Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

Abstract

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.

Fig. 1. KRAS and BRAF mutant cells predominantly take up DHA, the oxidized form of vitamin C, via GLUT1

(A) DHA, but not vitamin C, is transported into colorectal cancer cells (CRC) via GLUT1. [¹⁴C]-vitamin C was added to the culture media (2 mM glucose) for 30 minutes. [¹⁴C] Scintillation count per microgram of protein input was measured. Treating cells with GSH or STF31 (GLUT1 inhibitor) significantly reduced vitamin C uptake in all cases when compared to no GSH or STF31 treatment. One-way ANOVA followed by Dunnett’s post-test for multiple comparisons. *p < 0.01, **p < 0.001, n=3. (B) [¹⁴C]-vitamin C uptake was monitored in 2 mM glucose and signal normalized to total protein. P; Parental cells, WT-GLUT1; Exogenously expressed GLUT1 in WT cells, GLUT1 KO; GLUT1 knockout cells. Asterisks indicate significant decreases in vitamin C uptake of WT or GLUT1 KO cells relative to the parental lines, MUT, and WT-GLUT1. One-way ANOVA followed by Dunnett’s post-test. *p < 0.01, n=3. (C) LC/MS analysis of intracellular vitamin C and DHA in KRAS or BRAF isogenic cell lines derived from HCT116 and VACO432, respectively. Cells were treated with 1 mM (HCT116) or 2 mM (VAOC432) vitamin C for one hour before extracting vitamin C and DHA (Student’s t test, n=6). All data represent means ± s.d.

Fig. 2. Vitamin C is selectively toxic to cells with mutant KRAS or BRAF alleles

(A) Cell viability assay in 2 mM glucose or 2 mM glucose plus GSH in the presence of vitamin C (VC) for 48 hrs (HCT116, DLD1, RKO: 0.125 mM; VACO432: 0.375 mM) after cells were plated at a low density. Values were normalized to vehicle control. Parental (P) and MUT cells were significantly more sensitive than WT cells in the presence of vitamin C. One-way ANOVA (p<0.0001) with Dunnett’s post-test. *p < 0.0001, n=3. (B) HCT116 (KRAS: G13D/+) or VACO432 (BRAF: V600E/+) cells were injected subcutaneously into the flank of 6 to 8-week-old female athymic nude mice. After 7–10 days, mice were randomly divided into two groups. One group was treated with freshly prepared vitamin C in 400 ul PBS (4 g/kg) twice a day via IP injection (HCT116: n=6, VACO432: n=6). Control group mice were treated with PBS with the same dosing schedule (HCT116: n=4, VACO432: n=7). Tumor sizes were measured 2–3 times per week in an unblinded manner. Experiments were repeated twice independently. (C) At 7 weeks of age, Apc^flox/flox mice and Apc^flox/flox/LSL-Kras^G12D mice were treated with a single intraperitoneal injection (IP) of low dose tamoxifen (20 mg/kg) to activate the stem-cell-specific Cre and facilitate loss of Apc and activation of the Kras G12D allele. 3 weeks after tamoxifen injection, Apc^flox/flox mice (male=8 and female=9 mice) and Apc^flox/flox/LSL-Kras^G12D mice (male=7 and female=9) were divided into two groups (vitamin C at 4 g/kg or PBS) and treated daily with IP injections (5–6 times per week). Based on weight loss and Hemoccult score, all Apc^flox/flox mice were sacrificed at 6 weeks of treatment. Apc^flox/flox/LSL-Kras^G12D male mice were sacrificed at 5 weeks after treatment and Apc^flox/flox/LSL-Kras^G12D female mice were sacrificed at 7 weeks after treatment; average polyp numbers in the PBS group for female and male mice were similar. Apc^flox/flox/LSL-Kras^G12D mice experiments were repeated twice. Polyp number and volume was determined in whole mount tissue following methylene blue staining using a dissecting microscope in an unblinded manner. (D) Immunoblots of GLUT1 protein, phospo-ERK1/2, and total-ERK in tumors from Apc^flox/flox mice (n=4) and Apc^flox/flox/LSL-Kras^G12D mice (n=4). In. E.: normal intestinal epithelial cells. (E) Absolute amounts of intracellular vitamin C (VC) were measured in tumors derived from Apc^flox/flox mice and Apc^flox/flox/LSL-Kras^G12D mice treated with either vitamin C (4 g/kg) or PBS. Samples were harvested one hour post treatment. Two-way ANOVA (p =0.0002) followed by tukey’s test for multiple comparisons. All data represent means ± s.d.

Fig. 3. Vitamin C inhibits glycolysis thereby depleting ATP and selectively killing KRAS and BRAF mutant cells

(A) Heatmap depicting significantly changed glycolytic and PPP metabolite levels in mutant cells after a one-hour vitamin C or vehicle treatment as analyzed by LC-MS/MS. Red: increase; blue: decrease. PPP; Pentose phosphate pathway, TCA; tricarboxylic acid cycle. (B) Relative ratios of reduced to oxidized glutathione (GSH/GSSG) in KRAS and BRAF isogenic cell lines determined by LC-MS/MS as in (A). The ratio was significantly decreased following vitamin C in both MUT and WT cells (Student’s t test, *p <0.002, n=3) but the extent was greater in the MUT cells than in the WT cells. (C) Following a one-hour vitamin C (VC) treatment, cells were incubated with the ROS-sensitive fluorescent dye, DCF-DA, for 30 min and fluorescence measured by flow cytometry. Asterisks indicate significant increases in ROS following vitamin C treatment (Student’s t test, *p <0.01, n=3). (D) The extracellular acidification rate (ECAR) was monitored in KRAS and BRAF isogenic cell lines. Red arrows indicate the time of vitamin C (VC) or vehicle (CON) addition (n=6). (E) ATP levels were determined in KRAS and BRAF isogenic cell lines after a one-hour vitamin C (VC) treatment. Although ATP levels were significantly decreased in all cells (Student’s t test, *p<0.05, **p<0.002, n=3), the decrease was much more pronounced in MUT cells (two way ANOVA). (F) Cells were treated with vitamin C (VC) or vitamin C combined with N-acetyl cysteine (NAC) for one hour before immunoblotting for Thr172 phosphorylation (p-AMPK) or total AMPK (t-AMPK). (G) Cells were treated with vitamin C alone (VC) or vitamin C plus NAC, pyruvate (Pyr), or Trolox for 48 hours and viability measured with a CellTiter-Glo assay. Cell viability in parental (P) and MUT cells compared to WT cells was significantly decreased in vitamin C alone but not vitamin C combination treatments. One-way ANOVA (p<0.0001, VC group) with Dunnett’s post-test. *p <0.0001, n=3. (H) 8-week-old female athymic nude mice with subcutaneous tumors from parental HCT116 cells were treated with vitamin C (VC) alone (4 g/kg), NAC alone (30 mM in drinking water), VC plus NAC, or PBS twice a day via IP injection. Tumor sizes were measured once per week in an unblinded manner. Experiments were repeated twice independently. Vitamin C treatment alone significantly decreased tumor growth compared to PBS (p =0.016) but adding NAC to the vitamin C treatment abolished this effect (p=0.845). Mixed effect analysis followed by Tukey’s test. 1 and 2 mM Vitamin C was used for HCT116 and VACO432 cells, respectively (A–F). For viability assays at low cell densities, 0.125 and 0.375 mM vitamin C was used for HCT116 and VACO432 cells, respectively (G). All data represent means ± s.d.

Fig. 3. Vitamin C inhibits glycolysis thereby depleting ATP and selectively killing KRAS and BRAF mutant cells

(A) Heatmap depicting significantly changed glycolytic and PPP metabolite levels in mutant cells after a one-hour vitamin C or vehicle treatment as analyzed by LC-MS/MS. Red: increase; blue: decrease. PPP; Pentose phosphate pathway, TCA; tricarboxylic acid cycle. (B) Relative ratios of reduced to oxidized glutathione (GSH/GSSG) in KRAS and BRAF isogenic cell lines determined by LC-MS/MS as in (A). The ratio was significantly decreased following vitamin C in both MUT and WT cells (Student’s t test, *p <0.002, n=3) but the extent was greater in the MUT cells than in the WT cells. (C) Following a one-hour vitamin C (VC) treatment, cells were incubated with the ROS-sensitive fluorescent dye, DCF-DA, for 30 min and fluorescence measured by flow cytometry. Asterisks indicate significant increases in ROS following vitamin C treatment (Student’s t test, *p <0.01, n=3). (D) The extracellular acidification rate (ECAR) was monitored in KRAS and BRAF isogenic cell lines. Red arrows indicate the time of vitamin C (VC) or vehicle (CON) addition (n=6). (E) ATP levels were determined in KRAS and BRAF isogenic cell lines after a one-hour vitamin C (VC) treatment. Although ATP levels were significantly decreased in all cells (Student’s t test, *p<0.05, **p<0.002, n=3), the decrease was much more pronounced in MUT cells (two way ANOVA). (F) Cells were treated with vitamin C (VC) or vitamin C combined with N-acetyl cysteine (NAC) for one hour before immunoblotting for Thr172 phosphorylation (p-AMPK) or total AMPK (t-AMPK). (G) Cells were treated with vitamin C alone (VC) or vitamin C plus NAC, pyruvate (Pyr), or Trolox for 48 hours and viability measured with a CellTiter-Glo assay. Cell viability in parental (P) and MUT cells compared to WT cells was significantly decreased in vitamin C alone but not vitamin C combination treatments. One-way ANOVA (p<0.0001, VC group) with Dunnett’s post-test. *p <0.0001, n=3. (H) 8-week-old female athymic nude mice with subcutaneous tumors from parental HCT116 cells were treated with vitamin C (VC) alone (4 g/kg), NAC alone (30 mM in drinking water), VC plus NAC, or PBS twice a day via IP injection. Tumor sizes were measured once per week in an unblinded manner. Experiments were repeated twice independently. Vitamin C treatment alone significantly decreased tumor growth compared to PBS (p =0.016) but adding NAC to the vitamin C treatment abolished this effect (p=0.845). Mixed effect analysis followed by Tukey’s test. 1 and 2 mM Vitamin C was used for HCT116 and VACO432 cells, respectively (A–F). For viability assays at low cell densities, 0.125 and 0.375 mM vitamin C was used for HCT116 and VACO432 cells, respectively (G). All data represent means ± s.d.

Fig. 4. Vitamin C-induced ROS inhibits GAPDH by cysteine S-glutathionylation and depleting NAD⁺

(A) Cells were incubated with vehicle (CON) or vitamin C (VC) for one hour (HCT116: 1 mM, VACO432: 2 mM). Cell extracts were prepared in the presence of iodoacetic acid (IAA) to prevent S-thiolation during extraction, immunoprecipitated with a GAPDH antibody, and analyzed by non-reducing SDS-PAGE and probed with the indicated antibodies. (B) HCT116 cells were incubated with vehicle, vitamin C (VC) or H₂O₂ for one hour. Immunoblots were performed with the indicated antibodies as in (A). (C) Immunoblots for p(ADP)r (ADP-ribose polymers), Ser-139-phosphorylated, total H2AX, and β-actin on lysates from cells treated with vehicle (CON) or vitamin C (VC) for one hour. (D) Cells were treated with vitamin C alone (VC) (0.125 mM) or vitamin C plus Olaparib (10 uM) (VC + PARPi) or β-nicotinamide mononucleotide (NMN, 1 mM) (VC + NMN). Viability after 48 hours of treatment was measured using a CellTiter-glo assay and normalized to untreated controls. Asterisks indicate significant differences compared to MUT cells treated with vitamin C alone. Two-way ANOVA followed by Tukey’s test. *p <0.01, **p <0.001, MUT groups, n=3. (F) Schematic showing how vitamin C selectively kills cells KRAS or BRAF mutant cells.