Therapeutic Potential of Probiotic-Derived P8 Protein as an Anti-Metastatic Agent in Colorectal Cancer

We previously described the use of probiotics to deliver a Lactobacillus rhamnosus-derived therapeutic protein, P8, which has been identified as a candidate colorectal cancer (CRC) suppressor protein with anti-proliferation and anti-migration activities. P8 was found to penetrate cell membranes by endocytosis, suppressing cell proliferation through G₂ cell cycle arrest. Despite the ability of P8 to suppress cell migration in vitro, its mechanism of action in CRC is unclear. We profiled the P8-interacting partner proteins using the pull-down method with His-tagged bait P8 and then identified them by LC-MS/MS. Among the interacting targets, we focused on the mothers against decapentaplegic homolog 1 (Smad1), which is well known as one of the important modulators of the bone morphogenetic protein (BMP)-derived migration pathway in CRC. The present study discovers that P8 prevents the phosphorylation of Smad1 or heterologous complexes within the Smad family, interfering with the importation of Smad1 or its complexes into the nucleus. Thus, P8 significantly inhibits the up-regulation of epithelial-mesenchymal transition (EMT)-related genes mediated by Smad1. P8 also inhibits the morphological changes required for cell migration or adhesion. P8 induces morphologic changes in DLD-1 cells, and their spheroid surfaces, resulting in a significant reduction of the number and length of filopodia, as well as the down-regulation of the expression of myosin X and its accumulation in filopodia tips. This phenomenon seems to be a major negative regulator of cell motility that could be of key importance in metastasis. Use of a mouse model of human CRC metastasis confirmed that P8 significantly suppresses the liver metastatic rate. Probiotic-derived protein P8 significantly suppresses CRC metastasis through inhibition of the Smad1-EMT signal pathway and cell-cell adhesion.