Notch信号传导是细胞间信号转导中的关键分子通路之一。尽管NOTCH受体的作用机制已相对较为了解，但它们在头颈部鳞状细胞癌(HNSCC)的发生和发展过程中的生物学意义仍不明确。本研究通过比较RIN-1和CB-103等代表性"下一代"小分子Notch调节剂与DAPT等"经典"NOTCH干扰化合物(如γ-分泌酶抑制剂)在HNSCC上的生长和分化调控效应，揭示了它们在不同的细胞和组织培养条件下(包括2D单层培养、无黏附或球体培养、3D器官样培养以及斑马鱼体内模型)的作用。在这些通路中，Notch调节剂RIN-1表现出最显著的全部效应。在分子水平上，RIN-1依赖的Notch信号激活导致了NOTCH调控靶点表达的特征性改变，即转录抑制因子HES1和HEY1、细胞周期抑制剂p21(CDKN1A)和前凋亡标志物BAX的表达。这些改变导致了HNSCC细胞在2D培养中的增殖的限制、生长的抑制和运动能力的降低。因此，在细胞周期中G2-M期的停滞和凋亡诱导也可以观察到。类似的抗癌效果也在3D培养和斑马鱼模型中观察到。相反，RIN-1处理导致Notch信号的抑制，并在无附着的细胞培养条件下限制了HNSCC球体的生长。我们的结果表明，调控Notch信号可能可用作具有完整NOTCH信号传导的选定患者的化疗药物。©2023 Springer Nature Limited.

Notch signalling is one of the key molecular pathways involved in cell-to-cell signal transduction. Although the mechanisms of action of the NOTCH receptors are already relatively well known, their biological implications remain unclear, especially during the initiation and progression of head and neck squamous cell carcinoma (HNSCC). Here, we present the growth- and differentiation-modulating effects of various "next generation" small molecule Notch modulators represented by RIN-1, and CB-103, on HNSCC, compared to gamma secretase inhibitors as "conventional" NOTCH interfering compounds, like DAPT. These molecules were tested in different cell- and tissue culture conditions represented by 2D monolayer, non-adherent or spheroid culture, 3D organoid cultures, and zebrafish in vivo model. The most pronounced, pleiotropic effects were observed for the NOTCH modulator RIN-1. At the molecular level, RIN-1-dependent activation of Notch signalling led to characteristic changes in the expression of NOTCH-regulated targets, i.e., the transcriptional suppressors HES1 and HEY1, p21 (CDKN1A) cell cycle inhibitor, and pro-apoptotic BAX markers. These changes led to restriction of proliferation, growth, and reduced motility of HNSCC cells in 2D cultures. Consequently, cell cycle arrest in the G2-M phase and induction of apoptosis were observed. Similar anticancer effects were observed in 3D cultures and in the zebrafish model. In contrast, RIN-1 treatment resulted in inhibition of Notch signalling and the growth of HNSCC spheroids under non-adherent cell culture conditions. Our results suggest that modulation of Notch signalling could be used as a chemotherapeutic agent in selected patients with intact NOTCH signaling.© 2023. Springer Nature Limited.