NEDD4L Identified Through In Vivo Functional Screens Inhibits Colorectal Cancer Liver Metastasis

Colorectal cancer is the second leading cause of cancer-related deaths worldwide.More than 90% of colorectal cancer patients die due to metastases.Particularly,liver is the first target organ in colorectal cancer metastasis,and liver metastasis accounts for 45–71% of all metastatic cases in colorectal cancer.Molecular-targeted drugs,such as cetuximab,fruquintinib,and regorafenib,which have the advantages of high specificity and good clinical efficacy with weak side effects,have been widely used to treat colorectal cancer patients in recent years.However,molecular targets and effective drugs are lacking for the treatment of colorectal cancer liver metastases.E3 ubiquitin ligases,G-protein-coupled receptors,and protein kinases are the most frequently targeted biomolecules due to their high druggability and represent the largest groups of drug targets with a high potential of giving rise to effective drugs.Therefore,in this study,a lentiviral library of sh RNAs targeting these three families were expressed in poorly metastatic colorectal cancer cells,which were then xenografted into mice via splenic injection,a method that effectively mimics liver colonization of metastatic colorectal cancer cells.In this way,we focused on metastatic colonization,which is the most inefficient step in colorectal cancer liver metastasis.From this in vivo functional screening,the E3 ubiquitin ligase NEDD4L(Neural precursor cell expressed developmentally down-regulated 4-like)was found to inhibit colorectal cancer liver metastasis via its ubiquitin ligase activity.Colorectal cancer liver colonization is the process in which colorectal cancer cells adapt to the liver microenvironment and eventually outgrow into lethal macroscopic lesions.NEDD4 L inhibited the proliferation but not the stemness of colorectal cancer cells and thereby suppressed their colonization of the liver.Tumor cells have been reported to change their metabolism to acquire the nutrients and produce the energy needed for their rapid proliferation,for which they commonly activate m TOR.Accordingly,we found that NEDD4 L suppressed the proliferation of colorectal cancer cells by inhibiting the AKT/m TOR signaling pathway.To find the substrates downstream of NEDD4 L,we performed tandem immunoprecipitation and LC-MS/MS.Consequently,we identified PRMT5(Protein arginine methyltransferase 5)as a candidate substrate protein of NEDD4 L.By using MG132,an inhibitor of ubiquitin-dependent proteasomal degradation,we demonstrated that NEDD4 L mediated the ubiquitination and subsequent degradation of PRMT5.Using the PRMT5 PPNAY mutants,we demonstrated that NEDD4 L mediated the same process by recognizing the PPNAY motif of PRMT5.To confirm the function of PRMT5 in NEDD4L-mediated liver colonization of metastatic colorectal cancer cells,we generated a PRMT5-knockout colorectal cancer cell line and found that loss of PRMT5 suppressed the AKT/m TOR signaling pathway,inhibited the proliferation of the cells,and thereby suppressed their metastatic colonization of the liver.Using the PRMT5 inhibitor EPZ015666 and a methyltransferase mutant of PRMT5,we then demonstrated that PRMT5 used its methyltransferase activity to regulate the AKT/m TOR signaling pathway.Finally,we demonstrated that PRMT5 promoted AKT activation by catalyzing arginine methylation of AKT.In addition,analysis of clinical data showed that i)NEDD4L expression in the liver metastases of colorectal cancer patients was significantly lower than that in the primary tumors,ii)overall survival was significantly longer in the patients with high NEDD4 L expression in the colorectal tumors than in the patients with low expression,and iii)NEDD4L expression in the colorectal tumors was negatively correlated with the activity of the m TOR signaling pathway.In summary,via an in vivo functional screening,we identified that the E3 ubiquitin ligase NEDD4 L inhibited liver colonization of metastatic colorectal cancer cells.Furthermore,we characterized the underlying mechanism and found that NEDD4 L suppressed the proliferation of colorectal cancer cells by mediating ubiquitination and subsequent degradation of PRMT5 and thereby inhibiting the AKT/m TOR signaling pathway.Taken together,this study provides a theoretical basis and several potential molecular targets for the targeted treatment of colorectal cancer liver metastases.