Yokoi Ryoma, Mori Toshiki, Hirosawa Koichiro M, Kondo Ryojiro, Taguchi Tomohiko, Matsuhashi Nobuhisa, Suzuki Kenichi G N
Department of Gastroenterological Surgery and Pediatric Surgery, Graduate School of Medicine, Gifu University, Gifu 501-1193, Japan.
Institute for Glyco-Core Research (iGCORE), Gifu University, Gifu 501-1193, Japan.
iScience. 2025 Aug 14;28(9):113374. doi: 10.1016/j.isci.2025.113374. eCollection 2025 Sep 19.
Oncogenic KRAS mutations are frequent in colorectal cancer, presenting substantial challenges due to constitutive activation and resistance to molecular-targeted therapies driven by mutation-specific biochemical properties. In this study, using single-molecule imaging, we quantitatively analyzed diffusional behaviors of oncogenic KRAS mutants and associated signaling molecules to elucidate their signaling mechanisms and therapeutic implications. Upon EGF stimulation, KRAS molecules exhibited transient trapping and reduced diffusion due to interactions with SOS1 and BRAF, leading to the temporary formation of signaling complexes. Our results demonstrate that analysis of the temporal fraction and frequency of transient trapping events offers a more sensitive and precise evaluation of KRAS activation levels than western blotting. Furthermore, the study of dynamics of individual oncogenic KRAS molecules provides a more accurate assessment of the therapeutic efficacy of various molecular-targeted drugs. Consequently, we propose a highly sensitive strategy to enhance the therapeutic targeting of oncogenic KRAS in living colorectal cancer cells.
致癌性KRAS突变在结直肠癌中很常见，由于其组成性激活以及由突变特异性生化特性驱动的对分子靶向治疗的抗性，带来了巨大挑战。在本研究中，我们使用单分子成像技术，定量分析了致癌性KRAS突变体和相关信号分子的扩散行为，以阐明其信号传导机制和治疗意义。在表皮生长因子（EGF）刺激下，KRAS分子由于与SOS1和BRAF相互作用而表现出瞬时捕获和扩散减少，导致信号复合物的临时形成。我们的结果表明，与蛋白质免疫印迹法相比，对瞬时捕获事件的时间分数和频率进行分析，能更灵敏、精确地评估KRAS激活水平。此外，对单个致癌性KRAS分子动力学的研究，能更准确地评估各种分子靶向药物的治疗效果。因此，我们提出了一种高度灵敏的策略，以增强对活的结直肠癌细胞中致癌性KRAS的治疗靶向性。
