Downregulation of RECK by promoter methylation correlates with lymph node metastasis in non‐small cell lung cancer

Abstract

In the present study, we addressed the molecular mechanism of the downregulation of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), a critical tumor suppressor that can potently inhibit angiogenesis and metastasis, in non-small cell lung cancer and its clinical significance. The methylation status of the RECK gene promoter was studied by methylation-specific polymerase chain reaction. RECK mRNA and protein levels were investigated by reverse transcription-polymerase chain reaction and western blot analysis. Downregulation of RECK was observed in 60% of the 55 tumors analyzed. Using methylation-specific polymerase chain reaction analysis methylation of the RECK promoter was detected in 63.6% (35/55) of the tumor tissues. A strong correlation between downregulation and promoter methylation was found in these tumors (P = 0.000005). More importantly, downregulation of RECK significantly correlated with lymph node metastasis (P = 0.038). T he reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) gene was isolated as a transformation suppressor gene that induced flat reversion in v-K-ras-transformed NIH/3T3 cells.(1) This gene encodes a membrane-anchored glycoprotein that can negatively regulate matrix metalloproteinase-2 (MMP-2) and MMP-9 activities and inhibit tumor angiogenesis and metastasis.(2,3) Whereas RECK mRNA is highly expressed in most normal human tissues and untransformed cells, it is downregulated or undetectable in many tumor cell lines or in cells ectopically expressing active oncogenes.(2) Pathological studies have demonstrated that RECK downregulation is found in human cancers including pancreatic cancer, breast cancer, non-small cell lung cancer and colon cancer.(4 -7) In addition, reduced RECK expression correlates with poor prognosis in these cancers. However, the molecular mechanism that causes gene silencing of RECK in cancer cells is still unknown.Mutations in the K-ras proto-oncogene are found frequently in non-small cell lung cancer.(8) Aberrant activation of this oncogene has been implicated in many aspects of malignant phenotypes including proliferation, transformation, invasion and metastasis. Numerous studies have shown that oncogenic ras increases the metastatic ability of transformed cells.(9-11) However, the underlying mechanism is poorly characterized. Recently, we addressed the effect of oncogenic ras on RECK expression using an in vitro inducible system.(12) Our results showed that ras activation stimulates the expression of DNA methyltransferase 3b (DNMT3b), promotes DNMT3b binding to the RECK promoter and represses RECK expression via methylation of the promoter. We also found that human lung cancer cells harboring K-ras mutations exhibit increased promoter methylation and reduced expression of the RECK gene.(12) To verify the results observed in our previous study, we examined the methylation status of the RECK promoter in 55 lung tumor tissues. Moreover, we studied whether RECK promoter methylation correlated with K-ras mutation and other clinicopa...

The Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) Interacts with Membrane Type 1 Matrix Metalloproteinase and CD13/Aminopeptidase N and Modulates Their Endocytic Pathways

“…RECK has been shown to suppress the extracellular release of proMMP-9 (1) and competitively inhibit the proteolytic activity of active MMP-9 (1) and MMP-2 (3). Subsequently, the current investigation implicates that RECK contains domains functionally similar to the substrates of MMPs 5 as in the case of ␣2-macroglobulin, which is a major plasma inhibitor of metalloproteases (8). As compared with soluble MMP inhibitors represented by tissue inhibitor of metalloproteinases (TIMPs) (8), the most distinguishing feature of RECK is its ability to covalently anchor to the membrane surface via a post-translational modification (glycosylphosphatidylinositol (GPI) anchor) that is conserved among species (1).…”

“…4). RECK promoter was recently found to be frequently methylated in metastatic non-small cell lung carcinomas (5). In addition, RECK has been implicated in chronic inflammatory diseases (6,7).…”

Effects of green tea polyphenol on methylation status of RECK gene and cancer cell invasion in oral squamous cell carcinoma cells

“…Its downregulation has been shown in several types of human cancers. Recently, the decrease in RECK expression is reported to correlate with hypermethylation of the promoter region (Furumoto et al, 2000;Song et al, 2006;Chang et al, 2007;Cho et al, 2007). In human cancers, aberrant methylation of tumour suppressor genes is of comparable significance to classic genetic mutations (Lund and van Lohuizen, 2004;Ha and Califano, 2006;Stresemann et al, 2006;Shaw, 2006).…”

Reversion-inducing cysteine-rich protein with Kazal motifs interferes with epidermal growth factor receptor signaling

“…Furthermore, histone modification and DNA methylation have also been reported to be involved in the transcriptional regulation of RECK (Chang et al, 2004(Chang et al, , 2007. RECK expression is frequently correlated with a favorable prognosis in patients with various types of cancer (reviewed in Clark et al, 2006Clark et al, , 2007Noda and Takahashi, 2007).…”