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 4

AA leads to increased TET activity and 5hmC levels in ccRCC cells.

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AA leads to increased TET activity and 5hmC levels in ccRCC cells. (A) S...
(A) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. (B) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 (n = 2). (C and D) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid (n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. 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 0.025 (0.05/2 to account for multiple comparisons). (E) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. 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). (F) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. (G) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. (H) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.