Colorectal cancer is one of the most frequently occurring malignancies and a major cause of cancer death. Distant metastases in this disease most commonly develop in the liver and are often untreatable. Here, we use proteomics to characterise, qualitatively and quantitatively, extracellular matrix (ECM) from colorectal cancer liver metastases. We show that citrullination of the ECM by cancer cell derived peptidyl arginine deiminase 4 (PAD4) is important for the growth of liver metastases. Citrullination of proteins, a posttranslational conversion of arginine residues to citrulline, is well recognised in rheumatoid arthritis, but largely undocumented in cancer. PAD4, a key enzyme responsible for catalysing citrullination, is produced by metastatic colorectal cancer cells and found at higher levels in human liver metastases than in normal liver. Functional significance for citrullination in metastatic growth was evident in murine models where inhibition of citrullination, either globally by pharmacologic inhibition of PADs or specifically in colorectal cancer cells by PAD4 knockdown reduced liver metastatic burden by 3- to 4-fold (P < 0.05). Additionally, citrullination of key extracellular matrix (ECM) component collagen type I in vitro led to greater adhesion and 25-30% decreased migration of colorectal cancer cells (P < 0.05) along with increased expression of characteristic epithelial markers, indicating a role for citrullination in promoting mesenchymal-to-epithelial transition (MET). Overall, our study revealed PAD4- dependent citrullination of the ECM altering mesenchymal-epithelial plasticity in colorectal cancer cells and the progression of liver metastasis. These data indicate that inhibition of citrullination could be exploited to potentially prevent the development of liver metastases in colorectal cancer.</p
