Abstract
IntroductionDevelopment 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.MethodsTo 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 (TAMR), 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.ResultsSignificant differences were observed in global gene expression and DNA methylation profiles between the parental tamoxifen-sensitive cell line and the 4 tamoxifen-resistant TAMR sublines. The 4 TAMR 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 TAMR 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.ConclusionOur 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.
Highlights
Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer
Principal component analysis and unsupervised cluster analysis Principle component analysis of the modified DNA methylation-specific digital karyotyping (MMSDK) data, which depicts all variables without any a priori classification and data filtering in the three-dimensional space, showed that MCF-7/S0.5 separated from the four tamoxifen-resistant cell line model (TAMR) cell lines, indicating overall differences in global DNA methylation profiles between parental and resistant cell lines (Figure 2A)
MCF-7/S0.5 separated from the four TAMR cell lines for both MMSDK (Figure 2C) and digital gene expression (DGE) (Figure 2D)
Summary
Development of resistance to tamoxifen is an important clinical issue in the treatment of breast cancer. Around 80% of breast cancer patients present with primary breast tumors that are estrogen receptor (ER) alpha-positive, suggesting that the tumor is dependent on estrogen for growth [1,2]. Most of these patients are offered endocrine therapy, which currently consists of the anti-estrogen tamoxifen or aromatase inhibitors. These drugs can be used successfully both in the adjuvant and advanced disease settings. Disease progression eventually occurs in most patients receiving tamoxifen treatment
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