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. 2019 Jun 1;111(6):584-596.
doi: 10.1093/jnci/djy160.

Modulation of Redox Homeostasis by Inhibition of MTHFD2 in Colorectal Cancer: Mechanisms and Therapeutic Implications

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Free PMC article

Modulation of Redox Homeostasis by Inhibition of MTHFD2 in Colorectal Cancer: Mechanisms and Therapeutic Implications

Huai-Qiang Ju et al. J Natl Cancer Inst. .
Free PMC article

Abstract

Background: Overcoming oxidative stress is a critical step for tumor progression; however, the underlying mechanisms in colorectal cancer (CRC) remain unclear.

Methods: We investigated nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent enzyme methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) expression, clinical relevance, redox modification, and molecular mechanisms using the CRC cells and tissues (n = 462 paired samples). The antitumor effects of MTHFD2 inhibitor LY345899 on CRC tumorigenesis and metastasis were evaluated in vitro and in vivo. Data analysis used Kaplan-Meier, Pearson's correlation, and Student t test where appropriate. All statistical tests were two-sided.

Results: Here, we report that the patients with high expression of MTHFD2 have a shorter overall survival (HR = 1.62, 95% CI = 1.12 to 2.36, P = .01) and disease-free survival (HR = 1.55, 95% CI = 1.07 to 2.27, P = .02) than patients with low MTHFD2 expression. Suppression of MTHFD2 disturbs NADPH and redox homeostasis and accelerates cell death under oxidative stress, such as hypoxia or anchorage independence (P ≤ .01 for all). Also, genetic or pharmacological inhibition of MTHFD2 suppresses CRC cell growth and lung and peritoneal metastasis in cell-based xenografts (n = 5-8 mice per group). Importantly, LY345899 treatment statistically significantly suppresses tumor growth and decreases the tumor weight in CRC patient-derived xenograft models (n = 10 mice per group, mean [SD] tumor weight of the vehicle-treated group was 1.83 [0.19] mg vs 0.74 [0.30] mg for the LY345899-treated group, P < .001).

Conclusions: Our study presents evidence that MTHFD2 confers redox homeostasis and promotes CRC cell growth and metastasis. The folate analog LY345899 as MTHFD2 inhibitor displays therapeutic activity against CRC and warrants further clinical investigation for CRC treatment.

Figures

Figure 1.
Figure 1.
The expression and clinical significance of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in colorectal cancer (CRC). A) Expression profiling of various potential nicotinamide adenine dinucleotide phosphate (NADPH)-producing enzymes in The Cancer Genome Atlas (TCGA) CRC database. B) Quantitative polymerase chain reaction (qPCR) analysis of the indicated gene expression in 55 paired CRC tissues. C) MTHFD2 expression in multiple CRC microarray data sets is available from the Oncomine database. D and E) Immunoblotting analysis of MTHFD2 protein levels in paired CRC tissues, CRC cells, and colorectal epithelial cells (CCD112, CCD841). F) Representative immunohistochemical staining showing positive staining in primary CRC tumor tissues and paired liver metastatic tissues (scale bar = 100 µm). G) The immunohistochemical staining scores of MTHFD2 in paired primary CRC tumor tissues (N = 462), lymph node metastatic tissues (LNM, N = 119), or liver metastatic tissues (LM, N = 83). H) qPCR analysis of MTHFD2 expression in 28 paired liver metastatic tissues and 72 CRC tissues with or without liver metastasis. I) Kaplan–Meier analysis of 5-year overall survival or disease-free survival for CRC patients with low vs high expression of MTHFD2 (Kaplan–Meier analysis with the log-rank test). β-Actin was included as a loading control. Data in C, G, and H are presented as a box-and-whiskers graph (min-max), and the horizontal line across each box indicates the median. All statistical analyses were performed using Student paired t test. All statistical tests were two-sided. ME1 = malic enzyme 1; G6PD = glucose-6-phosphate dehydrogenase; MTHFD1/2 = methylene tetrahydrofolate dehydrogenase 1/2; N = adjacent normal tissues; T = tumor.
Figure 2.
Figure 2.
Effects of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) inhibition on colorectal cancer (CRC) cell survival during hypoxia. A) Gene set enrichment analysis revealed that the MTHFD2 expression is positively correlated with reactive oxygen species (ROS)-related gene signature available from The Cancer Genome Atlas CRC database. B) Immunoblotting evaluating the knockdown efficiency of MTHFD2 with three unique shRNAs (#1, #2, #3) in SW620 and LoVo cells. C) Cell death (Annexin V/PI positive cells) was measured by Annexin-V/PI assays in indicated cells treated with hydrogen peroxide (H2O2, 100 µM) for 48 hours. D and E) The nicotinamide adenine dinucleotide phosphate (NADP)/NADP+ and reduced glutathione (GSH)/oxidized glutathione (GSSG) levels were measured in the indicated CRC cells under normoxia or hypoxia. F) Representative histograms and quantification of cellular ROS levels in the indicated CRC cells exposed to hypoxia for 48 hours, as detected by the fluorescent probe 2′,7′–dichlorodihydrofluorescein diacetate (DCF-DA). G) Cell death was measured by Annexin-V/PI assays in the indicated CRC cells under normoxia or hypoxia for 72 hours (red numbers indicate subpopulation of cells positive for Annexin V/PI). H) Immunoblotting analysis of cleaved poly ADP-ribose polymerase (PARP) in the indicated CRC cells. I) Quantification of cell death in the indicated CRC cells after hypoxia for 72 hours (with or without 5 mM N-acetyl-L-cysteine). β-Actin was included as a loading control. Data in C–F and I are presented as the mean (SD) (n = 3), and statistical analyses were performed using Student unpaired t test. All statistical tests were two-sided. CT = control; NES = Normalized enrichment score; FDR = False discovery rate; shMTHFD2 = short hairpin RNA-MTHFD2.
Figure 3.
Figure 3.
Effects of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) inhibition on colorectal cancer (CRC) cell anoikis under detachment. A and B) Cellular nicotinamide adenine dinucleotide phosphate (NADP)/NADP+ and reduced glutathione (GSH)/oxidized glutathione (GSSG) levels were measured in the indicated CRC cells under attached or detached conditions for 24 hours. C and D) Representative histograms and quantification of cellular reactive oxygen species (ROS) levels in the indicated CRC cells under attached or detached conditions for 24 hours, as detected by the fluorescent probe 2′,7′–dichlorodihydrofluorescein diacetate (DCF-DA). E) Cell death was measured by Annexin-V/PI assays in the indicated CRC cells under attached or detached conditions for 72 hours (red numbers indicate subpopulation of cells positive for Annexin V/PI). F) Quantification of cell death in the indicated cells cultured under detached conditions for 72 hours (with or without 5 mM N-acetyl-L-cysteine). G-H) Phase-contrast micrographs and quantification of sphere formation by MTHFD2-knockdown and control CRC cells cultured under detached conditions in 6-well ultralow attachment plates (2.0 x 103 cells/well) (scale bar = 200 µm). Data in A, B, D, F, and H are presented as the mean (SD) (n = 3). All statistical analyses were performed using Student unpaired t test. All statistical tests were two-sided. CT = control.
Figure 4.
Figure 4.
Effects of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) inhibition on colorectal cancer (CRC) tumorigenesis and metastasis in vivo. A) Xenograft model was established in nude mice subcutaneously implanted with MTHFD2-knockdown and control CRC cells (N = 5). Representative images of tumor-bearing mice were taken at day 28. B) Tumor volumes recorded on the indicated days are shown. C) Photograph and comparison of excised tumor size. D) The tumor weights of the indicated group on day 32 were measured. These mouse experiments were repeated once. E) Paraffin-embedded tumor sections derived from the SW620 group were stained with hematoxylin and eosin (H&E) or MTHFD2, Ki67, and cleaved caspase 3 antibodies (scale bar = 100 µm). Apoptotic cells were visualized by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining (green) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) (scale bar = 10 µm). F) The proliferation index (Ki67 staining) and apoptotic index (TUNEL staining) in tumor sections were also quantified. G) Representative H&E staining and statistical results of metastatic lung nodules from mice injected via the tail vein with MTHFD2 knockdown and control SW620 cells for 60 days (five sections evaluated per lung). H) Representative H&E staining and statistical results of scattered tumors in the excised intestines of mice (N = 8) orthotopically implanted with the indicated cells. Arrows indicate the metastatic foci (scale bar = 100 µm). All statistical analyses were performed using Student unpaired t test. All statistical tests were two-sided. CT = control; shCT = short hairpin RNA-Control; shControl = short hairpin RNA-Control; shMTHFD2 = short hairpin RNA-MTHFD2.
Figure 5.
Figure 5.
Signaling pathway involved in methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) expression in colorectal cancer (CRC). A) Distribution of alteration frequency of MTHFD2 in multiple cancer types. Details in parentheses indicate the source of the corresponding tumor dataset (cBioPortal for Cancer Genomics). B) Gene set enrichment demonstrating the enrichment of Kras-related gene sets in the ranked gene list of MTHFD2 up vs MTHFD2 down available from The Cancer Genome Atlas CRC database. C) Quantitative polymerase chain reaction (qPCR) analysis of the MTHFD2 expression levels in Kras-mutant (Mu) vs Kras wild-type (WT) cells (NCI-60), or CRC specimens with wild-type Kras (N = 37) vs mutant Kras (N = 21). D) qPCR and immunoblotting analysis of MTHFD2 expression in 293T/iKras cells after Kras induction by 20 ng/mL doxycycline (Dox) for indicated time. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. E) Immunoblotting analysis of MTHFD2 expression in SW620 and LoVo cells transfected with Kras siRNAs (#1, #2). F) Immunoblotting analysis of MTHFD2 expression in SW620 or LoVo cells treated with Akt (also known as protein kinase B or PKB), which is a well know as the serine/threonine kinase or extracellular signal regulated kinase1/2 (ERK1/2). Inhibitor at the indicated concentration for 48 hours. G) MTHFD2 expression was inversely associated with p-Akt and p-ERK expression in 462 clinical CRC specimens. Shown are visualizations of two representative cases (scale bar = 100 µm). H) Percentages of samples showing low or high MTHFD2 expression relative to p-Akt and p-ERK. Chi-square test was used to study the association between MTHFD2 and p-Akt or p-ERK expression. All statistical tests were two-sided. CT = control; NES = Normalized enrichment score; FDR = False discovery rate; NC = Negative control; RNAi = RNA interference; iKras = Inducible kras; H&E = Haematoxylin and Eosin stain.
Figure 6.
Figure 6.
Transcriptional regulation of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in colorectal cancer (CRC). A) Gene set enrichment enrichment score and distribution of c-Myc-regulated genes along the rank of MTHFD2 up vs MTHFD2 down available from The Cancer Genome Atlas CRC database. B and C) Quantitative polymerase chain reaction (qPCR) and immunoblotting analysis of MTHFD2 expression in 293T/iKras cells after Kras induction and/or c-Myc depletion for 72 hours. D and E) qPCR and immunoblotting analysis of MTHFD2 expression in SW620 and LoVo cells transfected with c-Myc siRNAs (#1, #2). F) c-Myc DNA-binding sites are present in the human MTHFD2 promoter region. G) Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) in SW620 and LoVo cells. Representative of two independent experiments. H) Relative MTHFD2 luciferase promoter activity in indicated cells with c-Myc depletion or overexpression. I) Scatterplots of MTHFD2 vs c-Myc mRNA expression in CRC tissues (N = 48) analyzed by qPCR. J) Scatterplots of MTHFD2 vs c-Myc mRNA expression in cell lines (N = 62) available from CCLE database and CRC cell lines (N = 12). Pearson correlation coefficient (r) and P value are displayed. Data in B, D, and H are presented as the mean (SD) (n = 3), and statistical analyses were performed using Student unpaired t test. All statistical tests were two-sided. CT = control; DOX = doxycycline; NES = Normalized Enrichment Score; FDR = False Discovery Rate; NC = Negative Control; RNAi = RNA interference; IgG = Immunoglobulin G; si-c-Myc = Small interfering RNA-c-Myc.
Figure 7.
Figure 7.
Antitumor activity of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) inhibitor LY345899 in colorectal cancer (CRC). A) Chemical structure of LY345899. B) The cell viability of the indicated CRC cells treated with LY345899 for 72 hours was determined by MTS assay. C) The relative nicotinamide adenine dinucleotide phosphate (NADP)/NADP+ (NADP/NADP+) and reactive oxygen species (ROS) levels were measured in the indicated CRC cells treated with LY345899 (10 µM) for 24 hours. D and E) Representative and statistical results of annexin V/PI staining in LoVo and SW620 cells treated with LY345899 for 48 hours under hypoxia or detachment. F) The tumor growth curves and weights were measured and recorded for SW620-based xenograft mice treated with LY345899 (5 mg/kg or 10 mg/kg per mouse, intraperitoneal (ip, 5 d/wk). The mice were treated with LY345899 when the tumor volume reached 100 mm3. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison test. G) The tumor growth curves and the weights were measured and recorded for the patient derived xenograft (PDX) mice (#1, #2) treated with LY345899 (10 mg/kg per mouse, ip, 5 d/wk) (N = 5). H) Representative and quantification of TdT-mediated dUTP Nick-End Labeling staining for apoptosis analysis in the indicated groups (scale bar = 10 µm). I) Images and statistical results of scattered tumors in the excised intestines of LY345899-treated mice (N = 8) orthotopically implanted with CRC cells or PDX tissues (scale bar = 100 µm). Data in B, D, and H are presented as the mean (SD) (n = 3), and statistical analyses were performed using Student unpaired t test. All statistical tests were two-sided. DCF-DA = 2′,7′–dichlorodihydrofluorescein diacetate; H&E = haematoxylin and eosin staining; PBS = phosphate buffered saline.
Figure 8.
Figure 8.
Proposed working model of this study. Methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) confers redox homeostasis under hypoxia or extracellular matrix detachment and thus enhances malignancy and distant metastasis for colorectal cancer (CRC). Mechanistically, MTHFD2 is transcriptionally upregulated by c-Myc though Kras downstream, including PI3K/Akt and ERK pathways. GSH = reduced glutathione; GSSG = oxidized glutathione; NADP = nicotinamide adenine dinucleotide phosphate; ROS = reactive oxygen species; THF = tetrahydrofolate; PI3K = phosphatidylinositide 3-kinases; Akt = Protein kinase B (PKB); ERK = Extracellular signal regulated kinase.

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