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. 2012 Mar;55(3):833-45.
doi: 10.1002/hep.24736. Epub 2011 Dec 19.

AKT (v-akt murine thymoma viral oncogene homolog 1) and N-Ras (neuroblastoma ras viral oncogene homolog) coactivation in the mouse liver promotes rapid carcinogenesis by way of mTOR (mammalian _target of rapamycin complex 1), FOXM1 (forkhead box M1)/SKP2, and c-Myc pathways

Coral Ho  1 , Chunmei Wang, Sandra Mattu, Giulia Destefanis, Sara Ladu, Salvatore Delogu, Julia Armbruster, Lingling Fan, Susie A Lee, Lijie Jiang, Frank Dombrowski, Matthias Evert, Xin Chen, Diego F Calvisi
Affiliations

AKT (v-akt murine thymoma viral oncogene homolog 1) and N-Ras (neuroblastoma ras viral oncogene homolog) coactivation in the mouse liver promotes rapid carcinogenesis by way of mTOR (mammalian _target of rapamycin complex 1), FOXM1 (forkhead box M1)/SKP2, and c-Myc pathways

Coral Ho et al. Hepatology. 2012 Mar.

Abstract

Activation of v-akt murine thymoma viral oncogene homolog (AKT) and Ras pathways is often implicated in carcinogenesis. However, the oncogenic cooperation between these two cascades in relationship to hepatocellular carcinoma (HCC) development remains undetermined. To investigate this issue, we generated a mouse model characterized by combined overexpression of activated forms of AKT and neuroblastoma Ras viral oncogene homolog (N-Ras) protooncogenes in the liver by way of hydrodynamic gene transfer. The molecular mechanisms underlying crosstalk between AKT and N-Ras were assessed in the mouse model and further evaluated in human and murine HCC cell lines. We found that coexpression of AKT and N-Ras resulted in a dramatic acceleration of liver tumor development when compared with mice overexpressing AKT alone, whereas N-Ras alone did not lead to tumor formation. At the cellular level, concomitant up-regulation of AKT and N-Ras resulted in increased proliferation and microvascularization when compared with AKT-injected mice. Mechanistic studies suggested that accelerated hepatocarcinogenesis driven by AKT and N-Ras resulted from a strong activation of mammalian _target of rapamycin complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras mice, yet by way of mTORC1-independent mechanisms. The biological effects of coactivation of AKT and N-Ras were then recapitulated in vitro using HCC cell lines, which supports the functional significance of mTORC1, FOXM1/SKP2, and c-Myc signaling cascades in mediating AKT and N-Ras-induced liver tumor development.

Conclusion: Our data demonstrate the in vivo crosstalk between the AKT and Ras pathways in promoting liver tumor development, and the pivotal role of mTORC1-dependent and independent pathways in mediating AKT and Ras induced hepatocarcinogenesis.

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Figures

Figure 1
Figure 1
Co-expression of myristylated AKT1 (myr-AKT) and mutated N-Ras (N-RasV12) induces mouse liver tumor development. (A) Phenotype of the wild-type (WT) and AKT/Ras mouse (4 weeks post injection). (B) Survival curve of AKT only, Ras only, and AKT combined with Ras injected mice. (C) Liver weight of wild-type and AKT/Ras mice at different time points (1 to 6 weeks post injection). (D) Gross images of wild-type (WT) liver and AKT/Ras mice livers 4 or 6 weeks post injection (w.p.i).
Figure 2
Figure 2
Stepwise hepatocarcinogenesis in AKT/Ras mice. Three weeks after injection already about 60% of the liver tissue was occupied by lesions (A). The latter were mostly consisting of preneoplastic hepatocytes, that began to emerge few days after injection (higher magnification in A with arrow on mitosis; one week after injection), and were characterized by a clear cytoplasm due to an increase in fat and glycogen-storing (glycogen staining in the PAS reaction; inset). At this stage, the first small hepatocellular adenomas originated from the clear cell foci (arrows in B; higher magnification in C). (D) Four weeks after injection, many of these tumors have grown to a size of several millimeters in diameter, replacing much of the former preneoplastic and normal liver tissue. (E) Five weeks after injection, many tumors further progressed to frankly malignant neoplasms that have replaced most of the normal liver tissue. (F) Typical example of a hepatocellular carcinoma. Tumors often showed a mixed phenotype with dominating hepatocellular differentiation, trabecular growth intermingled with ductular/pseudoglandular areas and a lesser degree of fat storage than in preneoplasias. Several mitotic figures are indicated by arrows. All H&E staining, except for the inset (PAS reaction). The lower edge of the panel represents 0.3 mm in A, 0.5 mm in inset, 4.9 mm in B,D,E, 0.7 mm in C, and 0.3 mm in F.
Figure 3
Figure 3
Proliferation (A), apoptosis (B), and angiogenesis (C; microvessel density, MVD) indices in wild-type livers and preneoplastic and neoplastic livers developed in AKT and AKT/Ras mice. Ten samples per each group per each assay were analyzed. Each bar represents mean ± SD. Tukey-Kramer test: P < 0.0001 a, vs. wild-type liver; b, vs. AKT preneoplastic liver; c, vs. AKT tumors.
Figure 4
Figure 4
Levels of activation of N-Ras/MAPK, mTORC1, and mTORC2 pathways in wild-type, AKT, and AKT/Ras mice. (A) Representative immunoblotting of the Ras proteins and their major downstream effectors. Both total (t) and membranous (m; sign of activation) levels of N-Ras were determined. Levels of activated (Ras-GTP) N-Ras, H-Ras, and Ki-Ras were assessed. The abbreviation p-RAF-1(1) indicates phosphorylation/activation of RAF-1 at serine 338 by Ras. (B) Representative immunoblotting of AKT, mTOR and their downstream effectors in the mTORC1 (p-RPS6, p-4E-BP1, HIF-1α, VEGF-α, MCL-1, FASN, ACAC, SCD1) and mTORC2 (LIPIN1, SGK1) complexes. (C) Assessment of TSC2 regulators by immunoblotting. Six to nine samples per each group per each assay were analyzed. Expression patterns for the same proteins did not show significant difference between wild-type and N-Ras injected livers (not shown). Abbreviations: IB, immunoblotting; IP, immunoprecipitation; WT, wild-type; P, preneoplasia; T, tumor.
Figure 5
Figure 5
(A) Appearance of a subset of small cells (thin arrows) within clear-cell preneoplastic lesions (thick arrow) surrounded by normal liver tissue (asterisk) exhibiting strong phosphorylation/activation of ERK proteins (p-ERK1/2) during AKT/Ras neoplastic transformation. These cells are characterized by a high proliferation rate (inset). Maintenance of p-ERK1/2 overexpression in a resulting small tumor (B) and in subsequent, large hepatocellular carcinomas (C). (D,E) Immunohistochemical staining for phosphorylated/activated mTOR (p-mTOR) and its _target phosphorylated/activated RPS6 (p-RPS6) in serial sections, showing a HCC (left part), preneoplasias (arrows in D) and surrounding normal liver tissue (marked in D with an asterisk). (F) Overexpression of the lipogenic enzyme ACAC in the same mouse sample: HCC (left), preneoplastic cells (arrows) and normal tissue (asterisk). Note the strongest staining for p-mTOR and p-RPS6 in HCC and in preneoplastic liver for ACAC, respectively. The lower edge of the panel represents 0.4 mm in A, 0.3 mm in B, 2.5 mm in C, 0.5 mm in D-F.
Figure 6
Figure 6
(A-C) Effect of suppressing AKT and N-Ras, either alone or in combination, via siRNA on cell viability (A), apoptosis (B), and angiogenesis (C; assessed by VEGF-α secretion in the medium) in the AKT/Ras cell line. Equivalent results were obtained in SNU-389 cells (not shown). (D-F) Effect of overexpressing AKT and N-Ras, either alone or in combination, via transient transfection on cell viability (D), apoptosis (E), and angiogenesis (F; assessed by VEGF-α secretion in the medium) in the human HLF HCC cell line. Cells were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum, serum-starved for 24 h, and then treated with either specific siRNAs or respective cDNAs. Results at 24 and 48 hours are shown. Each bar represents mean ± SD of 3 independent experiments conducted in triplicate. Results from untreated and scramble treated cells did not differ significantly and are, thus, merged. Tukey-Kramer test: P < 0.0001 a, vs. control (untreated cells); b, vs. N-Ras siRNA/cDNA; c, vs. AKT siRNA/cDNA.
Figure 7
Figure 7
Activation of FOXM1 and c-Myc pathways in AKT/Ras tumors. (A) Levels of FOXM1 and its _targets in wild-type, AKT, and AKT/Ras mice as assessed by immunoblotting. (B) Representative immunoblotting of the proteins _targeted to SKP2-dependent ubiquitination following phosphorylation at specific sites. (C) Levels of c-Myc and its _targets as assessed by immunoblotting. Expression patterns for the same proteins did not show significant difference between wild-type and N-Ras injected livers (not shown). Abbreviations: WT, wild-type; P, preneoplasia; T, tumor. (D) Effect of suppressing N-Ras and AKT, either alone or in combination, via siRNA on the levels of FOXM1 and c-Myc pathways in the AKT/Ras cell line. Equivalent results were obtained in SNU-389 cells (not shown). (E) Effect of overexpressing Ras and AKT, either alone or in combination, via transient transfection on the levels of FOXM1 and c-Myc pathways in the human HLF HCC cell line. Cells were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum, serum-starved for 24 h, and then transfected with either specific siRNAs or respective cDNAs. Results at 48 hours are shown.
Figure 8
Figure 8
(A) Effect of suppressing Raptor via specific siRNA on the levels of mTORC1 (p-RPS6, HIF-1α), FOXM1 (FOXM1, SKP2, CKS1), and c-Myc (c-Myc, MAD1) _targets in the AKT/Ras cell line. (B) Effect of co-expressing myristylated AKT (myr-AKT) and mutated N-Ras (N-RasV12) either alone or in combination with Raptor silencing on the levels of mTORC1 (p-RPS6, HIF-1α), FOXM1 (FOXM1, SKP2, CKS1), and c-Myc (c-Myc, MAD1) _targets in the HLF cell line. (C) Effect of suppressing c-Myc and FOXM1 genes either alone or together with Raptor silencing via specific siRNAs in the AKT/Ras cell line. Cells were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum, serum-starved for 24 h, and then treated with either specific siRNAs or respective cDNAs. Results at 24 and 48 hours are shown. Each bar represents mean ± SD of 3 independent experiments conducted in triplicate. Results from untreated and scramble treated cells did not differ significantly and are, thus, merged. Tukey-Kramer test: P < 0.0001 a, vs. control (untreated cells); b, vs. c-Myc siRNA; c, vs. FOXM1-siRNA; d, vs. Raptor siRNA.
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