Search

Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. Results To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. Conclusions miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents, and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. To investigate if the miR-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. An upstream CpG island of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, cell xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2’-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma.

Gap junctions are specialized plasma membrane domains consisting of channels formed by members of the connexin protein family. Gap junctional intercellular communication is often lost in cancers due to aberrant localization or downregulation of connexins, and connexins are therefore suggested to act as tumor suppressor genes in various tissues. The aim of this study was to investigate the expression pattern and DNA promoter methylation status of connexins in colorectal cancer. Expression of six (GJA1, GJA9, GJB1, GJB2, GJC1 and GJD3) connexin genes was detected in normal colonic tissue samples. GJC1 expression was reduced in colorectal carcinomas compared to normal tissue samples. All analyzed connexins were hypermethylated in colon cancer cell lines, although at various frequencies. GJA4, GJB6 and GJD2 were hypermethylated in 60% (29/48), 25% (12/48) and 96% (46/48) of primary colorectal carcinomas, respectively. However, the methylation status was not associated with gene expression. GJC1 has two alternative promoters, which were methylated in 42% (32/76) and 38% (25/65) of colorectal tumors, and in none of the normal mucosa samples. Expression of GJC1 was significantly lower in methylated compared with unmethylated samples (p < 0.01) and was restored in cell lines treated with the demethylating drug 5-aza-2'deoxycytidine. Taken together, DNA hypermethylation of the promoter region of GJC1, encoding connexin45, is an important mechanism in silencing gene expression in colorectal cancer.

Cell lines are invaluable biomedical research tools, and recent literature has emphasized the importance of genotype authentication and characterization. In the present study, 24 out of 27 cell line identities were confirmed by short tandem repeat profiling. The molecular phenotypes of the 24 colon cancer cell lines were examined, and microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) were determined, using the Bethesda panel mononucleotide repeat loci and two epimarker panels, respectively. Furthermore, the BRAF, KRAS and PIK3CA oncogenes were analyzed for mutations in known hotspots, while the entire coding sequences of the PTEN and TP53 tumor suppressors were investigated. Nine cell lines showed MSI. Thirteen and nine cell lines were found to be CIMP positive, using the Issa panel and the Weisenberger et al. panel, respectively. The latter was found to be superior for CIMP classification of colon cancer cell lines. Seventeen cell lines harbored disrupting TP53 mutations. Altogether, 20/24 cell lines had the mitogen-activated protein kinase pathway activating mutually exclusive KRAS or BRAF mutations. PIK3CA and PTEN mutations leading to hyperactivation of the phosphoinositide 3-kinase/AKT pathway were observed in 13/24 cell lines. Interestingly, in four cell lines there were no mutations in neither BRAF, KRAS, PIK3CA nor in PTEN. In conclusion, this study presents molecular features of a large number of colon cancer cell lines to aid the selection of suitable in vitro models for descriptive and functional research.

OBJECTIVES: We recently identified a six-gene methylation-based biomarker panel suitable for early detection of colorectal cancer (CRC). In this study, we compared the performance of this novel epi-panel with that of previously identified DNA methylation markers in the same clinical tissue sample sets. METHODS: Quantitative methylation-specific PCR was used to analyze the promoter region of SEPT9 and VIM in a total of 485 tissue samples, divided into test and validation sets. ITGA4, NTRK2, OSMR, and TUBG2 were also included in the analyses. Receiver operating characteristic (ROC) curves were used to compare the performances of the individual biomarkers with that of the novel epi-panel. RESULTS: SEPT9 and VIM were methylated in 82 and 67% of CRCs (n=169) and in 88 and 54% of the adenomas (n=104). Only 3% of the normal mucosa samples (n=107) were methylated for these genes, confirming that the methylation was highly cancer-specific. Areas under the ROC curve (AUC), distinguishing CRCs from normal mucosa, were 0.94 for SEPT9 and 0.81 for VIM. AUC values for separating adenomas from normal mucosa samples were 0.96 and 0.81 for the same genes. In comparison, the novel epi-panel achieved an AUC of 0.98 (CRC) and 0.97 (adenomas). ITGA4, OSMR, NTRK2, and TUBG2 were methylated in 90, 78, 7, and 1% of the CRCs, and in 76, 77, 3, and 0% of the adenomas. Between 0 and 2% of the normal mucosa samples were methylated for the same genes. ITGA4 and OSMR achieved an AUC of 0.96 and 0.92 (CRC vs. normal mucosa), and 0.93 and 0.92 (adenomas vs. normal mucosa). CONCLUSIONS: We have confirmed the high performance of some of the previously identified DNA methylation markers. Furthermore, we showed that a recently reported epi-panel performed better than the individual DNA methylation biomarkers when analyzed in the same tissue samples. This observation was also true for VIM and SEPT9, which are included in commercially available noninvasive tests for CRC. These results further underscore the value of combining a manageable number of individual markers into a panel, which in addition to having a higher sensitivity and specificity might provide a more profound robustness to a noninvasive test compared with single markers.

We read with great interest the recent article of Moinova and colleagues reporting that aberrant methylation of exon-1 of the vimentin ( VIM ) gene is a highly common epigenetic alteration in neoplasia of the upper (esophagus and gastric) and the lower (colorectal) gastrointestinal tract ([1][1]).

Introduction Colorectal cancer is a common disease with a world-wide annual incidence of 1.2 million. The mortality rate is high, especially for patients diagnosed with an advanced tumor stage. We recently identified DNA methylation based biomarkers for early detection of colorectal cancer (and unpublished). In the beginning of 2012, these markers were licensed to the British biotech company Oxford Gene Technology for development into a non-invasive test suitable for colorectal cancer screening (http://www.ogt.co.uk/news_events/news/659_ogt_signs_licensing_deal_for_colorectal_ cancer_biomarkers). Two tests based on cancer specific aberrant DNA methylation are currently commercially available, including analyses of VIM and SEPT9 in stool and blood samples, respectively. The aim of the present study was to compare the performance of the in house biomarker panel with previously published epigenetic biomarkers using a large series of tissue samples. Methods Quantitative methylation-specific polymerase chain reaction was used to analyze the promoter methylation status of ITGA4, NTRK2, OSMR, TUBG2, SEPT9, and VIM in test and validation series comprising altogether 169 primary colorectal cancers, 105 matching normal mucosa samples, 104 adenomas, and 107 normal samples from colorectal cancer-free individuals. Receiver operating characteristic (ROC) curves were used to evaluate the overall performance of the biomarkers in tissue samples and the results were compared with the performance of the in house biomarker panel. Results Across the test and validation series, ITGA4, SEPT9, OSMR, VIM, NTRK2, and TUBG2 were methylated in 91%, 82%, 79%, 67%, 7%, and 1% of the colorectal carcinomas, and in 79%, 88%, 78%, 54%, 3%, and 0% of the adenomas. The promoter methylation was highly cancer-specific and only 1%, 3%, 2%, 3%, 0%, and 0% of the normal mucosa samples were methylated for the same genes. Resulting areas under the ROC curves for separating colorectal carcinomas from normal mucosa samples were 0.95, 0.93, 0.92, and 0.80 for ITGA4, SEPT9, OSMR, and VIM, respectively. In comparison, the in house biomarker panel achieved an area under the ROC curve of 0.97. Conclusion In a direct comparison performed by a single lab, using the same protocol and the same series of tissue samples, the performance of our recently identified in house biomarker panel was shown to be better than previously identified DNA methylation based biomarkers, including VIM and SEPT9. This underscores the value of combining a manageable number of individually promising biomarkers into a panel, which can achieve more robustness in addition to a high sensitivity and specificity. From this comparison we conclude that the in house biomarker panel is highly suitable for developing into a non-invasive test for early detection of colorectal cancer.

Epigenetic and genetic alterations of microRNAs (miRNAs) are frequently seen in cancer, and are responsible for the deregulation of differentiation and proliferation programs. The levels of transcription of miRNAs have been directly linked to epigenetic silencing, i.e. hypermethylation of CpG islands in tumors. Osteosarcoma is the most common primary malignant bone tumor and shows complex genomic aberrations. Using Illumina's Infinium technology we have profiled global promoter methylation of the EuroBoNeT osteosarcoma cell line panel (eurobonet.eu), in addition to a small panel of normal samples (osteoblast primary cultures and normal bone). A list of miRNAs that could be associated to a promoter CpG island was generated by identifying miRNAs that are encoded within transcripts of protein-coding host genes, or within 10 kb from a CpG island. Using an integrative approach, we have analyzed miRNA expression patterns together with DNA methylation data. Interestingly, hypermethylation of the upstream promoter seems to strongly suppress miRNA expression, and a high inverse correlation was observed between the degree of DNA methylation and expression of 17 miRNAs. The identified miRNAs were associated with hypermethylation in a high proportion of the osteosarcoma cell lines and hypomethylation in normal samples, suggesting that their transcription is regulated by promoter methylation, and that their silencing may play a role in osteosarcoma biology. To study this further, a subset of the cell lines was treated with a DNA demethylation agent. The relative expression levels of 8 miRNAs were found to increase upon treatment, as determined by quantitative RT-PCR. Thus, the transcription of these miRNAs seems to be regulated by epigenetic mechanisms. The methylation status of CpG islands associated with the most interesting miRNAs was then analyzed in more detail using methylation-specific PCR and direct bisulphite sequencing. We are currently expanding these investigations to a large tumor set where we will use quantitative methylation-specific PCR as a tool to determine the methylation status. Based on our findings, methylation of CpG islands seems to play a crucial role in controlling miRNA expression in osteosarcomas. This reveals new possibilities for activation of tumor-suppressing miRNAs by epigenetic treatment of osteosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 197. doi:1538-7445.AM2012-197