The Suppression Of N-3 PUFAs On MTORC1 Sinaling Pathway And Colorectal Tumorigenesis

Background and aimsColorectal cancer ranks second among women and third men in all cancers worldwide. The APC gene, the most important tumor suppressor gene in Wnt/β-catenin signaling pathway, had been currently widely studied in colorectal cancer.lt is closely related to not only familial adenomatous polyposis (FAP), but also sporadic colorectal cancer. Dove laboratory established a good experimental model---C57BL/6J-APCMin/+ mouse strains,with the two strands of the APC suffering nonsense mutations on codon 850,thereby resulting in multiple intestinal adenomas. Meanwhile, a large number of studies have shown that cancer patients benefit from dietary changes,and colorectal cancer is closely associated with dietary factors, including polyunsaturated fatty acids (PUFAs), a hot research point. PUFAs are fatty acids containing two or more unsaturated double bonds.n-3 PUFAs regulate cardiovascular function,as well as have anti-atherogenic, anti-inflammatory, anti-tumor and other special effects. Studies have demonstrated that imbalance dietary intake of fatty acids is one of the major risk factors for colorectal cancer, and what affects cellular homeostasis and normal growth process most is not the absolute level of n-6 PUFAs or n-3 PUFAs, but the ratio of n-6/n-3 PUFAs.The fat-1 gene, mainly found in some lower plants, microbes and nematodes in recent years,encoding an n-3 fatty acid desaturase. Since this enzyme can catalyze the conversion of n-6 PUFA to n-3 PUFA by introducing a double bond into fatty acyl chains, transgenic expression of fat-1 enables the host to produce n-3 PUFAs endogenously while concomitantly reducing the levels of n-6 PUFAs. The fat-1 transgenic mice will also enable the investigation of the biological properties of n-3 PUFAs without having to incorporate n-3 PUFAs in the diet, becoming a more reliable and intuitive model.The mechanistic target of rapamycin (mTOR) is a Ser/Thr kinase that regulates cell growth, metabolism, protein translation, lipid synthesis, mitochondrial energy metabolism, cell growth and proliferation, aging and other kind of significant physiological processes. mTOR works depending on the context of two functionally distinct signaling complexes, named mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTORCl consists of mTOR, mLST8/GβL, Raptor and PRAS40, and is sensitive to rapamycin. Cell growth factor and insulin activates and phosphorylates PI3K/Akt, which could inhibit tuberous sclerosis complex 1/2 (TSC 1/2), thereby weakening the suppression of TSC 1/2 on Rheb and further activating mTORCl.mTORC2 is composed of mTOR,mLST8,Rictor,PRR5 and mSinl.The function of mTORC2 is unclear, but it has been shown to phosphorylate Akt at serine 473 and to regulate cytoskeleton organization and cell motility. Recent evidence indicates that,mTOR sinaling is a key factor in promoting the development of a variety of tumors,which is closely related to the occurrence and development of colorectal cancer,currently about 40% of colorectal tumor specimens observed activation of mTORCl signaling pathway.However, the interaction of n-3 PUFAs,colorectal cancer and mTOR signaling is unknown.More and more clinicians now have found that there is a close relationship between intestinal tumors and lipids. Moser reported that APCMin/+ mice with severe anemia almost suffer from high hyperlipidemia. Lipoprotein lipase (LPL) and peroxisome proliferator-activated receptors (PPAR)have essential effects in regulating fatty substances. Recent reports suggest that, in adipose tissue,mTOR plays a key regulatory role in lipid uptake mediated by LPL and promoting lipid synthesis activated by activation of the PPAR.Therefore,we selected ApeMin/+ mouse as a model of colorectal cancer, together with fat-1 mouse that endogenously produces n-3 PUFAs,to explore the effects of n-3 PUFAs on colorectal cancer and relevant lipid changes,and on mTOR signaling pathway.Methods1. We selected ApcMin/+ mouse as a model of colorectal cancer, together with fat-1 mouse that endogenously produces n-3 PUFAs, to investigate the effects of endogenousing n-3 PUFAs on intestinal adenomas, lipids as well as mTORC1 signaling pathways.2. Colorectal tumor cell lines were treated with various growth factors, and at the same time, with DHA/the fat-1 cDNA, to assess the effect of DHA/the fat-1 cDNA on the activity and stability of mTORC1, and that on the proliferation and apoptosis of colorectal tumor cells.3. We treat normal colorectal cells and adipose-derived stem cells with APC-SiRNA or the fat-1 cDNA,to investigate the effects of fat-1 on mTORCl signaling pathways..Results1、Reduced mTORCl activities and suppression of colorectal tumorigenesis in fat-1 transgenic ApcMin/+ colorectal cancer mouse modelFat-1 transgenic ApcMin/+ colorectal cancer mouse models were created by mating these two kinds of mice.Fatty acid composition analysis confirmed an increased ratio of n-3/n- 6 PUFAs compared with the control mice. The number of intestinal adenomas in fat-1 -ApcMin/+ mice significantly decreased,and the volume reduced. Moreover, phosphorylation of S6 (S235/236) were reduced in the adenomas of fat-1 -ApcMin/+ mice. These data clearly show that endogenous n-3 PUFAs in mouse decreases mTORCl activities and prevents colorectal carcinogenesis.2、n-3 PUFAs inhibits mTORCl signaling in colorectal cancer cell lines.We first examined whether dietary and endogenous n-3 PUFAs inhibit mTORC1 in colorectal cancer cell lines. DHA or the fat-1 cDNA rapidly and dose-dependently suppressed insulin-, AA- and amino acids-stimulated mTORCl-directed phosphorylation of S6 (S235/235) in HCT116 and SW480 cells. In NCM460 cells transfected with APC-SiRNA and the fat-1 cDNA, the phosphorylation of S6 (S235/235) significantly reduced. These results suggest that mTORCl signaling pathways involves in the suppression of endogenous n-3 PUFAs on colorectal cancer.3、Reduced serum lipid levels in fat-1 transgenic ApcMin/+ colorectal cancer mouse model and reduced mTORCl activities in adipose-derived stem cells trasnfected with APC-SiRNASerum lipid levels in fat-1-ApcMin/+ mice significantly decreased, and the fat-1 cDNA is able to inhibit the activites of mTORC1 in adipose-derived stem cells transtected with APC-SiRNA. This shows that the ratio n-3/n-6 inhibits effectively betters hyperlipidemia associated with colorectal cancer and mTORC1 involves in the suppression of endogenous n-3 PUFAs on hyperlipidemia associated with colorectal cancer.Conclusion1. Both dietary and endogenous n-3 PUFAs prevented colorectal carcinogenesis, tumor growth.2. mTORC1 sinaling pathway involved in the suppression of endogenous n-3 PUFAs on colorectal cancer and lipid metabolism associated with colorectal cancer3. Our findings convincingly clarify the causal relationship between n-3 PUFAs and colorectal cancer prevention and provide evidence for n-3 PUFAs in colorectal cancer treatment.