Cushing's disease (CD) is a rare endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor. Pasireotide is the only pituitary-targeted drug approved for adult patients. Nevertheless, many side effects are encountered and curative therapy is still challenging. Ubiquitin-specific peptidase 8 (USP8) plays a crucial role in the modulation of corticotroph cells growth and ACTH secretion. Here, we explored the anticancer potential of the USP8 inhibitor RA-9 in USP8-WT human tumor corticotroph cells and murine AtT-20 cells. Our results showed that RA-9 causes cell proliferation decrease (-24.3 ± 5.2%, P < 0.01) and cell apoptosis increase (207.4 ± 75.3%, P < 0.05) in AtT-20 cells, as observed with pasireotide. Moreover, RA-9 reduced ACTH secretion in AtT-20 cells (-34.1 ± 19.5%, P < 0.01), as well as in AtT-20 cells transfected with USP8 mutants, and in one out of two primary cultures in vitro responsive to pasireotide (-40.3 ± 6%). An RA-9 mediated decrease of pERK1/2 levels was observed in AtT-20 cells (-52.3 ± 13.4%, P < 0.001), comparable to pasireotide, and in primary cultures, regardless of their in vitro responsiveness to pasireotide. Upregulation of p27 was detected upon RA-9 treatment only, both in AtT-20 cells (167.1 ± 36.7%, P < 0.05) and in one primary culture tested (168.4%), whilst pCREB level was similarly halved in AtT-20 cells by both RA-9 and pasireotide. Altogether, our data demonstrate that RA-9 is efficient in exerting cytotoxic effects and inhibitory actions on cell proliferation and hormone secretion by modulating the expression of pERK1/2, pCREB and p27. Inhibition of USP8 might represent a novel strategy to target both USP8-WT and USP8-mutated tumors in CD patients.

中文翻译：

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USP8 抑制剂 RA-9 可减少肿瘤促肾上腺皮质激素中的促肾上腺皮质激素释放和细胞生长。

库欣病 (CD) 是一种罕见的内分泌疾病，由分泌促肾上腺皮质激素 (ACTH) 的垂体肿瘤引起。Pasireotide 是唯一获批用于成年患者的垂体靶向药物。然而，会遇到许多副作用，并且治愈性治疗仍然具有挑战性。泛素特异性肽酶 8 (USP8) 在调节促肾上腺皮质激素细胞生长和促肾上腺皮质激素分泌中起关键作用。在这里，我们探索了 USP8 抑制剂 RA-9 在 USP8-WT 人肿瘤促皮质激素细胞和鼠 AtT-20 细胞中的抗癌潜力。我们的研究结果表明，RA-9 导致 AtT-20 细胞中的细胞增殖减少（-24.3 ± 5.2%，P < 0.01）和细胞凋亡增加（207.4 ± 75.3%，P < 0.05），如用帕瑞肽观察到的那样。此外，RA-9 减少 AtT-20 细胞中的 ACTH 分泌（-34.1 ± 19.5%，P < 0.01），以及用 USP8 突变体转染的 AtT-20 细胞，以及在体外对帕瑞肽有反应的两种原代培养物中的一种 (-40.3 ± 6%)。在 AtT-20 细胞中观察到 RA-9 介导的 pERK1/2 水平降低（-52.3 ± 13.4%，P < 0.001），与帕瑞肽相当，在原代培养物中，无论它们对帕瑞肽的体外反应如何。仅在 RA-9 处理后检测到 p27 的上调，在 AtT-20 细胞（167.1 ± 36.7%，P < 0.05）和测试的一种原代培养物中（168.4%），而在 AtT-20 细胞中 pCREB ​​水平同样减半通过 RA-9 和帕瑞肽。总之，我们的数据表明，RA-9 通过调节 pERK1/2、pCREB ​​和 p27 的表达有效地发挥细胞毒作用和对细胞增殖和激素分泌的抑制作用。