ADAMTS12 is a stromal modulator in chronic liver disease.

Adamalysins, a family of metalloproteinases containing a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTSs), belong to the matrisome and play important roles in various biological and pathological processes, such as development, immunity and cancer. Using a liver cancer dataset from the International Cancer Genome Consortium, we developed an extensive in silico screening that identified a cluster of adamalysins co-expressed in livers from patients with hepatocellular carcinoma (HCC). Within this cluster, ADAMTS12 expression was highly associated with recurrence risk and poorly differentiated HCC signatures. We showed that ADAMTS12 was expressed in the stromal cells of the tumor and adjacent fibrotic tissues of HCC patients, and more specifically in activated stellate cells. Using a mouse model of carbon tetrachloride-induced liver injury, we showed that Adamts12 was strongly and transiently expressed after a 24 h acute treatment, and that fibrosis was exacerbated in Adamts12-null mice submitted to carbon tetrachloride-induced chronic liver injury. Using the HSC-derived LX-2 cell line, we showed that silencing of ADAMTS12 resulted in profound changes of the gene expression program. In particular, genes previously reported to be induced upon HSC activation, such as PAI-1, were mostly down-regulated following ADAMTS12 knock-down. The phenotype of these cells was changed to a less differentiated state, showing an altered actin network and decreased nuclear spreading. These phenotypic changes, together with the down-regulation of PAI-1, were offset by TGF-ß treatment. The present study thus identifies ADAMTS12 as a modulator of HSC differentiation, and a new player in chronic liver disease.

ADAM and ADAMTS Proteins, New Players in the Regulation of Hepatocellular Carcinoma Microenvironment.

The tumor microenvironment plays a major role in tumor growth, invasion and resistance to chemotherapy, however understanding how all actors from microenvironment interact together remains a complex issue. The tumor microenvironment is classically represented as three closely connected components including the stromal cells such as immune cells, fibroblasts, adipocytes and endothelial cells, the extracellular matrix (ECM) and the cytokine/growth factors. Within this space, proteins of the adamalysin family (ADAM for a disintegrin and metalloproteinase; ADAMTS for ADAM with thrombospondin motifs; ADAMTSL for ADAMTS-like) play critical roles by modulating cell-cell and cell-ECM communication. During last decade, the implication of adamalysins in the development of hepatocellular carcinoma (HCC) has been supported by numerous studies however the functional characterization of most of them remain unsettled. In the present review we propose both an overview of the literature and a meta-analysis of adamalysins expression in HCC using data generated by The Cancer Genome Atlas (TCGA) Research Network.

Integration of miRNA-regulatory networks in hepatic stellate cells identifies TIMP3 as a key factor in chronic liver disease.

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSC) is a critical process involved in liver fibrosis. Several miRNAs are implicated in gene regulation during this process but their exact and respective contribution is still incompletely understood. Here we propose an integrative approach of miRNA-regulatory networks to predict new targets. METHODS: miRNA regulatory networks in activated HSCs were built using lists of validated miRNAs and the CyTargetLinker tool. The resulting graphs were filtered according to public transcriptomic data and the reduced graphs were analysed through GO annotation. A miRNA network regulating the expression of TIMP3 was further studied in human liver samples, isolated hepatic cells and mouse model of liver fibrosis. RESULTS: Within the up-regulated miRNAs, we identified a subnetwork of five miRNAs (miR-21-5p, miR-222-3p, miR-221-3p miR-181b-5p and miR-17-5p) that target TIMP3. We demonstrated that TIMP3 expression is inversely associated with inflammatory activity and IL1-ß expression in vivo. We further showed that IL1-ß inhibits TIMP3 expression in HSC-derived LX-2 cells. Using data from The Cancer Genome Atlas (TCGA), we showed that, in hepatocellular carcinoma (HCC), TIMP3 expression is associated with survival (P < .001), while miR-221 (P < .05), miR-222 (P < .01) and miR-181b (P < .01) are markers for a poor prognosis. CONCLUSIONS: Several miRNAs targeting TIMP3 are up-regulated in activated HSCs and down-regulation of TIMP3 expression is associated with inflammatory activity in liver fibrosis and poor prognosis in HCC. The regulatory network including specific miRNAs and TIMP3 is therefore central for the evolution of chronic liver disease.

A pipeline to create predictive functional networks: application to the tumor progression of hepatocellular carcinoma.

BACKGROUND: Integrating genome-wide gene expression patient profiles with regulatory knowledge is a challenging task because of the inherent heterogeneity, noise and incompleteness of biological data. From the computational side, several solvers for logic programs are able to perform extremely well in decision problems for combinatorial search domains. The challenge then is how to process the biological knowledge in order to feed these solvers to gain insights in a biological study. It requires formalizing the biological knowledge to give a precise interpretation of this information; currently, very few pathway databases offer this possibility. RESULTS: The presented work proposes an automatic pipeline to extract automatically regulatory knowledge from pathway databases and generate novel computational predictions related to the state of expression or activity of biological molecules. We applied it in the context of hepatocellular carcinoma (HCC) progression, and evaluate the precision and the stability of these computational predictions. Our working base is a graph of 3383 nodes and 13,771 edges extracted from the KEGG database, in which we integrate 209 differentially expressed genes between low and high aggressive HCC across 294 patients. Our computational model predicts the shifts of expression of 146 initially non-observed biological components. Our predictions were validated at 88% using a larger experimental dataset and cross-validation techniques. In particular, we focus on the protein complexes predictions and show for the first time that NFKB1/BCL-3 complexes are activated in aggressive HCC. In spite of the large dimension of the reconstructed models, our analyses over the computational predictions discover a well constrained region where KEGG regulatory knowledge constrains gene expression of several biomolecules. These regions can offer interesting windows to perturb experimentally such complex systems. CONCLUSION: This new pipeline allows biologists to develop their own predictive models based on a list of genes. It facilitates the identification of new regulatory biomolecules using knowledge graphs and predictive computational methods. Our workflow is implemented in an automatic python pipeline which is publicly available at https://github.com/LokmaneChebouba/key-pipeand contains as testing data all the data used in this paper.

Proteomic screening identifies the zonula occludens protein ZO-1 as a new partner for ADAM12 in invadopodia-like structures.

The epithelial mesenchymal transition (EMT) is a key process for cancer cell invasion and migration. This complex program whereby epithelial tumor cells loose polarity and acquire mesenchymal phenotype is driven by the regulation of cell-cell adhesion and cell-substrate interactions. We recently described the association of ADAM12 with EMT and we now use immunoprecipitation and proteomic approaches to identify interacting partners for ADAM12 during EMT. We identify twenty proteins that are involved in molecular mechanisms associated with adhesion/invasion processes. Integrative network analyses point out the zonula occludens protein ZO-1, as a new potential partner for ADAM12. In silico screening demonstrates that ZO-1 and ADAM12 are coexpressed in breast cancer cell lines sharing EMT signature. We validate the interaction between ZO-1 and ADAM12 in invasive breast cancer cell lines and show that ZO-1 and ADAM12 co-localize in actin- and cortactin-rich structures. Silencing either ADAM12 or ZO-1 inhibits gelatin degradation demonstrating that both proteins are required for matrix degradation. We further show that matrix metalloprotease 14, known to mediate degradation of collagen in invadopodia-like structures interacts with ZO-1. Depletion of PKCε that regulates the recruitment of ADAM12 and ZO-1 to cell membranes induces a decrease in ADAM12 and ZO-1 at invadopodia-like structures and degradation activity. Together our data provide evidence for a new interaction between ADAM12, a mesenchymal marker induced during TGF-ß-dependent EMT and ZO-1, a scaffolding protein expressed in tight junctions of epithelial cells, both proteins being redistributed at the invadopodia-like structures of mesenchymal invasive cells to promote PKCε-dependent matrix degradation.

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis.

Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

A posttranscriptional mechanism that controls Ptbp1 abundance in the Xenopus epidermis.

The output of alternative splicing depends on the cooperative or antagonistic activities of several RNA-binding proteins (RBPs), like Ptbp1 and Esrp1 in Xenopus. Fine-tuning of the RBP abundance is therefore of prime importance to achieve tissue- or cell-specific splicing patterns. Here, we addressed the mechanisms leading to the high expression of the ptbp1 gene, which encodes Ptbp1, in Xenopus epidermis. Two splice isoforms of ptbp1 mRNA differ by the presence of an alternative exon 11, and only the isoform including exon 11 can be translated to a full-length protein. In vivo minigene assays revealed that the nonproductive isoform was predominantly produced. Knockdown experiments demonstrated that Esrp1, which is specific to the epidermis, strongly stimulated the expression of ptbp1 by favoring the productive isoform. Consequently, knocking down esrp1 phenocopied ptbp1 inactivation. Conversely, Ptbp1 repressed the expression of its own gene by favoring the nonproductive isoform. Hence, a complex posttranscriptional mechanism controls Ptbp1 abundance in Xenopus epidermis: skipping of exon 11 is the default splicing pattern, but Esrp1 stimulates ptbp1 expression by favoring the inclusion of exon 11 up to a level that is limited by Ptbp1 itself. These results decipher a posttranscriptional mechanism that achieves various abundances of the ubiquitous RBP Ptbp1 in different tissues.

Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities.

Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.

Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down-regulation.

CUGBP1 is an RNA-binding protein controlling alternative splicing, mRNA translation and stability. In this work we used a motif scoring approach to identify putative CUGBP1 binding sites for genes located on the human chromosome 12. This allowed us to identify the gene CD9 as a presumptive target for CUGBP1-mediated regulation. In a number of cancers, the tetraspanin CD9 is down-regulated, an event correlated with a bad prognostic. Using a combination of biochemical approaches and CUGBP1 knockdown, we showed that CUGBP1 directly controls CD9 expression.

Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis.

Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.

Multiple roles of Condensins: a complex story.

Condensins are pentameric complexes that were initially described as being involved in the dynamics of chromosomes during mitosis. It has been recently established that two related complexes (Condensin I and Condensin II) contribute to this process. An increasing sum of studies, using different approaches in various organisms, leads to the paradigm that Condensins are required for the correct segregation of replicated chromosomes by cooperating somehow with Topoisomerase II in sister chromatid resolution. Depending on species and/or experimental studies, these complexes also contribute to some aspects of the assembly and compaction of mitotic chromosomes. Recent studies provided evidences that Condensins and related complexes also function in non-mitotic processes such as replication and transcription. Biochemical studies have highlighted mechanistic aspects of Condensin function and initiated a fine functional dissection of core and regulatory subunits. However, the exact contribution of each subunit remains largely elusive as well as the functional interplay between Condensin I and Condensin II.

Introduction to chromosome dynamics in mitosis.

To ensure that the genetic information, replicated in the S-phase of the cell cycle, is correctly distributed between daughter cells at mitosis, chromatin duplication and chromosome segregation are highly regulated events. Since the early 1980's, our knowledge of the mechanisms governing these two events has greatly increased due to the use of genetic and biochemical approaches. We present here, first, an overview of the replication process, highlighting molecular aspects involved in coupling replication with chromatin dynamics in mitosis. The second part will present the current understanding of chromosome condensation and segregation during mitosis in higher eukaryotes. Finally, we will underline the links that exist between replication and mitosis.

Expression and functional dynamics of the XCAP-D2 condensin subunit in Xenopus laevis oocytes.

The 13 S condensin complex plays a crucial role in the condensation and segregation of the two sets of chromosomes during mitosis in vivo as well as in cell-free extracts. This complex, conserved from yeast to human, contains a heterodimer of structural maintenance of chromosome (SMC) family proteins and three additional non-SMC subunits. We have investigated the expression of the non-SMC condensin component XCAP-D2 in Xenopus laevis oocytes. When studied during meiotic maturation, XCAP-D2 starts to accumulate at the time of germinal vesicle breakdown and reaches its maximal amount in metaphase II oocytes. This accumulation is specifically blocked by injection of antisense oligonucleotides. XCAP-D2 antisense-injected oocytes progress normally through meiosis until metaphase II. At this stage, however, chromosomes exhibit architecture defaults, and resolution of sister chromatids is impaired. Surprisingly, in mitotic extracts made from XCAP-D2 knocked-down oocytes, sperm chromatin normally condenses into compacted chromosomes, whereas the amounts of both free and chromosome-bound XCAP-D2 are markedly reduced. This apparent discrepancy is discussed in light of current knowledge on chromosome dynamics.

Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis condensin complex in interphase cells.

Cell cycle dynamics and localization of condensins--multiprotein complexes involved in late stages of mitotic chromosome condensation--were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to beta-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF, fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.

c-Jun ARE targets mRNA deadenylation by an EDEN-BP (embryo deadenylation element-binding protein)-dependent pathway.

In mammalian cells, certain mRNAs encoding cytokines or proto-oncogenes are especially unstable, because of the presence of a particular sequence element in their 3'-untranslated region named ARE (A/U-rich element). AREs cause this instability by provoking the rapid shortening of the poly(A) tail of the mRNA. The deadenylation of mRNAs mediated by AREs containing repeats of the AUUUA motif (class I/II AREs) is conserved in Xenopus embryos. Here, we first extend these observations by showing that c-Jun ARE, a representative of class III (non-AUUUA) AREs, also provokes the deadenylation of a reporter RNA in Xenopus embryos. Next, by immunodepletion and immunoneutralization experiments, we show that, in Xenopus, the rapid deadenylation of RNAs that contain the c-Jun ARE, but not an AUUUA ARE, requires EDEN-BP. This RNA-binding protein was previously shown to provoke the rapid deadenylation of certain Xenopus maternal RNAs. Finally, we show that CUG-BP, the human homologue of EDEN-BP, specifically binds to c-Jun ARE. Together, these results identify CUG-BP as a plausible deadenylation factor responsible for the post-transcriptional control of c-Jun proto-oncogene mRNA in mammalian cells.