Christine Toulas

αvβ3/αvβ5 Integrins-FAK-RhoB: A Novel Pathway for Hypoxia Regulation in Glioblastoma

The presence of hypoxic areas in glioblastoma is an important determinant in tumor response to therapy and, in particular, to radiotherapy. Here we have explored the involvement of integrins, up to now known as regulators of angiogenesis and invasion, in the regulation of tumor hypoxia driven from the tumor cell. We first show that hypoxia induces the recruitment of alpha(v)beta(3) and alpha(v)beta(5) integrins to the cellular membrane of U87 and SF763 glioblastoma cells, thereby activating the focal adhesion kinase (FAK). We then show that inhibiting alpha(v)beta(3) or alpha(v)beta(5) integrins in hypoxic cells with a specific inhibitor or with siRNA decreases the hypoxia-inducible factor 1alpha (HIF-1alpha) intracellular level. This integrin-dependent regulation of HIF-1alpha is mediated through the regulation of FAK, which in turn activates the small GTPase RhoB, leading to the inhibition of GSK3-beta. Furthermore, silencing this pathway in glioma cells of established xenografts dramatically reduces glioma hypoxia, associated with a significant decrease in vessel density. Our present results unravel a new mechanism of hypoxia regulation by establishing the existence of an alpha(v)beta(3)/alpha(v)beta(5) integrin-dependent loop of hypoxia autoregulation in glioma. Targeting this hypoxia loop may be crucial to optimizing radiotherapy efficiency.

Activation of RhoB by Hypoxia Controls Hypoxia-Inducible Factor-1α Stabilization through Glycogen Synthase Kinase-3 in U87 Glioblastoma Cells

Hypoxia is a crucial factor in tumor aggressiveness and resistance to treatment, particularly in glioma. Our previous results have shown that inhibiting the small GTPase RhoB increased oxygenation of U87 human glioblastoma xenografts, in part, by regulating angiogenesis. We investigated here whether RhoB might also control a signaling pathway that would permit glioma cells to adapt to hypoxia. We first showed that silencing RhoB with siRNA induced degradation and inhibition of the transcriptional activity of the hypoxia-inducible factor by the proteasome in U87 hypoxic cells. This RhoB-dependent degradation of hypoxia-inducible factor-1alpha in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3beta pathway. While investigating how hypoxia could activate this signaling pathway, using the GST-Rhotekin RBD pulldown assay, we showed the early activation of RhoB by reactive oxygen species under hypoxic conditions and, subsequently, its participation in the ensuing cellular adaptation to hypoxia. Overall, therefore, our results have not only highlighted a new signaling pathway for hypoxia controlled by the small GTPase RhoB, but they also strongly implicate RhoB as a potentially important therapeutic target for decreasing tumor hypoxia.

RhoB controls the 24 kDa FGF-2-induced radioresistance in HeLa cells by preventing post-mitotic cell death.

Farnesylated Ras oncoprotein induces a cellular resistance to ionizing radiation that can be reversed by farnesyltransferase inhibitors (FTI). We previously demonstrated that, expression of the 24 kDa FGF2 isoform in wild type ras bearing HeLa cells, induced radioresistance which was also reversed by FTI. We tested the hypothesis that wild type Ras or RhoB, which has been proposed as a potential FTI target, could control the FGF-2-induced radioresistance mechanisms. For this, we expressed inducible dominant negative forms of Ras (RasN17) and Rho (RhoBN19) in 24 kDa FGF2 transfected HeLa cells and analysed their survival after irradiation. While no cell survival modification was observed after RasN17 induction, the expression of RhoBN19 induced a radiosensitization of FGF2 radioresistant HeLa cells in the same range as the one observed after a 48 h treatment with the specific FTI, R115777. Moreover, we showed that activated RhoB but not RhoA induced radioresistance in NIH3T3 cells. The radiosensitizer effect of RhoBN19 expression was due to the induction of the radiation induced post-mitotic cell death. Taken together, these data demonstrate that 24 kDa FGF-2-induced radioresistance is controlled by Rho pathways and suggest that RhoB should be a major determinant in cellular resistance to ionizing radiation.

The radioprotective effect of the 24 kDa FGF-2 isoform in HeLa cells is related to an increased expression and activity of the DNA dependent protein kinase (DNA-PK) catalytic subunit.

We previously reported that overexpression of the 24 kDa basic fibroblast factor (or FGF-2) isoform provides protection from the cytotoxic effect of ionizing radiation (IR). DNA double-strand breaks (DSB), the IR-induced lethal lesions, are mainly repaired in human cells by non-homologous end joining system (NHEJ). NHEJ reaction is dependent on the DNA-PK holoenzyme (composed of a regulatory sub-unit, Ku, and a catalytic sub-unit, DNA-PKcs) that assembles at sites of DNA damage. We demonstrated here that the activity of DNA-PK was increased by twofold in two independent radioresistant cell lines, HeLa 3A and CAPAN A3, overexpressing the 24 kDa FGF-2. This increase was associated with an overexpression of the DNA-PKcs without modification of Ku expression or activity. This overexpression was due to an up-regulation of the DNA-PKcs gene transcription by the 24 kDa FGF-2 isoform. Finally, HeLa 3A cells exhibited the hallmarks of phenotypic changes associated with the overexpression of an active DNA-PKcs. Indeed, a faster repair rate of DSB and sensitization to IR by wortmannin was observed in these cells. Our results represent the characterization of a new mechanism of control of DNA repair and radioresistance in human tumor cells dependent on the overproduction of the 24 kDa FGF-2 isoform.

Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

High-grade gliomas, glioblastomas (GB), are refractory to conventional treatment combining surgery, chemotherapy, mainly temozolomide, and radiotherapy. This highlights an urgent need to develop novel therapies and increase the efficacy of radio/chemotherapy for these very aggressive and malignant brain tumors. Recently, tumor metabolism became an interesting potential therapeutic target in various cancers. Accordingly, combining drugs targeting cell metabolism with appropriate chemotherapeutic agents or radiotherapy has become attractive. In light of these perspectives, we were particularly interested in the anti-cancer properties of a biguanide molecule used for type 2 diabetes treatment, metformin. In our present work, we demonstrate that metformin decreases mitochondrial-dependent ATP production and oxygen consumption and increases lactate and glycolytic ATP production. We show that metformin induces decreased proliferation, cell cycle arrest, autophagy, apoptosis and cell death in vitro with a concomitant activation of AMPK, Redd1 and inhibition of the mTOR pathway. Cell sensitivity to metformin also depends on the genetic and mutational backgrounds of the different GB cells used in this study, particularly their PTEN status. Interestingly, knockdown of AMPK and Redd1 with siRNA partially, but incompletely, abrogates the induction of apoptosis by metformin suggesting both AMPK/Redd1-dependent and –independent effects. However, the primary determinant of the effect of metformin on cell growth is the genetic and mutational backgrounds of the glioma cells. We further demonstrate that metformin treatment in combination with temozolomide and/or irradiation induces a synergistic anti-tumoral response in glioma cell lines. Xenografts performed in nude mice demonstrate in vivo that metformin delays tumor growth. As current treatments for GB commonly fail to cure, the need for more effective therapeutic options is overwhelming. Based on these results, metformin could represent a potential enhancer of the cytotoxic effects of temozolomide and/or radiotherapy.

Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible involvement in radioresistance

Glioblastomas (GBM) are some bad prognosis brain tumors despite a conventional treatment associating surgical resection and subsequent radio-chemotherapy. Among these heterogeneous tumors, a subpopulation of chemo- and radioresistant GBM stem-like cells appears to be involved in the systematic GBM recurrence. Moreover, recent studies showed that differentiated tumor cells may have the ability to dedifferentiate and acquire a stem-like phenotype, a phenomenon also called plasticity, in response to microenvironment stresses such as hypoxia. We hypothesized that GBM cells could be subjected to a similar dedifferentiation process after ionizing radiations (IRs), then supporting the GBM rapid recurrence after radiotherapy. In the present study we demonstrated that subtoxic IR exposure of differentiated GBM cells isolated from patient resections potentiated the long-term reacquisition of stem-associated properties such as the ability to generate primary and secondary neurospheres, the expression of stemness markers and an increased tumorigenicity. We also identified during this process an upregulation of the anti-apoptotic protein survivin and we showed that its specific downregulation led to the blockade of the IR-induced plasticity. Altogether, these results demonstrated that irradiation could regulate GBM cell dedifferentiation via a survivin-dependent pathway. Targeting the mechanisms associated with IR-induced plasticity will likely contribute to the development of some innovating pharmacological strategies for an improved radiosensitization of these aggressive brain cancers.

Inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of human farnesyltransferase, BIM‐46228

Oncogenic mutations of the ras gene leading to constitutive activation of downstream effectors have been detected in a wide spectrum of human cancers (pancreas, thyroid, colon, non‐small‐cell lung cancer). Membrane anchorage of Ras, required for functional activity in signal transduction, is facilitated by post‐translational modifications resulting in covalent attachment of a farnesyl group to the cysteine in the C‐terminal CAAX motif. This attachment is mediated by farnesyltransferase (FTase). Here, we report a novel FTase inhibitor, BIM‐46228, which showed (i) specific inhibition of purified human FTase enzyme, (ii) inhibition of proliferation in vitro in a large spectrum of human tumor cell lines, (iii) inhibition of growth of human tumor xenografts in athymic nude mice treated by per os administration and (iv) the benefits of in vitro combination of its activity with chemotherapy or radiotherapy.

Breast cancer risk in BRCA1 and BRCA2 mutation carriers and polyglutamine repeat length in the AIB1 gene.

Marked variation in phenotypic expression among BRCA1 and BRCA2 mutation carriers may be partly explained by modifier genes that influence mutation penetrance. Variation in CAG/CAA repeat lengths coding for stretches of glutamines in the C‐terminus of the AIB1 protein (amplified in breast cancer 1, a steroid receptor coactivator) has been proposed to modify the breast cancer risk in women carrying germline BRCA1 mutations. We genotyped the AIB1 repeat length polymorphism from the genomic DNA of a group of 851 BRCA1 and 324 BRCA2 female germline mutation carriers to estimate an association with breast cancer risk modification. Hazard ratios (HR) were calculated using a Cox proportional hazards model. For BRCA1 and BRCA2 mutation carriers, analyzed separately and together, we found that women who carried alleles with 28 or more polyglutamine repeats had no increased risk of breast cancer compared to those who carried alleles with fewer repeats (HR for BRCA1/2 carriers = 0.88, 95% CI [confidence interval] = 0.75–1.04). Analyzing average repeat lengths as a continuous variable showed no excess risk of breast cancer (BC) in BRCA1 or BRCA2 mutation carriers (HR for average repeat length in BRCA1/2 carriers = 1.01, 95% CI = 0.92–1.11). These results strongly suggest that contrary to previous studies, there is no significant effect of AIB1 genetic variation on BC risk in BRCA1 mutation carriers and provide an indication that there is also no strong risk modification in BRCA2 carriers.

Basic Fibroblast Growth Factor-2/β3 Integrin Expression Profile: Signature of Local Progression After Chemoradiotherapy for Patients With Locally Advanced Non–Small-Cell Lung Cancer

PURPOSE No biologic signature of chemoradiotherapy sensitivity has been reported for patients with locally advanced non-small-cell lung cancer (NSCLC). We have previously demonstrated that basic fibroblast growth factor (FGF-2) and alphavbeta3 integrin pathways control tumor radioresistance. We investigated whether the expression of the proteins involved in these pathways might be associated with the response to treatment and, therefore, the clinical outcome. METHODS AND MATERIALS FGF-2, beta3 integrin, angiopoietin-2, and syndecan-1 expression was studied using immunohistochemistry performed on biopsies obtained, before any treatment, from 65 patients exclusively treated with chemoradiotherapy for locally advanced NSCLC. The response to treatment was evaluated according to the Response Evaluation Criteria in Solid Tumors criteria using computed tomography at least 6 weeks after the end of the chemoradiotherapy. Local progression-free survival, metastasis-free survival, and disease-free survival were studied using the log-rank test and Cox proportional hazard analysis. RESULTS Among this NSCLC biopsy population, 43.7% overexpressed beta3 integrin (beta3(+)), 43% FGF-2 (FGF-2(+)), 41.5% syndecan-1, and 59.4% angiopoietin-2. Our results showed a strong association between FGF-2 and beta3 integrin expression (p = .001). The adjusted hazard ratio of local recurrence for FGF-2(+)/beta3(+) tumors compared with FGF-2(-)/beta3(-) tumors was 6.1 (95% confidence interval, 2.6-14.6, p = .005). However, the risk of local recurrence was not increased when tumors overexpressed beta3 integrin or FGF-2 alone. Moreover, the co-expression of these two proteins was marginally associated with the response to chemoradiotherapy and metastasis-free survival. CONCLUSION The results of this study have identified the combined profile FGF-2/beta3 integrin expression as a signature of local control in patients treated with chemoradiotherapy for locally advanced NSCLC.

Radiation-induced mitotic cell death and glioblastoma radioresistance: A new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1alpha and survivin in U87 cells

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.