Untargeted Metabolomics Showed Accumulation of One-Carbon Metabolites to Facilitate DNA Methylation during Extracellular Matrix Detachment of Cancer Cells

Abstract

Tumor cells detached from the extracellular matrix (ECM) undergo anoikis resistance and metabolic reprogramming to facilitate cancer cell survival and promote metastasis. During ECM detachment, cancer cells utilize genomic methylation to regulate transcriptional events. One-carbon (1C) metabolism is a well-known contributor of SAM, a global substrate for methylation reactions, especially DNA methylation. DNA methylation-mediated repression of NK cell ligands MICA and MICB during ECM detachment has been overlooked. In the current work, we quantitated the impact of ECM detachment on one-carbon metabolites, expression of 1C regulatory pathway genes, and total methylation levels. Our results showed that ECM detachment promotes the accumulation of one-carbon metabolites and induces regulatory pathway genes and total DNA methylation. Furthermore, we measured the expression of well-known targets of DNA methylation in NK cell ligands in cancer cells, namely, MICA/B, during ECM detachment and observed low expression compared to ECM-attached cancer cells. Finally, we treated the ECM-detached cancer cells with vitamin C (a global methylation inhibitor) and observed a reduction in the promoter methylation of NK cell ligands, resulting in MICA/B re-expression. Treatment with vitamin C was also found to reduce global DNA methylation levels in ECM-detached cancer cells.

Keywords: DNA methylation; ECM detachment; NKG2DLs; anoikis; one-carbon metabolism.

Figure 1

Increasing the level of one-carbon metabolites in the ECM-detached condition. (a) Overall experimental design. (b) The one-carbon metabolites detected in the attached (AT) and 6 days (6D) ECM-detached HeLa cell line. (c) The one-carbon metabolites detected in the attached (AT) and 6 days (6D) ECM-detached MCF-7 cell line. The data are presented as mean ± SEM; ns, not significant; * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Figure 2

One-carbon metabolic genes and 5mC levels (%) in ECM-attached and -detached conditions. (a) The mRNA expression levels of one-carbon metabolic genes in the attached (AT) methionine cycles and 6 days detached (6D_DT) conditions in the HeLa cell lines. (b) The mRNA expression levels of one-carbon metabolic genes in folate cycles in attached (AT) and 6 days detached (6D_DT) conditions in the HeLa cell lines. (c) The mRNA expression levels of one-carbon metabolic genes in methionine cycles in attached (AT) and 6 days detached (6D_DT) conditions in the MCF-7 cell lines. (d) The mRNA expression levels of one-carbon metabolic genes in folate cycles in attached (AT) and 6 days detached (6D_DT) conditions in the MCF-7 cell lines. SHMT1, serine hydroxymethyltransferase 1; MTHFR, methylenetetrahydrofolate reductase. (e,f). HeLa and MCF-7 cell lines showed increased 5mC levels in the 6 days (6D_DT) ECM-detached condition compared to the attached (AT) condition. The data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p< 0.001, and **** p < 0.0001.

Figure 3

ECM detachment represses NKG2DLs–MICA/B expression. (a) Fold changes in mRNA expression of DNMTs in attached (AT) and 6 days ECM-detached (6D_DT) HeLa cell lines. (b) Fold changes in mRNA expression of DNMTs in attached (AT) and 6 days ECM-detached (6D_DT) MCF-7 cell lines. (c) Fold changes in mRNA expression of MICA/B in attached (AT) and 6 days ECM-detached (6D_DT) conditions in HeLa cell lines. (d) Fold changes in mRNA expression of MICA/B in attached (AT) and 6 days ECM-detached (6D_DT) conditions in MCF-7 cell lines. (e,f). MICA/B surface expression is repressed in the 6 days ECM-detached (6D_DT) condition compared to the attached (AT) condition in HeLa and MCF-7 cell lines. The data are presented as mean ± SEM; ns, not significant.

Figure 4

Vitamin C treatment mediated reductions in global DNA methylation, DNA methyltransferase (DNMT1/3a), and induction of MICA/B expression in ECM-detached HeLa cell lines. (a) Vitamin C treatment reduced 5mC levels. (b) Vitamin C treatment repressed DNMT1/3a mRNA expression. (c) HeLa cell line showing that 5-aza-dc and vitamin C did not reduce the methylation signal in gel electrophoresis compared to the untreated 6 Days ECM-detached condition. (d) Vitamin C treatment increased the fold changes in mRNA expression of MICA/B in the 6 days ECM-detached condition. (e) Vitamin C treatment increased surface MICA/B expression. 6D_Meth, 6 days detached, methylated; 6D_Unmrth, 6 days detached, unmethylated; 5Azadc_Meth, 5-aza-dc methyl-treated; 5Azadc_Unmeth, 5-aza-dc unmethylated; VC_meth, vitamin C methyl-treated; VC_Unmeth, vitamin C unmethylated. The data are presented as mean ± SEM; ns, not significant.

Figure 5

Vitamin C treatment-mediated reduction of global DNA methylation, DNA methyltransferase, and induction of MICA/B expression in ECM-detached MCF-7 cell lines. (a) Vitamin C treatment reduced 5mC levels. (b) Vitamin C treatment repressed DNMTs mRNA expression. (c) MCF-7 cell line showing 5-aza-dc and vitamin C reducing methylation signal in gel electrophoresis compared to untreated 6 Days ECM-detached condition. (d) Vitamin C treatment increased the fold change of MICA/B mRNA expression in the 6 days ECM-detached condition. (e) Vitamin C treatment increased surface MICA/B expression. 6D_Meth, 6 days detached methylated; 6D_Unmrth, 6 days detached unmethylated; 5Azadc_Meth, 5-aza-dc methyl-treated; 5Azadc_Unmeth, 5-aza-dc unmethylated; VC_meth, vitamin C methyl-treated; VC_Unmeth, vitamin C unmethylated. The data are presented as mean ± SEM; ns, not significant.