Abstract P4-06-10: Epigenetic silencing of glutamine synthetase (Glul) defines glutamine depletion therapy
| Autor: | V. Haley, Peter Schmid, Karen O’Leary, Mark R. Lackner, F. Cavicchioli, Alistair Thompson, A Shia, Tim Crook, Nigro C Lo |
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| Rok vydání: | 2012 |
| Předmět: | |
| Zdroj: | Cancer Research. 72:P4-06 |
| ISSN: | 1538-7445 0008-5472 |
| DOI: | 10.1158/0008-5472.sabcs12-p4-06-10 |
| Popis: | Background: Methylation-dependent transcriptional silencing of genes involved in amino acid synthesis can provide potential targets for novel synthetic lethality strategies. Glutamine synthetase (Glul) is the key enzyme in the biosynthesis of glutamine. We identified Glul as a novel gene subject to methylation-dependent transcriptional silencing in breast cancer cell lines using a combined functional screen with methylation reversal assays and methylation arrays. Methods: Methylation reversal assays were performed using 5-aza-2-deoxycytidine and/or trichostatin treatment coupled with whole genome mRNA microarrays (Illumina HT-12 v4 Expression BeadChip Kit). Expression of Glul with and without pharmacological methylation reversal with azacytidine and/or trichostatin was validated using qRT-PCR and Western Blot. Methylation of Glul was analysed using methylation microarrays (Illumina 450K Methylation BeadChip), bisulphite sequencing and pyrosequencing. Sensitivity to glutamine deprivation was assessed using an MTT assay after culturing cells in media with various glutamine concentrations or in complete absence of glutamine. We used a panel of 55 breast cancer cell lines and formalin-fixed paraffin-embedded tissue from a series of 116 stage I-III primary breast cancers with linked mature clinical outcome data that were randomly selected from the Cuneo Tissue Bank. Tissue samples were subject to histopathological review to ensure adequate representation of cancer cells. Results: Dense methylation of the CpG-island of Glul was detected in 45% of cell lines across all subtypes. Methylation of the CpG island was linked with absent or down-regulated expression of Glul in some but not all cell lines, and Glul expression could be reactivated by azacytidine and trichostatin in these cell lines. Methylation of shore areas was detected in several cell lines but was not associated with transcriptional silencing. Cells with methylation-dependent low or absent Glul expression were highly sensitive to glutamine deprivation, whereas cell lines without Glul methylation were rescued by compensatory up-regulation of Glul. Using pyrosequencing, dense methylation of the CpG island of Glul was found in 32.8% of patients, with an additional 17.2% of patients showing partial methylation. No significant association with a specific breast cancer subtype or outcome was found. Conclusions: This is the first report of methylation-dependent transcriptional silencing of Glul expression in cancer. Our data demonstrate that a significant proportion of primary breast cancers show methylation of Glul and suggest that glutamine deprivation could be a novel synthetic lethality strategy for these cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-10. |
| Databáze: | OpenAIRE |
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