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-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 (Δ ψ ) 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 Δ ψ 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.

High-dose intravenous vitamin C, a promising multi-targeting agent in the treatment of cancer

“…One notable example of the progress in VitC pre-clinical research is the recent work in hard-to-treat Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) driven tumours, such as KRAS mutant colorectal cancer (CRC) [ 25 , 27 , 32 ]. Based on prior studies by Yun et al [ 32 ] and Aguilera et al [ 25 ], Cenigaonandia-Campillo et al [ 27 ] used elevated doses of VitC (5–10 mM) in KRAS mutant CRC tumours, both in vitro and in vivo.…”

“…One notable example of the progress in VitC pre-clinical research is the recent work in hard-to-treat Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) driven tumours, such as KRAS mutant colorectal cancer (CRC) [ 25 , 27 , 32 ]. Based on prior studies by Yun et al [ 32 ] and Aguilera et al [ 25 ], Cenigaonandia-Campillo et al [ 27 ] used elevated doses of VitC (5–10 mM) in KRAS mutant CRC tumours, both in vitro and in vivo. They showed that VitC was able to target common metabolic aberrancies by decreasing adenosine triphosphate (ATP) and glucose transporter 1 (GLUT-1) levels, as well as by dissipating the mitochondrial membrane potential, which could sensitize KRAS mutant CRC cells to current treatments such as chemotherapy.…”

“…A vast number of studies have shown encouraging anti-cancer activity of VitC at millimolar concentrations (~ 1-20 mM) in pre-clinical models of various cancer types [15]. The most investigated have been leukaemia [20][21][22][23][24], colon cancer [25][26][27][28][29][30][31][32], melanoma [33][34][35][36][37], pancreatic cancer [14,31,38] and prostate cancer [39][40][41]. Similar results have been described for the treatment of non-small-cell lung cancer (NSCLC) [16], breast cancer [31,42], ovarian cancer [31,43,44], hepatocellular carcinoma [45,46], malignant mesothelioma [47,48], thyroid cancer [49,50], oral squamous cell carcinoma [51], neuroblastoma [52] and glioma, including the difficult-to-treat glioblastoma multiform (GBM) [16,53,54].…”

Ras Family of Small GTPases in CRC: New Perspectives for Overcoming Drug Resistance

“…It has been shown that Vitamin C inhibits GAPDH and impairs cell growth specifically in KRAS and BRAF-mutant CRC cell line by interfering with glycolysis [150]. Aguilera et al reported that Vitamin C is also implicated in KRAS uncoupling from the plasma membrane, disrupting the expression of key metabolic enzymes and promoting cell death in KRAS-mutated cells [151]. Recently, the same group demonstrated that this compound, at pharmacological doses, can also modulate Krebs Cycle through PDK-1 (pyruvate dehydrogenase kinase 1) inhibition, again killing cells harbouring KRAS mutations but not those that are wild type [152].…”

“…Targeting metabolism has emerged as a good strategy for managing KRAS-mutated CRC and, therefore, for overcoming anti-EGFR resistance. Indeed, it has been demonstrated in preclinical trials that Vitamin C is enough to sensitize KRAS-mutant cells to cetuximab in CRC [151] and Erlotinib in lung cancer [200], both in vivo and in vitro. Unfortunately, although Vitamin C is under clinical trials in combination with the anti-EGFR panitumumab in WT KRAS patients, no clinical trials targeting both metabolism and EGFR have been performed for KRAS-mutated CRC patients so far.…”