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HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway.

Xue M, Yao S, Hu M, Li W, Hao T, Zhou F, Zhu X, Lu H, Qin D, Yan Q, Zhu J, Gao SJ, Lu C - Nucleic Acids Res. (2014)

Bottom Line: Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV).Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3' UTR of its mRNA.Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, P.R. China Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, P.R. China Department of Microbiology, Nanjing Medical University, Nanjing 210029, P.R. China Department of Physiology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, P.R. China.

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PTEN/AKT/mTOR pathway mediates K1- and Nef-induced angiogenesis in vivo. (A) Overexpression of PTEN suppressed the synergetic effect of soluble Nef protein on K1-induced angiogenesis in the CAM model. HUVECs transduced with K1 and incubated with soluble Nef protein for 72 h were transfected with pPTEN and a control plasmid (pcDNA) for 24 h. The treated cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (B) Western blot analysis of the phosphorylation levels of AKT and mTOR in tumor tissues from the CAMs treated as in (A). (C) Inhibition of mTOR activity suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in the CAM model. EA.hy926 cells transduced with K1 and Nef were treated with rapamycin (100 nM) or the control reagent, DMSO. The collected cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (D) Overexpression of PTEN suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1 and Nef were transfected with pPTEN and pcDNA for 72 h. The collected cells were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the ‘Materials and Methods’ section. The hemoglobin content of the Matrigel plugs was determined with O.D. value at 540 nm. Data represent mean ± SD. n = 5 tumors per group. Three independent experiments were performed and gave similar results.
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Figure 3: PTEN/AKT/mTOR pathway mediates K1- and Nef-induced angiogenesis in vivo. (A) Overexpression of PTEN suppressed the synergetic effect of soluble Nef protein on K1-induced angiogenesis in the CAM model. HUVECs transduced with K1 and incubated with soluble Nef protein for 72 h were transfected with pPTEN and a control plasmid (pcDNA) for 24 h. The treated cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (B) Western blot analysis of the phosphorylation levels of AKT and mTOR in tumor tissues from the CAMs treated as in (A). (C) Inhibition of mTOR activity suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in the CAM model. EA.hy926 cells transduced with K1 and Nef were treated with rapamycin (100 nM) or the control reagent, DMSO. The collected cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (D) Overexpression of PTEN suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1 and Nef were transfected with pPTEN and pcDNA for 72 h. The collected cells were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the ‘Materials and Methods’ section. The hemoglobin content of the Matrigel plugs was determined with O.D. value at 540 nm. Data represent mean ± SD. n = 5 tumors per group. Three independent experiments were performed and gave similar results.

Mentions: To examine the role of PTEN/AKT/mTOR signaling in mediating the synergistic effect of Nef- and K1-induced angiogenesis, HUVECs expressing K1, HUVECs incubated with soluble Nef protein or both were transfected with pPTEN and subsequently implanted onto CAMs. Overexpression of PTEN completely inhibited angiogenesis induced by K1, soluble Nef or both in the HUVEC-implanted CAM model (Figure 3A). As expected, overexpression of PTEN led to the reduction of phosphorylated forms of AKT and mTOR, both of which are downstream of PTEN (Figure 3B). Next, we confirmed the above results with rapamycin, an mTOR inhibitor, in the EA.hy926 cell-implanted CAM model. The quantifications of blood vessels showed that rapamycin effectively suppressed the synergistic effect of Nef and K1 on angiogenesis (Figure 3C).


HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway.

Xue M, Yao S, Hu M, Li W, Hao T, Zhou F, Zhu X, Lu H, Qin D, Yan Q, Zhu J, Gao SJ, Lu C - Nucleic Acids Res. (2014)

PTEN/AKT/mTOR pathway mediates K1- and Nef-induced angiogenesis in vivo. (A) Overexpression of PTEN suppressed the synergetic effect of soluble Nef protein on K1-induced angiogenesis in the CAM model. HUVECs transduced with K1 and incubated with soluble Nef protein for 72 h were transfected with pPTEN and a control plasmid (pcDNA) for 24 h. The treated cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (B) Western blot analysis of the phosphorylation levels of AKT and mTOR in tumor tissues from the CAMs treated as in (A). (C) Inhibition of mTOR activity suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in the CAM model. EA.hy926 cells transduced with K1 and Nef were treated with rapamycin (100 nM) or the control reagent, DMSO. The collected cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (D) Overexpression of PTEN suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1 and Nef were transfected with pPTEN and pcDNA for 72 h. The collected cells were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the ‘Materials and Methods’ section. The hemoglobin content of the Matrigel plugs was determined with O.D. value at 540 nm. Data represent mean ± SD. n = 5 tumors per group. Three independent experiments were performed and gave similar results.
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Figure 3: PTEN/AKT/mTOR pathway mediates K1- and Nef-induced angiogenesis in vivo. (A) Overexpression of PTEN suppressed the synergetic effect of soluble Nef protein on K1-induced angiogenesis in the CAM model. HUVECs transduced with K1 and incubated with soluble Nef protein for 72 h were transfected with pPTEN and a control plasmid (pcDNA) for 24 h. The treated cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (B) Western blot analysis of the phosphorylation levels of AKT and mTOR in tumor tissues from the CAMs treated as in (A). (C) Inhibition of mTOR activity suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in the CAM model. EA.hy926 cells transduced with K1 and Nef were treated with rapamycin (100 nM) or the control reagent, DMSO. The collected cells were implanted onto the CAMs. The quantification of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing 10 technical replicates. (D) Overexpression of PTEN suppressed the synergetic effect of ectopic Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1 and Nef were transfected with pPTEN and pcDNA for 72 h. The collected cells were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the ‘Materials and Methods’ section. The hemoglobin content of the Matrigel plugs was determined with O.D. value at 540 nm. Data represent mean ± SD. n = 5 tumors per group. Three independent experiments were performed and gave similar results.
Mentions: To examine the role of PTEN/AKT/mTOR signaling in mediating the synergistic effect of Nef- and K1-induced angiogenesis, HUVECs expressing K1, HUVECs incubated with soluble Nef protein or both were transfected with pPTEN and subsequently implanted onto CAMs. Overexpression of PTEN completely inhibited angiogenesis induced by K1, soluble Nef or both in the HUVEC-implanted CAM model (Figure 3A). As expected, overexpression of PTEN led to the reduction of phosphorylated forms of AKT and mTOR, both of which are downstream of PTEN (Figure 3B). Next, we confirmed the above results with rapamycin, an mTOR inhibitor, in the EA.hy926 cell-implanted CAM model. The quantifications of blood vessels showed that rapamycin effectively suppressed the synergistic effect of Nef and K1 on angiogenesis (Figure 3C).

Bottom Line: Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV).Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3' UTR of its mRNA.Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, P.R. China Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, P.R. China Department of Microbiology, Nanjing Medical University, Nanjing 210029, P.R. China Department of Physiology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, P.R. China.

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Related in: MedlinePlus