New target proteins for drug discovery in colon cancer

Background: Colorectal cancer (CRC) is one of the most common causes of death in Western countries. Because of the high heterogeneity and incidence of this disease, it is crucial to improve the knowledge on its biology – a fundamental step for drug discovery - and to develop clinically-relevant diagnostic and prognostic biomarkers. Evidence suggests that MAGL [(a serine hydrolase that converts monoacylglycerols, such as the endocannabinoid 2-arachydonoyl glycerol (2-AG), in glycerol and fatty acid], NAAA [(a cysteine hydrolase responsible of the catabolism of palmitoylethanolamine (PEA)] and GPR35 (an orphan-G protein coupled receptor) affect biological events (e.g. proliferation, differentiation, survival) and regulates pathophysiological states (e.g. intestinal inflammation) which are suggestive of a possible involvement in CRC. Here, we explored the possible contribution of MAGL, NAAA and GPR35 to colon tumorigenesis. Material and Methods: The role of MAGL, NAAA and GPR35 was assessed in vivo, via genetic or pharmacological blockade, in APCmin mice as well as in the azoxymethane (AOM), AOM/dextran sodium sulfate (DSS) and xenograft models. Cell proliferation was evaluated in CRC cells, healthy colonic epithelial cells and colonic organoids 3D culture by using the BrdU and EdU incorporation; migration was examined in CRC and endothelial cells by using the scratch assay. Angiogenesis was assessed in tumour tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in the aortic ring model and in endothelial cells by using the tube formation assay. Cell metabolism was measured by the quantification of extracellular acidification rate (ECAR), oxygen consumption rate (OCR), lactate production and glucose uptake in bone marrow derived macrophages (BMDM). MAGL, NAAA and GPR35 expression was evaluated by RT-PCR and immunohistochemistry; 2-AG and PEA levels were measured by liquid chromatography mass spectrometry. Results: MAGL- The MAGL inhibitor URB602 reduced xenograft tumour growth, the effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. A direct antiangiogenic effect was observed in human endothelial cells, too. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated AOM-induced preneoplastic lesions and tumours in wild-type but not in MAGL-deficient mice. NAAA- NAAA expression was reduced in biopsies of clinically-diagnosed CRC patients as well as in CRC cells incubated with tumour secretome. The NAAA inhibitor AM9053 and its substrate PEA inhibited proliferation and migration in CRC cells. Increased PEA levels in vivo (via NAAA inhibition or by its exogenous administration) resulted in chemopreventive effects in the AOM model of colon carcinogenesis and reduced xenograft tumour growth. GPR35- GPR35 deletion resulted in a reduction of cell energetic demand and production in M0-, M1- and M2- BMDM. Also, depletion of GPR35 in M2-BMDM decreased their capability to produce the CXCL1 (pro-angiogenic chemokine) and to stimulate the tube formation of endothelial cells. Also, GPR35-/- mice showed a reduced intestinal turnover in physiological conditions and, importantly, a reduced colon tumorigenesis, as highlighted in two different experimental models of colon cancer. It is noteworthy that the protective role of GPR35 in APCmin mice was confirmed with the conditional deletion of Gpr35 (Villin-Cre) in the intestinal epithelium. Conclusions: In summary, by elucidating the physiopathological role of MAGL, NAAA and GPR35 in experimental colon tumorigenesis and by ascertain their dysregulation in intestinal tumours, this PhD thesis put forth such proteins as possible innovative prognostic markers in clinically-diagnosed CRC and as new molecular targets to be explored in drug discovery.