TYMS Enhances Colorectal Cell Antioxidant Capacity Via the KEAP1-NRF2 Pathway to Resist Ferroptosis

Abstract

Purpose: Thymidylate synthase (TYMS) is a key regulatory enzyme in DNA synthesis. We identified the biological effect and molecular mechanisms of TYMS in colorectal cancer (CRC). Methods: We employed western blot and immunohistochemistry for the assessment of TYMS expression in CRC samples. MTT and colony assay were carried out to illuminate the effect of TYMS on the proliferation of CRC cells. Xenograft models were constructed to evaluate the consequences of TYMS overexpression on CRC in vivo. Metabolomics was utilized to analyze the alterations in cellular molecular metabolites subsequent to TYMS overexpression. The impact of TYMS on NRF2 localization and KEAP1 expression was explored by means of western blot. The expression levels of GSH, ROS, MDA, and PTGS2 mRNA were measured to assess ferroptosis. Results: TYMS expression in CRC tumor tissues was upregulated compared to adjacent non-cancerous tissues. Cells overexpressing TYMS displayed enhanced proliferative capabilities. Metabolomic analysis revealed that overexpression of TYMS was associated with elevated levels of GSH within cells and a decrease in the lipid peroxidation product, 4-hydroxyhexenal. ROS detection assays further demonstrated a significant enhancement in cellular antioxidant capacity due to TYMS overexpression. Overexpression of TYMS downregulated KEAP1 expression and promoted NRF2 translocation into the nucleus. Consequently, transcription of downstream antioxidant genes was upregulated, enhancing cellular antioxidant capacity, reducing ROS levels, diminishing lipid peroxidation products, and heightening resistance to ferroptosis induced by erastin. Additionally, our study indicated that the TYMS inhibitor 5-fluorouracil (5-FU) exhibited favorable drug synergism with erastin. Conclusion: TYMS was overexpressed in CRC, which was correlated with poor prognosis of CRC patients. TYMS enhanced the antioxidant capacity of CRC cells via the KEAP1-NRF2 pathway, thereby increasing resistance to erastin-induced ferroptosis.

Keywords: Colorectal cancer; Kelch-like ECH-associated protein 1; Nuclear factor erythroid 2-related factor 2; ROS; Thymidylate synthase; ferroptosis.

Figure 1

TYMS expression in CRC tissues and correlations to survival. (A) Expression of TYMS in CRC patients analyzed with the GEPIA online tool. (B) Detection of TYMS expression by western blot in CRC tissues and adjacent tissues from eight CRC patients. (C, D) Representative IHC images and the scoring of IHC showing the level of TYMS in CRC tissues and adjacent tissues. Black color scale bars: 100 μm, blue color scale bars: 50 μm. (E) Pie chart showing the proportion of upregulation, unchanged and downregulation in TYMS for comparison between CRC tissues and corresponding noncancerous tissues. n= 100 pair of samples. (F, G) Kaplan-Meier analysis of overall survival in all CRC patients and patients not receiving chemotherapy with differential TYMS expression. **** p< 0.0001.

Figure 2

TYMS induced tumorigenesis. (A)Western blotting detected the expression of endogenous TYMS in different CRC cell lines. (B, C) Cell proliferation assay was performed in TYMS overexpressed cells (SW480 and SW620, B), knocked down cells (HT29 and HCT116, C), and correspondence control cells, respectively. (D, E) Colony formation assay was performed in TYMS overexpressed cells (D), TYMS knocked down cells (E), and correspondence control cells (vector or shcon). (F) Ectopic expressed TYMS in SW480 cells promoted tumor growth in xenograft mice model. (G) The volume and weight of tumors derived from TYMS ectopic expressed cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

TYMS enhanced the antioxidant capacity. (A) Comparison of differential metabolites between TYMS overexpress (TYMS) and control cells (vector) in SW480 cell by untargeted Metabolomics. GSH levels(B) and ROS levels(C) in TYMS overexpressed cells (SW480 and SW620), knocked down cells (HT29 and HCT116), and correspondence control cells. (D) DCFH-DA fluorescent probe detected the fluorescence intensity of cells in TYMS overexpressed cells and correspondence control cells. Scale bars: 50 μm. (E) HE and IHC assay for TYMS and PTGS2 were performed in isolated tumor tissues. Scale bars: 100 μm. *p < 0.05, **p < 0.01.

Figure 4

TYMS promoted the nuclear translocation of NRF2. (A)TYMS affected NRF2 expression in nucleus and cytoplasm. (B) RT-qPCR detected the expression of NRF2 dependent antioxidant enzymes. (C) KEAP1 protein levels in TYMS overexpressed cells, knocked down cells, and correspondence control cells. (D) RT-qPCR detected the expression of KEAP1 in TYMS overexpressed cells, knocked down cells, and correspondence control cells. (E) TYMS overexpression resulted in a greater accumulation of red fluorescence produced by NRF2 protein in the nucleus. Scale bars: 100 μm. *p < 0.05, **p < 0.01.

Figure 5

TYMS enhanced resistance to ferroptosis. (A) The IC50s of erastin were analyzed in CRC cells with TYMS overexpress or knockdown. ROS levels (B), GSH levels(C), GSH/GSSG(D), MDA levels(E) and PTGS2 mRNA levels (F) in TYMS overexpressed cells, knocked down cells, and correspondence control cells treated with different concentrations of erastin. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

The NRF2 inhibitor ML385 reversed the resistance to ferroptosis. (A) MTT assay was performed after treatment with ML385 and Ferrostatin-1(Fer) in the TYMS-overexpression cells and correspondence control cells. (B) The IC50s of erastin were analyzed after treatment with ML385 and Ferrostatin-1 in the TYMS-overexpression cells and correspondence control cells. ROS levels (C), MDA levels(D), PTGS2 mRNA levels (E)and cell viability(F) in TYMS overexpressed cells, knocked down cells, and correspondence control cells after treatment with ML385, Ferrostatin-1 and Z-VAD-FMK(ZVF). *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

Figure 7

Combination of erastin with 5-FU enhanced anti-tumor effects. (A) MTT assays showed combination of erastin with 5-FU treatment exhibited higher inhibitory effects on cell growth compared with mono treatment in SW480 and SW620. (B) Combination of erastin with 5-FU treatment exhibited higher inhibitory effects on clone formation compared with mono treatment in SW480 and SW620. **p < 0.01, ***p < 0.001, ****p< 0.0001.