Phosphoproteomics measurement of clonal endocrine therapy sensitive and resistant breast cancer populations

Around two thirds of breast tumors are characterized by the expression of estrogen receptor α and are therapeutically treated by blocking estrogen signaling. First line endocrine therapeutics are Tamoxifen which displaces estrogen form its receptor preventing receptor activation and aromatase inhibitors which prevent the formation of estrogen and thereby hormone-deprive the tumors. Therapy resistance remains a major clinical problem, especially for patients with late-stage diagnoses. Tumor heterogeneity may promote therapy resistance either through the selection of pre-existing rare clones or by providing a repertoire of cells that may develop resistance over time. In order to study endocrine therapy resistance on a clonal level, we have developed barcoded (allowing the tracking of single cells) endocrine therapy sensitive and resistant breast cancer cell lines. From these complex cell pools, multiple single cells characterized by a specific barcode were isolated and grown out. We then subjected the clones to phosphoproteomics measurement by Mass spectrometry. Based on their phosphorylation pattern, the kinase activity profiles of endocrine therapy resistant clones were determined to identify differentially activated kinases with implications in therapy resistance.

约三分之二的乳腺肿瘤以雌激素受体α（estrogen receptor α）的表达为特征，临床通过阻断雌激素信号通路开展治疗。一线内分泌治疗药物包括他莫昔芬（Tamoxifen）与芳香化酶抑制剂（aromatase inhibitors）：前者可竞争性结合雌激素受体，置换内源性雌激素以阻断受体激活；后者则抑制雌激素合成，从而使肿瘤处于雌激素剥夺状态。治疗耐药仍是临床面临的主要难题，尤其针对晚期确诊患者。肿瘤异质性可通过两种途径促进治疗耐药：一是筛选预先存在的稀有克隆亚群，二是提供具备随时间推移获得耐药性潜能的细胞群体储备。为在克隆水平研究内分泌治疗耐药机制，我们构建了带有条形码标记（可实现单细胞追踪）的内分泌治疗敏感型与耐药型乳腺癌细胞系。从这些复杂的细胞混合池中，我们分离出多个携带特定条形码的单细胞并进行扩增培养。随后我们通过质谱（Mass spectrometry）对这些克隆开展磷酸化蛋白质组学（phosphoproteomics）检测。基于各克隆的磷酸化修饰模式，我们分析了内分泌治疗耐药克隆的激酶活性谱，以鉴定出与治疗耐药相关的差异激活激酶。