1. Volume 29, Issue 4, Pages 523-535 (April 2016)
Spontaneous Hepatocellular Carcinoma after the Combined Deletion of Akt Isoforms 
Qi Wang, Wan-Ni Yu, Xinyu Chen, Xiao-ding Peng, Sang-Min Jeon, Morris J. Birnbaum, Grace Guzman, Nissim Hay 
Cancer Cell 
Volume 29, Issue 4, Pages (April 2016)
DOI: /j.ccell
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2. Cancer Cell 2016 29, 523-535DOI: (10.1016/j.ccell.2016.02.008)
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3. Figure 1
The Combined Deletion of Akt1 and Akt2 in Adult Mice Induces Rapid Mortality Preceded by Body Weight Loss and Hypoglycemia
(A) Breeding scheme to generate Akt1f/f;Akt2−/−;R26CreERT2 and Akt1f/f;Akt3−/−;R26CreERT2 mice.
(B) Kaplan-Meier plot showing the survival of 2-month-old Akt1f/f;Akt2−/−;R26CreERT2+/− (n = 20), Akt1f/f;Akt2−/−;R26CreERT2+/+ (n = 10), and Akt1f/f;Akt3-/;-R26CreERT2+/+ (n = 10) mice after tamoxifen injection for 5 consecutive days.
(C) Blood glucose levels in fed Akt1f/f;Akt2−/−, Akt1f/f;Akt2−/−R26CreERT2+/−, and Akt1f/f;Akt2−/−;R26CreERT2+/+ mice before and after tamoxifen injection (n = 6, ±SEM, p < 0.05 versus before tamoxifen).
(D) Body weights of Akt1f/f;Akt2−/−, Akt1f/f;Akt2−/−;R26CreERT2+/−, and Akt1f/f;Akt2−/−R26CreERT2+/+ mice after tamoxifen injection (n = 6, ±SEM, p < 0.05 versus Akt1f/f;Akt2−/− mice).
(E) Kaplan-Meier plot showing the survival of 2-month-old Akt1f/fAkt2−/−R26CreERT2+/+ mice after tamoxifen injection (n = 11).
(F) Glucose levels in Akt1f/f;Akt2−/−;R26CreERT2+/+ mice before and after the start of tamoxifen injection (n = 4, ±SEM, p < 0.05 tamoxifen versus corn oil).
(G) Serum insulin levels in Akt1f/f;Akt2−/−;R26CreERT2+/+ mice before and after the start of tamoxifen injection and before death (n = 4, ±SEM, p < 0.03 tamoxifen versus corn oil).
(H) Serum IL-6 levels in tamoxifen-injected Akt1f/f;Akt2−/−;R26CreERT2+/+ mice (n = 4, ±SEM, p < 0.03 tamoxifen versus corn oil).
See also Figure S1.
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4. Figure 2
Hepatic Deletion of Akt1 in Akt2−/− Mice Inhibits Growth Hormone Receptor Expression, STAT5 Activation, and IGF1 Expression, and Induces Early Onset of HCC
(A) qRT-PCR showing hepatic Igf1 mRNA levels in Akt2−/− and Akt1hep−/−;Akt2−/− mice (n = 4, ±SEM, p = ).
(B) Hepatic STAT5 phosphorylation on Tyr694 in Akt2−/− or Akt1hep−/−;Akt2−/− mice.
(C) Level of growth hormone receptor mRNA in Akt2−/− and Akt1hep−/−;Akt2−/− mouse livers (n = 4, ±SEM, ∗p = 0.013).
(D) Model illustrating the role of Akt in the GH/IGF1 axis (see text for details).
(E) Representative images of the livers of 6-month-old male mice of the indicated genotype.
(F) Quantification of the number of macroscopic tumors per liver in 6- to 7-month-old male mice (n = 10, ±SEM, ∗∗p < Akt1hep−/−;Akt2−/− mice versus Akt2−/−).
(G) Liver sections showing tumor lesions in 4- and 7-month-old Akt1hep−/−Akt2−/− mice (upper panel, low magnification; bottom panel, high magnification). The tumor areas are marked.
See also Figures S2 and S3.
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5. Figure 3
Hepatic Deletion of Akt1 in Akt2−/− Mice Induces Liver Injury, High Serum Levels of IL-6, Inflammation, Cell Death, and Cell Proliferation
(A) Serum AST levels in 3- and 6-month-old male mice with the indicated genotypes (n = 4–6, ±SEM, p < 0.05 Akt1hep−/−;Akt2−/− versus Akt1hep+/-;Akt2−/− or Akt2−/− mice).
(B) Serum ALT levels in 3- and 6-month-old male mice with the indicated genotypes (n = 4–6, ±SEM, p < 0.05 Akt1hep−/−;Akt2−/− versus Akt1hep+/-;Akt2−/− or Akt2−/− mice).
(C) Serum IL-6 levels in 4- to 6-month-old Akt1hep−/−;Akt2−/− and Akt2−/− male mice (n = 6, ±SEM, p < 0.05 Akt1hep−/−;Akt2−/− versus Akt2−/− mice).
(D) TNF-α mRNA levels as measured by qRT-PCR in the livers of 4- to 6-month-old Akt1hep−/−;Akt2−/− and Akt2−/− male mice (n = 4, ±SEM, p = 0.045).
(E) Representative images of H&E and F4/80 staining in liver sections from 3- to 4-month-old Akt1hep−/−;Akt2−/− and Akt2−/− mice (N, non-tumor area; T, tumor area). Scale bar, 100 μm.
(F) Images of H&E and F4/80 staining of liver sections from 7- to 8-month-old Akt1hep−/−;Akt2−/− and Akt2−/− mice. Scale bar, 100 μm. Bar graph shows the quantification of F4/80 staining (n = 4, ±SEM, ∗p = 0.024).
(G) Images of liver sections from 7-to 8-month-old Akt1hep−/−;Akt2−/− and Akt2−/− male mice showing immunohistochemistry with either anti-Ki67 or anti-cleaved caspase 3 (CC3). Scale bar, 100 μm. Bar graphs shows quantifications of the results (n = 4, ±SEM; ∗p = ∗∗p = ).
See also Figure S4.
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6. Figure 4
Liver Injury and Inflammation Induced by Hepatic Deletion of Akt1 and Akt2 Are FoxO1-dependent
(A) The mRNA level of known FoxO target genes in the livers of Akt1hep−/−;Akt2−/− compared with Akt2−/− mice was measured by qRT-PCR (n = 4 ±SEM, ∗p < 0.05, ∗∗p < 0.01).
(B) Fasl and Bcl2l11 mRNAs in the livers of Akt1hep−/−;Akt2−/− and Akt2−/− mice were measured by qRT-PCR (n = 4, ±SEM, ∗p < 0.05).
(C) Schematic depicting systemic injection of AAV-Tbg-Cre or control AAV-Tbg-GFP for the hepatic deletions of either both Akt1 and Akt2 or Akt1, Akt2, and Foxo1 in Akt1f/f;Akt2f/f and Akt1f/f;Akt2f/f;Foxo1f/f mice, respectively.
(D) Serum ALT levels after AAV-Tbg-Cre or AAV-Tbg-GFP injection (n = 4, ±SEM, ∗p < 0.05).
(E) Representative liver section images immunostained with anti-F4/80 after deletion of either both Akt1 and Akt2 or Akt1, Akt2, and FoxO1. Scale bar, 100 μm.
(F) Quantification of F4/80 immunostaining (n = 4, ±SEM, ∗∗p < 0.005, GFP and Akt1f/f;Akt2f/f versus Akt1f/f;Akt2f/f;Foxo1f/f mice).
See also Figures S5 and S6.
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7. Figure 5
Phospho-STAT3, and IL-6 Expression in Akt1hep−/−;Akt2−/− Liver
(A) Immunoblot showing the level of STAT3 phosphorylation in the livers of 6-month-old male mice with the indicated genotypes (NT, non-tumor area; T, tumor area).
(B) Representative liver tumor section showing immunohistochemistry with Akt1 antibodies.
(C) Immunoblot showing the expression and phosphorylation of Akt1, Akt2, and STAT3 in isolated hepatocytes derived from Akt1f/f;Akt2−/− liver and isolated hepatocytes derived from tumors in Akt1hep−/−;Akt2−/− liver.
(D) PCR genotyping showing the floxed and deleted Akt1 in hepatocytes isolated from Akt1f/f;Akt2−/− liver and from tumors in Akt1hep−/−;Akt2−/− liver. NS, non-specific band.
(E) Representative images of IHC for IL-6 of Akt1hep−/−;Akt2−/− liver sections from different liver samples. Scale bar, 100 μm.
(F) A representative image showing positively stained IL-6 cells and the location of the tumor (marked with a dashed line). Scale bar, 500 μm. Inset shows a higher magnification of the IL-6-positive cells. Scale bar, 100 μm.
See also Figure S7.
Cancer Cell  , DOI: ( /j.ccell )
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8. Figure 6
Characterization of Akt1hep−/−;Akt2−/− Liver-Bearing Tumors by Gene Expression
(A) Expression of genes that are frequently induced in mouse and human HCC in Akt1hep−/−;Akt2−/− livers versus Akt2−/− livers of 6-month old male mice was measured by qRT-PCR (n = 4, ±SEM, ∗p ≤ 0.05, ∗∗p < 0.005).
(B) The expression of Fosl2 and associated target genes in the liver of 6-month old male Akt1hep−/−;Akt2−/− and Akt2−/− mice was determined by qRT-PCR (n = 4, ±SEM, ∗p < 0.05).
(C) Expression of Igf2bp1 mRNA in Akt1hep−/−;Akt2−/− liver compared with Akt2−/− liver (n = 3, ±SEM, ∗p = 0.052).
(D) Expression of Igf2bp3 mRNA in Akt1hep−/−;Akt2−/− liver compared with Akt2−/− liver (n = 4, ±SEM, ∗∗p < 0.005).
(E) Expression of IGF2BP3 protein in Akt1hep−/−;Akt2−/− livers compared with Akt2−/− livers.
See also Figure S8.
Cancer Cell  , DOI: ( /j.ccell )
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9. Figure 7
DEN-Induced Hepatocarcinogenesis and Lung Metastasis in Akt1−/− and Akt2−/− Mice
(A) The incidence of macroscopic tumors ≥1 cm in diameter in the livers of Akt1−/−, Akt2−/−, and WT mice, 12 months after exposure to DEN.
(B) The incidence of macroscopic metastatic tumors in the lungs of Akt1−/−, Akt2−/−, and WT mice 12 months after exposure to DEN (results are presented as the mean ± SEM).
(C) Images depicting lungs with macroscopic lung tumors indicated by arrows. Scale bar, 10 mm.
(D) The number of microscopic metastatic tumors in the lung of Akt2−/−, and WT mice, 12 months after exposure to DEN. Data are presented as the mean ± SEM.
(E) H&E stained lung sections from WT, Akt1−/−, and Akt2−/− mice. Tumor is marked in the Akt2−/− lung section (Left panel-scale bar 500μM; Right panel-scale bar 100μM).
(F) RT-PCR analysis of Afp expression in tumors developed in the lung of Akt2−/− mice.
(G) Representative images of livers bearing tumors and corresponding lungs (tumors are indicated by arrowheads) from DEN treated Akt2−/− and WT mice (scale bars, 10 mm). Total number of macroscopic liver tumors and number of tumors ≥1 cm in diameter are shown in parentheses. Fed glucose and insulin levels in the corresponding mice are shown adjacent to the images.
(H) Immunoblot showing p-Akt1 in tumors derived from either WT or Akt2−/− livers.
(I) Immunoblot showing p-ERK in tumors derived from either WT or Akt2−/− livers. Upper panel, total Akt (t-Akt); middle panel, p-ERK; bottom panel, total ERK (t-ERK).
Cancer Cell  , DOI: ( /j.ccell )
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10. Figure 8
Illustration Depicting HCC Development and Progression in Akt1hep−/−;Akt2−/− and Akt2−/− Mice
(A) The deletion of hepatic Akt1 and Akt2 induces liver damage and apoptosis, which is largely dependent on the activation of FoxO. FoxO induces inflammation and IL-6 and TNF-α production by macrophages and plasma cells. IL-6 induces compensatory proliferation by activating STAT3 in the survived hepatocytes. Eventually, these cells give rise to HCC with gene signatures similar to human HCC. Autocrine production of IL-6 by hepatocytes was also observed. The high level of insulin in the mice may facilitate HCC progression.
(B) Exposure of Akt2−/− mice that display a high level of insulin to DEN-induced hepatocarcinogenesis induces a high incidence of metastasis to the lung.
Cancer Cell  , DOI: ( /j.ccell )
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