Zhou Changhua, Nitschke Ashley M, Xiong Wei, Zhang Qiang, Tang Yan, Bloch Michael, Elliott Steven, Zhu Yun, Bazzone Lindsey, Yu David, Weldon Christopher B, Schiff Rachel, McLachlan John A, Beckman Barbara S, Wiese Thomas E, Nephew Kenneth P, Shan Bin, Burow Matthew E, Wang Guangdi
Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA.
Breast Cancer Res. 2008;10(6):R105. doi: 10.1186/bcr2210. Epub 2008 Dec 16.
Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-alpha. Additionally, the relationship between TNF-alpha resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.
To compare differences in the proteome of the TNF-alpha resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-alpha resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.
Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (delta-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.
This study demonstrates that MEK5 over-expression promotes a TNF-alpha resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.
尽管对人类乳腺癌化疗耐药机制进行了深入研究，但很少有报告系统地研究化疗增敏剂肿瘤坏死因子（TNF）-α耐药的潜在机制。此外，由MEK5/Erk5信号介导的TNF-α耐药与上皮-间质转化（EMT）之间的关系，而EMT是一个与乳腺癌侵袭、转移和复发相关的过程，此前尚未得到研究。
为了比较TNF-α耐药的MCF-7乳腺癌细胞系MCF-7-MEK5（其中TNF-α耐药由MEK5/Erk5信号介导）及其亲代TNF-α敏感的MCF-7细胞系MCF-7-VEC蛋白质组的差异，使用了二维凝胶电泳和高效毛细管液相色谱联用串联质谱方法。使用RT-PCR分析在转录水平验证差异蛋白表达。使用免疫荧光染色和基因表达分析确认EMT表型。利用靶向Erk5的短发夹RNA策略研究EMT中MEK/Erk5途径的必要性。
蛋白质组学分析和PCR分析用于鉴定和确认蛋白质的差异表达。在MCF-7-MEK5与MCF-7-VEC细胞中，波形蛋白（VIM）、谷胱甘肽-S-转移酶P（GSTP1）和肌酸激酶B型（CKB）上调，而角蛋白8（KRT8）、角蛋白19（KRT19）和谷胱甘肽-S-转移酶Mu 3（GSTM3）下调。E-钙黏蛋白和波形蛋白的形态学及免疫荧光染色显示MCF-7-MEK5细胞中存在EMT表型。此外，与MCF-7-VEC细胞相比，MCF-7-MEK5细胞中EMT调节基因SNAI2（蛞蝓）、ZEB-1（δ-EF1）和N-钙黏蛋白（CDH2）上调，而E-钙黏蛋白（CDH1）下调。靶向Erk5的RNA干扰逆转了MEK5介导的EMT基因表达。
本研究表明，MEK5过表达促进了与不同蛋白质组变化相关的TNF-α耐药表型（VIM/vim、GSTP1/gstp1和CKB/ckb上调；KRT8/krt8、KRT_{19}/krt19和GSTM3/gstm3下调）。我们进一步证明，MEK5介导的向EMT表型的进展依赖于完整的Erk5，并与SNAI2和ZEB1表达上调相关。
