Research paperLRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication
Highlights
► We tested more than 230 tumor/normal pairs from different cancers. ► Methylation of the LRRC3B was studied using methylation specific NotI microarrays. ► Methylation of the LRRC3B gene was found in 28 (ovarian)–44 (prostate) % of samples for 7 different epithelial cancers. ► LRRC3B exhibited cell growth inhibiting activity in colony formation experiments. ► Expression of LRRC3B gene is strongly down-regulated at the latest stages of RCC and ovarian cancer.
Introduction
Epithelial tumors are the most prevalent and lethal cancers in the world. They cause more than 80% of all cancer deaths. For example, only lung cancer kills >150 000 patients each year in USA and many more around the world. Loss of heterozygosity (LOH) involving several chromosome 3p regions accompanied by chromosome 3p deletions is a characteristic feature of the major epithelial cancers (MEC). It is detected in almost 100% of small cell lung cancer (SCLC), clear cell renal cell carcinoma (RCC) and more than 90% of non-small cell lung cancer (NSCLC) [1]. Chromosome transfer experiments indicated that different regions of 3p could suppress the tumorigenic properties of cancer lines. The presence of several tumor suppressor genes (TSG) in 3p was suggested [2], [3].
Aberrant tumor acquired DNA promoter region methylation constitutes an important mechanism in carcinogenesis and can represent the main mechanism for inactivation of several TSGs. This mechanism of inactivation has been thoroughly studied during large-scale searches for chromosome 3 TSGs (see [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]).
In different types of tumors, aberrant or accidental methylation of CpG islands in the promoter region has been observed for many cancer-related genes resulting in the silencing of their expression. The genes involved include tumor suppressor genes, genes that suppress metastasis and angiogenesis, and genes that repair DNA suggesting that epigenetics plays an important role in tumorigenesis. The potent and specific inhibitor of DNA methylation, 5-aza-2-deoxycytidine (5-AZA-dC) has been demonstrated to reactivate the expression most of these malignancy suppressor genes in human tumor cell lines. These genes may be interesting targets for chemotherapy with inhibitors of DNA methylation in patients with cancer and this may help clarify the importance of this epigenetic mechanism in tumorigenesis. Spontaneous regression of malignant tumors enchanted researchers, and it is now noticed that genes inactivated by hypermethylation are frequently involved in tumors that relatively often undergo spontaneous regression. Carcinogenic mechanisms of some carcinogens seem to involve modifications of epigenetic switch, and some dietary factors also have the possibility to modify the switches.
The growing interest in understanding the methylation mechanisms is obvious from the fact that only in January-beginning of July 2011 more than 40 reviews were registered in PubMed. These reviews cover different aspects in the field and it becomes clear that methylation is a basic, vital feature/mechanism in mammalian cells.
New type of microarrays recently developed in our group open new possibilities for large-scale study of methylation patterns in normal and pathological cells [8]. The main objective is to prepare and to use NotI microarrays (NMA, i.e. glass microarrays with attached NotI DNA fragments) for comparison of normal and malignant cells at genomic level. The main idea of the approach is that NotI enzyme cuts only unmethylated CpG pairs inside the recognition site of the enzyme (5′-GCGGCCGC-3′) and only small fraction (0.1–0.05%) of the human genome containing NotI digested fragments is labeled. Thus in contrast to all other methods where undigested by methylation sensitive enzymes DNA fragments are labeled, we label only digested DNA fragments. As a consequence our probe contains 10-fold less repeats, it is more hot, not so sensitive to incomplete digestion and gives less background. Earlier to prove results of NMA hybridization we used Southern blotting, quantitative real time PCR, methyl-specific PCR (MSP) and bisulfite sequencing. We sequenced 28 genes from 105 tumors and results were always compatible with the NMA results.
Our estimation is that human genome contains 10.000–15.000 NotI sites and 5.000–9.000 of them are unmethylated in a particular cell. At present we use NMA for human chr.3 (188 NotI containing genes).
Leucine-rich repeat-containing 3B (LRRC3B) is an evolutionarily highly conserved leucine-rich repeat-containing protein located in 3p24, but its biological significance is unknown. It was suggested that human genome has more than 2000 LRR-containing proteins and they participate in many important processes, including plant and animal immunity, hormone–receptor interactions, cell adhesion, signal transduction, regulation of gene expression, and apoptosis. A number of microarray expression profiling studies on human cancers have shown that LRRC3B is down-regulated in gastric, breast, colon, testis, prostate, and brain cancers, suggesting LRRC3B involvement in carcinogenesis (see [15]).
DNA methylation is a key mechanism to inhibit the expression of tumor suppressor genes in cancer, and DNA methylation markers have been applied in cancer risk assessment, early detection, prognosis, and prediction of response to cancer therapy.
We decided to check methylation of the LRRC3B gene in different tumors after we discovered that it is frequently methylated in childhood acute lymphoblastic leukemia [16]. It was also shown by other authors that LRRC3B is frequently methylated in HeLa cervical cancer cell line, acute leukemia, gastric and colon cancer [15], [16], [17], [18], [19].
Expression of the gene was down-regulated in these tumors and restored by a demethylating agent, 5-aza-2′-deoxycytidine [15], [16], [17], [18], [19]. Stable transfection of LRRC3B in SNU-601 cells, a gastric cancer cell line, inhibited anchorage-dependent and anchorage-independent colony formation, and LRRC3B expression suppressed tumorigenesis in nude mice [15]. Thus it was suggested that LRRC3B is a putative tumor suppressor gene that is silenced in cancers by epigenetic mechanism.
In this study we further analyzed methylation of LRRC3B in major epithelial cancers: breast, cervical, lung, kidney, ovarian, colon and prostate.
Section snippets
Cell lines, tumor samples and general methods
Paired tumor/normal samples: breast (47 pairs), cervical (43 pairs), lung (40 pairs, non-small cell lung cancer, NSCLC), kidney (34 pairs, clear cell renal cell carcinoma, RCC), ovarian (25 pairs), colon (24 pairs) and prostate (18 pairs) were obtained from Blokhin Cancer Research Center, the Russian Academy of Medical Sciences. Adjacent morphologically normal tissues (conventional “normal” tissues) were obtained from patients after surgical resection of primary tumours. Top and bottom sections
Analysis of NSCLC samples for methylation of LRRC3B using NotI microarrays and bisulfite sequencing
Chromosome 3 specific NotI microarrays containing 188 genes was hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), cervical (43 pairs), lung (40 pairs), kidney (34 pairs), ovarian (25 pairs), colon (24 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. In breast cancer LRRC3B was methylated and/or deleted in 32% of breast samples, in
Conclusions
A few articles were dedicated to the LRRC3B gene. According to Kim et al. [15], LRRC3B is a target of aberrant methylation in gastric cancer. They showed LRRC3B silencing by epigenetic mechanisms in both gastric cancer cell lines and primary tumors and its tumor suppressor activity in vitro and in vivo as well. In this work we for the first time demonstrated that LRRC3B gene was methylated and/or deleted with high frequency in major epithelial cancers: breast, cervical, lung, kidney, ovarian,
Acknowledgments
This work was supported by research grants from the Swedish Cancer Society, the Swedish Institute, the Swedish Research Council and Karolinska Institute, State Contracts 02.740.11.5227 and 16.552.11.7034 with the Russian Ministry of Education and Science and by grants 10-04-01213-а and 11-04-00269 from the Russian Foundation for Basic Research.
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These authors contributed equally to this work.