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. 2016 Jul;19(3):359-71.
doi: 10.1007/s10456-016-9509-6. Epub 2016 Apr 22.

mTORC2 mediates CXCL12-induced angiogenesis

Mary E Ziegler  1 , Michaela M S Hatch  1 , Nan Wu  1 , Steven A Muawad  1 , Christopher C W Hughes  2   3   4
Affiliations

mTORC2 mediates CXCL12-induced angiogenesis

Mary E Ziegler et al. Angiogenesis. 2016 Jul.

Abstract

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.

Keywords: Akt; Angiogenesis; CXCL12; CXCR4; mTOR; mTORC2.

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Figures

Fig. 1
Fig. 1
CXCL12/CXCR4 activation regulates angiogenesis by triggering mTORC2 (a) EC were coated onto Cytodex beads and embedded in a fibrin gel. NHLFs were seeded on top of the gel, and twenty-four hours after embedding AMD3100 (1μM) was added to the medium. DMSO was used as a vehicle control and fresh inhibitor was added with each medium change. Beads were fixed at day 7 and imaged. Scale bar: 150 μm. (b) The number of sprouts per bead was quantified. (c–e) Serum-starved EC were stimulated with CXCL12 (50 ng/ml, 30 min) alone or after pretreatment with AMD3100 (1μM, 1 hr), rapamycin (100 nM, 24 hr), PP242 (600 nM, 24hr), PIK-75 (500 nM, 1 hr), TGX-221 (500 nM, 1 hr), PI-103 (250 nM, 1hr) or Pertussis Toxin (100ng/mL, 2 hr). DMSO was used as a vehicle control. The level of Akt phosphorylation (S473) was determined by Western blot. β-actin was used as a loading control. The phospho-bands was measured and normalized using ImageJ. The numbers below the blots indicate the level of phospho-Akt relative to the lowest expression (n = 3).
Fig. 2
Fig. 2
mTORC2 is required for sprouting angiogenesis (a) EC were transfected with control, raptor or rictor siRNA or (b) with control, Sin1 or Rheb siRNA. Twenty-four hours later a fibrin sprouting assay (as described above) was performed. The beads were fixed at day 7 and imaged. Scale bar: 150 μm. The number of sprouts per bead was quantified. The error bars represent the SEM (*p < 0.05; **p < 0.01). All experiments were repeated a minimum of 3 times with similar results.
Fig. 3
Fig. 3
Inhibiting mTORC2 signaling blocks angiogenic sprouting (a) A fibrin bead sprouting assay was performed as described in Figure 1 and twenty-four hours after embedding the cells were treated with rapamycin as indicated, with DMSO used as a vehicle control. The inhibitor was replaced with each medium change and the beads were fixed at day 7 for imaging. Scale bar: 150 μm. (b) The number of sprouts per bead was quantified. The error bars represent the SEM (***p < 0.001). The experiment was repeated a minimum of 3 times with similar results.
Fig. 4
Fig. 4
CXCL12-induced EC invasion is dependent on mTORC2 (a) EC were seeded onto collagen gels containing CXCL12 and incubated with vehicle (DMSO) control or AMD3100 (1μM), rapamycin (100nM) or PP242 (600nM) for 24 hrs after before fixing and imaging. Scale bar: 150 μm. (b) The average number of invading cells was calculated by counting 5 wells per condition. (c) ECs were transfected with either control, raptor or rictor siRNA. Forty-eight hours after transfection the cells were seeded onto collagen gels containing CXCL12 and incubated for 24 hrs after which time the gels were fixed and imaged. Scale bar: 150 μm. (d) The average number of invading cells was calculated by counting 5 wells per condition. The error bars represent the SEM (**p < 0.01; ***p < 0.001). All experiments were repeated a minimum of 3 times with similar results.
Fig. 5
Fig. 5
mTORC2 inhibition suppresses tumor angiogenesis in vivo (a) BALB/c mice were implanted subcutaneously with CT26 tumor cells. Once the tumor reached a volume of 150mm3 the mice were administered either a high or low dose of rapamycin (1.5mg/kg or 0.5μg/kg, respectively) or water as a control, delivered by intraperitoneal injection. Each group consisted of 5–6 mice and all mice were treated daily for 10 days. Tumors were paraffin embedded and sectioned and EC within the tumor sections were visualized by staining for CD31+ cells. Scale Bar: 150 μm. (b) The vascular density in the tumors was calculated using ImageJ by determining the area of CD31+ cells relative to the entire area of the section. (c) Tumor tissue from the mice was collected and homogenized in lysis buffer. The tissue lysates were run on an SDS-PAGE gel and a Western blot was performed. The membranes were probed for phoshpo-Akt (S473) and phospho-S6-RP (S240/244) and β-actin was used as a loading control. Bands were quantified using ImageJ and the expression of phospho-protein relative to the control mice is indicated below the blot. The images are all from the same blot and separated to present only the relevant treatment groups. (d) Tumor volume was monitored daily. Error bars represent the SEM *p < 0.05.
Fig. 6
Fig. 6
mTORC2 inhibition decreases the expression of PFKFB3. (a) Tumor tissue from the mice described in Figure 5 was collected and homogenized in lysis buffer and PFKFB3 expression was analyzed by Western blot. β-actin was used as a loading control. Bands were quantified using ImageJ and intensities are indicated below the blot as PFKFB3 expression relative to the control mice. (b) A fibrin bead angiogenesis assay was performed as described above and twenty-four hours after embedding the cells were treated with 3PO as indicated. DMSO was used as a vehicle control and the inhibitor was replaced with each medium change. The beads were fixed at day 7 and the number of sprouts per bead was quantified. Scale bar: 150 μm. (c) EC were transfected with either control or PFKFB3 siRNA and twenty-four hours later a fibrin bead angiogenic assay was performed. The beads were fixed at day 7 and imaged. Scale bar: 150 μm. The number of sprouts per bead was quantified. The error bars represent the SEM (**p < 0.01; ***p < 0.001). All experiments were repeated a minimum of 3 times with similar results.
Fig. 7
Fig. 7
CXCL12/CXCR4 signal transduction in EC is mTORC2 dependent The activation of CXCR4 on EC by CXCL12 initiates G protein signaling leading to PI3K activation, which then triggers mTORC2 to phosphorylate Akt at S473. CXCL12 stimulation does not potentiate signal transduction downstream of mTOR1. The disruption or inhibition of mTORC2 blocks angiogenesis both in vitro and in vivo. Inhibition of mTORC2 in vivo reduces the expression of PFKFB3 placing this metabolic regulator downstream of the mTORC2 signaling cascade and designates these targets as key regulators of angiogenesis.
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References

    1. Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med. 2011;17(11):1359–1370. doi: 10.1038/nm.2537. nm.2537 [pii] - DOI - PubMed
    1. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473(7347):298–307. doi: 10.1038/nature10144. nature10144 [pii] - DOI - PMC - PubMed
    1. Salcedo R, Oppenheim JJ. Role of chemokines in angiogenesis: CXCL12/SDF-1 and CXCR4 interaction, a key regulator of endothelial cell responses. Microcirculation. 2003;10(3–4):359–370. doi: 10.1038/sj.mn.7800200. 7800200 [pii] - DOI - PubMed
    1. Newman AC, Chou W, Welch-Reardon KM, Fong AH, Popson SA, Phan DT, Sandoval DR, Nguyen DP, Gershon PD, Hughes CC. Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived hepatocyte growth factor and fibronectin in angiogenesis. Arterioscler Thromb Vasc Biol. 2013;33(3):513–522. doi: 10.1161/ATVBAHA.112.300782. ATVBAHA.112.300782 [pii] - DOI - PMC - PubMed
    1. Strasser GA, Kaminker JS, Tessier-Lavigne M. Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood. 2010;115(24):5102–5110. doi: 10.1182/blood-2009-07-230284. blood-2009-07-230284 [pii] - DOI - PubMed

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