DIRAS2 Contributes to Radiation Resistance of Renal Cell Carcinoma Via Autophagy Induction and MKK4-JNK1 Pathway Activation

Radiation resistance has been regarded as a main obstacle to improve the definitive treatment of renal cell carcinoma (RCC), of which clear cell RCC (ccRCC) is the most common histological type. However, the molecular mechanism underlying the radiation resistance remains largely unclear. In this study, we investigated the effect of DIRAS2 on the response to ionizing radiation (IR) in human ccRCC cells. Here, we found the expression level of DIRAS2 was significantly upregulated in human ccRCC tissues using the Oncomine platform and the Cancer Genome Atlas (TCGA) database, which was further validated by immunohistochemistry. Overexpression of DIRAS2 promoted radiation resistance of ccRCC cells based on clonogenic survival assay and enhanced the levels of radiation induced-autophagy. Moreover, inhibition of autophagy by chloroquine (CQ) pre-treatment largely eliminated the effect of DIRAS2 overexpression on radiation-resistance. Finally, molecular mechanism investigation showed that overexpression of DIRAS2 upregulated the activity of mitogen-activated protein kinase (MAPK) kinase 4 (MKK4)- c-Jun NH2-terminal kinase 1 (JNK1)-Bcl-2 pathway in response to IR. Taken together, these results indicate that DIRAS2 may confer radiation resistance on human RCC via autophagy induction through MKK4-JNK1-Bcl-2 signaling pathway.

ATF4/CEMIP/PKCα promotes anoikis resistance by enhancing protective autophagy in prostate cancer cells

The survival of cancer cells after detaching from the extracellular matrix (ECM) is essential for the metastatic cascade. The programmed cell death after detachment is known as anoikis, acting as a metastasis barrier. However, the most aggressive cancer cells escape anoikis and other cell death patterns to initiate the metastatic cascade. This study revealed the role of cell migration-inducing protein (CEMIP) in autophagy modulation and anoikis resistance during ECM detachment. CEMIP amplification during ECM detachment resulted in protective autophagy induction via a mechanism dependent on the dissociation of the B-cell lymphoma-2 (Bcl-2)/Beclin1 complex. Additional investigation revealed that acting transcription factor 4 (ATF4) triggered CEMIP transcription and enhanced protein kinase C alpha (PKCα) membrane translocation, which regulated the serine70 phosphorylation of Bcl-2, while the subsequent dissociation of the Bcl-2/Beclin1 complex led to autophagy. Therefore, CEMIP antagonization attenuated metastasis formation in vivo. In conclusion, inhibiting CEMIP-mediated protective autophagy may provide a therapeutic strategy for metastatic prostate cancer (PCa). This study delineates a novel role of CEMIP in anoikis resistance and provides new insight into seeking therapeutic strategies for metastatic PCa.

The Effect of Autophagy Induction in Oncolytic Reovirus Replication in Mesenchymal Stem Cells

Background and Aims: Oncolytic reoviruses can infect and kill malignant cells while sparing their normal counterparts. Reoviral infection can induce or activate autophagy, even though metformin can induce autophagy. Identifying and regulating the cellular pathways important for reovirus replication and oncolysis can improve targeted-biological therapies for cancer. Here, the autophagic process was triggered via metformin, and we investigated the effect of autophagy activation on oncolytic reovirus replication in mesenchymal stem cells as primary cells and L929 cell lines. Materials and Methods: Adipose derived mesenchymal stem cells (AD-MSCs) and L929 cells were treated with metformin and reovirus type-3 strain Dearing (T3D). Twenty-four hours after infection, the viability of AD-MSCs and L929 cells were examined by MTT assay. Also, the effect of metformin-induced autophagy in the reovirus replication in these cells was determined by real-time polymerase-chain-reaction. Results: Our results show that treatment with metformin and reovirus reduced the viability of the cells compared to treatment with metformin or reovirus alone in both cells. Also, coadministration of metformin and reovirus significantly decreased the relative expression level of the Beclin-1 gene compared to treatment with metformin in both cells. However, the expression level of the reovirus L3 gene after treatment with metformin and reovirus in L929 cells increased significantly compared to AD-MSCs. Conclusion: Our data suggest that metformin-induced autophagy enhances reoviral replication in AD-MSCs and L929 cells. These findings represent the role of autophagy induction in facilitating reovirus replication and contribute to a better understanding of reovirus-host interactions.

Autophagy Induction by Cyclic Stretch and Its Effect on Cytokines in Retinal Pigment Epithelial Cells

Background: Strong evidence of the correlation between age-related macular degeneration (AMD) and vitreomacular interface abnormality (VMIA). Meanwhile, as a crucial mechanism of retinal pigment epithelial (RPE) cells’ homeostasis, autophagy induction by cyclic stretch appears to be particularly significant.Methods: Cultured ARPE-19 cells were subjected to cyclic stretch (20% elongation, 1HZ) for 1h, 2h, 6h, 12h,24h and 48h by FX-5000 Tension System. Then, we observed the expression levels of LC3I, LC3II, Beclin-1, SQSTM1/p62, LAMP-1, mTOR and phosphorylated mTOR(pmTOR), AMPK and pAMPK, NADPH oxidase 4 (NOX4), and vascular endothelial growth factor (VEGF) in RPE cells under stretch by western blot and immunofluorescence.Results: We found autophagic proteins mostly induced by cyclic stretch in a time-dependent fashion via mTOR suppression and AMPK activation, except for SQSTM1/p62. 3-Methyladenine(3-MA), an inhibitor for autophagy, could reduce the up-regulation of autophagy due to cyclic stretch, leading to higher level of VEGF release after 24h cyclic stretch. Rapamycin could narrow the increase degree of VEGF and NOX4 by cyclic stretch by raise autophagic level in RPE cells.Conclusion: Stretch might induce autophagy in RPE cells by mTOR or AMPK pathway. Autophagy might play the protective function for RPE cells away from mechanical stress derived from VMIA-related AMD.

3,3’-Diindolylmethane induces apoptosis and autophagy in fission yeast

3,3’-Diindolylmethane (DIM) is a compound derived from the digestion of indole-3-carbinol, found in the broccoli family. It induces apoptosis and autophagy in some types of human cancer. DIM extends lifespan in the fission yeast Schizosaccharomyces pombe. The mechanisms by which DIM induces apoptosis and autophagy in humans and expands lifespan in fission yeasts are not fully understood. Here, we show that DIM induces apoptosis and autophagy in log-phase cells, which is dose-dependent in fission yeast. A high concentration of DIM disrupted the nuclear envelope (NE) structure and induced chromosome condensation at an early time point. In contrast, a low concentration of DIM induced autophagy but did not disrupt NE structure. The mutant defective in autophagy was more sensitive to a low concentration of DIM, demonstrating that the autophagic pathway contributes to the survival of cells against DIM. Moreover, our results showed that the lem2 mutant is more sensitive to DIM. NE in the lem2 mutant was disrupted even at the low concentration of DIM. Our results demonstrate that the autophagic pathway and NE integrity are important to maintain viability in the presence of a low concentration of DIM. The mechanism of apoptosis and autophagy induction by DIM might be conserved in fission yeast and humans. Further studies will contribute to the understanding of the mechanism of apoptosis and autophagy by DIM in fission yeast and humans.

Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells

Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

Vinexin contributes to autophagic decline in brain ageing across species

Autophagic decline is considered a hallmark of ageing. The activity of this intracytoplasmic degradation pathway decreases with age in many tissues and autophagy induction ameliorates ageing in many organisms, including mice. Autophagy is a critical protective pathway in neurons and ageing is the primary risk factor for common neurodegenerative diseases. Here, we describe that autophagosome biogenesis declines with age in mouse brains and that this correlates with increased expression of the SORBS3 gene (encoding vinexin) in older mouse and human brain tissue. We characterise vinexin as a negative regulator of autophagy. SORBS3 knockdown increases F-actin structures, which compete with YAP/TAZ for binding to their negative regulators, angiomotins, in the cytosol. This promotes YAP/TAZ translocation into the nucleus, thereby increasing YAP/TAZ transcriptional activity and autophagy. Our data therefore suggest brain autophagy decreases with age in mammals and that this is likely, in part, mediated by increasing levels of vinexin.