A novel crosstalk between BRCA1 and poly (ADP-ribose) polymerase 1 in breast cancer

Abstract

BRCA mutations are the main known hereditary factor for breast cancer. Notably, poly (ADP-ribose) polymerase 1 (PARP1) expression status plays a critical role in breast cancer progression and the clinical development of PARP1 inhibitors to treat BRCA-mutated breast cancer has advanced rapidly. However, dynamic crosstalk between BRCA1 and PARP1 remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation, promoter methylation, or knockdown) were accompanied by increased PARP1 and nicotinamide adenine dinucleotide (NAD) levels, and a subsequent increase in NAD-dependent PARP1 activity in MDA-MB-231 and primary breast cancer cells; (ii) the overexpression of BRCA1 resulted in decreased PARP1 and NAD levels, and a subsequent impairment in NAD-dependent PARP1 activity in MDA-MB-231 and primary breast cancer cells; and (iii) intracellular NAD levels were largely responsible for regulating PARP1 activity in breast cancer cells, and NAD levels were positively correlated with PARP1 activity in human breast cancer specimens (R = 0.647, P < 0.001). Interestingly, the high efficiency of PARP1 triggered by BRCA1 inactivation may further inhibit BRCA1 transcription by NAD depletion. These results highlight a novel interaction between BRCA1 and PARP1, which may be beneficial for the dynamic balance between BRCA1 and PARP1-related biologic processes, especially for maintaining stable DNA repair ability. All of this may improve our understanding of the basic molecular mechanism underlying BRCA1- and PARP1-related breast cancer progression.

Keywords: BRCA1; CtBP, C-terminal binding proteins; DMEM, Dulbecco's Modified Eagles Medium; DNA repair; ER, endoplasmic reticulum; ETS1, protein C-ets-1; NAD; NAD, nicotinamide adenine dinucleotide; Nampt, nicotinamide phosphoribosyltransferase; PARP1; PARP1, poly (ADP-ribose) polymerase 1; PCR, polymerase chain reaction; SD, standard deviations; TNBC, triple-negative breast cancer; breast cancer; shRNAs, short hairpin RNAs.

Figure 1.

Intracellular NAD levels, PARP1 levels and activity in non-mutated and BRCA1-mutated breast cancer. (A—C) NAD and NADH levels, and the NAD/NADH ratio were measured in 41 pairs of non-mutated and BRCA1-mutated breast cancer and their adjacent normal tissue. (D—F) PARP1 protein and mRNA levels, and PARP1 activity were measured in 41 pairs of non-mutated and BRCA1-mutated breast cancer samples and the adjacent normal tissue. Bar graphs show mean ± SD.

Figure 2.

Intracellular NAD levels, PARP1 levels and activity in breast 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 the BRCA1 RefSeq gene (exon 1 is shown as a blue box and intron 1 is shown as an arrowed line). The arrow indicates the direction of transcription. (B) Summary of the methylation patterns of the BRCA1 promoter; the y-axis shows the mean methylation sites. (C—F) BRCA1 levels, NAD levels, PARP1 levels and activity were measured in a breast cancer sample with a hypermethylated BRCA1 promoter, compared with adjacent normal tissue (unmethylated BRCA1 promoter). Bar graphs show mean ± SD (Each group, n = 15). (G and H) Correlation between the BRCA1 levels, and PARP1 levels or activity in breast cancer tissues, respectively (Each group, n = 53). (I and J) Correlation between the NAD levels, and PARP1 levels or activity in breast cancer tissues, respectively (Each group, n = 53).

Figure 3.

Effects of BRCA1 on intracellular NAD levels, PARP1 levels and activity. (A—C) NAD levels, PARP1 levels and activity after knockdown or overexpression of BRCA1 in MDA-MB-231 and MCF-7 cells (repeated 12 times), and primary non-mutated and BRCA1-mutated breast cancer cells (n = 12). Bar graphs show mean ± SD. Sh, shRNAs; Op, overexpression. *P < 0.05 vs. control.

Figure 4.

Effects of intracellular NAD on PARP1 expression and activity. (A—C) NAD levels, PARP1 levels and activity after incubation with different concentrations of NAD in MDA-MB-231 (repeated 12 times), and primary non-mutated and BRCA1-mutated breast cancer cells (n = 12). 1–5: incubation with 0, 1, 10, 100, or 1000 μM NAD. Bar graphs show mean ± SD. *P < 0.05 vs. control. (D—F) NAD levels, PARP1 levels and activity after knockdown or overexpression of Nampt in MDA-MB-231 (repeated 12 times), and primary non-mutated and BRCA1-mutated breast cancer cells (n = 12). Bar graphs show mean ± SD. Sh, shRNAs; Op, overexpression. *P < 0.05 vs. control.

Figure 5.

Effects of PARP1-mediated intracellular NAD consumption on BRCA1 levels. (A) BRCA1 levels after incubation with different concentrations of NAD in MDA-MB-231 and MCF-7 cells (repeated 12 times), and primary non-mutated and BRCA1-mutated breast cancer cells (n = 12). 1–5: incubation with 0, 1, 10, 100, or 1000 μM NAD. Bar graphs show mean ± SD. *P < 0.05 vs. control. (B and C) NAD and BRCA1 levels after knockdown or overexpression of PARP1 in MDA-MB-231 and MCF-7 cells (repeated 12 times), and primary non-mutated and BRCA1-mutated breast cancer cells (n = 12). Bar graphs show mean ± SD. Sh, shRNAs; Op, overexpression. *P < 0.05 vs. control.

Figure 6.

Proposed model of crosstalk between NAD-dependent BRCA1 and PARP1 in breast cancer. (A) BRCA1 inactivation events (mutation, promoter methylation, or other pathways) will induce PARP1 expression and NAD-dependent PARP1 activity. The high efficiency of PARP1-related NAD consumption may further inhibit BRCA1 expression, and prevent BRCA1 inactivation-mediated increases in NAD levels. (B) balancing mechanism between BRCA1 and PARP1, a proposed model to maintain stable DNA repair ability.