Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry
Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry
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DOI：
10.1002/pmic.200500617
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发表时间：
2006-06-01
期刊：
影响因子：
3.4
通讯作者：
Shao, Zhi-Min
中科院分区：
文献类型：
--
作者：
Li, Da-Qiang;Wang, Lei;Shao, Zhi-Min
为了更好地理解乳腺癌转移的分子机制并寻找转移进展的潜在标志物，我们通过在无胸腺小鼠中逐步选择由亲代MDA-MB-435细胞引起的肺转移细胞，开发了人MDA-MB-435乳腺癌细胞系的高转移变体。使用2-DE和LC-IT-MS的比较蛋白质组学分析显示，102个蛋白质点可重复地改变了三倍以上的选择的变体和它的父母之间的对应物。11个差异表达的蛋白质点，确定了高置信度使用SEQUEST与未解释的串联质量的原始数据。组织蛋白酶D前体，过氧化物酶6（PDX 6），热休克蛋白27（HSP 27），HSP 60，原肌球蛋白1（TPM 1），TPM 2，TPM 3，TPM 4，14-3-3蛋白激酶和肿瘤蛋白D54在高转移性变异中上调，而α B-晶体（CRAB）仅在其亲本中检测到。在我们的模型中，通过实时定量PCR和Western blotting分析证实了四种蛋白质的差异表达，包括PDX 6，CRAB，TPM 4和HSP 60。80例乳腺癌供体的免疫组织化学分析表明，TPM 4（p = 0.002）、HSP 60（p = 0.001）、PDX 6（p = 0.002）而非CRAB（p = 0.113）染色与淋巴结转移的存在显著相关。此外，TPM 4染色也与临床分期相关（p = 0.000），但在TPM 4、PDX 6、CRAB和HSP 60表达与肿瘤大小、激素受体和HER-2状态之间未发现显著相关性（p > 0.05）。并对这些蛋白的功能意义进行了讨论。这些蛋白质组学数据对于了解乳腺癌转移和寻找转移进展的潜在标志物是有价值和信息的。
To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4,14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.