Abstract B22: The epigenetic regulation of X-linked FOXP3 function in breast cancer

Background: The X-linked FOXP3 is well-recognized as a nuclear transcription factor expressed by regulatory T cells, but FOXP3 is also the first X-linked tumor suppressor identified in breast epithelial cells. Indeed, aging mice with a Scurfy heterozygous mutation of Foxp3 have a high rate of spontaneous breast cancers. The significance of FOXP3 as a tumor suppressor in humans is supported by the prevalence of FOXP3 somatic mutations and gene deletions. However, these genetic alterations were identified in the minority of breast cancer samples, but most breast cancer samples detected the lack of nuclear FOXP3, suggesting a potential epigenetic mechanism of FOXP3 inactivation in breast cancer cells. In this study, we have investigated the potential mechanism of DNA methylation regulating X-linked FOXP3 inactivation and tested the reactivation of X-linked FOXP3 in breast cancer cells. Methods and Material: The DNA methylation status and miRNA expression of FOXP3 were determined by using pyrosequencing with quantitative PCR in both normal breast epithelial cells and breast cancer cells. Anisomycin was used to induce the DNA demethylation and transcription of FOXP3 in breast cancer cells. Results: First, promoter hypermethylation at the FOXP3 5′CpG Island was identified in breast cancer cells. A small CpG motif in intron 1 of FOXP3 had been reported as a T regulatory cell-specific demethylated region (TSDR) involved in regulating FOXP3 expression in T cells, but a prominent 5′CpG island was also identified in the promoter region of FOXP3. Our data revealed that both the CpG Island and TSDR are approximately 50% methylated in primary breast epithelial cells. Surprisingly, the CpG Island was almost 100% methylated in MDA-MB453 cells, which do not express FOXP3; 60% to 90% methylated in MCF7 and MDA-MB231 cells, which express low levels FOXP3; and approximately 50% methylated in MCF10A breast epithelial cells, which express normal levels of FOXP3. However, there was no difference in TSDR methylation (approximately 50%) between benign and malignant breast epithelial cells. In contrast, both the CpG Island and TSDR were nearly 100% methylated in male Jurkat T cells. Thus, epigenetic inactivation of FOXP3 in breast cancer cells differs from the epigenetic inactivation in T cells, and the 5′CpG Island appears to be a critical methylation site in breast cancer cells. Second, DNA demethylation in the FOXP3 CpG Island was regulated by anisomycin in breast cancer cells. Our data showed that anisomycin can reduce the DNA methylation in the FOXP3 CpG Island but not TSDR, and induce the transcription of FOXP3 in breast cancer cells, but not in male Jurkat T cells. Interestingly, anisomycin-induced expression of FOXP3 was dramatically raised after Xist silencing, which appears to enhance anisomycin-induced expression of FOXP3 in breast cancer cells. Thus, the combination of Xist siRNA and anisomycin is likely to be a more effective approach for reactivation of FOXP3 in breast cancer cells, but additional studies are needed to explore the molecular mechanisms underlying this reactivation. Conclusion: There is an epigenetic mechanism during FOXP3 inactivation in breast cancer. Importantly, tissue-specific FOXP3 reactivation can be epigenetically regulated in breast cancer cells, which may provide a new approach for designing effective targeted therapies for breast cancer patients with FOXP3 defects. Note: This abstract was not presented at the conference. Citation Format: Silin Li, Runhua Liu, Lizhong Wang. The epigenetic regulation of X-linked FOXP3 function in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B22.