Combined inhibition of the FAK-Rho-ROCK signaling cascade, one of the important players in mechanotransduction, in colorectal cancers

Abstract

Purpose: Colorectal cancer (CRC) is one of the most common cancer types globally, with a high mortality rate. The FAK-Rho-ROCK successive signaling cascade promotes growth, migration and invasion of cancer cells. Focal adhesions are major sites of interactions between extracellular mechanical environments and intracellular biochemical signaling molecules/cytoskeleton and therefore focal adhesion proteins have been proposed to play important roles in mechanotransduction. This study aims to evaluate the effects of combination treatments with Focal Adhesion Kinase (FAK), Rho-ROCK, and YAP/TAZ inhibitors on the proliferative and epithelial-mesenchymal transition (EMT)-related metastatic characteristics of colorectal cancer cells. Material and Methods: In vitro experiments were performed using the HCT-116 colon cancer cell line. The effects of Y-15 (FAK inhibitor), ROCK inhibitor-2, and YAP/TAZ inhibitor-2, either applied alone or in combination, on cell proliferation were analyzed using the WST-1 cell viability assay. Epithelial-mesenchymal transition (EMT) markers, E-cadherin and N-cadherin, were evaluated via immunofluorescence staining, and fluorescent intensity was analyzed using ImageJ software. Results: Y-15, when applied alone or in combination with other inhibitors, significantly reduced cell proliferation (p≤0.005). Moreover, the combination of Y-15 and ROCK inhibitor-2 increased E-cadherin levels while decreasing N-cadherin levels (p≤0.0159, p≤0.0286). While the effect of YAP/TAZ inhibitor-2 alone was limited, specific effects were observed in combination treatments. Conclusion: This study demonstrates the potential of FAK-Rho-ROCK pathway inhibitors in the treatment of colorectal cancer. The ability of Y-15, in particular, to inhibit cell viability/proliferation and metastatic processes suggests that combination strategies targeting these pathways could contribute to the development of new therapeutic approaches for CRC.

Keywords

• Colorectal cancer
• Focal Adhesion Kinase
• Rho-ROCK
• YAP/TAZ

References

1. Dekker E, Tanis PJ, Vleugels JLA, et al. Colorectal cancer. The Lancet. 2019; 394(10207):1467-1480.
2. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71(3):209-249.
3. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. (2018) 68:394–424.
4. Brás MM, Sousa SR, Carneiro F, et al. Mechanobiology of Colorectal Cancer. Cancers (Basel) 2022;14(8):1945.
5. Guan G, Cannon RD, Coates DE, Mei L. Effect of the Rho-Kinase/ROCK Signaling Pathway on Cytoskeleton Components. Genes (Basel). 2023 Jan 20;14(2):272.
6. Ma WW. Development of focal adhesion kinase inhibitors in cancer therapy. Anticancer Agents Med Chem. 2011 Sep;11(7):638-42.
7. Zebda N, Dubrovskyi O, Birukov KG. Focal adhesion kinase regulation of mechanotransduction and its impact on endothelial cell functions. Microvasc Res 2012;83(1):71-81.
8. Huang G, Ho B, Conroy J, et al. The microarray gene profiling analysis of glioblastoma cancer cells reveals genes affected by FAK inhibitor Y15 and combination of Y15 and temozolomide. Anticancer Agents Med Chem 2014;14(1):9-17.

9. McLeod R, Kumar R, Papadatos-Pastos D, et al. First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors. Clin Cancer Res 2020;26(18):4777-4784.
10. Vennin C, Rath N, Pajic M, Olson MF, Timpson P. Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy. Small GTPases. 2020;11(1):45-52.
11. Otkur W, Liu X, Chen H, et al. GPR35 antagonist CID-2745687 attenuates anchorage-independent cell growth by inhibiting YAP/TAZ activity in colorectal cancer cells. Front Pharmacol 20231;14:1126119.
12. Chang L, Azzolin L, Di Biagio D, et al. The SWI/SNF complex is a mechanoregulated inhibitor of YAP and TAZ. Nature 2018;563(7730):265-269.
13. Lee BY, Timpson P, Horvath LG, Daly RJ. FAK signaling in human cancer as a target for therapeutics. Pharmacol Ther 2015;146:132-49.
14. Dawson JC, Serrels A, Stupack DG, Schlaepfer DD, Frame MC. Targeting FAK in anticancer combination therapies. Nat Rev Cancer. 2021;21(5):313-324.
15. Vennin C, Rath N, Pajic M, Olson MF, Timpson P. Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy. Small GTPases 2020;11(1):45-52.
16. Nam GH, Lee EJ, Kim YK, et al. Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer. Nat Commun 2018;9(1):2165.
17. Golubovskaya V, O'Brien S, Ho B, et al. Down-regulation of ALDH1A3, CD44 or MDR1 sensitizes resistant cancer cells to FAK autophosphorylation inhibitor Y15. J Cancer Res Clin Oncol 2015;141(9):1613-31.
18. Golubovskaya V, Curtin L, Groman A, Sexton S, Cance WG. In vivo toxicity, metabolism and pharmacokinetic properties of FAK inhibitor 14 or Y15 (1, 2, 4, 5-benzenetetramine tetrahydrochloride). Arch Toxicol 2015;89(7):1095-101.
19. Zheng Y, Zhou R, Cai J, et al. Matrix Stiffness Triggers Lipid Metabolic Cross-talk between Tumor and Stromal Cells to Mediate Bevacizumab Resistance in Colorectal Cancer Liver Metastases. Cancer Res 2023;83(21):3577-3592.