Department of Hepatobiliary Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei, China.
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Yangtze University, Jingzhou, China.
Cancer Chemother Pharmacol. 2021 Feb;87(2):217-228. doi: 10.1007/s00280-020-04199-6. Epub 2020 Nov 23.
The survival benefit of sorafenib, the most used drug for advanced hepatocellular carcinoma (HCC), is unsatisfactory due to the development of adaptive resistance. Exploring the mechanisms underlying sorafenib resistance is important to develop sensitizing strategy. Sphingomyelin synthase (SMS) plays a critical role in sphingolipid metabolism which is involved in oncogenesis and drug resistance.
SMS1 and SMS2 levels in HCC cells in response to prolonged chemotherapy were analyzed using ELISA. mRNA and protein levels of SMS in HCC and adjacent normal tissues were analyzed by ELISA and real-time PCR. The roles of SMS and its downstream targets were investigated using cellular and biochemical assays and mass spectrometry.
SMS1, but not SMS2, was upregulated in HCC in response to sorafenib treatment, although HCC displayed similar RNA and protein level of SMS1 compared to adjacent normal liver tissues. Overexpression of SMS1 promoted HCC growth and migration, and alleviated sorafenib's toxicity. SMS1 inhibition via genetic and pharmacological approaches consistently resulted in inhibition of growth and migration, and apoptosis induction in sorafenib-resistance HCC cells. SMS1 inhibition also augmented the efficacy of sorafenib in sensitive HCC cells. SMS1 inhibition disrupted sphingolipid metabolism via accumulating ceramide and decreasing sphingomyelin, inducing mitochondrial dysfunction and oxidative stress, and decreasing Ras activity in resistant cells. Overexpression of constitutively active Ras reversed the inhibitory effects of SMS1 inhibition. Although SMS1 overexpression did not affect Ras expression and activity, Pearson correlation coefficient analysis of SMS1 and Ras expression demonstrated that there was positive correlation between SMS1 and RAS (NRAS, R = 0.55, p < 0.01; KRAS, R = 0.44, p < 0.01).
Our work is the first to suggest that SMS1 plays a more important role in sorafenib resistance than tumorigenesis, and provides preclinical evidence to overcome sorafenib resistance with SMS1 inhibition in HCC.
索拉非尼是治疗晚期肝细胞癌（HCC）最常用的药物，但由于适应性耐药的发展，其生存获益并不理想。探索索拉非尼耐药的机制对于开发增敏策略很重要。神经酰胺合成酶（SMS）在神经酰胺代谢中起着关键作用，而神经酰胺代谢与肿瘤发生和耐药有关。
采用 ELISA 法分析 HCC 细胞对长期化疗的反应中 SMS1 和 SMS2 的水平。采用 ELISA 和实时 PCR 法分析 HCC 及相邻正常组织中 SMS 的 mRNA 和蛋白水平。采用细胞和生化测定法以及质谱法研究 SMS 及其下游靶标的作用。
尽管 HCC 与相邻正常肝组织相比，SMS1 的 RNA 和蛋白水平相似，但 HCC 对索拉非尼治疗的反应中，SMS1 而不是 SMS2 上调。SMS1 的过表达促进 HCC 的生长和迁移，并减轻了索拉非尼的毒性。通过遗传和药理学方法抑制 SMS1，可一致抑制耐药 HCC 细胞的生长和迁移，并诱导凋亡。SMS1 抑制也增强了敏感 HCC 细胞中索拉非尼的疗效。SMS1 抑制通过积累神经酰胺和减少神经鞘磷脂，诱导线粒体功能障碍和氧化应激，降低 Ras 活性，从而破坏鞘脂代谢。过表达组成型激活 Ras 逆转了 SMS1 抑制的抑制作用。尽管 SMS1 过表达不影响 Ras 的表达和活性，但 SMS1 和 Ras 表达的 Pearson 相关系数分析表明，SMS1 与 Ras 之间存在正相关（NRAS，R=0.55，p<0.01；KRAS，R=0.44，p<0.01）。
我们的工作首次表明，SMS1 在索拉非尼耐药中比在肿瘤发生中发挥更重要的作用，并提供了克服 HCC 中 SMS1 抑制耐药的临床前证据。
