Basal Autophagy Is Required for Herpes simplex Virus-2 Infection

Autophagy is a conserved catabolic process of the cell, which plays an important role in regulating plethora of infections. The role of autophagy in Herpes simplex virus-2 (HSV-2) infection is unknown. Here, we found that HSV-2 does not allow induction of an autophagic response to infection, but maintains basal autophagy levels mostly unchanged during productive infection. Thus, we investigated the importance of basal autophagy for HSV-2 infection, using pharmacological autophagy suppression or cells genetically deficient in an autophagy-essential gene (ATG5). Interference with basal autophagy flux in cells significantly reduced viral replication and diminished the infection. These results indicate that basal autophagy plays an indispensable role required for a productive infection. Importantly, this study draws a sharp distinction between induced and basal autophagy, where the former acts as a viral clearance mechanism abrogating infection, while the latter supports infection

Characterization of changes in the proteome in different regions of 3D multicell tumor spheroids.

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required

mTORC1-independent autophagy regulates receptor tyrosine kinase phosphorylation in colorectal cancer cells via an mTORC2-mediated mechanism

The intracellular autophagic degradative pathway can have a tumour suppressive or tumour-promoting role depending on the stage of tumour development. Upon starvation or targeting of oncogenic receptor tyrosine kinases (RTKs), autophagy is activated owing to the inhibition of PI3K/AKT/mTORC1 signalling pathway and promotes survival, suggesting that autophagy is a relevant therapeutic target in these settings. However, the role of autophagy in cancer cells where the PI3K/AKT/mTORC1 pathway is constitutively active remains partially understood. Here we report a role for mTORC1-independent basal autophagy in regulation of RTK activation and cell migration in colorectal cancer (CRC) cells. PI3K and RAS-mutant CRC cells display basal autophagy levels despite constitutive mTORC1 signalling, but fail to increase autophagic flux upon RTK inhibition. Inhibition of basal autophagy via knockdown of ATG7 or ATG5 leads to decreased phosphorylation of several RTKs, in particular c-MET. Internalised c-MET colocalised with LAMP1-negative, LC3-positive vesicles. Finally, autophagy regulates c-MET phosphorylation via an mTORC2-dependent mechanism. Overall, our findings reveal a previously unappreciated role of autophagy and mTORC2 in regulation of oncogenic RTK activation, with implications for understanding of cancer cell signalling.Cell Death and Differentiation advance online publication, 5 May 2017; doi:10.1038/cdd.2017.41