microRNA expression signatures of gastrointestinal stromal tumours: associations with imatinib resistance and patient outcome.

BACKGROUND: Gastrointestinal stromal tumour (GIST) is mainly initialised by receptor tyrosine kinase gene mutations. Although the tyrosine kinase inhibitor imatinib mesylate considerably improved the outcome of patients, imatinib resistance still remains a major therapeutic challenge in GIST therapy. Herein we evaluated the clinical impact of microRNAs in imatinib-treated GISTs. METHODS: The expression levels of microRNAs were quantified using microarray and RT-qPCR in GIST specimens from patients treated with neoadjuvant imatinib. The functional roles of miR-125a-5p and PTPN18 were evaluated in GIST cells. PTPN18 expression was quantified by western blotting in GIST samples. RESULTS: We showed that overexpression levels of miR-125a-5p and miR-107 were associated with imatinib resistance in GIST specimens. Functionally, miR-125a-5p expression modulated imatinib sensitivity in GIST882 cells with a homozygous KIT mutation but not in GIST48 cells with double KIT mutations. Overexpression of miR-125a-5p suppressed PTPN18 expression, and silencing of PTPN18 expression increased cell viability in GIST882 cells upon imatinib treatment. PTPN18 protein levels were significantly lower in the imatinib-resistant GISTs and inversely correlated with miR-125a-5p. Furthermore, several microRNAs were significantly associated with metastasis, KIT mutational status and survival. CONCLUSIONS: Our findings highlight a novel functional role of miR-125a-5p on imatinib response through PTPN18 regulation in GIST.

Hydrolysis and composition of recovered fibres fractionated from solid recovered fuel.

Fibres fractionated from solid recovered fuel (SRF), a standardised market combustion fuel produced from sorted waste, were considered as a source of lignocellulosic fermentable sugars. The fibre yield from four samples of SRF was 25-45%, and the separated material consisted of 52-54% carbohydrates, mainly glucan, with a high content of ash (12-17%). The enzymatic digestibility of recovered fibres was studied at low and high solids loading and compared with model substrates containing only chemical and mechanical pulps. Above 80% hydrolysis yield was reached at 20% solids loading in 48 h, but variation was observed between different samples of recovered fibres. Surfactants were found to improve the hydrolysis yield of recovered fibres especially in tumbling-type of mixing at low solids loading, where hydrolysis was found to stagnate without surfactants. The results suggest that SRF is a potential source of easily digestible lignocellulosic carbohydrates for use in biorefineries.

Fibroblast-dependent regulation of the stem cell properties of cancer cells.

There is emerging evidence that cancer stem cells (CSCs), like normal tissue stem cells, are regulated by a niche formed of mesenchymal cells. In this review we summarize the current knowledge of the role of cancer associated fibroblasts (CAFs) in a tentative CSC niche. We also discuss findings from our own studies showing that CAF derived factors have a strong stimulatory effect on the stem cell properties of breast cancer cells. Based on recent literature we conclude that CAFs are strong modulators of the stem cell properties of cancer cells. This effect is likely to be particularly relevant under circumstances of early stages of tumor cell dissemination and metastasis.

Expression profiling of microdissected cell populations selected from basal cells in normal epidermis and basal cell carcinoma.

BACKGROUND: Basal cell carcinomas (BCCs) are prevalent tumours with uniform histology that develop without any known precursor lesion. Alterations in the sonic hedgehog-patched1 signalling pathway are accepted as necessary events for tumorigenesis, and mutations in the patched1 gene are frequently present in tumours. OBJECTIVES: To analyse transcript profiles in BCC. METHODS: We used laser-assisted microdissection to isolate and collect cell populations defined under the microscope. Peripheral cells from nests of BCC were selected to represent tumour cells, and normal keratinocytes from epidermis basal layer were used as control. Extracted RNA was amplified and hybridized on to a cDNA microarray. Results Our results show that BCC cells express a transcript signature that is significantly different from that of normal keratinocytes, and over 350 genes with various functions were identified as differentially expressed. The compiled data suggest an upregulation of the Wnt signalling pathway as a major event in BCC cells. Furthermore, tumour cells appear to have an increased sensitivity to oxygen radicals and dysregulated genes involved in antigen presentation. RESULTS: were validated at both the transcriptional level using real-time polymerase chain reaction and at the protein level using immunohistochemistry. CONCLUSIONS: We show that microdissection in combination with robust strategies for RNA extraction, amplification and cDNA microarray analysis allow for reliable transcript profiling and that antibody-based proteomics provides an advantageous strategy for the analysis of corresponding differentially expressed proteins. We found that expression patterns were significantly altered in BCC cells compared with basal keratinocytes and that the Wnt signalling pathway was upregulated in tumour cells.

DEP-1 protein tyrosine phosphatase inhibits proliferation and migration of colon carcinoma cells and is upregulated by protective nutrients.

The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.

Characterization of an imatinib-sensitive subset of high-grade human glioma cultures.

High-grade gliomas, including glioblastomas, are malignant brain tumors for which improved treatment is urgently needed. Genetic studies have demonstrated the existence of biologically distinct subsets. Preliminary studies have indicated that platelet-derived growth factor (PDGF) receptor signaling contributes to the growth of some of these tumors. In this study, human high-grade glioma primary cultures were analysed for sensitivity to treatment with the PDGF receptor inhibitor imatinib/Glivec/Gleevec/STI571. Six out of 15 cultures displayed more than 40% growth inhibition after imatinib treatment, whereas seven cultures showed less than 20% growth inhibition. In the sensitive cultures, apoptosis contributed to growth inhibition. Platelet-derived growth factor receptor status correlated with imatinib sensitivity. Supervised analyses of gene expression profiles and real-time PCR analyses identified expression of the chemokine CXCL12/SDF-1 (stromal cell-derived factor 1) as a predictor of imatinib sensitivity. Exogenous addition of CXCL12 to imatinib-insensitive cultures conferred some imatinib sensitivity. Finally, coregulation of CXCL12 and PDGF alpha-receptor was observed in glioblastoma biopsies. We have thus defined the characteristics of a novel imatinib-sensitive subset of glioma cultures, and provided evidence for a functional relationship between imatinib sensitivity and chemokine signaling. These findings will assist in the design and evaluation of clinical trials exploring therapeutic effects of imatinib on malignant brain tumors.

Smad2 suppresses the growth of Mv1Lu cells subcutaneously inoculated in mice.

Smad2 and Smad3 are intracellular signal transduction proteins of importance in transforming growth factor-beta (TGFbeta)-mediated inhibition of epithelial cell proliferation. Inactivating mutations in the Smad2 and Smad3 genes have been found in various human malignancies. Here, we show that expression of Smad2 leads to the inhibition of growth of Mv1Lu cells inoculated with Matrigel subcutaneously (s.c.) in severe combined immunodeficient (SCID) mice. In histological appearance, the Matrigel plugs with Smad2-transfected cells showed strongly reduced cell density, proliferation and angiogenesis compared with the small tumour nodules of similar size formed by the vector- or Smad3-transfected cells. The histological appearance of vector- and Smad3-transfected cells inoculated in mice was identical. Overexpression of Smad2 and Smad3 in Mv1Lu cells led to the inhibition of cell growth in three-dimensional cultures when compared with vector-transfected cells. Overexpression of Smad2 and Smad3 also decreased the hyperphosphorylation of pRb in Smad-transfected cells. Thus, increased expression of Smad2 leads to inhibition of Mv1Lu cell proliferation and a reduction in the growth of the Smad2-expressing cells inoculated in mice.

An extracellular ligand increases the specific activity of the receptor-like protein tyrosine phosphatase DEP-1.

Cellular growth, differentiation and migration is regulated by protein tyrosine phosphorylation. Receptor-like protein tyrosine phosphatases are thus likely to be key regulators of vital cellular processes. The regulation of these enzymes is in general poorly understood. Ligands have been identified only for a small subset of the receptor-like protein tyrosine phosphatases and in no case has upregulation of the specific activity by extracellular ligands been demonstrated. Prompted by earlier findings of ligands for receptor-like protein tyrosine phosphatases in extracellular matrix we investigated if Matrigel, a preparation of extracellular matrix proteins, contained modulators of the specific activity of the receptor-like protein tyrosine phosphatase DEP-1. Matrigel stimulation of cells increased the specific activity of immunoprecipitated DEP-1. Also, incubation of immunoprecipitated DEP-1 with Matrigel led to an increase in DEP-1 activity, which was blocked by soluble DEP-1 extracellular domain. Finally, immunoprecipitated DeltaECD-DEP-1, a mutant form of DEP-1 lacking most of the extracellular domain, failed to respond to Matrigel stimulation. These experiments identify Matrigel as a source of DEP-1 agonist(s) and provide the first evidence for upregulation of the specific activity of receptor-like protein tyrosine phosphatases by extracellular ligands.

Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis.

Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.

Ligand stimulation reduces platelet-derived growth factor beta-receptor susceptibility to tyrosine dephosphorylation.

Ligand binding to the platelet-derived growth factor (PDGF) beta-receptor leads to increased receptor tyrosine phosphorylation as a consequence of dimerization-induced activation of the intrinsic receptor tyrosine kinase activity. In this study we asked whether ligand-stimulated PDGF beta-receptor tyrosine phosphorylation, to some extent, also involved reduced susceptibility to tyrosine dephosphorylation. To investigate this possibility we compared the sensitivity of ligand-stimulated and non-stimulated forms of tyrosine-phosphorylated PDGF beta-receptors to dephosphorylation using various preparations containing protein-tyrosine phosphatase activity. Ligand-stimulated or unstimulated tyrosine-phosphorylated receptors were obtained after incubation of cells with pervanadate only or pervanadate, together with PDGF-BB, respectively. Dephosphorylation of receptors immobilized on wheat germ agglutinin-Sepharose, as well as of receptors in intact cell membranes, was investigated under conditions when rephosphorylation did not occur. As compared with unstimulated receptors the ligand-stimulated PDGF beta-receptors showed about 10-fold reduced sensitivity to dephosphorylation by cell membranes, a recombinant form of the catalytic domain of density-enhanced phosphatase-1, or recombinant protein-tyrosine phosphatase 1B. We conclude that ligand-stimulated forms of the PDGF beta-receptor display a reduced susceptibility to dephosphorylation. Our findings suggest a novel mechanism whereby ligand stimulation of PDGF beta-receptor, and possibly other tyrosine kinase receptors, leads to a net increase in receptor tyrosine phosphorylation.

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases.

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor.

The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.

Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors.

Most solid malignancies display interstitial hypertension and a poor uptake of anticancer drugs. Platelet-derived growth factor (PDGF) and the cognate tyrosine kinase receptors are expressed in many tumors. Signaling through PDGFbeta receptors was shown recently to increase interstitial fluid pressure (IFP) in dermis after anaphylaxis-induced lowering of IFP. In this study, we show that treatment with the selective PDGF receptor kinase inhibitor, STI571, formerly known as CGP57148B, decreased the interstitial hypertension and increased capillary-to-interstitium transport of 51Cr-EDTA in s.c. growing rat PROb colonic carcinomas. Furthermore, treatment with an antagonistic PDGF-B oligonucleotide aptamer decreased interstitial hypertension in these tumors. PDGFbeta receptors were expressed in blood vessels and stromal cells but not in the tumor cells of PROb colonic carcinomas. Our study indicates a previously unrecognized role of PDGF receptors in tumor biology, although similar effects of PDGF on IFP have been demonstrated previously in the dermis. The data suggest interference with PDGF receptors, or their ligands, as a novel strategy to increase drug uptake and therapeutic effectiveness of cancer chemotherapy.

Involvement of platelet-derived growth factor in disease: development of specific antagonists.

Platelet-derived growth factor (PDGF) is a family of dimeric isoforms that stimulates, e.g., growth, chemotaxis and cell shape changes of various connective tissue cell types and certain other cells. The cellular effects of PDGF isoforms are exerted through binding to two structurally related tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation. This enables a number of SH2 domain containing signal transduction molecules to bind to the receptors, thereby initiating various signaling pathways. PDGF isoforms have important roles during the embryonic development, particularly in the formation of connective tissue in various organs. In the adult, PDGF stimulates wound healing. Overactivity of PDGF has been implicated in certain disorders, including fibrotic conditions, atherosclerosis, and malignancies. Different kinds of PDGF antagonists are currently being developed and evaluated in different animal disease models, as well as in clinical trials.

Antiangiogenic effects of latent antithrombin through perturbed cell-matrix interactions and apoptosis of endothelial cells.

Antithrombin is a plasma protein of the serpin superfamily that may occur as several conformational variants. The native form of antithrombin is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat-denatured form, denoted latent antithrombin. Fibroblast growth factor (FGF)-induced angiogenesis in the chick embryo and angiogenesis in mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrombin were less efficient in these respects. Treatment with latent antithrombin induced apoptosis of cultured endothelial cells and inhibited cell migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF receptor kinase activity was unaffected. However, actin reorganization, activation of focal adhesion kinase, and focal adhesion formation were disturbed by latent antithrombin treatment of FGF-2-stimulated endothelial cells. These data indicate that latent antithrombin induces apoptosis of endothelial cells by disrupting cell-matrix interactions through uncoupling of focal adhesion kinase.

Intimal hyperplasia recurs after removal of PDGF-AB and -BB inhibition in the rat carotid artery injury model.

Several antagonists specific for platelet-derived growth factor (PDGF) or its receptors have recently been developed and shown to inhibit intimal hyperplasia formation in various animal models, but data investigating the durability of this intervention is limited. The present study was designed to investigate the potency of PDGF B-chain aptamer, a novel type of PDGF-AB and -BB antagonist, in the rat carotid model and to characterize intermediate-term effects on lesion formation. One hundred thirty-four animals were randomized to aptamer treatment or placebo. Daily treatment with the antagonist resulted in a 50% reduction in lesion size at 2 weeks (P<0.001). The beneficial effect involved increased apoptosis and possibly an interference with smooth muscle cell migration. Discontinuing administration 1 week earlier did not give any significant benefit compared with phosphate-buffered saline-treated controls. When the antagonist was administered for 2 weeks and the vessels analyzed 6 weeks later, the beneficial effect was lost and the treated lesions had a higher intima-media and area-cell ratio compared with the treated lesions in the 2-week-endpoint study. Our findings confirm a role of PDGF B-chain in intimal hyperplasia, but the successful use of PDGF antagonists may require either prolonged treatment or combination therapy with other agents.

Site-selective dephosphorylation of the platelet-derived growth factor beta-receptor by the receptor-like protein-tyrosine phosphatase DEP-1.

Ligand stimulation of PDGF beta-receptors leads to autophosphorylation of the regulatory tyrosine 857 and of tyrosine residues that in their phosphorylated form serve as docking sites for Src homology 2 domain-containing proteins. Regulation of the PDGF beta-receptor by protein-tyrosine phosphatases is poorly understood. We have investigated PDGF beta-receptor dephosphorylation by receptor-like protein-tyrosine phosphatase DEP-1 using a cell line with inducible DEP-1 expression and by characterizing in vitro dephosphorylation of the PDGF beta-receptor and of receptor-derived phosphopeptides by DEP-1. After DEP-1 induction PDGF beta-receptor.DEP-1 complexes and reduced receptor tyrosine phosphorylation were observed. Phosphopeptide analysis of the PDGF beta-receptors from DEP-1-expressing cells and of the receptors dephosphorylated in vitro by DEP-1 demonstrated that dephosphorylation of autophosphorylation sites of the receptor differed and revealed that the regulatory Tyr(P)(857) was not a preferred site for DEP-1 dephosphorylation. When dephosphorylation of synthetic receptor-derived peptides was analyzed, the selectivity was reproduced, indicating that amino acid sequence surrounding the phosphorylation sites is the major determinant of selectivity. This notion is supported by the observation that the poorly dephosphorylated Tyr(P)(562) and Tyr(P)(857), in contrast to other analyzed phosphorylation sites, are surrounded by basic amino acid residues at positions -4 and +3 relative to the tyrosine residue. Our study demonstrates that DEP-1 dephosphorylation of the PDGF beta-receptor is site-selective and may lead to modulation, rather than general attenuation, of signaling.

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.