Autophagy In Cell Contact Inhibtion

Autophagy is an intracellular catabolic degradation process in which cytoplasmic contents like misfolded proteins and damaged organelles are engulfed by double-membrane autophagosomes and degraded in lysosomes.This process is an important cellular stress response which helps maintain the homeostasis of cells.Autophagy dysfunction disturbs cell growth and metabolism which lead to the occurrence of many diseases as diabetes,neurodegeneration and cancer.Accumulating evidence shows that autophagy can eliminate p62,damaged mitochondria,protein aggregates and ROS to suppress tumorigenesis.However,autophagy is hijacked by some tumors to serve as a survival mechanism that plays a vital role in tumor cell growth.The loss of contact inhibition is a primary character of many cancers.Hippo-YAP pathway plays an important role in the regulation of contact inhibition and tumorigenesis.In high cell density,cells contact with each other and activate Hippo pathway which lead to YAP degradation and cell growth inhibition.In this study,we found that,in high cell density,WT MEFs stopped growing and cultured as a single layer,but Atg5-/-MEFs clearly grew on top of each other and piled up suggesting a function of autophagy in cell contact inhibition.To further exclude the possibility of the cell specificity in the contact inhibition,two autophagy deficient cell lines,ATG5-/-and ATG7-/-HEK293s were constructed by CRISPR-Cas9 system.We found that ATG5-/-and ATG7-/-HEK293s were also piled up in high density.Autophagy inhibited by Wortmannin or CQ induces a significant accumulation of cell clones in multilayer.These results demonstrate autophagy deficiency suppresses cell contact inhibition.Further mechanism analysis indicated that,in Atg5-/-and Atg7-/-cells,YAP nuclear localization rate is increased and the protein level of YAP was dramatically up-regulated with no change in its mRNA level.This data suggests YAP can be degraded through autophagy-lysosome pathway.Consistently,we found that inhibition of autophagic degradation by CQ induces dose and time-dependently accumulating of YAP.And a significant colocalization of YAP and autophagosome marker LC3 were observed by immune fluorescent test.Conclusion:in this study,our results revealed that autophagy dysfunction can promote YAP activity which leading the normal cells to lose contact inhibition and get transforming properties.This may be a new mechanism involved in the regulation of tumorigenesis by autophagy.