DNMT1 mediates metabolic reprogramming induced by Epstein-Barr virus latent membrane protein 1 and reversed by grifolin in nasopharyngeal carcinoma

Abstract

Cancer cells frequently adapt fundamentally altered metabolism to support tumorigenicity and malignancy. Epigenetic and metabolic networks are closely interactive, in which DNA methyltransferases (DNMTs) play important roles. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (EBV-LMP1) is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis because it can trigger multiple cell signaling pathways that promote cell transformation, proliferation, immune escape, invasiveness, epigenetic modification, and metabolic reprogramming. Our current findings reveal for the first time that LMP1 not only upregulates DNMT1 expression and activity, but also promotes its mitochondrial translocation. This induces epigenetic silencing of pten and activation of AKT signaling as well as hypermethylation of the mtDNA D-loop region and downregulation of oxidative phosphorylation (OXPHOS) complexes, consequently, leading to metabolic reprogramming in NPC. Furthermore, we demonstrate that grifolin, a natural farnesyl phenolic compound originated from higher fungi, is able to attenuate glycolytic flux and recover mitochondrial OXPHOS function by inhibiting DNMT1 expression and activity as well as its mitochondrial retention in NPC cells. Therefore, our work establishes a mechanistic connection between epigenetics and metabolism in EBV-positive NPC and provides further evidence for pathological classification based on CpG island methylator phenotype (CIMP) in EBV-associated malignancies. In addition, grifolin might be a promising lead compound in the intervention of high-CIMP tumor types. The availability of this natural product could hamper tumor cell metabolic reprogramming by targeting DNMT1.

Fig. 1. LMP1 promotes reprogramming of glucose metabolism in NPC cells.

a The mRNA and (b) protein levels of LMP1 in CNE1 and CNE1-LMP1 cells. c Glucose consumption and (d) lactate generation in CNE1 and CNE1-LMP1 cells. e GC/MS-based metabolome analysis of the indicated metabolite levels after labeling CNE1 and CNE1-LMP1 cells with ¹³C₆-d-glucose (“m” represents the mass of the metabolite fragment ion without any ¹³C; “m + n” (n = 2, 4, 6), where “n” represents the number of ¹³C atoms in the metabolite). f The mRNA and (g) protein levels of LMP1 in C666-1 control (con) or C666-1 shLMP1 cells. h Glucose consumption and (i) lactate generation in C666-1 con or C666-1 shLMP1 cells. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **) indicate significant differences (p < 0.05, p < 0.01, respectively). NS not significant (Student’s t test)

Fig. 2. DNMT1 mediates the downregulation of PTEN by LMP1.

The mRNA levels of the pten gene in (a) CNE1 and CNE1-LMP1 cells and (b) C666-1 con and C666-1 shLMP1 cells. The protein levels of (c) PTEN and (d) p-AKT in CNE1 and CNE1-LMP1 cells and C666-1 con and C666-1 shLMP1 cells. Methylated and unmethylated levels of the pten gene in (e) CNE1 and CNE1-LMP1 cells and (f ) C666-1 con and C666-1 shLMP1 cells. The mRNA and protein levels of DNMT1 in (g) CNE1 and CNE1-LMP1 cells and (h) C666-1 con and C666-1 shLMP1 cells. DNMT1 activity levels in (i) CNE1 and CNE1-LMP1 cells and ( j) C666-1 con and C666-1 shLMP1 cells. k The mRNA levels of pten and DNMT1 genes in CNE1-LMP1 cells treated with control siRNA (CON) or 2 different DNMT1 siRNAs (1# and 2#). l The protein levels of DNMT1, PTEN, and p-AKT were detected by western blot assay in CNE1 and CNE1-LMP1 cells. m Glucose consumption and (n) lactate generation in CNE1 and CNE1-LMP1 cells treated with control siRNA or DNMT1 siRNAs (1# and 2#). Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **, ***) indicate significant differences (p < 0.05, p < 0.01, p < 0.001, respectively)

Fig. 3. LMP1 promotes DNMT1 mitochondrial localization to inhibit OXPHOS complex genes expression.

The expression levels of DNMT1 in mitochondrial fractions, cytosolic fractions, and total cell lysates of (a) CNE1 and CNE1-LMP1 cells and (b) C666-1 con and C666-1 shLMP1 cells. VDAC serves as a mitochondria-specific marker and actin is a loading control for western blot analysis. c CNE1 and CNE1-LMP1 cells and (d) C666-1 con and C666-1 shLMP1 cells were seeded on coverslips overnight and the co-localization of DNMT1 (green) and mitochondria (red) was determined by confocal microscopy, when nuclei were stained blue. Mitochondria were stained using Mitotracker. Pearson’s correlation coefficients for the co-localization of DNMT1 and mitochondria are shown as bar graphs (scale bar, 10 μm). e The DNA levels of mitochondria-encoded genes mt-ND6, mt-ATPase6, and mt-COXII in each designated group. In the CNE1-LMP1 + 5-aza-dC group, cells were incubated in basal medium containing 5% serum containing 5-aza-dC (10 μM) for 5 days. f The DNA levels of mt-ND6, mt-ATPase6, and mt-COXII in C666-1 con and C666-1 shLMP1 cells. g The protein levels of OXPHOS complex proteins in CNE1 and CNE1-LMP1 and C666-1 con and C666-1 shLMP1 cells. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **, ***) indicate significant differences (p < 0.05, p < 0.01, p < 0.001, respectively)

Fig. 4. Grifolin targets DNMT1 to inhibit glycolysis.

Grifolin treatment inhibits (a) glucose consumption and (b) lactate generation, and (c) the proportion of glucose utilization in CNE1 and CNE1-LMP1 cells. d Effect of grifolin on PTEN expression and phosphorylation level of AKT in CNE1 and CNE1-LMP1 cells. Effect of grifolin on (e) mRNA and (f ) methylated/unmethylated levels of the pten gene in CNE1 and CNE1-LMP1 cells. Grifolin treatment decreases DNMT1 (g) protein expression and (h) enzymatic activity in CNE1 and CNE1-LMP1 cells. CNE1 and CNE1-LMP1 cells were incubated in basal medium containing 5% serum with DMSO, grifolin (10 μM), or 5-aza-dC (10 μM) for 5 days. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (**, ***) indicate significant differences (p < 0.01, p < 0.001, respectively)

Fig. 5. Grifolin blocks DNMT1 mitochondrial localization to restore OXPHOS.

Mitochondrial respiration was determined by measuring oxygen consumption rate (OCR) without (DMSO) or with grifolin treatment in (a) CNE1 and (b) CNE1-LMP1 cells. Left, seahorse extracellular flux analyzer measurements of OCR metabolic profile using the mito stress cell assay. Traces shown are representative of two independent experiments in which each data point represents replicates of five wells. Data are shown as mean values ± S.D. Middle, quantitative determination of basal OCR value of each designated group. Right, cellular energy phenotype of each designated group. c CNE1 and CNE1-LMP1 cells were treated with DMSO or grifolin (10 μM) for 3 days and intracellular NADH levels were detected using NADH-specific sensor Frex-Mit at the excitation wavelength of 485 nm. cpYFP-Mit was used as a negative control and the light imaging of each field as cell density control. Quantification of the fluorescent intensity of Frex-Mit imaging is shown as bar graphs. d Grifolin treatment rescues CI activity downregulated by LMP1. CNE1 and CNE1-LMP1 cells were treated with DMSO or grifolin (10 μM) for 5 days and CI activity was measured. e Grifolin treatment inhibits translocation of DNMT1 to mitochondria. CNE1 and CNE1-LMP1 cells were seeded on coverslips overnight and treated with DMSO or grifolin (10 μM) for 5 days, respectively. The co-localization of DNMT1 (green) and mitochondria (red) was detected by confocal microscopy, and nuclei are stained blue. Pearson’s correlation coefficients for the co-localization of DNMT1 and mitochondria are shown as bar graphs. Scale bar, 10 μm. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **, ***) indicate significant differences (p < 0.05, p < 0.01, p < 0.001, respectively)

Fig. 6. Grifolin treatment restores OXPHOS complexes expression.

CNE1 and CNE1-LMP1 cells were treated with DMSO, 5-aza-dC (10 μM) or grifolin (10 μM) for 5 days. a The methylated/unmethylated levels of the D-loop region in mitochondrial DNA was determined by MSP analysis. b–d The DNA levels of mitochondria-encoded ND6, COXII, and ATPase6 genes were examined by real-time PCR in each designated group in CNE1 and CNE1-LMP1 cells. Data are shown as mean values ± S.D. of independent, triplicate experiments. The asterisks (*, **, ***) indicate significant differences (p < 0.05, p < 0.01, p < 0.001, respectively). The OXPHOS complex I–V proteins were detected by western blot analysis in each designated group in (e) CNE1 and (f) CNE1-LMP1 cells. UM unmethylation, M methylation