Fig. 5
From: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer
PPARγ/RXRαS427F confers partial resistance to immunotherapies. a RT-qPCR analysis of chemokines/cytokines in T24 lines engineered to overexpress RXRA-WT, RXRA-S427F, RXRA-S427Y (upper), and PPARG (lower). Controls are RXRA-WT for RXRA mutant lines and vector control (Vec) for PPARG overexpressing line. Expression normalized to GAPDH and data presented as mean fold change vs. control ± SEM of three biological replicates. b Chemokine array analysis of conditioned media collected from T24 lines engineered to overexpress PPARG (PPARγ) vs. control (Vec). Dotted boxes represent controls. Significant changes in secretion are outlined. One representative of three independent experiments is shown. c FACS based quantitation of infiltrating CD3 + CD8 + double positive T cells into subcutaneously implanted MBT2 tumors overexpressing RXRA-WT (n = 6) or RXRA-S427F (n = 6). Data presented as percent of total tumor-derived cells following dissociation. d Left, individual MBT2-RXRαWT tumor volumes in response to PBS (red, n = 12) or anti-CTLA4 (blue, n = 12). P = 0.0189 at day 7. Right, individual MBT2-RXRαS427F tumor volumes in response to PBS (red, n = 12) or anti-CTLA4 (blue, n = 12). P > 0.05 at day 7. One-way ANOVA followed by Tukey’s post-hoc test performed. e Heatmap presenting pathway level analysis (activation, red; suppression, blue) of differentially expressed genes in PPARγ knockdown lines (PPARγ-sh#4 and -sh#9 engineered in SV-HUC-1 line expressing RXRA-S427Y) relative to vector control. The analysis was based on three biological replicates. f Left, Knockdown of PPARγ or RXRα by shRNAs in HT-1197 cells. GAPDH was used as the control. Right, RT-qPCR analysis of inflammatory genes CCL2 and CXCL10 following inducible knockdown of PPARγ and RXRα in HT-1197 cells. Data normalized to GAPDH and presented as mean fold change (Dox treated vs. untreated) ± SEM of three biological replicates. g RT-qPCR analysis of IL8 and CCL2 following treatment with PPARγ agonist rosiglitazone (Rosi) or PPARγ antagonist T0070907 in 5637 cells. Data normalized to GAPDH and presented as mean fold change ± SEM of three biological replicates. h Schematic representation of the role of tumor-intrinsic PPARγ/RXRαS427F/Y in transcriptional regulation and immunosurveillance. CoA, co-activator complex; CoR, co-repressor complex; ITF, inflammation-related transcription factors
