Integrin α5ß1 plays a critical role in resistance to temozolomide by interfering with the p53 pathway in high-grade glioma.

Integrins play a role in the resistance of advanced cancers to radiotherapy and chemotherapy. In this study, we show that high expression of the α5 integrin subunit compromises temozolomide-induced tumor suppressor p53 activity in human glioblastoma cells. We found that depletion of the α5 integrin subunit increased p53 activity and temozolomide sensitivity. However, when cells were treated with the p53 activator nutlin-3a, the protective effect of α5 integrin on p53 activation and cell survival was lost. In a functional p53 background, nutlin-3a downregulated the α5 integrin subunit, thereby increasing the cytotoxic effect of temozolomide. Clinically, α5ß1 integrin expression was associated with a more aggressive phenotype in brain tumors, and high α5 integrin gene expression was associated with decreased survival of patients with high-grade glioma. Taken together, our findings indicate that negative cross-talk between α5ß1 integrin and p53 supports glioma resistance to temozolomide, providing preclinical proof-of-concept that α5ß1 integrin represents a therapeutic target for high-grade brain tumors. Direct activation of p53 may remain a therapeutic option in the subset of patients with high-grade gliomas that express both functional p53 and a high level of α5ß1 integrin.

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo.

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.

alpha5beta1 integrin antagonists reduce chemotherapy-induced premature senescence and facilitate apoptosis in human glioblastoma cells.

The alpha5beta1 integrin represent a new therapeutic target for glioblastoma, which are malignant brain tumors difficult to cure with conventional therapies. Glioblastoma are known to be highly resistant to chemotherapy. We, therefore, investigated whether blocking alpha5beta1 integrin with specific nonpeptidic antagonists concomitantly with chemotherapy (ellipticine and temozolomide) may impact the response to chemotherapy of human glioblastoma. Here we show that inhibiting alpha5beta1 integrin with 2 selective ligands (SJ749 and K34c) decreases chemotherapy-induced premature senescence and facilitates cell apoptosis in a functional p53 background (U87MG cells). When p53 is mutated and inactive (U373 cells), chemotherapy induces p53-independent cell apoptosis instead of senescence that is not improved by integrin antagonists. Silencing p53 in U87MG cells with siRNA as well as evaluating HCT116 p53+/+ and p53-/- colon carcinoma cell behavior support the hypothesis of an as yet unknown effect of alpha5beta1 integrin antagonists on the control of chemotherapy-induced premature senescence and apoptosis. alpha5beta1 integrin antagonists modulate the p53 signaling induced by chemotherapy. Our results highlight a new role of the alpha5beta1 integrin in the control of glioblastoma aggressiveness and responsiveness to chemotherapy, which may have a crucial impact in the clinical management of patients suffering from brain tumors.

Caveolin-1 regulates glioblastoma aggressiveness through the control of alpha(5)beta(1) integrin expression and modulates glioblastoma responsiveness to SJ749, an alpha(5)beta(1) integrin antagonist.

Caveolin-1 plays a checkpoint function in the regulation of processes often altered in cancer. Although increased expression of caveolin-1 seems to be the norm in the glioma family of malignancies, populations of caveolin-1 positive and negative cells coexist among glioblastoma specimens. As no data are available to date on the contribution of such cells to the phenotype of glioblastoma, we manipulated caveolin-1 in the glioblastoma cell line U87MG. We showed that caveolin-1 plays a critical role in the aggressiveness of glioblastoma. We identified integrins as the main set of genes affected by caveolin-1. We reported here that the phenotypic changes observed after caveolin-1 modulation were mediated by alpha(5)beta(1) integrins. As a consequence of the regulation of alpha(5)beta(1) levels by caveolin-1, the sensitivity of cells to the specific alpha(5)beta(1) integrin antagonist, SJ749, was affected. Mediator of caveolin-1 effects, alpha(5)beta(1) integrin, is also a marker for glioma aggressiveness and an efficient target for the treatment of glioma especially the ones exerting the highest aggressive phenotype.

Detection of a hypersialylated beta1 integrin endogenously expressed in the human astrocytoma cell line A172.

Gliomas are the most common deadly brain tumors. Human cerebral tumors express high level of alpha5beta1 integrins. As a potential new target, alpha5beta1 was investigated here in two human astrocytoma cell lines, A172 and U87MG. We found that a hypersialylated beta1 integrin was endogenously expressed in A172 cells. It forms heterodimers with alpha5 subunits, localizes at the cell membrane and allows adhesion to fibronectin. This form of beta1 integrin was only recognized by the 9EG7 anti-beta1 antibody and appeared devoid of other specific antibody epitopes (12G10, TS2/16 and mAb13 shown here to be N-glycosylation sensitive). Overexpression of the beta1 integrin subunit in A172 cells not only increased the hypersialylated form but also led to the appearance of a non-hypersialylated beta1 form also addressed to the cell surface. Compared to wild-type A172 cells, beta1-A172 cells showed increased adhesion to fibronectin and decreased sensitivity to SJ749, a non-peptidic alpha5beta1 antagonist. In addition, beta1-A172 cells exhibited increased matrix dependence for normal cell cycling. Collectively, the data add new evidence for the role of beta1 glycosylation/sialylation in the regulation of integrin functions.

The small alpha5beta1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells.

The potential role of alpha5beta1 integrins in cancer has recently attracted much interest. However, few alpha5beta1-selective antagonists have been developed compared with other integrins. The most specific nonpeptidic alpha5beta1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of alpha5beta1. SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G0-G1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more alpha5beta1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing alpha5. Treatment of alpha5-A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of alpha5beta1 expression. Our study highlights the importance of alpha5beta1 as an anticancer target and shows for the first time that a small nonpeptidic alpha5beta1-specific antagonist affects proliferation of tumor cells.