Inactivation of LLC1 gene in nonsmall cell lung cancer

Abstract

Serial analysis of gene expression studies led us to identify a previously unknown gene, c20orf85, that is present in the normal lung epithelium but absent or downregulated in most primary nonsmall cell lung cancers and lung cancer cell lines. We named this gene LLC1 for Low in Lung Cancer 1. LLC1 is located on chromosome 20q13.3 and has a 70% GC content in the promoter region. It has 4 exons and encodes a protein containing 137 amino acids. By in situ hybridization, we observed that LLC1 message is localized in normal lung bronchial epithelial cells but absent in 13 of 14 lung adenocarcinoma and 9 out of 10 lung squamous carcinoma samples. Methylation at CpG sites of the LLC1 promoter was frequently observed in lung cancer cell lines and in a fraction of primary lung cancer tissues. Treatment with 5-aza deoxycytidine resulted in a reduced methylation of the LLC1 promoter concomitant with the increase of LLC1 expression. These results suggest that inactivation of LLC1 by means of promoter methylation is a frequent event in nonsmall cell lung cancer and may play a role in lung tumorigenesis.

Figure 1

LLC1 gene and its expression in lung tumor and normal tissues. (a) LLC1 protein sequence and its homologues. Mouse (BC050790), bovine (BC109834) and canine (XM_849590). LLC1 amino acid sequences had about 70% homology to the human sequence (NM178456). The bold characters represent identical amino acids. (b) The level of LLC1 expression was determined by real-time RT-PCR. The expression of LLC1 was decreased in 9 out of 16 adenocarcinoma tissues. (c) In human tissues, LLC1 expression was determined in ovary (Ov), fetal lung (Lu), stomach (St), liver (Lv), breast (Br), colon (Co), heart (Ht), skeletal muscle (Ms), placenta (Pl), brain (BR), spleen (Sp), bladder (Bl), skin (Sk) and kidney (Kd). Y-axis is the relative level of LLC1 expression (on a log scale) in tumor samples compared with paired non-tumor tissues (b) or the median Ct difference of LLC1 and GUSB (−3.4) for nontumor lung tissues (c).

Figure 2

The expression of LLC1 in lung tissue and lung adenocarcinoma by RNA in situ hybridization. The LLC1 was expressed in lung epithelial cells (a and b). (c) Lack of LLC1 expression in primary lung cancers. (d) Lung cancer with a weak LLC1 expression. Tissue samples were counterstained with hematoxylin, and photographed under ×100 (a and c) or ×400 (b and d) magnification. Arrow heads (▲) indicate tissue areas of LLC1 expression. Arrow (→) indicates tumor regions without LLC1 expression.

Figure 3

The expression by real-time RT-PCR and the promoter methylation status of LLC1 in NSCLC cell lines. (a) The promoter region of LLC1 is shown with the CpG islands (*); and the PCR primer sites for the MSBE assay (underlined). The ATG start site of LLC1 is boxed, as well as the selected CpG site for the MSBE assay. (b) Typical results from MSBE for the methylation of LLC1 promoter are shown for cell lines, H23 and H522. The relative peak heights (M/U ratios) are indicated. (c) The increased methylation (upper panel) is correlated with the decreased LLC1 expression in most lung cancer cell lines (lower panel). (d) The methylation status, as assessed by MSBE method (upper panel), directly correlates with those observed by MSP (lower panel). Cell lines are indicated on the X-axis and Y-axis as the methylation/unmethylation peak ratio (M/U) from the MSBE assay or the relative Ct value in Q-PCR analysis as described in the Material and Method.

Figure 4

The restoration of LLC1 expression in NSCLC cell lines and methylation of LLC1 promoter in primary lung cancers. (a) Promoter methylation status in A549, H1299 and HOP92 cells before and after treatment with 5-aza dC. (b) The expression of LLC1 in indicated cell lines before and after 5-aza dC treatment. (c) The methylation of LLC1 promoter in 67 primary NSCLC and 29 nontumor lung samples. Y-axis is the methylation/unmethylation peak ratio (M/U) from MSBE assay. The average M/U ratios for tumor and nontumor groups are indicated by the horizontal lines along with standard deviations. p value was determined by pair-wise t test.