Supplementary Material for: Inhibition of ceramide kinase is effectively against cisplatin-resistant ovarian cancer cells by regulating ceramide and C1P levels

Objective: Chemoresistance in ovarian cancer results in treatment failure, yet underlying mechanisms that regulate chemoresistance remain largely unclear. There is emerging evidence relating ovarian cancer drug resistance with bioactive sphingolipids and regulation of sphingolipid metabolism. This work investigated the expression and function of ceramide kinase (CerK), a lipid kinase that regulate central bioactive sphingolipids, in ovarian cancer, as well as the therapeutic potential of targeting CERK. Design: The level of ceramide, C1P and Cerk in ovarian cancer and normal counterpart were measured. Functions of Cerk in ovarian cancer was examined. Materials, Setting, Methods: Immunohistochemistry, ELISA and mass spectrometry methods were used to measure the level of ceramids, C1P and CerK in primary tissues. Proliferation and apoptosis assays were performed in ovarian cancer cells after CERK depletion, CERK overexpression and NVP-231 treatment in the absence or presence of cisplatin. Results: Compared to normal ovarian cells, CerK and its-mediated bioactive sphingolipids ceramide and ceramide 1-phosphate (C1P) were decreased in ovarian cancer tissues. Interestingly, cisplatin-resistant ovarian cancer cells displayed increased CerK, decreased ceramide and increased C1P, and furthermore that CerK level was closely associated with ceramide and C1P levels in ovarian cancer cells. Functional analysis demonstrated that CerK overexpression was sufficient to promote growth and confer chemo-resistance in ovarian cancer cells. CerK inhibition via both genetic and pharmacological approaches suppressed growth and induced apoptosis in cisplatin-resistant cells, and furthermore that significantly augmented cisplatin’s efficacy. Limitations: The functional analysis of C1P was performed in in vitro ovarian cancer cells. In vivo studies were needed to further confirm the effects of CERK inhibition. Conclusions: Our work is the first to show the critical role of CerK as the underlying mechanism of ovarian cancer chemoresistance, through regulating ceramide and C1P.

研究目的：卵巢癌的化疗耐药会导致治疗失败，但其调控化疗耐药的潜在分子机制仍未完全阐明。越来越多的证据表明，卵巢癌耐药与生物活性鞘脂以及鞘脂代谢调控密切相关。本研究旨在探讨神经酰胺激酶（ceramide kinase, CerK）——一种调控核心生物活性鞘脂的脂质激酶——在卵巢癌中的表达与功能，以及靶向CERK的治疗潜力。
实验设计：检测卵巢癌组织及其正常对照组织中神经酰胺、C1P及CerK的表达水平，并分析CerK在卵巢癌中的生物学功能。
材料、实验设置与方法：采用免疫组化、酶联免疫吸附试验（ELISA）及质谱法检测原发组织中神经酰胺、C1P及CerK的表达水平；分别在顺铂存在或缺失的条件下，对CERK敲低、CERK过表达以及NVP-231处理后的卵巢癌细胞进行增殖与凋亡检测。
实验结果：与正常卵巢细胞相比，卵巢癌组织中CerK及其介导的生物活性鞘脂——神经酰胺（ceramide）与神经酰胺1-磷酸（ceramide 1-phosphate, C1P）的表达水平均显著降低。值得注意的是，顺铂耐药卵巢癌细胞中CerK表达上调、神经酰胺水平降低而C1P水平升高，且卵巢癌细胞中CerK的表达水平与神经酰胺及C1P的水平密切相关。功能实验结果显示，过表达CerK即可促进卵巢癌细胞增殖并赋予其化疗耐药性；通过基因及药理学手段抑制CerK，可抑制顺铂耐药细胞的增殖并诱导其凋亡，同时还能显著增强顺铂的抗肿瘤效果。
研究局限性：本研究中关于C1P的功能实验均基于卵巢癌细胞体外模型开展，后续需通过体内实验进一步验证靶向CERK的治疗效应。
结论：本研究首次证实，CerK通过调控神经酰胺与C1P的代谢，在卵巢癌化疗耐药的发生发展中发挥关键作用。