Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells.

Breast cancer prognosis and response to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively). Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in hormone-free condition is not fully understood. We used ER-/PR-positive breast cancer cells to investigate the role of PR in ESR1 regulation in the absence of hormones. We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and DNA methylation of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. As a consequence, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistently, DNA methylation impedes PR binding to consensus progesterone responsive elements. These findings contribute to understanding the complex crosstalk between PR and ER and suggest that the analysis of ESR1 promoter methylation in breast cancer cells can help to design more appropriate targeted therapies for breast cancer patients.

Simvastatin Enhances the Effects of Radiotherapy and Cetuximab on a Cell Line (FaDu) Derived from a Squamous Cell Carcinoma of Head and Neck.

Radiotherapy (XRT) delivered with the antibody cetuximab is a standard treatment option for squamous cell carcinomas of head and neck (SCCNH). Cetuximab acts by blocking epidermal growth factor receptor (EGFR) signaling to inhibit cancer progression. However, a significant percentage of patients will not respond to XRT and cetuximab. Statins reduce the synthesis of cholesterol and isoprenoid derivates that may be required for efficient EGFR signaling. We assessed whether the statin simvastatin could improve this combined therapy. In vitro, simvastatin enhanced the effects of XRT alone and in combination with cetuximab in wound healing, cell proliferation, and clonogenic assays in FaDu cells. These results were reflected in xenoimplanted tumors growing into subcutaneous tissue of athymic mice where concomitant treatment with simvastatin decreased tumor growth. Consistently, lower levels of phosphorylated extracellular signal-regulated kinases 1 and 2, phosphatidylinositol 3-kinase/AKT-protein kinase B, and signal transducer and activator of transcription 3 oncoproteins and higher levels of caspase-3 and apoptosis in cell cultures and xenografts were observed. The EGFR-overexpressing A431 cell line was used to reproduce these antitumor effects of simvastatin. Our findings suggest that simvastatin may improve the efficiency of concomitant XRT and cetuximab. Further investigation in the treatment of SCCNH is warranted.

Development and characterization of an isogenic cell line with a radioresistant phenotype.

INTRODUCTION: Radiation resistance is a major cause of death in cancer patients. Cancer cells react during radiotherapy by re-programming specific cell functions that may confer resistance to radiation. The understanding of this complex process is hindered due to the lack of appropriate study models. We describe an experimental development of a radioresistant isogenic cancer cell line, and its molecular characterization. MATERIALS AND METHODS: A431-cultured cells were irradiated for 7 month until 85 Gy. Then, a selected single cell was left to grow as stable A431-R cell line. Clonogenic assay was used to determine cell survival, the α and ß parameters of the LQ model, and the mean inactivation dose. The DNA repair ability of cells was evaluated by pulsed-field electrophoresis method. Differential effect of fractionated radiation was ultimately tested in xenografts. Furthermore, we used a wound healing assay, Western blot for EGFR, AKT and ERK1/2 and ELISA test for vascular endothelial growth factor (VEGF) secretion. Finally we explored CD44 marker and cell cycle distribution. RESULTS: The established A431-R cell line showed radiation resistance in clonogenic assays, repair of radiation-induced DNA fragmentation and xenografted tumours. The radiation resistance was associated with in vitro higher cell growth and migration, increased levels of former oncoproteins, and secretion of VEGF. CONCLUSIONS: In this model, the emergence of radiation resistance was associated with the acquisition of biological traits that support more aggressive behaviour of cancer cells. We have generated a model that will be useful for mechanistic studies and development of rational treatments against radiation resistance in cancer.

Development and refinement of a technique using a medical radiation therapy facility to irradiate immunodeficient mice bearing xenografted human tumours.

The need for using immunodeficient mice for xenoimplantation of tumours is increasing in translational research in radiation oncology. However, adverse effects of radiation and infectious diseases may ruin the experimental work, in particular when appropriate facilities are not available. In this report, we describe a procedure to deliver fractionated radiotherapy to xenoimplanted tumours in immunodeficient mice using a medical linear accelerator, a method that was devised as an alternative to the lack of facilities devoted to radiation research. The mice were irradiated under anaesthesia and aseptic conditions. Thirty Gray in 10 days using a 6 MV photon beam were delivered only to the right thigh of the mice where tumours were implanted. The mice were evaluated twice a week up to planned euthanasia. The follow-up of mice was completed without premature interruption due to toxicities or infectious diseases, an observation which demonstrates the feasibility of the method.

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis.

BACKGROUND: Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. METHODS: Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. RESULTS: We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. CONCLUSIONS: The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay.