Abstract：
Cushing disease is a life-threatening disorder caused by autonomous secretion of ACTH from pituitary neuroendocrine tumors (PitNETs). Few drugs are indicated for inoperative Cushing disease, in particular that due to aggressive PitNETs. To explore agents that regulate ACTH-secreting PitNETs, we conducted high-throughput screening (HTS) using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. For the HTS, we constructed a live cell-based ACTH reporter assay for high-throughput evaluation of ACTH changes. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, with high-throughput acquisition of fluorescence images. We treated AtT-20 cells with compounds and assessed ACTH concentrations in the conditioned media using the reporter assay. Of 2480 screened bioactive compounds, over 50% inhibition of ACTH secreted from AtT-20 cells was seen with 84 compounds at 10 μM and 20 compounds at 1 μM. Among these hit compounds, we focused on thiostrepton (TS) and determined its antitumor effects in both in vitro and in vivo xenograft models of Cushing disease. Transcriptome and flow cytometry analyses revealed that TS administration induced AtT-20 cell cycle arrest at the G2/M phase, which was mediated by FOXM1-independent mechanisms including downregulation of cyclins. Simultaneous TS administration with a cyclin-dependent kinase 4/6 inhibitor that affected the cell cycle at the G0/1 phase showed cooperative antitumor effects. Thus, TS is a promising therapeutic agent for Cushing disease. Our list of hit compounds and new mechanistic insights into TS effects serve as a valuable foundation for future research.
摘要：
库欣病是由垂体神经内分泌肿瘤（PitNET）自主分泌促肾上腺皮质激素（ACTH）引起的一种危及生命的疾病。很少有药物可用于不手术的库欣病，特别是由于积极的PitNET。探索调节分泌ACTH的PitNETs的药物，我们使用AtT-20进行了高通量筛选（HTS），AtT-20是一种以ACTH分泌为特征的鼠垂体肿瘤细胞系。对于HTS,我们构建了一个基于活细胞的ACTH报告基因检测方法,用于高通量评估ACTH的变化.该测定基于过表达ACTH受体成分的HEK293T细胞和荧光cAMP生物传感器，与荧光图像的高通量采集。我们用化合物处理AtT-20细胞,并使用报告基因测定评估条件培养基中的ACTH浓度。在2480种筛选的生物活性化合物中，用10μM的84种化合物观察到AtT-20细胞分泌的ACTH抑制超过50％，和20种1μM的化合物。在这些被击中的化合物中，我们专注于硫链菌素（TS），并确定了其在库欣病的体外和体内异种移植模型中的抗肿瘤作用。转录组和流式细胞术分析显示，TS给药诱导AtT-20细胞周期停滞在G2/M期，这是由FOXM1非依赖性机制介导的，包括细胞周期蛋白的下调。在G0/1期与影响细胞周期的CDK4/6抑制剂同时施用TS显示出协同抗肿瘤作用。因此，TS是一种有前途的治疗库欣病的药物。我们的命中化合物列表和对TS效应的新机理见解为未来的研究奠定了宝贵的基础。
