1 Akt1 and Akt3 exert opposing roles in the regulation of vascular ...

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Akt1 and Akt3 exert opposing roles in the regulation of vascular tumor growth

Thuy L. Phung1#, Wa Du1, Qi Xue2, Sriram Ayyaswamy1, Damien Gerald2, Zeus Antonello3,

Sokha Nhek2, Carole A. Perruzzi2, Isabel Acevedo3, Rajesh Ramanna-Valmiki1, Paul Rodriguez-

Waitkus1, Ladan Enayati1, Marcelo L. Hochman4, Dina Lev5, Sandaruwan Geeganage6 and

Laura E. Benjamin2,3,6#

1Department of Pathology, Texas Children’s Hospital and Baylor College of Medicine, Houston,

Texas, USA; 2ImClone Systems, New York, NY, USA; 3Department of Pathology, Beth Israel

Deaconess Medical Center, Boston, Massachusetts, USA; 4Hemangioma International

Treatment Center, Charleston, South Carolina, USA; 5Department of Cancer Biology, MD

Anderson Cancer Center, Houston, Texas, USA; and 6Eli Lilly and Company, Indianapolis, IN,

USA

on April 6, 2018. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 11, 2014; DOI: 10.1158/0008-5472.CAN-13-2961

http://cancerres.aacrjournals.org/

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Running title: Differential regulation of vascular tumors by Akt isoforms

Key words: Akt3, vascular tumors, mTOR, S6-Kinase

Funding: This work was supported in part by NIH grants R01 CA106263 and P01 CA09264401

(LEB); American Heart Association 11BGIA5590018, NIH K08 HL087008, NIH R03 AR063223,

American Cancer Society 122019-RSG-12-054-01-CSM and the Baylor Clinical and

Translational Research Program (TLP).

# Corresponding authors:

Laura E. Benjamin, PhD

ImClone Systems, a wholly-owned subsidiary of Eli Lilly

450 East 29th Street, 12th Floor

New York, NY 10016, USA

Phone: 646-638-6381

Email: [email protected]

Thuy L. Phung, MD, PhD

Texas Children’s Hospital

1102 Bates Avenue, Suite 830

Houston, TX 77030, USA

Phone: 832-824-5202

Email: [email protected]

Potential conflict of interest: QX, SN, DG, CAP, SG and LEB are employees of ImClone

Systems, a wholly-owned subsidiary of Eli Lilly and Company. The other authors report no

conflict of interest.



mailto:[email protected]


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ABSTRACT

Vascular tumors are endothelial cell neoplasms whose mechanisms of tumorigenesis

are poorly understood. Moreover, current therapies, particularly those for malignant lesions,

have little beneficial effect on clinical outcomes. In this study, we show that endothelial

activation of the Akt1 kinase is sufficient to drive de novo tumor formation. Mechanistic

investigations uncovered opposing functions for different Akt isoforms in this regulation, where

Akt1 promotes and Akt3 inhibits vascular tumor growth. Akt3 exerted negative effects on tumor

endothelial cell growth and migration by inhibiting activation of the translation regulatory kinase

S6K through modulation of Rictor expression. S6K in turn acted through a negative feedback

loop to restrain Akt3 expression. Conversely, S6K signaling was increased in vascular tumor

cells where Akt3 was silenced, and the growth of these tumor cells was inhibited by a novel S6K

inhibitor. Overall, our findings offer a preclinical proof-of-concept for the therapeutic utility of

treating vascular tumors, such as angiosarcomas, with S6K inhibitors.




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INTRODUCTION

Vascular tumors are endothelial cell (EC) neoplasms with a wide spectrum of clinical

presentations, ranging from benign infantile hemangiomas in children to low-grade malignant

hemangioendotheliomas and highly aggressive angiosarcomas in adults. To date, the

molecular pathogenesis of vascular tumors is poorly understood and current therapies,

particularly those for malignant vascular tumors, do not significantly improve patient outcome

(1).

Akt is a major signaling pathway activated by vascular endothelial growth factor (VEGF)

that regulates EC survival (2). In infantile hemangioma (IH), hemangioma-derived endothelial

cells (hemeEC) have constitutively active VEGF receptor-2 signaling with high phosphorylation

levels of ERK1/2 and Akt (3). Human angiosarcoma (AS) expresses VEGF-A and the VEGF

receptors (4). We have shown increased phosphorylation of Akt and 4E-BP1 in AS (5).

Hyperactivation of PI3-kinase results in hemangiosarcoma formation in chicken chorioallantoic

membrane (6). Akt1, Akt2 and Akt3 are isoforms that have shared as well as distinct functions

in cancer cells. Both Akt1 and Akt2 promote cancer cell survival and growth. However, in

breast and ovarian cancer, Akt1 decreases cell motility and metastasis and blocks epithelial-to-

mesenchymal phenotype, whereas Akt2 enhances these processes (7-9). Akt3 is preferentially

required for the growth of triple-negative breast cancer (10), and a gene fusion of Akt3 with

MAGI3 leads to constitutive Akt3 activation and is enriched in these tumors (11). Interestingly,

there is some evidence suggesting that Akt3 exerts inhibitory effects in cancer. N-Cadherin

promotes breast cancer metastasis by inhibiting Akt3, and Akt3 has been shown to inhibit lung

tumor growth in mice (12-14). Studies of animal models of breast cancer with simultaneous

deletion or overexpression of Akt1, Akt2 and Akt3 lend further support to Akt isoform-specific

roles in cancer (8, 9).




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Mammalian target of rapamycin (mTOR) complex-1 (mTORC1) and complex-2

(mTORC2) are composed of multiple subunits, including mTOR and Raptor (in mTORC1), and

mTOR and Rictor (in mTORC2) (15, 16). Akt activates mTORC1, which phosphorylates the

translational regulators 4E-BP1 and p70 S6-Kinase (S6K). S6K in turn activates S6 ribosomal

protein (S6) (17, 18). mTORC2 directly activates Akt by phosphorylating it at serine 473,

thereby exerting feedback regulation on the Akt signaling pathway (15). The S6K pathway is

important in protein synthesis and cell growth, and acts as a regulator of actin cytoskeleton

dynamics in cell migration (19, 20).

In this study, we showed that endothelial Akt1 drives vascular tumor growth.

Importantly, we have uncovered the opposing functions of Akt1 and Akt3 in the regulation of

tumor growth, which is mediated through S6K, and found a novel negative feedback regulation

on Akt3 by S6K. We also demonstrated the clinical utility of a novel S6K inhibitor in the

treatment of vascular lesions.




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MATERIALS AND METHODS

Animals. Animal studies were conducted in compliance with the Beth Israel Deaconess

Medical Center (BIDMC) Institutional Animal Care and Use Committee guidelines. Double

transgenic myristoylated Akt1 mice in mixed FVB genetic background have been previously

described (21). C57 Bl/6 Akt3-/- mice were from Argiris Efstratiadis and Morris Birnbaum (22).

Cell lines and reagents. The use of human tissues was approved by the Institutional Review

Boards at BIDMC and Baylor College of Medicine. Primary human dermal microvascular

endothelial cells (EC), infantile hemangioma and mouse EC were isolated as described (21, 23).

ASM.5 cells were from Vera Krump-Konvalinkov and EOMA cells were from ATCC as

previously published (23-25). Cell line authentication and validation by short tandem repeats

was performed. HA-tagged-MyrAkt3 and constitutively active S6K (R3A) have been described

(26, 27). LY2584702 (Eli Lilly, Inc.) was prepared in 0.25% Tween-80 and 0.05% antifoam, and

administered orally to mice (12.5 mg/kg twice daily). All antibodies used were from Cell

Signaling Technologies, except for antibodies to smooth muscle actin, β-actin and -tubulin

(Sigma), CD31 (BD Biosciences) and glucose transporter-1 (Dr. Morris Birnbaum).

Mouse hemangioma skin graft model. Ten-mm circular pieces of flank skin from donor

myrAkt1 mice were grafted onto the back of recipient mice with absorbable Vicryl sutures

(Ethicon). Recipient animals were maintained on 1.5 mg/ml tetracycline in the drinking water as

described (21) to turn off myrAkt1 while the grafts were left to heal for 2 weeks. After this time,

half of the recipients continued to receive tetracycline for 4 weeks, while the other half was

taken off tetracycline to turn on myrAkt1 expression.




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Immunoprecipitation and western blots. For Akt isoform immunoprecipitations, cells were

lysed in RIPA buffer with protease/phosphatase inhibitors. Cell lysates were precleared with

Protein A/G Agarose, 1 hour, 40C, followed with addition of primary antibodies to 0.8 mg protein

lysates and rotated overnight, 40C. Protein A/G Agarose was then added to the lysates and

rotated for 2 hours, 40C. Immunoprecipitated proteins were denatured in Laemmli buffer and

analyzed by western blotting as described (23).

Tumor growth. EOMA cells (0.3 x 106) were injected subcutaneously in 6-8 week old nu/nu

female mice (2 sites/mouse, 4-5 mice/group). Tumor size was measured daily. For drug

treatment, when tumors reached 0.01 cm3 in size, the animals were treated with vehicle control

or LY2584702 (12.5 mg/kg twice daily, oral dosing). Tumor size was measured every 3-4 days.

Lentiviral shRNA and quantitative real-time PCR. shRNA clones used are listed in

Supplementary Table S2. qPCR primers are provided in Supplementary Materials and

Methods. Lentivirus packaging and qPCR were performed as described (23).

Statistical analysis. Data were presented as mean ± SD. The difference between multiple

experimental groups was assessed by one-way ANOVA, and the difference between multiple

experimental groups at different time points (two independent variables) was assessed by two-

way ANOVA using GraphPad Prism.

Other standard reagents and methods are provided in Supplementary Materials and

Methods.




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RESULTS

Akt is activated in human vascular tumors. Clinical specimens of 17 benign infantile

hemangioma (IH), 6 kaposiform hemangioendothelioma (KHE), 16 Kaposi’s sarcoma (KS) and

9 angiosarcoma (AS) were stained for phosphorylated Akt Serine 473 (pAkt S473). All of the

tumors expressed increased pAkt levels compared to adjacent normal blood vessels in the

same tissue sections (Figure 1A). We also compared the levels of pAkt in these tumors with

blood vessels in 20 normal skin specimens as the normal counterpart of neoplastic vessels.

The percent positively stained tumor cells (stain reactivity) and the stain intensity per group

were calculated. Higher pAkt stain reactivity was seen in vascular tumors than in normal skin

(73.3±2.1% in normal skin vs. 92.3±1.3%* in IH, 95.5±1.2%* in KHE, 92.3±1.3%* in KS and

89.7±1.9%* in AS; *P<0.0001) (Figure 1B). To evaluate the stain intensity, the staining was

scored using a 3-tier system (1, low; 2, moderate; and 3, high) by two pathologists and the

average score was graphed. Representative pictures of low and high stain intensity are shown

(Supplementary Figure S1). Significantly higher pAkt stain intensity was seen in tumors (1.6 ±

0.1 in normal skin vs. 2.1 ± 0.2* in IH, 2.2 ± 0.3* in KHE, 2.5 ± 0.2* in KS and 3.0 ± 0.03* in AS;

*P<0.01) (Figure 1C and Supplementary Figure S1). These results showed increased Akt

activation across different types of human vascular tumors.

To determine whether Akt is hyperactivated in neoplastic EC, we focused on IH, which is

a common soft tissue tumor of infancy and fresh tissues are available for studies. We purified

hemangioma-derived endothelial cells (hemeEC) from IH using CD31-magnetic bead isolation

and stained for endothelial markers (Supplementary Figure S2A). HemeEC showed a 1.9-fold

increase in pAkt as compared with normal human dermal microvascular endothelial cells

(HDMEC) by western blot (Figure 1D). Consistent with a previous report of constitutive VEGFR-

2 activation in IH (3), we observed increased phosphorylated VEGFR-2 in a subset of hemeEC




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samples. Interestingly, PTEN levels were also reduced in some hemangiomas. These findings

showed increased Akt activation in IH, which is associated with decreased PTEN and increased

VEGFR-2 activation.

We next examined Akt activation in malignant vascular tumors. We utilized ASM.5 cells

isolated from a spontaneous human angiosarcoma (24), and EOMA cells derived from a

spontaneous mouse hemangioendothelioma (25). ASM.5 and EOMA cells had increased Akt

activation as compared with normal EC (Figures 1E-F) (1.0 ± 0.2 in HDMEC vs. 2.8 ± 1.1* in

ASM.5, *P<0.05, N=3), (1.0 ± 0.5 in mouse EC vs. 6.7 ± 2.0* in EOMA, *P<0.05, N=4).

Furthermore, lower levels of PTEN were seen in both cell types. These findings indicate

common aberrations in the PTEN/Akt pathway in both benign and malignant vascular tumors.

Akt1 promotes the growth and migration of vascular tumor cells. HemeEC and ASM.5

cells were transduced with lentiviral short-hairpin RNA to Akt1 (shAkt1). Significant Akt1

knockdown was achieved using two independent shAkt1 clones as compared with pLKO

scramble shRNA control (Figures 2A-B). Akt1 knockdown significantly reduced the growth of

hemeEC and ASM.5 cells as assessed by cellular DNA content (Figures 2C-D). Since Akt1 is

known to affect cell survival (28), we determined the effects of shAkt1 on apoptosis. Akt1

knockdown resulted in increased tumor cell apoptosis in response to serum-starvation as

determined by the apoptotic marker Annexin V (Supplementary Figures S2B-C). Akt1

knockdown decreased basal and VEGF-stimulated migration of hemeEC and ASM.5 cells in

Boyden chamber migration assay (Figures 2E-F). Akt1 knockdown also significantly inhibited

basal and VEGF-induced cord formation in hemeEC (Figures 2G-H). These findings showed

that Akt1 plays a key role in promoting the survival and migration of vascular tumor cells.




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Endothelial Akt1 activation induces hemangioma formation in mice. To determine whether

Akt1 activation in EC is sufficient to drive de novo vascular tumor formation, we utilized a double

transgenic mouse model that expresses tetracycline-inducible and endothelial cell-specific

activated myristoylated Akt1 (myrAkt1) (21). myrAkt1 transgene induction increased Akt1

expression by 3.9-fold, but did not affect Akt2 and Akt3 levels in EC isolated from these mice

(Supplementary Figures S3A-B). myrAkt1 mice have systemic pathological angiogenesis, but

they do not develop vascular tumors due to shortened lifespan from systemic edema (21). To

study the long-term effects of endothelial Akt1 activation, we developed a skin graft model in

which 10-mm circular pieces of skin from myrAkt1 donors (FVB background) were transplanted

onto the back skin of immunodeficient nu/nu mice (Figure 3A). Recipient animals were

maintained on tetracycline to turn off myrAkt1 while the grafts were left to heal for 2 weeks.

After this time, animals were either kept on tetracycline (myrAkt1 expression off) or taken off

tetracycline (myrAkt1 on) for 4 weeks. Without myrAkt1 expression, a dermal scar was present

but no tumor was seen (Figure 3B). However, induction of myrAkt1 resulted in the development

of red tumor masses at the graft sites. The tumors measured 0.39 ± 0.14 cm3 in size and had

histologic features consistent with benign hemangiomas (Figure 3C). The tumor vessels co-

expressed the endothelial marker CD31 and HA-tagged myrAkt1 (Figure 3D). Abundant pAkt

was seen in tumor vessels, some of which contained a covering of smooth muscle actin (SMA)

while many were SMA-deficient. Interestingly, the expression of glucose transporter-1 (Glut-1),

a biomarker found uniquely in human IH (29), was seen in CD31+ vessels. We also performed

myrAkt1 skin grafts in syngeneic immunocompetent FVB recipients and observed similar tumor

development in these mice with tumors measuring 0.49 ± 0.21 cm3 in size (Supplementary

Figures S3C-D).

Sustained endothelial Akt1 activation is necessary to maintain hemangioma growth. Skin

graft transplantation was performed and myrAkt1 expression was turned on to allow




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hemangioma to develop. After 4 weeks, myrAkt1 expression was turned off in half of the

animals while myrAkt1 expression continued to be on in the other half. Tumors in animals with

sustained myrAkt1 expression continued to grow, but those in animals with myrAkt1 turned off

regressed dramatically over the next 3.5 weeks and had few vessels and more fibrofatty tissue

(Figures 3E-F). Thus, sustained endothelial Akt1 activation is required to maintain the integrity

of blood vessels in this vascular tumor model, and supports our previous finding of the

“plasticity” of the microvasculature in response to Akt1 signaling (21).

Akt3 expression is reduced in vascular tumors. Immunostains for Akt1, Akt2 and Akt3 in 15

IH and 10 AS samples showed that the levels of Akt1 and Akt2 in these tumors were similar to

those present in adjacent normal blood vessels in the same tissue sections (Figure 4A). By

contrast, Akt3 levels were reduced in tumor tissues. We next compared the levels of Akt

isoforms in vascular tumors with the blood vessels in 6 normal human skin specimens (Figure

4B). The stain reactivity and stain intensity were calculated as described for pAkt stains in

Figure 1. Representative pictures of low and high Akt isoform stain intensity are shown in

Supplementary Figure S4 at different magnifications. The stain reactivity (%) and stain intensity

for endogenous Akt1 were similar in normal skin and vascular tumors (for Akt1 stain reactivity,

85.6 ± 4.4% in normal skin vs. 77.0 ± 5.4% in IH and 87.7 ± 1.5% in AS; P-values = not

significant. For Akt1 stain intensity, 2.3 ± 0.4 in normal skin vs. 1.5 ± 0.2 in IH and 2.1 ± 0.3 in

AS; P-values = not significant). Likewise, endogenous Akt2 levels were similar in normal skin

and vascular tumors (for Akt2 stain reactivity, 78.0 ± 13.5% in normal skin vs. 89.1 ± 1.7% in IH

and 86.0 ± 3.2% in AS; P-values = not significant. For Akt2 stain intensity, 1.8 ± 0.5 in normal

skin vs. 2.3 ± 0.2 in IH and 2.3 ± 0.2 in AS; P-values = not significant). In contrast, endogenous

Akt3 levels were reduced in vascular tumors as compared to normal skin (for Akt3 stain

intensity, 2.4 ± 0.3 in normal skin vs. 1.1 ± 0.1* in IH and 1.4 ± 0.2* in AS; *P<0.05). The stain




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reactivity for Akt3 was lower in IH (88.7 ± 2.3% in normal skin vs. 69.5 ± 8.3%* in IH; *P<0.05),

but not in AS (88.7 ± 2.3% in normal skin vs. 87.8 ± 2.0% in AS; P-value = not significant).

Immunoblots for Akt3 in ASM.5 and EOMA cells showed that they had significantly lower

Akt3 levels than normal EC, which is consistent with the findings in patient tumor tissues

(Supplementary Figure S5). We did not observe a significant change in Akt3 in hemeEC. To

examine the phosphorylation status of Akt isoforms, ASM.5 cells were immunoprecipitated with

antibodies specific for Akt1, Akt2 and Akt3, and immunoblotted for each Akt isoform and

phospho-Akt. Akt1 and Akt3 were phosphorylated at both threonine 308 (T308) and S473

residues, sites that are required for full Akt activation (Figure 4C). Interestingly, Akt2 did not

appear to be phosphorylated in these studies, indicating that only Akt1 and Akt3 are the two

active isoforms in these tumor cells.

Akt1 promotes, whereas Akt3 inhibits vascular tumor growth. We assessed the effects of

loss of each Akt isoform on sprouting angiogenesis in IH. Since Akt2 does not appear to be

activated in vascular tumor cells (Figure 4C), we chose to focus on Akt1 and Akt3. Western blot

analysis and quantitation by densitometry showed effective knockdown of each Akt isoform in

hemeEC by shRNA (Supplementary Figure S6A). Of note, the levels of Akt3 appeared higher

than Akt1 and Akt2 in the blots. However, because the antibodies to detect Akt1, Akt2 and Akt3

were different antibodies, it would not be possible to cross compare the levels of one Akt

isoform to another by western blots.

In spheroid sprouting assay, loss of Akt1 reduced basal and VEGF-A-stimulated sprout

formation as compared with pLKO (Figures 5A-B). However, loss of Akt3 significantly increased

sprout formation under basal conditions and with VEGF-A using independent shRNAs. Similar

findings were observed in ASM.5 and EOMA cells. Effective knockdown of each Akt isoform




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was achieved as shown by densitometric quantitation of western blots (Supplementary Figures

S6B-C). Compared with pLKO, loss of Akt1 reduced tumor cell migration in scratch wound

assays (Figures 5C-D). In contrast, loss of Akt3 significantly increased cell migration. Akt1 and

Akt3 also exert opposing effects on cell growth – loss of Akt1 reduced, whereas loss of Akt3

increased EOMA growth in vitro (Figure 5E).

To evaluate the functions of Akt isoforms in vivo, Akt1, Akt2 and Akt3 were knocked-

down in EOMA cells using independent shRNA clones for each isoform (Supplementary Figure

S6D). Cells were then injected subcutaneously in nu/nu mice and tumor size was monitored for

12 days. Loss of Akt1 reduced tumor growth; loss of Akt2 had no effect, whereas loss of Akt3

enhanced tumor growth (Figure 5F and Supplementary Figure S6E). These findings

demonstrate the opposing roles of Akt1 and Akt3 in vascular tumor growth.

The distinct functions of Akt1 and Akt3 are mediated through p70 S6-Kinase. To evaluate

downstream effectors of Akt isoforms, Akt1 and Akt3 were knocked-down in hemeEC and

EOMA cells and analyzed for Akt, S6K and S6. pAkt levels were normalized to total Akt levels

and calculated relative to that in pLKO. Knockdown of either Akt1 or Akt3 decreased pAkt in

hemeEC (Figure 6A) (1.0 ± 0.0 in pLKO vs. 0.6 ± 0.1* in shAkt1 and 0.7 ± 0.04* in shAkt3;

*P<0.05, N=4). Similar reduction in pAkt was found in EOMA cells (1.0 ± 0.0 in pLKO vs. 0.4 ±

0.02* in shAkt1 and 0.7 ± 0.1* in shAkt3; *P<0.05, N=3). These results indicate that both Akt1

and Akt3 contribute to the total pool of phosphorylated Akt in these cells.

Knockdown of Akt1 in hemeEC decreased levels of phosphorylated S6K (pS6K) and its

downstream effector phosphorylated S6 (pS6). By contrast, loss of Akt3 increased pS6K and

pS6 (Figure 6A). Quantitative analysis of western blots showed that pS6K levels in hemeEC

were 1.0 ± 0.0 in pLKO vs. 0.5 ± 0.2* in shAkt1 and 3.3 ± 0.5* in shAkt3; *P<0.05, N=3.




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Similarly, the levels of pS6 were 1.0 ± 0.0 in pLKO vs. 0.5 ± 0.1* in shAkt1 and 1.9 ± 0.3* in

shAkt3; P<0.05. We also evaluated EOMA cells and found similar opposing effects of Akt1 and

Akt3 on S6K and S6 activation (Figure 6A). pS6K levels in EOMA cells were 1.0 ± 0.0 in pLKO

vs. 0.3 ± 0.1* in shAkt1 and 3.2 ± 0.3* in shAkt3; *P<0.05, N=3. Similarly, levels of pS6 were

1.0 ± 0.0 in pLKO vs. 0.3 ± 0.2* in shAkt1 and 2.2 ± 0.7* in shAkt3; *P<0.05. Endothelial cells

from Akt3-/- mice similarly showed increased levels of pS6K and pS6 (Figure 6A). pAkt levels

were 1.0 ± 0.0 in wild type (WT) cells vs. 0.8 ± 0.03* in Akt3-/- cells; *P<0.05, N=3. pS6K levels

were 1.0 ± 0.0 in WT cells vs. 4.8 ± 0.8* in Akt3-/- cells; *P<0.05. Levels of pS6 were 1.0 ± 0.0

in WT cells vs. 1.8 ± 0.3* in Akt3-/- cells; *P<0.05. Thus, we have observed in vascular tumor

cells and confirmed in Akt3-/- cells that Akt1 and Akt3 exert opposite effects on S6K signaling

pathway, in which Akt1 promotes whereas Akt3 inhibits S6K activation.

To determine whether S6K mediates the biological effects observed in knockdown

studies, we rescued Akt1 knockdown cells with over-expression of constitutively activated S6K

R3A, and rescued Akt3 knockdown cells with concurrent knockdown of S6K. S6K rescued

shAkt1 and shAkt3 effects on the migration and proliferation of EOMA and ASM.5 cells (Figures

6B-E and Supplementary Figure S7). These findings showed that S6K is a mediator of the

inhibitory effects of Akt3 in vascular tumor cells.

We also utilized a small molecule inhibitor of S6K, LY2584702. LY2584702 is highly

selective for S6K1 when tested against 83 different kinases and 45 cell surface receptors. In

S6K1 enzyme assay, the drug’s IC50 = 2 nM. For pS6 inhibition in cells, the IC50 = 100 nM

(Supplementary Table S1). The drug has some activity against the S6K-related kinases MSK2

and RSK at high concentrations (enzyme assay IC50 = 58-176 nM). LY2584702 inhibits S6K

activity in EOMA cells, as determined by the phosphorylation of its downstream effector S6, in a

dose dependent manner (Figure 6F). To examine the role of S6K in vivo, EOMA cells




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expressing shAkt3 were implanted in nu/nu mice, then treated for 14 days with LY2584702 or

rapamycin, which is a potent inhibitor of mTORC1 and S6K activation (30). Analysis of tumors

removed after 14 days showed that LY2584702 inhibited S6 phosphorylation almost as

effectively as rapamycin (Figure 6G). Loss of Akt3 increased tumor growth as compared with

pLKO (Figure 6H). LY2584702 treatment alone did not significantly affect the growth of pLKO

tumors. However, it significantly reduced the growth of tumors with shAkt3. These findings

showed that down-regulation of Akt3 increased S6K activation in vascular tumors, and

enhanced the anti-tumor efficacy of S6K inhibition with LY2584702.

Akt3 modulates Rictor levels. We evaluated the effects of Akt3 knockdown on the

protein components of mTOR complexes in EOMA and ASM.5 cells. The levels of mTOR and

Raptor (a key component of mTORC1) were not affected by loss of Akt1 or Akt3 (Figures 7A-B).

However, the levels of Rictor (a key component of mTORC2) were significantly reduced in cells

with loss of Akt3, but not Akt1. Rictor immunoblots were quantified by densitometry, normalized

to β-actin and calculated relative to pLKO. In EOMA cells, Rictor levels were 1.0 ± 0.0 in pLKO

vs. 1.4 ± 0.2 in shAkt1 and 0.4 ± 0.2* in shAkt3; *P<0.05, N=9. Similarly in ASM.5 cells, Rictor

levels were 1.0 ± 0.0 in pLKO vs. 1.1 ± 0.2 in shAkt1 and 0.5 ± 0.03* in shAkt3; *P<0.05, N=3.

We observed that knockdown of Akt3, but not Akt1, significantly reduced Rictor mRNA (Figure

7C). These findings indicate that Akt3 positively regulates Rictor levels, at least in part by

modulating Rictor mRNA expression.

To determine whether Rictor affects S6K phosphorylation, we knocked down Rictor and

immunoblotted for pS6K and pS6 (mTORC1 activity) and pAkt S473 (mTORC2 activity). Rictor

knockdown decreased pAkt levels in both EOMA and ASM.5 cells (Figure 7D and

Supplementary Figure S8A). Importantly, loss of Rictor resulted in increased pS6K in both cell

lines. Densitometric analysis showed that in EOMA cells, pS6K levels were 1.0 ± 0.0 in pLKO




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vs. 1.6 ± 0.2* in shRictor; *P<0.05, N=4. pS6 downstream of S6K also increased with Rictor

knockdown. pS6 Levels were 1.0 ± 0.0 in pLKO vs. 1.7 ± 0.3* in shRictor; *P<0.05, N=4. Rictor

knockdown appeared to recapitulate the effects of Akt3 knockdown on S6K and S6, suggesting

that Akt3 regulates S6K activation by modulating Rictor levels.

To more definitely demonstrate that the inhibitory effects of Akt3 on S6K is mediated by

Rictor, we performed a rescue experiment in which constitutively activated myristoylated Akt3

(myrAkt3) was overexpressed in EOMA cells. Expression of HA-tagged myrAkt3 was confirmed

by immunoblotting (Figure 7E). Overexpression of myrAkt3 increased pAkt (T308 and S473)

and Rictor, but decreased pS6K. Concurrent knockdown of Rictor in cells with myrAkt3

overexpression rescued the inhibitory effects of myrAkt3 on S6K phosphorylation.

Densitometric analysis showed that pS6K levels were 1.0±0.0 in vector vs. 0.3±0.04* in HA-

myrAkt3 and 1.5±0.5* in HA-myrAkt3 + shRictor; *P<0.05, N=3. These findings further support

our hypothesis that Akt3 is a negative regulator of S6K signaling pathway and Rictor is a

potential mediator of the effects of Akt3.

S6K exerts negative feedback regulation on Akt3. It has been shown that S6K exerts

negative feedback regulation on Akt signaling via down-regulating IRS-1 and receptor tyrosine

kinase signaling (31). Given the observed differential effects of Akt1 and Akt3 on S6K pathway,

we investigated whether S6K exerts differential feedback regulation on Akt isoforms.

Knockdown of S6K increased pAkt (T308 and S473) in EOMA cells, consistent with the relief of

feedback inhibition of Akt signaling by S6K (Figure 7F). S6K knockdown increased Akt3, but

not Akt1 levels, in EOMA and ASM.5 cells (Figure 7F and Supplementary Figure S8B). Akt3

levels in EOMA cells were 1.0±0.0 in pLKO vs. 1.9±0.5* in shS6K; *P<0.05, N=3. Inhibition of

S6K activation with rapamycin showed a significant increase in Akt3, but not Akt1

(Supplementary Figure S8C). In immunoprecipitation experiments, more Akt3 was pulled down




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in cells with S6K knockdown as compared with pLKO (1.0 in pLKO vs. 1.8 in shS6K, N=2)

(Figure 7G). No changes were seen with Akt1 (1.0 in pLKO vs. 0.9 in shS6K). Conversely,

overexpression of constitutively active S6K reduced levels of Akt3 (Figure 7H). These findings

show that Akt3 inhibits S6K activation, which in turn exerts negative feedback regulation on Akt3

itself.




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DISCUSSION

Our studies showed increased Akt activation and decreased PTEN levels in both benign

and malignant vascular tumors. These tumors are composed of multiple cell types, including

neoplastic endothelial cells, inflammatory and stromal cells. It is not known whether the

endothelial cellular component alone is sufficient for vascular tumor development. We showed

in myrAkt1 animal model that de novo hemangioma formation is driven by endothelial Akt1

activation, and is “endothelial-cell autonomous.” Tumor regression was observed upon loss of

endothelial myrAkt1 in our animal model, indicating that sustained Akt signaling is required for

tumor maintenance. Spontaneous regression is a distinctive characteristic of infantile

hemangioma that is biologically programmed in the natural progression of the tumor. It is

conceivable that a pre-programmed network that switches off downstream VEGF signaling

pathways, such as Akt, may be a mechanism of hemangioma regression.

Little is known about Akt3 function. Limited studies on Akt3 showed that it is required for

VEGF stimulation of mitochondrial biogenesis and autophagy in EC, and is important for growth-

factor induced angiogenic responses (32). Akt3 is the dominant isoform in melanoma and

ovarian cancer and regulates cellular senescence, VEGF secretion and angiogenesis in these

tumors (33-35). Our studies of vascular tumors and the emerging literature on Akt3 provide a

new perspective on Akt signaling: there is a “check-and-balance” by different Akt isoforms to

modulate overall Akt signaling output and limit unchecked growth signals downstream of Akt.

Thus, one Akt isoform may regulate growth-promoting biological output, while another isoform

may regulate growth-inhibitory output in order to ensure homeostatic regulation of Akt signaling.

We have found that Akt1 and Akt3 have unique opposing roles, in which Akt1 promotes,

whereas Akt3 inhibits tumor endothelial cell growth. It is possible that the net balance of Akt

signaling output drives vascular tumors. Akt3 is expressed at lower levels in vascular tumors

than in normal blood vessels. Such a scenario has also been observed in malignant glioma, in




19

which the expression of Akt3 is lower in the tumor than in normal brain tissue; however, the

remaining Akt3 has kinase activity (36). This suggests that although the level of endogenous

Akt3 is reduced, it still retains the functional capacity as an active kinase.

Different Akt isoforms can signal via distinct downstream pathways that may vary

depending on the cellular context and subcellular localization (37). Akt1 and Akt3, but not Akt2

ablation in mice predominantly influences GSK-3 α/β signaling in a mouse lung tumor model

(14). Akt3, but not Akt1, is crucial for the activation of the mTORC1/S6K signaling pathway in

the brain, thus reinforcing the differential effects of Akt isoforms on the S6K pathway (22). We

have found that Akt1 and Akt3 exert distinct effects on S6K signaling: Akt1 stimulates, whereas

Akt3 inhibits S6K activation. To determine how Akt3 regulates S6K activation, we showed that

knockdown of Akt3 leads to reduced Rictor protein levels, which is due at least in part to a

reduction of Rictor mRNA expression.

Overexpression of myrAkt3 increases Rictor levels and reduces S6K activation.

Importantly, knockdown of Rictor in cells with myrAkt3 overexpression rescues the effects of

myrAkt3 on S6K. Taken together, these findings suggest a potential mechanism by which Akt3

regulates S6K activation through Rictor. We postulate that by regulating Rictor levels, Akt3 can

affect the formation of mTOR complexes and the balance of mTORC1 and mTORC2 activities

in the cell. Published studies lend support to this hypothesis. The formation of TOR complexes

in Caenorhabditis elegans is controlled by semaphorin-plexin signaling, in which semaphorin

promotes the formation of TORC1 and inhibits TORC2 by promoting a shift of TOR from Rictor

towards Raptor, thereby altering the ratio of TORC1 and TORC2 (38). Another mechanism of

regulation of mTOR complex assembly involves the mTOR inhibitor rapamycin. Rapamycin

mainly inhibits mTORC1, but long-term drug treatment can lead to the inhibition of mTORC2, in

which rapamycin may sequester mTOR and interfere with the assembly of mTORC2 (39).




20

Emerging studies have highlighted the importance of negative feedback loops that

normally operate to dampen various types of signaling, and therefore ensure homeostatic

regulation of signals in the cell. We have found that S6K exerts negative feedback regulation on

Akt3. This is consistent with the known S6K-mediated negative feedback on the PI3 Kinase-Akt

pathway (31). The selective feedback effects of S6K on Akt3 may reflect the collective pressure

in tumor cells to lower Akt3 levels as a way to counteract the inhibitory effects of active Akt3.

Improved understanding of the unique roles of Akt isoforms has led to the recent

development of Akt inhibitors that preferentially block Akt1 and Akt2, such as Akti-1/2 (40). We

have found that inhibition of S6K activity with LY2584702 was more effective in reducing the

growth of vascular tumors with loss of Akt3 than tumors with normal levels of Akt3. These

findings highlight the potential clinical utility of treating vascular tumors, such as angiosarcoma,

with agents that block S6K signaling. While our studies are selective for vascular tumors at this

time, the findings provide an impetus for further investigation of Akt isoforms in other tumor

types, which could potentially improve our ability to integrate molecular data with therapeutic

treatment regimens.




21

ACKNOWLEDGEMENTS

We wish to thank the following people for providing reagents and technical support:

Argiris Efstratiadis, Morris Birnbaum, Vera Krump-Konvalinkov, Rebecca Chin, Alex Toker,

William Hahn, Andrew Aplin, Milton Finegold, Cecilia Rosales, Bhuvaneswari Krishnan,

Ningning Zheng, Benjamin Hopkins, Rafael Rojano and Tareq Qdaisat.




22

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26

FIGURE LEGENDS

Figure 1. Akt phosphorylation in benign and malignant human vascular tumors. (A)

Immunohistochemical stains of vascular tumors for pAkt (S473). Scale bar, 100 μm. Insets

show staining in normal blood vessels adjacent to tumor. (B) pAkt reactivity in normal human

skin and vascular tumors. *P<0.01 vs. NL skin. (C) The stain intensity was scored using a 3-tier

system (see main text) with median values (red lines). *P<0.01 vs. NL skin. NL Skin, normal

skin; IH, infantile hemangioma; KHE, kaposiform hemangioendothelioma; KS, Kaposi’s

sarcoma; AS, angiosarcoma. (D) Western blots of human dermal microvascular endothelial

cells (HDMEC) and primary infantile hemangioma EC (HemeEC). Graph shows densitometric

quantitation of pAkt / total Akt ratios, normalized to HDMEC as control. *P<0.05, N=6. (E-F)

Western blots of (E) HDMEC and ASM.5 cells, and (F) normal mouse EC and EOMA cells.

Figure 2. Akt1 promotes vascular tumor cell growth and migration. (A) HemeEC and (B)

ASM.5 expressing pLKO or shAkt1 (independent clones #1 and #2) were analyzed by western

blot. (C-D) HemeEC and ASM.5 DNA content, expressed as fluorescence units relative to

“Day=0”. *P<0.05, N=3. (E-F) Transwell migration assays of (E) hemeEC and (F) ASM.5 cells

expressing shAkt1 ± VEGF-A (50 ng/ml) for 5 hours normalized to “pLKO-VEGF” control.

*P<0.05 vs. pLKO-VEGF; **P<0.05 vs. pLKO+VEGF, N=3. (G) Representative bright field

images of HemeEC cord formation on Collagen I matrix ± VEGF (50 ng/ml) for 14 hours. (H)

Quantitation of cord length. *P<0.01 vs. pLKO-VEGF; **P<0.01 vs. pLKO+VEGF, N=3.

Figure 3. Endothelial myrAkt1 activation drives to hemangioma formation in vivo. (A)

Schematic of myrAkt1 skin graft model of hemangioma (see text for details). (B) Vascular

tumors developed in the grafts 4 weeks following myrAkt1 induction. (C) Microscopic features

of the tumor (scale bar, 100 μm). Arrows indicate tumor boundary. Graph of tumor volume in

myrAkt1 mice is shown. (D) Immunofluorescence stains of myrAkt1 tumor for CD31 (red) and




27

HA-tagged myrAkt1 (green); smooth muscle actin (SMA, red) and phospho-Akt (green); glucose

transporter-1 (Glut-1, red) and CD31 (green); nuclei (blue). (E-F) myrAkt1-induced

hemangiomas regressed when myrAkt1 was turn off for 3.5 weeks. Images are representative

of 6 tumors per group. Red arrows, blood vessels; black arrows, fibrofatty tissue. Scale bar,

0.5 cm.

Figure 4. Akt1, Akt2 and Akt3 expression in human vascular tumors. (A) Immunostains of

infantile hemangioma and angiosarcoma tissues for Akt isoforms. Insets show staining in

normal blood vessels adjacent to tumor. (B) Plots of semi-quantitative analysis of Akt1, Akt2

and Akt3 immunostains in normal human skin and vascular tumors. The stain reactivity is the

percentage of tissues with positive staining. The stain intensity was scored using a 3-tier

system (see main text) with median values (red lines). NL Skin, normal skin; IH, infantile

hemangioma; AS, angiosarcoma. *P<0.05 vs. NL Skin. (C) Akt isoforms were

immunoprecipitated from ASM.5 cell lysates and immunoblotted.

Figure 5. Differential effects of Akt isoforms in vascular tumor cells. (A) Spheroids of hemeEC

expressing pLKO, shAkt1 or shAkt3 were cultured in Matrigel/Collagen I matrix ± VEGF (50

ng/ml) for 24 hours. Arrowheads indicate endothelial sprouts. (B) Total sprout length per

spheroid was quantified and normalized to “pLKO -VEGF” control. *P<0.01 vs. pLKO -VEGF;

**P<0.01 vs. pLKO+VEGF; N = 3, independent shAkt clones #1 and #2 for each Akt isoform.

(C) ASM.5 and (D) EOMA cells with Akt1 or Akt3 knockdown were assessed for cell migration in

scratch wound assays. The area of wound closure was quantified and shown as percent

closure relative to t=0 hour. *P<0.05, N= 9. (E) EOMA cells expressing pLKO, shAkt1 or shAkt3

were assessed for cell growth (fluorescence units of DNA content). *P<0.01, N=6. (F) EOMA

cells with stable Akt1, Akt2 or Akt3 knockdown were implanted in mice and monitored for tumor

growth. *P<0.05, N = 8.




28

Figure 6. Akt1 and Akt3 exert opposing effects on S6K pathway. (A) HemeEC and EOMA cells

expressing pLKO, shAkt1 or shAkt3 were immunoblotted. Lung microvascular EC from wild

type (WT) and Akt3-/- mice were similarly analyzed. (B) EOMA cells expressing pLKO, shAkt1,

shAkt1+S6K R3A, shAkt3, or shAkt3+shS6K were assessed for cell migration by scratch wound

assay. Bright-field images of scratch wounds are shown. (C) EOMA cell migration was

assessed in Boyden chamber transwell assay. *P<0.01 vs. pLKO; **P<0.01 vs. shAkt1;

***p<0.01 vs. shAkt3, N=4. (D-E) ASM.5 cells expressing shAkt3 or shAkt3+shS6K were

assessed for (D) cell migration in transwell assay, and (E) cell growth (relative to “Day=0”).

*P<0.05 vs. pLKO; **P<0.05 vs. shAkt3, N=3. (F) Chemical structure of LY2584702. EOMA

cells were treated ± LY2584702 for 4 hours and then immunoblotted. Cells in 0.1% FBS and

10% FBS served as controls. (G) EOMA tumors grown in mice treated ± Rapamycin (2

mg/kg/day, i.p. injections) or LY2584702 (12.5 mg/kg twice daily, oral dosing) for 14 days and

immunoblotted. Two tumor samples (#1 and #2) per treatment condition were analyzed. (H)

Mice with EOMA tumors expressing pLKO or shAkt3 were treated ± LY2584702. *P<0.05 vs.

pLKO; **P<0.05 vs. shAkt3+Vehicle, N=6.

Figure 7. Akt3 regulates Rictor levels and is under negative feedback regulation by S6K. (A-B)

EOMA and ASM.5 cells expressing pLKO, shAkt1 or shAkt3 were immunoblotted. (C) qPCR for

Rictor mRNA in EOMA cells with Akt1 or Akt3 knockdown, calculated relative to pLKO.

*P<0.01, N=4. (D) EOMA cells expressing pLKO or shRictor were immunoblotted to assess for

Akt/S6K pathway activation. (E) EOMA cells expressing HA-myrAkt3 or HA-myrAkt3 + shRictor

were immunoblotted. (F) Immunoblots of EOMA cells expressing pLKO or shS6K. (G) EOMA

cells were immunoprecipitated for Akt1 and Akt3, and blotted for these isoforms. (H)

Immunoblots of cells expressing pLKO or constitutively active S6K.




Figure 1

Infantile

Hemangioma Kaposiform

Hemangioendothelioma Kaposi’s Sarcoma Angiosarcoma A

Phosphorylated Akt

* * *

pAkt % Reactivity

*

Perc

en

tag

e

B * * * *

Sta

in S

co

re

C pAkt Intensity

pAkt

Total Akt

pVEGFR-2

Total VEGFR-2

PTEN

β-Actin

HDMEC HemeEC D

0.0

0.5

1.0

1.5

2.0

2.5

pA

kt / T

ota

l A

kt

(N

orm

aliz

ed to

HD

ME

C)

*

β-Actin

F Mouse

EC EOMA

pAkt

Total Akt

PTEN

pAkt

Total Akt

PTEN

β-Actin

HDMEC ASM.5 E




Figure 2

A

Akt1

Akt2

Akt3

β-Actin

HemeEC

Akt1

Akt2

Akt3

β-Actin

ASM.5 B

ASM.5

0.0

1.0

2.0

3.0

4.0

0 1 2 3 4

pLKO

shAkt1 #1

shAkt1 #2

Flu

ore

scence U

nits

(Re

lative

to

Da

y 0

)

* * *

*

Days

*

HemeEC C D

0.0

1.0

2.0

3.0

4.0

0 1 2 3 4

pLKO

shAkt1

Flu

ore

scence U

nits

(Re

lative

to

Da

y 0

)

* *

*

Days

G HemeEC Cord Formation

+V

EG

F

pLKO shAkt1 #1

-VE

GF

shAkt1 #2 E

F H




Figure 3

A B C

myrAkt1OFF 4 wks

TET:myrAkt1

VE-C dh i TTA

X

A

0.6

0.8

(cm

3)

B CmyrAkt1 OFF myrAkt1 ON

TUMORmyrAkt1

OFF2 k

Nu/Nu Recipients

OFF 4 wks

myrAkt1 ON 4 wks

Double Transgenic Donor

Cadherin:TTA

0.0

0.2

0.4

Tum

or V

olum

e 2 wks

myrAkt1OFF2 wks

RecipientsTransgenic Donor

myrAkt1 ONmyrAkt1 ON for another 3 5 wks Tumor H&E

ED

myrAkt1OFF

myrAkt1ON

myrAkt1 ON another 3.5 wks Tumor H&EDCD31 HA-Tag Nuclei SMA pAkt Nuclei Glut-1 CD31 Nuclei

myrAkt1 ONmyrAkt1 OFF for another 3.5 wks Tumor H&EF

on April 6, 2018. ©

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Figure 4

Infantile Hemangioma Angiosarcoma

Akt1A Akt1 % Reactivity

age

ore

Akt1 IntensityB

Infantile Hemangioma Angiosarcoma

NL Skin IH AS NL Skin IH AS

Akt2 % Reactivity Akt2 I t it

Perc

enta

Stai

n Sc

o

Akt2 Stai

n Sc

ore

Akt2 % Reactivity Akt2 Intensity

Perc

enta

ge

NL Skin IH AS

P

Akt3 Intensity

core

* *

Akt3 % Reactivity

age

*

NL Skin IH AS

Akt3 NL Skin IH AS NL Skin IH AS

Stai

n S * *

Perc

enta

pAkt (S473)

IP:

pAkt (T308)

C

Akt1

Akt2

Akt3

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Figure 5

1200 pLKO

E*

EOMA

6pLKOshAkt1 #1shAkt1 #2 **

BpLKO shAkt1 shAkt3

A HemeEC

600

800

1000

pLKOshAkt1shAkt3

cenc

e U

nits

*

*3

4

5shAkt3 #1shAkt3 #2

out L

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pLK

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*

*

pLKO shAkt1 shAkt3

-VEGF

0

200

400

Fluo

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***0

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2

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*

* **+ VEGF

0 2 4 6 8Days

-VEGF +VEGF

CpLKO shAkt1 shAkt3

t = 0h

ASM.5

0.5LKO

F EOMA Tumor Growth

DpLKO shAkt1 shAkt3

EOMA

t 0h

t = 16h0.3

0.4

0.5pLKOshAkt1 #1shAkt2 #1shAkt3 #1

me

(cm

3 )

* *

*

60%

80%

100%

nt C

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*

*

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0.2

* *

Tum

or V

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60%

80%

100%

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*

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20%

40%

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Per

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Per

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Figure 6

**CB pLKO shAkt1

shAkt1 + S6K R3A shAkt3

shAkt3 + shS6K

pAkt

HemeECA

20406080

100

*

****

Mig

rate

d C

ellst = 0 h

pS6

pS6K

Total S6K

Total Akt

F

t = 16 h0#

M

120D

Total S6

EOMA

F

020406080

100120

***

Mig

rate

d C

ells

EOMA Tumor GrowthHpLKO + VehiclepLKO + LY2584702

pAkt

Total Akt

pS6K

Total S6K

10%

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S

1600

800

400

200

100

β Actin

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LY2584702 (nM)0

pLKO shAkt3 shAkt3 + shS6K

# M

0 06

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e (c

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Total S6

Mouse ECβ-Actin

G#1 #2 #1 #2 #1 #2

Vehicle RAPA LY2584702Tumor: 0.02

0.04

0.06

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Figure 7

A EOMA

Akt1

Akt2

β-Actin

Raptor

Rictor

Akt3

mTOR

IP: Akt1 IP: Akt3

Akt1

Akt3

G

Akt3

Akt1

Total Akt

pS6

Total S6

H

Akt1

Akt2

β-Actin

Rictor

Akt3

Raptor

ASM.5 B C

0.0

0.4

0.8

1.2

Ric

tor

mR

NA

Fold

Chan

ge

(Rela

tive t

o p

LK

O)

*

D

E F

Total S6K

Akt3

Akt1

β-Actin

pAkt (T308)

pAkt (S473)

Total Akt




Published OnlineFirst November 11, 2014.Cancer Res Thuy L Phung, Wa Du, Qi Xue, et al. tumor growthAkt1 and Akt3 exert opposing roles in the regulation of vascular

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