Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies

Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization

An approach to analyse the specific impact of rapamycin on mRNA-ribosome association

<p>Abstract</p> <p>Background</p> <p>Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background.</p> <p>Methods</p> <p>We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation.</p> <p>Results</p> <p>High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug.</p> <p>Conclusion</p> <p>The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.</p

Activation of PI3K/mTOR pathway occurs in most adult low-grade gliomas and predicts patient survival

Recent evidence suggests the Akt-mTOR pathway may play a role in development of low-grade gliomas (LGG). We sought to evaluate whether activation of this pathway correlates with survival in LGG by examining expression patterns of proteins within this pathway. Forty-five LGG tumor specimens from newly diagnosed patients were analyzed for methylation of the putative 5′-promoter region of PTEN using methylation-specific PCR as well as phosphorylation of S6 and PRAS40 and expression of PTEN protein using immunohistochemistry. Relationships between molecular markers and overall survival (OS) were assessed using Kaplan-Meier methods and exact log-rank test. Correlation between molecular markers was determined using the Mann-Whitney U and Spearman Rank Correlation tests. Eight of the 26 patients with methylated PTEN died, as compared to 1 of 19 without methylation. There was a trend towards statistical significance, with PTEN methylated patients having decreased survival (P = 0.128). Eight of 29 patients that expressed phospho-S6 died, whereas all 9 patients lacking p-S6 expression were alive at last follow-up. There was an inverse relationship between expression of phospho-S6 and survival (P = 0.029). There was a trend towards decreased survival in patients expressing phospho-PRAS40 (P = 0.077). Analyses of relationships between molecular markers demonstrated a statistically significant positive correlation between expression of p-S6(235) and p-PRAS40 (P = 0.04); expression of p-S6(240) correlated positively with PTEN methylation (P = 0.04) and negatively with PTEN expression (P = 0.03). Survival of LGG patients correlates with phosphorylation of S6 protein. This relationship supports the use of selective mTOR inhibitors in the treatment of low grade glioma

Involvement of mTOR in CXCL12 Mediated T Cell Signaling and Migration

CXCL12 is a pleiotropic chemokine involved in multiple different processes such as immune regulation, inflammatory responses, and cancer development. CXCL12 is also a potent chemokine involved in chemoattraction of T cells to the site of infection or inflammation. Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that modulates different cellular processes, such as metabolism, nutrient sensing, protein translation, and cell growth. The role of mTOR in CXCL12-mediated resting T cell migration has yet to be elucidated.Rapamycin, an inhibitor of mTOR, significantly inhibits CXCL12 mediated migration of both primary human resting T cells and human T cell leukemia cell line CEM. p70(S6K1), an effector molecule of mTOR signaling pathway, was knocked down by shRNA in CEM cells using a lentiviral gene transfer system. Using p70(S6K1) knock down cells, we demonstrate the role of mTOR signaling in T cell migration both in vitro and in vivo.Our data demonstrate a new role for mTOR in CXCL12-induced T cell migration, and enrich the current knowledge regarding the clinical use of rapamycin

Bicyclic triterpenoid Iripallidal induces apoptosis and inhibits Akt/mTOR pathway in glioma cells

<p>Abstract</p> <p>Background</p> <p>The highly resistant nature of glioblastoma multiforme (GBM) to chemotherapy prompted us to evaluate the efficacy of bicyclic triterpenoid Iripallidal against GBM in vitro.</p> <p>Methods</p> <p>The effect of Iripallidal on proliferation and apoptosis in glioma cell lines was evaluated by MTS, colony formation and caspase-3 activity. The effect of iripallidal to regulate (i) Akt/mTOR and STAT3 signaling (ii) molecules associated with cell cycle and DNA damage was evaluated by Western blot analysis. The effect of Iripallidal on telomerase activity was also determined.</p> <p>Results</p> <p>Iripallidal (i) induced apoptosis, (ii) inhibited Akt/mTOR and STAT3 signaling, (iii) altered molecules associated with cell cycle and DNA damage, (iv) inhibited telomerase activity and colony forming efficiency of glioma cells. In addition, Iripallidal displayed anti-proliferative activity against non-glioma cancer cell lines of diverse origin.</p> <p>Conclusion</p> <p>The ability of Iripallidal to serve as a dual-inhibitor of Akt/mTOR and STAT3 signaling warrants further investigation into its role as a therapeutic strategy against GBM.</p

PDGF-C Induces Maturation of Blood Vessels in a Model of Glioblastoma and Attenuates the Response to Anti-VEGF Treatment

Recent clinical trials of VEGF inhibitors have shown promise in the treatment of recurrent glioblastomas (GBM). However, the survival benefit is usually short-lived as tumors escape anti-VEGF therapies. Here we tested the hypothesis that Platelet Derived Growth Factor-C (PDGF-C), an isoform of the PDGF family, affects GBM progression independent of VEGF pathway and hinders anti-VEGF therapy.We first showed that PDGF-C is present in human GBMs. Then, we overexpressed or downregulated PDGF-C in a human GBM cell line, U87MG, and grew them in cranial windows in nude mice to assess vessel structure and function using intravital microscopy. PDGF-C overexpressing tumors had smaller vessel diameters and lower vascular permeability compared to the parental or siRNA-transfected tumors. Furthermore, vessels in PDGF-C overexpressing tumors had more extensive coverage with NG2 positive perivascular cells and a thicker collagen IV basement membrane than the controls. Treatment with DC101, an anti-VEGFR-2 antibody, induced decreases in vessel density in the parental tumors, but had no effect on the PDGF-C overexpressing tumors.These results suggest that PDGF-C plays an important role in glioma vessel maturation and stabilization, and that it can attenuate the response to anti-VEGF therapy, potentially contributing to escape from vascular normalization

Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines