Study On Prevention Of Endometrial Cancer By N-3PUFAs And Its Mechanisms

BackgroundEndometrial cancer is the most common gynecologic malignancy and a major cause of morbidity and mortality for women worldwide, with nearly200000cases diagnosed every year and a rising incidence in postmenopausal women. Although the precise cause of the endometrial cancer is unknown, various associated risk factors for the disease have been identified. The main risk factors for the development of endometrial carcinoma are obesity and chronic unopposed oestrogen stimulation of the endometrium. Previous publications demonstrate that parity, oral contraception, body mass index (BMI), physical activity and diet may explain up to80%of the risk of endometrial cancer, emphasizing the importance of lifestyle modification for prevention of this disease. On the other hand, although it is highly treatable by surgery when diagnosed at an early stage and grade, therapies for advanced and recurrent disease are rarely curative. Currently, treatment of metastatic or recurrent disease is based on conventional chemotherapy combination regimens. Advances in the understanding of the molecular pathology of endometrial carcinoma lead to the development and testing of targeted therapies. Of the potential therapeutic targets identified to date, the mammalian target of rapamycin (mTOR) signaling pathway is a major target for treatment of the disease.Mammalian target of rapamycin (mTOR) is a highly conserved Ser/Thr kinase which integrates diverse signals including nutrients, growth factors, energy and stresses to control cell growth, proliferation, survival, and metabolism. mTOR elicits its pleiotropic functions in the context of two functionally distinct signaling complexes, termed mTOR complex1(mTORCl) and complex2(mTORC2). mTORCl plays a key role in translation initiation by directly phosphorylating p70S6kinase1(S6K1) and4E-BP1, and is sensitive to rapamycin. mTORC2is not susceptible to acute rapamycin inhibition. The function of mTORC2is less clear, but it has been shown to phosphorylate Akt (S473) and to regulate cell survival and cell motility. As a critical drug target, mTOR signaling is up-regulated in endometrial cancer and plays key roles in the carcinogenesis and progression of the disease. Clinical trails show that responses to indirect (allostric) mTORCl inhibitors are modest, which in part, relate to positive feedback from mTORC2on the Akt pathway that can continue upon inhibition of mTORCl. Therefore, second-generation catalytic mTORCl/2inhibitors that can act directly on mTOR are being developed and have entered clinical trials.Omega-3(n-3) and omega-6(n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids necessary for human health. Laboratory and animal studies have shown that long-chain n-3PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), inhibit tumorigenesis for various cancer sites, n-6PUFAs such as arachidonic acid (AA), on the other hand, have been shown to promote tumor growth and progression. Total fat intake and the ratio of n-6to-3PUFAs in the Western diet have increased significantly since the Industrial Revolution, which is thought to contribute to obesity, inflammation and cancer. Studies in human populations have linked high consumption of fish or fish oil (n-3PUFAs) to reduce risk of colon, prostate, and breast cancer, combined with demonstrated effects of n-3PUFAs on cancer from preclinical (animal and in vitro) models, has motivated the development of clinical interventions using n-3PUFAs in the prevention and treatment of these cancers.Although the role of dietary PUFAs in various cancers has received great attention in the literature, and the preventive effect of n-3PUFAs on obesity has been established, the evidence on the association of n-3PUFAs with endometrial cancer is limited. A recent epidemiological study suggests that higher dietary intake of the EPA and DHA in food and supplements were associated with lower risk of endometrial cancer. No previous study has examined the effect of n-3PUFAs on endometrial cancer in preclinical models. We have previously developed a transgenic mouse model that expresses fat-1, a desaturase that catalyzes the conversion of n-6to n-3PUFAs and produces n-3PUFAs endogenously, which enables the investigation of the biological properties of n-3PUFAs without having to incorporate n-3PUFAs such as DHA in the diet. Our recent work also identified a critical role for the n-6PUFA (AA) in the activation of mTORC1/2signaling in breast carcinogenesis and angiogenesis. This study aimed to identify the effects of n-3PUFAs and transgenic fat-1expression on mTORCl/2signaling and the pathogenesis and progression of endometrial cancer in preclinical (cell and mouse) models.Results1. Exogenous n-3PUFAs inhibit endometrial cancer cell growth in vitro and in xenograft modelsTo investigate the potential protective role of n-3PUFAs against endometrial cancer, we first examined the effect of DHA on proliferation of cultured endometrial carcinoma HEC-1-A, HEC-1-B and RL95-2cell lines. We found that DHA dose-and time-dependently (12.5,25and50μM) inhibited cell proliferation in the tested endometrial cancer cells. The colony formation assay further confirmed that DHA prevented colony formation of these cell lines in a dose-dependent manner. These results demonstrate that DHA effectively inhibits endometrial cancer cell growth in vitro.To further identify the role of n-3PUFAs in endometrial cancer cell growth in vivo, we established endometrial tumor xenograft models by subcutaneously implanting HEC-1-A and RL95-2cells into nude mice. Data show that dietary n-3PUFAs (1g/kg/d) efficiently prevented the tumor growth and reduced the average tumors weights and tumors volumes. This result indicates that dietary n-3PUFAs suppresses endometrial cancer cell growth in vivo.Together, these evidences demonstrate that the DHA or dietary n-3PUFAs effectively inhibit endometrial cancer cell growth in animal and cell culture models.2. Endogenously produced n-3PUFAs inhibit endometrial cancer cell growth in vitro and in xenograft modelsDiets or nutritional supplements contain many nutrients and other components that may interact, which adds a layer of complexity to their evaluation. Transgenic expression of fat-1is capable of converting n-6to n-3PUFAs, leading to an increase in n-3PUFAs and a decrease in n-6/n-3ratio, allows well-controlled studies to be performed in the absence of restricted diets. To further identify the role of n-3PUFAs in endometrial tumor growth, we established endometrial cancer cell culture and tumor xenograft models producing endogenous n-3PUFAs. Firstly, proliferation and colony formation of HEC-1-A, HEC-1-B and RL95-2cells transfected with recombinant adenovirus that carries with or without fat-1gene (Ad-fat-1or Ad) were assessed. The results showed that both proliferation rates and colony formation of fat-1expressing cells were significantly decreased compared with cells transfected with empty virus in all tested endometrial cancer cell lines.Next, we established fat-1transgenic severe combined immune deficiency (SCID) mice. Fatty acid composition analysis identified a significantly increased ratio of n-3/n-6PUFAs in these mice compared with control SCID mice lacking fat-1expression. Interestingly, although xenograft tumors with an average volume of223mm3were observed within31days in control SCID mice, we failed to observe tumor growth in any fat-1SCID animals. We suggest that endometrial tumor cells are unable to proliferate or survive in the presence of high levels of endogenously-produced n-3PUFAs in vivo. Taken together, these data unequivocally demonstrate that endogenously produced n-3PUFAs suppress endometrial cancer cell growth in vitro and in vivo.3. n-3PUFAs prevent endometrial cancer cell migrationMetastasis is the major cause of mortality and morbidity among endometrial cancer patients. Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. This requires migration of cancer cells. To investigate the potential role of n-3PUFAs in endometrial cancer cell migration, effects of DHA on cell migration were examined by using a wound healing assay in a serum-free medium.25μM DHA-treated HEC-1-A and HEC-1-B cells filled the gap more slowly than vehicle control cells did, suggesting that DHA prevented endometrial cancer cell migration. We further confirmed the result by cell migration transwell assay. Cells were serum-starved to inhibit proliferation thus facilitating cell motility analysis and cells migrated to the lower chamber were quantified24h after DHA incubation. The results showed that DHA significantly prevented HEC-1-A and HEC-1-B cells migration as measured by crystal violet staining. Moreover, fat-1expressing HEC-1-A and HEC-1-B cells migrate more slowly than the vehicle control cells did, indicating that endogenous n-3PUFAs inhibit endometrial cancer cell migration. These findings demonstrate that n-3PUFAs could prevent endometrial cancer cell migration efficiently.4. n-3PUFAs promote endometrial cancer cell apoptosis in vivo and in vitron-3PUFAs have been shown to promote apoptosis in a variety of cancer cells. We next examined if n-3PUFAs potentiate apoptosis in endometrial cancer cells and xenograft. It was found that DHA increased the number of dead cells dose-dependently in serum-starved HEC-1-A, HEC-1-B and RL95-2cell lines. Cleavage of poly (ADP-ribose) polymerase (PARP) was also enhanced significantly by DHA in a dose-and time-dependent manner. It is suggested that DHA promotes endometrial cancer cell apoptosis in vitro. The effect of n-3PUFAs on apoptosis was further examined in xenograft models. The results revealed that n-3PUFAs administration promoted xenografted endometrial tumor cell apoptosis as manifested by the enhanced cleavage of PARP in tumors burder in n-3PUFAs-treated mice. We conclude that n-3-PUFAs promote endometrial cancer cell apoptosis in vivo and in vitro.5. n-3PUFAs inhibit mTORC1/2signaling in endometrial cancer cell lines and xenograft modelPrevious studies have shown mTOR signaling plays important roles in tumorigenesis and progression of endometrial cancer and have revealed a clinical advantage in targeting this pathway. We have recently reported a critical role for n-6 PUFA-activated mTORCl/2signaling in mammary tumorigenesis and angio genesis. We then asked if n-6PUFAs stimulate mTORCl/2activity in endometrial cancer cells. As expected, AA (n-6PUFA) acutely stimulated mTORC1-directed phosphorylation of S6(S235/236) and mTORC2-directed phosphorylation of Akt at position Ser473. It has been established that n-3PUFAs often exert functions by antagonizing the effects of n-6PUFAs. We next examined whether n-3PUFA DHA inhibits mTORCl/2in endometrial cancer cell lines. In HEC-1-A cells, DHA rapidly and dose-dependency suppressed AA-stimulated phosphorylation of S6(S235/236) and Akt (S473). We next examined the role of endogenously produced n-3PUFAs on mTORC1/2signaling. In HEC-1-A cells transfect with the fat-1cDNA, the phosphorylation of S6(S235/236) and Akt (S473) were significantly reduced compared with cells transfected with the control vector. These results suggest that both mTORCl and mTORC2signaling pathways are targets of exogenous and endogenous n-3PUFAs in endometrial cancer cells.We further determine if n-3PUFAs suppress mTORC1/2in vivo. Interestingly, high dietary n-3PUFAs repressed both mTORCl and mTORC2signaling pathways endometrial tumor xenograft model, as manifest by decreased phosphorylation levels of S6(S235/236) and Akt (S473) in mice with high n-3/n-6PUFAs diet. Moreover, levels of phosphorylated S6(S235/236) and Akt (S473), were lower in the livers of fat-1SCID mice compared to control mice. We suggest that mTORCl and mTORC2signaling are targets of n-3PUFAs in vivo. We suggest that the suppression of mTORCl/2signaling by n-3PUFAs may contribute to their inhibitory effects on endometrial tumor growth.Taken together, these data demonstrate that DHA and endogenously produced n-3PUFAs target mTORCl/2pathways in vitro and in vivo.Conclusion1. This study provides comprehensive preclinical evidences that both dietary and endogenously produced n-3PUFAs efficiently prevent endometrial cancer in vitro and in vivo through inhibition of cell growth, migration and promotion of apoptosis.2. mTORC1/2signaling pathway is target of n-3PUFAs in endometrial cancer. 3. Our results provide rationale for the development of clinical interventions using n-3PUFAs in the prevention and treatment of endometrial cancer.