Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma
Niraj Shenoy, … , Yiyu Zou, Amit Verma
Niraj Shenoy, … , Yiyu Zou, Amit Verma
Published January 31, 2019
Citation Information: J Clin Invest. 2019;129(4):1612-1625. https://doi.org/10.1172/JCI98747.
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Research Article Oncology

Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma

Abstract

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

Authors

Niraj Shenoy, Tushar D. Bhagat, John Cheville, Christine Lohse, Sanchari Bhattacharyya, Alexander Tischer, Venkata Machha, Shanisha Gordon-Mitchell, Gaurav Choudhary, Li-Fan Wong, LouAnn Gross, Emily Ressigue, Bradley Leibovich, Stephen A. Boorjian, Ulrich Steidl, Xiaosheng Wu, Kith Pradhan, Benjamin Gartrell, Beamon Agarwal, Lance Pagliaro, Masako Suzuki, John M. Greally, Dinesh Rakheja, R. Houston Thompson, Katalin Susztak, Thomas Witzig, Yiyu Zou, Amit Verma

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Figure 6

High-dose AA treatment leads to inhibition of ccRCC growth in vitro via non–free radical mechanisms.

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High-dose AA treatment leads to inhibition of ccRCC growth in vitro via ...
(A) ccRCC (769-P) cells were treated with AA for 4 hours with and without catalase treatment (to abrogate H2O2), followed by incubation in fresh media for 24 hours. The acute cytotoxicity of AA was reversed by catalase treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid (n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). Results were similar with 786-O (Supplemental Figure 5). (B and C) ccRCC cells were treated with AA and catalase and followed for longer time points. AA led to dose-dependent and progressive loss of viability despite catalase control, suggesting non–free radical mechanism of proliferation inhibition. t test. Data are shown as mean ± SEM (n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed at each time point via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). *P < 0.05. (D–F) ccRCC cells (786-O) were treated with catalase and AA (5 mM) and assessed for apoptosis by FACS. AA treatment led to significant increase in apoptosis after 48 hours of treatment. AA exposure for D1 (24 hours) and D3 (48–72 hours) with apoptosis assay at 96 hours. Representative flow figures are shown. n = 2. Data are shown as means with individual data points overlaid in D. (G–I) ccRCC cells (786-O) were treated with catalase and AA (5 mM) and assessed for cell cycle by FACS. AA treatment led to significant increase in G0/G1 arrest after 48 hours of treatment. AA exposure for D1 (24 hours) and D3 (48–72 hours) with apoptosis assay at 96 hours. Representative flow figures are shown. n = 2. Data are shown as means with individual data points overlaid in G.