Identification of Novel Predictive Biomarkers for Endometrial Malignancies: N-Acylethanolamines

Abstract

Objective: To identify new biochemical markers for endometrial cancer (EC). Recent evidence suggests that members of the endocannabinoid system ( N -acylethanolamines) that bind to and activate receptors that are dysregulated in EC are involved in this tumour's biology. These observations suggest increased N -acylethanolamine levels in the tissue that might appear in plasma and could be used as disease biomarkers. Methods: N -arachidonoylethanolamine (anandamide, AEA) and the N -acylethanolamine substances, N -oleoylethanolamine (OEA), and N -palmitoylethanolamine (PEA) were quantified in plasma and endometrial tissue collected from 31 EC and seven atrophic controls using UHPLC-MS/MS. Receiver-operating characteristics (ROC) and logistic regression were used to determine diagnostic accuracy. Cannabinoid receptor 1 (CB1) and 2 (CB2) protein levels were determined by specific immunohistochemistry and histomorphometric analyses. Correlations between plasma and tissue levels of the three N -acylethanolamines and tissue levels of the three N -acylethanolamines and CB1 and CB2 receptor expression levels were determined using correlation analysis. Results: Plasma and tissue AEA and PEA levels were significantly ( p < 0.05) higher in EC than controls whilst OEA levels were significantly elevated in type 1 EC tissues but not in plasma. There were significant positive correlations between plasma and tissue levels of AEA ( R 2 = 0.302, p = 0.008) and PEA ( R 2 = 0.182, p = 0.047), but not for OEA ( R 2 = 0.022, p = 0.506). The diagnostic accuracies for EC were: sensitivity of 53.3%, specificity of 100% for plasma AEA (>1.36 nM); sensitivity of 73.3%, specificity of 100% for plasma PEA (>27.5 nM); and sensitivity of 93.3%, specificity of 28.6% for plasma OEA (>4.97 nM). Logistic regression increased the area under the ROC curve (AUC) from 0.781 for AEA, 0.857 for PEA, and 0.543 for OEA to a combined AUC of 0.933 for EC diagnosis. Significant inverse correlations between tissue AEA ( R 2 = 0.343, p = 0.003) and PEA ( R 2 = 0.384, p < 0.0001) levels and CB1 expression were observed. No correlation between tissue levels of OEA and CB1 and tissue levels of any of the three N -acylethanolamines and CB2 protein expression were observed, except in the type 1 EC patients. Conclusion: Since plasma AEA and PEA are significantly elevated in patients with EC and a reflection of production by the endometrial tumour, then these lipids have the ...

The Interplay between Cancer Biology and the Endocannabinoid System—Significance for Cancer Risk, Prognosis and Response to Treatment

“…The expression of CB1R, AEA and palmitoylethanolamine lipid (PEA) were not significantly different between normal and tumour tissue although AEA and PEA showed elevated levels in EMC [76]. Statistical significance was reached in the study by Ayakanny et al who demonstrated that plasma and tissue AEA and PEA levels were significantly higher in EMC than in controls [78]. Since their levels are significantly higher in plasma of EMC patients than in the healthy controls, these biomarkers can be useful in early and non-invasive diagnosis of endometrial cancer.…”

Expression and Function of the Endocannabinoid Modulating Enzymes Fatty Acid Amide Hydrolase and N-Acylphosphatidylethanolamine-Specific Phospholipase D in Endometrial Carcinoma

“…atrophic controls; FAAH was decreased whilst NAPE-PLD was increased. Since these metabolizing enzymes are responsible for controlling "steady state" levels of the NAEs, particularly AEA (17), they could be responsible for the increased AEA and PEA levels we previously reported for EC tissues (1). The lack of correlation between plasma NAE concentrations and lymphocytic FAAH activity in all the tumor grades (Figure 1), suggest that a concomitant loss of FAAH expression and an increase in NAPE-PLD expression in the endometrium is a possible reason for the observed higher levels of these NAEs in endometrial cancer, especially Type 1 EC.…”

“…We therefore, examined the possibility that protein expression does not exactly mirror the pattern of transcript expression and found that this indeed was the case for NAPE-PLD here, where the transcripts were not significantly higher in EC, but the protein levels were. These data suggest that only FAAH expression is likely to be involved in the modulation of tissue AEA, OEA and PEA levels in EC (1). We could have used immunoblotting techniques to complement the transcript experiments, but immunoblotting techniques do not indicate where a protein is synthesized or might act.…”

“…Only type 1 EC patients were included for the measurement of lymphocytic FAAH enzyme activity in EC, because these women had been shown to have the highest plasma AEA and PEA concentrations (1). There were no differences in the ages and BMIs of the type 1 EC patients (cases) and controls (nonmalignant cases) ( Table 1A).…”

“…The levels of the endocannabinoids N-arachidonylethanolamine (anandamide; AEA), N-palmityolethanolamine (PEA), and Noleoylethanolamine (OEA) have been shown to be higher in endometrial cancer tissue of post-menopausal women when compared to that of controls (1). The key enzymes of the endocannabinoid system, N-acylphoshatidylethanolaminespecific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), which are respectively involved in the synthesis and degradation of some of the endocannabinoids, have also been shown to be expressed and regulated in normal endometrium (2)(3)(4).…”