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Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer.

ABSTRACT: Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1? involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (??) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)?, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial ?? dissipation and diminished pyruvate dehydrogenase E1-? phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.

SUBMITTER: Cenigaonandia-Campillo A

PROVIDER: S-EPMC7914362 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer.

Cenigaonandia-Campillo Aiora A Serna-Blasco Roberto R Gómez-Ocabo Laura L Solanes-Casado Sonia S Baños-Herraiz Natalia N Puerto-Nevado Laura Del LD Cañas Jose Antonio JA Aceñero María Jesús MJ García-Foncillas Jesús J Aguilera Óscar Ó

Theranostics 20210125 8

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC ...[more]

PMID: 33664850

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Project description:Goal and Objectives: Here we first time report that p-PDH A1 (S293 in mouse but in Human is S264), and PKM2 can regulates the many genes upon Insulin in Human Heptocellular Carcinoma HepG2 cells Line. The goal of this study is to identify the target genes, of which the promoters are associated with p-PDHA1 and PKM2, which may regulate target genes' trascription. We performed Chromatin immunoprecipitation-sequencing (ChIP-Seq) experiments by using antibodies agaist p-PDHA1 and PKM2. Methods: HepG2 cells were cultured in DMEM media, supplemented with 10% FBS and 1% penicillin/Streptomycin antibiotics. In 100 mm (diameter) dishes, the cells were seeded (1×107 – 3×107 cells per dish) and then incubated for 24 h. The media was exchanged for DMEM without serum for 12 h and the cells were then treated with 100 nM insulin for 48 h. The cells were then washed with PBS. To crosslink proteins to DNA, we added formaldehyde drop-wise directly to PBS containing samples to a final concentration of 0.75% and incubated at room temperature for 1 h. The crosslinking was stopped by adding glycine to the samples to a final concentration of 125 mM, followed by incubation for 10 min at RT. The cells were then rinsed twice with ice cold PBS and were harvested. The cell pellets were suspended in ChIP lysis buffer with freshly added cocktail of protease/phosphatase inhibitors and incubated for 10 min on ice. Sonication of the sample was then performed with an OMNI Sonic Ruptor-400 sonicator (OMNI International, Kennesaw, GA, USA) on ice for 20 impulses of 20 sec each. This was repeated five times with a 30 sec interval for each. The sonicated sample was centrifuged at 13,000 rpm for 15 min and 200 μg of the protein–chromatin complex was used for each round of immunoprecipitation {Cap, 2020, 32046944}, performed with anti-p-PDH A1 (S293), and PKM2 antibodies overnight at 4°C. IgG antibody used as a control. The formed antibody–protein complex was captured by incubation with pre-blocked Pierce Protein A/G Beads (Pierce). After sequential washing the beads with wash buffer I, II, III and IV several times and DNA sequencing of ChIP was performed by eBiogen (Seoul, Korea). Results: From ChIP Seq data analysis, we found that the promoters of many genes are associated with both p-PDH and PKM2. p-PDHA1 and PKM2 alone or together may regulate transcription of many genes upon insulin. In our current manuscript, we sort out the common genes which may be regulated p-PDHA1/PKM2 complex common genes in hepatocellular carcinoma cell line in response to insulin stimulation. Conclusions: Our study represents the first detailed analysis of p-PDHA1/PKM2 complex regarding to transcriptional regulation through ChIP-seq. The optimized data analysis workflows reported here may provide a framework for comparative transcritional investigations of p-PDHA1 and PKM2. We conclude that ChIP-Seq data of p-PDHA1 and PKM2 would expedite genetic network analyses and permit the dissection of complex biologic functions as well as cancer biology studies.

Dataset Information

Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer.

Publications

Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic <i>KRAS</i> mutant colon cancer.

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