BRCA1 Deficiency Upregulates NNMT, Which Reprograms Metabolism and Sensitizes Ovarian Cancer Cells to Mitochondrial Metabolic Targeting Agents

Abstract

BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. SIGNIFICANCE: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

Figure 1.. CDK12 depletion downregulates BRCA1, which causes decreased ATP levels and suppresses mitochondrial respiration.

(A) Analysis of ATP levels in CDK12-depleted cells. OVCAR-8 (top panel) and PEA1 (bottom panel) cells were transfected with control luciferase (siLuc) or CDK12 siRNAs (siCDK12 #1 and siCDK12 #2). 48 h after transfection, ATP levels were measured (right panels), and CDK12 and tubulin levels were analyzed by immunoblotting (left panels). Data are means ± SEM, n = 3 independent experiments. *P < 0.05, **P < 0.01, unpaired t test. (B) Effect of CDK12 depletion on oxygen consumption rate (OCR). OVCAR-8 or PEA1 cells were transfected with control siLuc or CDK12 siRNAs. 48 h later, OCRs were measured under basal conditions and following the sequential additions of oligomycin, FCCP, and rotenone/antimycin A using a Seahorse XFp extracellular flux analyzer. Data are representative of 3 independent experiments. (C) BRCA1 depletion also disrupts OCR. OVCAR-8 and PEA1 cells were transfected with control siLuc or BRCA1 siRNAs (siBRCA1 #1 and siBRCA1 #2). 48 h later, BRCA1 and TUBULIN were analyzed by immunoblotting (left panels), and OCR was measured as described in B (right panels). Data are means ± SEM, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test. (D) Ectopic BRCA1expression rescues the OCR defect induced by BRCA1. OVCAR-8 (top panel) and PEA1 (bottom panel) cells were transfected with siLuc or CDK12 siRNAs plus empty vector (EV) or SFB-BRCA1 plasmid (SFB-BRCA1). OCR was measured as described in B. Data are means ± SEM, n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test.

Figure 2.. BRCA1 depletion induces NNMT upregulation, which causes decreased OCR.

(A) OVCAR-8 and PEA1 cells were transfected with control luciferase (siLuc), BRCA1, or NNMT siRNAs. 48 h later, the cells were analyzed by qPCR for NNMT and BRCA1 mRNA levels, which are expressed relative to GAPDH mRNA levels as an internal control (left panels) and immunoblotted for NNMT, BRCA1, and tubulin (right panel). Data are means ± SEM, n = 3 independent experiments. **P < 0.01, ***P < 0.001, unpaired t test. (B) BRCA1 and CDK12 mRNA and protein levels are inversely correlated with NNMT mRNA and protein. Scatter plots of NNMT mRNA expression as a function of BRCA1 and CDK12 mRNA expression in HGSOC tumors from patients and PDX models (left two panels). Scatter plots of NNMT protein as a function of BRCA1 mRNA and CDK12 protein levels in HGSOC tumors from patients. Spearman or Pearson correlations are shown in the images. (C) Co-depletion of NNMT reverses the OCR defect induced by BRCA1 depletion. OVCAR-8 (top panel) and PEA1 (bottom panel) cells were transfected with siLuc, BRCA1, or NNMT siRNAs individually or co-transfected with BRCA1 and NNMT siRNAs. OCR was measured as described in Fig. 1B and NNMT, BRCA1, and tubulin levels were measured by immunoblotting. Data are means ± SEM, n = 3 independent experiments. *P <0.05, **P <0.01, ***P <0.001, unpaired t test. (D) NNMT overexpression phenocopies the OCR defect induced by BRCA1 depletion. OVCAR-8 cells were transiently transfected with empty vector (EV) or a plasmid that expresses Myc-DDK-tagged NNMT. After 24 h, OCR was analyzed as described in Fig. 1B and cells were immunoblotted for NNMT and tubulin. Data are means ± SEM, n = 3 independent experiments. *P <0.05, **P <0.01, ***P <0.001, unpaired t test.

Figure 3.. BRCA1 depletion sensitizes ovarian cancer cells to agents that disrupt metabolism.

(A-C) OVCAR-8 and PEA1 cells were transfected with control luciferase (siLuc) or BRCA1 siRNAs. 48 h later, the cells were trypsinized, immunoblotted for BRCA1 and tubulin (bottom panels), and subjected to colony formation assays. The indicated concentrations of VLX600 (A), tigecycline (B), or WZB117 (C) were added 12 h after plating, and the cells were cultured for 8–10 days to allow colony formation. Data are representative of 3 independent experiments. Error bars: means ± SEM of three technical replicates in the representative experiment.

Figure 4.. NNMT overexpression sensitizes ovarian cancer cells to agents that disrupt metabolism.

(A and B) Clones of OVCAR-8 cells stably transfected with empty vector (pcDNA3) or the Myc-DDK-NNMT expression plasmid were subjected to immunoblotting for NNMT and tubulin (A, top panel), examined for OCR as described in Fig. 1B (A, bottom panel), and treated with VLX600 (B, top panel) or tigecycline (B, top panel), which were added 12 h after plating the cells. The cells were cultured for 8–10 days to allow colony formation. Data in A (bottom panel) are means ± SEM, n = 3 independent experiments, **P <0.01, unpaired t test. Data in B are representative of 3 independent experiments. Error bars: means ± SEM of three technical replicates for each data point in the representative experiment. (C) VLX600 further reduces ATP levels in NNMT overexpressing cells (top panel) and BRCA1-depleted cells (bottom panel). Stable clones of the OVCAR-8-EV or the OVCAR-8-Myc-DDK-NNMT cells were treated with 50 nM VLX600 for 24 h, and OCR was measured as described in Fig. 1B (top panel). Control luciferase (siLuc)- or BRCA1 siRNA-transfected OVCAR-8 cells were treated with indicated concentrations of VLX600 for 24 h and OCR was measured (bottom panel) Data are means ± SEM, n = 3 independent experiments, *P <0.05, **P <0.01, unpaired t test. (D) Model for the increased cytotoxic effect of metabolic inhibitors in BRCA1-deficient cells. Left panel: In BRCA1-proficient cells low NNMT levels lead to higher ATP levels so that these cells are less sensitive to metabolic inhibitors. Right panel: In BRCA1-deficient cells, NNMT levels are increased, leading to reduction in OXPHOS and ATP levels. These cells are more sensitive to OXPHOS inhibitors, because they are already metabolically stressed and unable to meet their energy demands when challenged with inhibitors that further reduce ATP levels.