MIEN1 promoter ablation provides novel evidence for colorectal cancer genome editing-based therapeutics.




Ranade, Payal


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Purpose: Colorectal cancer (CRC) is one of the leading causes of cancer associated mortalities worldwide. It starts with hyperproliferation of epithelial cells forming a polyp & leads to stages 3 & 4 which are associated with acquisition of metastasis. Chemotherapy majorly focuses on attenuating symptoms with no promising cure. Hence, studying the underlying mechanism of CRC progression & identifying novel therapeutic targets is critical for early diagnosis & treatment.

The process of metastasis involves a complex interplay of signaling pathways, where various proteins play crucial roles. One such important protein that aids the process of migration is Migration & Invasion ENhancer 1 (MIEN1). In CRC, MIEN1 expression is predominantly upregulated in cancerous tissue in comparison to normal colorectal tissue, which is closely associated with invasive behavior. But the exact mechanism involved in the process of metastasis is yet unexplored.

Methods: It is established that MIEN1 overexpression is a result of 17q12 chromosomal amplification, & such dysregulated expression is linked to the trans-regulation of its minimal promoter region. Therefore, we aim to investigate the effect of MIEN1 promoter ablation on CRC migration properties. CRISPR-Cas9 gene editing technology was used for deleting MIEN1 promoter region in the CRC cell line HT29. RNA seq & bioinformatics tools were employed to assess the transcriptomic consequences of genome-editing. Several DEGs discerned by RNA seq analysis were involved in different biological processes & molecular pathways such as cell adhesion, migration, invasion, & angiogenesis. Out of these the genes vital to CRC biogenesis were evaluated at both RNA & protein levels.

Results: We analyzed the effect of MIEN1 knock-out on CRC metastatic potential using functional assays such as wound healing, Matrigel invasion, hanging drop cell – cell adhesion, & found that migration potential of HT29 cells was significantly reduced in absence of MIEN1 protein. We also successfully demonstrated that MIEN1 deletion disrupts the cytoskeletal rearrangement by affecting F-actin reorganization using phalloidin staining. Confocal staining of different proteins participating in actin cytoskeleton rearrangement such as paxillin, FAK, MIEN1 gave us an insight about the role of MIEN1 in mediating phosphorylation of FAK at different phosphorylation sites such as Tyr 397 & 925. Immunoblotting analysis of an array of proteins further confirmed the role of MIEN1 in actin cytoskeleton dynamics.

Conclusion: MIEN1 is an important metastatic protein that is specifically overexpressed in cancerous cells. Taken together, our results prove that MIEN1 is involved in different signaling pathways responsible for CRC migration & its deletion leads to perturbation of several biological processes especially the actin cytoskeleton rearrangement, involved in metastasis. Hence, targeting MIEN1 would be a potentially effective therapeutic strategy for CRC patients.