Recently, there has been a great interest in elucidating the role of epigenetic gene regulation and its potential impact in tumor progression. Epigenetic regulation can dictate gene transcription globally through chromatin remodeling, or locally by DNA methylation. Chromatin remodeling is achieved by the actions of histone deacetylases that promote chromatin condensation into facultative heterochromatin and histone acetylases that facilitate chromatin relaxation and the formation of euchromatin that can undergo active gene transcription.
22 Upregulation of de novo DNA methyltransferases suppresses gene transcription by methylating cytosine-rich regions called CpG islands located within the gene promoter regions.
22,25,26 Together, these two epigenetic mechanisms have been recognized as important pathways in tumor development and metastasis. We recently reported that immunized mice against P815 murine mastocytoma cells could reject a subcutaneous tumor challenge of wild-type P815 tumor cells, but were unable to reject P815 tumor cells that were isolated from the anterior chamber of the eye.
27 Treatment of the eye-derived P815 cells with 5-Aza reversed the escape phenotype and the treated cells were rejected in P815 immunized mice. We determined that factors in the aqueous humor, primarily TGFβ, upregulated expression of de novo methyltransferases and histone deacetylases in tumor cells that were injected into the anterior chamber (Chen PW, unpublished data, 2011). Cells that were injected into the eye experienced increased global DNA methylation and histone deacetylation. Interestingly, studies by Mori et al. demonstrated that treating malignant cutaneous melanoma cells with either the demethylating agent 5-Aza or TSA upregulated expression of CXCR4 and enhanced cell migration.
20 In this study, we demonstrate that CXCR4
+ LS174T colon cancer cells downregulate CXCR4 gene transcription and receptor expression when these cells are introduced into the eye. Expression of CXCR4 by CXCR4
+ LS174T cells injected subcutaneously into the skin, the cecal wall, or intrasplenically to induce liver metastasis remained unchanged. Treatment of the LS174T-AC tumor cells with demethylating agent 5-Aza restored expression of CXCR4 by the eye-derived tumor cells but did not affect CXCR4 expression in the LS174T-SC, -CEC, or -LM tumor cells. Our results suggest that suppression of CXCR4 expression is epigenetically regulated by methylation of the CXCR4 gene. Studies by Bogani et al. demonstrated that suppression of CXCR4 expression by CD34
+ cells from patients with primary myelofibrosis was due to hypermethylation of a CpG island in the CXCR4 gene promoter, and treatment of the CD34
+ cells with 5-Aza restored expression of CXCR4 and showed improved migration in response to CXCL12.
28 Our findings are in congruence with these data since we demonstrate an increase in DNA methyltransferases in the LS174T-AC tumor cells, which is absent in the wild-type and also the LS174T cell lines derived from the different organs. Moreover, we determined that the CXCR4 promoter region of LS174T-SC and LS174T-LM showed both unmethylated and methylated CpG motifs in a pattern similar to the wild-type LS174T cells, whereas, LS174T-AC cells demonstrated almost complete methylation within a CpG island within the CXCR4 promoter. A possible reason why the parent LS174T cells and the LS174T cells derived from the skin and liver expressed both unmethylated and methylated sites in the CXCR4 promoter could be due to heterogeneity of the cells within the tumor. Since the skin and liver cannot induce epigenetic changes through methylation, we observed both unmethylated and methylated CXCR4 in these cell lines. By contrast, eye-derived LS174T cells undergo methylation within the ocular environment, which results in methylation of unmethylated CpG regions in CXCR4 promoter. It is interesting to note that we also examined other CpG islands within the CXCR4 promoter by bisulfite conversion and PCR, and we did not observe differential expression of unmethylated and methylated CXCR4 PCR products (data not shown), suggesting that methylation of one CXCR4 CpG island is sufficient to downregulate expression of this gene in anterior chamber–derived LS174T cells. Together, these results indicate that factors in the ocular environment induce gene-specific methylation that is sufficient for the epigenetic downregulation of CXCR4 expression. The initial characterization of the mechanisms responsible for epigenetic downregulation are discussed below and also merit future investigation.