Data from Cytoglobin, the Newest Member of the Globin Family, Functions as a Tumor Suppressor Gene

Abstract

<div>Abstract<p>Cytoglobin (<i>CYGB)</i> is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. <i>CYGB</i> is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the <i>CYGB</i> gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of <i>CYGB</i> as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the <i>CYGB</i> promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of <i>CYGB</i> methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of <i>CYGB</i>, we performed the following: (<i>a</i>) RNA interference–mediated knockdown of <i>CYGB</i> gene on colony formation in a <i>CYGB</i> expression–positive lung cancer cell line, resulting in increased colony formation; (<i>b</i>) enforced gene expression in <i>CYGB</i> expression–negative lung and breast cancer cell lines, reducing colony formation; and (<i>c</i>) identification of potential proximate targets down-stream of the <i>CYGB</i> genes. Our data constitute the first direct functional evidence for <i>CYGB</i>, the newest member of the globin family, as a tumor suppressor gene. [Cancer Res 2008;68(18):7448–56]</p></div>