Angela Maria Cozzolino

ERK5/MAPK is activated by TGFβ in hepatocytes and required for the GSK-3β-mediated Snail protein stabilization

Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, specifically activated by MEK5, and involved in the regulation of many cellular functions including proliferation, survival, differentiation and apoptosis. MEK5/ERK5 module is an important element of different signal transduction pathways. The aim of this study was to investigate whether ERK5 participates to the signalling of the multifunctional cytokine TGFbeta, known to play an important role in the regulation of hepatic growth. Here, we reported that ERK5 is phosphorylated and activated by TGFbeta in hepatocytes, with a rapid and sustained kinetic, through a Src-dependent pathway. Moreover, we demonstrated that ERK5 participates to the TGFbeta-induced Snail protein regulation being required for its stabilization. We also found that the functional inactivation of ERK5 impedes the TGFbeta-mediated glycogen synthase kinase-3beta inactivation suggesting this as mechanism responsible for ERK5-mediated Snail stabilization. Thus, results presented in this study uncovered for the first time a role for ERK5 in the TGFbeta-induced cellular responses.

An epistatic mini-circuitry between the transcription factors Snail and HNF4α controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs.

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4α, directly represses the expression of the epithelial microRNAs (miRs)-200c and -34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4α, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and -200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4α and miRs-200a, b, c and -34a as epistatic elements controlling hepatic stem cell maintenance/differentiation.

Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that, in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines as in vitro models of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

TGFβ overrides HNF4α tumor suppressing activity through GSK3β inactivation: implication for hepatocellular carcinoma gene therapy

BACKGROUND & AIMS The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity. METHODS Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity. CONCLUSIONS Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.

Analysis of the miR-34a locus in 62 patients with familial cutaneous melanoma negative for CDKN2A/CDK4 screening

MicroRNAs are small non-coding RNAs, which inhibit expression of specific target genes at the post-transcriptional level and are often misregulated in human cancer. Among them, miR-34a is considered a tumor suppressor with a hypothetical role in melanoma tumorigenesis. In this work, 62 Italian index patients with familial melanoma and negative for CDKN2A/CDK4 screening were investigated for miR-34a germline mutations. Eight novel miR-34a sequence variants were identified at both the heterozygous (c.+259G>A, c.+424G>A, c.+1465C>T, c.+1769C>T, c.+2456T>G, c.+2603C>T, c.+2972T>A, c.+3069T>C) and homozygous (c.+424G>A, c.+1465C>T, c.+1769C>T) states. Molecular screening identified all nucleotide changes in a healthy population of 150 controls and demonstrated that they are common polymorphisms. However, statistically significant differences of allele and genotype frequencies were detected for c.+1465C>T and c.+1769C>T, and borderline values for c.+2456T>G. By stratifying patients by relevant clinical features (presence/absence of multiple primary melanoma, Breslow’s thickness, phototype and number of nevi), no significant findings were noted except for an association between the c.+424G>A (heterozygous individual GA) and multiple primary melanoma and phototype III–IV. Our preliminary study suggests that miR-34a, although having a role in late tumorigenesis, does not contribute to the inherited susceptibility to cutaneous melanoma. A function as phenotypic modulator in familial melanoma cannot be excluded.

AXIN2 germline mutations are rare in familial melanoma

In a recent study published in ‘‘Genes Chromosomes and Cancer’’ (Castiglia et al., 2008), one germline Axis inhibition protein 2 (AXIN2) mutation was first described in a sporadic melanoma patient with healthy and noncarrier daughters. This preliminary result made us hypothesize that deregulation of the Wnt/ß-catenin signaling pathway could promote melanoma development and/ or progression and that AXIN2 mutations could be involved in familial melanoma (FAM) susceptibility. AXIN2 is a negative regulator gene of Wnt/ß-catenin signaling, and it is a putative tumor suppressor gene in chromosome band 17q24. The gene product is a scaffold protein that takes part in a multiprotein complex that facilitates ßcatenin phosphorylation and destruction (Kishida et al., 1998; Ikeda et al., 2000). Hence, a role of AXIN2 in human cancer pathogenesis could be suspected. However, available data do not provide a clear picture and the possible role of AXIN2 somatic/germline allelic variants in disease development and/or progression is still largely unknown. Somatic heterozygous mutations in AXIN2 have been described in human cancer including endometrioid adenocarcinoma (Wu et al., 2001), hepatocellular carcinoma (Taniguchi et al., 2002), gastric carcinoma (Kim et al., 2009), and medulloblastoma (Koch et al., 2007). AXIN2 germline truncating mutations were found in patients with syndromic tooth agenesis and colorectal cancer (Lammi et al., 2004). Specific AXIN2 single nucleotide polymorphisms have been reported to correlate with isolated tooth agenesis (Callahan et al., 2009) and to be associated with lung cancer in Japanese and Turkish patients (Kanzaki et al., 2006; Emine et al., 2009). However, no AXIN2 germline mutation was found in non-syndromic colorectal cancer (Peterlongo et al., 2005) and no association was demonstrated with multiple polyposis (Lejeune et al., 2006). However, with the exception of the work by Castiglia et al. (2008), no further study has investigated the role of AXIN2 in human melanoma. In this study, a cohort of 84 Italian FAM patients, 22 of whom with multiple primary melanoma (MPM), were recruited from the Oncological Dermatology Service at the IFO-IRCCS Hospital in Rome (Italy). Former molecular analyses of CDKN2A and CDK4 genes have excluded the presence of pathogenic mutations in all these patients (Majore et al., 2008; Binni et al., 2010). After having obtained informed consent, the entire coding region and splice site junctions of AXIN2 were amplified from the constitutional DNA using 11 primer pairs that anneal on flanking introns, as previously described (Peterlongo et al., 2005). PCR products were purified using Biomek 3000 (Beckman Coulter, Fullerton, CA) according to the manufacturer’s protocol, and sequenced in both directions using the 3130xl Genetic Analyzer (Applied Biosystem). Genomic DNAs from 150 unrelated ethnically matched healthy individuals (corresponding to 300 control chromosomes) were screened for the presence of each identified sequence variation to assess whether it was a real mutation or a polymorphism. Consequences of sequence variations on primary and secondary protein structure were investigated using POLYPHEN (http://genetics. bwh.harvard.edu/pph/), SIFT (blocks.fhcrc.org/ sift/SIFT.html), NetSurfP (Petersen et al., 2009) and Hierarchical Neural Network (http://www. expasy.ch/tools/#second ary) in silico predictors. Putative splice-site mutation effects were analyzed by using the splice site prediction program NNSPLICE ver.0.9 (http://www.fruitfly.org/seq_ tools/splice. html) and ESEFINDER 3.0 (rulai. cshl.edu/tools/ESE/). Molecular analysis disclosed six distinct heterozygous AXIN2 variations in FAM subjects without MPM and with an early age at onset (16–34 years). Two of these, c.1201-84delG and c.1489T >A (p.C497S), were novel heterozygous polymorphic variants, being detected in healthy controls. Moreover, no difference in allele frequencies between the patient group and healthy subjects

Spike-in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload

Hepatitis C virus (HCV)‐induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal‐restricted histological distribution of pathological iron deposits has hampered the attempt to perform large‐scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload‐induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike‐in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron‐associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV‐infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV‐infected patients without iron overload.

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation

BackgroundDespite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation.ResultsWe apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation.ConclusionsTaken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

New Tools for Molecular Therapy of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, arising from neoplastic transformation of hepatocytes or liver precursor/stem cells. HCC is often associated with pre-existing chronic liver pathologies of different origin (mainly subsequent to HBV and HCV infections), such as fibrosis or cirrhosis. Current therapies are essentially still ineffective, due both to the tumor heterogeneity and the frequent late diagnosis, making necessary the creation of new therapeutic strategies to inhibit tumor onset and progression and improve the survival of patients. A promising strategy for treatment of HCC is the targeted molecular therapy based on the restoration of tumor suppressor proteins lost during neoplastic transformation. In particular, the delivery of master genes of epithelial/hepatocyte differentiation, able to trigger an extensive reprogramming of gene expression, could allow the induction of an efficient antitumor response through the simultaneous adjustment of multiple genetic/epigenetic alterations contributing to tumor development. Here, we report recent literature data supporting the use of members of the liver enriched transcription factor (LETF) family, in particular HNF4α, as tools for gene therapy of HCC.