Frequent aberrant methylation of the promoter region of sterile alpha motif domain 14 in pulmonary adenocarcinoma

Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor-suppressor and tumor-related genes. In order to identify novel hypermethylated genes in early stage lung adenocarcinoma, we carried out methylated CpG island amplification, modified suppression subtractive hybridization, and methylation-specific polymerase chain reaction to identify aberrant methylation of CpG islands in the A/J mouse lung adenoma model, which histologically mimics the early stage of human pulmonary adenocarcinoma. Through methylated CpG island amplification, suppression subtractive hybridization, and differential screening, we detected five genes, three of which have human homologs. Two of them showed downregulation of their expression in human lung adenocarcinoma. Of these two genes, we selected sterile alpha motif domain 14 (SAMD14) and further analyzed its methylation status and expression level by methylation-specific polymerase chain reaction and quantitative real-time polymerase chain reaction. Most of the lung adenocarcinoma cell lines showed suppressed expression of SAMD14 together with hypermethylation at the promoter region, although an immortalized bronchial epithelium cell line (PL16B) did not show hypermethylation and did express SAMD14. The expression of SAMD14 in A549 was rescued by treatment with the demethylation agent 5-aza-2'-deoxycytidine. These data indicate that hypermethylation of the SAMD14 gene promoter region is associated with silencing of its expression. Hypermethylation at the CpG site of the SAMD14 promoter region was detected frequently in early invasive adenocarcinoma (8/24, 33.3%) but not in in situ adenocarcinoma (0/7, 0%) or normal lung tissue (0/31, 0%). Hypermethylation of the SAMD14 gene is a specific event in pulmonary adenocarcinogenesis and malignant progression.     

NORE1A,a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers

We recently demonstrated that RASSF1A, a new tumour-suppressor gene located at 3p21.3 is frequently inactivated by promoter region hypermethylation in a variety of human cancers including lung, breast, kidney and neuroblastoma. We have identified another member of the RASSF1 gene family by in silico sequence analysis using BLAST searches. NORE1 located at 1q32.1 exists in three isoforms (NORE1Aalpha, NORE1Abeta and NORE1B). Both NORE1A and NORE1B isoforms have separate CpG islands spanning their first exons. NORE1Aalpha Produces a 418 aa protein containing a Ras-association (RA) domain and a diacylglycerol (DAG) binding domain. NORE1Abeta produces a C-terminal truncation of the RA domain. NORE1B also contains the RA domain but not the DAG domain. NORE1 is the human homologue of the mouse Ras effector Nore1. No inactivating somatic mutations were found in lung tumour lines; however, NORE1A promoter region CpG island was hypermethylated in primary tumours and tumour cell lines. NORE1A promoter was methylated in 10/25 breast, 4/40 SCLC, 3/17 NSCLC, 1/6 colorectal and 3/9 kidney tumour cell lines, while NORE1B promoter was unmethylated in the same tumour cell lines. While 24% (6/25) of primary NSCLC underwent NORE1A methylation, methylation in SCLC was a rare event (0/22); (P = 0.0234). NORE1A expression in tumour cell lines was reactivated after treatment with a demethylating agent. There was no correlation between NORE1A and RASSF1A methylation status in NSCLC. Our results demonstrate that NORE1A is inactivated in a subset of human cancers by CpG island promoter hypermethylation, and in lung cancer this hypermethylation may be histological type specific.     

CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer.

CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.     

Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers

A variety of tumor suppressor genes are down-regulated by hypermethylation during carcinogenesis. Using methylated CpG amplification-representation difference analysis, we identified a DNA fragment corresponding to the Tazarotene-induced gene 1 (TIG1) promoter-associated CpG island as one of the genes hypermethylated in the leukemia cell line K562. Because TIG1 has been proposed to act as a tumor suppressor, we tested the hypothesis that cytosine methylation of the TIG1 promoter suppresses its expression and causes a loss of responsiveness to retinoic acid in some neoplastic cells. We examined TIG1 methylation and expression status in 53 human cancer cell lines and 74 primary tumors, including leukemia and head and neck, breast, colon, skin, brain, lung, and prostate cancer. Loss of TIG1 expression was strongly associated with TIG1 promoter hypermethylation (P < 0.001). There was no correlation between TIG1 promoter methylation and that of retinoid acid receptor beta2 (RARbeta2), another retinoic-induced putative tumor suppressor gene (P = 0.78). Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 5 days restored TIG1 expression in all eight silenced cell lines tested. TIG1 expression was also inducible by treatment with 1 micro M all-trans-retinoic acid for 3 days except in densely methylated cell lines. Treatment of the K562 leukemia cells with demethylating agent combined with all-trans-retinoic acid induced apoptosis. These findings indicate that silencing of TIG1 promoter by hypermethylation is common in human cancers and may contribute to the loss of retinoic acid responsiveness in some neoplastic cells.     

Hierarchical clustering of lung cancer cell lines using DNA methylation markers.

Recent analyses of global and gene-specific methylation patterns in cancer cells have suggested that cancers from different organs demonstrate distinct patterns of CpG island hypermethylation. Although certain CpG islands are frequently methylated in many different kinds of cancer, others are methylated only in specific tumor types. Because distinct patterns of CpG island hypermethylation can be seen in tumors from different organs, it seems likely that histological subtypes of cancer within a given organ may exhibit distinct methylation patterns as well. The goal of our study was to determine whether the patterns of CpG island hypermethylation could be used to distinguish between different histological subtypes of lung cancer. We analyzed the methylation status of 23 loci in 91 lung cancer cell lines using the quantitative real-time PCR method MethyLight. Genes PTGS2 (COX2), CALCA, MTHFR, ESR1, MGMT, MYOD1, and APC showed statistically significant differences in the level of CpG island methylation between small cell lung cancer (SCLC) and non-small cell lung cancer cell lines (NSCLC). Hierarchical clustering using a panel consisting of these seven loci yielded two major groups, one of which contained 78% of the SCLC lines. Within this group, a large cluster consisted almost exclusively of SCLC cell lines. Our results show that DNA methylation patterns differ between NSCLC and SCLC cell lines and suggest that these patterns could be developed into a powerful molecular marker to achieve accurate diagnosis of lung cancer.     

Epigenetic silencing of maspin gene expression in human breast cancers

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.