Functional succinate dehydrogenase deficiency and loss of ascorbic acid transporter SLC23A1 are pathognomonic adverse features of clear cell renal cancer
Authors: Aggarwal Ritesh K. , Zou Yiyu , Luchtel Rebecca A. , Pradhan Kith , Ashai Nadia , Ramachandra Nandini , Albanese Joseph M. , Yang Jung-in , Wang Xiaoyang , Aluri Srinivas , Gordon Shanisha , Machha Venkata , Tischer Alexander , Aboumohamed Ahmed , Gartrell Benjamin A. , Hafizi Sassan , Pullman James , Shenoy Niraj 
Published: July 2020

ABSTRACTBackgroundReduced 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 germlineSDHmutations (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 theSDHDtranscript. We demonstrate that reduced expression ofSDHsubunits 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.SDHsubunits under-expression had a striking association withCDHI(E-cadherin) loss in ccRCC tumors, in keeping with succinate-inducedCDH1hypermethylation 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,CDH1hypermethylation 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]. LowerSLC23A1was associated with worse survival in ccRCC (TCGA). Lastly, intravenous AA significantly prolonged survival in a metastatic ccRCC xenograft model with increased succinate and reducedSLC23A1expression.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 ‘SDHgermline 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 germlineSDHmutations. 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.Graphical abstract depicting the consequential adverse downregulation of Succinate Dehydrogenase in ccRCC and its central role in oxidative phosphorylation