The lncRNA HOTAIR: a pleiotropic regulator of epithelial cell plasticity.

The epithelial-to-mesenchymal transition (EMT) is a trans-differentiation process that endows epithelial cells with mesenchymal properties, including motility and invasion capacity; therefore, its aberrant reactivation in cancerous cells represents a critical step to gain a metastatic phenotype. The EMT is a dynamic program of cell plasticity; many partial EMT states can be, indeed, encountered and the full inverse mesenchymal-to-epithelial transition (MET) appears fundamental to colonize distant secondary sites. The EMT/MET dynamics is granted by a fine modulation of gene expression in response to intrinsic and extrinsic signals. In this complex scenario, long non-coding RNAs (lncRNAs) emerged as critical players. This review specifically focuses on the lncRNA HOTAIR, as a master regulator of epithelial cell plasticity and EMT in tumors. Molecular mechanisms controlling its expression in differentiated as well as trans-differentiated epithelial cells are highlighted here. Moreover, current knowledge about HOTAIR pleiotropic functions in regulation of both gene expression and protein activities are described. Furthermore, the relevance of the specific HOTAIR targeting and the current challenges of exploiting this lncRNA for therapeutic approaches to counteract the EMT are discussed.

Extracellular signal-Regulated Kinase 5 (ERK5) is required for the Yes-associated protein (YAP) co-transcriptional activity.

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration. However, recent observations indicate that YAP activity can be also modulated by Hippo independent/integrating pathways, still largely unexplored. In this study, we demonstrated the role of the extracellular signal-regulated kinase 5 (ERK5)/mitogen-activated protein kinase in the regulation of YAP activity. By means of ERK5 inhibition/silencing and overexpression experiments, and by using as model liver stem cells, hepatocytes, and hepatocellular carcinoma (HCC) cell lines, we provided evidence that ERK5 is required for YAP-dependent gene expression. Mechanistically, ERK5 controls the recruitment of YAP on promoters of target genes and its physical interaction with the transcriptional partner TEAD; moreover, it mediates the YAP activation occurring in cell adhesion, migration, and TGFß-induced EMT of liver cells. Furthermore, we demonstrated that ERK5 signaling modulates YAP activity in a LATS1/2-independent manner. Therefore, our observations identify ERK5 as a novel upstream Hippo-independent regulator of YAP activity, thus unveiling a new target for therapeutic approaches aimed at interfering with its function.

Exosome-Associated circRNAs as Key Regulators of EMT in Cancer.

Epithelial-to-mesenchymal transition (EMT) is a dynamic program of cell plasticity aberrantly reactivated in cancer. The crosstalk between tumor cells and the tumoral microenvironment (TME) has a pivotal importance for the induction of the EMT and the progression toward a malignant phenotype. Notably, exosomes are key mediators of this crosstalk as vehicles of specific molecular signals that include the class of circular RNAs (circRNAs). This review specifically focuses on the role of exosome-associated circRNAs as key regulators of EMT in cancer. The relevance of these molecules in regulating the intercellular communication in TME and tumor progression is highlighted. Moreover, the here-presented evidence indicates that exosome-associated circRNA modulation should be taken in account for cancer diagnostic and therapeutic approaches.

SYNCRIP Modulates the Epithelial-Mesenchymal Transition in Hepatocytes and HCC Cells.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) control gene expression by acting at multiple levels and are often deregulated in epithelial tumors; however, their roles in the fine regulation of cellular reprogramming, specifically in epithelial-mesenchymal transition (EMT), remain largely unknown. Here, we focused on the hnRNP-Q (also known as SYNCRIP), showing by molecular analysis that in hepatocytes it acts as a "mesenchymal" gene, being induced by TGFß and modulating the EMT. SYNCRIP silencing limits the induction of the mesenchymal program and maintains the epithelial phenotype. Notably, in HCC invasive cells, SYNCRIP knockdown induces a mesenchymal-epithelial transition (MET), negatively regulating their mesenchymal phenotype and significantly impairing their migratory capacity. In exploring possible molecular mechanisms underlying these observations, we identified a set of miRNAs (i.e., miR-181-a1-3p, miR-181-b1-3p, miR-122-5p, miR-200a-5p, and miR-let7g-5p), previously shown to exert pro- or anti-EMT activities, significantly impacted by SYNCRIP interference during EMT/MET dynamics and gathered insights, suggesting the possible involvement of this RNA binding protein in their transcriptional regulation.

Design and Functional Validation of a Mutant Variant of the LncRNA HOTAIR to Counteract Snail Function in Epithelial-to-Mesenchymal Transition.

HOTAIR is a lncRNA overexpressed in several epithelial cancers and strongly correlated with invasion. This lncRNA was proven a pivotal element of the epithelial-to-mesenchymal transition (EMT), a transdifferentiation process triggering metastasis. Snail, master inducer of EMT, requires HOTAIR to recruit EZH2 on specific epithelial target genes (i.e., HNF4α, E-cadherin, and HNF1α) and cause their repression. Here, we designed a HOTAIR deletion mutant form, named HOTAIR-sbid, including the putative Snail-binding domain but depleted of the EZH2-binding domain. HOTAIR-sbid acted as a dominant negative of the endogenous HOTAIR. In both murine and human tumor cells, HOTAIR-sbid impaired the ability of HOTAIR to bind Snail and, in turn, trigger H3K27me3/EZH2-mediated repression of Snail epithelial target genes. Notably, HOTAIR-sbid expression was proven to reduce cellular motility, invasiveness, anchorage-independent growth, and responsiveness to TGFß-induced EMT. These data provide evidence on a lncRNA-based strategy to effectively impair the function of a master EMT-transcriptional factor. SIGNIFICANCE: This study defines an innovative RNA-based strategy to interfere with a pivotal function of the tumor-related lncRNA HOTAIR, comprising a dominant negative mutant that was computationally designed and that impairs epithelial-to-mesenchymal transition.

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.

The lncRNA HOTAIR transcription is controlled by HNF4α-induced chromatin topology modulation.

The expression of the long noncoding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is largely deregulated in epithelial cancers and positively correlates with poor prognosis and progression of hepatocellular carcinoma and gastrointestinal cancers. Furthermore, functional studies revealed a pivotal role for HOTAIR in the epithelial-to-mesenchymal transition, as this RNA is causal for the repressive activity of the master factor SNAIL on epithelial genes. Despite the proven oncogenic role of HOTAIR, its transcriptional regulation is still poorly understood. Here hepatocyte nuclear factor 4-α (HNF4α), as inducer of epithelial differentiation, was demonstrated to directly repress HOTAIR transcription in the mesenchymal-to epithelial transition. Mechanistically, HNF4α was found to cause the release of a chromatin loop on HOTAIR regulatory elements thus exerting an enhancer-blocking activity.

Exosome-Mediated Signaling in Epithelial to Mesenchymal Transition and Tumor Progression.

Growing evidence points to exosomes as key mediators of cell⁻cell communication, by transferring their specific cargo (e.g., proteins, lipids, DNA and RNA molecules) from producing to receiving cells. In cancer, the regulation of the exosome-mediated intercellular communication may be reshaped, inducing relevant changes in gene expression of recipient cells in addition to microenvironment alterations. Notably, exosomes may deliver signals able to induce the transdifferentiation process known as Epithelial-to-Mesenchymal Transition (EMT). In this review, we summarize recent findings on the role of exosomes in tumor progression and EMT, highlighting current knowledge on exosome-mediated intercellular communication in tumor-niche establishment, migration, invasion, and metastasis processes. This body of evidence suggests the relevance of taking into account exosome-mediated signaling and its multifaceted aspects to develop innovative anti-tumoral therapeutic approaches.

The lncRNA H19 positively affects the tumorigenic properties of glioblastoma cells and contributes to NKD1 repression through the recruitment of EZH2 on its promoter.

The still largely obscure molecular events in the glioblastoma oncogenesis, a primary brain tumor characterized by an inevitably dismal prognosis, impel for investigation. The importance of Long noncoding RNAs as regulators of gene expression has recently become evident. Among them, H19 has a recognized oncogenic role in several types of human tumors and was shown to correlate to some oncogenic aspects of glioblastoma cells. Here we, hypothesyze that in glioblastoma H19 exerts its function through the interaction with the catalytic subunit of the PRC2 complex, EZH2. By employing a factor analysis on a SAGE dataset of 12 glioblastoma samples, we show that H19 expression in glioblastoma tissues correlates with that of several genes involved in glioblastoma growth and progression. H19 knock-down reduces viability, migration and invasiveness of two distinct human glioblastoma cell lines. Most importantly, we provide a mechanistic perspective about the role of H19 in glioblastoma cells, by showing that its expression is inversely linked to that of NKD1, a negative regulator of Wnt pathway, suggesting that H19 might regulate NKD1 transcription via EZH2-induced H3K27 trimethylation of its promoter. Indeed, we showed that H19 binds EZH2 in glioblastoma cells, and that EZH2 binding to NKD1 and other promoters is impaired by H19 silencing. In this work we describe H19 as part of an epigenetic modulation program executed by EZH2, that results in the repression of Nkd1. We believe that our results can provide a new piece to the complex puzzle of H19 function in glioblastoma.

A cryptic RNA-binding domain mediates Syncrip recognition and exosomal partitioning of miRNA targets.

Exosomal miRNA transfer is a mechanism for cell-cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip's amino-terminal domain, which was previously thought to mediate protein-protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip's RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5' to this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences.

Functional Roles and Therapeutic Applications of Exosomes in Hepatocellular Carcinoma.

Exosomes are important in intercellular communication. They assure the horizontal transfer of specific functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA) from donor to recipient cells. Notably, tumor-derived exosomes (TDEs) appear to be an important vehicle of specific signals in cancer, impacting on tumor growth and metastasis. Recent researches point to the characterization of exosomes in Hepatocellular Carcinoma (HCC), the major adult liver malignancy. In this review, we summarize current findings on HCC exosomes, focusing on the identification of noncoding RNAs as exosome-enriched functional regulators and new potential biomarkers. The great potential of exosomes in future HCC diagnostic and therapeutic approaches is underlined.

The RNA-Binding Protein SYNCRIP Is a Component of the Hepatocyte Exosomal Machinery Controlling MicroRNA Sorting.

Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is deregulated in pathological conditions, the mechanisms controlling specific RNA sorting into extracellular vesicles are still poorly understood. Here, we identified the RNA binding protein SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein; also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP knockdown impairs sorting of miRNAs in exosomes. Furthermore, SYNCRIP directly binds to specific miRNAs enriched in exosomes sharing a common extra-seed sequence (hEXO motif). The hEXO motif has a role in the regulation of miRNA localization, since embedment of this motif into a poorly exported miRNA enhances its loading into exosomes. This evidence provides insights into the mechanisms of miRNA exosomal sorting process. Moreover, these findings open the way for the possible selective modification of the miRNAs exosomal cargo.

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.

Molecular Mechanisms Underlying Peritoneal EMT and Fibrosis.

Peritoneal dialysis is a form of renal replacement alternative to the hemodialysis. During this treatment, the peritoneal membrane acts as a permeable barrier for exchange of solutes and water. Continual exposure to dialysis solutions, as well as episodes of peritonitis and hemoperitoneum, can cause acute/chronic inflammation and injury to the peritoneal membrane, which undergoes progressive fibrosis, angiogenesis, and vasculopathy, eventually leading to discontinuation of the peritoneal dialysis. Among the different events controlling this pathological process, epithelial to mesenchymal transition of mesothelial cells plays a main role in the induction of fibrosis and in subsequent functional deterioration of the peritoneal membrane. Here, the main extracellular inducers and cellular players are described. Moreover, signaling pathways acting during this process are elucidated, with emphasis on signals delivered by TGF-ß family members and by Toll-like/IL-1ß receptors. The understanding of molecular mechanisms underlying fibrosis of the peritoneal membrane has both a basic and a translational relevance, since it may be useful for setup of therapies aimed at counteracting the deterioration as well as restoring the homeostasis of the peritoneal membrane.

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 TGFß-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.

Epigenetic regulation in hepatocellular carcinoma requires long noncoding RNAs.

Recent evidence has proven the relevance of epigenetic changes in the development of hepatocellular carcinoma (HCC), the major adult liver malignancy. Moreover, HCC onset and progression correlate with the deregulation of several long noncoding RNAs (lncRNAs), exhibiting great biological significance. As discussed in this review, many of these transcripts are able to specifically act as tumor suppressors or oncogenes by means of their role as molecular platforms. Indeed, these lncRNAs are able to bind and recruit epigenetic modifiers on specific genomic loci, ultimately resulting in regulation of the gene expression relevant in cancer development. The evidence presented in this review highlights that lncRNAs-mediated epigenetic regulation should be taken into account for potential targeted therapeutic approaches.

Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte.

The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumours), is mainly because of the phenomenon of epithelial-to-mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non-tissue-specific, master transcriptional regulators. The reverse process, the mesenchymal-to-epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus must include tissue-specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. In particular, we will focus on hepatocyte and describe the pivotal role in the control of EMT/MET dynamics exerted by a tissue-specific molecular mini-circuitry. Recent evidence, indeed, highlighted as two transcriptional factors, the master gene of EMT Snail, and the master gene of hepatocyte differentiation HNF4α, exhorting a direct reciprocal repression, act as pivotal elements in determining opposite cellular outcomes. The different balances between these two master regulators, further integrated by specific microRNAs, in fact, were found responsible for the EMT/METs dynamics as well as for the preservation of both hepatocyte and stem/precursor cells identity and differentiation. Overall, these findings impact the maintenance of stem cells and differentiated cells both in in vivo EMT/MET physio-pathological processes as well as in culture.

TGFß overrides HNF4α tumor suppressing activity through GSK3ß 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 ß (TGFß) 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 TGFß 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. GSK3ß activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS: We demonstrated that the presence of TGFß impairs the efficiency of HNF4α as tumor suppressor. We found that TGFß induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3ß kinase as one of the TGFß targets mediating HNF4α functional inactivation: GSK3ß chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3ß mutant impairs the TGFß-induced inhibitory effect on HNF4α tumor suppressor activity. CONCLUSIONS: Our data identify in the dominance of TGFß a limit for the HNF4α-mediated gene therapy of HCC.