Epigenetic loss of AOX1 expression via EZH2 leads to metabolic deregulations and promotes bladder cancer progression

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Advanced Bladder Cancer (BLCA) remains a clinical challenge that lacks effective therapeutic measures. Here, we show that distinct, stage-wise metabolic alterations in BLCA are associated with the loss of function of aldehyde oxidase (AOX1). AOX1 associated metabolites have a high predictive value for advanced BLCA and our findings demonstrate that AOX1 is epigenetically silenced during BLCA progression by the methyltransferase activity of EZH2. Knockdown (KD) of AOX1 in normal bladder epithelial cells re-wires the tryptophan-kynurenine pathway resulting in elevated NADP levels which may increase metabolic flux through the pentose phosphate (PPP) pathway, enabling increased nucleotide synthesis, and promoting cell invasion. Inhibition of NADP synthesis rescues the metabolic effects of AOX1 KD. Ectopic AOX1 expression decreases NADP production, PPP flux and nucleotide synthesis, while decreasing invasion in cell line models and suppressing growth in tumor xenografts. Further gain and loss of AOX1 confirm the EZH2-dependent activation, metabolic deregulation, and tumor growth in BLCA. Our findings highlight the therapeutic potential of AOX1 and provide a basis for the development of prognostic markers for advanced BLCA.

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This research was fully supported by American Cancer Society (ACS) Award 127430-RSG-15-105-01-CNE (N.P.), NIH/NCI R01CA220297 (N.P), and NIH/NCI R01CA216426 (N.P.), partially supported by the following grants: NIH 1RO1CA133458-01 (A.S.K.), and NIH U01 CA167234, Komen CCR award to S.M.K. (CCR16380599) as well as funds from Alkek Center for Molecular Discovery (A.S.K.). This project was also supported by the Agilent Technologies Center of Excellence in Mass Spectrometry at Baylor College of Medicine, Metabolomics Core, Human Tissue Acquisition and Pathology at Baylor College of Medicine with funding from the NIH (P30 CA125123), CPRIT Proteomics and Metabolomics Core Facility (D.P.E.), (RP170005), and Dan L. Duncan Cancer Center. Imaging for this project was supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from NIH (DK56338, CA125123, and 1S10OD020151-01), CPRIT (RP150578), the Dan L. Duncan Comprehensive Cancer Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics. CAM assay was supported by the Patient-Derived Xenograft and Advanced in vivo Models Core Facility at Baylor College of Medicine with funding from the Cancer Prevention and Research Institute of Texas (CPRIT) grant #170691. Imaging for this project was supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from NIH (DK56338, and CA125123), CPRIT (RP150578, RP170719), the Dan L. Duncan Comprehensive Cancer Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics. Research reported in this study was supported by the National Cancer Institute of the National Institutes of Health under award number 5 P30 CA142543 09.

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V.V., V.P., A.S. and N.P., conceived the project and wrote the manuscript with editorial input from all of the authors. V.V., V.P., D.A.B., S.M.K., Q.C. and N.P. designed the experiments. V.V., V.P., D.A.B., V.D., S.M.K., S.R.D., J.D., A.K.J., F.S. and B.K. performed the experiments. S.M., J.M., J.A., K.R. and C.C. assisted with the dataset analysis. A.S. and N.P. assisted with mass spectroscopy measurements and functional study. F.V.R. and Y.L. provided clinical specimens. F.V.R. and Y.L. provided clinical input on data interpretation. A.G.S. and H.V. performed the OVIVO experiments.

Correspondence to Nagireddy Putluri.

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S.M.K. is stake holder of NeoZenome Therapeutics Inc.

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