Instituto de Investigação e Inovação em Saúde (i3S), Rua Alfredo Allen 208, 4200-135 Porto, Portugal.
Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Rua Júlio Amaral de Carvalho 45, 4200-135 Porto, Portugal.
Cells. 2022 Jun 21;11(13):1988. doi: 10.3390/cells11131988.
Understanding how mutant KRAS signaling is modulated by exogenous stimuli is of utmost importance to elucidate resistance mechanisms underlying pathway inhibition failure, and to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at perceiving KRAS-autonomous versus -non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) upon KRAS silencing and treatment with rhTGFβ1-activated fibroblasts secretome. A proteomic analysis revealed that rhTGFβ1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs 43% and 38% of these alterations in HCT116 and LS174T cells, respectively. These KRAS-dependent proteins were localized and displayed molecular functions that were common to both cell lines (e.g., extracellular exosome, RNA binding functions). Moreover, 67% and 78% of the KRAS-associated proteome of HCT116 and LS174T cells, respectively, was controlled in a KRAS-non-autonomous manner, being dependent on fibroblast-secreted factors. In HCT116 cells, KRAS-non-autonomously controlled proteins were mainly involved in proteoglycans in cancer, p53, and Rap1 signaling pathways; whereas in LS174T cells, they were associated with substrate adhesion-dependent cell-spreading and involved in metabolic processes. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of integrating the tumor microenvironment in the study of KRAS-associated effects.
了解外源性刺激如何调节突变型 KRAS 信号对于阐明导致通路抑制失败的抵抗机制以及为突变型 KRAS 患者发现新的治疗靶点至关重要。因此，为了感知 KRAS 自主和非自主机制，我们研究了两种突变型 KRAS 结直肠癌细胞系（HCT116 和 LS174T）在 KRAS 沉默和用 rhTGFβ1 激活的成纤维细胞分泌组处理后的反应。蛋白质组学分析显示，rhTGFβ1 激活的成纤维细胞分泌的因子触发了细胞系特异性的蛋白质组改变，并且突变型 KRAS 分别控制 HCT116 和 LS174T 细胞中 43%和 38%的这些改变。这些依赖 KRAS 的蛋白质被定位并显示出两种细胞系共有的分子功能（例如，细胞外外泌体、RNA 结合功能）。此外，HCT116 和 LS174T 细胞的 KRAS 相关蛋白质组的 67%和 78%分别以 KRAS 非自主的方式受到控制，依赖于成纤维细胞分泌的因子。在 HCT116 细胞中，KRAS 非自主控制的蛋白质主要参与癌症中的蛋白聚糖、p53 和 Rap1 信号通路；而在 LS174T 细胞中，它们与底物粘附依赖性细胞扩展有关，并参与代谢过程。这项工作强调了 KRAS 相关信号的上下文依赖性，并强调了将肿瘤微环境纳入 KRAS 相关效应研究的重要性。
