KRAS Mutations in Colorectal Cancer Drive a Unique Mechanotransduction Response to Peristalsis-Associated Forces

50% of all colorectal cancer (CRC) cases will metastasize which greatly reduces the 5-year survival rates¹. Expanding our understanding of early CRC progression is vital to finding new therapeutic targets to improve patient outcomes. CRCs begin as polyps in the inner lining of the colon, which progress malignantly, especially when they harbor genetic mutations in the KRAS gene². Many other microenvironmental factors also drive tumor progression and metastasis, including mechanical forces. In the CRC tumor microenvironment, cancerous and precancerous cells are continuously exposed to mechanical forces associated with peristalsis (shear stress and multi-axial strain). Therefore, we hypothesized that peristalsis contributes to malignant progression in CRC, especially if they harbor oncogenic KRAS mutations. We defined malignant progression by three major hallmarks: i) cancer stem cell enrichment, ii) increased epithelial to mesenchymal transition (EMT) gene signatures, and iii) emergence of an invasive morphology (elongation and filopodial protrusions). All of these collectively allow CRC to progress and metastasize. The objective of this work was to leverage our custom-built peristalsis bioreactor³ to evaluate if peristalsis-associated mechanotransduction results in malignant progression of KRAS mutant 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). Two cell lines were tested: (1) KRAS mutant HCT116; and (2) KRAS wild type (WT) KM12. Cells were exposed to peristalsis or maintained as static controls for 24 hours and then collected to evaluate malignant progression via three methods: i) flow cytometry for cancer stem cell (CSC) emergence, ii) qPCR for EMT gene expression analysis, and iii) immunofluorescence imaging to identify invasive cell morphology (elongation and filopodia density). Additionally, gene expression analysis of the Rho/ROCK mechanotransduction pathway and the Wnt pathway were tested via qPCR to determine their involvement in cellular response to peristalsis. Subsequent inhibition and activation of ROCK and Wnt were employed in the assessment of malignant progression as a result of peristalsis-associated mechanotransduction.

Results and Discussion:

Peristalsis enriched LGR5+ cancer stem cells (CSCs) in KRAS mutant HCT116 cells (1.8 fold increase), but not WT KM12 cells exposed to peristalsis, compared to cells in static controls (Fig. 1B). This immediately indicated that KRAS mutation was an important factor in determining peristalsis-associated mechanotransduction leading to CSC emergence. EMT gene expression: Exposure to peristalsis robustly increased EMT gene expression in KRAS mutant cells relative to static controls (increases in SNAIL1, SNAIL2, ZEB1, ETS-1, MMP2, and decrease in E-CAD). In contrast, WT KM12 cells demonstrated minimal changes in EMT gene expression (Fig. 1C). Cell morphology (elongation and filopodia density): Actin filaments were visualized with fluorescent phalloidin to quantify filopodia density (hairy protrusions; Fig 1D) and cell elongation. Peristalsis significantly increased filopodia density and cell elongation in KRAS mutant HCT116 cells compared to static controls. Mechanotransduction: Peristalsis activated the mechanotransductive Rho/ROCK pathway in KRAS mutant HCT116, compared to static controls, implicating its involvement in the acquisition of invasive morphologies. Peristalsis also significantly increased gene expression of several Wnt ligands (Wnt1, Wnt4, Wnt5a, Wnt5b, Wnt7a, and Wnt7b) that are involved in the enrichment of LGR5+ CSCs. Inhibition of both these pathways resulted in loss of malignant progression hallmarks downstream of peristalsis.
This work demonstrates that active KRAS mutations play an important role in the mechanotransduction of peristalsis in CRC progression. KRAS mutations result in deadly, chemo resistant and metastatic CRC. Therefore, our future work will explore why KRAS mutant cells respond uniquely to peristalsis-associated mechanotransduction. Ultimately, this work will illuminate mechanical avenues that can be targeted to curb progression and invasion in KRAS mutant CRC.

Acknowledgements:

This work was supported by the CPRIT TREC Award RP230204, NIH R37CA269224-01A1, and the Texas A&M Department of Biomedical Engineering.

References:

1. Rawla P. Prz Gastroenterol. 2019;14:89-103; 2. Huang D. Cancer Metastasis Rev. 2018;37:173-187; 3. Clevenger AJ. Cells Tissues Organs. 2023;212:96-110