8434

RECK

ST15;reversion-inducing-cysteine-rich protein with kazal motifs;RECK;reversion-inducing-cysteine-rich protein with kazal motifs

membrane-anchored glycoprotein (metastasis and invasion)|reversion-inducing cysteine-rich protein with Kazal motifs|suppression of tumorigenicity 15 (reversion-inducing-cysteine-rich protein with kazal motifs)|suppression of tumorigenicity 5 (reversion-in

9p13.3

protein-coding

Count: 3; Pubmed_search,UniProt,Generif

Downregulation of the tumor suppressor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) has been reported under hypoxic conditions (Lee et al., 2010); however, the signaling pathways involved in this downregulation have not yet been identified. Hypoxia causes the silencing of RECK mRNA expression, but treatment with inhibitors of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated kinase (MAPK) (PD98059, SP600125, and SB203580 respectively) or their dominant negative mutants recovered RECK suppression induced by hypoxia as analyzed with semiquantitative RT-PCR analysis and a RECK promoter luciferase assay. Hypoxia increased phosphorylation of ERK1/2, JNK and p38 MAPKs. The activities of matrix metalloproteinase (MMP)-9 and MMP-2 were increased under hypoxic conditions but treatment with PD98059, SP600125 and SB203580 inhibited their activation in cancer cells, as seen by zymography. Moreover, treatment with the inhibitors blocked cancer cell migration induced by hypoxia in H-Ras transformed MCF10A mammary cells. RECK suppression under hypoxic conditions was inversely related to HIF-1alpha expression; however, treatment with PD98059, SP600125 and SB203580 did not influence binding of HIF-1alpha to the reverse hypoxia responsive element site of the RECK promoter in a DNA precipitation assay. These results suggest that the ERK, JNK and p38 MAPK signaling pathways are indirectly involved in RECK suppression but are not involved in the binding activity of HIF-1alpha to the reverse hypoxia responsive element site on the RECK promoter under hypoxic conditions.

The membrane-anchored matrix metalloproteinase-regulator RECK is often downregulated in various types of cancers; the levels of residual RECK in resected tumors often correlate with better prognosis. Forced expression of RECK in cancer cells suppresses tumor angiogenesis, invasion, and metastasis in xenograft models. RECK is therefore a promising marker for benignancy and a potential effector in cancer therapy. We established a cell line containing two transgene systems: (1) the secreted alkaline phosphatase (SEAP) gene fused to Reck promoter and (2) the HRAS(12V) oncogene driven by the Tet-off promoter system. This cell line exhibits transformed phenotype in regular medium and flat morphology with increased SEAP activity in the presence of doxycycline, allowing the assessment of RECK-inducing activity of chemicals in the contexts of both transformed and untransformed cells. Our pilot experiments with 880 known bioactive compounds detected 34 compounds that activate RECK promoter; among these, 10 were authentic anticancer drugs. Four selected compounds up-regulated endogenous RECK protein in several human cancer cell lines. The top-ranking compound, disulfiram, strongly suppressed spontaneous lung-metastasis of human fibrosarcoma cells in nude mice. Our data demonstrate the value of this screen in discovering effective cancer therapeutics.

Our previous study demonstrates that HER-2/Neu oncogene inhibits a matrix metalloproteinase inhibitor and tumor metastasis suppressor RECK to promote metastasis. Conversely, the effect of RECK on the oncogenic function of HER-2/Neu is unknown. Ectopic expression of RECK in 293T cells and HER-2/Neu-overexpressing breast cancer cells shows that RECK and HER-2/Neu are co-localized and these two proteins can be co-immunoprecipitated. RECK inhibits HER-2/Neu receptor dimerization and autophosphorylation, which causes reduction of ERK and AKT kinase activity and down-regulation of HER-2/Neu target genes. RECK expression is reduced in 58.8% of breast cancer tissues and is associated with lymph node invasion supporting its anti-metastatic role. Collectively, we provide the first evidence that RECK can negatively regulate oncogenic activity of HER-2/Neu by inhibiting receptor dimerization.CI - Copyright (c) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

expression cloning is a powerful approach to finding genes that induce appreciable changes in cultured cells. One way to use this technique in cancer research is to isolate cDNAs that induce flat reversion in transformed cells. Such screening, however, is inherently artificial, and therefore requires independent validation of the clinical relevance of isolated genes. Studies of the mechanisms of actions, physiological functions and mechanisms of regulation of these genes at various levels may enrich our knowledge of cancer biology and supplement our toolbox in developing new cancer diagnoses and therapies. In this article we discuss the promise, limitations and recent innovations in this approach, taking one transformation suppressor gene, RECK, as an example.

cancer cells show characteristic gene expression profiles. Recent studies support the potential importance of microRNA (miRNA) expression signatures as biomarkers and therapeutic targets. The membrane-anchored protease regulator RECK is downregulated in many cancers, and forced expression of RECK in tumor cells results in decreased malignancy in animal models. RECK is also essential for mammalian development. In this study, we found that RECK is a target of at least three groups of miRNAs (miR-15b/16, miR-21 and miR-372/373); that RECK mutants lacking the target sites for these miRNA show augmented tumor/metastasis-suppressor activities; and that miR-372/373 are upregulated in response to hypoxia through HIF1alpha and TWIST1, whereas miR-21 is upregulated by RAS/ERK signaling. These data indicate that the hypoxia- and RAS-signaling pathways converge on RECK through miRNAs, cooperatively downregulating this tumor suppressor and thereby promoting malignant cell behavior.

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a tumor suppressor and the suppression of RECK is induced by Ras or Her-2/neu oncogenes. However, regulation of RECK under hypoxic microenvironment is largely unknown. Here, we identified that hypoxia significantly downregulates RECK mRNA and protein expression using semiquantitative RT-PCR, real-time RT-PCR and western blot analysis. This repression was reversed by the HDAC inhibitor, trichostatin A (TSA) and HIF-1 inhibitor, YC-1. Hypoxia-induced downregulation of RECK was abolished by knockdown of HDAC1 and HIF-1alpha with respective small interfering RNAs (siRNAs), whereas overexpression of HDAC1 and HIF-1alpha suppressed RECK expression similar to the level under hypoxic conditions. Transfection of a deletion mutant of the second reverse HRE (rHRE2, -2345 to -2333) site of RECK promoter completely removed RECK suppression under hypoxia, indicating that the rHRE2 site is responsible for the inhibition of RECK. Chromatin immunoprecipitation and DNA affinity precipitation assays demonstrated that HDAC1 and HIF-1alpha were recruited to the rHRE2 region of RECK promoter under hypoxic conditions, but the treatment of TSA or YC-1 inhibited their binding to the rHRE2 site. Moreover, TSA and YC-1 inhibited hypoxia-induced cancer cell migration, invasion and MMPs secretion. Taken together, we can conclude that hypoxia induces RECK downregulation through the recruitment of HDAC1 and HIF-1alpha to the rHRE2 site in the promoter and the inhibition of hypoxic RECK silencing would be a therapeutic and preventive target for early tumorigenesis.CI - Copyright 2010 Elsevier B.V. All rights reserved.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. SFRP1 (the secreted frizzled-related protein 1), a putative tumor suppressor gene mapped onto chromosome 8p12-p11.1, the frequent loss of heterozygosity (LOH) region in human HCC, encodes a Wingless-type (Wnt) signaling antagonist and is frequently inactivated by promoter methylation in many human cancers. However, whether the down-regulation of SFRP1 can contribute to hepatocarcinogenesis still remains unclear. METHODS: We investigated the expression of SFRP1 through real time RT-PCR and immunohistochemistry staining. The cell growth and colony formation were observed as the overexpression and knockdown of SFRP1. The DNA methylation status within SFRP1 promoter was analyzed through methylation-specific PCR or bisulphate-treated DNA sequencing assays. Loss of heterozygosity was here detected with microsatellite markers. RESULTS: SFRP1 was significantly down-regulated in 76.1% (35/46) HCC specimens at mRNA level and in 30% (30/100) HCCs indicated by immunohistochemistry staining, as compared to adjacent non-cancerous livers. The overexpression of SFRP1 can significantly inhibit the cell growth and colony formation of YY-8103, SMMC7721, and Hep3B cells. The RNA interference against the constitutional SFRP1 in the offspring SMMC7721 cells, which were stably transfected by ectopic SFRP1, can markedly promote cell growth of these cells. LOH of both microsatellite markers D8S532 and D8SAC016868 flanking the gene locus was found in 13% (6 of 46 HCCs) and 6.5% (3 of 46 HCCs) of the informative cases, respectively, where 5 of 8 HCC specimens with LOH showed the down-regulation of SFRP1. DNA hypermethylation within SFRP1 promoter was identified in two of three HCC specimens without SFRP1 expression. Moreover, the DNA methylation of SFRP1 promoter was significantly reduced, along with the re-expression of the gene, in those HCC cell lines, Bel7404, QGY7701, and MHCC-H, as treated by DAC. CONCLUSION: Our data suggested that the down-regulation of SFRP1 as a candidate tumor suppressor gene, triggered by the epigenetic and/or genetic events, could contribute to the oncogenesis of HCC.

The extracellular matrix (ECM) is important for both structural integrity and functions of the brain. Matrix metalloproteinases (MMPs) play major roles in ECM-remodeling under both physiological and pathological conditions. Reversion-inducing cysteine-rich protein with Kazal motifs (Reck) is a membrane-anchored MMP-regulator implicated in coordinated regulation of pericellular proteolysis. Although patho-physiological importance of MMPs and another group of MMP-regulators, tissue inhibitor of metalloproteinases, in brain ischemia has been demonstrated, little is known about the role of Reck in this process. In this study, we found that Reck is up-regulated in hippocampus and penumbra of subventricular zone after transient cerebral ischemia in mice. Most of the Reck-positive cells found at day 2 after ischemia are positive for Nestin as well as Ki67 and localized to the CA2 region of the hippocampus. At day 7 after ischemia, the Reck-positive cells increased in number, extended processes, expressed the reactive astrocyte marker GFAP and the neuronal marker NF200, and were widely distributed in the hippocampus. In the mutant mice carrying single functional Reck allele (Reck+/-), tissue damage and cell death after cerebral ischemia were augmented, the recovery of long-term potentiation in the hippocampus was compromised, NR2C subunit of NMDA receptor was up-regulated, gelatinolytic activity of MMPs were up-regulated and laminin-immunoreactivity was reduced. Our data implicate Reck in protection of ECM/tissue integrity and promotion of functional recovery in the brain after transient cerebral ischemia.CI - (c) 2010 The Authors. Journal Compilation (c) 2010 International Society for Neurochemistry.

BACKGROUND: Developmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear. RESULTS: We found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos. CONCLUSIONS: Our findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyonic tissues.

Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.