mTOR and HDAC Inhibitors Converge on the TXNIP/Thioredoxin Pathway to Cause Catastrophic Oxidative Stress and Regression of RAS-Driven Tumors - PubMed (original) (raw)

Figure 3. HDAC and mTOR Inhibitors function by converging on the Thioredoxin Interacting Protein (TXNIP) and activating Apoptosis Signal-regulating kinase 1 (ASK1)

A) Microarray analysis of 90-8TLs after 24 hours of treatment with vehicle, sapanisertib (100nM), vorinostat (2μM), or both sapanisertib and vorinostat. Heatmap depicts the uniquely upregulated genes (dark blue) or downregulated genes (light blue) from cells treated with sapanisertib plus vorinostat, as compared with all other treatment groups, with a_p_ < 0.001 and a fold change of 1.5 or more. TXNIP is highlighted in red, as a gene of particular interest within this signature. B). Immunoblot depicts protein levels of TXNIP, phosphorylated S6 and Acetylated H3K9 after 16 hours of treatment with sapanisertib (200nM), vorinostat (2μM), or sapanisertib and vorinostat in S462s. Actin serves as a control. Below, numbers indicate relative TXNIP protein levels quantified and normalized to actin levels, with vehicle levels set to 1. C) 90-8TLs were treated with sapanisertib (100nM), vorinostat (2μM), or sapanisertib and vorinostat and analyzed as in B. D) S462s were treated with vehicle, sapinsertib (200nM), panobinostat (20nM), or sapinsertib + panobinostat and analyzed as in B. E) S462s were infected with lentiCRISPRv2 expressing guides against LACZ or TXNIP as indicated. Immunoblot depicts TXNIP, phosphorylated S6 (pS6), and acetylated histone H3 at lysine 9 (Ac. H3K9) protein levels after 16 hours of treatment as in B and D. Actin and S6 serve as controls. F) As in E, S462s were infected with lentiCRISPRv2 expressing guides against LACZ or TXNIP as indicated and treated with sapanisertib (200nM) and vorinostat (2μM). Left y-axis indicates the log2 of fold change in cell number after 3 days. The Log2 values on the left axis have been converted to the actual percent increase or decrease in cell number after 72 hours to best appreciate relative changes, shown on the right y-axis. Error bars indicate SD of technical triplicates.*_p_=0.000007, **p<0.000001 G) S462s with indicated CRISPR guides were treated with vehicle or sapinsertib (200nM) + panobinostat (20nM), and analyzed as in E. *_p_=0.000004, **_p_=0.000001 H) 90-8TLs were treated with vehicle, sapinsertib (100nM), auranofin (750nM) or auranofin + sapinsertib. Left y-axis indicates the log2 of fold change in cell number after 3 days. The Log2 values on the left axis have been converted to the actual percent increase or decrease in cell number after 72 hours to best appreciate relative changes, shown on the right y-axis. Error bars indicate SD of technical triplicates. *_p_=0.000004, **p<0.000001 I) Immunoblot depicting phosphorylated P38, TXNIP, acetylated histone H3K9, and phosphorylated S6 levels in S462s after 16 hours of treatment with sapanisertib (200nM) and panobinostat (20nM). P38 serves as a control J) Immunoblot depicting phosphorylated P38, TXNIP, acetylated histone H3K9, and phosphorylated S6 levels in 90-8TLs after 16 hours of treatment with sapanisertib (100nM) and vorinostat (2μM). P38 serves as a control K) 90-8TLs (left) and S462s (right) were transfected with pooled siRNAs targeting MAP3K5 (ASK1) or non-targeting (CTRL) and treated with vehicle, sapanisertib (100nM 90-8TL, 200nM S462), vorinostat (2μM), or sapanisertib and vorinostat. Left y-axis indicates the log2 of fold change in cell number after 3 days. The Log2 values on the left axis have been converted to the actual percent increase or decrease in cell number after 72 hours to best appreciate relative changes, shown on the right y-axis. Error bars indicate SD of technical triplicates. *_p_=0.003935, **p<0.000001 L) 90-8TLs were transfected with pooled siRNAs targeting SLC2A1 (GLUT1) or non-targeting (CTRL), and treated with vehicle, sapanisertib (100nM), vorinostat (2μM), or sapanisertib and vorinostat. Left y-axis indicates the log2 of fold change in cell number after 3 days. The Log2 values on the left axis have been converted to the actual percent increase or decrease in cell number after 72 hours to best appreciate relative changes, shown on the right y-axis. Error bars indicate SD of technical triplicates.