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Vitamin C uncouples the Warburg metabolic switch in KRAS mutant colon cancer.

ABSTRACT: KRAS mutation is often present in many hard-to-treat tumors such as colon and pancreatic cancer and it is tightly linked to serious alterations in the normal cell metabolism and clinical resistance to chemotherapy.In 1931, the winner of the Nobel Prize in Medicine, Otto Warburg, stated that cancer was primarily caused by altered metabolism interfering with energy processing in the normal cell. Increased cell glycolytic rates even in the presence of oxygen is fully recognized as a hallmark in cancer and known as the Warburg effect.In the late 1970's, Linus Pauling and Ewan Cameron reported that vitamin C may have positive effects in cancer treatment, although deep mechanistic knowledge about this activity is still scarce.We describe a novel antitumoral mechanism of vitamin C in KRAS mutant colorectal cancer that involves the Warburg metabolic disruption through downregulation of key metabolic checkpoints in KRAS mutant cancer cells and tumors without killing human immortalized colonocytes.Vitamin C induces RAS detachment from the cell membrane inhibiting ERK 1/2 and PKM2 phosphorylation. As a consequence of this activity, strong downregulation of the glucose transporter (GLUT-1) and pyruvate kinase M2 (PKM2)-PTB dependent protein expression are observed causing a major blockage of the Warburg effect and therefore energetic stress.We propose a combination of conventional chemotherapy with metabolic strategies, including vitamin C and/or other molecules targeting pivotal key players involved in the Warburg effect which may constitute a new horizon in anti-cancer therapies.

SUBMITTER: Aguilera O

PROVIDER: S-EPMC5216991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 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 (Δψ) 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)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α 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.

Project description:BackgroundOncogenic KRAS mutations are prevalent in human cancers, but effective treatment of KRAS-mutant malignancies remains a major challenge in the clinic. Increasing evidence suggests that aberrant metabolism plays a central role in KRAS-driven oncogenic transformation. The aim of this study is to identify selective metabolic dependency induced by mutant KRAS and to exploit it for the treatment of the disease.MethodWe performed an integrated analysis of RNAi- and CRISPR-based functional genomic datasets (n = 5) to identify novel genes selectively required for KRAS-mutant cancer. We further screened a customized library of chemical inhibitors for candidates that are synthetic lethal with NOP56 depletion. Functional studies were carried out by genetic knockdown using siRNAs and shRNAs, knockout using CRISPR/Cas9, and/or pharmacological inhibition, followed by cell viability and apoptotic assays. Protein expression was determined by Western blot. Metabolic ROS was measured by flow cytometry-based quantification.ResultsWe demonstrated that nucleolar protein 5A (NOP56), a core component of small nucleolar ribonucleoprotein complexes (snoRNPs) with an essential role in ribosome biogenesis, confers a metabolic dependency by regulating ROS homeostasis in KRAS-mutant lung cancer cells and that NOP56 depletion causes synthetic lethal susceptibility to inhibition of mTOR. Mechanistically, cancer cells with reduced NOP56 are subjected to higher levels of ROS and rely on mTOR signaling to balance oxidative stress and survive. We also discovered that IRE1α-mediated unfolded protein response (UPR) regulates this process by activating mTOR through p38 MAPK. Consequently, co-targeting of NOP56 and mTOR profoundly enhances KRAS-mutant tumor cell death in vitro and in vivo.ConclusionsOur findings reveal a previously unrecognized mechanism in which NOP56 and mTOR cooperate to play a homeostatic role in the response to oxidative stress and suggest a new rationale for the treatment of KRAS-mutant cancers.

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Vitamin C uncouples the Warburg metabolic switch in KRAS mutant colon cancer.

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