Promoter methylation study of the H37/RBM5 tumor suppressor gene from the 3p21.3 human lung cancer tumor suppressor locus.

Loss of heterozygosity (LOH) at chromosome 3p21.3 is one of the most prevalent genetic disturbances occurring at the earliest stage of tumor development for a wide variety of human cancers, culminated in lung cancer. The 19 genes residing at 3p21.3 have been vigorously characterized for tumor suppressor activity and gene inactivation mechanism because of their potentially significant merits of clinical applications. Many of these 19 genes have been shown to manifest various growth inhibitory properties, however none of them are inactivated by coding mutations in their remaining allele as in the Knudson's two- hits hypothesis. Thus far the most prevailing, alternative gene inactivation mechanism known for the 3p21.3 TSGs is epigenetic silencing by promoter hypermethylation. Previously, we have focused our investigation on one of the 19 genes at 3p21.3, H37/RBM5, and demonstrated its tumor suppressor activity both in vitro and in vivo as well as its mRNA/protein expression loss from the remaining allele in a majority of the primary lung tumors examined. The current study tested our hypothesis that the H37 inactivation in primary lung tumors may, as seen in most of the other 3p21.3 TSGs, be due to hypermethylation in its promoter CpG islands. Contrary to this most plausible postulation, however, we found no evidence of epigenetic gene silencing for the H37 TSG. Here we suggest some of the possible, further- alternative means of the H37 gene expression loss in tumor, including defects in transcription and post-transcriptional/translational modifications as well as mechanisms related to haploinsufficiency.

3p21.3 tumor suppressor gene H37/Luca15/RBM5 inhibits growth of human lung cancer cells through cell cycle arrest and apoptosis.

Deletion at chromosome 3p21.3 is the earliest and the most frequently observed genetic alteration in lung cancer, suggesting that the region contains tumor suppressor gene(s) (TSG). Identification of those genes may lead to the development both of biomarkers to identify high-risk individuals and novel therapeutics. Previously, we cloned the H37/Luca15/RBM5 gene from 3p21.3 and showed its TSG characteristics. To investigate the physiologic function of H37 in the lung and its mechanism of tumor suppression, we have stably transfected H37 into A549 non-small cell lung cancer cells. A549/H37 cells show significant growth inhibition compared with the vector controls by in vitro and in vivo cell proliferation assays. Using this lung cancer cell model, we have found that the molecular mechanism of H37 tumor suppression involves both cell cycle (G(1)) arrest and apoptosis. To further define H37's function in cell cycle/apoptotic pathways, we investigated differential expression profiles of various cell cycle and apoptosis regulatory proteins using Western blot analysis. Both cyclin A and phophorylated RB levels were decreased in H37-transfected cells, whereas expression of Bax protein was increased. Mitochondrial regulation of apoptosis further downstream of Bax was investigated, showing change in the mitochondrial membrane potential, cytochrome c release into the cytosol, and enhanced caspase-9 and caspase-3 activities. We also report that H37 may mediate apoptosis in a p53-independent manner, and Bax knockdown by small interfering RNA suggests Bax plays a functional role downstream of H37. Lastly, we proposed a tumor suppression model of H37 as a post-transcriptional regulator for cell cycle/apoptotic-related proteins.

The two single nucleotide polymorphisms in the H37/RBM5 tumour suppressor gene at 3p21.3 correlated with different subtypes of non-small cell lung cancers.

Allele loss and genetic alteration in chromosome 3p, particularly in 3p21.3 region, are the most frequent and the earliest genomic abnormalities found in lung cancer. Multiple 3p21.3 genes exhibit various degrees of tumour suppression activity suggesting that 3p21.3 genes may function as an integrated tumour suppressor region through their diverse biological activities. We have previously demonstrated growth inhibitory effects and tumour suppression mechanism of the H37/RBM5 gene which is one of the 19 genes residing in the 370kb minimal overlap region at 3p21.3. In the current study, in an attempt to find, if any, mutations in the H37 coding region in lung cancer cells, we compared nucleotide sequences of the entire H37 gene in tumour versus adjacent normal tissues from 17 non-small cell lung cancer (NSCLC) patients. No mutations were detected; instead, we found the two silent single nucleotide polymorphisms (SNPs), C1138T and C2185T, within the coding region of the H37 gene. In addition, we found that specific allele types at these SNP positions are correlated with different histological subtypes of NSCLC; tumours containing heterozygous alleles (C+T) at these SNP positions are more likely to be associated with adenocarcinoma (AC), whereas, homozygous alleles (either C or T) are associated with squamous cell carcinoma (SCC) (p=0.0098). We postulate that, these two silent polymorphisms may be in linkage disequilibrium (LD) with a disease causative allele in the 3p21.3 tumour suppressor region which is packed with a large number of important genes affecting lung cancer development. In addition, because of prevalent loss of heterozygosity (LOH) detected at 3p21.3 which precedes lung cancer initiation, these SNPs may be developed into a marker screening for the high risk individuals.

A candidate tumor suppressor gene, H37, from the human lung cancer tumor suppressor locus 3p21.3.

Frequent allelic loss and homozygous deletions within chromosome 3p in human lung cancers have suggested that the 3p21.3 (370-kb) region contains a critical tumor suppressor gene(s) (TSG). With the exact identity/characteristics of such a gene(s) still unconfirmed, a lack of inactivating structural mutations in the expressed genes contained within this region may indicate that the 3p TSG(s) do not fit into the classical "two-mutation" model. This report characterizes a candidate 3p TSG, H37, located within the 370-kb region. Reduced expression of the H37 transcript was found in 9 of 11 (82%) of primary non-small cell lung cancers (NSCLCs) when compared with adjacent normal tissues. Generation of an H37 antibody followed by immunohistochemical analysis of primary NSCLC specimens demonstrated that 46 of 62 (73%) of these cancers contain reduced levels of H37 protein when compared with adjacent normal bronchial cells. Moreover, introduction of the H37 cDNA into human breast cancer cells deleted of 3p21-22 reduced both anchorage-dependent and -independent cell growth in vitro. Subsequent transfection of H37 cDNA into one of the human lung cancer cell lines homozygously deleted in this region resulted in a very low yield of H37-expressing clones. H37 also suppressed anchorage-dependent and -independent growth of A9 mouse fibrosarcoma cells and inhibited tumor formation in nude mice. These data indicate a potential role for H37 as one of the 3p TSGs in human lung cancer.

RBM5/H37 tumor suppressor, located at the lung cancer hot spot 3p21.3, alters expression of genes involved in metastasis.

The RBM5/H37 gene is located at the most 'sought-after' tumor suppressor locus in lung cancer, 3p21.3. This region of most frequent chromosomal deletion found at the earliest stage in lung cancer development houses 19 genes, many of which may act together as a 'tumor suppressor group', representing one of the most promising opportunities for development of new diagnostics/prognostics and therapeutics for lung cancer as well as for many other types of cancers. For the past decade, we have demonstrated tumor suppressor function of RBM5 in vitro and in vivo involving cell cycle arrest and apoptosis, as well as loss of RBM5 mRNA and protein expression in primary lung tumors. Here we report our latest data suggesting that RBM5 may regulate inhibition of metastasis in lung cancer. We performed cDNA microarray to identify global gene expression changes caused by RBM5 gene knockdown. In order to identify "consensus" pathways consistently deregulated by RBM5 loss irrespective of genetic background, the experiments were repeated in three different lung cancer cell lines of varying RBM5 expression levels, a normal lung epithelial cell line, and a normal breast epithelial cell line. Both Gene Set Enrichment Analysis (GSEA) and individual gene analysis identified consistent, statistically significant gene expression changes common to all five cell pairs examined. Genes involved in the functions of cell adhesion, migration and motility, known to be important in the metastatic process, were upregulated with RBM5-knockdown. These genes include Rac1, ß-catenin, collagen, laminin and the overall gene set of the gene ontology group "proteinaceous extracellular matrix". Among these, we have focused on Rac1 and ß-catenin which play essential roles in cell movement downstream of Wnt signaling. We have confirmed increased protein expression of ß-catenin and increased protein activation of Rac1 with RBM5-knockdown. In addition, we found that RBM5 protein expression loss in primary lung tumors is correlated with increased lymph node metastasis in a small number of lung cancer patients. These data are corroborated by an independent report showing RBM5 as part of a 17-gene signature of metastasis in primary solid tumors. Taken together, the accumulated evidence suggests that RBM5 expression loss may increase the metastatic potential of tumors. Further study is warranted to evaluate the potential clinical utility of RBM5 in lung cancer diagnostics, prognostics and therapeutics.