Introduction: Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Tamoxifen resistance may be the result of acquisition of epigenetic regulation within breast cancer cells, such as DNA methylation, resulting in changed mRNA expression of genes pivotal for estrogen-dependent growth. Alternatively, tamoxifen resistance may be due to selection of pre-existing resistant cells, or a combination of the two mechanisms.
Methods: To evaluate the contribution of these possible tamoxifen resistance mechanisms, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massive parallel sequencing to analyze a well-established tamoxifen-resistant cell line model (TAM(R)), consisting of 4 resistant and one parental cell line. Another tamoxifen-resistant cell line model system (LCC1/LCC2) was used to validate the DNA methylation and gene expression results.
Results: Significant differences were observed in global gene expression and DNA methylation profiles between the parental tamoxifen-sensitive cell line and the 4 tamoxifen-resistant TAM(R) sublines. The 4 TAM(R) cell lines exhibited higher methylation levels as well as an inverse relationship between gene expression and DNA methylation in the promoter regions. A panel of genes, including NRIP1, HECA and FIS1, exhibited lower gene expression in resistant vs. parental cells and concurrent increased promoter CGI methylation in resistant vs. parental cell lines. A major part of the methylation, gene expression, and pathway alterations observed in the TAM(R) model were also present in the LCC1/LCC2 cell line model. More importantly, high expression of SOX2 and alterations of other SOX and E2F gene family members, as well as RB-related pocket protein genes in TAMR highlighted stem cell-associated pathways as being central in the resistant cells and imply that cancer-initiating cells/cancer stem-like cells may be involved in tamoxifen resistance in this model.
Conclusion: Our data highlight the likelihood that resistant cells emerge from cancer-initiating cells/cancer stem-like cells and imply that these cells may gain further advantage in growth via epigenetic mechanisms. Illuminating the expression and DNA methylation features of putative cancer-initiating cells/cancer stem cells may suggest novel strategies to overcome tamoxifen resistance.