Abstract
Recent clinical trials with histone deacetylase inhibitors (HDACi) have shown increased progression free survival by re-sensitizing resistant estrogen receptor positive (ER+) breast cancer cells to hormone suppressive therapies (HT). However, these trials lacked a sensitive, specific assay to identify and monitor HDACi/HT sensitive or resistant tumors. We tested detection of ER expression and histone acetylation of chromatin at the growth regulation by estrogen in breast cancer 1 (GREB1) gene, an estrogen-responsive gene involved in ER expression, in circulating tumor cell (CTC) as potential candidate assays for HDACi/HT sensitivity. ER+ and ER- CTC were detected and isolated from breast cancer patient peripheral blood by high speed fluorescence activated cell sorting (FACS) for use in mRNA analysis and anti-acetylated histone-mediated Chromatin Immunoprecipitation (ChIP). cDNA from mRNA and DNA extracted from the ChIP isolates were quantified by real-time PCR for GREB1. CTC isolates from patients who had an ER+ breast cancer primary contained both ER+ and ER- cells. More ER+ than ER- CTC was found in HT sensitive patients compared to HT resistant patients (p = 0.0559). GREB1 was found in acetylated histone chromatin from both ER+ and ER- CTC. The number of ER+ and ER- CTC found in peripheral blood appears to parallel patient outcomes as to their sensitivity to HT. Acetylated histone analysis can detect chromatin containing GREB1 in CTC, suggesting it may be useful as a more specific measure of HDACi effects on breast tumor cells. A larger, longitudinal data set following patients through HT/HDACi trials is needed to confirm these observations and their development for clinical use.
Highlights
Breast cancer incidence in the United States is currently reported by the National Institutes for Health as between 63.51 to 98.69 cases per 100,000 among women, with a marked decline in mortality in the last decade [1]
We aseptically collect live circulating tumor cell (CTC) from the peripheral blood of breast cancer patients with known estrogen receptors (ER)+ metastatic breast cancer to analyze histone acetylation of GREB1, a gene expressed in response to estrogen and involved in regulation of ER in breast and breast cancer cells [19] [20], to determine if such estrogen-responsive gene activation indicator analyses could afford a better, more specific monitoring assay for following the mechanism and efficacy of histone deacetylase inhibitors (HDACi) treatment in re-sensitizing hormone suppressive therapies (HT) resistant breast tumor cells and promoting better efficacy of adjuvant HT treatment in preventing relapse and metastatic recurrence of ER+ tumors
Higher estradiol levels were found in patients resistant to HT and/or having ER− CTC numbers greater or equal to ER+ CTC numbers compared with patients who had predominantly ER+ CTC and remained sensitive to HT (Figure 1(b))
Summary
Breast cancer incidence in the United States is currently reported by the National Institutes for Health as between 63.51 to 98.69 cases per 100,000 among women, with a marked decline in mortality in the last decade [1]. Great advancements have been made in early detection and treatment of primary breast cancer through greater awareness of the value of regular prognostic screening, and advancements in surgical, chemotherapeutic, hormonal, and radiation therapies. Detection and efficient monitoring of metastasis could mean the difference between recovering quality of life and severely debilitating terminal illness. Once tumor cells enter the circulation, they can de-differentiate into stem cell-like morphologies conducive to metastatic invasion [2]. This means their character changes, often to the point of becoming resistant to original treatment given to a patient
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