From the Department of Integrative Biology and Pharmacology, McGovern Medical School University of Texas Health Science Center at Houston, Houston, Texas 77030.
the Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435, and.
J Biol Chem. 2018 Aug 31;293(35):13696-13706. doi: 10.1074/jbc.RA118.003907. Epub 2018 Jul 3.
Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examine the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalizes HRAS and KRAS from the PM with similar potency and disrupts the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays. Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in Madin-Darby canine kidney (MDCK) cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryo fibroblasts expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less mouse embryo fibroblasts expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 selectively inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.
致癌性 RAS 蛋白通常在人类癌症中表达。为了发挥功能，RAS 蛋白必须经历翻译后修饰并定位于质膜（PM）。因此，防止 RAS PM 靶向的化合物具有作为潜在 RAS 抑制剂的潜力。在这里，我们研究了最近描述的 KRAS PM 定位抑制剂黄烷醌 G01（G01）的作用机制。我们表明，G01 以相似的效力将 HRAS 和 KRAS 从 PM 中错误定位，并破坏了留在 PM 上的 RAS 蛋白的空间组织。G01 还抑制了表皮生长因子受体和转铁蛋白受体的循环，但不损害霍乱毒素的内化，表明抑制了循环内体功能。在寻找受损的内体循环的机制时，我们观察到 G01 还增强了细胞神经鞘磷脂（SM）和神经酰胺水平，并破坏了几种脂质和胆固醇报告器的定位，表明 G01 的分子靶标可能涉及 SM 代谢。事实上，G01 在 KRAS 定位测定中与其他靶向 SM 代谢的化合物表现出很强的协同作用。此外，G01 显著阻断了表达组成性激活的致癌性突变 RASG12V 的 Madin-Darby 犬肾（MDCK）细胞中的 RAS-RAF-MAPK 信号。G01 还抑制了表达致癌性突变 KRASG12V 或 KRASG12D 的 RAS 缺失小鼠胚胎成纤维细胞的增殖，但不抑制表达致癌性突变 BRAFV600E 的 RAS 缺失小鼠胚胎成纤维细胞的增殖。与这些作用一致，G01 选择性抑制了 KRAS 转化的胰腺、结肠和子宫内膜癌细胞的增殖。总之，这些结果表明 G01 应该作为一种潜在的抗 RAS 治疗药物进行进一步评估。
