, Volume 19, Issue 3, pp 359–371 | Cite as

mTORC2 mediates CXCL12-induced angiogenesis

  • Mary E. Ziegler
  • Michaela M. S. Hatch
  • Nan Wu
  • Steven A. Muawad
  • Christopher C. W. HughesEmail author
Original Paper


The chemokine CXCL12, through its receptor CXCR4, positively regulates angiogenesis by promoting endothelial cell (EC) migration and tube formation. However, the relevant downstream signaling pathways in EC have not been defined. Similarly, the upstream activators of mTORC2 signaling in EC are also poorly defined. Here, we demonstrate for the first time that CXCL12 regulation of angiogenesis requires mTORC2 but not mTORC1. We find that CXCR4 signaling activates mTORC2 as indicated by phosphorylation of serine 473 on Akt and does so through a G-protein- and PI3K-dependent pathway. Significantly, independent disruption of the mTOR complexes by drugs or multiple independent siRNAs reveals that mTORC2, but not mTORC1, is required for microvascular sprouting in a 3D in vitro angiogenesis model. Importantly, in a mouse model, both tumor angiogenesis and tumor volume are significantly reduced only when mTORC2 is inhibited. Finally, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which is a key regulator of glycolytic flux, is required for microvascular sprouting in vitro, and its expression is reduced in vivo when mTORC2 is targeted. Taken together, these findings identify mTORC2 as a critical signaling nexus downstream of CXCL12/CXCR4 that represents a potential link between mTORC2, metabolic regulation, and angiogenesis.


CXCL12 CXCR4 Angiogenesis mTOR Akt mTORC2 



We thank Nazilya Gasanova, Sarah M. Sukardi, and Kimberly Lim for their help in quantifying the immunohistochemical specimens; Dr. Kehui Wang of the Pathology Research Services Core at UCI for processing the tissue sections for analysis; and Dr. David Fruman from the Department of Molecular Biology and Biochemistry at UCI for his helpful discussions during the course of these experiments. This work was supported by the National Institutes of Health/National Cancer Institute Institutional Training Grant Fellowship T32CA009054 to M.E.Z and RO1 HL60067 to C.C.W.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. C.C.W.H. receives support from the Chao Family Comprehensive Cancer Center through a National Cancer Institute Center Grant, P30A062203.

Supplementary material

10456_2016_9509_MOESM1_ESM.docx (2.3 mb)
Supplementary material 1 (DOCX 2323 kb)


  1. 1.
    Weis SM, Cheresh DA (2011) Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med 17(11):1359–1370. doi: 10.1038/nm.2537 CrossRefPubMedGoogle Scholar
  2. 2.
    Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473(7347):298–307. doi: 10.1038/nature10144 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Salcedo R, Oppenheim JJ (2003) Role of chemokines in angiogenesis: CXCL12/SDF-1 and CXCR4 interaction, a key regulator of endothelial cell responses. Microcirculation 10(3–4):359–370. doi: 10.1038/ CrossRefPubMedGoogle Scholar
  4. 4.
    Newman AC, Chou W, Welch-Reardon KM, Fong AH, Popson SA, Phan DT, Sandoval DR, Nguyen DP, Gershon PD, Hughes CC (2013) Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived hepatocyte growth factor and fibronectin in angiogenesis. Arterioscler Thromb Vasc Biol 33(3):513–522. doi: 10.1161/ATVBAHA.112.300782 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Strasser GA, Kaminker JS, Tessier-Lavigne M (2010) Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 115(24):5102–5110. doi: 10.1182/blood-2009-07-230284 CrossRefPubMedGoogle Scholar
  6. 6.
    Teicher BA, Fricker SP (2010) CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 16(11):2927–2931. doi: 10.1158/1078-0432.CCR-09-2329 CrossRefPubMedGoogle Scholar
  7. 7.
    Duda DG, Kozin SV, Kirkpatrick ND, Xu L, Fukumura D, Jain RK (2011) CXCL12 (SDF1alpha)-CXCR4/CXCR7 pathway inhibition: an emerging sensitizer for anticancer therapies? Clin Cancer Res 17(8):2074–2080. doi: 10.1158/1078-0432.CCR-10-2636 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149(2):274–293. doi: 10.1016/j.cell.2012.03.017 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Bracho-Valdes I, Moreno-Alvarez P, Valencia-Martinez I, Robles-Molina E, Chavez-Vargas L, Vazquez-Prado J (2011) mTORC1- and mTORC2-interacting proteins keep their multifunctional partners focused. IUBMB Life 63(10):896–914. doi: 10.1002/iub.558 CrossRefPubMedGoogle Scholar
  10. 10.
    Laplante M, Sabatini DM (2009) mTOR signaling at a glance. J Cell Sci 122(Pt 20):3589–3594. doi: 10.1242/jcs.051011 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Liu L, Parent CA (2011) Review series: TOR kinase complexes and cell migration. J Cell Biol 194(6):815–824. doi: 10.1083/jcb.201102090 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chen G, Chen SM, Wang X, Ding XF, Ding J, Meng LH (2012) Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells. J Biol Chem 287(15):12132–12141. doi: 10.1074/jbc.M111.302299 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hashimoto I, Koizumi K, Tatematsu M, Minami T, Cho S, Takeno N, Nakashima A, Sakurai H, Saito S, Tsukada K, Saiki I (2008) Blocking on the CXCR4/mTOR signalling pathway induces the anti-metastatic properties and autophagic cell death in peritoneal disseminated gastric cancer cells. Eur J Cancer 44(7):1022–1029. doi: 10.1016/j.ejca.2008.02.043 CrossRefPubMedGoogle Scholar
  14. 14.
    Carretero-Ortega J, Walsh CT, Hernandez-Garcia R, Reyes-Cruz G, Brown JH, Vazquez-Prado J (2010) Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac activation, endothelial cell migration, and in vitro angiogenesis. Mol Pharmacol 77(3):435–442. doi: 10.1124/mol.109.060400 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hernandez-Negrete I, Carretero-Ortega J, Rosenfeldt H, Hernandez-Garcia R, Calderon-Salinas JV, Reyes-Cruz G, Gutkind JS, Vazquez-Prado J (2007) P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration. J Biol Chem 282(32):23708–23715. doi: 10.1074/jbc.M703771200 CrossRefPubMedGoogle Scholar
  16. 16.
    Kim EK, Yun SJ, Ha JM, Kim YW, Jin IH, Yun J, Shin HK, Song SH, Kim JH, Lee JS, Kim CD, Bae SS (2011) Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis. Oncogene 30(26):2954–2963. doi: 10.1038/onc.2011.22 CrossRefPubMedGoogle Scholar
  17. 17.
    Nakatsu MN, Hughes CC (2008) An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods Enzymol 443:65–82. doi: 10.1016/S0076-6879(08)02004-1 CrossRefPubMedGoogle Scholar
  18. 18.
    Koh W, Stratman AN, Sacharidou A, Davis GE (2008) In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis. Methods Enzymol 443:83–101. doi: 10.1016/S0076-6879(08)02005-3 CrossRefPubMedGoogle Scholar
  19. 19.
    Hemmings BA, Restuccia DF (2012) PI3K-PKB/Akt pathway. Cold Spring Harb Perspect Biol 4(9):a011189. doi: 10.1101/cshperspect.a011189 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J (2005) Rheb binds and regulates the mTOR kinase. Curr Biol 15(8):702–713. doi: 10.1016/j.cub.2005.02.053 CrossRefPubMedGoogle Scholar
  21. 21.
    Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B (2006) SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127(1):125–137. doi: 10.1016/j.cell.2006.08.033 CrossRefPubMedGoogle Scholar
  22. 22.
    Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, Markhard AL, Sabatini DM (2006) Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22(2):159–168. doi: 10.1016/j.molcel.2006.03.029 CrossRefPubMedGoogle Scholar
  23. 23.
    De Bock K, Georgiadou M, Schoors S, Kuchnio A, Wong BW, Cantelmo AR, Quaegebeur A, Ghesquiere B, Cauwenberghs S, Eelen G, Phng LK, Betz I, Tembuyser B, Brepoels K, Welti J, Geudens I, Segura I, Cruys B, Bifari F, Decimo I, Blanco R, Wyns S, Vangindertael J, Rocha S, Collins RT, Munck S, Daelemans D, Imamura H, Devlieger R, Rider M, Van Veldhoven PP, Schuit F, Bartrons R, Hofkens J, Fraisl P, Telang S, Deberardinis RJ, Schoonjans L, Vinckier S, Chesney J, Gerhardt H, Dewerchin M, Carmeliet P (2013) Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 154(3):651–663. doi: 10.1016/j.cell.2013.06.037 CrossRefPubMedGoogle Scholar
  24. 24.
    Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9(6):669–676. doi: 10.1038/nm0603-669nm0603-669 CrossRefPubMedGoogle Scholar
  25. 25.
    Mazzieri R, Pucci F, Moi D, Zonari E, Ranghetti A, Berti A, Politi LS, Gentner B, Brown JL, Naldini L, De Palma M (2011) Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells. Cancer Cell 19(4):512–526. doi: 10.1016/j.ccr.2011.02.005 CrossRefPubMedGoogle Scholar
  26. 26.
    Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121(3):335–348. doi: 10.1016/j.cell.2005.02.034 CrossRefPubMedGoogle Scholar
  27. 27.
    Zhang F, Zhang X, Li M, Chen P, Zhang B, Guo H, Cao W, Wei X, Cao X, Hao X, Zhang N (2010) mTOR complex component Rictor interacts with PKCzeta and regulates cancer cell metastasis. Cancer Res 70(22):9360–9370. doi: 10.1158/0008-5472.CAN-10-0207 CrossRefPubMedGoogle Scholar
  28. 28.
    Kuehn HS, Jung MY, Beaven MA, Metcalfe DD, Gilfillan AM (2011) Prostaglandin E2 activates and utilizes mTORC2 as a central signaling locus for the regulation of mast cell chemotaxis and mediator release. J Biol Chem 286(1):391–402. doi: 10.1074/jbc.M110.164772 CrossRefPubMedGoogle Scholar
  29. 29.
    Liu L, Das S, Losert W, Parent CA (2010) mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion. Dev Cell 19(6):845–857. doi: 10.1016/j.devcel.2010.11.004 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Feil C, Augustin HG (1998) Endothelial cells differentially express functional CXC-chemokine receptor-4 (CXCR-4/fusin) under the control of autocrine activity and exogenous cytokines. Biochem Biophys Res Commun 247(1):38–45. doi: 10.1006/bbrc.1998.8499 CrossRefPubMedGoogle Scholar
  31. 31.
    Gupta SK, Lysko PG, Pillarisetti K, Ohlstein E, Stadel JM (1998) Chemokine receptors in human endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem 273(7):4282–4287CrossRefPubMedGoogle Scholar
  32. 32.
    Salcedo R, Wasserman K, Young HA, Grimm MC, Howard OM, Anver MR, Kleinman HK, Murphy WJ, Oppenheim JJ (1999) Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: in vivo neovascularization induced by stromal-derived factor-1alpha. Am J Pathol 154(4):1125–1135CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Volin MV, Joseph L, Shockley MS, Davies PF (1998) Chemokine receptor CXCR4 expression in endothelium. Biochem Biophys Res Commun 242(1):46–53. doi: 10.1006/bbrc.1997.7890 CrossRefPubMedGoogle Scholar
  34. 34.
    Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ (2006) A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med 203(9):2201–2213. doi: 10.1084/jem.20052144 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Zabel BA, Wang Y, Lewen S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ (2009) Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands. J Immunol 183(5):3204–3211. doi: 10.4049/jimmunol.0900269 CrossRefPubMedGoogle Scholar
  36. 36.
    Naumann U, Cameroni E, Pruenster M, Mahabaleshwar H, Raz E, Zerwes HG, Rot A, Thelen M (2010) CXCR7 functions as a scavenger for CXCL12 and CXCL11. PLoS One 5(2):e9175. doi: 10.1371/journal.pone.0009175 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Gan X, Wang J, Su B, Wu D (2011) Evidence for direct activation of mTORC2 kinase activity by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 286(13):10998–11002. doi: 10.1074/jbc.M110.195016 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Sotsios Y, Whittaker GC, Westwick J, Ward SG (1999) The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes. J Immunol 163(11):5954–5963PubMedGoogle Scholar
  39. 39.
    Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M, Bruns CJ, Zuelke C, Farkas S, Anthuber M, Jauch KW, Geissler EK (2002) Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 8(2):128–135. doi: 10.1038/nm0202-128nm0202-128 CrossRefPubMedGoogle Scholar
  40. 40.
    Phung TL, Ziv K, Dabydeen D, Eyiah-Mensah G, Riveros M, Perruzzi C, Sun J, Monahan-Earley RA, Shiojima I, Nagy JA, Lin MI, Walsh K, Dvorak AM, Briscoe DM, Neeman M, Sessa WC, Dvorak HF, Benjamin LE (2006) Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin. Cancer Cell 10(2):159–170. doi: 10.1016/j.ccr.2006.07.003 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Sharma SG, Nanda S, Longo S (2010) Anti-angiogenic therapy in renal cell carcinoma. Recent Pat Anticancer Drug Discov 5(1):77–83CrossRefPubMedGoogle Scholar
  42. 42.
    Battelli C, Cho DC (2011) mTOR inhibitors in renal cell carcinoma. Therapy 8(4):359–367. doi: 10.2217/thy.11.32 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kornakiewicz A, Solarek W, Bielecka ZF, Lian F, Szczylik C, Czarnecka AM (2014) Mammalian target of rapamycin inhibitors resistance mechanisms in clear cell renal cell carcinoma. Curr Signal Transduct Ther 8(3):210–218. doi: 10.2174/1574362409666140206222746 CrossRefPubMedGoogle Scholar
  44. 44.
    Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci USA 105(45):17414–17419. doi: 10.1073/pnas.0809136105 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Deprez J, Vertommen D, Alessi DR, Hue L, Rider MH (1997) Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades. J Biol Chem 272(28):17269–17275CrossRefPubMedGoogle Scholar
  46. 46.
    Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB, Hay N (2001) Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev 15(11):1406–1418. doi: 10.1101/gad.889901 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Kohn AD, Summers SA, Birnbaum MJ, Roth RA (1996) Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271(49):31372–31378CrossRefPubMedGoogle Scholar
  48. 48.
    Van Schaftingen E, Hue L, Hers HG (1980) Fructose 2,6-bisphosphate, the probably structure of the glucose- and glucagon-sensitive stimulator of phosphofructokinase. Biochem J 192(3):897–901CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Manes NP, El-Maghrabi MR (2005) The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via inhibition by phosphoenolpyruvate. Arch Biochem Biophys 438(2):125–136. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  50. 50.
    Xu Y, An X, Guo X, Habtetsion TG, Wang Y, Xu X, Kandala S, Li Q, Li H, Zhang C, Caldwell RB, Fulton DJ, Su Y, Hoda MN, Zhou G, Wu C, Huo Y (2014) Endothelial PFKFB3 plays a critical role in angiogenesis. Arterioscler Thromb Vasc Biol 34(6):1231–1239. doi: 10.1161/ATVBAHA.113.303041 CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Monterrubio M, Mellado M, Carrera AC, Rodriguez-Frade JM (2009) PI3Kgamma activation by CXCL12 regulates tumor cell adhesion and invasion. Biochem Biophys Res Commun 388(2):199–204. doi: 10.1016/j.bbrc.2009.07.153 CrossRefPubMedGoogle Scholar
  52. 52.
    Wang S, Amato KR, Song W, Youngblood V, Lee K, Boothby M, Brantley-Sieders DM, Chen J (2015) Regulation of endothelial cell proliferation and vascular assembly through distinct mTORC2 signaling pathways. Mol Cell Biol. doi: 10.1128/MCB.00306-14 Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Mary E. Ziegler
    • 1
  • Michaela M. S. Hatch
    • 1
  • Nan Wu
    • 1
  • Steven A. Muawad
    • 1
  • Christopher C. W. Hughes
    • 1
    • 2
    • 3
    Email author
  1. 1.The Department of Molecular Biology and BiochemistryUniversity of California IrvineIrvineUSA
  2. 2.The Department of Biomedical EngineeringUniversity of California IrvineIrvineUSA
  3. 3.The Edwards Lifesciences Center for Advanced Cardiovascular TechnologyUniversity of California IrvineIrvineUSA

Personalised recommendations