Prolonged Glutamine Starvation Reactivates mTORfor Autophagiclysosome Reformation to Maintain Cell Survival in Oral Cancer

Abstract

Autophagy, a cellular recycling mechanism, requires lysosomes as primary degradative compartments and maintaining lysosomal homeostasis during prolonged stress is vital for sustaining continuous autophagy. Lysosomal repopulation can be achieved through de novo biosynthesis, but autophagic lysosome reformation (ALR) can also replenish the lysosomal pool. During ALR, the cell undertakes significant autolysosomal membrane remodelling and scission to form membrane fragments, which mature into active lysosomes. Glutamine-deprived oral cancer cells orchestrate a fluctuation in autophagy due to inhibiting the mammalian target of rapamycin (mTOR) during autophagy induction but subsequently reactivate due to prolonged starvation. Further, time kinetic studies indicate the inhibition in autophagy flux and lysosomal pool in the initial phase but restored later during glutamine starvation. This recovery is due to pro-lysosome formation from the autolysosome membrane through the extending reformation tubules. In glutamine-starved oral cancer cells, mTOR is the central modulator in regulating tubule formation. Upon pharmacological inhibition with rapamycin, it led to ALR inhibition, as evidenced by failure in tubule elongation from autolysosomes. Moreover, RAB7 and clathrin are essential to induce ALR, and it showed that siRNA targeting RAB7 and clathrin inhibits tubule formation in glutamine-starved oral cancer cells. In this setting, we have examined the physiological relevance of ALR during prolonged glutamine deprivation, and it was found that genetic and pharmacological inhibition of critical proteins involved in ALR promotes cell death in oral cancer cells, establishing ALR is essential for oral cancer survival during cellular stress.