Altered radiation responses of breast cancer cells resistant to hormonal therapy

Abstract

Endocrine therapy agents (the selective estrogen receptor (ER) modulators such as tamoxifen or the selective ER down-regulators such as ICI 182,780) are key treatment regimens for hormone receptor-positive breast cancers. While these drugs are very effective in controlling ER-positive breast cancer, many tumors that initially respond well to treatment often acquire drug resistance, which is a major clinical problem. In clinical practice, hormonal therapy agents are commonly used in combination or sequence with radiation therapy. Tamoxifen treatment and radiotherapy improve both local tumor control and patient survival. However, tamoxifen treatment may render cancer cells less responsive to radiation therapy. Only a handful of data exist on the effects of radiation on cells resistant to hormonal therapy agents. These scarce data show that cells that were resistant to tamoxifen were also resistant to radiation. Yet, the existence and mechanisms of cross-resistance to endocrine therapy and radiation therapy need to be established. Here, we for the first time examined and compared radiation responses of MCF-7 breast adenocarcinoma cells (MCF-7/S0.5) and two antiestrogen resistant cell lines derived from MCF-7/S0.5: the tamoxifen resistant MCF-7/TAMR-1 and ICI 182,780 resistant MCF-7/182R-6 cell lines. Specifically, we analyzed the radiation-induced changes in the expression of genes involved in DNA damage, apoptosis, and cell cycle regulation. We found that the tamoxifen-resistant cell line in contrast to the parental and ICI 182,780-resistant cell lines displayed a significantly less radiation-induced decrease in the expression of genes involved in DNA repair. Furthermore, we show that MCF-7/TAMR-1 and MCF-7/182R-6 cells were less susceptible to radiation-induced apoptosis as compared to the parental line. These data indicate that tamoxifen-resistant breast cancer cells have a reduced sensitivity to radiation treatment. The current study may therefore serve as a roadmap to the future analysis of the mechanisms of cross-resistance between hormonal therapy and radiation.

Figure 1. Gene expression profiling of MCF-7/S0.5, MCF-7/TAM^R-1 and MCF-7/182^R-6

The Venn diagram shows the number of significantly changed genes in the MCF-7/S0.5, MCF-7/TAM^R-1 and MCF-7/182^R-6 cell lines upon radiation in comparison to their corresponding un-irradiated controls, as identified by the gene expression profiling analysis. The arrows beside the numbers in brackets represent the direction of genes alteration (up- or down-regulation).

Figure 2. Fold change in the levels of CCNA2, CCNB2, CDC20, PTTG1 and BAX transcripts detected by qRT-PCR

Each treatment group was compared to its corresponding control. Actin was used as a reference gene (calculated by Pfaffl). * - significant, p<0.001; ** - significant, p<0.01. (Student's t-test).

Figure 3. Radiation-induced H2AX phosphorylation in MCF-7/S0.5, MCF-7/TAM^R-1 and MCF-7/182^R-6 cells

The results on the pictures and a figure below are presented as an average number of γH2AX foci per cell ± SE, n = 200. * - significantly different from the respective control; p < 0.05; # - significantly different from MCF-7/S0.5 cell line. Controls were not significantly different between three cell lines; p<0.05. Magnification, × 100. Blue – DAPI, green – γH2AX.

Figure 4. Radiation-induced DNA damage in MCF-7/S0.5, MCF-7/TAM^R-1, and MCF-7/182^R-6 cells as determined by the Alkaline Comet assay

The graphs represent the percentage of DNA in the comet tails (tail intensity) obtained by the Alkaline Comet assay performed on MCF-7/S0.5, MCF-7/TAM^R-1, and MCF-7/182^R-6 cells 30 minutes, 6 and 24 hours after X-ray irradiation. Tail intensity levels are represented as mean ± SD; * - significantly different from the respective control, p < 0.01; ** - significantly different from the respective control, p<0.05. (Student's t-test). Comet representative pictures of tail intensity are located beside the charts.

Figure 5. Radiation-induced apoptosis in MCF-7/S0.5, MCF-7/TAM^R-1 and MCF-7/182^R-6 cells

The number of cells in early apoptosis was measured using the Annexin V-FITC assay for control cells (CT) and cells irradiated with 0.5 Gy and 5 Gy of X-rays. M1 – AnnexinV- positive cells; Viable cells - AnnexinV- and PI-negative (the lower left quadrants); Cells in the early apoptosis state - AnnexinV-positive and PI-negative (the lower right quadrants); Cells in the late apoptosis state or already dead cells - both Annexin V- and PI-positive (the upper right quadrants).