Roles of mTOR-regulating GTPases in regulating drug resistance in senescence-like hepatoma cells

Radiotherapy and chemotherapy can arrest cancer cells in a senescence-like state, which can lead to therapy resistance and cancer relapse. Despite the cell cycle arrest, senescence-like cells have persistent mTOR activity that is insensitive to nutrient starvation. The mechanisms and functions of mTOR activation in senescence-like cells remains unclear. mTOR is regulated by several small GTPases including the lysosome-localized Rag complex, ER-Golgi-localized Arf1 and Rab1, and endosome-localized Rab5. In this study, we knocked down these GTPases in both proliferating and senescence-like HepG2 cells induced by X-ray radiation. We then compared mTOR activity and drug resistance to MEK inhibitors. We also examined the roles of autophagy and lysosomal activity in mTOR activation. In addition, by analyzing the Cancer Genome Atlas (TCGA) database, we studied the relationship between the expression levels of these GTPases and the survival of liver hepatoma carcinoma (LIHC) patients. Our results showed that although all GTPases were required for optimal mTOR activation in proliferating HepG2 cells, only Rag is required in senescent-like counterparts. Consistently, the drug resistance of senescent-like cells can be reduced by knocking down of Rag but not other GTPases. Autophagic and lysosomal activity were increased in senescent cells; pharmacological inhibition of autophagy-lysosome decreased mTOR activity and preferentially sensitized the senescence-like HepG2 cells to MEK inhibitors. Therefore, recycling of intracellular materials could be a key mechanism to maintain mTOR activity and promote drug resistance in senescence-like state. In LIHC patients, expression of Rag but not Rab5 or Arf1 was associated with unfavorable prognosis. Our study therefore has defined a key role of Rag GTPase in mediating mTOR activation and drug resistance in senescent-like HepG2 cells, which could have important implications in developing second-line treatments for liver cancer.