliciter par « Les écureuils vivent partout, sauf [qu’ils ne vivent pas] en Australie »), la préposition semble en effet capable d’impliquer le prédicat principal avec signe inverti, et de bâtir sur une telle implication une sorte de sous énoncé qui, à la rigueur, est totalement inconséquent avec celui qui le précède (si « les écureuils ne vivent pas en Australie », le fait qu’ils « vivent partout » est faux). Néanmoins, l’analyse montre qu’alors que certaines de ces oppositions peuvent enfin être dépassées, d’autres ne le peuvent pas. C’est, respectivement, le cas des relations limitatives et des relations exceptives. La relation limitative, impliquant le rapport « tout » - « partie », permet de résoudre le conflit dans les termes d’une somme algébrique entre deux sous énoncés pourvus de différent poids informatif et de signe contraire. Les valeurs numériques des termes de la somme étant déséquilibrées, le résultat est toujours autre que zéro. La relation exceptive, au contraire, qui n’implique pas le rapport « tout » - « partie », n’est pas capable de résoudre le conflit entre deux sous énoncés pourvus du même poids informatif et en même temps de signe contraire : les valeurs numériques des termes de la somme étant symétriques et égales, le résultat sera toujours équivalent à zéro

The Effect of a DNA Repair Gene on Cellular Invasiveness: Xrcc3 Over-Expression in Breast Cancer Cells

Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point

Distribution, cellular localization, and therapeutic potential of the tumor-associated antigen Ku70/80 in glioblastoma multiforme.

Antibodies specifically targeting tumor-associated antigens have proved to be important tools in the treatment of human cancer. A desirable target antigen should be unique to tumor cells, abundantly expressed, and readily available for antibody binding. The Ku70/80 DNA-repair protein is expressed in the nucleus of most cells; it is, however, also present on the cell surface of tumor cell lines, and antibodies binding Ku70/80 at the cell surface were recently shown to internalize into tumor cells. To evaluate the potential of Ku70/80-antigen as a therapeutic target for immunotoxins in glioblastoma multiforme, we investigated binding and localization of Ku70/80-specific antibodies in tissue samples from glioblastomas and normal human brains, and in glioma cell cultures. Furthermore, the internalization and drug-delivery capacity were evaluated by use of immunotoxicity studies. We demonstrate that Ku70/80 is localized on the cell plasma membrane of glioma cell lines, and is specifically present in human glioblastoma tissue. Antibodies bound to the Ku70/80 antigen on the cell surface of glioma cells were found to internalize via endocytosis, and shown to efficiently deliver toxins into glioblastoma cells. The data further imply that different antibodies directed against Ku70/80 possess different abilities to target the antigen, in relation to its presentation on the cell surface or intracellular localization. We conclude that Ku70/80 antigen is uniquely presented on the plasma membrane in glioblastomas, and that antibodies specific against the antigen have the capacity to selectively bind, internalize, and deliver toxins into tumor cells. These results imply that Ku70/80 is a potential target for immunotherapy of glioblastoma multiforme