Protective role of miR-155 in breast cancer through RAD51 targeting impairs homologous recombination after irradiation

Abstract

Cell survival after DNA damage relies on DNA repair, the abrogation of which causes genomic instability and development of cancer. However, defective DNA repair in cancer cells can be exploited for cancer therapy using DNA-damaging agents. DNA double-strand breaks are the major lethal lesions induced by ionizing radiation (IR) and can be efficiently repaired by DNA homologous recombination, a system that requires numerous factors including the recombinase RAD51 (RAD51). Therapies combined with adjuvant radiotherapy have been demonstrated to improve the survival of triple-negative breast cancer patients; however, such therapy is challenged by the emergence of resistance in tumor cells. It is, therefore, essential to develop novel therapeutic strategies to overcome radioresistance and improve radiosensitivity. In this study we show that overexpression of microRNA 155 (miR-155) in human breast cancer cells reduces the levels of RAD51 and affects the cellular response to IR. miR-155 directly targets the 3'-untranslated region of RAD51. Overexpression of miR-155 decreased the efficiency of homologous recombination repair and enhanced sensitivity to IR in vitro and in vivo. High miR-155 levels were associated with lower RAD51 expression and with better overall survival of patients in a large series of triple-negative breast cancers. Taken together, our findings indicate that miR-155 regulates DNA repair activity and sensitivity to IR by repressing RAD51 in breast cancer. Testing for expression levels of miR-155 may be useful in the identification of breast cancer patients who will benefit from an IR-based therapeutic approach.

Keywords: TNBC; gamma-rays; noncoding RNA.

Fig. 1.

miR-155 protective role in TNBC through RAD51 targeting. (A) Overall survival of TNBC patients according to miR-155 expression; the cohort was dichotomized using the median expression as cutoff. (B) Positions of miR-155 putative binding sites on RAD51 transcript. (C) Psicheck2 vector with RAD51 WT insert (full-length RAD51 transcript) with Mut miR-155 CDS and Mut miR-155 3′-UTR containing a deletion of the miR-155 target site in the CDS (position 367) and in the 3′-UTR (position 1594), respectively, were cotransfected with miR-155 or scrambled miR in 293HEK cells. Luciferase activity was recorded after 24 h. Data represent the mean ± SD from at least three determinations from four independent transfections.

Fig. 2.

miR-155 is anti-correlated to RAD51 expression. (A) Bar graph represents anticorrelation between miR-155 and RAD51 expression levels analyzed by real-time PCR in a breast cancer cell panel. (B) Box plots represent miR-155 and RAD51 H-score anticorrelation in the TNBC cohort (Materials). (C) Decreasing H-score of RAD51 in lymph node (LN) metastasis compared with primary tumors. (D) Increasing miR-155 expression level in LN metastasis compared with primary tumors. (E) Representative RAD51 staining in primary tumor tissue (Upper) and LN metastasis (Lower). (F) Overall survival of TNBC patients according to anticorrelation miR-155 and RAD51 IHC data expression; the best outcome is given by the group of patients with high miR-155 and low RAD51 expression.

Fig. 3.

miR-155 affects survival through homologous recombination impairment. (A) Clonogenic survival assay performed on stably miR-155–overexpressing cells after increasing dose of gamma-rays shows decreased clonogenic capability owing to miR-155 overexpression. (B) HR assay shows impairment of the HR process owing to miR-155 overexpression. The comparisons between MCF7 scrambled and MCF7 miR-155 are statistically significant and represent the average values of three independent experiments.

Fig. 4.

miR-155 inhibits gamma-rays-induced RAD51 foci formation. (A) Stably miR-155–overexpressing MCF7 cells were treated with IR (8 Gy) and then fixed for immunofluorescent staining of RAD51, 30 min after IR. Representative image of RAD51 immunostaining (Upper Left); GFP signal represents the miR-155 overexpression in the stable clones (Upper Right); Lower Left represents the counterstain with DAPI; Lower Right shows the overlay of the three signals. (B) Bar graph shows relative quantification of RAD51 foci in cells with at least 10 foci after IR (8 Gy) at different time points; miR-155 overexpression induces a decrease in RAD51 foci formation compared with scrambled clones.

Fig. 5.

miR-155 increases gamma-rays-induced γ-H2AX foci formation. (A) Bar graph shows relative quantification of γ-H2AX foci in cells with at least 10 foci after IR (8 Gy) at different time points; overall miR-155 overexpression induced a marked increase in γ-H2AX foci compared with scrambled clones. (B) Stably miR-155–overexpressing MCF7 cells were treated with IR (8 Gy) and then fixed for immunofluorescent staining of γ-H2AX foci, 30 min after IR. Representative image of γ-H2AX immunostaining in scrambled stably transfected MCF7 cells is shown Upper Right; Upper Left shows an increase in γ-H2AX foci in MCF7 stably overexpressing miR-155. Lower panels represent the respective counterstain with DAPI. (C) Graph shows the detection of γ-H2AX by flow cytometry (phospho-histone H2AX, Alexa Fluor 488 conjugate). MCF7 24 h after transfection with scrambled or miR-155 were exposed to IR (8 Gy) and harvesting at different time points. Data are normalized to untransfected and unirradiated cells.

Fig. 6.

miR-155 induces a delay in DNA damage repair through RAD51 targeting. MCF7 were transfected with scramble or miR-155 and after 48 h were treated with IR (8 Gy) and harvested for Western blot at three different time points. Rad51 has a threshold level that is not affected by miR-155 overexpression (30 min and 2 h); on the contrary, overexpression of RAD51 full-length and miR-155 shows an unequivocal targeting from the miR. The time point at 4 h shows a RAD51 induction owing to IR exposure; here is also evident the miR-155 targeting. The γ-H2AX signal persists much longer in miR-155–overexpressing cells, showing a delay in DNA damage repair.