Abstract LB-128: Reversible epigenetic regulation of X-linked FOXP3 function in breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: FOXP3, the nuclear transcription factor expressed by regulatory T cells, is recently identified by our and other groups as an X-linked tumor suppressor gene in Breast Cancer. The lack of nuclear FOXP3 was detected in the most breast cancer samples. However, FOXP3 genetic alterations were only identified in the minority of breast cancer samples, suggesting a potential epigenetic mechanism of FOXP3 inactivation in breast cancer cells. Methods and Material: The DNA methylation status and mRNA 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, 5’CpG Island in FOXP3 promoter was found to be differently methylated in primary breast epithelial cells and cancer cells.A small CpG motif in intron 1 of FOXP3 has been reported as a T regulatory cell-specific demethylated region (TSDR) involved in regulating FOXP3 expression in T cells. Our data revealed that both the CpG Island and TSDR of FOXP3 are approximately 50% methylated in primary breast epithelial cells. Surprisingly, the CpG Island was almost 100% methylated in MDA-MB453 cells with no expression of FOXP3, 60% to 90% methylated in MCF7 and MDA-MB231 cells with low expression FOXP3, and approximately 50% methylated in MCF10A breast epithelial cells with normal expression 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. Citation Format: Jin Lu, Lizhong Wang. Reversible epigenetic regulation of X-linked FOXP3 function in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-128. doi:10.1158/1538-7445.AM2014-LB-128