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Epigenetic activation of the prostaglandin receptor EP4 promotes resistance to endocrine therapy for breast cancer [RNA-seq]

ABSTRACT: Approximately 75% of breast cancers express estrogen receptor α (ERα) and depend on estrogen signals for continued growth. Aromatase inhibitors (AIs) prevent estrogen production and inhibit estrogen receptor signaling, resulting in decreased cancer recurrence and mortality. Advanced tumors treated with AIs almost always develop resistance to these drugs via the up-regulation of alternative growth signals. The mechanisms that drive this resistance - especially epigenetic events that alter gene expression - are however not well understood. Here we performed a genome-wide DNA methylation and expression analysis of cell line models to find epigenetically regulated genes involved in acquired aromatase inhibitor resistance. We discovered that prostaglandin E2 receptor 4 (PTGER4) is up-regulated after demethylation and promotes phosphorylation and activation of ERα. Knockdown and inhibitor studies demonstrate that PTGER4 promotes AI resistance via ligand independent activation of the ERα-cofactor CARM1. We believe that we have discovered a novel epigenetic mechanism for altering cell signaling and acquiring endocrine therapy resistance. Our findings indicate that PTGER4 is a potential drug target in AI resistant cancers. Additionally, the epigenetic component of PTGER4 regulation suggests that further study of PTGER4 may yield valuable insights into how DNA methylation-targeted diagnoses and/or treatments can improve AI resistant breast cancer treatment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE74941 | GEO | 2017/06/19

SECONDARY ACCESSION(S): PRJNA301976

REPOSITORIES: GEO

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Project description:Despite the success of the aromatase inhibitors (AIs) in treating estrogen receptor positive breast cancer, 15-20% of patients receiving adjuvant AIs will relapse within 5-10 years of treatment initiation. Long term estrogen deprivation (LTED) of breast cancer cells in culture has been successfully used to mimic AI-induced estrogen depletion to dissect mechanisms of AI resistance. However, we hypothesized that a subset of patients receiving AI therapy may maintain low circulating concentrations of estrogens that influence the development of endocrine resistance. We sought to expand established LTED models to account for these observations. MCF-7 cells were grown in medium with charcoal stripped serum supplemented with defined concentrations of E2 or the estrogenic androgen metabolite 3βAdiol. Cells were selected in the EC10 and EC90 of E2 or 3βAdiol, or estrogen deprived. Estrogen-independence was evaluated during selection by assessing cell growth in the absence or presence of E2 or 3βAdiol. Following >7 months of selection, estrogen-independence developed in estrogen-deprived cells and cells maintained in low concentrations of steroid hormone. Functional analyses demonstrated that estrogen-deprived and low-steroid selected cells developed estrogen-independence via unique mechanisms, independent and dependent of ERα, respectively. Estrogen-independent growth in low-steroid selected cells could be blocked by kinase inhibitors. However, these cells were completely resistant to kinase inhibition in the presence of low steroid hormone concentrations. These data offer a novel perspective on the development of resistance to AI therapy, and may yield novel approaches to treat AI-resistant tumors. MCF-7 cells were selected for >7months in IMEM+10% Charcoal Stripped FBS, supplmented with defined steroid hormone conditions. Following outgrowth of estrogen-independent cells, cell lines were grown in estrogen-free conditions and gene expression analysis was performed to identify drivers of estrogen-independent growth. Cells were assessed in biological triplicates.

Project description:Postmenopausal breast cancer patients benefit from aromatase inhibitors (AIs) that reduce the levels of estrogens critical for the growth of estrogen receptor (ER)-positive tumors. Unfortunately, many tumors are resistant to AI, and we are only beginning to understand the complex mechanisms underlying treatment resistance. Here we set out to determine whether epigenetic changes could contribute to therapy resistance. For AI-resistance models, we used previously established MCF-7 cell clones, termed C4-12 and long-term estrogen deprivation (LTED), that were isolated after being cultured in estrogen-free media for 9 months and 18M-bM-^@M-^S24 months, respectively. Methyl CpG Binding Domain (MBD) - pull down followed by affymetrix promoter array was used to detect promoter methylation patterns in these cell lines. These studies identified widespread genomic hyper- and hypomethylation events, with a significant enrichment of promoter hypermethylation of development-associated genes in both cell lines. A developmentally regulated gene that was heavily methylated and lost expression in both cell-line systems was HOXC10. Moreover, we found that HOXC10 is an estrogen-regulated gene and lack of ER regulation is associated with progressive epigenetic silencing through EZH2/H3K27me3 and DNA hypermethylation. Stable knockdown of HOXC10 in MCF-7 cells resulted in increased cell growth, reduced cell apoptosis, and increased cell motility. A preliminary study using AI-treated breast tumors did not show significant associations, however, the numbers were small. We identified HOXC10 methylation as a novel determinant of endocrine resistance. Also, we revealed that epigenetic reprogramming of genes involved in development may be a fundamental phenomenon in hormone resistance. Therefore, our study might provide the basis for the expansion of clinical markers for endocrine resistance and future clinical trials such as combination of endocrine and epigenetic therapies. Long-term estrogen deprived previously established MCF-7 cell clones termed C4-12 and LTED genomic DNA subjected to Methyl CpG Binding Domain (MBD) - pull down followed by affymetrix promoter array to detect promoter methylation patterns

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Epigenetic activation of the prostaglandin receptor EP4 promotes resistance to endocrine therapy for breast cancer [RNA-seq]

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