Martins Flávia, Machado Ana L, Carvalho Joana, Almeida Catarina R, Beck Hans C, Carvalho Ana S, Backman Vadim, Matthiesen Rune, Velho Sérgia
Institute for Research and Innovation in Health, University of Porto, Porto, Portugal.
Faculty of Medicine, University of Porto, Porto, Portugal.
Sci Rep. 2025 Apr 24;15(1):14329. doi: 10.1038/s41598-025-94549-2.
Despite the development of mutant-selective KRAS inhibitors, colorectal cancer (CRC) responses remain limited, with stable disease and rapid recurrence being common outcomes. The molecular mechanisms enabling CRC cells to tolerate KRAS inhibition and ultimately develop resistance remain poorly understood. Here, we investigated early transcriptional and proteomic responses to KRAS silencing in 3D CRC cell line spheroid models, aiming to identify pathways associated with sensitivity or resistance to KRAS blockade. Cell lines were stratified into KRAS silencing-sensitive (HCT116 and SW480) and -resistant (LS174T and SW837) groups based on spheroid growth, cell cycle progression, and apoptosis induction. Transcriptional profiling revealed the unfolded protein response (UPR) and WNT/β-catenin signaling as pathways specifically upregulated in KRAS silencing-sensitive cells and downregulated in resistant cells. Proteomic analysis of membrane-enriched fractions further supported UPR deregulation, showing a pronounced downregulation of translation-related proteins in sensitive cells. Functional assays validated that the sensitive cell line HCT116 exhibits reduced protein aggregation and lower translational capacity upon KRAS knockdown, consistent with UPR activation. Pharmacological inhibition of IRE1α-mediated UPR signaling did not revert KRAS silencing-induced cell cycle arrest or apoptosis in this cell line. Collectively, our results highlight the UPR activation as an early adaptive response of KRAS-dependent CRC cells to KRAS silencing.
尽管已经开发出了突变选择性KRAS抑制剂，但结直肠癌（CRC）的反应仍然有限，疾病稳定和快速复发是常见的结果。CRC细胞耐受KRAS抑制并最终产生耐药性的分子机制仍知之甚少。在此，我们研究了3D CRC细胞系球体模型中对KRAS沉默的早期转录和蛋白质组学反应，旨在确定与对KRAS阻断的敏感性或耐药性相关的途径。根据球体生长、细胞周期进展和凋亡诱导，将细胞系分为KRAS沉默敏感组（HCT116和SW480）和耐药组（LS174T和SW837）。转录谱分析显示，未折叠蛋白反应（UPR）和WNT/β-连环蛋白信号通路在KRAS沉默敏感细胞中特异性上调，在耐药细胞中下调。对富含膜的组分进行蛋白质组学分析进一步支持了UPR失调，显示敏感细胞中与翻译相关的蛋白质明显下调。功能分析证实，敏感细胞系HCT116在KRAS敲低后表现出蛋白质聚集减少和翻译能力降低，这与UPR激活一致。药理学抑制IRE1α介导的UPR信号并未逆转该细胞系中KRAS沉默诱导的细胞周期停滞或凋亡。总的来说，我们的结果突出了UPR激活是KRAS依赖性CRC细胞对KRAS沉默的早期适应性反应。
