Betulinic Acid Exerts Antimetastatic Activity Through Targeting Skp2/E-cadherin Axis In NSCLC

Objective:Lung cancer is the leading cause of cancer-related mortality worldwide,and non-small cell lung cancer(NSCLC)accounts for approximately 85%of all lung cancers.Platinumbased chemotherapy remains the mainstay treatments for NSCLC patients.However,chemoresistance frequently occurs that leads to treatment failure.A majority of patients succumb to the disease due to drug resistance and cancer metastasis.Skp2 is overexpressed in multiple cancers and plays a critical role in tumor development through ubiquitin/proteasome-dependent degradation of its substrate proteins.Drugs targeting Skp2 have exhibited promising anticancer activity by inhibiting the activity of Skp2-SCF E3 ligase.Under the guidance of holistic concepts of Traditional Chinese medicine(TCM),combined with modern pharmacological researches,TCM has advantages in the prevention and treatment of lung cancer metastasis.In recent years,although some progress has been made in the treatment of lung cancer metastasis using TCM,more experiments and clinical trails are still needed for exploration.The numerous phytochemicals exhibit promising antitumor activities.Hence,these are considered suitable candidate drugs for novel anticancer therapies.Here,we identified the Skp2 inhibitor via high-throughput virtual screening of a phytochemical library and studied the underlying mechanism of antimetastatic effect of the inhibitor in NSCLC.Methods:In this study,we analyzed the expression level of Skp2 in NSCLC using online databases.And then,we used the high-throughput molecular docking to identified the Skp2 inhibitor and used co-immunoprecipitation to tested whether the compounds could block the binding of Skp2 to Skp1.In vivo ubiquitination assay was used to test the effect of BA on Skp2-SCF E3 ligase activity.The cellular thermal shift assay(CETSA)was performed and the Lys145 of Skp2 was mutated to Ala 145(Skp2 K145A)to probe the direct binding of BA to Skp2.CHX and Western blot were used to test the effect of BA on the expression of Skp2 and related proteins.Wound-healing,transwell,and sphere formation assay were used to test whether BA could affect migration and stemness in NSCLC.Stable overexpression of Skp2 was established in NSCLC cells to examine whether the inhibitory effects of BA on cell migration was linked to its effect on Skp2.We established the spontaneous metastasis model and the experimental metastasis mouse model to test the effect of BA in vivo.A cisplatin-resistant lung cancer cell line was established to determine the ability of BA to attenuate the chemoresistance-associated migration.Results:We found that Skp2 mRNA level in lung tumors is higher than that in adjacent tissues,and the expression level of Skp2 was negatively associated with patient’s prognosis in NSCLC.And the result of high-throughput structure-based virtual screening and co-IP showed that BA prevents drastically the interactions between Skp2 and Skp1.CETS A and the Lys145 of Skp2 was mutated to Ala145(Skp2 K145A)demonstrated that BA directly interacts with Skp2 at the Lys145 residue.We found that BA significantly decreased Skp2 protein stability and inhibited the activity of Skp2-SCF E3 ligase in NSCLC.We found that BA also significantly increased the protein level of p27 and induced cell cycle arrest in G1 phase,leading to inhibition of proliferation of NSCLC.In vivo ubiquitination assay revealed that BA markedly inhibited Skp2-mediated E-cadherin ubiquitination,leading to the inhibition of metastasis of NSCLC.Wound-healing,transwell,and sphere formation assay showed that BA efficiently inhibit the migratory and stem-like properties of lung cancer cells.We found that Skp2 is involved in BA-mediated inhibition of migration and invasion of NSCLC by establishing stable overexpression of Skp2 in NSCLC cells.The result of spontaneous metastasis model found that BA significantly inhibits primary tumor growth,as well as spontaneous lung metastasis by targeting the Skp2-SCF E3 ligase/Ecadherin axis in vivo.And the result of the experimental metastasis mouse model also showed that BA strongly inhibits the lung metastatic by down regulating Skp2/E-cadherin axis in vivo.We found that BA inhibited the proliferation and metastasis of NSCLC in vitro and in vivo.And we found that BA inhibits chemoresistance-associated migration via targeting Skp2 in NSCLC.In addition,compared with A549,enhanced migration capacity,and increased Skp2 expression were observed in A549/CDDP cells.What is more,we found that the migration and invasive capacities,as well as the expression of Skp2 of A549/CDDP cells were efficiently reduced by BA.These data suggest that BA can be further utilized to overcome metastasis in NSCLC.Conclusion:In conclusion,our study demonstrated that BA could bind to residue Lys145 of Skp2 located in the core part of Skp1-Skp2 binding interface.And BA inhibited migration and invasion of NSCLC both in vivo and in vitro by targeting Skp2 and suppressing Skp2mediated E-cadherin ubiquitination.Furthermore,our data suggest that the metastasis of NSCLC is accompanied by the development of cisplatin resistance.And the high levels of Skp2 is key to concomitant development of chemoresistance and metastasis.While BA also inhibits the migration of cisplatin-resistant NSCLC.Therefore,our study provides novel evidence supporting the antimetastatic potential of BA in NSCLC that is worth further clinical investigation.