ABSTRACT
DDX3 is a DEAD box RNA helicase with oncogenic properties. RK-33 is developed as a small-molecule inhibitor of DDX3 and showed potent radiosensitizing activity in preclinical tumor models. This study aimed to assess DDX3 as a target in breast cancer and to elucidate how RK-33 exerts its anti-neoplastic effects. High DDX3 expression was present in 35% of breast cancer patient samples and correlated with markers of aggressiveness and shorter survival. With a quantitative proteomics approach, we identified proteins involved in the mitochondrial translation and respiratory electron transport pathways to be significantly downregulated after RK-33 or DDX3 knockdown. DDX3 localized to the mitochondria and DDX3 inhibition with RK-33 reduced mitochondrial translation. As a consequence, oxygen consumption rates and intracellular ATP concentrations decreased and reactive oxygen species (ROS) increased. RK-33 antagonized the increase in oxygen consumption and ATP production observed after exposure to ionizing radiation and reduced DNA repair. Overall, we conclude that DDX3 inhibition with RK-33 causes radiosensitization in breast cancer through inhibition of mitochondrial translation, which results in reduced oxidative phosphorylation capacity and increased ROS levels, culminating in a bioenergetic catastrophe.
Subject(s)
Breast Neoplasms/pathology , DEAD-box RNA Helicases/metabolism , Mitochondria/metabolism , Protein Biosynthesis/drug effects , Radiation-Sensitizing Agents/pharmacology , Azepines/pharmacology , Azepines/therapeutic use , Breast/pathology , Breast Neoplasms/mortality , Breast Neoplasms/therapy , Cell Line, Tumor , DEAD-box RNA Helicases/antagonists & inhibitors , DEAD-box RNA Helicases/genetics , Down-Regulation , Female , Gene Knockdown Techniques , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Mitochondria/drug effects , Mitochondria/genetics , Mitochondria/radiation effects , Oncogenes/drug effects , Proteomics , Radiation-Sensitizing Agents/therapeutic use , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/radiation effects , Survival AnalysisABSTRACT
Autophagy is a conserved proteolytic mechanism required for maintaining cellular homeostasis. The role of this process in vertebrate neural development is related to metabolic needs and stress responses, even though the importance of its progression has been observed in a number of circumstances, both in embryonic and in postnatal differentiating tissues. Here we show that the proautophagic proteins Ambra1 and Beclin 1, involved in the initial steps of autophagosome formation, are highly expressed in the adult subventricular zone (SVZ), whereas their downregulation in adult neural stem cells in vitro leads to a decrease in cell proliferation, an increase in basal apoptosis and an augmented sensitivity to DNA-damage-induced death. Further, Beclin 1 heterozygosis in vivo results in a significant reduction of proliferating cells and immature neurons in the SVZ, accompanied by a marked increase in apoptotic cell death. In sum, we propose that Ambra1- and Beclin 1-mediated autophagy plays a crucial role in adult neurogenesis, by controlling the survival of neural precursor cells.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Brain/metabolism , Lateral Ventricles/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Autophagy , Beclin-1 , Brain/pathology , Cell Proliferation , Cells, Cultured , Heterozygote , Lateral Ventricles/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , NeurogenesisABSTRACT
BACKGROUND AND PURPOSE: Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-A (PDGF-AA) are potent modulators of oligodendrocytes, the main responsible cells for myelin regeneration. We measured FGF-2 and PDGF-AA in the sera and cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RR-MS) and compared these values with control subjects. METHODS: Twenty-three patients with RR-MS and 23 subjects without inflammatory and demyelinating diseases were included. Serum samples of the patients were collected in both relapse and remission phases and were analyzed with ELISA method. CSF was drawn during the relapse period. Blood and CSF were drawn from control subjects for comparison. Wilcoxon and Mann-Whitney U-test and Spearman's rank correlation were used for analysis. P values of <0.05 were considered significant. RESULTS: Age and sex distribution were similar in both groups. Serum values of FGF-2 were higher in relapse phase compared with remission phase, with a trend toward significance (P=0.052). CSF PDGF-AA showed significant negative correlation with disease duration (correlation coefficient=-0.58, P=0.004). Serum levels of PDGF did not differ between two phases significantly. There was no difference in serum and CSF values of these factors between patients and controls. When we compared patients with prolonged disease with controls, a significant difference between the CSF levels of PDGF-AA was observed (mean±SEM 2.78±0.8 in controls vs. 0.55±0.29 in patients with MS≥2 years, P<0.05). CONCLUSION: Our study was the first to show that CSF PDGF-AA is related to disease duration. Supporting previous findings we showed that serum and CSF levels of these factors are weak indicators of disease severity.
