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 April 1, 2019; First 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 3

L2HGDH deletions and underexpression are seen in ccRCC and are significantly associated with adverse prognosis.

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L2HGDH deletions and underexpression are seen in ccRCC and are significa...
(A) Analysis of TCGA data set revealed that TET2 is mutated (heterozygous) only in 2.2% of ccRCC tumors. The mutations seen are predominantly missense (green) or nonsense (black) and are predicted to result in truncated proteins. (B) TCGA analysis revealed no significant difference in TET2 expression between ccRCC and normal kidney. (C) TET2 IHC revealed no difference in expression patterns between high-grade and low-grade ccRCC. Parts A, B and C, taken together, indicate that neither the mutation rate nor the expression of TET2 explains the loss of 5hmC in higher grade ccRCC. (D) L2HGDH gene-expression comparison between matched normal kidney and ccRCC tumor from TCGA (n = 72). The mean normalized log2 expression for the normal group is 9.28 and that of the tumor group is 7.75 (t test, P < 0.001). (E) Copy number variation (CNV) data for 528 patients (TCGA) with ccRCC are as follows: deletion, 217 (41%); no copy number variation, 295 (56%); amplification, 16 (3%). (F) TCGA analysis of methylation status of ccRCC tumors based on low L2HGDH and high L2HGDH. (G) L2HGDH IHC (n = 40; 20 with high 5hmC and 20 with low 5hmC) demonstrating that loss of L2HGDH is associated with loss of 5hmC (t test, P = 0.009). Box plots have horizontal lines at the 25th percentile, the median, and the 75th percentile. Vertical lines extend to the 10th and 90th percentiles. (H) Representative photographs showing loss of L2HDH in lower 5hmC ccRCC. (I) Lower L2HGDH expression is associated with shorter survival (P < 0.001). n = 533 ccRCC patients, grouped based on L2HGDH expression less than or greater than median. Survival analysis performed with log-rank test. The median survival (in days) for the low L2HGDH group is 1980. Median not reached for the high L2HGDH group.