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ASC/TMS1, a caspase-1 activating adaptor, is downregulated by aberrant methylation in human melanoma
Xin Guan1, Junji Sagara, Taro Yokoyama
1Department of Molecular Oncology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan.
Abstract:
ASC/TMS1 is an adaptor protein activating caspase-1 that stimulates processing of proIL-1beta and proIL-18. ASC was reported to be aberrantly methylated and silenced in human breast cancers. In our present study, ASC expression was examined in 12 melanoma cell lines by Western blot analysis and in 18 benign melanocytic nevi and 32 melanoma tissues by immunohistochemical staining. ASC expression was absent or reduced in 7 of 12 (58.3%) cell lines and in 20 of 32 (62.5%) melanoma tissues, whereas all 18 benign melanocytic nevi showed intensive ASC expression. To investigate whether ASC silencing in melanoma is involved in aberrant methylation, methylation specific PCR was carried out. Five of ten (50%) melanoma tissues exhibited methylation in CpG island of ASC companied with reduced ASC expression. Six of twelve (50%) melanoma cell lines showed aberrant methylation in the ASC gene, and 4 of the 6 (66.7%) methylation positive cell lines exhibited reduced ASC expression. We characterized methylation patterns in melanoma cell lines by using bisulfite genomic sequencing, and found that the degree of aberrant methylation correlated with the level of reductive ASC expression. Treatment with demethylating agent 5-aza-2'-deoxycytidine resulted in both demethylation of the ASC gene and the upregulation of ASC expression in the methylation positive melanoma cell lines. Our study shows that ASC is downregulated in melanoma, and that its suppression is partially mediated by aberrant methylation.
Insights
Apoptosis-associated speck-like protein containing a CARD (ASC) is downregulated in melanoma. Aberrant methylation of the ASC gene contributes to its silencing, suggesting a potential therapeutic target for melanoma treatment.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- ASC/TMS1 is an adaptor protein crucial for caspase-1 activation, involved in processing pro-inflammatory cytokines IL-1beta and IL-18.
- Aberrant methylation and silencing of ASC have been previously observed in human breast cancers.
Purpose of the Study:
- To investigate the expression levels of ASC in melanoma cell lines and tissues.
- To determine the role of aberrant methylation in the silencing of ASC in melanoma.
- To explore the potential of demethylating agents in restoring ASC expression in melanoma.
Main Methods:
- Western blot analysis and immunohistochemical staining were used to assess ASC expression in melanoma cell lines, benign nevi, and melanoma tissues.
- Methylation-specific PCR and bisulfite genomic sequencing were employed to analyze ASC gene methylation patterns.
- Treatment with the demethylating agent 5-aza-2'-deoxycytidine was performed to evaluate its effect on ASC methylation and expression.
Main Results:
- ASC expression was found to be absent or reduced in a significant proportion of melanoma cell lines (58.3%) and tissues (62.5%), while consistently high in benign nevi.
- Aberrant methylation of the ASC gene was detected in 50% of melanoma tissues and 50% of melanoma cell lines, correlating with reduced ASC expression.
- Demethylation treatment with 5-aza-2'-deoxycytidine successfully restored ASC gene demethylation and upregulated ASC expression in methylation-positive melanoma cell lines.
Conclusions:
- ASC is frequently downregulated in melanoma.
- Aberrant methylation of the ASC gene is a significant mechanism contributing to its suppression in melanoma.
- Restoring ASC expression through demethylation presents a potential therapeutic strategy for melanoma.