Functional succinate dehydrogenase deficiency is a pathognomonic adverse feature of clear cell renal cancer

Abstract

BackgroundReduced 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 SDH mutations (categorized SDH-deficient Renal Cell Carcinoma in the 2016 WHO classification) ResultsWe 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 SDHD transcript. We demonstrate that reduced expression of SDH 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. SDH subunits under-expression had a striking association with CDHI (E-cadherin) loss in ccRCC tumors, in keeping with succinate-induced CDH1 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, CDH1 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 SLC23A1 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 SLC23A1 expression. ConclusionsTaken 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 SDH germline mutation-associated RCC. Furthermore, oncogenic accumulation of succinate can be abrogated by TET modulation with AA. STATEMENT OF SIGNIFICANCEIn 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 SDH 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. Furthermore, we demonstrate that oncogenic accumulation of succinate in ccRCC can be countered by TET modulation with ascorbic acid, and that ccRCC is characterized by a marked loss of ascorbic acid transporter SLC23A1. O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/188433v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@728d5eorg.highwire.dtl.DTLVardef@9f2e24org.highwire.dtl.DTLVardef@8e7011org.highwire.dtl.DTLVardef@15f8d33_HPS_FORMAT_FIGEXP M_FIG Graphical abstract depicting the consequential adverse downregulation of Succinate Dehydrogenase in ccRCC and its central role in oxidative phosphorylation C_FIG