Identification of 33 candidate oncogenes by screening for base-specific mutations.

BACKGROUND: Genes with recurrent codon-specific somatic mutations are likely drivers of tumorigenesis and potential therapeutic targets. Hypermutable cancers may represent a sensitive system for generation and selection of oncogenic mutations. METHODS: We utilised exome-sequencing data on 25 sporadic microsatellite-instable (MSI) colorectal cancers (CRCs) and searched for base-specific somatic mutation hotspots. RESULTS: We identified novel mutation hotspots in 33 genes. Fourteen genes displayed mutations in the validation set of 254 MSI CRCs: ANTXR1, MORC2, CEP135, CRYBB1, GALNT9, KRT82, PI15, SLC36A1, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1 and TTPAL. A database search found examples of the hotspot mutations in multiple cancer types. CONCLUSIONS: This work reveals a variety of new recurrent candidate oncogene mutations to be further scrutinised as potential therapeutic targets.

Mutation analysis of components of the Mediator kinase module in MED12 mutation-negative uterine leiomyomas.

BACKGROUND: Kinase module of Mediator complex ('CDK8 submodule') consists of four subunits: CDK8, Cyclin C, MED12, and MED13. Recently, we reported recurrent MED12 mutations in 70% of uterine leiomyomas. The aim of this study was to analyse whether mutations in other components of the module contribute to the development of these lesions. METHODS: Mutation screening of altogether 70 MED12 mutation-negative uterine leiomyomas was carried out by direct sequencing. RESULTS: None of the tumours displayed somatic mutations in the coding regions of CDK8/CDK19, CCNC, or MED13. CONCLUSIONS: Mutations in CDK8/CDK19, CCNC, and MED13 do not frequently contribute to genesis of uterine leiomyomas.

Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis.

The tumor-suppressor p53 can induce various biological responses. Yet, it is not clear whether it is p53 in vivo promoter selectivity that triggers different transcription programs leading to different outcomes. Our analysis of genome-wide chromatin occupancy by p53 using chromatin immunoprecipitation (ChIP)-seq revealed 'p53 default program', that is, the pattern of major p53-bound sites that is similar upon p53 activation by nutlin3a, reactivation of p53 and induction of tumor cell apoptosis (RITA) or 5-fluorouracil in breast cancer cells, despite different biological outcomes. Parallel analysis of gene expression allowed identification of 280 novel p53 target genes, including p53-repressed AURKA. We identified Sp1 as one of the p53 modulators, which confer specificity to p53-mediated transcriptional response upon RITA. Further, we found that STAT3 antagonizes p53-mediated repression of a subset of genes, including AURKA.

Patched acts catalytically to suppress the activity of Smoothened.

Mutations affecting the transmembrane proteins Patched (Ptc) or Smoothened (Smo) that trigger ligand-independent activity of the Hedgehog (Hh) signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma. Despite extensive genetic studies demonstrating the importance of these receptor components in embryonic patterning and cancer, the mechanism by which Ptc regulates Smo is not understood. Here we report that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, we show that free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; we demonstrate that the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumour suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule.

The Hedgehog and Wnt signalling pathways in cancer.

The Wnt and Hedgehog (Hh) signalling pathways have long been known to direct growth and patterning during embryonic development. Recent evidence also implicates these pathways in the postembryonic regulation of stem-cell number in epithelia such as those of the skin and intestine, which undergo constant renewal. A pathological role for the Wnt and Hh pathways has emerged from studies showing a high frequency of specific human cancers associated with mutations that constitutively activate the transcriptional response of these pathways. This article focuses on Hh and Wnt signal transduction and reviews evidence suggesting that tumorigenesis associated with pathway activation may result from mis-specification of cells towards stem-cell or stem cell-like fates.

Deprivation of liberty in psychiatric treatment: a Finnish perspective.

This article is concerned with the deprivation of patients' liberty while undergoing psychiatric treatment, with special reference to the situation in Finland. It is based on a review of Finnish law, health care statistics, and empirical and theoretical studies. Relevant research findings from other countries are also discussed. In Finland, it is required that patients are cared for by mutual understanding with themselves; coercive measures may be applied only if they are necessary for the treatment of the illness, or for safeguarding patients' safety or the safety of others. Involuntary psychiatric hospitalization is closely regulated by the Mental Health Act. However, the rules concerning the deprivation of liberty during inpatient treatment (by seclusion, restraint and restricted leave) are formulated in very general terms. Therefore, Finnish psychiatric hospitals have their own policies concerning when and how seclusion may be used. The practice of seclusion and the use of restraint therefore vary among the psychiatric hospitals in Finland.

Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine.

Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer.

Recently, monoclonal antibodies against the human vascular endothelial growth factor receptor VEGFR-3 were shown to provide a specific antigenic marker for lymphatic endothelium in various normal tissues. In this study we have investigated the expression of VEGFR-3 and its ligand VEGF-C in normal breast tissue and in breast tumors by immunohistochemistry. VEGFR-3 was weakly expressed in capillaries of normal breast tissue and in fibroadenomas. In intraductal breast carcinomas, VEGFR-3 was prominent in the "necklace" vessels adjacent to the basal lamina of the tumor-filled ducts. VEGF receptor 1 and 2 as well as blood vessel endothelial and basal lamina markers were colocalized with VEGFR-3 in many of these vessels. Antibodies against smooth muscle alpha-actin gave a weak staining of the necklace vessels, suggesting that they were incompletely covered by pericytes/smooth muscle cells. A highly elevated number of VEGFR-3 positive vessels was found in invasive breast cancer in comparison with histologically normal breast tissue (P < 0.0001, the Mann-Whitney test). VEGF-C was located in the cytoplasm of intraductal and invasive cancer cells. The results demonstrate that the expression of VEGFR-3 becomes up-regulated in the endothelium of angiogenic blood vessels in breast cancer. The results also suggest that VEGF-C secreted by the intraductal carcinoma cells acts predominantly as an angiogenic growth factor for blood vessels, although this paracrine signaling network between the cancer cells and the endothelium may also be involved in modifying the permeabilities of both blood and lymphatic vessels and metastasis formation.

Hepatocyte growth factor releases mink epithelial cells from transforming growth factor beta1-induced growth arrest by restoring Cdk6 expression and cyclin E-associated Cdk2 activity.

Transforming growth factor beta (TGF-beta) potently suppresses Mv1Lu mink epithelial cell growth, whereas hepatocyte growth factor (HGF) counteracts TGF-beta-mediated growth inhibition and induces Mv1Lu cell proliferation (J. Taipale and J. Keski-Oja, J. Biol. Chem. 271:4342-4348, 1996). By addressing the cell cycle regulatory mechanisms involved in HGF-mediated release of Mv1Lu cells from TGF-beta inhibition, we show that increased DNA replication is accompanied by phosphorylation of the retinoblastoma protein and alternative regulation of cyclin-Cdk-inhibitor complexes. While TGF-beta treatment decreased the expression of Cdk6, this effect was counteracted by HGF, followed by partial restoration of cyclin D2-associated kinase activity. Notably, HGF failed to prevent TGF-beta induction of p15 and its association with Cdk6. However, HGF reversed the TGF-beta-mediated decrease in Cdk6-associated p27 and cyclin D2-associated Cdk6, suggesting that HGF modifies the TGF-beta response at the level of G1 cyclin complex formation. Counteraction of TGF-beta regulation of Cdk6 by HGF may in turn affect the association of p27 with Cdk2-cyclin E complexes. Though HGF did not differentially regulate the total levels of p27 in TGF-beta-treated cells, p27 immunodepletion experiments suggested that upon treatment with both growth factors, less p27 is associated with Cdk2-cyclin E complexes, in parallel with restoration of the active form of Cdk2 and the associated kinase activity. The results demonstrate that HGF intercepts TGF-beta cell cycle regulation at multiple points, affecting both G1 and G1-S cyclin kinase activities.

Latent transforming growth factor-beta binding proteins (LTBPs)--structural extracellular matrix proteins for targeting TGF-beta action.

Growth factors of the transforming growth factor-beta family are potent regulators of the extracellular matrix formation, in addition to their immunomodulatory and regulatory roles for cell growth. TGF-beta s are secreted from cells as latent complexes containing TGF-beta and its propeptide, LAP (latency-associated peptide). In most cells LAP is covalently linked to an additional protein, latent TGF-beta binding protein (LTBP), forming the large latent complex. LTBPs are required for efficient secretion and correct folding of TGF-beta s. The secreted large latent complexes associate covalently with the extracellular matrix via the N-termini of the LTBPs. LTBPs belong to the fibrillin-LTBP family of extracellular matrix proteins, which have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. Currently four different LTBPs and two fibrillins have been identified. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures. LTBPs are now known to exist both as soluble molecules and in association with the extracellular matrix. An important consequence of this is LTBP-mediated deposition and targeting of latent, activatable TGF-beta into extracellular matrices and connective tissues. LTBPs have a dual function, they are required both for the secretion of the small latent TGF-beta complex as well as directing bound latent TGF-beta to extracellular matrix microfibrils. However, it is not known at present whether LTBPs are capable of forming microfibrils independently, or whether they are a part of the fibrillin-containing fibrils. Most LTBPs possess RGD-sequences, which may have a role in their interactions with the cell surface. At least LTBP-1 is chemotactic to smooth muscle cells, and is involved in vascular remodelling. Analyses of the expressed LTBPs have revealed considerable variations throughout the molecules, generated both by alternative splicing and utilization of multiple promoter regions. The significance of this structural diversity is mostly unclear at present.

Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.

Recombinant latent transforming growth factor beta-binding protein 2 assembles to fibroblast extracellular matrix and is susceptible to proteolytic processing and release.

Latent transforming growth factor beta-binding protein 2 (LTBP-2) belongs to the fibrillin-LTBP gene family and is a component of 10-nm microfibrils. LTBP-2 consists mainly of domains of 8-cysteine and EGF-like repeats linked by proline-rich regions. To characterize the biochemical properties of LTBP-2, its assembly to the extracellular matrix, and its proteolytic release from the matrix, LTBP-2 was expressed recombinantly in Chinese hamster ovary cells and purified to homogeneity under nondenaturing conditions. Purified LTBP-2 bound calcium and was glycosylated at the central domain of EGF-like repeats. Antibodies made against the recombinant LTBP-2 decorated fibrillar structures in fibroblast extracellular matrix. Treatment of matrices with plasmin or elastase released a soluble approximately 160-kDa LTBP-2 fragment. Processing of LTBP-2 was studied by treating purified LTBP-2 with plasmin or porcine pancreatic elastase. LTBP-2 was processed with these proteases initially to a approximately 160-kDa fragment, and with higher concentrations to a protease-resistant approximately 120-kDa fragment. Processing sites were localized by amino acid sequencing to proline-rich regions at the N-terminal part of LTBP-2, suggesting that the matrix binding sites locate to the N-terminal approximately 500 amino acids of LTBP-2. Purified and biotinylated LTBP-2 could be assembled to fibrillar structures in fibroblast extracellular matrix during cell cultivation, indicating that LTBP-2 assembly to the matrix is not strictly linked to cells that make it and suggesting that microfibril assembly may involve soluble intermediates.

Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion.

Growth factors of the transforming growth factor-beta (TGF-beta) family inhibit the proliferation of epithelial, endothelial, and hematopoietic cells, and stimulate the synthesis of extracellular matrix components. TGF-beta s are secreted from cells in high-molecular-mass protein complexes that are composed of three proteins, the mature TGF-beta-dimer, the TGF-beta propeptide dimer, or latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Mature TGF-beta is cleaved from its propeptide during secretion, but the proteins remain associated by noncovalent interactions. LTBP is required for efficient secretion and processing of latent TGF-beta and it binds to LAP via disulfide bond(s). LTBP is a component of extracellular matrix microfibrils, and it targets the latent TGF-beta complex to the extracellular matrix. TGF-beta signaling is initiated by proteolytic cleavage of LTBP that results in the release of the latent TGF-beta complex from the extracellular matrix. TGF-beta is activated by dissociation of LAP from the mature TGF-beta. Subsequent signaling involves binding of active TGF-beta to its type II cell surface receptors, which phosphorylate and activate type I TGF-beta receptors. Type I receptors, in turn, phosphorylate cytoplasmic transcriptional activator proteins Smad2 and Smad3, inducing their translocation to the nucleus. Recent evidence suggests that acquisition of resistance to TGF-beta growth inhibition plays a major role in the progression of epithelial and hematopoietic cell malignancies. The role of secretion of TGF-beta in tumorigenesis is more complex. The secretion of TGF-beta s by tumor cells may contribute to autocrine growth inhibition, but on the other hand, it may also promote invasion, metastasis, angiogenesis, and even immunosuppression. Tumor cells may also fail to deposit LTBP:TGF-beta complexes to the extracellular matrix. The elucidation of the mechanisms of the release of TGF-beta from the matrix and its subsequent activation aids the understanding of the pathophysiologic roles of TGF-beta in malignant growth, and allows the development of therapeutic agents that regulate the activity of TGF-beta.

Identification and characterization of a new latent transforming growth factor-beta-binding protein, LTBP-4.

Transforming growth factor betas (TGF-betas) are secreted by most cell types as latent high molecular weight complexes consisting of TGF-beta and its latency associated peptide (LAP) propeptide dimers, covalently linked to latent TGF-beta-binding proteins (LTBPs). Currently, three different LTBPs are known (LTBPs 1, 2, and 3), all with highly similar protein domain structure consisting of epidermal growth factor-like and 8-Cys repeats. The 3rd 8-Cys repeat of LTBP-1 mediates its association with TGF-beta1.LAP. By using an expressed sequence tag homologous to the 3rd 8-Cys repeat of human LTBP-1 as a probe, a novel cDNA similar to known LTBPs was cloned from human heart cDNA library. This cDNA was named LTBP-4 and found to exist in at least four different forms, generated by alternative splicing at the amino terminus and at the central epidermal growth factor repeat domain. One of the alternative amino-terminal forms contained an RGD sequence, indicating possible cell-surface interactions with integrins. LTBP-4 gene was localized to chromosomal position 19q13. 1-19q13.2. The major LTBP-4 mRNA form is about 5.1 kilobase pairs in size and is predominantly expressed in the heart, aorta, uterus, and small intestine. Immunoblotting analysis indicated that LTBP-4 was secreted from cultured human lung fibroblasts both in a free form and in a disulfide bound complex with a TGF-beta. LAP-like protein. Both LTBP-4 forms were also found to be deposited in the extracellular matrix. The matrix-associated LTBP-4 was susceptible to proteolytic release with plasmin. LTBP-4 is a new member of the growing LTBP-fibrillin family of proteins and offers an alternative means for the secretion and targeted matrix deposition of TGF-betas or related proteins.

Growth factors in the extracellular matrix.

Much recent research has focused on the study of the expression of growth factor genes and on the identification of growth factor signaling mechanisms inside cells. However, growth factor signaling can also be regulated outside of cells by extracellular matrix proteins and proteolytic enzymes. The ability of extracellular proteins to process complex information in the absence of new protein synthesis is illustrated in blood clotting and complement pathways. An increasing number of growth factors, including IGFs, FGFs, TGF-beta's, and HGF, have been found to associate with the extracellular matrix proteins or with heparan sulfate. Rapid and localized changes in the activity of these factors can be induced by release from matrix storage and/or by activation of latent forms. These growth factors, in turn, control cell proliferation, differentiation, and synthesis and remodeling of the extracellular matrix. It is therefore likely that much of the information processing necessary for construction of complex multicellular organisms occurs in the extracellular environment. This suggests that extracellular matrix plays a major role in the control of growth factor signaling.

Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils.

We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.

Hepatocyte growth factor releases epithelial and endothelial cells from growth arrest induced by transforming growth factor-beta1.

Human lung fibroblasts and Mv1Lu mink lung epithelial cells were used as a model to study the role of extracellular matrix in epithelial-mesenchymal interactions. Extracellular matrices of fibroblasts were found to contain growth promoting activity that reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). The majority of the activity was identified as hepatocyte growth factor/scatter factor (HGF) by inhibition with specific antibodies and by reconstitution of the effect by recombinant HGF. HGF induced cell proliferation when contact-inhibited Mv1Lu cells were trypsinized and plated in the presence of TGF-beta1. The effect was valid also in assays where Madin-Darby canine kidney epithelial cells or bovine capillary endothelial cells were used. The multiplication of chronically TGF-beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 and HGF require proteolysis for activation, the results imply that proteolytic activity of epithelial and endothelial cells directs their responses to signals from mesenchymal-type extracellular matrices, and that during development, matrix-bound growth and invasion promoting and suppressing factors are activated in a coordinated manner.

Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1.

Transforming growth factor-betas (TGF-betas) are produced by most cells in large latent complexes of TGF-beta and its propeptide (LAP) associated with a binding protein. The latent TGF-beta binding proteins (LTBPs-1, -2 and -3) mediate the secretion and, subsequently, the association of latent TGF-beta complexes with the extracellular matrix (ECM). The association of beta1-LAP with LTBP-1 was characterized at the molecular level with an expression system in mammalian cells, where TGF-beta1 and various fragments of LTBP-1 were co-expressed and secreted with the aid of a signal peptide synthesized to the LTBP-1 constructs. Immunoblotting of the fusion protein complexes indicated that the third 8-Cys repeat of LTBP-1 bound covalently to the LAP region of TGF-beta1. The cysteine required for the association between LTBP-1 and beta1-LAP was mapped to Cys33 of beta1-LAP. The N-terminal region of LTBP-1 consisting of the first 400 amino acids was found to associate covalently with the ECM. The data indicate that an 8-Cys repeat of LTBP is capable of covalent and specific protein-protein interactions. These interactions are mediated by exchanging cysteine disulfide bonds between the core 8-Cys repeat and an optionally associated protein during the secretion. This is, to our knowledge, the first demonstration of an extracellular protein module that is able to exchange cysteine disulfide bonds with heterologous ligand proteins.

Human mast cell chymase and leukocyte elastase release latent transforming growth factor-beta 1 from the extracellular matrix of cultured human epithelial and endothelial cells.

Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta 1 (TGF-beta 1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta 1, its propeptide (beta 1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta 1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta 1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta 1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta 1 but did not dissociate high M(r) latent TGF-beta 1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta 1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.