The Role Of HIV-1 Nef Protein In Regulation Of KSHV K1-induced Angiogenesis And Tumorigenesis And Its Possible Molecular Mechanisms

Background:Kaposi’s sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8/HHV-8) has been causally linked to the development of Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV infection is necessary but not sufficient for KS development without other factors. One potentially important cofactor is human immunodeficiency virus type 1 (HIV-1). Individuals dually infected with KSHV and HIV-1 have a greatly enhanced prevalence of KS compared with those infected with KSHV alone. A lot of studies have continued to focus on the possible mechanisms by now. We have previously showed that HIV-1 Tat not only regulates KSHV latency/replication by modulating the JAK/STAT signaling pathway, but also promotes KSHV Kaposin A-induced tumorigenesis. Accessory negative factor (Nef) is another early and secretory HIV-1-encoded protein, which may also influence angiogenesis and tumorigenesis induced by KSHV Kl.Objective:To explore the possible roles of HIV-1 Nef in KSHV K1-induced angiogenesis and tumorigenesis on chick chorioallantoic membrane (CAM) and in athymic nu/nu mice and investigate the possible signaling pathways involved in this process.Methods:At first, EA.hy926 cells were infected by recombinant lentivirus containing KSHV Kl gene or its control (Mock), respectively. Got cells named EA/K1 or EA/Mock expressing green fluorescent protein (GFP) by flow cytometry assay (FCM) screening. Next, explored the possible roles of Nef in K1-induced EA.hy926 cells proliferation, colony formation, cells cycle, microtube formation ability by cell counting kit-8 (CCK-8), plate culturing assay, FCM analysis, and microtube formation analysis/tumorigenesis and angiogenesis on chick chorioallantoic membrane (CAM) and in athymic nude mice. Furthermore, detected the possible mechanisms by the cDNA constructs or the dominant-negative (DN) constructs in PI3K/PTEN/Akt/mTOR signal pathway-related kinases and Rapamycin (inhibitor of mTOR). At last, Western blot was used to detect the phosphorylation level of PI3K/PTEN/Akt/mTOR signal pathway-related kinases and IHC staining to test the expression of CD31, SMA and vascular endothelial growth factor (VEGF) in K1-induced tumor tissues on CAM and in nude mice.Results:By using CCK-8, plate culturing assay, FCM analysis, and microtube formation analysis, we demonstrated that Nef enhanced proliferation, colony formation, and G1-S phase transition of the cell cycle induced by K1 in EA.hy926 cells. Animal experiments further demonstrated that Nef could accelerate tumorigenesis and agiogenesis induced by K1 on CAM and in nude mice. Mechanistic studies demonstrated that it can suppress the cooperation of Nef and Kl in vivo to block the PI3K/PTEN/Akt/mTOR signaling pathway by PTEN-cDNA, Akt-DN, PI3K-DN or Rapamycin (mTOR inhibitor). Finally, immunohistochemistry (IHC) staining manifested that the expression of CD31, SMA and VEGF in K1-induced tumor tissues on CAM and in nude mice were increased by Nef protein and inhibited by Rapamycin.Conclusion:These data suggest that HIV-1 Nef can accelerate proliferation and vascular tube formation ability/tumorigenesis and angiogenesis induced by K1 in vitro/in vivo. PI3K/PTEN/Akt/mTOR signal pathways contributed partially to the roles of Nef protein in K1-induced angiogenesis and tumorigenesis; Meanwhile, Nef protein upregulated the expression of CD31, SMA and VEGF in K1-induced tumor tissues.