Estrogen induces RAD51C expression and localization to sites of DNA damage

Abstract

Homologous recombination (HR) is a conserved process that maintains genome stability and cell survival by repairing DNA double-strand breaks (DSBs). The RAD51-related family of proteins is involved in repair of DSBs; consequently, deregulation of RAD51 causes chromosomal rearrangements and stimulates tumorigenesis. RAD51C has been identified as a potential tumor suppressor and a breast and ovarian cancer susceptibility gene. Recent studies have also implicated estrogen as a DNA-damaging agent that causes DSBs. We found that in ERα-positive breast cancer cells, estrogen transcriptionally regulates RAD51C expression in ERα-dependent mechanism. Moreover, estrogen induces RAD51C assembly into nuclear foci at DSBs, which is a precursor to RAD51 complex recruitment to the nucleus. Additionally, disruption of ERα signaling by either anti-estrogens or siRNA prevented estrogen induced upregulation of RAD51C. We have also found an association of a worse clinical outcome between RAD51C expression and ERα status of tumors. These findings provide insight into the mechanism of genomic instability in ERα-positive breast cancer and suggest that individuals with mutations in RAD51C that are exposed to estrogen would be more susceptible to accumulation of DNA damage, leading to cancer progression.

Keywords: DNA double-strand break repair; ERα; RAD51C; breast cancer; estrogen.

Figure 1.

ERα regulates RAD51C expression in an estrogen dependent manner. (A) MDA-MB-231, −468, −436, MCF7, T47D and ZR 75-1 cells were grown in phenol red-free media with 10% charcoal-stripped FBS for 3 d and either serum starved or treated with estrogen for 30 min as indicated. Lysates were generated and the indicated proteins were detected by immunoblot. (B) MDA-MB-231, −468, −436, MCF7, T47D and ZR 75-1 cells were grown in phenol red-free media with 10% charcoal-stripped FBS for 3 d and either serum starved or treated with 10nM estrogen for 24 hr as indicated. Lysates were generated as described in “Materials and Methods” and the indicated proteins were detected by immunoblot. (C) Quantification of RAD51C protein levels normalized to actin from (B) was performed using Odyssey Image Studio Version 4.0 and graphed using Excel.

Figure 2.

Estrogen induces RAD51C foci assembly. (A) T47D cells were serum-starved or stimulated with estrogen for 24 hr as indicated. Immunofluorescence was performed as described in “Materials and Methods.” Scale bar represents 50 μm. (B) MCF7 cells were treated and processed as described in (A). (C) MDA-MB-231 cells were treated and processed as described in (A).

Figure 3.

ERα transcriptionally regulates RAD51C expression in estrogen-dependent manner. (A) MCF7 cells were transfected with scambled siRNA or siRNA against ERα and treated with estrogen for 48 hr, as indicated. RT-qPCR was performed as described in “Materials and Methods” and data was plotted using Excel. **p < 0.001; NS, non-statistically significant; n = 3. (B) MCF7 cells were treated as described in (A), RT-qPCR was performed as described in “Materials and Methods” and data was plotted using Excel. **p < 0.001; NS, non-statistically significant; n = 3. (C) MDA-MB-231, −468, −436, MCF7, T47D and ZR 75-1 cells were serum starved or treated with estrogen for 24 hr, as indicated. Lysates were generated as described in “Materials and Methods” and the indicated proteins were detected by immunblot. (D) MCF7 cells were transfected, stimulated with estrogen for 24 hr as indicated and Luciferase reporter assay was performed as described in “Materials and Methods.” Data was plotted using Excel. **p < 0.001. (E) MCF7 cells were transfected, stimulated with estrogen for 24 hr as indicated and Luciferase reporter assay was performed as described in “Materials and Methods.” Data was plotted using Excel. **p < 0.001; n = 3. (F) MDA-MB-231 and MCF7 cells were either serum starved, treated with estrogen for 24 hr or pre-treated with estrogen for 30 min following treatment with tamoxifen for 24 hr. Lysates were generated as described in “Materials and Methods” and indicated proteins were detected by immunoblot. (G) MDA-MB-231 and MCF7 cells were either serum starved, treated with estrogen for 24 hr or pre-treated with estrogen for 30 min following treatment with fulvestrant for 24 hr. Lysates were generated as described in “Materials and Methods” and indicated proteins were detected by immunoblot.

Figure 4.

Estrogen induces RAD51C foci assembly in ERα dependent manner. (A) T47D cells were transfected with scrambled siRNA, or siRNAs against ERα and either serum-starved or stimulated with estrogen for 24 hr. Immunofluorescence was performed as described in “Materials and Methods.” Scale bar represents 50 μm. (B) MCF7 cells were treated and processed as described in (A).

Figure 5.

GSA-Tumor analysis of RAD51C shows worse clinical prognosis for ERα-positive tumors. (A) MDA-MB-231, MCF7 and T47D cells were transfected with scrambled siRNA or siRNA against RAD51C and treated with estrogen for 48 hr, as indicated. Lysates were generated as described in “Materials and Methods” and the indicated proteins were detected by immunblot. (B) Kaplan-Meier analysis using DMFS of ERα-positive tumors separated into 3 categories with respect to RAD51C expression. (B) Kaplan-Meier analysis using RFS of Luminal A tumors separated into 3 categories with respect to RAD51C expression. (C) Kaplan-Meier analysis using RFS of Luminal A tumors separated into 3 categories with respect to RAD51C expression.