Cyclin D1 is Associated with Radiosensitivity of Triple-Negative Breast Cancer Cells to Proton Beam Irradiation

Abstract

Proton therapy offers a distinct physical advantage over conventional X-ray therapy, but its biological advantages remain understudied. In this study, we aimed to identify genetic factors that contribute to proton sensitivity in breast cancer (BC). Therefore, we screened relative biological effectiveness (RBE) of 230 MeV protons, compared to 6 MV X-rays, in ten human BC cell lines, including five triple-negative breast cancer (TNBC) cell lines. Clonogenic survival assays revealed a wide range of proton RBE across the BC cell lines, with one out of ten BC cell lines having an RBE significantly different from the traditional generic RBE of 1.1. An abundance of cyclin D1 was associated with proton RBE. Downregulation of RB1 by siRNA or a CDK4/6 inhibitor increased proton sensitivity but not proton RBE. Instead, the depletion of cyclin D1 increased proton RBE in two TNBC cell lines, including MDA-MB-231 and Hs578T cells. Conversely, overexpression of cyclin D1 decreased the proton RBE in cyclin D1-deficient BT-549 cells. The depletion of cyclin D1 impaired proton-induced RAD51 foci formation in MDA-MB-231 cells. Taken together, this study provides important clues about the cyclin D1-CDK4-RB1 pathway as a potential target for proton beam therapy in TNBC.

Keywords: CDK4/6 inhibitor; breast cancer; cyclin D1; proton therapy; relative biological effectiveness.

Figure 1

Comparison of radiosensitivity and proton relative biological effectiveness (RBE) in breast cancer cell lines. (A) Comparison of the survival fraction at 2 Gy (SF2) of X-ray or proton treatment across 11 human breast cancer cell lines. Data are presented as the mean ± SEM from three independent experiments and the differences between proton and X-ray results are evaluated by a student t-test; * p < 0.05; ** p < 0.01. (B) Comparison of SF2 for X-rays and protons between non-TNBC and TNBC groups. No significant differences between groups are seen. (C) Proton RBE values relative to megavoltage X-rays for ten BC cell lines at SF = 0.5 and SF = 0.1. (D) Comparison of RBE₁₀ values for BC cell lines. RBE₁₀ was calculated as the ratio of megavoltage X-ray doses to proton doses that yield 10% survival. RBE of each cell line was compared to 1.10 using a one-sample t-test; * p < 0.05.

Figure 2

The possible implication of the RB1 pathway in the proton sensitivity of TNBC cells. (A) Dose-response curves of three TNBC cell lines, MDA-MB-231, Hs578T, and BT-549, which were irradiated with X-rays or protons. Data are presented as the mean ± SEM for three independent experiments. Curves were fitted with a linear quadratic model. The differences are evaluated by a two-way ANOVA; * p < 0.05; *** p < 0.001. Dashed lines indicate SF = 0.1. (B) Expression of RB pathway-related proteins among the three TNBC cell lines. Western blot analysis was performed as described in the materials and methods. β-Actin was used as a loading control. (C) Scatter plot for correlation between RBE₁₀ values and CCND1 mRNA expression in human BC cell lines. Log2-transformed RMA values were obtained from the Cancer Cell Line Encyclopedia (CCLE) database. Pearson’s correlation coefficients (r) between gene expression and proton RBE₁₀ across all samples were presented.

Figure 3

RB1 downregulation via siRNA or CDK4/6 inhibitor does not increase proton RBE in MDA-MB-231 cells. (A) Western blot analysis confirms siRNA-mediated RB1 knockdown in MDA-MB-231 cells. β-Actin was used as a loading control. Neither radiation treatment nor RB1 knockdown affected the expression of cyclin D1 (upstream effector) or E2F1 (downstream of RB1). (B) Effect of RB1 depletion on the clonogenic survival of MDA-MB-231 cells after X-ray or proton irradiation. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; *** p < 0.001. (C) The RB1 depletion augmented radiation-induced apoptosis. Apoptotic cell death was assessed by flow cytometry as described in the materials and methods. Data are presented as the mean ± SEM of two independent experiments. The differences were evaluated by a two-way ANOVA; ** p < 0.01; *** p < 0.001. (D) PD-0332991, a selective CDK4/6 inhibitor, decreased total RB and its phosphorylated form in a dose-dependent manner. β-Actin was used as a loading control. (E) The effect of PD-0332991 on clonogenic survival. MDA-MB-231 cells were pre-treated with 100 nM PD-0332991 for 3 h, followed by irradiation with the indicated doses of X-rays or protons. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; * p < 0.05; *** p < 0.001. (F) PD-0332991 enhanced proton radiation-induced apoptosis. MDA-MB-231 cells were pre-treated with 500 nM PD-0332991, followed by exposure to 4 Gy of X-rays or protons. Data are presented as the mean ± SEM of three independent experiments and the differences are evaluated by a two-way ANOVA; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Cyclin D1 depletion increases proton RBE in MDA-MB-231 cells. (A) Western blot analysis confirms siRNA-mediated cyclin D1 knockdown in MDA-MB-231 cells. β-Actin was used as a loading control. (B) Effect of cyclin D1 knockdown on the clonogenic survival of MDA-MB-231 cells after X-ray or proton irradiation. The cells were transfected with either control siRNA or CCND1 siRNA, followed by irradiation with the indicated doses of X-rays or protons. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; * p < 0.05. (C) Effect of cyclin D1 knockdown on cell cycle progression in MDA-MB-231 cells. (D) Cyclin D1 knockdown promoted radiation-induced apoptosis. MDA-MB-231 cells transfected with control siRNA or cyclin D1 siRNA were irradiated with 4 Gy of X-rays or protons. Apoptosis was determined using flow cytometry 72 h post-irradiation. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; * p < 0.05; *** p < 0.001.

Figure 5

Depletion or re-expression of cyclin D1 affects proton RBE in TNBC cells. (A) The knockdown of cyclin D1 via siRNA in Hs578T cells. (B) Depletion of cyclin D1 increased proton RBE in the Hs578T cell line, as judged by a clonogenic survival assay. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; ** p < 0.01; *** p < 0.001. Dashed lines indicate SF = 0.1. (C) Western blot analysis confirmed transient overexpression of HA-tagged cyclin D1 in BT-549 cells. Ectopic expression of cyclin D1 did not affect expression of other genes, such as RB1 and E2F1. (D) Overexpression of cyclin D1 in BT-549 cells decreased RBE₁₀ from 1.40 to 1.14. Data are presented as the mean ± SEM of two independent experiments and the differences are evaluated by a two-way ANOVA; *** p < 0.001.

Figure 6

Depletion of cyclin D1 impairs radiation-induced formation of RAD51 foci. (A) Representative images of radiation-induced RAD51 foci in MDA-MB-231 cells. The MDA-MB-231 cells were transfected with either control siRNA or CCND1 siRNA, followed by irradiation with 4 Gy of X-rays or protons. The cells were fixed at the indicated times and stained with DAPI (blue) and RAD51 (green), as described in the materials and methods. (B) Kinetics of cells with RAD51 foci (n ≥ 5) after 4 Gy of X-rays or protons. Data are presented as the mean ± SEM of two independent experiments (n = 40). (C) Depletion of cyclin D1 decreased RAD51 expression. β-Actin was used as a loading control.