Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones.

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

4EBP1/eIF4E and p70S6K/RPS6 axes play critical and distinct roles in hepatocarcinogenesis driven by AKT and N-Ras proto-oncogenes in mice.

UNLABELLED: Concomitant expression of activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and Ras in mouse liver (AKT/Ras) leads to rapid tumor development through strong activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 functions by regulating p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/ eukaryotic translation initiation factor 4E (4EBP1/eIF4E) cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of the RPS6 pathway by rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras-induced hepatocarcinogenesis. Combined treatment with rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong antineoplastic effect was successfully recapitulated by ablating regulatory associated protein of mTORC1, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the proto-oncogene whose activity is specifically inhibited by 4EBP1, resulted in hepatocellular carcinoma (HCC) development in cooperation with activated Ras. Mechanistically, we identified the ectonucleoside triphosphate diphosphohydrolase 5/ adenylate kinase 1/cytidine monophosphate kinase 1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues. CONCLUSIONS: Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras proto-oncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human HCC.

Cell turnover in the repopulated rat liver: distinct lineages for hepatocytes and the biliary epithelium.

The dynamics of cell renewal in the normal adult liver remains an unresolved issue. We investigate the possible contribution of a common biliary precursor cell pool to hepatocyte turnover in the chimeric long-term repopulated rat liver. The retrorsine (RS)-based model of massive liver repopulation was used. Animals not expressing the CD26 marker (CD26(-)) were injected with RS, followed by transplantation of 2 million syngeneic hepatocytes isolated from a normal CD26-expressing donor. Extensive (80-90%) replacement of resident parenchymal cells was observed at 1 year post-transplantation and persisted at 2 years, as expected. A panel of specific markers, including cytokeratin 7, OV6, EpCAM, claudin 7 and α-fetoprotein, was employed to locate the in situ putative progenitor and/or biliary epithelial cells in the stably repopulated liver. No overlap was observed between any of these markers and the CD26 tag identifying transplanted cells. Exposure to RS was not inhibitory to the putative progenitor and/or biliary epithelial cells, nor did we observe any evidence of cell fusion between these cells and the transplanted cell population. Given the long-term (>2 years) stability of the donor cell phenotype in this model of liver repopulation, the present findings suggest that hepatocyte turnover in the repopulated liver is fuelled by a cell lineage distinct from that of the biliary epithelium and relies largely on the differentiated parenchymal cell population. These results support the solid biological foundation of liver repopulation strategies based on the transplantation of isolated hepatocytes.

Hepatocyte senescence in vivo following preconditioning for liver repopulation.

UNLABELLED: In the retrorsine (RS)-based model of massive liver repopulation, preexposure to this naturally occurring alkaloid is sufficient to prime normal host parenchymal cells to be slowly replaced by transplanted normal hepatocytes. The basis for this striking effect is yet to be fully elucidated. In the present studies the possible involvement of cell senescence was investigated. Fischer 344 rats were treated according to the RS-based protocol for hepatocyte transplantation, i.e., two doses of RS, 2 weeks apart, and were killed at 4 or 8 weeks after treatment. Control groups were given saline. Expression of senescence-associated beta-galactosidase was greatly induced in hepatocytes exposed to RS. In addition, several other changes that have been related to cell senescence were observed: these included markers of persistent activation of a DNA damage response, an increased expression of mammalian target of rapamycin, and positive regulators of the cell cycle, together with the induction of p21 and p27 cyclin-dependent kinase inhibitors. Furthermore, RS treatment increased levels of interleukin-6 in the liver, consistent with the activation of a senescence-associated secretory phenotype. CONCLUSION: These findings indicate that RS induces hepatocyte senescence in vivo. We propose that cell senescence and the associated secretory phenotype can contribute to the selective growth of transplanted hepatocytes in this system.

Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation.

UNLABELLED: Hu antigen R (HuR) is a central RNA-binding protein regulating cell dedifferentiation, proliferation, and survival, which are well-established hallmarks of cancer. HuR is frequently overexpressed in tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2-mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation. CONCLUSION: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy.

Hepatic differentiation of amniotic epithelial cells.

UNLABELLED: Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, naïve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. CONCLUSION: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.

The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive primary liver tumor with increasing incidence worldwide, dismal prognosis, and few therapeutic options. Mounting evidence underlines the role of the Hippo pathway in this disease; however, the molecular mechanisms whereby the Hippo cascade contributes to cholangiocarcinogenesis remain poorly defined. METHODS: We established novel iCCA mouse models via hydrodynamic transfection of an activated form of transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo pathway downstream effector, either alone or combined with the myristoylated AKT (myr-AKT) protooncogene, in the mouse liver. Hematoxylin and eosin staining, immunohistochemistry, electron microscopy, and quantitative real-time RT-PCR were applied to characterize the models. In addition, in vitro cell line studies were conducted to address the growth-promoting roles of TAZ and its paralog YAP. RESULTS: Overexpression of TAZ in the mouse liver triggered iCCA development with very low incidence and long latency. In contrast, co-expression of TAZ and myr-AKT dramatically increased tumor frequency and accelerated cancer formation in mice, with 100% iCCA incidence and high tumor burden by 10 weeks post hydrodynamic injection. AKT/TAZ tumors faithfully recapitulated many of the histomolecular features of human iCCA. At the molecular level, the development of the cholangiocellular lesions depended on the binding of TAZ to TEAD transcription factors. In addition, inhibition of the Notch pathway did not hamper carcinogenesis but suppressed the cholangiocellular phenotype of AKT/TAZ tumors. Also, knockdown of YAP, the TAZ paralog, delayed cholangiocarcinogenesis in AKT/TAZ mice without affecting the tumor phenotype. Furthermore, human preinvasive and invasive iCCAs and mixed hepatocellular carcinoma/iCCA displayed widespread TAZ activation and downregulation of the mechanisms protecting TAZ from proteolysis. CONCLUSIONS: Overall, the present data underscore the crucial role of TAZ in cholangiocarcinogenesis.

Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors.

Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.

The growth pattern of transplanted normal and nodular hepatocytes.

Overt neoplasia is often the end result of a long biological process beginning with the appearance of focal lesions of altered tissue morphology. While the putative clonal nature of focal lesions has often been emphasized, increasing attention is being devoted to the possible role of an altered growth pattern in the evolution of carcinogenesis. Here we compare the growth patterns of normal and nodular hepatocytes in a transplantation system that allows their selective clonal proliferation in vivo. Rats were pre-treated with retrorsine, which blocks the growth of resident hepatocytes, and were then transplanted with hepatocytes isolated from either normal liver or hepatocyte nodules. Both cell types were able to proliferate extensively in the recipient liver, as expected. However, their growth pattern was remarkably different. Clusters of normal hepatocytes integrated in the host liver, displaying a normal histology; however, transplanted nodular hepatocytes formed new hepatocyte nodules, with altered morphology and sharp demarcation from surrounding host liver. Both the expression and distribution of proteins involved in cell polarity, cell communication, and cell adhesion, including connexin 32, E-cadherin, and matrix metalloproteinase-2, were altered in clusters of nodular hepatocytes. Furthermore, we were able to show that down-regulation of connexin 32 and E-cadherin in nodular hepatocyte clusters was independent of growth rate. These results support the concept that a dominant pathway towards neoplastic disease in several organs involves defect(s) in tissue pattern formation.

Universal toxin-based selection for precise genome engineering in human cells.

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.

The degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the disease.

Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21(WAF1), p27(KIP1), p57(KIP2) and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21(WAF1), p27(KIP1), p57(KIP), p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21(WAF1), P27(KIP1), P57(KIP2) and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators.

SKP2 and CKS1 promote degradation of cell cycle regulators and are associated with hepatocellular carcinoma prognosis.

BACKGROUND & AIMS: The cell cycle regulators P21(WAF1), P27(KIP1), P57(KIP2), P130, RASSF1A, and FOXO1 are down-regulated during hepatocellular carcinoma (HCC) pathogenesis. We investigated the role of the ubiquitin ligase subunits CKS1 and SKP2, which regulate proteasome degradation of cell cycle regulators, in HCC progression. METHODS: Human HCC tissues from patients with better (HCCB, >3 years survival) and poorer prognosis (HCCP, <3 years survival) and HCC cell lines were analyzed. RESULTS: The promoters of P21(WAF1), P27(KIP1), and P57(KIP2) were more frequently hypermethylated in HCCP than HCCB. Messenger RNA levels of these genes were up-regulated in samples in which these genes were not methylated; protein levels increased only in HCCB because of CKS1- and SKP2-dependent ubiquitination of these proteins in HCCP. The level of SKP2 expression correlated with rate of HCC cell proliferation and level of microvascularization of samples and was inversely correlated with apoptosis and survival. In HCCB, SKP2 activity was balanced by degradation by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-CDH1 and up-regulation of SKP2 suppressor histidine triad nucleotide binding protein 1 (HINT1). In HCCP, however, SKP2 was not degraded because of down-regulation of the phosphatase CDC14B, CDK2-dependent serine phosphorylation (which inhibits interaction between CDH1 and SKP2), and HINT1 inactivation. In HCC cells, small interfering RNA knockdown of SKP2 reduced proliferation and ubiquitination of the cell cycle regulators, whereas SKP2 increased proliferation and reduced expression of cell cycle regulators. CONCLUSIONS: Ubiquitination and proteasome degradation of P21WAF1, P27KIP1, P57KIP2, P130, RASSF1A, and FOXO1 and mechanisms that prevent degradation of SKP2 by APC/C-CDH1 contribute to HCC progression. CKS1-SKP2 ligase might be developed as a therapeutic target or diagnostic marker.

Ras-driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control.

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.

Potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors based on the benzoisoxazole moiety: application of a bioisosteric scaffold hopping approach to flufenamic acid.

The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data.

Aging promotes neoplastic disease through effects on the tissue microenvironment.

A better understanding of the complex relationship between aging and cancer will provide important tools for the prevention and treatment of neoplasia. In these studies, the hypothesis was tested that aging may fuel carcinogenesis via alterations imposed in the tissue microenvironment. Preneoplastic hepatocytes isolated from liver nodules were orthotopically injected into either young or old syngeneic rats and their fate was followed over time using the dipeptidyl-peptidase type IV (DPPIV) system to track donor-derived-cells. At 3 months post-Tx, the mean size of donor-derived clusters was 11±3 cells in young vs. 42±8 in old recipients. At 8 months post-Tx, no visible lesion were detected in any of 21 young recipients, while 17/18 animals transplanted at old age displayed hepatic nodules, including 7 large tumors. All tumors expressed the DPPIV marker enzyme, indicating that they originated from transplanted cells. Expression of senescence-associated ß-galactosidase was common in liver of 18-month old animals, while it was a rare finding in young controls. Finally, both mRNA and IL6 protein were found to be increased in the liver of aged rats compared to young controls. These results are interpreted to indicate that the microenvironment of the aged liver promotes the growth of pre-neoplastic hepatocytes.

Rat-derived amniotic epithelial cells differentiate into mature hepatocytes in vivo with no evidence of cell fusion.

Amniotic epithelial cells (AEC) derived from human placenta represent a useful and noncontroversial source for liver-based regenerative medicine. Previous studies suggested that human- and rat-derived AEC differentiate into hepatocyte-like cells upon transplantation. In the retrorsine (RS) model of liver repopulation, clusters of donor-derived cells engrafted in the recipient liver and, importantly, showed characteristics of mature hepatocytes. The aim of the current study was to investigate the possible involvement of cell fusion in the emergence of hepatocyte clusters displaying a donor-specific phenotype. To this end, 4-week-old GFP(+)/DPP-IV(-) rats were treated with RS and then transplanted with undifferentiated AEC isolated from the placenta of DPP-IV(+) pregnant rats at 16-19 days of gestational age. Results indicated that clusters of donor-derived cells were dipeptidyl peptidase type IV (DPP-IV) positive, but did not express the green fluorescent protein (GFP), suggesting that rat amniotic epithelial cells (rAEC) did not fuse within the host parenchyma, as no colocalization of the two tags was observed. Moreover, rAEC-derived clusters expressed markers of mature hepatocytes (eg, albumin, cytochrome P450), but were negative for the expression of biliary/progenitor markers (eg, epithelial cell adhesion molecule [EpCAM]) and did not express the marker of preneoplastic hepatic nodules glutathione S-transferase P (GST-P). These results extend our previous findings on the potential of AEC to differentiate into mature hepatocytes and suggest that this process can occur in the absence of cell fusion with host-derived cells. These studies support the hypothesis that amnion-derived epithelial cells can be an effective cell source for the correction of liver disease.

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

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 ß-catenin (ΔN90-ß-catenin) protooncogenes, via hydrodynamic gene delivery. We found that forced overexpression of SKP2, N-RasV12 or ΔN90-ß-catenin alone as well as co-expression of SKP2 and ΔN90-ß-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 ß-catenin mutation or activation. Altogether, the present data indicate that SKP2 cooperates with N-Ras and AKT proto-oncogenes to promote hepatocarcinogenesis in vivo.

Co-activation of PIK3CA and Yap promotes development of hepatocellular and cholangiocellular tumors in mouse and human liver.

Activation of the PI3K and Yes-associated protein (Yap) signaling pathways has been independently reported in human hepatocellular carcinoma (HCC). However, the oncogenic interactions between these two cascades in hepatocarcinogenesis remain undetermined. To assess the consequences of the crosstalk between the PI3K and Yap pathways along liver carcinogenesis, we generated a mouse model characterized by combined overexpression of activated mutant forms of PIK3CA (PIK3CAH1047R) and Yap (YapS127A) in the mouse liver using hydrodynamic transfection (PIK3CA/Yap). In addition, suppression of PI3K and Yap pathways was conducted in human HCC and cholangiocarcinoma (CCA) cell lines. We found that concomitant activation of PI3K and Yap pathways triggered rapid liver tumor development in mice. Histologically, tumors were pure HCC, CCA, or mixed HCC/CCA. At the molecular level, PIK3CA/Yap tumors were characterized by activation of the mTORC1/2, ERK/MAPK, and Notch pathways. Simultaneous activation of PI3K and Yap pathways frequently occurred in human liver tumor specimens and their combined suppression was highly detrimental for the growth of HCC and CCA cell lines. In conclusion, our study demonstrates the oncogenic cooperation between PI3K and Yap pathways along liver carcinogenesis. The PIK3CA/Yap mouse represents an important preclinical liver tumor model for the development of novel therapeutics against this malignancy.