Functional succinate dehydrogenase deficiency and loss of ascorbic acid transporter SLC23A1 are pathognomonic adverse features of clear cell renal cancer

Abstract

<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously thought to be relevant only in 0.05-0.5% of kidney cancers associated with germline <jats:italic>SDH</jats:italic> mutations (categorized ‘SDH-deficient Renal Cell Carcinoma’ in the 2016 WHO classification)</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>We show that under-expression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) tumors during pathogenesis and progression, with a marked adverse impact on survival in a large cohort (n=516) of ccRCC patients. From a mechanistic standpoint, we show that von Hippel-Lindau (VHL) loss induced hypoxia-inducible factor (HIF) dependent upregulation of mir-210 in ccRCC causes direct inhibition of the <jats:italic>SDHD</jats:italic> transcript. We demonstrate that reduced expression of <jats:italic>SDH</jats:italic> subunits is associated with genome-wide increase in methylation and enhancement of epithelial mesenchymal transition (EMT) in ccRCC tumors, consistent with succinate-induced inhibition of TET activity and increase in invasiveness/ migratory ability of ccRCC cells. TET-2 inhibition-induced global regulatory DNA hypermethylation drives SDH loss-induced enrichment of EMT. <jats:italic>SDH</jats:italic> subunits under-expression had a striking association with <jats:italic>CDHI</jats:italic> (E-cadherin) loss in ccRCC tumors, in keeping with succinate-induced <jats:italic>CDH1</jats:italic> hypermethylation and under-expression in ccRCC cells. Next, in conformity with recombinant TET-2 fluorescence quenching dynamics with succinate and ascorbic acid (AA, a TET enzyme co-factor), AA treatment led to reversal of succinate-induced inhibition of TET activity, <jats:italic>CDH1</jats:italic> hypermethylation and under-expression, as well as enhanced invasiveness in ccRCC cells. Furthermore, using immunohistochemical analysis and artificial intelligence quantitation, we report that ccRCC is characterized by a marked loss of ascorbic acid transporter SLC23A1 [median percent positive cells in ccRCC primary tumors (n=104) and normal kidney cortex (n=7) was 0.7 and 32.4 respectively; p=0.0012]. Lower <jats:italic>SLC23A1</jats:italic> was associated with worse survival in ccRCC (TCGA). Lastly, intravenous AA significantly prolonged survival in a metastatic ccRCC xenograft model with increased succinate and reduced <jats:italic>SLC23A1</jats:italic> expression.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Taken together, these findings strongly indicate that functional SDH deficiency is a pathognomonic adverse feature of ccRCC (which accounts for ∼80% of all kidney cancers), and that the WHO category ‘SDH-deficient RCC’ should be re-named ‘<jats:italic>SDH</jats:italic> germline mutation-associated RCC’. Furthermore, oncogenic accumulation of succinate can be abrogated by TET modulation with pharmacologic AA.</jats:p></jats:sec><jats:sec><jats:title>STATEMENT OF SIGNIFICANCE</jats:title><jats:p>In this study, we show that under-expression of succinate dehydrogenase (SDH) subunits resulting in the accumulation of oncogenic succinate is a common, adverse, epigenetic modulating feature occurring in a vast majority of clear cell renal cell carcinoma (ccRCC), during pathogenesis and progression. Functional SDH deficiency is therefore a pathognomonic feature of ccRCC (which accounts for ∼80% of all kidney cancers), and not just limited to the 0.05-0.5% of kidney cancer patients with germline <jats:italic>SDH</jats:italic> mutations. Based on the findings reported, we propose that the ‘SDH-deficient RCC’ category in the 2016 WHO classification of kidney tumors be renamed ‘SDH germline mutation-associated RCC’.</jats:p><jats:p>Furthermore, we show that the oncogenic accumulation of succinate in ccRCC can be countered by TET modulation with pharmacologic AA.</jats:p><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:caption><jats:title>Graphical abstract depicting the consequential adverse downregulation of Succinate Dehydrogenase in ccRCC and its central role in oxidative phosphorylation</jats:title></jats:caption><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="188433v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Immune evasion in renal cell carcinoma: biology, clinical translation, future directions

“…L2HG competitively inhibits the Ten-Eleven-Translocation (TET) enzymes, including TET2, which has been shown to have an important role in cancer immunity 15,45 . ccRCC is also characterized by loss of succinate dehydrogenase resulting in the accumulation of oncogenic succinate, which also inhibits the TET enzymes, further contributing to the widespread genomic aberrant hypermethylation in the malignancy 46,47 .…”

Oncometabolites—A Link between Cancer Cells and Tumor Microenvironment

“…They confirmed that succinate increased HIF-1α protein and mRNA expression with an intensity dependent on the incubation time. An important effect of succinate accumulation is the hypermethylation of histones and DNA cytosine [ 67 , 83 ] as a result of the succinate-mediated inhibition of histone lysine demethylases (KDMs) and TETs [ 74 , 84 ]. Succinate-mediate hypermethylation changes the expression profile of genes, leading to the activation of epithelial-to-mesenchymal transition.…”

Targeting Mitochondrial Oncometabolites: A New Approach to Overcome Drug Resistance in Cancer

“…As mentioned before, mitochondrial oncometabolites cause epigenetic changes in cancer cells, boosting oncogenesis and cancer progression. SDH deficient tumors are characterized by hypermethylation of histones and DNA cytosine [14,43], as a result of the succinate-mediated inhibition of histone lysine demethylases (KDMs) and TETs [26,44]. The hypermethylation changes the expression profile of specific genes, leading for instance to the activation of the EMT program, as demonstrated in pheochromocytomas and paragangliomas knocked-down for SDHB [45].…”