Ali Naveid A, Wu Jianmin, Hochgräfe Falko, Chan Howard, Nair Radhika, Ye Sunny, Zhang Luxi, Lyons Ruth J, Pinese Mark, Lee Hong Ching, Armstrong Nicola, Ormandy Christopher J, Clark Susan J, Swarbrick Alexander, Daly Roger J
Breast Cancer Res. 2014 Sep 9;16(5):437. doi: 10.1186/s13058-014-0437-3.
Although aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets.
We applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growth factor receptor 2, Her2 (HER2); mouse mammary tumour virus-polyoma virus middle T antigen (PyMT); and the p53-/- transplant model (p53). Pathways and protein-protein interaction networks were identified by bioinformatics analysis. Molecular mechanisms underpinning differences in tyrosine phosphorylation were characterised by Western blot analysis and array comparative genomic hybridisation. The functional role of mesenchymal-epithelial transition factor (Met) in a subset of p53-null tumours was interrogated using a selective tyrosine kinase inhibitor (TKI), small interfering RNA (siRNA)-mediated knockdown and cell proliferation assays.
The three models could be distinguished on the basis of tyrosine phosphorylation signatures and signalling networks. HER2 tumours exhibited a protein-protein interaction network centred on avian erythroblastic leukaemia viral oncogene homologue 2 (Erbb2), epidermal growth factor receptor and platelet-derived growth factor receptor α, and they displayed enhanced tyrosine phosphorylation of ERBB receptor feedback inhibitor 1. In contrast, the PyMT network displayed significant enrichment for components of the phosphatidylinositol 3-kinase signalling pathway, whereas p53 tumours exhibited increased tyrosine phosphorylation of Met and components or regulators of the cytoskeleton and shared signalling network characteristics with basal and claudin-low breast cancer cells. A subset of p53 tumours displayed markedly elevated cellular tyrosine phosphorylation and Met expression, as well as Met gene amplification. Treatment of cultured p53-null cells exhibiting Met amplification with a selective Met TKI abrogated aberrant tyrosine phosphorylation and blocked cell proliferation. The effects on proliferation were recapitulated when Met was knocked down using siRNA. Additional subtypes of p53 tumours exhibited increased tyrosine phosphorylation of other oncogenes, including Peak1/SgK269 and Prex2.
This study provides network-level insights into signalling in the breast cancer models utilised and demonstrates that comparative phosphoproteomics can identify conserved oncogenic signalling pathways. The Met-amplified, p53-null tumours provide a new preclinical model for a subset of triple-negative breast cancers.
尽管异常的酪氨酸激酶信号传导是特定乳腺癌亚型的特征，但迄今为止尚未对乳腺癌小鼠模型中的酪氨酸磷酸化进行全面分析。这可能会识别出保守的致癌途径和潜在的治疗靶点。
我们将免疫亲和/质谱工作流程应用于三种小鼠模型：表达截短型Neu（人表皮生长因子受体2 Her2（HER2）的大鼠同源物）的鼠干细胞病毒-Neu；小鼠乳腺肿瘤病毒-多瘤病毒中间T抗原（PyMT）；以及p53基因敲除移植模型（p53）。通过生物信息学分析确定途径和蛋白质-蛋白质相互作用网络。通过蛋白质印迹分析和阵列比较基因组杂交来表征酪氨酸磷酸化差异的分子机制。使用选择性酪氨酸激酶抑制剂（TKI）、小干扰RNA（siRNA）介导的敲低和细胞增殖试验来研究间充质-上皮转化因子（Met）在一部分p53缺失肿瘤中的功能作用。
这三种模型可以根据酪氨酸磷酸化特征和信号网络进行区分。HER2肿瘤表现出以禽成红细胞白血病病毒癌基因同源物2（Erbb2）、表皮生长因子受体和血小板衍生生长因子受体α为中心的蛋白质-蛋白质相互作用网络，并且它们显示出ERBB受体反馈抑制剂1的酪氨酸磷酸化增强。相比之下，PyMT网络显示磷脂酰肌醇3-激酶信号通路的成分有显著富集，而p53肿瘤表现出Met以及细胞骨架成分或调节因子的酪氨酸磷酸化增加，并且与基底样和claudin低表达乳腺癌细胞具有共同的信号网络特征。一部分p53肿瘤显示细胞酪氨酸磷酸化和Met表达明显升高，以及Met基因扩增。用选择性Met TKI处理表现出Met扩增的培养p53缺失细胞可消除异常的酪氨酸磷酸化并阻断细胞增殖。当使用siRNA敲低Met时，对增殖的影响得以重现。p53肿瘤的其他亚型表现出包括Peak1/SgK269和Prex2在内的其他致癌基因的酪氨酸磷酸化增加。
本研究提供了对所使用的乳腺癌模型中信号传导的网络层面见解，并证明比较磷酸化蛋白质组学可以识别保守的致癌信号通路。Met扩增的p53缺失肿瘤为一部分三阴性乳腺癌提供了一种新的临床前模型。
