Role of Increased n-acetylaspartate Levels in Cancer

Abstract

Background: The clinical and biological effects of metabolic alterations in cancer are not fully understood.

Methods: In high-grade serous ovarian cancer (HGSOC) samples (n = 101), over 170 metabolites were profiled and compared with normal ovarian tissues (n = 15). To determine NAT8L gene expression across different cancer types, we analyzed the RNA expression of cancer types using RNASeqV2 data available from the open access The Cancer Genome Atlas (TCGA) website (http://www.cbioportal.org/public-portal/). Using NAT8L siRNA, molecular techniques and histological analysis, we determined cancer cell viability, proliferation, apoptosis, and tumor growth in in vitro and in vivo (n = 6-10 mice/group) settings. Data were analyzed with the Student's t test and Kaplan-Meier analysis. Statistical tests were two-sided.

Results: Patients with high levels of tumoral NAA and its biosynthetic enzyme, aspartate N-acetyltransferase (NAT8L), had worse overall survival than patients with low levels of NAA and NAT8L. The overall survival duration of patients with higher-than-median NAA levels (3.6 years) was lower than that of patients with lower-than-median NAA levels (5.1 years, P = .03). High NAT8L gene expression in other cancers (melanoma, renal cell, breast, colon, and uterine cancers) was associated with worse overall survival. NAT8L silencing reduced cancer cell viability (HEYA8: control siRNA 90.61% ± 2.53, NAT8L siRNA 39.43% ± 3.00, P < .001; A2780: control siRNA 90.59% ± 2.53, NAT8L siRNA 7.44% ± 1.71, P < .001) and proliferation (HEYA8: control siRNA 74.83% ± 0.92, NAT8L siRNA 55.70% ± 1.54, P < .001; A2780: control siRNA 50.17% ± 4.13, NAT8L siRNA 26.52% ± 3.70, P < .001), which was rescued by addition of NAA. In orthotopic mouse models (ovarian cancer and melanoma), NAT8L silencing reduced tumor growth statistically significantly (A2780: control siRNA 0.52 g ± 0.15, NAT8L siRNA 0.08 g ± 0.17, P < .001; HEYA8: control siRNA 0.79 g ± 0.42, NAT8L siRNA 0.24 g ± 0.18, P = .008, A375-SM: control siRNA 0.55 g ± 0.22, NAT8L siRNA 0.21 g ± 0.17 g, P = .001). NAT8L silencing downregulated the anti-apoptotic pathway, which was mediated through FOXM1.

Conclusion: These findings indicate that the NAA pathway has a prominent role in promoting tumor growth and represents a valuable target for anticancer therapy.Altered energy metabolism is a hallmark of cancer (1). Proliferating cancer cells have much greater metabolic requirements than nonproliferating differentiated cells (2,3). Moreover, altered cancer metabolism elevates unique metabolic intermediates, which can promote cancer survival and progression (4,5). Furthermore, emerging evidence suggests that proliferating cancer cells exploit alternative metabolic pathways to meet their high demand for energy and to accumulate biomass (6-8).

Figure 1.

Relative metabolite levels in high-grade serous ovarian cancer (HGSOC) and metabolic profiling. A) The hierarchical clustering of 172 metabolites in HGSOC and normal ovaries. B) A comparison of the median levels of all metabolites in HGSOC and in normal ovary. C) NAT8L is the rate-limiting enzyme in NAA biosynthesis; NAA is metabolized by aspartoacyclase (ASPA). D) Spearman correlation (r) of relative NAA levels in 101 HGSOC samples and expression levels of genes in the NAA pathway (NAT8L and ASPA). All statistical tests were two-sided.

Figure 2.

Kaplan-Meier survival curves for patients according to NAA metabolism. A) Overall survival rates according to NAA levels in high-grade serous ovarian cancer (HGSOC) samples used for metabolic profiling. B) NAA levels in HGSOC samples used for nuclear magnetic resonance (NMR) spectroscopy. C) NAT8L gene expression in HGSOC samples from The Cancer Genome Atlas (TCGA). D) NAT8L gene expression in HGSOC samples from the Tothill database. E) Polymerase chain reaction NAT8L gene expression in HGSOC samples. F) NAT8L protein expression in HGSOC samples used for tissue microarray. G) NAT8L gene expression in breast adenocarcinoma, lung squamous cancer, kidney renal cell carcinoma, uterine endometrioid carcinoma, and colorectal adenocarcinoma from TCGA. H) NAT8L gene expression in melanoma from two different cohorts are shown. All statistical tests were two-sided.

Figure 3.

Effect of NAT8L on glucose and glutamine metabolism. 13C-based isotope tracer analysis using gas chromatography/mass spectrometry (GC/MS). A) Relative metabolite levels compared between NAT8L knockdown and control (n = 8). B) U-¹³C₆ glucose isotope labeling map of glycolysis and TCA cycle. Mass isotopomer distribution of different metabolites compared between NAT8L knockdown and control (n = 4): (C) aspartate, (D) citrate, (E) fumarate, (F) alpha-ketoglutarate, (G) glutamate. H) The percentage of U-¹³C₆ glucose contribution to TCA cycle metabolites. I) U-¹³C₅ glutamine isotope labeling map of glycolysis and TCA cycle. J) Mass isotopomer distribution of M5 glutamate, alpha-ketoglutarate, M4 fumarate, asparate, citrate, and M5 citrate. K) The percentage of U-¹³C₅ glutamine contribution to TCA cycle metabolites. Data are represented as mean ± SD (*P < .001). All statistical tests were two-sided (n = 4 from two independent experiments).

Figure 4.

Silencing NAT8L in ovarian cancer cell lines. Comparison of treatment with control (NT) siRNA and NAT8L siRNA in (A) cell proliferation, (B) cell cycle progression, (C) cell viability, and (D) apoptosis in ovarian cancer cell lines (HEYA8 AND A2780). F) Administration of 100nM NAA in A2780 cells transfected with NAT8L siRNA (*P < .001). Data are presented as mean ± SD. Unpaired t test was used. All tests were two-sided. Graphs represent replicates of multiple repeated experiments. ptx = paclitaxel.

Figure 5.

Computational Pathway Analysis and in vitro validation of the effect of NAT8L silencing on expression of anti-apoptotic genes. A) Pathway analysis using IPA software. B) The effect of NAT8L silencing on FOXM1 expression level in A2780 cells. Cells were transfected with NAT8L siRNA (100nM) for six hours before exposing to complete media with or without NAA (50nM). FOXM1 levels were assessed at 24 hours post-transfection. Data are presented as mean ± SD (*P < .001). Unpaired t test was used. All tests were two-sided. Graphs represent replicates of a single experiment.

Figure 6.

Silencing NAT8L in orthotopic mouse models of ovarian cancer and melanoma. A) A2780: siRNA NT (n = 6), siRNA NAT8L siRNA NT (n = 6); HEYA8: siRNA NT (n = 8), siRNA NAT8L siRNA NT (n = 7). B) Combination = siRNA NAT8L + paclitaxel. HEYA8: siRNA NT (n = 8), paclitaxel (n = 7), siRNA NAT8L (n = 7), siRNA combination (n = 7). C) A375-SM: siRNA NT (n = 10), siRNA NAT8L siRNA NT (n = 10). siRNA NT = control siRNA. Data are presented as mean ± SD. Unpaired t test was used. All tests were two-sided. Graphs represent replicates of a single experiment.