RECK Promoter Research Articles

Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

LINC01419 facilitates hepatocellular carcinoma growth and metastasis through targeting EZH2-regulated RECK.

Long non-coding RNAs (lncRNAs) have been reported to play significant roles in human tumorigenesis, for example, in hepatocellular carcinoma (HCC). This study explored the role of LINC01419, a new lncRNA, in HCC. In vitro study revealed that LINC01419 promotes growth and migration of HCC cells. Genes that affected cell proliferation and cell migration were identified using RNA-sequence. Subsequently, it was confirmed that LINC01419 binds to EZH2, leading to histone methylation of the RECK promoter. Interaction between LINC01419 and FUS stabilized EZH2 mRNA thereby enhancing EZH2 expression. Conclusively, the results of this study confirm that LINC01419 may serve as a potential target for HCC diagnosis and treatment.

LncRNA SNHG8 accelerates proliferation and inhibits apoptosis in HPV-induced cervical cancer through recruiting EZH2 to epigenetically silence RECK expression.

Infection of human papillomaviruses (HPVs), such as subtypes HPV16 and HPV18 is carcinogenic to human and is prominent cause of HPV-positive cervical carcinoma (CC). A closer investigation into the mechanism of HPV-induced CC may stimulate the generation of an improved therapy treating cervical cancer. Our study herein interrogated the function of a small nucleolar RNA host gene 8 (SNHG8) in HPV-induced CC. As a result, a notable increase of SNHG8 in HPV-induced CC cells was found compared with HPV-negative CC cells. Functionally, it identified that SNHG8 aggravated the cell proliferation and migration in Cell Counting Kit-8 and transwell assays. Besides, flow cytometry apoptosis assay displayed that blockade of SNHG8 exacerbated apoptosis of HPV-positive CC cells. As detected by fluorescence in situ hybridization analysis and subcellular fractionation assay, SNHG8 was primarily expressed in the nucleus and exerted suppressive role on reversion inducing cysteine-rich protein with kazal motifs (RECK) expression, which implied a potential transcriptional regulation of SNHG8 on RECK level. Mechanically, SNHG8 was disclosed to interact with enhancer of zeste homolog 2 (EZH2) based on RNA immunoprecipitation assay. ChIP assay further unveiled the occupancy of EZH2 in the promoter region of RECK. An additional chromatin immunoprecipitation assay highlighted that SNHG8 intensified the enrichment of EZH2 and H3K27me3 in RECK promoter region. Altogether, it reflected that SNHG8 recruited EZH2 to downregulate RECK expression, leading to HPV-induced CC aggravation.

Dephosphorylation-induced EZH2 activation mediated RECK downregulation by ERK1/2 signaling.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene, a widely known cancer inhibitor, could effectively suppress cancer metastasis and angiogenesis. Downregulation or loss of RECK expression frequently occurs during cancer progression. However, the mechanism underlying RECK dysregulation has not been fully elucidated. Herein, we reportedfor the first time that enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, could epigenetically attenuate RECK expression via catalyzingH3K27 trimethylation (H3K27me3) within the RECK promoter. Furthermore, we also proved, for the first time, the involvement of EZH2 in the inhibition of RECK byextracellular signal-related kinases (ERK)-1/2 signaling. Next, we revealed that the modulation of the enzymic activity of EZH2 resulting from posttranslational phosphorylation at the serine-21 site was responsible for the increased enrichment of H3K27me3 at the RECK promoter region by ERK1/2 signaling. Collectively, the results of our study shed more light on the mechanisms responsible for the dysregulation of RECK by the ERK1/2 pathway.

Casticin inhibits the activity of transcription factor Sp1 and the methylation of RECK in MGC803 gastric cancer cells.

The present study investigated the effect of casticin on reversion-inducing-cysteine-rich protein with kazal motifs (RECK) gene expression and intracellular methylation levels in MGC803 gastric cancer cells. Cells were treated with 1, 10 and 30 µmol/l casticin. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were performed to determine the protein expression and mRNA levels of RECK and DNA methyltransferase 1 (DNMT1), respectively. High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was used to detect RECK methylation. In addition, MGC803 cell proliferation was measured by an MTT assay and the DNA-binding activity of transcription factor Sp1 was determined using an enzyme-linked immunosorbent assay. The results demonstrated that treatment with 1, 10 and 30 µmol/l casticin significantly increased RECK protein expression and mRNA levels. In addition, casticin (30 µmol/l) decreased RECK promoter methylation levels by 31%, global DNA methylation levels by 39% and nuclear methylation activity by 71.6%. Furthermore, casticin downregulated the mRNA levels and protein expression of DNMT1. The MTT assay demonstrated that MGC803 cell proliferation was inhibited by casticin treatment and DNA binding assays indicated that casticin reduced the DNA-binding activity of Sp1. The present study therefore indicated that casticin inhibits the proliferation of gastric cancer MGC803 cells by upregulating RECK gene expression and reducing intracellular methylation levels.

Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

Histone acetylation/deacetylation plays a key role in the epigenetic regulation of multiple pro-fibrotic genes. Here we investigated the effects of histone deacetyltransferase (HDAC) inhibition on angiotensin (Ang)-II-induced pro-fibrotic changes in adult mouse cardiac fibroblasts (CF). CF express class I HDACs 1 and 2, and Ang-II induces their activation. Notably, silencing HDAC1 or HDAC2 attenuated Ang-II induced CF proliferation and migration. Under basal conditions, HDAC1 dimerizes with HDAC2 in CF and Ang-II reversed this interaction. Treatment with Trichostatin A (TSA), a broad-spectrum HDAC inhibitor, restored their physical association, and attenuated Ang-II-induced MMP9 expression, IL-18 induction, and extracellular matrix (collagen I, collagen III and fibronectin) production. Further, TSA inhibited Ang-II-induced MMP9 and Il18 transcription by blocking NF-κB and AP-1 binding to their respective promoter regions. By inhibiting Sp1 binding to RECK promoter, TSA reversed Ang-II-induced RECK suppression, collagen and fibronectin expression, and CF migration and proliferation. The class I-specific HDAC inhibitor Mocetinostat (MGCD) recapitulated TSA effects on Ang-II-treated CF. Together, these results demonstrate that targeting HDACs attenuates the pro-inflammatory and pro-fibrotic effects of Ang-II on CF.

Epigallocatechin‑3‑gallate inhibits the invasion of salivary adenoid cystic carcinoma cells by reversing the hypermethylation status of the RECK gene.

Epigallocatechin‑3‑gallate (EGCG) is an active and major constituent of green tea. As a non‑nucleoside inhibitor of DNA methylation, EGCG is able to inhibit the hypermethylation of newly synthesised DNA, resulting in the reversal of hypermethylation and recovery in expression of the silenced genes. Reversion‑inducing cysteine‑rich protein with Kazal motifs (RECK) is a novel tumour suppressor gene, which negatively regulates matrix metalloproteinases, and inhibits tumour invasion, angiogenesis and metastasis. The present study aimed to examine the effects of EGCG on the methylation status of the RECK gene and tumour invasion in a salivary adenoid cystic carcinoma (SACC) cell line in vitro. Marked levels of methylated and weak levels of unmethylated RECK promoter were detected in the SACC83 cells, which was determined using methylation‑specific polymerase chain reaction (PCR). In addition, the treatment of SACC83 cells with EGCG partially reversed the hypermethylation status of the RECK gene. Western blot analysis and reverse transcription‑PCR demonstrated that EGCG significantly enhanced the protein and mRNA expression levels of RECK, and significantly reduced the invasive ability of the SACC83 cells, as determined using a Transwell assay. These results suggested that EGCG possesses novel anti‑metastatic therapeutic potential for the treatment of SACC.

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

BackgroundMutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors.MethodsSuppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-rasval12 up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7–4) harboring the inducible Ha-rasval12 oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters.ResultsRetinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-rasval12 up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells “7-4” by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model.ConclusionsWe confirmed that RbAp46 is a Ha-rasval12 up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1155-7) contains supplementary material, which is available to authorized users.

Clinical significance of RECK promoter methylation in pancreatic ductal adenocarcinoma

The aim of this study was to analyze the clinical significance of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) promoter methylation in pancreatic ductal adenocarcinoma (PDA). Methylation-specific polymerase chain reaction was used to examine the promoter methylation status of RECK in 60 pairs of PDA tissue samples and adjacent non-cancerous tissue samples. Statistical analyses were applied to test the associations between RECK promoter methylation status, clinicopathologic factors, and prognosis. The rate of RECK promoter methylation was significantly higher in PDA tissues than in adjacent non-cancerous tissues (P < 0.001). RECK methylation status was significantly associated with clinical stage (P = 0.017), histological differentiation (P = 0.046), and lymph node metastasis (P = 0.003), but was not associated with gender, age, and tumor location (all P > 0.05). Additionally, RECK promoter methylation is associated with malignant behavior and poor prognosis. In conclusion, determination of RECK promoter methylation status in tumor tissues may assist in the identification of patients who require aggressive postoperative intervention in order to improve prognosis.

RECKing MMP: Relevance of Reversion-inducing Cysteine-rich Protein with Kazal Motifs as a Prognostic Marker and Therapeutic Target for Cancer (A Review)

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored glycoprotein that negatively regulates the activities of matrix metalloproteinases (MMPs) and inhibits tumor invasion, metastasis, and angiogenesis. RECK is essential for normal development and is a key mediator of tissue remodeling and stabilization of tissue architechture. Downregulation of RECK documented in a wide range of malignant neoplasms correlates with poor prognosis, and tumor metastasis. The RECK gene is a common negative target for oncogenic signals that act on the Sp1-binding site of the RECK promoter. Both natural and synthetic agents have been identified as upregulators of RECK. Several strategies have been proposed to enhance RECK expression including forced expression of RECK, use of mimetics, recombinant peptides, microRNA antagonists, and gene therapy. Upregulation of RECK could be a valuable therapeutic option to improve prognosis and block tumor progression. This review addresses the potential value of RECK as a prognostic marker and as a molecular target for cancer therapy.

Histone deacetylase inhibitor, apicidin, inhibits human ovarian cancer cell migration via class II histone deacetylase 4 silencing

This study examined the molecular mechanisms of apicidin in the modulation of human ovarian cancer SKOV-3 cells invasion and migration. Apicidin markedly decreased histone deacetylase 4 (HDAC4) expression and blocked cell migration and invasion. Cell migration was inhibited via down-regulation of matrix metalloproteinase-2 (MMP-2) and up-regulation of RECK in the HDAC4-blocked SKOV-3 cells. Apicidin significantly suppressed the binding of HDAC4 to Sp1 binding elements of the RECK promoter via repression of HDAC4. In an in vivo model, apicidin suppressed the growth of transplanted SKOV-3 cells by down-regulating HDAC4 and MMP-2. Apicidin may potentially be used as an anti-cancer agent for inhibition of cancer cell migration and invasion through the repression of MMP-2 which is related to the reduction of HDAC4.

Effect of RECK Gene Polymorphisms on Hepatocellular Carcinoma Susceptibility and Clinicopathologic Features

BackgroundThe reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) down-regulation has been confirmed in numerous human cancers and is clinically associated with metastasis. This study investigates the potential associations of RECK single-nucleotide polymorphisms (SNPs) with hepatocellular carcinoma (HCC) susceptibility and its clinicopathologic characteristics.Methodology/Principal FindingsA total of 135 HCC cancer patients and 501 cancer-free controls were analyzed for four RECK SNPs (rs10814325, rs16932912, rs11788747, and rs10972727) using real-time PCR and PCR-RFLP genotyping analysis. After adjusting for other co-variants, the individuals carrying RECK promoter rs10814325 inheriting at least one C allele had a 1.85-fold [95% confidence interval (CI), 1.03–3.36] risk of developing HCC compared to TT wild type carriers. The HCC patients, who carried rs11788747 with at least one G allele, had a higher distant metastasis risk than wild type probands.ConclusionsRECK gene polymorphisms might be a risk factor increasing HCC susceptibility and distant metastasis in Taiwan.

The Silencing of RECK Gene is Associated with Promoter Hypermethylation and Poor Survival in Hepatocellular Carcinoma

Background: To evaluate the promoter methylation status of RECK gene and mRNA expression in patients with hepatocellular carcinoma (HCC).Methods: We analyzed RECK methylation by MSP, and RECK mRNA by real-time PCR in 74 HCC. The liver cell lines (7721, Chang and Hep-G2) were treated with 5-Aza-CdR and TSA.Results: RECK mRNA were lower in HCC tissues (Mean -∆Ct = -3.29) than that in Non-Hcc tissues (Mean -∆Ct = -2.42). Expression of RECK was elevated in only 24 (32.43%) of the 74 HCC patients but decreased (-∆∆Ct<0) in 50 (67.57%) of the patients. RECK promoter was hypermethylated in 55.4% (41/74) of HCCs, and in only 17.6% (13/74) of Non-Hcc samples. RECK mRNA were lower in HCC patients with hypermethylation (∆MI>=0.5) (Mean -∆∆Ct = -1.75) than those with demethylation (∆MI<0.5) (Mean -∆∆Ct = 0.05), and there is a decreased tendency for RECK mRNA in HCC patients with promoter hypermethylation (p = 0.002). There was a significantly correlation found between RECK mRNA and poor survival after surgery. After treated by 5-Aza-CdR and TSA, we found that RECK mRNA induced different changes in 7721, Chang and Hep-G2 cells. And RECK demethylation also induced by epigenetic inhibitors.Conclusion: The results suggested that the hypermethylation may lead to promoter silencing of RECK mRNA and associated with poor survival in HCC.

Attenuated expression and function of the RECK tumor suppressor under hypoxic conditions is mediated by the MAPK signaling pathways

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-1α expression; however, treatment with PD98059, SP600125 and SB203580 did not influence binding of HIF-1α 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-1α to the reverse hypoxia responsive element site on the RECK promoter under hypoxic conditions.

Effects of DNMT and MEK inhibitors on the expression of RECK, MMP-9, -2, uPA and VEGF in response to arsenite stimulation in human uroepithelial cells

It has been shown that the ingestion of arsenic-contaminated drinking water is closely correlated with risk of several cancers. The mechanism of arsenic-induced carcinogenesis is still unclear. The RECK, MMP-9, -2, uPA and VEGF are the most common dysregulation in human tumors and cancer cell lines. However, the effect of arsenite on these markers expression and the molecular mechanism are still unclear. The purpose of the study was to investigate the relationship between the expression of RECK, MMP-9, -2, uPA and VEGF in arsenite-treated human and rat uroepithelial cells. In addition, we also observed and compared the expression of these markers in urothelial carcinoma (UC) from Blackfoot disease (BFD) areas and non-Blackfoot disease (non-BFD) areas. We analyzed the arsenite causing cell proliferation, RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, immunocytochemistry (ICC), RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using methylation-specific PCR (MSP). Our results show that arsenite downregulation of RECK is caused by epigenetic inactivation via promoter hypermethylation, and that levels of MMP-9, -2, uPA and VEGF were increased in human uroepithelial cells (SV-HUC-1). However, when the cells were pretreated with inhibitors (5-aza-CdR or U0126) for 24 h, the effects of arsenite on RECK, MMP-9, -2, uPA and VEGF expression were suppressed. Indeed, we also found significant differences between the expression of RECK, MMP-9, -2, uPA and VEGF in UC from the BFD areas and non-BFD areas ( p = 0.006, 0.007, 0.003, <0.001 and 0.001 respectively), as detected by immunohistochemistry (IHC). In in vivo study, our results showed the RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups. In conclusion, our results support the notion that arsenite might cause the histologic changes, RECK, MMP-9, -2, uPA and VEGF dysregulation through epigenetic inactivation and ERK1/2 activation in SV-HUC-1 cells. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.

A novel screen using the Reck tumor suppressor gene promoter detects both conventional and metastasis-suppressing anticancer drugs.

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.

Tumor reversion holds promise.

In the present issue of the journal, Makoto Noda and colleagues present a screening assay for anti-cancer drugs based on tumor reversion, identifying a series of hitherto unsuspected compounds that are of potential therapeutic interest. More specifically, the assay is based upon triggering the promoter of Reck, which functions as an inhibitor of metalloproteinases. Among the pharmaceutical agents that were able to activate the Reck promoter, 1/3 were known anti-cancer drugs which act through different cytopathic mechanisms. The second group comprises drugs that inhibit growth of bacteria, plasmodium falciparum, fungi and worms. The third category consists of “drugs related to the function of the central nervous system”. Now, here comes the interesting and original idea of the paper. Activation of Reck induces “flat revertants” and Noda used this system already to clone the K-rev gene [1,2]. Flat revertants have a history of their own that started when investigators in the early 1960s used the NIH3T3 cells to assay the transforming potential of oncoviruses and oncogenes. A couple of years later in 1968, when Robert Pollack stepped into the field, he made a remarkable observation. Some of the cells infected with SV40 or polyoma viruses no longer showed the typical oncogenic phenotype, but instead, acquired a flat morphology [3]. These cells had also lost their oncogenic potential and were therefore named “flat revertants”. This reversion of the malignant phenotype was in line with the experiments initiated by Askanazy in teratocarcinoma cells at the beginning of the 20th century and later expanded to different species and cell types [4]. The most spectacular tumor reversion experiments have been carried out in plants by Braun [4]. Various approaches, using different biological systems [5,6,7] led to the identification of at least 300 genes that could be implicated in the tumor reversion process, including siah1, PS1, TSAP6, TCTP, Integrin receptors, and Nodal. Interestingly, Nodal is an embryonic morphogen recently found to be a key plasticity gene in aggressive tumor cells. Down-regulation of Nodal in this plastic phenotype results in reversion to a lineage specific cell type and tumor suppression [8]. Targeting tumor reversion genes in order to induce a suppression of the malignant phenotype has already led to the identification of a series of anti-histaminic, neuroleptic and anti-depressive drugs that inhibit intra-cellular levels of TCTP, a key gene in tumor reversion [9]. Other genes such as PS1, a predisposition gene to familial Alzheimer’s disease targeted by gamma-secretase inhibitors are tested in Kaposi sarcoma patients [4]. Tumor reversion as used here in Noda’s study [10] can be a fast-track for the identification of new anti-cancer drugs when we are still far away from understanding how reversion functions at the molecular level, and this is a major future challenge.

A novel screen using the Reck tumor suppressor gene promoter detects both conventional and metastasis-suppressing anticancer drugs.

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.

Hypermethylation of the RECK gene predicts poor prognosis in oral squamous cell carcinomas

The RECK gene is a novel tumor suppressor gene that regulates matrix metalloproteinases (MMPs) to inhibit tumor angiogenesis, invasion and metastasis. We investigated the methylation status of the RECK gene in 40 primary oral squamous cell carcinomas (OSCC) and 20 paired adjacent normal mucosa by methylation-specific PCR. Furthermore, we determined the prognostic importance of RECK hypermethylation in OSCC patients. Our findings showed that the RECK gene was methylated in 52.5% (21 of 40) of the primary OSCC. Among the 20 cases with corresponding normal tissues, RECK hypermethylation was detected in both primary tumor (55%, 11 of 20) and adjacent normal mucosa (30%, 6 of 20). Methylation of the RECK gene was not detected in all normal oral mucosa samples of the 12 healthy controls. In univariate analysis, RECK hypermethylation was inversely correlated with recurrence-free survival (p=0.027) and overall survival (p=0.023) of the OSCC patients. Multivariate analysis showed that the methylation status of the RECK gene was the only independent prognostic factor affecting overall survival (p=0.037). The result indicates that hypermethylation of RECK promoter is a common event in human OSCC, occurs concurrently in tumor-adjacent normal mucosa and is correlated with poor prognosis in OSCC patients. Although additional work is needed, hypermethylation of the RECK gene is a promising biomarker in early detection and prognosis for oral cancer patients.

Epigenetic inactivation of the metastasis suppressor RECK enhances invasion of human colon cancer cells

Down-regulation of RECK, an important metastasis suppressor gene, has been found in human colon cancer. However, the molecular mechanism for this down- regulation and its biological significance are still unclear. In the present study, we investigated whether down-regulation of RECK is caused by epigenetic inactivation via promoter methylation and tested the effect of DNA methyltransferase (DNMT) inhibitor on RECK expression and cell invasion. The mRNA and protein levels of RECK in colon tumor tissues and their normal counterparts were compared. We found that down-regulation of RECK was found in 48% of the twenty five tumors analyzed. MSP analysis demonstrated that methylation of RECK promoter was detected in 44% (11/25) of the tumor tissues and a strong correlation between down-regulation and promoter methylation was found (P = 0.028). Promoter methylation was also found in SW480 and SW620 human colon cancer cell lines. DNA methyltransferase (DNMT) inhibitor 5'-azacytidine reversed promoter methylation, restored RECK expression and suppressed invasion by these two cell lines. Restoration of RECK is critical for 5'-azacytidine-mediated suppression of cell invasion because inhibition of RECK by a specific antibody significantly attenuated the anti-invasive ability of 5'-azacytidine. Taken together, our results suggest that down-regulation of the metastasis suppressor RECK in colon cancer is associated with promoter methylation and that a DNMT inhibitor may restore RECK expression to inhibit cell invasion.