Stefano Cairo

The tripartite motif family identifies cell compartments

A functional genomic approach, based on systematic data gathering, was used to characterize a family of proteins containing a tripartite motif (TRIM). A total of 37 TRIM genes/proteins were studied, 21 of which were novel. The results demonstrate that TRIM proteins share a common function: by means of homo‐multimerization they identify specific cell compartments.

Hepatic Stem-like Phenotype and Interplay of Wnt/β-Catenin and Myc Signaling in Aggressive Childhood Liver Cancer

Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.

TAK1 Suppresses a NEMO-Dependent but NF-κB-Independent Pathway to Liver Cancer

The MAP3-kinase TGF-beta-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-kappaB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-kappaB-independent functions of the I kappaB-kinase (IKK)-subunit NF-kappaB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-kappaB-independent function of NEMO in parenchymal liver cells.

Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties

BackgroundThe TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.ResultsHere we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures.ConclusionWe found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.

Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer.

Myc activation has been implicated in the pathogenesis of hepatoblastoma (HB), a rare embryonal neoplasm derived from liver progenitor cells. Here, microRNA (miR) expression profiling of 65 HBs evidenced differential patterns related to developmental stage and Myc activity. Undifferentiated aggressive HBs overexpressed the miR-371–3 cluster with concomitant down-regulation of the miR-100/let-7a-2/miR-125b-1 cluster, evoking an ES cell expression profile. ChIP and Myc inhibition assays in hepatoma cells demonstrated that both miR clusters are regulated by Myc in an opposite manner. We show that the two miR clusters exert antagonistic effects on cell proliferation and tumorigenicity. Moreover, their combined deregulation cooperated in modulating the hepatic tumor phenotype, implicating stem cell-like regulation of Myc-dependent miRs in poorly differentiated HBs. Importantly, a four-miR signature representative of these clusters efficiently stratified HB patients, and when applied to 241 hepatocellular carcinomas (HCCs), it identified invasive tumors with a poor prognosis. Our data argue that Myc-driven reprogramming of miR expression patterns contributes to the aggressive phenotype of liver tumors originating from hepatic progenitor cells.

The Hepatitis B Virus X Protein Functionally Interacts with CREB-binding Protein/p300 in the Regulation of CREB-mediated Transcription

The hepatitis B virus infects more than 350 million people worldwide and is a leading cause of liver cancer. The virus encodes a multifunctional regulator, the hepatitis B virus X protein (HBx), that is essential for virus replication. HBx is involved in modulating signal transduction pathways and transcription mediated by various factors, notably CREB that requires the recruitment of the co-activators CREB-binding protein (CBP)/p300. Here we investigated the role of HBx and its potential interaction with CBP/p300 in regulating CREB transcriptional activity. We show that HBx and CBP/p300 synergistically enhanced CREB activity and that CREB phosphorylation by protein kinase A was a prerequisite for the cooperative action of HBx and CBP/p300. We further show that HBx interacted directly with CBP/p300 in vitro and in vivo. Using chromatin immunoprecipitation, we provide evidence that HBx physically occupied the CREB-binding domain of CREB-responsive promoters of endogenous cellular genes such as interleukin 8 and proliferating cell nuclear antigen. Moreover expression of HBx increased the recruitment of p300 to the interleukin 8 and proliferating cell nuclear antigen promoters in cells, and this is associated with increased gene expression. As recruitment of CBP/p300 is known to represent the limiting event for activating CREB target genes, HBx may disrupt this cellular regulation, thus predisposing cells to transformation.

Tbx3 Is a Downstream Target of the Wnt/β-Catenin Pathway and a Critical Mediator of β-Catenin Survival Functions in Liver Cancer

Tbx3 encodes a transcriptional repressor that is important for diverse patterning events during development, and Tbx3 mutation in humans causes the ulnar-mammary syndrome. Here, we describe the identification of Tbx3 in array-based search for genes downstream Wnt/beta-catenin that are implicated in liver tumorigenesis. Overexpression of Tbx3 is closely associated with the mutational status of beta-catenin in murine liver tumors induced by Myc as well as in human hepatocellular carcinomas and hepatoblastomas. Moreover, Tbx3 transcription is activated by ectopic expression of beta-catenin in mouse liver and in human tumor cell lines. Evidence that Tbx3 transcription is directly regulated by beta-catenin is provided by chromatin immunoprecipitation and reporter assays. Although HepG2 cells stably transfected with Tbx3 display moderately enhanced growth rate, the dominant negative mutant Tbx3-Y149S drastically inhibits hepatoma cell growth in vitro and in vivo. Moreover, small interfering RNAs (siRNA) directed against Tbx3 inhibit anchorage-independent growth of liver and colon carcinoma cells. We further show that inhibition of Tbx3 expression by specific siRNAs blocks beta-catenin-mediated cell survival and renders cells sensitive to doxorubicin-induced apoptosis. Conversely, ectopic expression of Tbx3 inhibits apoptosis induced by beta-catenin depletion. Marked overexpression of Tbx3 in a subset of hepatoblastomas is associated with chemotherapy-resistant phenotype and unfavorable patient outcome. These results reveal an unsuspected role of Tbx3 as a mediator of beta-catenin activities on cell proliferation and survival and as an important player in liver tumorigenesis.

Wnt signaling and hepatocarcinogenesis: The hepatoblastoma model

The Wnt/β-catenin pathway plays a key role in liver development, regeneration and tumorigenesis. Among human cancers tightly linked to abnormal Wnt/β-catenin signaling, hepatoblastoma (HB) presents with the highest rate (50-90%) of β-catenin mutations. HB is the most common malignant tumor of the liver in childhood. This embryonic tumor differs from hepatocellular carcinoma by the absence of viral etiology and underlying liver disease, and by distinctive morphological patterns evoking hepatoblasts, the bipotent precursors of hepatocytes and cholangiocytes. Recent studies of the molecular pathogenesis of hepatoblastoma have led to identify two major tumor subclasses resembling early and late phases of prenatal liver development and presenting distinctive chromosomal alterations. It has been shown that the molecular signature of Wnt/β-catenin signaling in hepatoblastoma is mainly imposed by liver context, but differs according to developmental stage. Finally, the differentiation stage of tumor cells strongly influences their invasive and metastatic properties, therefore affecting clinical behavior.

Mlx, a new Max-like bHLHZip family member: the center stage of a novel transcription factors regulatory pathway?

The Myc proto-oncogene family members have been identified as the cellular homologs of the transforming oncogene of avian retroviruses. They encode central regulators of mammalian cell proliferation and apoptosis, and they associate with the bHLHZip protein Max to bind specific DNA sequences and regulate the expression of genes important for cell cycle progression. The other family members, Mad1, Mxi1, Mad3, Mad4 and Rox (Mnt) antagonize their activities. The Mads and Rox compete with Myc in heterodimerizing with Max and in binding to the same specific target sequences. These Mads:Max and Rox:Max dimers repress transcription through binding to the mSIN3 corepressor protein and by tethering histone deacetylase-containing complexes to the DNA. In a screen for Rox interactors we isolated Mlx, a bHLHZip protein previously identified in a screen for Mad1 interactors. In the present work we extend the known dimerization partners of Mlx by demonstrating its ability to interact with Rox. Moreover, we show that contrary to previous reports Mlx is able to homodimerize and to bind E-box sequences at low concentration levels. The possible role of Mlx in an emerging regulatory pathway and acting parallel to the Max driven network is discussed.

The genomic landscape of hepatoblastoma and their progenies with HCC-like features

BACKGROUND & AIMS Hepatoblastoma (HB) is the most common childhood liver cancer and occasionally presents with histological and clinical features reminiscent of hepatocellular carcinoma (HCC). Identification of molecular mechanisms that drive the neoplastic continuation towards more aggressive HCC phenotypes may help to guide the new stage of targeted therapies. METHODS We performed comprehensive studies on genetic and chromosomal alterations as well as candidate gene function and their clinical relevance. RESULTS Whole-exome sequencing identified HB as a genetically very simple tumour (2.9 mutations per tumour) with recurrent mutations in ß-catenin (CTNNB1) (12/15 cases) and the transcription factor NFE2L2 (2/15 cases). Their HCC-like progenies share the common CTNNB1 mutation, but additionally exhibit a significantly increased mutation number and chromosomal instability due to deletions of the genome guardians RAD17 and TP53, accompanied by telomerase reverse-transcriptase (TERT) promoter mutations. Targeted genotyping of 33 primary tumours and cell lines revealed CTNNB1, NFE2L2, and TERT mutations in 72.5%, 9.8%, and 5.9% of cases, respectively. All NFE2L2 mutations affected residues of the NFE2L2 protein that are recognized by the KEAP1/CUL3 complex for proteasomal degradation. Consequently, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signalling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumours was significantly associated with metastasis, vascular invasion, the adverse prognostic C2 gene signature, as well as poor outcome. CONCLUSIONS Our study demonstrates the importance of CTNNB1 mutations and NFE2L2-KEAP1 pathway activation in HB development and defines loss of genomic stability and TERT promoter mutations as prominent characteristics of aggressive HB with HCC features.