﻿Cells were cultured in DMEM:F12 media (Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) FBS (Scientifix, Cheltenham, VIC, Australia) and penicillin-streptomycin answer (Sigma, St Louis, MO, USA), at 37?C and 5% CO2

﻿Cells were cultured in DMEM:F12 media (Life Technologies, Carlsbad, CA, USA) containing 10% (v/v) FBS (Scientifix, Cheltenham, VIC, Australia) and penicillin-streptomycin answer (Sigma, St Louis, MO, USA), at 37?C and 5% CO2. and cell growth, but did not affect spheroid area. Ishikawa cells express higher levels of the glutamine transporter SNAT1 compared to HEC1A cells, suggesting these cells may rely on both ASCT2 and SNAT1 for glutamine uptake. Since SNAT1 is also significantly upregulated in the endometrioid and serous subtypes, these data show that ASCT2 and SNAT1 could be used as markers of malignancy, and/or potential therapeutic targets in patients with endometrial carcinoma. Introduction Endometrial carcinoma is usually a malignancy arising from the lining of the uterus. RCAN1 There are various subtypes of endometrial carcinoma, with endometrioid adenocarcinoma representing about 80% of cases. Tumours of the endometrioid subtype are usually hormone responsive and generally have a good prognosis. The second most common subtype is usually serous DAA-1106 carcinoma, which accounts for 5C10% of cases of endometrial carcinoma.1 The serous subtype is generally not hormone responsive, and is associated with a poorer prognosis. The treatment for endometrial carcinoma typically consists of medical procedures including hysterectomy and salpingo-oophorectomy.2 Treatment of higher-grade tumours often also involves locoregional lymph node dissection as well as radiotherapy and/or chemotherapy.3, 4 In developed countries, endometrial carcinoma is the most common cancer of the female reproductive tract.5 Human papilloma virus (HPV)-associated cervical cancer is more prevalent in developing countries, due to the lack of effective screening programmes which have dramatically reduced rates in developed nations.6 However, this alone does not account for the high relative risk of endometrial carcinoma, DAA-1106 which has an increased incidence of over 10-fold in first world countries.7 Several other cancers, including breast, ovary, prostate, colon, pancreas and kidney, have also shown a similar increase in incidence in Western countries, irrespective of the patients genetic background. This increase has been associated with changes in diet, in particular an increase in dietary animal products which are inherently high in protein,8, 9, 10 suggesting that availability of nutrients such as amino acids may play a role in promoting cancer cell growth. In mammalian cells, there are three classes of amino acids: nonessential, essential and conditionally essential. nonessential amino acids can be synthesised by cells while essential amino acids must be obtained from external sources. Conditionally essential amino acids can be endogenously produced by cells, DAA-1106 but under certain conditions, demand can outweigh supply. Cancer cells have a particularly high demand for amino acids due to their high proliferative rate, often upregulating amino acid transporters on their cell surface to facilitate increased cellular uptake (reviewed in Bhutia in four separate endometrial cancer cell lines. Analysis of ASCT2 function by shRNA knockdown showed HEC1A cells rely on ASCT2-mediated glutamine uptake, while Ishikawa cells have only a partial reliance on ASCT2 function. These data demonstrate the importance of glutamine uptake in endometrial carcinoma, indicating that drugs targeting glutamine transporters such as ASCT2, and potentially SNAT1, may represent a novel therapeutic avenue. Results ASCT2 and SNAT1 are upregulated in endometrial carcinoma DAA-1106 To determine the expression levels of the amino acid transporter, ASCT2/SLC1A5, in endometrial carcinoma we compared gene expression in serous and endometrioid subtype tumours with normal age-matched endometrium using published gene expression data.29 These data showed a significant increase in ASCT2 mRNA expression in serous and endometrioid endometrial carcinoma samples in comparison to normal endometrium (we used 3D culture conditions32 to produce endometrial spheroids, and subsequently performed immunofluorescence. Under these growth conditions, ASCT2 predominantly localised to the apical cell membrane, though some faint basal localisation was also present in Ishikawa and HEC1A cells (Figure 3h). Chemical inhibition of glutamine transport inhibits endometrial cancer cell growth In order to determine if glutamine transport is critical for endometrial cancer cell growth we treated DAA-1106 each endometrial cancer cell line with two chemical inhibitors: BenSer and GPNA. Treatment with BenSer was able to significantly reduce cell growth in all cell lines tested, while GPNA inhibited cell growth of Ishikawa, HEC1A and KLE cell lines but not RL95-2 (Figures 4aCd). BenSer was more effective than GPNA at inhibiting growth of Ishikawa (day 10 ASCT2 knockdown has lower effects on cell growth, compared to knockdown. This most likely relates to the abundance of amino acids in tissue culture media compared to poorly vascularised tumours in vivo.20, 21 In this study we have shown that ASCT2 function contributes to cancer cell proliferation in a subset of.