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

Introduction: Colorectal cancer (CRC) is the second most common cause of cancer related deaths in the United States. To best improve patient outcomes with targeted therapeutics, it is important to first understand the initial stages of CRC development. CRC begins as an adenomatous polyp on the inner lining of the colon, which can progress malignantly over time. In CRC, mutations in the Kirsten rat sarcoma virus (KRAS) gene typically occur in the early stages of the polyp to malignant carcinoma transition with 35-40% of CRC patients harboring a KRAS mutation. Oncogenic mutations in the KRAS gene are known to amplify cancer cell mechanosensing in breast and pancreatic cancers, especially as it relates to cytoskeletal tensional homeostasis. This led us to hypothesize that CRC cells harboring activating oncogenic KRAS mutations will have uniquely altered mechanotransduction leading to malignant CRC progression. We previously developed and validated a peristalsis bioreactor that mimics the forces associated with colonic peristalsis and demonstrated that HCT116 KRASG13D cells exposed to peristalsis developed invasive signatures. In this work, multiple cell lines and patient-derived xenograft lines were employed to test the hypothesis that activating KRAS mutations change mechanotransduction via cancer stem cell enrichment. The objective of this work was to identify a connection between activating KRAS mutations and altered cellular response to peristalsis to improve our understanding of the role of peristalsis mechanics and targeted therapeutics in the treatment of CRC.

Materials and

Methods: The peristalsis bioreactor was tuned to replicate the mechanical parameters of the colon (0.4 Pa shear and 15% strain; Fig. 1A). Six APC wild type lines were tested: KRAS mutant (1) HCT116, (2) LS174T, and (3) PDX1; KRAS wild type (4) RKO and (5) PDX2, and (6) PDX3. Cells were exposed to peristalsis or maintained as static controls for 24 hours and then collected for downstream analysis. Flow cytometry methods were employed to evaluate LGR5 cancer stem cell emergence and unbiased gene set enrichment evaluated enriched pathways downstream of peristalsis. In vivo subcutaneous tumor growth was evaluated for tumorigenicity comparisons between KRAS mutant and wild type cells exposed to peristalsis or maintained as static controls. Previous work demonstrated LGR5 as a downstream target of Wnt activation so, Wnt-ligand gene expression was evaluated via qPCR, as well as associated β-catenin nuclear localization via immunofluorescence. To confirm the involvement of mutant KRAS in the altered mechanotransduction response to peristalsis, we employed gain and loss of function techniques for KRAS and its downstream effectors.

Results, Conclusions, and Discussions: LGR5 enrichment downstream of peristalsis was not universal and confined to oncogenic KRAS mutant cells with 2.5-3.8-fold increase in LGR5 (Fig. 1B). Wild type lines resulted in no LGR5 enrichment compared to static controls (Fig. 1B). To further support this result, we introduced the KRASG13D mutation into RKO KRASWT. Interestingly, when mutant KRASG13D was induced into the WT background cell line, exposure to peristalsis led to LGR5 enrichment. This result was backed by unbiased RNA-Seq analysis, where there were 6 unique pathways between HCT116 KRASG13D and RKO KRASWT and 5 unique pathways between RKO KRASWT and RKO KRASG13D out of the top 9 GO terms in response to peristalsis (Fig. 1C). As expected, in vivo analysis demonstrated increased growth rates in KRAS mutant cells exposed to peristalsis compared to static controls. Wnt-ligand gene expression in peristalsis was highly enriched in both HCT116 KRASG13D and RKO KRASG13D cells with mild enrichment in RKO KRASWT cells. However, Wnt inhibition (LGK974) did not impact LGR5 enrichment demonstrating a Wnt-ligand independent LGR5 response to peristalsis. Further, β-catenin nuclear localization analysis resulted in significant increases in KRAS mutant cells independent of Wnt-ligand inhibition (Fig. 1D). To directly evaluate the dependency of KRAS downstream effectors in regulating LGR5 expression in response to peristalsis, a MEK inhibitor was employed. Ultimately, the LGR5 enrichment and β-catenin activation phenotype downstream of peristalsis were sensitive to MEK inhibition in KRAS mutant cells.
This work demonstrates that that oncogenic KRAS mutations dictate a unique mechanotransduction response to peristalsis in CRC. With the application of peristalsis forces, CRC cells harboring a KRAS mutation demonstrated an altered functional phenotype and genotype that portends to increased tumor growth in vivo. Importantly, KRAS targeted therapeutic options are growing in the treatment of KRAS mutant cancers and can be used uniquely with other pharmacologic modulators that regulate colonic peristalsis to achieve favorable therapeutic outcomes.

Acknowledgements (Optional): This work was supported by the CPRIT TREC Award RP230204 (SAR) and NIH R37CA26922 (SAR).