DataSheet_4_Global signalling network analysis of luminal T47D breast cancer cells in response to progesterone.docx

BackgroundBreast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear.
MethodsIn this study we used antibody microarrays and phosphoproteomics to reveal a dynamic global signalling map that reveals new key regulated proteins and phosphor-sites and links between previously known and novel pathways. T47D breast cancer cells were used, and phospho-sites and pathways highlighted were validated using specific antibodies and phenotypic assays. Bioinformatic analysis revealed an enrichment in novel signalling pathways, a coordinated response between cellular compartments and protein complexes.
ResultsDetailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, epithelial to mesenchyme transition (EMT), cell adhesion, as well as transcription factors previously not associated with breast cancer cell proliferation. Pathway analysis confirmed the key role of the MAPK signalling cascade following progesterone and additional hormone regulated phospho-sites were identified. Full network analysis shows the activation of new signalling pathways previously not associated with progesterone signalling in T47D breast cancer cells such as ERBB and TRK. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins.
ConclusionsThis study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management.

### 研究背景
乳腺癌细胞在暴露于孕激素后，可通过激活信号级联反应进入细胞周期：该级联反应涉及大量酶类、转录因子与辅因子，可将外源信号从细胞膜传递至染色质，最终引发基因表达程序的改变。尽管该信号网络中的诸多事件已被单独阐明，但其如何协同发挥全局作用以最终引发细胞应答的机制仍不明确。

### 研究方法
本研究采用抗体芯片（antibody microarrays）与磷酸化蛋白质组学（phosphoproteomics）技术，构建了动态全局信号图谱，以此鉴定出关键的受调控蛋白与磷酸化位点（phosphorylation sites），并揭示了已知通路与新通路之间的关联。实验采用T47D乳腺癌细胞系，通过特异性抗体与表型实验对筛选出的磷酸化位点及信号通路进行验证。生物信息学分析显示，本研究筛选出的信号通路存在显著富集，且呈现出细胞区室间与蛋白质复合物间的协同应答特征。

### 研究结果
对数据的详细分析显示，参与细胞核结构构建、上皮间质转化（EMT）、细胞黏附的蛋白质复合物，以及此前未被发现与乳腺癌细胞增殖相关的转录因子，均发生了引人关注的表达变化。通路分析证实了丝裂原活化蛋白激酶（MAPK）信号级联反应在孕激素刺激后的关键调控作用，同时还鉴定出了更多受激素调控的磷酸化位点。全网络分析显示，本研究还发现了此前未被报道与孕激素信号通路相关的全新信号通路在T47D乳腺癌细胞中被激活，包括ERBB与TRK信号通路。由于不同的翻译后修饰可介导复杂的信号串扰机制，且孕激素可快速诱导大规模的多聚ADP核糖基化（PARylation），本研究详细阐明了同时包含磷酸化蛋白与多聚ADP核糖基化蛋白的重要染色质调控复合物。

### 研究结论
本研究为科研共同体提供了一项重要的研究资源：其鉴定出了与乳腺癌细胞生物学密切相关的全新调控因子与信号关联，且这些发现或可应用于癌症诊疗领域。