Discovery of IL-18 As a Novel Secreted Protein Contributing to Doxorubicin Resistance by Comparative Secretome Analysis of MCF-7 and MCF-7/Dox

BackgroundResistance to chemotherapy is the major cause of failure in breast cancer treatment. Recent studies suggest that secreted proteins may play important roles in chemoresistance. We sought to systematically characterize secreted proteins associated with drug resistance, which may represent potential serum biomarkers or novel drug targets.
Methodology/Principal FindingsIn the present work, we adopted the proteomic strategy of one-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry to compare the secretome of MCF-7 and doxorubicin-resistant MCF-7/Dox. A total of 2,084 proteins were identified with at least two unique peptides in the conditioned media of two cell lines. By quantification with label-free spectral counting, 89 differentially expressed secreted proteins (DESPs) between the two cell lines were found. Among them, 57 DESPs were first found to be related to doxorubicin resistance in this work, including 24 extracellular matrix related proteins, 2 cytokines and 31 unclassified proteins. We focused on 13 novel DESPs with confirmed roles in tumor metastasis. Among them, the elevated expression of IL-18 in doxorubicin-resistant cell lines and breast tumor tissues was validated and its role in doxorubicin resistance was further confirmed by cell viability experiments in the presence or absence of this protein.
Conclusions/SignificanceComparative analysis of the secretome of MCF-7 and MCF-7/Dox identified novel secreted proteins related to chemotherapy resistance. IL-18 was further validated to contribute to doxorubicin resistance, in addition to its confirmed role in breast cancer metastasis. Due to its dual roles in both drug resistance and tumor metastasis, IL-18 may represent a useful drug target for breast cancer therapy.

背景 化疗耐药是乳腺癌治疗失败的核心诱因。近期研究显示，分泌蛋白可能在化疗耐药进程中发挥重要调控作用。本研究旨在系统表征与药物耐药相关的分泌蛋白，此类蛋白或可作为潜在血清生物标志物或新型药物靶点。

方法与主要结果 本研究采用一维凝胶电泳联合液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry，LC-MS/MS）的蛋白质组学策略，对比分析亲本乳腺癌细胞MCF-7与阿霉素（doxorubicin）耐药细胞MCF-7/Dox的分泌组（secretome）。在两种细胞系的条件培养基（conditioned media）中，共鉴定出2084种蛋白，且每种蛋白至少包含2条独特肽段。通过无标记光谱计数（label-free spectral counting）进行定量分析，共筛选得到89种在两细胞系间存在差异表达的分泌蛋白（differentially expressed secreted proteins，DESPs）。其中，57种差异表达分泌蛋白为本研究首次发现与阿霉素耐药相关，涵盖24种细胞外基质相关蛋白、2种细胞因子及31种未分类蛋白。本研究重点聚焦于13种在肿瘤转移中已被证实功能的新型差异表达分泌蛋白，其中白细胞介素-18（IL-18）在阿霉素耐药细胞系与乳腺癌肿瘤组织中的表达水平显著上调；通过在培养体系中添加或抑制该蛋白的细胞活力实验，进一步验证了其在阿霉素耐药中的功能。

结论与意义 对MCF-7与MCF-7/Dox分泌组的对比分析，成功鉴定出与化疗耐药相关的新型分泌蛋白。除已证实的乳腺癌转移调控作用外，IL-18在阿霉素耐药中的功能亦得到验证。鉴于其在化疗耐药与肿瘤转移过程中的双重调控作用，IL-18或可成为乳腺癌治疗的有效药物靶点。