BRCA1 as a nicotinamide adenine dinucleotide (NAD)-dependent metabolic switch in ovarian cancer

Abstract

Both hereditary factors (e.g., BRCA1) and nicotinamide adenine dinucleotide (NAD)-dependent metabolic pathways are implicated in the initiation and progression of ovarian cancer. However, whether crosstalk exists between BRCA1 and NAD metabolism remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation and promoter methylation) were accompanied by elevated levels of NAD; (ii) the knockdown or overexpression of BRCA1 was an effective way to induce an increase or decrease of nicotinamide phosphoribosyltransferase (Nampt)-related NAD synthesis, respectively; and (iii) BRCA1 expression patterns were inversely correlated with NAD levels in human ovarian cancer specimens. In addition, it is worth noting that: (i) NAD incubation induced increased levels of BRCA1 in a concentration-dependent manner; (ii) Nampt knockdown-mediated reduction in NAD levels was effective at inhibiting BRCA1 expression; and (iii) the overexpression of Nampt led to higher NAD levels and a subsequent increase in BRCA1 levels in primary ovarian cancer cells and A2780, HO-8910 and ES2 ovarian cancer cell lines. These results highlight a novel link between BRCA1 and NAD. Our findings imply that genetic (e.g., BRCA1 inactivation) and NAD-dependent metabolic pathways are jointly involved in the malignant progression of ovarian cancer.

Keywords: BRCA1; BRCA1, breast cancer type 1 susceptibility protein; CtBP, C-terminal binding proteins; NAD; NAD, nicotinamide adenine dinucleotide; Nampt, nicotinamide phosphoribosyltransferase; NADH; Nampt; PCR, polymerase chain reaction; ovarian cancer; shRNAs, short hairpin ribonucleic acids.

Figure 1.

Intracellular NAD levels in non-BRCA1-mutated, BRCA1-mutated, and BRCA2-mutated ovarian cancer. (A–C), NAD levels, NADH levels, and NAD/NADH ratio were measured in 28 pairs of non-BRCA1-mutated and BRCA1-mutated ovarian cancer and their adjacent normal tissue. Bar graphs show mean ±SD. (D) BRCA1 mRNA levels and NAD levels were measured in A2780, SKOV-3, CAOV-3, HO-8910, and ES2 ovarian cancer cell lines (repeated 12 times). Bar graphs show mean ± SD. (E) correlation between BRCA1 mRNA levels and NAD levels in 40 non-BRCA1-mutated ovarian cancer specimens. (F–H) NAD levels, NADH levels, and NAD/NADH ratio were measured in 23 pairs of non-BRCA2-mutated and BRCA2-mutated ovarian cancer and their adjacent normal tissue. Bar graphs show mean ±SD.

Figure 2.

Intracellular NAD levels in ovarian cancer with hypermethylated promoter-mediated BRCA1 inactivation. (A) the location of CpG sites in the core promoter region of BRCA1. Genomic coordinates are shown, along with the primer-amplified fragments, GC percentage, location of individual CpG dinucleotides (dashes), and BRCA1 RefSeq gene (exon 1 is shown as a blue box and the intron is shown as an arrowed line). The arrow indicates the direction of transcription. (B, C) comparative analysis of methylation patterns in the core promoter region of BRCA1 in ovarian cancer and adjacent normal tissue. The circles correspond to the CpG sites denoted by black dashes in (A). Closed circles, methylation; open circles, unmethylated. Ten individual clones were sequenced for each sample. (D, G) summary of the methylation levels of BRCA1 core promoter from the measurements shown in B and C, respectively. (E, H) relative BRCA1 mRNA levels were measured in ovarian cancer with identified hypermethylated or unmethylated BRCA1 promoter, compared with their adjacent normal tissue. (F, I) intracellular NAD levels were measured in ovarian cancer with identified BRCA1 inactivation or not, respectively. Bar graphs show mean ± SD. Each group, n = 19.

Figure 3.

Effects of BRCA1 on intracellular NAD levels. Relative NAD levels after knockdown or overexpression of BRCA1 in 293T cells, A2780, SKOV-3, CAOV-3, HO-8910, and ES2 ovarian cancer cell lines, all wild-type for BRCA1 (repeated 12 times), and primary non-BRCA1-mutated (wild-type for BRCA1) and BRCA1-mutated (mutant-type for BRCA1) ovarian cancer cells (n = 28). Bar graphs show mean ± SD. Sh, shRNAs; Op, overexpression. * P < 0.05 vs. control.

Figure 4.

Effects of BRCA1 on intracellular Nampt levels. (A, B) Nampt mRNA and protein levels were measured in 23 pairs of non-mutated, 28 pairs of BRCA1-mutated, and 23 pairs of BRCA2-mutated ovarian cancer and their adjacent normal tissue. (C) Nampt mRNA levels after knockdown or overexpression of BRCA1 in 293T cells, A2780, SKOV-3, CAOV-3, HO-8910, and ES2 ovarian cancer cell lines, all wild-type for BRCA1 (repeated 12 times), and primary non-BRCA1-mutated (wild-type for BRCA1) and BRCA1-mutated (mutant-type for BRCA1) ovarian cancer cells (n = 12). Bar graphs show mean ± SD. Sh, shRNAs; Op, overexpression. * P < 0.05 vs. control.

Figure 5.

Effects of intracellular NAD on BRCA1 levels. (A, B) relative NAD or BRCA1 levels after incubation with different concentrations of NAD in 293T cells, A2780, SKOV-3, CAOV-3, HO-8910, and ES2 ovarian cancer cell lines (repeated 12 times), and primary non-BRCA1-mutated and BRCA1-mutated ovarian cancer cells (n = 28). One to five: incubation with 0, 1, 10, 100, or 1000 μM NAD. Bar graphs show mean ±SD. * P < 0.05 vs. control. (C, D) relative NAD or BRCA1 levels after knockdown or overexpression of Nampt in 293T cells, A2780, SKOV-3, CAOV-3, HO-8910, and ES2 ovarian cancer cell lines (repeated 12 times), and primary non-BRCA1-mutated and BRCA1-mutated ovarian cancer cells (n = 28). Bar graphs show mean ± SD. Sh, shRNAs; Op, overexpression. *P < 0.05 vs. control.