Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan.
Medical Research Institute, Kitano Hospital, PIIF Tazuke-kofukai, Kita-ku, Osaka 530-8480, Japan.
Endocrinology. 2024 Jul 26;165(9). doi: 10.1210/endocr/bqae089.
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.
库欣病是一种危及生命的疾病，由垂体神经内分泌肿瘤（PitNETs）自主分泌 ACTH 引起。很少有药物适用于无法手术的库欣病，特别是由于侵袭性 PitNETs 引起的库欣病。为了探索调节 ACTH 分泌性 PitNETs 的药物，我们使用 AtT-20 进行了高通量筛选（HTS），AtT-20 是一种具有 ACTH 分泌特征的鼠垂体肿瘤细胞系。对于 HTS，我们构建了一种基于活细胞的 ACTH 报告基因测定法，用于高通量评估 ACTH 的变化。该测定法基于过表达 ACTH 受体成分和荧光 cAMP 生物传感器的 HEK293T 细胞，通过高速获取荧光图像。我们用化合物处理 AtT-20 细胞，并使用报告基因测定法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，有 84 种化合物在 10 μM 时和 20 种化合物在 1 μM 时对 AtT-20 细胞分泌的 ACTH 有超过 50%的抑制作用。在这些命中化合物中，我们重点关注硫链丝菌素（TS），并确定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 细胞周期在 G2/M 期停滞，这是通过 FOXM1 非依赖性机制介导的，包括细胞周期蛋白的下调。同时给予 TS 和细胞周期蛋白依赖性激酶 4/6 抑制剂（影响 G0/1 期的细胞周期）显示出协同的抗肿瘤作用。因此，TS 是库欣病的一种有前途的治疗药物。我们的命中化合物列表和对 TS 作用的新机制见解为未来的研究提供了有价值的基础。
