乳腺癌骨转移的蛋白质组学特性及代谢途径活化研究

Elucidating the proteomic characteristics and metabolic pathway activation in breast cancer bone metastasis

    摘要
  • 摘要:
    目的 探究乳腺癌骨转移细胞的蛋白表达特征及调控骨转移的分子机制。
    方法 制备过表达萤火虫荧光素酶的人乳腺癌细胞系MCF-7-luc和骨转细胞亚系MCF-7-BOM-luc；左心室注射细胞建立乳腺癌骨转移裸鼠模型；应用小动物活性成像仪、微计算机断层扫描技术（micro-computed tomography，Micro-CT）检测骨转移及骨小梁变化；采用Transwell小室法评估细胞增殖、迁移和侵袭能力。通过蛋白质组学、蛋白质印迹（Western blot）和实时荧光定量PCR（qPCR）技术分析差异蛋白表达、上皮细胞-间充质转化（epithelial-mesenchymal transition，EMT）指标和信号通路活化情况；使用2-NBDG、ELISA法和Seahorse能量代谢仪分别检测葡萄糖摄取、L-乳酸含量和细胞能量代谢参数。
    结果 MCF-7-BOM-luc细胞迁移、侵袭、EMT特性以及骨骼转移能力方面优于MCF-7-luc。蛋白质组学发现MCF-7-BOM-luc中S100钙结合蛋白A4（S100 calcium-binding protein A，S100A4）表达增加，且差异蛋白主要集中在代谢途径。MCF-7-BOM-luc表现出E盒结合锌指蛋白1/2（zinc finger E-box binding homeobox 1/2，ZEB1/2）和波形蛋白（Vimentin）表达上升，E-钙黏蛋白（E-cadherin）下降，葡萄糖摄取、L-乳酸生成和糖酵解速率增强，及细胞外调节蛋白激酶1/2（extracellular signal-regulated kinases1/2，ERK1/2）和信号转导和转录激活因子3（signal transducer and activator of transcription 3，STAT3）的磷酸化水平提升。
    结论 MCF-7-BOM-luc通过激活ERK1/2和STAT3信号通路促进S100A4表达，从而增强EMT进程和糖酵解速率，具备骨转移恶性。通过蛋白质组学解析乳腺癌骨转移细胞的蛋白特征将为乳腺癌骨转移的诊断、预防和治疗提供潜力靶点。

     

    Abstract:
    Objective To investigate the protein expression characteristics of bone metastatic breast cancer cells and the underlying molecular mechanism driving bone metastasis.
    Methods A firefly luciferase-overexpressing human breast cancer cell line, MCF-7-luc, and its bone metastasis subline, MCF-7- BOM-luc, were established. Additionally, a nude mouse model of breast cancer bone metastasis was established by injecting the aforementioned cells into the left ventricle. Bone metastasis and trabecular changes were assessed using micro-computed tomography (Micro-CT). Cell migration, and invasion were evaluated using the Transwell assays. Differential protein expression and epithelial-mesenchymal transition (EMT) were analyzed using proteomics, Western blot, and reverse transcription-quantitative PCR (qPCR). We measured the glucose uptake, L-lactic acid content, and cell energy metabolism parameters using 2-NBDG, ELISA, and a Seahorse energy metabolizer.
    Results MCF-7-BOM-luc cells exhibited stronger migration, invasion, and EMT characteristics as well as more pronounced bone metastasis capabilities than the MCF-7-luc cells. Proteomic analysis revealed increased S100 calcium-binding protein A (S100A4) expression in MCF-7-BOM-luc cells, with other differentially expressed proteins being primarily involved in metabolic pathways. Additionally, MCF-7-BOM-luc cells displayed increased expression of E-box binding homeobox 1/2 (zinc finger E-box binding homeobox 1/2, ZEB1/2) and Vimentin and reduced E-cadherin expression. MCF-7-BOM-luc cells also exhibited enhanced glucose uptake, L-lactic acid production, and glycolysis rates as well as increased phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3).
    Conclusions MCF-7-BOM-luc cells promote S100A4 expression through ERK1/2 and STAT3 signaling pathway activation, which in turn enhances the EMT process and glycolysis rate, thereby contributing to malignant bone metastasis. Proteomic analysis of breast cancer bone metastasis-related protein characteristics could offer potential targets for the diagnosis, prevention, and treatment of breast cancer bone metastasis.

     

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