Pal Purab, Millner Alec, Semina Svetlana E, Huggins Rosemary J, Running Logan, Aga Diana S, Tonetti Debra A, Schiff Rachel, Greene Geoffrey L, Atilla-Gokcumen G Ekin, Frasor Jonna
Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Department of Chemistry, University at Buffalo, The State University of New York (SUNY), Buffalo, NY 14260, USA.
Cancers (Basel). 2022 May 12;14(10):2380. doi: 10.3390/cancers14102380.
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.
雌激素受体阳性（ER+）乳腺癌中，雌激素治疗耐药是一个关键问题。在本研究中，我们探究了鞘脂的改变如何促进雌激素治疗耐药乳腺癌中的细胞存活。我们对他莫昔芬敏感和耐药的MCF-7乳腺癌细胞系进行了基于液相色谱-质谱联用的靶向鞘脂组学分析。后续研究包括用小分子抑制剂处理细胞系和患者来源的异种移植类器官（PDxO）；通过细胞计数分析来测量细胞死亡、增殖和凋亡；RNA干扰介导的基因敲低；用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测基因和蛋白表达；靶向脂质分析；以及脂质回补实验。我们发现，与他莫昔芬敏感细胞相比，他莫昔芬耐药细胞中的神经酰胺和己糖神经酰胺水平较低。在用关键酶的小分子抑制剂干扰鞘脂途径后，我们发现神经酰胺激酶（CERK）对他莫昔芬耐药乳腺癌细胞的存活至关重要，对氟维司群耐药的PDxO也同样重要。抑制CERK可在他莫昔芬耐药细胞中诱导神经酰胺介导的细胞死亡。1-磷酸神经酰胺（C1P）可部分逆转抑制CERK诱导的他莫昔芬耐药细胞的死亡，可能是通过降低内源性神经酰胺水平来实现的。我们的研究结果表明，雌激素治疗耐药的乳腺癌细胞维持较低的神经酰胺水平是一种重要的促存活机制。因此，雌激素治疗耐药的乳腺癌模型对CERK具有独特的依赖性，因为其活性可以抑制神经酰胺的从头合成。
