Interestingly, the level of mRNA in the LTEDaro cells was much higher than that in MCF7aro cells at baseline, which is consistent with the up-regulation of MYC in LTEDaro cells (Fig

Interestingly, the level of mRNA in the LTEDaro cells was much higher than that in MCF7aro cells at baseline, which is consistent with the up-regulation of MYC in LTEDaro cells (Fig. sensitive and resistant cells, and performed RNA-sequencing to investigate mechanisms of MYC-mediated glutamine utilization in AI resistance. We found that glutamine metabolism was independent of estrogen but still required ER in AI resistant breast cancer cells. The expression of oncogene was up-regulated through the cross-talk between estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) in AI resistant breast cancer cells. Moreover, the glutamine transporter solute carrier family (SLC)1A5 was significantly up-regulated in AI resistant breast cancer cells. ER down-regulator fulvestrant inhibited MYC, SLC1A5, glutaminase (GLS) and glutamine consumption in AI resistant breast cancer cells. Inhibition of MYC, SLC1A5 and GLS decreased AI resistant breast cancer cell proliferation. Our study has uncovered that MYC expression is up-regulated by the cross-talk between ER and HER2 in AI resistant breast cancer cells. MYC-mediated glutamine metabolism is associated with AI resistance of breast cancer. resistance or ultimately develop acquired resistance to endocrine therapies [2]. Cross-talk between ER and human epidermal growth factor receptor 2 (HER2) signaling pathways has been implicated in endocrine therapy resistance [3-5]. HER2 signaling is up-regulated in breast tumors of patients treated with AIs [6]. Up-regulation of HER2 signaling pathways, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase LSM6 antibody (PI3K)/AKT, can phosphorylate and activate ER in a ligand-independent manner [7, 8]. Many groups, including us, have showed that ER is up-regulated and constitutively activated in endocrine resistant Nitisinone breast cancer cells [9-11]. The oncogene, which encodes c-MYC (MYC) protein, is a well-known ER-regulated Nitisinone gene [12, 13]. MYC is a transcription factor and plays a critical role in cell proliferation, growth, survival, differentiation and apoptosis [14]. Interestingly, MYC has been linked to endocrine resistance of breast cancer [15-18]. Glutamine is the most abundant amino acids in the body and plays an important role in cell proliferation. It is first converted to glutamate through the enzyme glutaminase (GLS), and then catabolized to -ketoglutarate, an intermediate of the tricarboxylic acid (TCA) cycle [19]. Glutamine can be a source Nitisinone of both carbon and nitrogen for the synthesis of lipid, protein and nucleotide [20]. Although the growth and survival of most cancers depend on a high rate of aerobic glycolysis, some cancer cells cannot survive in the absence of exogenous glutamine, termed glutamine addiction [20]. Estrogen stimulation has been found to increase glutamine consumption in ER-positive breast cancer MCF7 cells [21], suggesting that glutamine metabolism is essential for estrogen-dependent cell proliferation. Oncogenic levels of MYC have also been linked to elevated glutamine uptake and metabolism in human cancers [22, 23]. Given the association between MYC and endocrine resistance [15, 16] as well as the regulation of glutamine metabolism by MYC in cancer cells[22, 23], we hypothesized that MYC-mediated glutamine metabolism is also associated with AI resistance. We studied the expression and regulation of MYC and the effects of inhibition of MYC expression in both AI sensitive and resistant breast cancer cells. We evaluated the contribution of glutamine to cell proliferation, and the association between glutamine consumption and hormone in breast cancer cells. Finally, we performed RNA-sequencing and investigated mechanisms of MYC-mediated glutamine metabolism in AI resistance. 2. Materials and methods 2.1. Cell culture Human breast cancer cell line MCF7 derived cell lines MCF7aro and LTEDaro were generated in this laboratory and have been characterized and described previously [9, 24]. MCF7aro was routinely cultured in minimal Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, and 0.1 mg/mL G418. LTEDaro was maintained in phenol redCfree MEM containing 10% charcoal/dextran-treated FBS with identical supplements as parental MCF7aro cells. For experiments under testosterone treatment, MCF7aro cells were cultured in phenol redCfree MEM medium containing 10% charcoal/dextran-treated FBS for 72 hr before treatment. Experiments under deprived glutamine or glucose culture conditions were performed by using phenol redCfree Dulbecco’s Modified Eagle Medium (DMEM) without glucose and glutamine (GIBCO A14430-01) containing 10% charcoal/dextran-treated FBS. 2.2. Antibodies and reagents Antihuman MYC (#5605), p-MAPK (#9101), MAPK (#9102), p-AKT (Ser473) (#9271), AKT (#9272), p-ER (Ser167) (#5587), GAPDH (#2118) antibodies were obtained from Cell Signaling Technology. Antihuman HER2 (#06-562) and p-ER (Ser118) (ab32396) antibodies were from Abcam Inc. Antihuman ER (HC-20) antibody (sc-543) was from Santa Cruz Biotechnology. The ER antagonist fulvestrant (#14409) and the SLC1A5 inhibitor L-Glutamic acid -(p-nitroanilide) (GPNA) (G6133) were obtained from Sigma-Aldrich. The glutaminase inhibitor compound 968 (#352010) was from EMD Millipore. The AKT inhibitor MK-2206 (S1078) was from Selleck Chemicals. The nontargeting control siRNA (sc-37007) and MYC siRNA (sc-29226) were obtained from Santa Cruz Biotechnology. The HER2 siRNA (L-003126-00).

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