MIEN1 derived peptides exhibit anti-cancer activities by inhibiting the key pathways viz. epithelial-mesenchymal transition (EMT) and NF-kB signaling in MDA-MB-231 breast Cancer Cells

Purpose MIEN1 is a tumor-specific target protein that promotes cancer cell migration and invasion. MIEN1 is overexpressed in human breast, prostate, colorectal, gastric, ovarian, squamous cell carcinoma and non-small cell lung cancer (NSCLC) with minimal or no expression in normal cells, which makes it an excellent therapeutic target The overall goal of this project is to identify small inhibitory or interference peptides (iPeps) from the native MIEN1 protein, which could achieve the high selectivity for the native MIEN1 protein as well as being able to enter cells and inhibit the intracellular targets Methods Online tools like CASTp server were used to identify pockets or empty concavities on the MIEN1 protein surface into which solvent and other potential inhibitory molecules can gain access. Peptides were designed from the primary sequence of MIEN1 protein using various biophysical parameters and peptide designing tools such as AntiCp and Cancer PPD. Chimera program was used to check if these peptides can bind to MIEN1 protein. The peptides were synthesized and correct molecular weights were ascertained. Biological experiments like MTT and scratch assays were performed. RT-PCR was done to check the ability of the small inhibitory or interference peptides (iPeps) to inhibit the key pathways like the epithelial-mesenchymal transition (EMT) by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells. Western blot experiments were done to study the inhibition of the NFκB signaling pathway which is involved extensively in cancer development and progression. To show the specificity of these peptides for MIEN1 protein, circular dichroism (CD) experiments were performed. Results Two peptides designed based on MIEN1 protein sequence showed inhibition of MDA-MB-231 breast cancer cell proliferation in the MTT assay. Wound healing and transwell invasion assays show that these peptides inhibited the migration and invasion of MDA-MB-231 cells in a dose-dependent manner. RT-PCR results showed that the key molecules of epithelial-mesenchymal transition (EMT) were inhibited which can inhibit the migratory capacities of the cells. The nuclear translocation of p50 and p65 subunits of NFκB were also inhibited. The bio-active peptides attained a β-sheet structure in MIEN1 protein environment, in the circular dichroism (CD) experiments indicating the specific interaction with MIEN1 protein while the non-active peptides remained random coiled. Conclusions This work is the first report of the inhibitors designed for targeting MIEN1 protein. The peptides inhibitors were able to inhibit the key epithelial-to-mesenchymal transition transcription factors (EMT-TFs) like SNAIL, SLUG and TWIST1 which play a vital role in the metastatic process of breast cancer. Besides this the also downregulated nuclear factor-κB (NF-κB) activation. By inhibiting these two key processes aggressiveness and metastatic potential of MDA-MB-231 breast cancer cells were decreased.