Oncogenic KRAS Mutations Confer a Unique Mechanotransduction Response to Peristalsis in Colorectal Cancer Cells

Colorectal cancer (CRC) tumors start as precancerous polyps on the inner lining of the colon or rectum, where they are exposed to the mechanics of colonic peristalsis. Our previous work leveraged a custom-built peristalsis bioreactor to demonstrate that colonic peristalsis led to cancer stem cell enrichment in colorectal cancer cells. However, this malignant mechanotransductive response was confined to select CRC lines that harbored an oncogenic mutation in the KRAS gene. In this work, therefore, we explored the involvement of activating KRAS mutations on peristalsis-associated mechanotransduction in CRC. Peristalsis enriched the cancer stem cell marker LGR5 in KRAS mutant (G13D, etc.) lines, in a Wnt-independent manner. Conversely, LGR5 enrichment in wild type KRAS lines exposed to peristalsis were minimal. LGR5 enrichment downstream of peristalsis translated to increased tumorigenicity in vivo in KRAS mutant vs. wild type lines. Differences in mechanotransduction response was additionally apparent via unbiased gene set enrichment analysis, where many unique pathways were enriched in wild type vs. mutant lines, in response to peristalsis. Interestingly, peristalsis also triggered ß-catenin nuclear localization independent of Wnt, particularly in KRAS mutant lines. The central involvement of KRAS in the mechanotransductive responses was validated via gain and loss of function strategies. ß-catenin activation and LGR5 enrichment downstream of peristalsis converged to the activation of the MEK/ERK kinase cascade, that remains active in cells that harbor oncogenic KRAS mutations. Taken together, our results demonstrated that oncogenic KRAS mutations conferred a unique peristalsis-associated mechanotransduction response to colorectal cancer cells, resulting in cancer stem cell enrichment and increased tumorigenicity. These mechanosensory connections can be leveraged in improving the sensitivity of emerging therapies that target oncogenic KRAS. Overall design: To investigate the difference in KRAS mutant CRC cellular response to peristalsis, three cell lines were used: 1) HCT116 KRAS mutant, 2) RKO KRAS wild type and 3) RKO KRAS mutant. We performed gene expression analysis on all three cell lines in static and peristalsis conditions.

结直肠癌（Colorectal cancer, CRC）肿瘤起源于结肠或直肠内壁的癌前息肉，此时病变组织会暴露于结肠蠕动的力学刺激中。我们此前的研究借助定制的蠕动生物反应器（peristalsis bioreactor）证实，结肠蠕动可促使结直肠癌细胞内的癌症干细胞（cancer stem cell）富集，但该恶性机械转导响应仅局限于携带KRAS基因致癌突变的特定结直肠癌细胞系。本研究据此探索了激活型KRAS突变在结直肠癌蠕动相关机械转导过程中的作用。研究发现，蠕动可通过不依赖Wnt信号通路的方式，在携带KRAS突变（如G13D等）的细胞系中富集癌症干细胞标志物LGR5。与之相反，暴露于蠕动环境的KRAS野生型细胞系中，LGR5的富集程度极低。蠕动诱导的LGR5富集，在KRAS突变型细胞系中对应了体内致瘤性的增强，而野生型细胞系则无此显著变化。通过无偏基因集富集分析（gene set enrichment analysis, GSEA）也可观察到机械转导响应的差异：蠕动刺激下，野生型与突变型细胞系分别富集了大量独特的信号通路。有趣的是，蠕动还可触发β-连环蛋白（β-catenin）的核定位，且该过程不依赖Wnt信号，在KRAS突变型细胞系中尤为明显。KRAS在机械转导响应中的核心作用通过功能获得与功能缺失实验得到了验证。蠕动下游的β-连环蛋白激活与LGR5富集，最终汇聚至MEK/ERK激酶级联反应的激活，而该级联反应在携带致癌KRAS突变的细胞中始终处于激活状态。综上，本研究结果表明，致癌KRAS突变赋予了结直肠癌细胞独特的蠕动相关机械转导响应，最终导致癌症干细胞富集与致瘤性增强。这类机械感知关联可被用于提升靶向致癌KRAS的新兴治疗手段的敏感性。
实验整体设计：为探究KRAS突变型结直肠癌细胞对蠕动刺激的响应差异，本研究使用了3种细胞系：1）HCT116 KRAS突变型细胞系；2）RKO KRAS野生型细胞系；3）RKO KRAS突变型细胞系。我们对这3种细胞系分别在静态培养与蠕动培养条件下进行了基因表达分析。