Biology subjects

Aggarwal, R. K.

Publications and source records attributed to Aggarwal, R. K..

3 recordsLinked to original sources

bioRxiv2020

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

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

cancer biology↗

bioRxiv2020

Deconstructing glucose-mediated catabolite repression of the lac operon of Escherichia coli: II. Positive feedback exists and drives the repression

The expression of the lac operon of E. coli is subject to positive feedback during growth in the presence of gratuitous inducers, but its existence in the presence of lactose remains controversial. The key question in this debate is: Do the lactose enzymes, Lac permease and β-galactosidase, promote accumulation of allolactose? If so, positive feedback exists since allolactose does stimulate synthesis of the lactose enzymes. Here, we addressed the above question by developing methods for determining the intracellular allolactose concentration as well as the kinetics of enzyme induction and dilution. We show that during lac induction in the presence of lactose, the intracellular allolactose concentration increases with the lactose enzyme level, which implies that lactose enzymes promote allolactose accumulation, and positive feedback exists. We also show that during lac repression in the presence of lactose + glucose, the intracellular allolactose concentration decreases with the lactose enzyme levels, which suggests that under these conditions, the positive feedback loop turns in the reverse direction. The induction and dilution rates derived from the transient data show that the positive feedback loop is reversed due to a radical shift of the steady state induction level. This is formally identical to the mechanism driving catabolite repression in the presence of TMG + glucose.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2019

Deconstructing glucose-mediated catabolite repression of the lac operon of Escherichia coli: I. Inducer exclusion, by itself, cannot account for the repression.

The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to CRP-mediated transcriptional inhibition and EIIAGlc-mediated inducer exclusion. The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. The literature shows that in fully induced cells, inducer exclusion reduces the permease activity only 2-fold. However, it is conceivable that inducer exclusion drastically reduces the permease activity in partially induced cells. We measured the decline of lactose permease activity due to inducer exclusion in partially induced cells, but found that the permease activity decreased no more than 6-fold. We show that the repression is small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONS AND NOTATIONScAMP3’,5’-cyclic adenosine monophosphateCGSCColi Genetic Stock Centre, Yale UniversityCRPcAMP receptor proteinEIIAGlcEnzyme III of glucose transport system, PTS (Previously EIIIGlc)gdwGrams of cell dry weight, gIPTGIsopropyl β-D-thiogalactopyranoside or MeSHLBLuria-Bertani brothMUMiller unitsOD600Optical density at 600 nmONPGortho-nitrophenol-β-D-galactopyranosideRNAPRibonucleic acid (RNA) polymeraseTMGMethyl- β-D-1-thiogalactopyranoside[14C]TMGCarbon-14 labelled isotope of TMGUVUltraviolet (radiation)X-gal5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideNOTATIONSIntracellular concentration of phosphorylated EIIAglc in the absence of glucoseIntracellular concentration of dephosphorylated EIIAglc in the presence of glucoseEgSpecific activity of β-galactosidaseSteady state specific activity of β-galactosidase in the presence of glucoseSteady state specific activity of β-galactosidase in the presence of glycerolEpSpecific activity of Lac permeaseSteady state specific activity of Lac permeaseEp,tSpecific activity of free Lac permease in the absence of glucoseφpFractional reduction of permease activity in the presence of glucose, 1 - Ep/Ep,tk1, k2Proportionality constantsKdDissociation constant for complex OD600Optical density at 600 nmSp. rate of β-galactosidase synthesis in the presence of glucose + glycerol + TMGSp. rate of β-galactosidase synthesis in the presence of glycerol + TMGSpecific rate of diffusive efflux of TMGTIntracellular TMG concentrationSteady-state intracellular TMG concentrationSteady-state intracellular TMG concentration in the presence of glucoseSteady-state intracellular TMG concentration in the presence of glucoseTeExtracellular TMG concentrationµglcSpecific growth rate of E. coli cells on glucoseµglySpecific growth rate of E. coli cells on glycerolXBiomass or cell density, gdw l-1View Full Text

systems biology↗