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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.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.

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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Liver tumor development in SKP2/N-RasV12 mice by hydrodynamic gene delivery(A) Macroscopic and microscopic (B,C) appearance of SKP2-injected livers showing the absence of any gross or histological alterations 40 weeks post hydrodynamic injection. Scattered hepatocytes positive for nuclear HA-tagged SKP2 immunolabeling were present in the liver parenchyma of SKP2 injected mice (B, inset). (D-I) In striking contrast, concomitant overexpression of SKP2 and N-RasV12 resulted in the development of multiple tumors (E-I) in SKP2/N-RasV12 mice. In particular, small tumors (E,F) and large tumors (G-I) were constituted of small cells that were either glycogen-poor (F, as indicated by the PAS staining) or glycogen–rich (H, left part of the picture, demarcated by a dotted line). Tumors were classified as either hepatocellular adenomas (HCA; E,G) or carcinomas (HCC; I). Original magnifications: 40X in G; 100X in B; 200X in inset, E, and I; 400X in C, F, and H.
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Figure 1: Liver tumor development in SKP2/N-RasV12 mice by hydrodynamic gene delivery(A) Macroscopic and microscopic (B,C) appearance of SKP2-injected livers showing the absence of any gross or histological alterations 40 weeks post hydrodynamic injection. Scattered hepatocytes positive for nuclear HA-tagged SKP2 immunolabeling were present in the liver parenchyma of SKP2 injected mice (B, inset). (D-I) In striking contrast, concomitant overexpression of SKP2 and N-RasV12 resulted in the development of multiple tumors (E-I) in SKP2/N-RasV12 mice. In particular, small tumors (E,F) and large tumors (G-I) were constituted of small cells that were either glycogen-poor (F, as indicated by the PAS staining) or glycogen–rich (H, left part of the picture, demarcated by a dotted line). Tumors were classified as either hepatocellular adenomas (HCA; E,G) or carcinomas (HCC; I). Original magnifications: 40X in G; 100X in B; 200X in inset, E, and I; 400X in C, F, and H.

Mentions: To determine whether SKP2 contributes to hepatocarcinogenesis in vivo, we hydrodynamically delivered the pT3-EF1α-HA-SKP2 plasmid to the mouse liver either alone or in combination with an oncogenic form of human N-Ras (N-RasV12). Overexpression of SKP2 alone did not lead to any tumor formation or histological alteration in mice up to 40 weeks post injection (Fig. 1A-C). At this time point, scattered, single cells positive for HA-tagged SKP2 staining were detected in the liver parenchyma of SKP2 mice (Fig. 1B, inset). Similarly, overexpression of N-RasV12 alone did not result in any liver anomaly when harvested at the same time point (not shown). In striking contrast, co-expression of SKP2 and N-RasV12 genes triggered the development of multiple liver tumors by 20 weeks post injection in all injected mice (Fig. 1D-I). Tumors varied in size and were classified as HCA or HCC based on previously published criteria [24, 25] (Fig.1E-I; Supplementary Table 2). Tumors developed in SKP2/N-RasV12 mice were either composed of basophilic, glycogen-poor cells with small nuclei (Fig. 1E,F) or glycogen-rich cells (Fig. 1G,H). No lesions with cholangiocellular/ductular differentiation were detected in SKP2/N-RasV12 mice. Preneoplastic lesions developed in SKP2/N-RasV12 mice were morphologically equivalent to preneoplastic foci of altered hepatocytes that have been previously described in rat models of chemically-induced hepatocarcinogenesis [26]. The foci developed in these mice were exclusively basophilic, either pure or exhibiting the presence of a small component of clear cell hepatocytes (5-10%) located in the periphery of the focal lesion (Supplementary Fig. 1). Preneoplastic basophilic cells showed cytoplasmic basophilia and were smaller or, more rarely, bigger than normal hepatocytes. The basophilic phenotype was already evident in single hepatocytes or clusters of preneoplastic cells (Supplementary Fig. 1). To confirm that preneoplastic and neoplastic lesions were indeed induced by the ectopically injected oncogenes, we performed immunohistochemistry on SKP2/N-RasV12 lesions by using an anti-HA-tag and an anti-human N-Ras antibody (Fig. 2). As expected, preneoplastic lesions and tumors -but not the normal liver tissue-exhibited strong expression of HA-tag and human N-Ras proteins, implying that tumors originate from the transfected hepatocytes (Fig. 2C,D). Importantly, HA-tag immunoreactivity was localized in the nucleus of transfected cells (Fig. 2C, inset), in accordance with previous findings showing that SKP2 exerts its oncogenic potential at the nuclear level in human HCC [14, 15].


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)

Liver tumor development in SKP2/N-RasV12 mice by hydrodynamic gene delivery(A) Macroscopic and microscopic (B,C) appearance of SKP2-injected livers showing the absence of any gross or histological alterations 40 weeks post hydrodynamic injection. Scattered hepatocytes positive for nuclear HA-tagged SKP2 immunolabeling were present in the liver parenchyma of SKP2 injected mice (B, inset). (D-I) In striking contrast, concomitant overexpression of SKP2 and N-RasV12 resulted in the development of multiple tumors (E-I) in SKP2/N-RasV12 mice. In particular, small tumors (E,F) and large tumors (G-I) were constituted of small cells that were either glycogen-poor (F, as indicated by the PAS staining) or glycogen–rich (H, left part of the picture, demarcated by a dotted line). Tumors were classified as either hepatocellular adenomas (HCA; E,G) or carcinomas (HCC; I). Original magnifications: 40X in G; 100X in B; 200X in inset, E, and I; 400X in C, F, and H.
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Related In: Results  -  Collection

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Figure 1: Liver tumor development in SKP2/N-RasV12 mice by hydrodynamic gene delivery(A) Macroscopic and microscopic (B,C) appearance of SKP2-injected livers showing the absence of any gross or histological alterations 40 weeks post hydrodynamic injection. Scattered hepatocytes positive for nuclear HA-tagged SKP2 immunolabeling were present in the liver parenchyma of SKP2 injected mice (B, inset). (D-I) In striking contrast, concomitant overexpression of SKP2 and N-RasV12 resulted in the development of multiple tumors (E-I) in SKP2/N-RasV12 mice. In particular, small tumors (E,F) and large tumors (G-I) were constituted of small cells that were either glycogen-poor (F, as indicated by the PAS staining) or glycogen–rich (H, left part of the picture, demarcated by a dotted line). Tumors were classified as either hepatocellular adenomas (HCA; E,G) or carcinomas (HCC; I). Original magnifications: 40X in G; 100X in B; 200X in inset, E, and I; 400X in C, F, and H.
Mentions: To determine whether SKP2 contributes to hepatocarcinogenesis in vivo, we hydrodynamically delivered the pT3-EF1α-HA-SKP2 plasmid to the mouse liver either alone or in combination with an oncogenic form of human N-Ras (N-RasV12). Overexpression of SKP2 alone did not lead to any tumor formation or histological alteration in mice up to 40 weeks post injection (Fig. 1A-C). At this time point, scattered, single cells positive for HA-tagged SKP2 staining were detected in the liver parenchyma of SKP2 mice (Fig. 1B, inset). Similarly, overexpression of N-RasV12 alone did not result in any liver anomaly when harvested at the same time point (not shown). In striking contrast, co-expression of SKP2 and N-RasV12 genes triggered the development of multiple liver tumors by 20 weeks post injection in all injected mice (Fig. 1D-I). Tumors varied in size and were classified as HCA or HCC based on previously published criteria [24, 25] (Fig.1E-I; Supplementary Table 2). Tumors developed in SKP2/N-RasV12 mice were either composed of basophilic, glycogen-poor cells with small nuclei (Fig. 1E,F) or glycogen-rich cells (Fig. 1G,H). No lesions with cholangiocellular/ductular differentiation were detected in SKP2/N-RasV12 mice. Preneoplastic lesions developed in SKP2/N-RasV12 mice were morphologically equivalent to preneoplastic foci of altered hepatocytes that have been previously described in rat models of chemically-induced hepatocarcinogenesis [26]. The foci developed in these mice were exclusively basophilic, either pure or exhibiting the presence of a small component of clear cell hepatocytes (5-10%) located in the periphery of the focal lesion (Supplementary Fig. 1). Preneoplastic basophilic cells showed cytoplasmic basophilia and were smaller or, more rarely, bigger than normal hepatocytes. The basophilic phenotype was already evident in single hepatocytes or clusters of preneoplastic cells (Supplementary Fig. 1). To confirm that preneoplastic and neoplastic lesions were indeed induced by the ectopically injected oncogenes, we performed immunohistochemistry on SKP2/N-RasV12 lesions by using an anti-HA-tag and an anti-human N-Ras antibody (Fig. 2). As expected, preneoplastic lesions and tumors -but not the normal liver tissue-exhibited strong expression of HA-tag and human N-Ras proteins, implying that tumors originate from the transfected hepatocytes (Fig. 2C,D). Importantly, HA-tag immunoreactivity was localized in the nucleus of transfected cells (Fig. 2C, inset), in accordance with previous findings showing that SKP2 exerts its oncogenic potential at the nuclear level in human HCC [14, 15].

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.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.

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