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.

Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications.

Collagen plays a critical role in regulating breast cancer progression and therapeutic resistance. An improved understanding of both the features and drivers of tumor-permissive and -restrictive collagen matrices are critical to improve prognostication and develop more effective therapeutic strategies. In this study, using a combination of in vitro, in vivo and in silico experiments, we show that type III collagen (Col3) plays a tumor-restrictive role in human breast cancer. We demonstrate that Col3-deficient, human fibroblasts produce tumor-permissive collagen matrices that drive cell proliferation and suppress apoptosis in noninvasive and invasive breast cancer cell lines. In human TNBC biopsy samples, we demonstrate elevated deposition of Col3 relative to type I collagen (Col1) in noninvasive compared to invasive regions. Similarly, in silico analyses of over 1000 breast cancer patient biopsies from The Cancer Genome Atlas BRCA cohort revealed that patients with higher Col3:Col1 bulk tumor expression had improved overall, disease-free and progression-free survival relative to those with higher Col1:Col3 expression. Using an established 3D culture model, we show that Col3 increases spheroid formation and induces formation of lumen-like structures that resemble non-neoplastic mammary acini. Finally, our in vivo study shows co-injection of murine breast cancer cells (4T1) with rhCol3-supplemented hydrogels limits tumor growth and decreases pulmonary metastatic burden compared to controls. Taken together, these data collectively support a tumor-suppressive role for Col3 in human breast cancer and suggest that strategies that increase Col3 may provide a safe and effective modality to limit recurrence in breast cancer patients.

Cell morphology and mechanosensing can be decoupled in fibrous microenvironments and identified using artificial neural networks.

Cells interpret cues from and interact with fibrous microenvironments through the body based on the mechanics and organization of these environments and the phenotypic state of the cell. This in turn regulates mechanoactive pathways, such as the localization of mechanosensitive factors. Here, we leverage the microscale heterogeneity inherent to engineered fiber microenvironments to produce a large morphologic data set, across multiple cells types, while simultaneously measuring mechanobiological response (YAP/TAZ nuclear localization) at the single cell level. This dataset describing a large dynamic range of cell morphologies and responses was coupled with a machine learning approach to predict the mechanobiological state of individual cells from multiple lineages. We also noted that certain cells (e.g., invasive cancer cells) or biochemical perturbations (e.g., modulating contractility) can limit the predictability of cells in a universal context. Leveraging this finding, we developed further models that incorporate biochemical cues for single cell prediction or identify individual cells that do not follow the established rules. The models developed here provide a tool for connecting cell morphology and signaling, incorporating biochemical cues in predictive models, and identifying aberrant cell behavior at the single cell level.

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.

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

[This corrects the article DOI: 10.18632/oncotarget.25106.].

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.

Cellular Cholesterol Distribution Influences Proteolytic Release of the LRP-1 Ectodomain.

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional matricellular receptor composed of a large ligand-binding subunit (515-kDa α-chain) associated with a short trans-membrane subunit (85-kDa ß-chain). LRP-1, which exhibits both endocytosis and cell signaling properties, plays a key role in tumor invasion by regulating the activity of proteinases such as matrix metalloproteinases (MMPs). LRP-1 is shed at the cell surface by proteinases such as membrane-type 1 MMP (MT1-MMP) and a disintegrin and metalloproteinase-12 (ADAM-12). Here, we show by using biophysical, biochemical, and cellular imaging approaches that efficient extraction of cell cholesterol and increased LRP-1 shedding occur in MDA-MB-231 breast cancer cells but not in MDA-MB-435 cells. Our data show that cholesterol is differently distributed in both cell lines; predominantly intracellularly for MDA-MB-231 cells and at the plasma membrane for MDA-MB-435 cells. This study highlights the relationship between the rate and cellular distribution of cholesterol and its impact on LRP-1 shedding modulation. Altogether, our data strongly suggest that the increase of LRP-1 shedding upon cholesterol depletion induces a higher accessibility of the sheddase substrate, i.e., LRP-1, at the cell surface rather than an increase of expression of the enzyme.