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 5

Fluorescence quenching of recombinant TET-2 protein is unaffected by L2HG in the presence of AA.

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Fluorescence quenching of recombinant TET-2 protein is unaffected by L2H...
(A) Fluorescence spectra of 0.5 μM TET-2 are shown after excitation at 280 nm with increasing amounts of AA (from top to bottom). (B) The relative fluorescence intensity at 328 nm is shown as a function of AA. A concentration of 132 μM AA reduces the fluorescence signal by 90 %. (C) Quenching effect on 0.5 μM TET-2 for 2OG (black circles), L2HG (white circles), and L2HG in the presence of 23 μM 2OG (triangles). Quenching efficiency of 2OG is higher than that of L2HG (P < 0.001). L2HG quenching is less efficient with 2OG than without (paired t test, P = 0.002). The substrate specificity of TET-2 is 2OG over L2HG. (D) The fluorescence quenching effect of AA on 0.5 μM TET-2 is shown. Symbols are as follows: black circles, TET-2; white squares, TET-2 in the presence of 100 μM L2HG; triangles, TET-2 in the presence of 23 μM 2OG; and inverted triangles, quenching of TET-2 in the presence of 23 μM L2HG and 23 μM 2OG. The overlapping curves indicate that TET-2 is unaffected by L2HG in the presence of AA. (E) Comparison of the Stern-Volmer constants ± SEM obtained from the linear range of the data in parts C and D. P values are as indicated in the figure (paired t test). In the equation y = m × x + c, m indicates the Stern-Volmer constant. The (y, x) points are obtained from C and D. For the Stern-Volmer constants derived from C, 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 less than 0.017 (0.05/3 to account for multiple comparisons).