YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation

Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases

Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29

Background and aims: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. Methods: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. Results: HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR- 29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. Conclusions: HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, \u3b3-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients

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

Effects of iron on the aggregation propensity of the N-terminal fibrillogenic polypeptide of human apolipoprotein A-I

Specific mutations in APOA1 gene lead to systemic, hereditary amyloidoses. In ApoA-I related amyloidosis involving the heart, amyloid deposits are mainly constituted by the 93-residue N-terminal region of the protein, here indicated as [1-93]ApoA-I. Oxidative stress is known to be an enhancing factor for protein aggregation. In healthy conditions, humans are able to counteract the formation and the effects of oxidative molecules. However, aging and atmospheric pollution increase the concentration of oxidative agents, such as metal ions. As the main effect of iron deregulation is proposed to be an increase in oxidative stress, we analysed the effects of iron on [1-93]ApoA-I aggregation. By using different biochemical approaches, we demonstrated that Fe(II) is able to reduce the formation of [1-93]ApoA-I fibrillar species, probably by stabilizing its monomeric form, whereas Fe(III) shows a positive effect on polypeptide fibrillogenesis. We hypothesize that, in healthy conditions, Fe(III) is reduced by the organism to Fe(II), thus inhibiting amyloid formation, whereas during ageing such protective mechanisms decline, thus exposing the organism to higher oxidative stress levels, which are also related to an increase in Fe(III). This alteration could contribute to the pathogenesis of amyloidosis

Signaling networks controlling HCC onset and progression: influence of microenvironment and implications for cancer gene therapy

Hepatocarcinogenesis, as other epithelial malignancies, has been proved to be a multistep process that, starting from mutagenic events, allows the transformed liver cell to evolve towards a more aggressive phenotype, characterized by the acquisition of migratory/invasive and stem-cell-like properties. Hepatocellular carcinoma (HCC) can originate from both mature hepatocytes and liver precursor/stem cells. Whatever its origin, a common feature of advanced-stage HCC is the reduction or lack of expression of master genes of epithelial/hepatocyte differentiation, i.e. members of the liver enriched transcription factors (LEFTs) family like HNF4α, and conversely an increased expression of epithelial- to-mesenchymal transition (EMT) master genes, i.e. the transcriptional repressors belonging to the Snail family. Recently, it has emerged as members of these families are capable to directly repress each other and to regulate in opposite manner target genes involved in stemness and in hepatocyte differentiation, thus influencing cell outcome between epithelial/differentiated/poor aggressive and mesenchymal/undifferentiated/aggressive phenotype. Consequently, the restoration of LEFT functions in invasive HCC could represent an important goal for anti-cancer therapies. However, any strategy based on gene transfer needs to take in account the influence of micro-environmental factors in HCC tumor niche, like TGFβ, responsible for shifting the described balance in tumor cell towards the acquisition of stem-cell like properties and invasiveness, through Snail/EMT induction and LEFTs downregulation. The presence of this cytokine, indeed, was showed to override both anti-EMT and tumor suppressor activity of the ectopically expressed HNF4α protein. In this review, the rationale to propose implementation of HCC gene therapy will be discussed

TGFbeta overrides HNF4alpha tumor suppressing activity through GSK3beta inactivation: implication for gene therapy of HCC

The loss of transcriptional factor HNF4α is a predominant mechanism through which hepatocellular carcinoma (HCC) progress to a less differentiated and more aggressive phenotype and, therefore, HNF4α has been suggested as therapeutic tool for HCC gene therapy. The rationale of the proposed therapeutic strategy has been supported by several experimental data showing that its re-expression reduced tumor growth/invasivity in vitro and tumor formation in mice. Furthermore, we recently showed HNF4α expression in low-expressing HCC cell lines can directly hamper the epithelial-to-mesenchymal transition (EMT) program, through the transcriptional repression of EMT master genes, Snail and Slug (1).The tumor progression final output, however, comes not only from cell autonomous properties, acquired through mutations, but also from microenvironmental cues influencing tumor niche. The transforming growth factor β (TGFβ) is a major microenvironmental factor for HCC influencing tumor dedifferentiation, induction of EMT and acquisition of metastatic properties. Its pathway was found constitutively activated in HCC patients and correlated with a poor prognosis. In the attempt to investigate the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity, we demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α post-translational modifications (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 and PTM. The GSK3β chemical inhibition results, indeed, in HNF4α DNA binding impairment and correlates with its dephosphorylation while a constitutively active GSK3β mutant significantly impairs the TGFβ-induced inhibitory effect on HNF4α transcriptional and tumor suppressive activity. Overall, our data indicate that the presence of TGFβ represents a limit for the HNF4α-mediated gene therapy of HCC and suggest the need to develop a TGFβ-insensitive HNF4α protein in order to circumvent the influence of tumor microenvironment

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

New insights on the functional role of URG7 in the cellular response to ER stress

Hepatitis B virus (HBV) induces stress in the host cell endoplasmic reticulum and through HBx, a transactivating viral protein, activates the Unfolded Protein Response (UPR) pathways, favouring virus replication and cell survival1. Among others, HBx protein activates the expression of the Up- regulated Gene clone 7 (URG7), a protein localized into the ER2, with an anti-apoptotic activity, due to the PI3K/AKT signalling activation3. In order to shed light on the molecular mechanisms underlying URG7 activity, we studied its putative role in the ER stress response. Our main results demonstrate that URG7 is able to modulate the expression of UPR markers toward survival outcomes. The analysis of its interactome suggests that URG7 is able to interact with proteins involved in the synthesis, folding and protein degradation, in concert with an increase of total protein ubiquitination. All together, these data suggest that URG7 plays a pivotal role as a reliever of ER stress-induced apoptosis