Mechanistic Analysis Of Treatments With Combination Of Autophagy And MTOR Or Pentose Phosphate Pathway Inhibitors In Renal Cell Carcinoma

PartⅠMechanistic Analysis of the Dual Inhibition of Autophagy and mTOR Activity in Renal Cell CarcinomaAim To investigate the autophagy level and mTOR activity in RCC cells and corresponding mechanism of dual inhibition in tumour cell growth.Methods RCC4, ACHN,7860, and 769P clear cell RCC cell lines and HK2 normal renal tubule cells were acquired from ATCC cell bank and were detected for basal autophagy level and mTOR activity. Inhibitory effects on mTOR and autophagic pathways were compared between mono-therapies with mTOR or autophagy inhibition alone and combinations. Crystal violet assays were used to profile cell proliferation and apoptosis was studied between treatment groups.Results Compared with HK2 cells, the mTOR activity was obviously elevated in all RCC cell lines. The basal autophagy level of 7860 and 769P cells were slightly elevated compared to HK2 cells while basal autophagy level of RCC4 and ACHN cells were substantially increased with high mTOR activity. Dual inhibition did not further enhance the autophagy level of such cells indicating partially independent mediation of autophagy by mTOR. Rapamycin inhibits proliferation of 786O and RCC4 cells with synergistic effects in combination with genetic or pharmaceutical inhibition of autophagy. Dual inhibition induced apoptosis in 7860 cells and possibly necroptosis in RCC4 cells.Conclusion Elevated mTOR activity and basal autophagy level existed in all RCC cell lines, in which some cells exhibited both high mTOR and autophagic level. Mono-therapy with either mTOR or autophagy inhibitors could suppress cell proliferation whilst combination synergistically inhibited cells with low basal autophagy level (7860) and lost synergism in cells with high basal autophagy level (RCC4). Dual inhibition induced apoptosis in 786O cells and possibly induced necroptosis in RCC4 cells.Part ⅡMechanistic Analysis of the Dual Inhibition of Autophagy and Pentose Phosphate Pathway in Renal Cell CarcinomaAim To investigate the metabolic profile of RCC cells with high basal autophagy level (RCC4) after autophagy inhibition and the therapeutic effects of combination of dual inhibition of autophagy and targeted metabolic inhibition.Methods The metabolic profiling was performed in RCC4 cells treated and untreated with Chloroquine (CQ) and the Metabolite Set Enrichment Analysis (MSEA) was applied to reveal major changes in metabolic pathways. In vitro assays were performed to test the combination of CQ and metabolic inhibitor in RCC4 cells. The ROS stress and nutrient consumption was studied to validate the metabolic changes. The pNFxB level was studied to discuss the possible mechanism of dual inhibition in RCC4 cells.Results The metabolic status of RCC4 cells changed drastically after inhibition of autophagy in which the pentose phosphate pathway (PPP) changed the most. The metabolites and activity of PPP were substantially increased following autophagy inhibition. Combination of CQ and the PPP inhibitor 6-Aminonicotinamide (6AN) showed synergistic inhibitory effects on the proliferation of RCC4 cells. The uptake of glucose was increased with unchanged release of lactate in RCC4 cells further validated specific activation of PPP following CQ treatment. Combination treatment also increased pNFxB level indicating that inflammasome could paly a role in the inhibition of RCC4 cells.Conclusion RCC4 cells had hyperactive mTOR and basal autophagy level that render RCC4 relatively insensitive to combination of mTOR and autophagy inhibition. Metabolic profiling after CQ treatment revealed PPP activation and combination of 6AN and CQ synergistically inhibited proliferation of RCC4 cells possibly via induction of inflammasome.Part ⅢExpressions and Prognostic Contributions of Autophagy and PPP Related Genes in Renal Cell CarcinomaAim To investigate the expressions and prognostic contributions of autophagy and PPP related genes in renal cell carcinoma.Methods We studied the expressions of autophagy and PPP related genes in silico by analysing the TCGA (The Cancer Genome Atlas) dataset. We investigated all samples with complete RNA-seq data and analyzed with the MSKCC (Memorial Sloan-Kettering Cancer Center) online platform, cBioPortal for Cancer Genomics. We chose Provisional for clear cell renal cell carcinoma and studied expressions of autophagy related genes (ULK1, ATG5, BECN1, ATG7, ATG12, ATG13, MAP1LC3B, MAP1LC3C), and PPP related genes (G6PD, PGLS, PGD, RPIA, RPE, TALDO1, TKT). The expressions, relations between gene expression and prognosis, and crosstalks between genes were plotted.Results The cBioPortal included 469 complete samples in TCGA dataset. Prognosis was significantly poorer when expressions of autophagy related genes (ULK1, ATG7, MAP1LC3C) were altered. Prognosis was significantly poorer when expressions of PPP related genes (G6PD, PGLS, PGD, RPIA, TALDO1, TKT) were altered. The gene sets of autophagy and PPP were significantly associated with prognosis in renal clear cell carcinoma. There were extensive crosstalks between autophagy and PPP related genes.Conclusion Expressions of autophagy and PPP related genes were closely associated with prognosis of clear cell renal cell carcinoma. There was extensive network between autophagy and PPP gene sets indicating critical roles for autophagy and PPP in renal cell carcinoma.