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Simple Summary: Metastatic breast cancer remains a major clinical problem, contributing to significant patient mortality, which is partly due to a lack of understanding around the early changes within the primary tumor. Tumors frequently become more aggressive and less treatable due to the activation of other signaling pathways, and, in ER+ disease, one of these pathways is NFκB. The coactivation of ER and NFκB (via IKKβ) promotes invasion and metastasis, and, here, we identify the signatures that are associated with these phenotypes. These signatures improve our understanding of how ER can drive aggressive disease, and may lead to the identification of key drivers, which could potentially be targeted with future therapies. Most metastatic breast cancers arise from estrogen receptor α (ER)-positive disease, and yet the role of ER in promoting metastasis is unclear. Here, we used an ER+ breast cancer cell line that is highly invasive in an ER- and IKKβ-dependent manner. We defined two ER-regulated gene signatures that are specifically regulated in the subpopulations of invasive cells. The first consists of proliferation-associated genes, which is a known function of ER, which actually suppress rather than enhance invasion. The second signature consists of genes involved in essential biological processes, such as organelle assembly and vesicle trafficking. Importantly, the second subpopulation-specific signature is associated with aggressive disease and poor patient outcome, independently of proliferation. These findings indicate a complex interplay between ER-driven proliferation and invasion, and they define new ER-regulated gene signatures that are predictive of aggressive ER+ breast cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Endocrine therapy (ET) resistance is a major problem in estrogen receptor-positive breast cancer patients. Since there have been few lipidomic studies in ET resistance and sphingolipids are heavily implicated in multidrug-resistant and chemotherapy-resistant cancers, we aimed to investigate the sphingolipidome of tamoxifen-resistant breast cancer cells in search of a unique sphingolipid profile that can potentially be exploited therapeutically. We found that ET-resistant breast cancer cells maintain a lower level of ceramides for their survival. In order to achieve this, they are dependent on ceramide kinase (CERK), the activity of which helps maintain low endogenous ceramide levels, therefore promoting tamoxifen-resistant cell survival. Targeting CERK can therefore represent an opportunity to target therapy-resistant breast tumors and improve the patient outcome for women with ET-resistant disease. ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production. [ABSTRACT FROM AUTHOR]

Exosomes are small vesicles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The main goal of the work was to study the exosome involvement in the cell transfer of hormonal resistance. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells and MCF-7 sublines resistant to SERM tamoxifen and/or biguanide metformin, which exerts its anti-proliferative effect, at least in a part, via the suppression of estrogen machinery. The exosomes were purified by differential ultracentrifugation, cell response to tamoxifen was determined by MTT test, and the level and activity of signaling proteins were determined by Western blot and reporter analysis. We found that the treatment of the parent MCF-7 cells with exosomes from the resistant cells within 14 days lead to the partial resistance of the MCF-7 cells to antiestrogen drugs. The primary resistant cells and the cells with the exosome-induced resistance were characterized with these common features: decrease in ERα activity and parallel activation of Akt and AP-1, NF-κB, and SNAIL1 transcriptional factors. In general, we evaluate the established results as the evidence of the possible exosome involvement in the transferring of the hormone/metformin resistance in breast cancer cells. [ABSTRACT FROM AUTHOR]