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SKP2 cooperates with N-Ras or AKT to induce liver tumor development in mice.

Delogu S, Wang C, Cigliano A, Utpatel K, Sini M, Longerich T, Waldburger N, Breuhahn K, Jiang L, Ribback S, Dombrowski F, Evert M, Chen X, Calvisi DF - Oncotarget (2015)

Bottom Line: We found that forced overexpression of SKP2, N-RasV12 or ΔN90-β-catenin alone as well as co-expression of SKP2 and ΔN90-β-catenin did not induce liver tumor development.Noticeably, the tumor suppressor proteins whose levels have been shown to be downregulated by SKP2-dependent degradation in various tumor types, including p27, p57, Dusp1, and Rassf1A were not decreased in liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice.In human HCC specimens, nuclear translocation of SKP2 was associated with activation of the AKT/mTOR and Ras/MAPK pathways, but not with β-catenin mutation or activation.

View Article: PubMed Central - PubMed

Affiliation: Institut für Pathologie, Universitätsmedizin Greifswald, Greifswald, Germany.

ABSTRACT
Mounting evidence indicates that S-Phase Kinase-Associated Protein 2 (SKP2) is overexpressed in human hepatocellular carcinoma (HCC). However, the role of SKP2 in hepatocarcinogenesis remains poorly delineated. To elucidate the function(s) of SKP2 in HCC, we stably overexpressed the SKP2 gene in the mouse liver, either alone or in combination with activated forms of N-Ras (N-RasV12), AKT1 (myr-AKT1), or β-catenin (ΔN90-β-catenin) protooncogenes, via hydrodynamic gene delivery. We found that forced overexpression of SKP2, N-RasV12 or ΔN90-β-catenin alone as well as co-expression of SKP2 and ΔN90-β-catenin did not induce liver tumor development. Overexpression of myr-AKT1 alone led to liver tumor development after long latency. In contrast, co-expression of SKP2 with N-RasV12 or myr-AKT1 resulted in early development of multiple hepatocellular tumors in all SKP2/N-RasV12 and SKP2/myr-AKT1 mice. At the molecular level, preneoplastic and neoplastic liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice exhibited a strong induction of AKT/mTOR and Ras/MAPK pathways. Noticeably, the tumor suppressor proteins whose levels have been shown to be downregulated by SKP2-dependent degradation in various tumor types, including p27, p57, Dusp1, and Rassf1A were not decreased in liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice. In human HCC specimens, nuclear translocation of SKP2 was associated with activation of the AKT/mTOR and Ras/MAPK pathways, but not with β-catenin mutation or activation. Altogether, the present data indicate that SKP2 cooperates with N-Ras and AKT proto-oncogenes to promote hepatocarcinogenesis in vivo.

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Tumors from SKP2/N-RasV12 mice exhibit strong upregulation of AKT inhibitors (Pten, Phlpp1, and Phlpp2) and putative SKP2 targets (p27, p57, Dusp1, and Rassf1A)Sections of a small hepatocellular tumor already depicted in Fig. 3 are shown as an example in the present figure. Original magnification: 200X in all pictures. Abbreviation: HE, hematoxylin and eosin staining.
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Figure 4: Tumors from SKP2/N-RasV12 mice exhibit strong upregulation of AKT inhibitors (Pten, Phlpp1, and Phlpp2) and putative SKP2 targets (p27, p57, Dusp1, and Rassf1A)Sections of a small hepatocellular tumor already depicted in Fig. 3 are shown as an example in the present figure. Original magnification: 200X in all pictures. Abbreviation: HE, hematoxylin and eosin staining.

Mentions: To elucidate the molecular mechanisms underlying liver tumor development in SKP2/N-RasV12 mice, we assayed the Ras/MAPK and AKT/mTOR signaling cascades, which have been shown to be prominently activated in human HCC, by immunohistochemistry (Fig. 3). Noticeably, we found that both Ras/MAPK (as indicated by immunoreactivity of activated/phosphorylated ERK1/2) and AKT/mTOR cascades were strongly induced in liver lesions from SKP2/N-RasV12 mice (Fig. 3). In particular, as a consequence of AKT pathway activation, a strong induction of the downstream effectors of this cascade in SKP2/N-RasV12 mice, including proteins involved in glycolysis (hexokinase II, lactate dehydrogenase A/C), de novo fatty acid synthesis (fatty acid synthase, stearoyl-coA-desaturase 1, sterol regulatory element binding protein 1), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase), and protein translation (phosphorylated/activated ribosomal protein S6 and phosphorylated/inactivated 4E-binding protein 1) was detected, implying an important role of these signaling pathways in SKP2/Ras-driven hepatocarcinogenesis (Fig. 3). Subsequently, since AKT phosphorylation is negatively regulated by the phosphatase and tensin homolog (Pten) as well as the PH domain and leucine rich repeat protein phosphatase 1 and 2 (Phlpp1 and 2) tumor suppressors [27], we determined the levels of these proteins by immunohistochemistry. Interestingly, we found that Pten, Phlpp1, and Phlpp2 were not downregulated, but rather induced, in lesions from SKP2/N-RasV12 mice when compared with the other mouse groups (Fig. 4). These data suggest that activation/phosphorylation of AKT is not a consequence of downregulation of Pten, Phlpp1, and Phlpp2 proteins in SKP2/N-RasV12 mice. Western blot analysis of Ras/MAPK and AKT/mTOR pathways as well as AKT inhibitors revealed a similar pattern to that detected by immunohistochemistry, although the differences were less remarkable presumably due to the “dilution” effect of unaffected liver in protein lysates from SKP2/N-RasV12 mice (Supplementary Fig. 2). Another recognized way whereby SKP2 might induce activation of AKT is through AKT ubiquitination [28]. However, we did not find a significant increase of Akt ubiquitinylation in SKP2/N-RasV12 livers when compared with the other mouse groups (Supplementary Fig. 2), indicating that the latter mechanism is not responsible for increased AKT activity in SKP2/N-RasV12 mice.


SKP2 cooperates with N-Ras or AKT to induce liver tumor development in mice.

Delogu S, Wang C, Cigliano A, Utpatel K, Sini M, Longerich T, Waldburger N, Breuhahn K, Jiang L, Ribback S, Dombrowski F, Evert M, Chen X, Calvisi DF - Oncotarget (2015)

Tumors from SKP2/N-RasV12 mice exhibit strong upregulation of AKT inhibitors (Pten, Phlpp1, and Phlpp2) and putative SKP2 targets (p27, p57, Dusp1, and Rassf1A)Sections of a small hepatocellular tumor already depicted in Fig. 3 are shown as an example in the present figure. Original magnification: 200X in all pictures. Abbreviation: HE, hematoxylin and eosin staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385847&req=5

Figure 4: Tumors from SKP2/N-RasV12 mice exhibit strong upregulation of AKT inhibitors (Pten, Phlpp1, and Phlpp2) and putative SKP2 targets (p27, p57, Dusp1, and Rassf1A)Sections of a small hepatocellular tumor already depicted in Fig. 3 are shown as an example in the present figure. Original magnification: 200X in all pictures. Abbreviation: HE, hematoxylin and eosin staining.
Mentions: To elucidate the molecular mechanisms underlying liver tumor development in SKP2/N-RasV12 mice, we assayed the Ras/MAPK and AKT/mTOR signaling cascades, which have been shown to be prominently activated in human HCC, by immunohistochemistry (Fig. 3). Noticeably, we found that both Ras/MAPK (as indicated by immunoreactivity of activated/phosphorylated ERK1/2) and AKT/mTOR cascades were strongly induced in liver lesions from SKP2/N-RasV12 mice (Fig. 3). In particular, as a consequence of AKT pathway activation, a strong induction of the downstream effectors of this cascade in SKP2/N-RasV12 mice, including proteins involved in glycolysis (hexokinase II, lactate dehydrogenase A/C), de novo fatty acid synthesis (fatty acid synthase, stearoyl-coA-desaturase 1, sterol regulatory element binding protein 1), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase), and protein translation (phosphorylated/activated ribosomal protein S6 and phosphorylated/inactivated 4E-binding protein 1) was detected, implying an important role of these signaling pathways in SKP2/Ras-driven hepatocarcinogenesis (Fig. 3). Subsequently, since AKT phosphorylation is negatively regulated by the phosphatase and tensin homolog (Pten) as well as the PH domain and leucine rich repeat protein phosphatase 1 and 2 (Phlpp1 and 2) tumor suppressors [27], we determined the levels of these proteins by immunohistochemistry. Interestingly, we found that Pten, Phlpp1, and Phlpp2 were not downregulated, but rather induced, in lesions from SKP2/N-RasV12 mice when compared with the other mouse groups (Fig. 4). These data suggest that activation/phosphorylation of AKT is not a consequence of downregulation of Pten, Phlpp1, and Phlpp2 proteins in SKP2/N-RasV12 mice. Western blot analysis of Ras/MAPK and AKT/mTOR pathways as well as AKT inhibitors revealed a similar pattern to that detected by immunohistochemistry, although the differences were less remarkable presumably due to the “dilution” effect of unaffected liver in protein lysates from SKP2/N-RasV12 mice (Supplementary Fig. 2). Another recognized way whereby SKP2 might induce activation of AKT is through AKT ubiquitination [28]. However, we did not find a significant increase of Akt ubiquitinylation in SKP2/N-RasV12 livers when compared with the other mouse groups (Supplementary Fig. 2), indicating that the latter mechanism is not responsible for increased AKT activity in SKP2/N-RasV12 mice.

Bottom Line: We found that forced overexpression of SKP2, N-RasV12 or ΔN90-β-catenin alone as well as co-expression of SKP2 and ΔN90-β-catenin did not induce liver tumor development.Noticeably, the tumor suppressor proteins whose levels have been shown to be downregulated by SKP2-dependent degradation in various tumor types, including p27, p57, Dusp1, and Rassf1A were not decreased in liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice.In human HCC specimens, nuclear translocation of SKP2 was associated with activation of the AKT/mTOR and Ras/MAPK pathways, but not with β-catenin mutation or activation.

View Article: PubMed Central - PubMed

Affiliation: Institut für Pathologie, Universitätsmedizin Greifswald, Greifswald, Germany.

ABSTRACT
Mounting evidence indicates that S-Phase Kinase-Associated Protein 2 (SKP2) is overexpressed in human hepatocellular carcinoma (HCC). However, the role of SKP2 in hepatocarcinogenesis remains poorly delineated. To elucidate the function(s) of SKP2 in HCC, we stably overexpressed the SKP2 gene in the mouse liver, either alone or in combination with activated forms of N-Ras (N-RasV12), AKT1 (myr-AKT1), or β-catenin (ΔN90-β-catenin) protooncogenes, via hydrodynamic gene delivery. We found that forced overexpression of SKP2, N-RasV12 or ΔN90-β-catenin alone as well as co-expression of SKP2 and ΔN90-β-catenin did not induce liver tumor development. Overexpression of myr-AKT1 alone led to liver tumor development after long latency. In contrast, co-expression of SKP2 with N-RasV12 or myr-AKT1 resulted in early development of multiple hepatocellular tumors in all SKP2/N-RasV12 and SKP2/myr-AKT1 mice. At the molecular level, preneoplastic and neoplastic liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice exhibited a strong induction of AKT/mTOR and Ras/MAPK pathways. Noticeably, the tumor suppressor proteins whose levels have been shown to be downregulated by SKP2-dependent degradation in various tumor types, including p27, p57, Dusp1, and Rassf1A were not decreased in liver lesions from SKP2/N-RasV12 and SKP2/myr-AKT1 mice. In human HCC specimens, nuclear translocation of SKP2 was associated with activation of the AKT/mTOR and Ras/MAPK pathways, but not with β-catenin mutation or activation. Altogether, the present data indicate that SKP2 cooperates with N-Ras and AKT proto-oncogenes to promote hepatocarcinogenesis in vivo.

Show MeSH
Related in: MedlinePlus