Frequent Inactivation of RAMP2, EFEMP1 and Dutt1 in Lung Cancer by Promoter Hypermethylation

Abstract

Purpose: The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer. Experimental Designs: Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2 ¶-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth. Results: Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2 ¶-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor^containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation. Conclusions: Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.

EFEMP1 Expression Promotes In vivo Tumor Growth in Human Pancreatic Adenocarcinoma

“…Taken together with the observation that EFEMP1 overexpression stimulates VEGF production in vivo, our findings are concordant with reports of the up-regulation of VEGF in pancreatic cancer (34)(35)(36). In contrast to our data that support a role of EFEMP1 in tumor progression in pancreatic adenocarcinoma, EFEMP1 expression was found down-regulated in lung cancer (37) and up-regulated in only 10% of different solid cancer entities (18). These discordant data may be caused by differences in the tumor entities examined; however, all published data on EFEMP1 expression in human tumor samples are limited by small sample size, thus bear a rather preliminary character.…”

Role of fibulin-3 in lung cancer: In vivo and in vitro analyses

“…Both in our previous study (10) and the present study, fibulin-3 expression was revealed to be associated with the regulation of tumor cell growth and invasiveness in A549 cells by suppressing the expression of MMP-9 and -2. Furthermore, the mechanism of fibulin-3 was identified by using western blotting.…”

“…The fibulin-3 gene, also known as EFEMP1 (epidermal growth factorcontaining fibulin-like extracellular matrix protein 1) located at chromosome 2p16, is one of the 7 members of the fibulin gene family of extracellular glycoproteins (8). The level of fibulin-3 expression was found to be decreased in many cancer types due to aberrant promoter methylation and is correlated with poor survival of patients with breast cancer (9) lung cancer (10) and hepatocellular carcinoma (11). Lecka-Czernik et al (12) predicted that this protein has 2 possible isoforms, a ̔longʼ form of 53 to 55 kDa, with an NH2-terminal Ca 2+ -binding epidermal growth factor-like repeat, and a ̔shortʼ form of 40 to 43 kDa without this domain.…”

Assessing the association between EFEMP1 rs3791679 polymorphism and risk of glioma in a Chinese Han population

“…Previous studies have reported that the EFEMP1 gene plays a role in several types of cancers, such as lung cancer, liver cancer, breast cancer, prostate cancer, and nasopharyngeal cancer (Yue et al, 2007;Sadr-Nabavi et al, 2009;Hwang et al, 2010;Nomoto et al, 2010;Kim et al, 2011;Zhang et al, 2011). Yue et al (2007) reported that alterations in the expression of EFEMP1 could inhibit lung cancer-cell growth, resulting in a high tumor grade.…”