N-Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

Abstract

Background and purpose: N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme accountable for the breakdown of N-acylethanolamines (NAEs) and its pharmacological inhibition has beneficial effects in inflammatory conditions. The knowledge of NAAA in cancer is fragmentary with an unclarified mechanism, whereas its contribution to colorectal cancer (CRC) is unknown to date.

Experimental approach: CRC xenograft and azoxymethane models were used to assess the in vivo effect of NAAA inhibition. Further, the tumour secretome was evaluated by an oncogenic array, CRC cell lines were used for in vitro studies, cell cycle was analysed by cytofluorimetry, NAAA was knocked down with siRNA, human biopsies were obtained from surgically resected CRC patients, gene expression was measured by RT-PCR and NAEs were measured by LC-MS.

Key results: The NAAA inhibitor AM9053 reduced CRC xenograft tumour growth and counteracted tumour development in the azoxymethane model. NAAA inhibition affected the composition of the tumour secretome inhibiting the expression of EGF family members. In CRC cells, AM9053 reduced proliferation with a mechanism mediated by PPAR-α and TRPV1. AM9053 induced cell cycle arrest in the S phase associated with cyclin A2/CDK2 down-regulation. NAAA knock-down mirrored the effects of NAAA inhibition with AM9053. NAAA expression was down-regulated in human CRC tissues, with a consequential augmentation of NAE levels and dysregulation of some of their targets.

Conclusion and implications: Our results show novel data on the functional importance of NAAA in CRC progression and the mechanism involved. We propose that this enzyme is a valid drug target for the treatment of CRC growth and development.

Keywords: acylethanolamides; colon cancer; endocannabinoid system.

FIGURE 1

N‐acylethanolamine acid amidase (NAAA) selective inhibition reduces CRC growth in vivo. (a) Effect of the NAAA inhibitor AM9053 (20 mg·kg⁻¹, peritumourally) on in vivo tumour growth, measured as tumour volume (mm³), over a 15‐day period (seven measurements). Each dot represents the mean ± SD of 13 mice for vehicle and 15 mice for AM9053 experimental groups; *P < 0.05 versus vehicle by two‐way ANOVA. (b) Representative macroscopic pictures of tumour‐bearing mice (upper images) as well as of the explanted tumours (lower images), with the respective treatments, at the day of the kill. (c) Weights (reported in mg) of the xenograft tumours explanted from tumour‐bearing mice treated (n = 15) or not (n = 13) with AM9053 (20 mg·kg⁻¹, peritumourally). (d–f) Endogenous‐level measurements of NAEs: N‐oleoylethanolamine, OEA (d), N‐palmitoylethanolamine, PEA (e) and anandamide (AEA) (f) in mouse xenograft tumours treated (n = 6) or not (n = 7) with AM9053 (20 mg·kg⁻¹, peritumourally). The levels of NAEs are reported as pmol·mg⁻¹ of wet tissue weight. (g) Representative high‐end fluorescence images of Ki‐67 expression in frozen sections of tumours explanted from tumour‐bearing mice treated (lower images) or not (upper images) with AM9053 (20 mg·kg⁻¹, peritumourally). Ki‐67 staining is visualized in red, and nuclei were counterstained with DAPI in blue. Magnification: 20× (white scale bar 50 μm). (h) Graph showing the intensity mean value (±SD) of red Ki‐67‐positive cells. Values are expressed as means ± SD; *P < 0.05 versus vehicle, as assessed by Student's t test; n.s., not significant

FIGURE 2

The N‐acylethanolamine acid amidase (NAAA) selective inhibitor AM9053 affects tumour cell proliferation. (a) Cell viability assay in HCT116 and (b) in healthy human colonic epithelial cells (HCEC) alone or in the presence of AM9053 (0.1–3 μM, 24 h) (n = 5). Results are expressed as mean ± SD. (c) Cell proliferation rate of HCT116 cells incorporating bromodeoxyuridine (BrdU), alone or in the presence of AM9053 (0.1–3 μM, 24 h). Results are expressed as percentage of cell proliferation (n = 5 independent experiments). Values are expressed as means ± SD; *P < 0.05 versus control, as assessed by one‐way ANOVA followed by Dunnett's multiple comparisons test. (d) Cell proliferation rate of HCEC incorporating BrdU, alone or in the presence of AM9053 (3 μM, 24 h). Results are expressed as percentage of cell proliferation (n = 6). Values are expressed as means ± SD; n.s., not significant as assessed by unpaired Student's t‐test. (e) The antiproliferative effect of AM9053 (3 μM) was also evaluated in the presence of GW6471 (3 μM, PPAR‐α antagonist), 5′‐iodoresiniferatoxin (I‐RTX 0.1 μM, TRPV1 antagonist), AM251 (1 μM, CB₁ antagonist) and AM630 (1 μM, CB₂ antagonist). All results are expressed as percentage of cell proliferation (n = 5 independent experiments) and as means ± SD; *P < 0.05 versus control and/or AM9053 as assessed by one‐way ANOVA followed by Tukey's multiple comparisons test

FIGURE 3

The inhibitor AM9053 blocks tumoural cell cycle. (a) Cell cycle analysis by flow cytometry of HCT116 cells treated or not with AM9053 (3 μM, 24 h). Results are expressed as fold change of the cells in each cell cycle phase (n = 8 independent experiments) in order to uniform the % values of the cells in each phase of the cell cycle (n = 1 outlier has been removed by ROUT test). Values are expressed as means ± SD; *P < 0.05 versus control, as assessed by unpaired Student's t test and/or Mann–Whitney test. (b) Representative density plots and cell percentage, indicating the G0/G1‐, G2/M‐ and S‐phase distribution of HCT116 cells treated (right figure) or not (left figure) with AM9053 (3 μM, 24 h). Gene expression, in HCT116 cells, alone or in the presence of AM9053 (3 μM, 24 h), of cyclin A2 (c), cyclin‐dependent kinase 2 (CDK2) (d), cyclin B1 (e) and cyclin‐dependent kinase 1 (CDK1) (f). Gene expression was measured by qRT‐PCR and calculated by using the 2^−ΔCt formula (n = 6 independent experiments). Values are expressed as means ± SD; *P < 0.05 versus control and analysed by Student's t‐test; n.s., not significant

FIGURE 4

Transient knock‐down of N‐acylethanolamine acid amidase (NAAA) is associated with an antiproliferative effect. (a) NAAA gene expression in HCT116 cells alone (untransfected) or in the presence of scramble small interfering (si)RNA and/or NAAA siRNA (n = 6 independent experiments). NAAA gene expression was measured by qRT‐PCR and calculated by using the 2^−ΔCt formula. Values are expressed as means ± SD; *P < 0.05 versus scramble siRNA and/or NAAA siRNA as assessed by one‐way ANOVA followed by Kruskal–Wallis comparisons test; n.s., not significant. (b) Cell proliferation rate of HCT116 cells incorporating BrdU in the following conditions: alone (untransfected), untransfected in the presence of AM9053 (3 μM, 24 h), scramble siRNA, NAAA siRNA and NAAA siRNA in the presence of AM9053 (3 μM, 24 h). Results are expressed as percentage of cell proliferation (n = 5 independent experiments) and as means ± SD; *P < 0.05 versus untransfected and/or NAAA siRNA, as assessed by one‐way ANOVA followed by Tukey's multiple comparisons test; n.s., not significant. (c) Cell cycle analysis by flow cytometry of HCT116 cells in the presence of scramble siRNA and/or NAAA siRNA. Results are expressed as fold change of the cells in each cell cycle phase (n = 6) in order to uniform the % values of the cells in each phase of the cell cycle. Values are expressed as means ± SD; *P < 0.05 versus scramble siRNA, as assessed by unpaired Student's t‐test. (d) Representative density plots and cell percentage, indicating the G0/G1‐, G2/M‐ and S‐phase distribution of HCT116 cells in the presence of scramble siRNA (upper figure) and/or NAAA siRNA (lower figure). Gene expression, in HCT116 cells in the presence of scramble siRNA and/or NAAA siRNA, of cyclin A2 (e), CDK2 (f), cyclin B1 (g) and CDK1 (h). Gene expression was measured by qRT‐PCR and calculated by using the 2^−ΔCt formula (n = 6) (n = 1 outlier has been removed by ROUT test for cyclin B1 analysis). Values are expressed as means ± SD; *P < 0.05 versus scramble siRNA, as assessed by Student's t‐test; n.s., not significant

FIGURE 5

N‐acylethanolamine acid amidase (NAAA) expression and N‐acylethanolamine (NAE) levels in human colorectal cancer tissues. NAAA gene expression in (a) human colon cancer tissues versus adjacent non‐tumour colon specimens (defined as ‘healthy’) (n = 16) and (b) different tumour stages (pT2 [n = 5], pT3 [n = 6] and pT4 [n = 5]) (n = 3 outliers have been removed by ROUT test). NAAA expression was measured by qRT‐PCR and calculated by using the 2^−ΔCt formula. (c) NAAA relative transcript levels to housekeeping genes (expressed as log2) in healthy (n = 393) and colorectal cancer patients (n = 3769) extrapolated from Gene Expression across Normal and Tumour tissue (GENT2) database interrogation (n = 9 outliers have been removed by ROUT test). Endogenous‐level measurements of N‐acylethanolamines (NAEs): N‐palmitoylethanolamine (PEA) (d), N‐oleoylethanolamine (OEA) (e) and anandamide (AEA) (f) in human colon cancer tissues (n = 8) and adjacent non‐tumour colon specimens (n = 8) (n = 1 outlier has been removed by ROUT test). The levels of NAEs are reported as pmol·mg⁻¹ of wet tissue weight. All the data are expressed as means ± SD; *P < 0.05 versus ‘healthy’, as assessed by paired Student's t‐test; n.s., not significant