. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up-regulated by transforming growth factor ␤ (TGF-␤) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-␤, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3-and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40-and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression. I ncreased expression and activities of matrix metalloproteinase (MMP) has been shown widely in malignant phenotypes facilitating the breakdown of extracellular matrix component and cell evasion, but also unmasking bioactive cryptic fragments and releasing active growth factors which, in turn, favor tumor growth. 1 The ADAMs (a disintegrin and metalloproteinase-containing proteins) are a family of multidomain glycoproteins highly homologous to the class III snake venom metalloprotease-disintegrins. 2 The common extracellular part of the proteins includes a regulatory prodomain and metalloprotease, disintegrin-like and cystein-rich domains. Further, ADAMs are characterized by an epidermal growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic tail. More than 30 members have been identified in the ADAM family with a broad tissue distribution and have been involved in specific cellular processes including sperm-egg interaction, 3 myocyte fusion, 4 neurogenesis, 5 and adipogenesis. 6 Recently, ADAM12 gene therapy was shown to rescue the pathology of mdx-gene deficient dystrophic mice.

Hepatocellular carcinomas (HCCs) exhibit a diversity of molecular phenotypes, raising major challenges in clinical management. HCCs detected by surveillance programs at an early stage are candidates for potentially curative therapies (local ablation, resection, or transplantation). In the long term, transplantation provides the lowest recurrence rates. Treatment allocation is based on tumor number, size, vascular invasion, performance status, functional liver reserve, and the prediction of early (<2 years) recurrence, which reflects the intrinsic aggressiveness of the tumor. Well‐differentiated, potentially low‐aggressiveness tumors form the heterogeneous molecular class of nonproliferative HCCs, characterized by an approximate 50% β‐catenin mutation rate. To define the clinical, pathological, and molecular features and the outcome of nonproliferative HCCs, we constructed a 1,133‐HCC transcriptomic metadata set and validated findings in a publically available 210‐HCC RNA sequencing set. We show that nonproliferative HCCs preserve the zonation program that distributes metabolic functions along the portocentral axis in normal liver. More precisely, we identified two well‐differentiated, nonproliferation subclasses, namely periportal‐type (wild‐type β‐catenin) and perivenous‐type (mutant β‐catenin), which expressed negatively correlated gene networks. The new periportal‐type subclass represented 29% of all HCCs; expressed a hepatocyte nuclear factor 4A–driven gene network, which was down‐regulated in mouse hepatocyte nuclear factor 4A knockout mice; were early‐stage tumors by Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, and tumor–node–metastasis staging systems; had no macrovascular invasion; and showed the lowest metastasis‐specific gene expression levels and TP53 mutation rates. Also, we identified an eight‐gene periportal‐type HCC signature, which was independently associated with the highest 2‐year recurrence‐free survival by multivariate analyses in two independent cohorts of 247 and 210 patients. Conclusion: Well‐differentiated HCCs display mutually exclusive periportal or perivenous zonation programs. Among all HCCs, periportal‐type tumors have the lowest intrinsic potential for early recurrence after curative resection. (Hepatology 2017;66:1502–1518).

Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix components. In fibrotic livers, there is a markedly increased activity of matrix metalloproteinase 2 (MMP2), a major enzyme involved in extracellular matrix remodeling. We have previously shown that hepatic stellate cells secrete latent MMP2 and that MMP2 activation occurs in coculture of hepatic stellate cells and hepatocytes concomitantly with matrix deposition. In the present work we investigated the effects of various extracellular matrix components and concanavalin A, an inducer of immunemediated liver injuries, on MMP2 activation in cultured human hepatic stellate cells. Collagen I induced a dosedependent MMP2 activation, which was not blocked by both actinomycin and cycloheximide. Collagen VI, laminin, and a reconstituted basement membrane (matrigel) were ineffective in inducing activation. Specific antibodies against the subunits of ␣2␤1 integrins, the major collagen I receptor, induced partial inhibition of MMP2 activation. Fibrosis is the common response of chronic liver injury from various origins, including metabolic diseases, viral infections, and alcohol consumption. 1 Fibrosis is often associated with inflammatory infiltrates and regulated through an immune-mediated response. Hepatic stellate cells (HSC) play a pivotal role in the cellular and molecular events that lead to fibrosis. 2 Following liver injury, these cells undergo changes toward a myofibroblast-like phenotype, they proliferate and secrete abundant extracellular matrix (ECM) components. In addition, activated HSC are involved in ECM degradation by providing matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). 3 MMPs form a family of proteases, which are collectively capable of degrading most if not all ECM components. 4 They can be divided into groups of intersticial collagenases, gelatinases, stromelysins, elastases, and membrane-type matrix metalloproteinases (MT-MMPs) according to their substrate specifities and structural features. The 5 currently characterized MT-MMPs differ from all other MMPs by having transmembrane domains and short cytoplasmic tails. 5 Gelatinase A, (i.e., MMP2) degrades gelatin, types IV, V, VII, and X collagens and elastin, fibronectin, and vitronectin, and its activity is associated with ECM remodeling in wound healing, development, inflammation, fibrosis, angiogenesis, and tumor invasion. 6 MMP2 is secreted as an inactive proenzyme, which is activated by a membrane-linked process that involves MT1-MMP 5 and tissue inhibitor of metalloproteinases-2 (TIMP2). 7 MT1-MMP complexed with TIMP2 has been found to serve as a cell surface receptor for MMP2. 8 The activation of bound MMP2 has been proposed to occur through an initial cleavage by other (free) MT1-MMP molecules to an intermediate form followed by autoproteolytic activation of MMP2 to a fully active enzyme. An excess of TIMP2 inhibits MMP2 activation by binding to all MT1-MMP m...

Type XVIII collagen is a recently discovered nonfibrillar collagen associated with basement membranes in mice and expressed at high levels in human liver. We studied the origin, distribution, and RNA levels of type XVIII collagen in normal and fibrotic human livers by in situ hybridization, immunohistochemistry, and Northern and dot blots and compared procollagen ␣1(XVIII) RNA levels with those of procollagen ␣1(IV) and laminin ␥1, the two major components of liver basement membranes. In normal liver, type XVIII collagen was heavily deposited in perisinusoidal spaces and basement membrane zones. The major source of type XVIII collagen was hepatocytes and, to a lesser extent, endothelial, biliary epithelial, and vascular smooth muscle cells and peripheral nerves. In cirrhosis, type XVIII collagen formed a thick deposit along capillarized sinusoids. Grain counts after in situ hybridization showed myofibroblasts to increase their expression 13-fold in active and twofold in quiescent fibrosis, whereas hepatocytes increased their expression only twofold in both active and quiescent fibrosis. Activated stellate cells in vitro expressed type XVIII collagen at high levels. These data indicate that type XVIII collagen is a component of the perisinusoidal space and is associated with basement membrane remodeling. Hepatocytes and activated stellate cells are important sources of type XVIII collagen in normal and fibrotic liver respectively, which suggests tissue-specific regulation of its expression. (HEPATOLOGY 1998;28:98-107.)The collagen superfamily comprises at least 19 molecules that share repeating -Gly-X-Y-sequences and fold into triplehelical structures. 1,2 Specific collagen types arrange within extracellular matrices in precise aggregates, maintaining a delicate equilibrium in specialized tissues. 1,3,4 In the liver, collagen breakdown and deposition occur in fibrosis and cirrhosis as a result of the cooperation of injured or regenerating hepatocytes with stellate cells. Activated stellate cells are a major source of extracellular matrix proteins in fibrotic liver. 5-10 Indeed, upon activation during liver injury, stellate cells undergo phenotypic changes characteristic of myofibroblasts, express ␣-smooth muscle actin (␣-SMA), and produce extracellular matrix components at high levels. 11,12 The newly discovered type XVIII collagen [13][14][15] belongs to the nonfibrilar collagens, with frequent interruptions in the triple-helical structure, which possibly confer flexibility to the molecules. Type XVIII collagen is present in highly vascularized organs such as the liver, lung, kidney, and placenta. [16][17][18] Muragaki et al. 17 detected type XVIII collagen in vascular basement membrane zones in mice, and it has been hypothesized that this collagen type could be a new component of basement membranes, 19 the highly specialized extracellular matrices underlying epithelial or endothelial cells and surrounding muscle cells, fat, and peripheral nerve cells. Three variant N-terminal ends for mouse procollagen ␣1(XVIII...

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (Mr = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate.

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Matrix metalloproteinase-2 (MMP2) activation is associated with basement membrane remodeling that occurs in injured tissues and during tumor invasion. The newly described membrane-type MMPs (MT-MMPs) form a family of potential MMP2 activators. We investigated the localization and steady-state levels of MT1-MMP and MT2-MMP mRNA, compared with those of MMP2 and tissue inhibitor of MMP-2 in 22 hepatocellular carcinomas, 12 liver metastases from colonic adenocarcinomas, 13 nontumoral samples from livers with metastases, 10 benign tumors, and 6 normal livers. MMP2 activation was analyzed by zymography in the same series. The expression of MT1-MMP mRNA and the activation of MMP-2 were increased in hepatocellular carcinomas, metastases, and cholestatic nontumoral samples. MT2-MMP mRNA was rather stable in the different groups. MT1-MMP mRNA levels, but not MT2-MMP mRNA, correlated with MMP-2 and tissue inhibitor of MMP-2 mRNA levels and with MMP2 activation. In situ hybridization showed that MT1-MMP mRNA was expressed in stromal cells, and MT2-MMP mRNA was principally located in both hepatocytes and biliary epithelial cells. Consistently, freshly isolated hepatocytes expressed only MT2-MMP mRNA, and culture-activated hepatic stellate cells showed high levels of MT1-MMP mRNA. These results indicate that in injured livers, MMP2 activation is related to a coordinated high expression of MMP2, tissue inhibitor of MMP-2, and MT1-MMP. Furthermore, the finding of a preferential expression of MT2-MMP in hepatocytes, together with our previous demonstration that the activation of stellate cell-derived MMP2 in co-culture requires interactions with hepatocytes (Am J Pathol 1997, 150:51-58), suggests that parenchymal cells might play a pivotal role in the MMP2 activation process.

Constitutive activation of Wnt/b-catenin signaling in cancer results from mutations in pathway components, which frequently coexist with autocrine Wnt signaling or epigenetic silencing of extracellular Wnt antagonists. Among the extracellular Wnt inhibitors, the secreted frizzled-related proteins (SFRPs) are decoy receptors that contain soluble Wnt-binding frizzled domains. In addition to SFRPs, other endogenous molecules harboring frizzled motifs bind to and inhibit Wnt signaling. One of such molecules is V3Nter, a soluble SFRP-like frizzled polypeptide that binds to Wnt3a and inhibits Wnt signaling and expression of the b-catenin target genes cyclin D1 and c-myc. V3Nter is derived from the cell surface extracellular matrix component collagen XVIII. Here, we used HCT116 human colon cancer cells carrying the DS45 activating mutation in one of the alleles of b-catenin to show that V3Nter and SFRP-1 decrease baseline and Wnt3a-induced b-catenin stabilization. Consequently, V3Nter reduces the growth of human colorectal cancer xenografts by specifically controlling cell proliferation and cell cycle progression, without affecting angiogenesis or apoptosis, as shown by decreased