We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of ␤-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1␣. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of ␤-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated ␤-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated ␤-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.␤-catenin ͉ hepatocyte nuclear factor 1␣ ͉ liver cancer ͉ MET ͉ mouse ͉ hepatocellular carcinoma

In cells capable of entering the cell cycle, including cancer cells, ␤-catenin has been termed a master switch, driving proliferation over differentiation. However, its role as a transcriptional activator in terminally differentiated cells is relatively unknown. Herein we utilize conditional, cardiac-specific deletion of the ␤-catenin gene and cardiac-specific expression of a dominant inhibitory mutant of Lef-1 (Lef-1⌬20), one of the members of the T-cell factor/lymphocyte enhancer factor (Tcf/Lef) family of transcription factors that functions as a coactivator with ␤-catenin, to demonstrate that ␤-catenin/Tcf/Lef-dependent gene expression regulates both physiologic and pathological growth (hypertrophy) of the heart. Indeed, the profound nature of the growth impairment of the heart in the Lef-1⌬20 mouse, which leads to very early development of heart failure and premature death, suggests ␤-catenin/Tcf/Lef targets are dominant regulators of cardiomyocyte growth. Thus, our studies, employing complementary models in vivo, implicate ␤-catenin/Tcf/Lef signaling as an essential growth-regulatory pathway in terminally differentiated cells.

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Liver cancer comprises a group of malignant tumors, among which hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common. ICC is especially pernicious and associated with poor clinical outcome. Studies have shown that a subset of human ICCs may originate from mature hepatocytes. However, the mechanisms driving the trans-differentiation of hepatocytes into malignant cholangiocytes remain poorly defined. We adopted lineage tracing techniques and an established murine hepatocyte-derived ICC model by hydrodynamic injection of activated forms of AKT (myr-AKT) and Yap (YapS127A) proto-oncogenes. Wild-type, Notch1flox/flox, and Notch2flox/flox mice were used to investigate the role of canonical Notch signaling and Notch receptors in AKT/Yap-driven ICC formation. Human ICC and HCC cell lines were transfected with siRNA against Notch2 to determine whether Notch2 regulates biliary marker expression in liver tumor cells. We found that AKT/Yap-induced ICC formation is hepatocyte derived and this process is strictly dependent on the canonical Notch signaling pathway in vivo. Deletion of Notch2 in AKT/Yap-induced tumors switched the phenotype from ICC to hepatocellular adenoma-like lesions, while inactivation of Notch1 in hepatocytes did not result in significant histomorphological changes. Finally, in vitro studies revealed that Notch2 silencing in ICC and HCC cell lines down-regulates the expression of Sox9 and EpCAM biliary markers. Notch2 is the major determinant of hepatocyte-derived ICC formation in mice.

Shortening of the 3' untranslated regions (3'UTR) of mRNA is an important mechanism for oncogene activation. However, 3'UTR alteration events, their pathologic functions, and underlying mechanisms in human urothelial carcinoma of the bladder (UCB) are not clear. Here, we combine RNA sequencing, bioinformatics, and clinical studies in two independent cohorts of patients with UCB to identify a novel shorter 3'UTR isoform that is frequently expressed in UCB and is critical in the tumorigenesis and acquisition of a poor prognostic phenotype in patients. Short 3'UTR isoform of substantially upregulated RAC1 expression by escaping from miRNA-targeted repression and played an essential oncogenic role in UCB pathogenesis. An important cleavage/polyadenylation factor, cleavage stimulation factor 2 (CSTF2), induced 3'UTR shortening of in UCB by mediating slow transcriptional elongation at Cotranscriptional recruitment of CSTF2 on the GUAAU motif at proximal polyadenylation site of attenuated the recruitment of two transcription factors AFF1 and AFF4, causing the defects in elongation. CSTF2 regulated the tumorigenic functions of the shorter isoform in UCB cells, enhancing cell proliferation, migration, and invasion. The combination of high expression of CSTF2 and high usage of short-3'UTR isoform may be used as a powerful biomarker to predict poor prognosis in UCB. Our findings also suggest a CSTF2-regulated-3'UTR shortening program as an exploitable therapeutic strategy for patients with UCB. These findings demonstrate that the short isoform of is critical in UCB tumorigenesis and may have implications for developing new therapeutic strategies to treat this disease..

Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. Conclusion These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.

BackgroundHematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown.ResultsTo examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2−/− hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2−/− hESCs displayed attenuated generation of CD34+CD43+ hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2−/− hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2−/− hESCs in generation of CD34+CD43+ HPCs and granulocytes. Furthermore, we found that GATA2−/− hESCs restored the granulocyte potential in the presence of Notch signaling.ConclusionOur findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13619-015-0018-7) contains supplementary material, which is available to authorized users.

Primary liver cancer consists mainly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). A subset of human HCCs expresses a ICC-like gene signature and is classified as ICC-like HCC. The Hippo pathway is a critical regulator of normal and malignant liver development. However, the precise function(s) of the Hippo cascade along liver carcinogenesis remain to be fully delineated. The role of the Hippo pathway in a murine mixed HCC/ICC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated. The authors demonstrated the inactivation of Hippo in AKT/Ras liver tumors leading to nuclear localization of Yap and TAZ. Coexpression of AKT/Ras with Lats2, which activates Hippo, or the dominant negative form of TEAD2 (dnTEAD2), which blocks Yap/TAZ activity, resulted in delayed hepatocarcinogenesis and elimination of ICC-like lesions in the liver. Mechanistically, Notch2 expression was found to be down-regulated by the Hippo pathway in liver tumors. Overexpression of Lats2 or dnTEAD2 in human HCC cell lines inhibited their growth and led to the decreased expression of ICC-like markers, as well as Notch2 expression. Altogether, this study supports the key role of the Hippo cascade in regulating the differentiation status of liver tumors.