LCD313:a Phosphorylation Inhibitor Of ACAP4Suppresses Metastasis In Hepatocellular Carcinoma In Vivo

Background and objective：Hepatocellular Carcinoma brings great harm to human health around the world, forits severity in both incidence and mortality among all malignancies. Despite greatprogress in HCC therapy mainly with surgery section, metastasis still restrains theoutcome of HCC therapy, putting nearly90%of HCC sufferers into death. Thus it hasposed a great significance to explore molecular mechanism of HCC migration andmetastasis, and to develop both new paths and new drugs.A variety of molecules and their signaling network are involved in the progress ofHCC. ACAP4, a novel Arf6-specific GAP, plays a significant role in Arf6-dependentmigration and metastasis in HCC, and is regulated by phosphorylation of some of its amino acids. We proposed a suppressive effect of LCD313, a small synthetic peptide, onthe phophorylation of ACAP4, discussing whether inhibition of phosphorylation ofACAP4impacts on metastasis in HCC in vivo, as well as LCD313’s clinical probabilityas an anti-HCC drug.Methods and Results：A Hepatocellular Carcinoma cell line MHCC97-H-LUC expressing luciferase wasestablished and transplanted into nude mice in the liver. Afterwards the location andextent of transplanted tumors in nude mice were detected by bioluminescent imagingsystem. Consequently, an orthotopically transplanted nude mouse model withMHCC97-H-LUC can be subjected in to measurement dynamically and quantitatively.Based on the methods above, LCD313was injected into HCC transplanted nudemice in doses, observing differences of metastasis among nude mice with different dosesof LCD313, in order to discuss suppressive effect of LCD313on metastasis of HCC innude mice. Using bioluminescent imaging technology, an impact of LCD313on themetastasis in MHCC97-H-LUC transplanted in nude mice was confirmed. However, incertain range, different doses of LCD313performed similar suppressive effect. WhenLCD313was over-delivered, it tended to some side and toxic effects on nude mice.Conclusion：As a practical tool, Hepatocellular Carcinoma cell line MHCC97-H-LUC can bewell applied to detect the location and extent of transplanted tumor in vivo, and toexamine transplanted tumor dynamically and quantitatively, via bioluminescent imagingtechnology.Our analyses show that LCD313suppressed the phophorylation of some specificamino acids, especially Tyr733. Under this circumstance, ACAP4is moleculeconformational changed, its activity site is enveloped, and finally ACAP4’s activity issuppressed. In that case, the active/inactive Arf6recycling and Arf6-dependent membrane remodeling is blocked. Moreover, ACAP4mediated regulation of integrin β1stimulated by EGF signaling activated Grb2was repressed. Therefore, suppressingphosphorylation of ACAP4by LCD313inhibits the cytoskeleton and migration of HCC,which plays an important role in HCC metastasis.We prospect that in the near future, with the development of LCD313, an increasingnumber of new drugs targeting the inhibition of phosphorylation of ACAP4be applied toanti-HCC therapy.