Novel Compound Suppresses Tumor Metastasis Via Binding Myoferlin And Enhancing RhoA Activity

Tumor metastasis refers to the process in which cancer cells fall off from the primary tumor,metastasize to different sites through the blood or lymphatic vessels,and grow at the distal site.Cancer metastasis is the major cause of cancer morbidity and mortality,and accounts for about 90%of cancer deaths.Metastasis is one of the basic characteristics of malignant tumors.Metastasis is closely related to tumor size,quality of life,and survival time.Metastasis results in the failure of many tumor treatments.Until recently,cancer research has primarily focused on the development of methods/agents that can detect tumor at the early stage,and on agents that inhibit tumor growth.Due to a lack of under-standing of the mechanisms that underlie the metastatic process,limited success has been made on prevention and inhibition of cancer metastasis.Drugs that can specifically inhibited tumor metastasis have not been reported worldwide.In our previous work,4-methyl-3-nitrobenzoic acid was screened from the compound library by high-throughput method,which can effectively inhibit the metastasis of various tumor cells with low cytotoxicity.To improve its poor water solubility and enhance its pharmaceutical activity,TCI04 was screened from the derivative of 4-methyl-3-nitrobenzoic acid.At first,we optimized the synthesis and purification methods of TCI04 to ensure that its purity is greater than 99.9%,in line with preclinical drug research standards.The safety of TCI04 was then evaluated via MTT assay,cell cycle measured by FCM,acute toxicity experiments in mouse and sub-acute toxicity experiments in rats.The IC50 of TCI04inhibiting breast cancer cells MDA-MB-231 and 4T1 were 2449.07 and 2897.64?M,respectively,and had no significant effect on cell cycle.In pharmacodynamic evaluation,TCI04 can effectively inhibit the migration and invasion of various tumor cells such as breast cancer,lung cancer,melanoma,and glioma,and enhance cell-matrix adhesion to reduce tumor cells metastasis.In addition,TCI04 can also decrease its ability to form tubes in vitro by inhibiting the motility of endothelial cells.In the B16-F10/C57 melanoma metastasis model and breast cancer model,TCI04 can effectively inhibit tumor metastasis and angiogenesis,and prolong the survival of tumor-bearing mice.To analyze the target of TCI04,Biacore T200 based fishing assay was performed and found its target protein,Myoferlin,via MS.TCI04 binds to the C2B domain of Myoferlin(K_D=1.694±0.608?M),inhibits the translocation of Myoferlin from the perinuclear compartment to cytomembrane,and mimics the phenotype induced by downregulation of Myoferlin via siRNA,such as reversing EMT,enlarging focal adhesion,and promoting stress fiber formation.To further elucidate the molecular mechanism by which TCI04 inhibits cell migration via binding Myoferlin,we resolved all proteins that interact with Myoferlin by proteomics,which are mainly enriched in organelle localization,stress fiber formation and depolymerization,and Rho GTPases related signaling pathways.Further studies revealed that TCI04 increased the activity of RhoA through two pathways.Firstly,TCI04 impairs the binding of Myoferlin to Caveolin-1 and affects the formation of lipid rafts,which further inactivates lipid-dependent RND1 and reduces the activity of p190 RhoGAP.The activity of RhoA was enhanced by decreasing the hydrolysis of RhoA-GTP.Secondly,the Tyr 14 residue was exposed and phosphorylated due to the alteration of caveolin-1 homo-oligomerization after TCI04 binding with Myoferlin.The increased phosphorylation of caveolin-1 at Tyr 14promotes the activity of VAV2,which functions as a RhoGEF of RhoA,and enhances the formation of RhoA-GTP.Taken together,TCI04 is a low-toxicity small molecule compound that enhances the activity of RhoA by binding to Myoferlin and affecting its downstream pathways,thereby enhancing the adhesion of tumor cells to the matrix,promoting the formation of stress fibers,and inhibiting tumor cell metastasis.TCI04has a potential to be developed as a drug that specifically blocks tumor metastasis,representing a novel low-toxic anti-tumor therapy.