Elucidating the role of MIEN1 in colorectal cancer metastasis

Purpose: Colorectal cancer (CRC) ranks among the foremost contributors to global cancer-related fatalities, with its progression marked by hyperproliferation of epithelial cells culminating in polyp formation. Stages 3 and 4 of CRC are notably associated with metastasis. Despite existing chemotherapy focusing on symptom attenuation, a definitive cure remains elusive. This study addresses the imperative need to comprehend the underlying mechanisms of CRC progression for timely diagnosis and effective treatment. The process of metastasis involves a complex interplay of signaling pathways, where various proteins play crucial roles. Migration and Invasion ENhancer 1 (MIEN1) is a significant protein facilitating migration, and its expression is notably upregulated in CRC tissue compared to normal colorectal tissue, correlating with invasive behavior. The exact mechanism of MIEN1 involvement in metastasis remains unexplored. MIEN1 overexpression results from 17q12 chromosomal amplification, and dysregulated expression is associated with trans-regulation of its minimal promoter region. This study aims to investigate the impact of MIEN1 promoter ablation on CRC migration properties. Methods: Utilizing CRISPR-Cas9 gene editing, we deleted the MIEN1 promoter region in the CRC cell line HT29. RNA sequencing and bioinformatics tools revealed differentially expressed genes (DEGs) associated with essential biological processes and molecular pathways, including cell adhesion, migration, invasion, and angiogenesis. Evaluation of critical genes in CRC biogenesis at RNA and protein levels demonstrated that MIEN1 knock-out significantly reduced the migration potential of HT29 cells, as evidenced by functional assays such as wound healing and Matrigel invasion. Furthermore, MIEN1 deletion disrupted cytoskeletal rearrangement, affecting F-actin reorganization, confirmed through phalloidin staining. Confocal staining of proteins involved in actin cytoskeleton rearrangement provided insights into MIEN1's role in mediating phosphorylation of FAK at different sites. Immunoblotting analysis further substantiated MIEN1's involvement in actin cytoskeleton dynamics. Results: Our findings indicate a significant reduction in the migration potential of HT29 cells in the absence of MIEN1 protein. Additionally, we demonstrated the disruption of cytoskeletal rearrangement, specifically affecting F-actin reorganization, upon MIEN1 deletion using phalloidin staining. Confocal staining of various proteins involved in actin cytoskeleton rearrangement, such as FAK and cofilin, further strengthened the evidence of MIEN1 in actin reorganization. Immunoblotting analysis of a diverse set of proteins further confirmed the involvement of MIEN1 in the dynamics of actin cytoskeleton. Conclusion: MIEN1, an influential metastatic protein overexpressed in cancerous cells, plays a crucial role in various signaling pathways governing CRC migration. Deletion of MIEN1 perturbs biological processes, particularly actin cytoskeleton rearrangement crucial for metastasis. Thus, targeting MIEN1 emerges as a promising therapeutic strategy for CRC patients.