High-grade serous ovarian cancer (HGSOC), which makes up approximately 70 percent of ovarian cancer cases, is currently thought to originate in the epithelium of the fallopian tubes and other locations. This histological form of the disease also has among the lowest survival rates. To obtain a better understanding of the unique metabolism associated with the disease, a team led by Anil K. Sood, MD, Director of the Blanton-Davis Ovarian Cancer Research Program, Department of Gynecologic Oncology and Reproductive Medicine, Division of Surgery, The University of Texas MD Anderson Cancer Center, Houston, undertook a large-scale metabolic profiling study. Analysis revealed that striking abnormalities were present in lipid, amino acid, and carbohydrate metabolic pathways. Part of this research was recently presented at the Society of Gynecologic Oncology's 2017 Annual Meeting on Women's Cancer (Abstract 20) and part was published last year (J Natl Cancer Inst 2016; doi:10.1093/jnci/djv426).
HGSOC Metabolic Profiling
In this initial part of the study, tumor samples were obtained from 101 treatment-naïve HGSOC patients at the time of cytoreduction surgery. Sood noted, “It was important that we utilized samples from patients who had not undergone any sort of chemotherapy, so that we could obtain an accurate picture of the cancer's unaltered metabolic state.” The profiles from these HGSOC samples were compared to those of 15 noncancerous ovarian or fallopian tube tissue samples.
In the untargeted metabolic profiling stage, isolated samples were analyzed using gas chromatography mass spectrometry and ultrahigh performance liquid chromatography-tandem mass spectrometry optimized either for acidic or basic species.
Comparison of these samples revealed that 172 metabolites were present in statistically-significantly different amounts. For these metabolites, 30 were present in statistically-significantly lower levels in the HGSOC samples, while 142 were present at lower levels in the normal samples. Of the 142 metabolites more prominent in the HGSOC samples, the majority were included in lipid (53.5%) or amino acid (19.0%) metabolic pathways.
In the lipid pathway, the metabolite that showed the greatest excess in the HGSOC samples relative to the noncancerous ovarian tissue was cytidine 5'-diphosphocholine, a phosphatidylcholine intermediate, which was present at levels more than 30 times that found in the normal samples. For the amino acid pathway, the most prominent metabolite was N-acetylaspartate (NAA), which was present at levels approximately 23-fold greater in tumor than in the corresponding normal tissues.
NAA & NAT8L
The amounts of NAA in tumor samples were quantified using 1H nuclear magnetic resonance spectroscopy. The peak corresponding to the acetate's methyl group was rather distinct, being located at 2.005 ± 0.002 ppm. This structural assignment was confirmed using 1H-correlation spectroscopy (1H-COSY). NAA quantification was done by comparison of the tissue sample's 2.005 ppm NMR peak with that of a known NAA standard. Interestingly, there were no resonances for other N-acetylated amino acid derivatives in these samples, further implying the formation of NAA was not the result of widespread acetylation with the tumor environment.
To gauge the effect of NAA levels on overall survival (OS) for ovarian cancer, patients were dichotomized into those having greater than or lower than the median NAA values. Those patients having NAA levels lower than the median value had an OS duration of 5.1 years, while for those having higher-than-median levels, the figure was 3.6 years.
To determine the levels of aspartate N-acetyltransferase (NAT8L) expression in these samples, quantitative real-time polymerase chain reaction analyses were utilized. “This assay does not measure the levels of the enzyme present in the sample, rather, it quantifies the mRNA present that codes for the enzyme,” Sood clarified. The selection of NAT8L as the enzyme of choice to monitor was governed by the fact that, while its levels correlated with the elevated NAA levels in the ovarian tumor samples, the expression levels for aspartoacylase (the other main enzyme involved in the synthesis and breakdown of NAA) did not.
As with the NAA levels, HGSOC patients showed higher levels of NAT8L expression than those found in normal ovarian tissues. Cox proportional hazards model analysis showed there was a statistically-significant association between NAT8L expression levels and disease-specific survival duration when dichotomized around the median NAT8L value in these patients, affording a hazard ratio of 2.32 (95% CI-1.37-3.95; P=0.002).
NAT8L siRNA Silencing & Subsequent NAA Rescue
Reduction of NAA in ovarian cancer cells (HEYA8 & A2780 cell lines) was accomplished via siRNA-based silencing of NAT8L. It was hoped that, in doing so, a better understanding of the role NAA plays could be obtained. Initial tests showed this methodology did have the desired effects on both NAT8L and NAA levels. This silencing was shown to diminish cell proliferation as well as induce both G1 and S arrests. Additionally, apoptosis was increased and cell viability was decreased in statistically-significant manners.
In a separate experiment, A2780 cells were treated with NAT8L siRNA for 48 and 72 hours, followed by treatment with NAA. When treated with NAA, the previously-silenced samples displayed partial and complete recovery from apoptosis. This experiment highlighted the extent to which cellular proliferation was dependent upon the presence of NAA.
NAA Biological Role
A microarray was performed on A2780 cells that had been subjected to NAT8L siRNA treatment to determine the means by which that NAA mediates its anti-apoptotic effects. NAT8L siRNA was shown to downregulate the anti-apoptotic pathway in a statistically-significant manner via pathway enrichment analysis. Upstream analysis revealed the potential upstream regulator genes CREBBP, FOXM1, LEF1, and POLR2A were downregulated in a statistically-significant manner post-NAT8L siRNA treatment.
In the next experiment, A2780 cells were NAT8L-silenced in the presence and absence of NAA; the expression levels of these enzymes were then determined. In the absence of NAA with NAT8L siRNA, FOXM1 was significantly downregulated (7.7-fold; P=0.03); however, after the addition of NAA, this effect was rescued (13.8-fold; P < 0.001). This conclusively showed FOXM1 plays a critical role in the anti-apoptotic effect that NAT8L exerts.
Carbohydrate Metabolic Abnormalities
In addition to the abnormalities present in the lipid and amino acid metabolic pathways, the same profiling study also revealed anomalies within carbohydrate metabolism, specifically around the glycolytic and pentose phosphate pathways.
Once the obtained metabolites from the 101 HGSOC and 15-non-cancerous samples were classified into the proper pathways, full-scale gene expression systems-based analyses were completed. The genomic testing performed was coupled with synthetic lethal screening data for three chemotherapy-resistant cell lines. Integrated analysis of these data revealed that the carbohydrate metabolic pathway was one of the most enriched. In particular, intermediates within the glycolytic and pentose phosphate pathways were particularly prominent. Sood revealed, “Our analyses, which were capably led by Rebecca Previs, MD, showed that the key driver for these pathways was glucose-6-phosphate isomerase (GPI).”
GPI, also known as phosphohexose isomerase (PHI) or phosphoglucose isomerase, catalyzes the reversible conversion of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Because this isomerization is reversible, the direction of the conversion is dependent upon the relative concentrations of G6P and F6P.
Within the cell, GPI is involved in the glycolysis and gluconeogenesis pathways. However, this enzyme also can be secreted from the cell, and once in the extracellular domain, this enzyme is known by a number of other names: neuroleukin, maturation factor, and autocrine motility factor (AMF). For cancer cells, the phosphorylated AMF is thought to serve as a growth factor and play an important role in metastasis. Raz, et al, speculate that the phosphorylation is related to the enzyme's secretion rather than its function, as in the bacterial form of the enzyme, the phosphorylated serine (Ser-185) is replaced by a glycine residue (Biochimica et Biophysica Acta 2000;1480:235-244).
AMF, which is a secreted protein, has been shown to effect cell motility by receptor-mediated signaling, and consequently, has been implicated in tumor invasion and metastatic processes. As early as 1954, PHI was recognized as an important serum marker for the presence of metastatic breast cancer.
GPI-siRNA In Vivo Study
To assess the effect of silencing GPI upon cancer, an in vivo siRNA mouse model was utilized. “We sought to see how inhibiting the production of the enzyme would affect ovarian cancer tumors in an in vivo model,” Sood explained. “To accomplish this, we utilized an orthotopic murine model with an SKOV3ip1 ovarian cancer cell line and nano-liposomal siRNA delivery.” The GPI-siRNA group displayed significantly decreased metastases (P < 0.0001) slower tumor growth (0.0007) relative to the control siRNA group.
To determine the effects of GPI levels on the overall survival of ovarian cancer patients, a separate cohort of 75 patients were evaluated. Of these patients, 93.3 percent had advanced stage disease and more than half (56%) had high-grade serous histopathology. The median age in this cohort was 58 years.
For these patients, GPI levels were assessed using analyses and compared to the results obtained for normal ovarian and fallopian tube tissue samples.
“There was a clear association of lower overall survival for those patients having higher levels of GPI,” Sood commented. When asked about the publication of this data, he commented, “Dr. Previs, who will be the first author on this paper, is currently drafting the manuscript; we hope to have this submitted within the next month.”
Richard Simoneaux is a contributing writer.