Rag GTPase but not Rab1A, Rab5, Arf1 was required for persistent mTOR activity in senescence-like hepatoma cells
mTOR was persistently active and insensitive to short-term amino acid (AA) starvation in senescent primary human fibroblasts , but and mechanisms remain unclear. We aimed to characterize the roles of small GTPase activators of mTOR including Rag, Rab1A, Rab5, and Arf1 in senescence-like hepatoma cell line HepG2. We first applied x-ray radiation to HepG2 cells to obtain senescence-like cells. After 2 weeks post-radiation, cells are arrested and showed significant β-galactosidase activity, as determined by flow cytometry using Click-iT EdU indicator and CellEvent Senescence Green probe, respectively (Figure 1A and 1B). Second, we examined mTOR activity in response to AA starvation by Western blotting of S6 kinase phosphorylation (S6K-T389). The results showed that in senescence-like HepG2 cells mTOR activity was much higher than proliferating cells after 15, 30, and 60 min of AA starvation (Figure 1C and 1D). Third, we knocked down Rag, Rab1A, Rab5, and Arf1 through specific siRNAs in senescence-like and proliferating HepG2 cells. All GTPase knockdowns reduced mTOR activity in proliferating cells. Interestingly however, in the senescence-like HepG2 cells, only RagC siRNA significantly decreased S6K-T389 levels (Figure 1E-H), suggesting that Rag GTPase was specifically required for mTOR activity in senescence-like state.
Knocking down Rag but not Rab1A, Rab5, Arf1 increased sensitivity of senescence-like hepatoma cells to MEK inhibitors
Persistent mTOR activity contributes to chemotherapy resistance, including MEK inhibitors. Since our findings showed a specific requirement of Rag but not other GTPases in senescence-like cancer cells, we asked if Rag could be also specifically required for drug resistance. To do this, we treated senescence-like HepG2 cells and proliferating controls with 10 nM trametinib, a well-known MEK inhibitor for 3 hours. We then examined apoptosis and cell death through staining cells with Annexin V and propidium iodide (PI), respectively, followed by flow cytometry analysis. Consistently with previous studies, senescence-like cells were much resistant than proliferating cells to the MEK inhibitor. With the siRNA treatments, only RagC knockdown significantly increased the sensitivity to the MEK inhibitor in senescence-like HepG2 cells. Long term survival was examined by CellTiter-glo after treating cells with 10 nM trametinib for 3 days. Consistently, only RagC knockdown significantly reduced the survival in senescence-like HepG2 cells. Therefore, we concluded that Rag but not the other GTPases were required for drug resistance phenotype in senescence-like HepG2 cells.
Inhibiting of lysosomal activity reduced mTOR activity and sensitized senescence-like hepatoma cells to MEK inhibitor.
In response to nutrient limitation, cells activate autophagy to engulf intracellular materials in autophagosome, which is then digested in lysosome to generate free AAs and lipids for cell maintenance. Rag complex is localized to the lysosome and play essential roles in amino acid sensing and mTOR activation. The specific requirement of Rag GTPase in senescent-like cells suggested that hyperactive mTOR and drug resistance could be due to increased lysosomal activity. To test this possibility, we first examined if autophagic and lysosomal activity was increased in senescence-like HepG2 cells. By incubating cells with a lysosome-specific self-quenching substrate (which will emit fluorescence when degraded by lysosomal proteases), our flow cytometry experiments showed that lysosomal activity was robustly increased upon senescence induction (Figure 3A and 3B). Second, we inhibited autophagic and lysosomal activity by hydroxychloroquine (HCQ) and Bafilomycin-A1 (BAF), two well-known autophagy-lysosome inhibitors. Both drugs successfully reduced lysosomal activity as revealed by flow cytometry and accumulation of autophagic marker LC3 proteins (Figure 3A-D). Indeed, for both inhibitors, 1-hour treatment reduced mTOR activity in senescence-like HepG2 cells but had only slight effect on proliferating counterparts (Figure 3E). Consistently, these autophagy-lysosome inhibitors sensitized senescence-like HepG2 cells to MEK inhibitors, but have only slight effect on proliferating HepG2 cells (Figure 3F). Together, these results suggested that senescence-like cancer cells had increased dependency on autophagy-lysosome for their nutrient supply and cellular maintenance.
Elevated lysosomal activity increased Rag-mediated mTOR activation
Another situation relying on autophagy-lysosome for nutrient supply is starvation. We reasoned if the specific dependency of mTOR on Rag GTPase in senescence-like HepG2 cells was due to autophagic and lysosomal recycling of intracellular components, activating lysosomal activity in proliferating cells should increase the dependency of mTOR on Rag GTPase. To test this, we applied a partial AA starvation strategy, allowing for examination of S6K-T389 that was difficult to detect in full starvation. By decreasing the AAs to 5% of normal concentration in the medium for 18 hours, we found a significant increase in lysosomal activity in HepG2 cells compared to those cultured in full AA (Figure 4A-B). We knocked down RagC, Rab1A, Rab5, and Arf1 by siRNA and examined S6K-T389 levels by Western blot. All siRNA knockdowns fully inhibited S6K-T389 phosphorylation in normally cultured HepG2 cells (Figure 4C-D). Under partial starvation however, only RagC siRNA robustly decreased S6K-T389 levels; other siRNAs showed much reduced effect. Therefore, the results confirmed that cells with elevated autophagic and lysosomal activity increased the dependency on Rag GTPase for mTOR activation, lending further supports to the specific functions of Rag GTPase in promoting mTOR activity and drug resistance in senescence-like HepG2 cancer cells.
RagC was stronger than Rab1A, Rab5 or Arf1 GTPases as a prognostic predictor for unfavorable outcome in LIHC patients
We investigated into the clinical data regarding the association of the GTPases with liver cancer and overall survival of LIHC patients. RNAseq and survival data from NIH TGCA database were analyzed by using GEPIA, a recently developed web-based bioinformatics tool . By examining the differential expression of RagC, Rab1A, Rab5, and Arf1 in cancer tissues (n = 369) and non-cancer controls (n = 160), we found that RagC and Arf1 but not Rab1A or Rab5 were significantly elevated in cancer tissues (Figure 4E). The LIHC patients had gone through different treatments including chemotherapy and radiotherapy, which induced senescence-like cancer cells. If our discovery that RagC but not other mTOR regulating GTPases was specifically important for maintaining the survival and drug resistance of the senescence-like HepG2 cells, then higher levels of RagC but not other GTPases should predict poor survival. Indeed, by plotting the Kaplan Meier curve for LIHC patients with the 25% highest and 25% lowest RagC expression, we showed that RagC-high patients had significantly worse overall survival (Figure 4F). The hazard ratio was 2.8, indicating that the RagC-high patients were 2.8 times more likely to die than RagC-low patients. By similar standards, higher expression of Rab1A, Rab5, and Arf1 did not show significant (P<0.01) different survival than lower expressing controls.