Unknown,Transcriptomics,Genomics,Proteomics

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Targeted inhibition of ubiquitin signaling reverses the metabolic reprogramming and suppresses Glioblastoma growth

ABSTRACT: Glioblastoma (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population, and the high recurrence rate and resistance to current therapeutics urgently demand a better therapy for this disease. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. Deregulation of UPS is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, UPS system represents a valuable target for GBM treatment. Here, we find that the E3 ligase praja2 selectively marks primary IDH1-wild type GBM. By using an integrated approach, including proteomics, transcriptomics and metabolic profiling, we identify praja2 as a main component of a signaling network that regulates cancer cell growth and metabolism. We show that praja2 binds and regulates the stability of the Kinase Suppressor of Ras 2 (KSR2) and as consequence, the activity of the downstream AMP-dependent protein kinase (AMPK) in GBM cells. By degrading KSR2, praja2 attenuates the oxidative metabolism and promotes the aerobic glycolysis (Warburg effect). Brain delivery of siRNAs targeting praja2 upregulates KSR2 and negatively impacted on GBM growth, reducing the tumor size and significantly improving the survival rate of treated mice. These data highlight the role of praja2 as an essential regulator of cancer cell metabolism, and as a preferential therapeutic target to suppress GBM growth

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Giorgio Giurato

PROVIDER: E-MTAB-11137 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to such treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and -5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased of cell proliferation, and increased cell cycle arrest and apoptosis in comparison to only irradiated cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation and cell cycle regulation. To our knowledge, this is the first study to describe role of miR-338-5p in GBM and its potential to improve sensitivity of GBM to radiation. Study was performed on three glioblastoma multiforme cell lines A172, T98G and U87MG. This experiment was performed on Affymetrix GeneChip Human Gene ST 1.0 to elucidate the targets of miRNA-338-5p. Cell lines were seeded 24 hours prior transfection. After transfection with pre-miR338-5p or negative control cell were cultured for 24 hours and harvested. RNA was isolated using MirVana miRNA Isolation Kit (Ambion, USA) and checked for RNA integrity by Bioanalyzer 2100 and purity by ratios 260/280>1.8 and 260/230>1.8 by Nanodrop2000.

Project description:Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase, is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 due to compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which demonstrates a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human Phase I trial. The PTEN-deficient U87MG glioblastoma cell line was treated for 6 hours with vehicle control (DMSO) or to different concentrations of AT13148 and CCT128930 (0.1uM, 1xGI50 and 3XGI50).

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Targeted inhibition of ubiquitin signaling reverses the metabolic reprogramming and suppresses Glioblastoma growth

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