Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.     

Use of NotI microarrays in analysis of epigenetic and structural changes in epithelial tumor genomes by the example of human chromosome 3

A new comparative genome hybridization technology using NotI microarrays is described (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-enriched probes from tumor and normal genomic DNA with radically new NotI microarrays. A total of 181 NotI-binding loci of human chromosome 3 were assayed in 200 human malignant tissue samples from various organs: kidney, lung, breast, ovary, cervix, and prostate. The most significant portion (above 30%) of aberrations (deletions and methylation) were detected in NotI sites located in the MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, and ZIC4 genes. This indicates that they may be associated with cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results confirm that the proposed method can contribute to cancer genomics.     

Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and transcriptional level

In this work for the first time copy number and expression changes of the tumor suppressor gene RBSP3 (3p21.3) were investigated. The study was performed on HPV-positive squamous cervical carcinoma (SCC) using real-time PCR. Deletions were detected in 42% of cases (19 of 45 studied biopsies). Frequency of deletions was significantly higher in SCC samples with metastases (64%) than in tumors without metastases (32%, P < 0.05). In a few cases amplification of RBSP3 was also found. Altogether copy number changes of RBSP3 were detected in 51% of cases (23 of 45). Expression of RBSP3 was decreased in 64% of SCC samples (21 of 33). Again decreased expression of RBSP3 was detected significantly more frequently (83%) in tumors with metastases compared with SCC without metastases (52%, P < 0.05). In several cases however increased expression was observed. Altogether changes in expression of RBSP3 were detected in 79% (26 of 33) of SCC biopsies. Comparison of copy number and expression changes showed that in 23% of SCC cases decreased expression of RBSP3 was detected in samples with deletions and in 36% cases such decrease was not associated with copy number changes. Rarely more complicated SCC cases were found. For example in some tumors increased expression of RBSP3 was detected in samples with deletions or without changes in copy number. Results of the study suggested that RBSP3 is involved in the progression of SCC and complex mechanisms for inactivation of RBSP3. We also hypothesize that these data indicate that RBSP3 in addition to dephosphorylation of pRb has other functions important for malignant transformation because pRb is almost absent in HPV-positive SCC.     

Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial–mesenchymal transition

Background

DNA methylation is associated with aberrant gene expression in cancer, and has been shown to correlate with therapeutic response and disease prognosis in some types of cancer. We sought to investigate the biological significance of DNA methylation in lung cancer.

Results

We integrated the gene expression profiles and data of gene promoter methylation for a large panel of non-small cell lung cancer cell lines, and identified 578 candidate genes with expression levels that were inversely correlated to the degree of DNA methylation. We found these candidate genes to be differentially methylated in normal lung tissue versus non-small cell lung cancer tumors, and segregated by histologic and tumor subtypes. We used gene set enrichment analysis of the genes ranked by the degree of correlation between gene expression and DNA methylation to identify gene sets involved in cellular migration and metastasis. Our unsupervised hierarchical clustering of the candidate genes segregated cell lines according to the epithelial-to-mesenchymal transition phenotype. Genes related to the epithelial-to-mesenchymal transition, such as AXL, ESRP1, HoxB4, and SPINT1/2, were among the nearly 20% of the candidate genes that were differentially methylated between epithelial and mesenchymal cells. Greater numbers of genes were methylated in the mesenchymal cells and their expressions were upregulated by 5-azacytidine treatment. Methylation of the candidate genes was associated with erlotinib resistance in wild-type EGFR cell lines. The expression profiles of the candidate genes were associated with 8-week disease control in patients with wild-type EGFR who had unresectable non-small cell lung cancer treated with erlotinib, but not in patients treated with sorafenib.

Conclusions

Our results demonstrate that the underlying biology of genes regulated by DNA methylation may have predictive value in lung cancer that can be exploited therapeutically.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1079) contains supplementary material, which is available to authorized users.     

Epigenetic Modulations Induction Using DSCR1 Ectopic Expression in Breast Cancer Cells

Today, prognosis, diagnosis and treatment of cancers are progressing with non-invasive methods, including investigation and modification of the DNA methylation profile in cancer cells. One of the effective factors in regulating gene expression in mammals is DNA methylation. Methylation alterations of genes by external factors can change the expression of genes and inhibit the cancer. In the present study, we investigated the effect of Down syndrome critical region 1 gene (DSCR1) ectopic expression on the methylation status of the BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines. The effect of DSCR1 ectopic expression on cell viability in MCF7, MDA-MB-468, MDA-MB-231 and MCF10A cell lines was evaluated using MTT assay after the cells treated by lentivirus vectors harboring DSCR1 for 72 hours. Methylation status of BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines was assessed by Restriction Enzyme PCR (REP) method. Also, methylation changes of these genes in breast cancer cell lines after treatment by lentivirus vectors harboring DSCR1 for 7 days were analyzed by REP method. To confirm the effect of DSCR1 on methylation of genes, Real-time PCR was performed. The MTT assay results indicated that DSCR1 ectopic expression reduced cell viability in all three human breast cancer cell lines. Our results showed that DSCR1 ectopic expression after 6 days reversed the hypomethylation status of the BCL-XL, ITGA6, TCF3, VIM and CXCR4 genes and hypermethylation of RASSF1A and DOK7 genes. The expression levels of BCL-XL, ITGA6, TCF3, VIM and CXCR4 mRNA significantly reduced (P<0.05) and the expression levels of RASSF1A and DOK7 mRNA significantly increased (P<0.05). Our findings reveal for the first time the impact of DSCR1 ectopic expression on the methylation status of breast cancer cells and identify a novel agent for epigenetic therapy.     

High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

Background

Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas.

Methodology/Principal Findings

We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1–2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3–5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines.

Conclusions/Significance

This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread.     

Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.     

RASSF2 methylation is a strong prognostic marker in younger age patients with Ewing sarcoma

Ras-association domain family of genes consist of 10 members (RASSF1-RASSF10), all containing a Ras-association (RA) domain in either the C- or the N-terminus. Several members of this gene family are frequently methylated in common sporadic cancers; however, the role of the RASSF gene family in rare types of cancers, such as bone cancer, has remained largely uninvestigated. In this report, we investigated the methylation status of RASSF1A and RASSF2 in Ewing sarcoma (ES). Quantitative real-time methylation analysis (MethyLight) demonstrated that both genes were frequently methylated in Ewing sarcoma tumors (52.5% and 42.5%, respectively) as well as in ES cell lines and gene expression was upregulated in methylated cell lines after treatment with 5-aza-2′-deoxcytidine. Overexpression of either RASSF1A or RASSF2 reduced colony formation ability of ES cells. RASSF2 methylation correlated with poor overall survival (p = 0.028) and this association was more pronounced in patients under the age of 18 y (p = 0.002). These results suggest that both RASSF1A and RASSF2 are novel epigenetically inactivated tumor suppressor genes in Ewing sarcoma and RASSF2 methylation may have prognostic implications for ES patients.     

Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis

Aberrant DNA methylation patterns have been reported in inflamed tissues and may play a role in disease. We studied DNA methylation and gene expression profiles of purified intestinal epithelial cells from ulcerative colitis patients, comparing inflamed and non-inflamed areas of the colon. We identified 577 differentially methylated sites (false discovery rate <0.2) mapping to 210 genes. From gene expression data from the same epithelial cells, we identified 62 differentially expressed genes with increased expression in the presence of inflammation at prostate cancer susceptibility genes PRAC1 and PRAC2. Four genes showed inverse correlation between methylation and gene expression; ROR1, GXYLT2, FOXA2, and, notably, RARB, a gene previously identified as a tumor suppressor in colorectal adenocarcinoma as well as breast, lung and prostate cancer. We highlight targeted and specific patterns of DNA methylation and gene expression in epithelial cells from inflamed colon, while challenging the importance of epithelial cells in the pathogenesis of chronic inflammation.     

DNA Methylation Profiling for the Diagnosis and Prognosis of Patients with Nontuberculous Mycobacterium Lung Disease

The incidence of nontuberculous Mycobacterium (NTM) lung disease is rapidly increasing; however, its diagnosis and prognosis remain unclear while selecting patients who will respond to appropriate treatment. Differences in DNA methylation patterns between NTM patients with good or poor prognosis could provide important therapeutic targets. We used the Illumina MethylationEPIC (850k) DNA methylation microarray to determine the pattern between differentially methylated regions (DMRs) in NTM patients with good or poor prognosis (n = 4/group). Moreover, we merged and compared 20 healthy controls from previous Illumina Methylation450k DNA methylation microarray data. We selected and visualized the DMRs in the form of heatmaps, and enriched terms associated with these DMRs were identified by functional annotation with the “pathfinder” package. In total, 461 and 293 DMRs (|Log2 fold change| > 0.1 and P < 0.03) were more methylated in patients with four poor and four good prognoses, respectively. Furthermore, 337 and 771 DMRs (|Log2 fold change| > 0.08 and P < 0.001) were more methylated in eight NTM patients and 20 healthy controls, respectively. TGFBr1 was significantly less methylated, whereas HLA-DR1 and HLA-DR5 were more methylated in patients with poor prognosis (compared to those with good prognosis). LRP5, E2F1, and ADCY3 were the top three less-methylated genes in NTM patients (compared with the controls). The mTOR and Wnt signaling pathway-related genes were less methylated in patients with NTM. Collectively, genes related to Th1- cell differentiation, such as TGFBr1 and HLA-DR, may be used as biomarkers for predicting the treatment response in patients with NTM lung disease.     

Differential expression of an ensemble of the key genes involved in cell-cycle regulation in lung cancer

Targeted cancer therapy directed at individual targets is often accompanied by the rapid development of drug resistance. The development of a new generation of antitumor drugs involves the search for many targets simultaneously to block or, conversely, restore their activity. In this regard, simultaneous analysis of gene expression in a complex network of interactions, primarily cell cycle control elements, is relevant for the search of specific molecular markers for the differential diagnosis of adenocarcinoma (ADC) and squamous cell lung cancer (SCC), as well as new targets for therapy. In this paper we performed an extended quantitative analysis of the expression of two suppressor genes, CTDSPL and its target RB1, as well as 84 genes of the main participants of the p16^INK4A-Cdk/cyclin D1-Rb and p53/p21^Waf1 signaling pathways in the histological types of non-small-cell lung cancer (NSCLC), i.e., ADC and SCC, using the special panel of the Human Cell Cycle Regulation Panel. The expression profile of some genes shows the specificity to the histological type of NSCLC and the presence of metastases. The genes with a significantly increased expression that affect the activity of Rb (cyclins, cyclin-dependent kinases, their activators, inhibitors, etc.) can serve as potential targets for combined therapy of both ADC and SCC.     

Tumor Suppressor Function of the SEMA3B Gene in Human Lung and Renal Cancers

The SEMA3B gene is located in the 3p21.3 LUCA region, which is frequently affected in different types of cancer. The objective of our study was to expand our knowledge of the SEMA3B gene as a tumor suppressor and the mechanisms of its inactivation. In this study, several experimental approaches were used: tumor growth analyses and apoptosis assays in vitro and in SCID mice, expression and methylation assays and other. With the use of the small cell lung cancer cell line U2020 we confirmed the function of SEMA3B as a tumor suppressor, and showed that the suppression can be realized through the induction of apoptosis and, possibly, associated with the inhibition of angiogenesis. In addition, for the first time, high methylation frequencies have been observed in both intronic (32-39%) and promoter (44-52%) CpG-islands in 38 non-small cell lung carcinomas, including 16 squamous cell carcinomas (SCC) and 22 adenocarcinomas (ADC), and in 83 clear cell renal cell carcinomas (ccRCC). Correlations between the methylation frequencies of the promoter and the intronic CpG-islands of SEMA3B with tumor stage and grade have been revealed for SCC, ADC and ccRCC. The association between the decrease of the SEMA3B mRNA level and hypermethylation of the promoter and the intronic CpG-islands has been estimated in renal primary tumors (P < 0.01). Using qPCR, we observed on the average 10- and 14-fold decrease of the SEMA3B mRNA level in SCC and ADC, respectively, and a 4-fold decrease in ccRCC. The frequency of this effect was high in both lung (92-95%) and renal (84%) tumor samples. Moreover, we showed a clear difference (P < 0.05) of the SEMA3B relative mRNA levels in ADC with and without lymph node metastases. We conclude that aberrant expression and methylation of SEMA3B could be suggested as markers of lung and renal cancer progression.