NF-κB regulates radioresistance mediated by β1-integrin in three-dimensional culture of breast cancer cells

Abstract

β1-integrin induction enhances breast cancer cell survival after exposure to ionizing radiation (IR), but the mechanisms of this effect remain unclear. Although NF-κB initiates prosurvival signaling pathways post-IR, the molecular function of NF-κB with other key elements in radioresistance, particularly with respect to extracellular matrix-induced signaling, is not known. We discovered a typical NF-κB-binding site in the β1-integrin promoter region, indicating a possible regulatory role for NF-κB. Using three-dimensional laminin-rich extracellular matrix (3D lrECM) culture, we show that NF-κB is required for β1-integrin transactivation in T4-2 breast cancer cells post-IR. Inhibition of NF-κB reduced clonogenic survival and induced apoptosis and cytostasis in formed tumor colonies. In addition, T4-2 tumors with inhibition of NF-κB activity exhibit decreased growth in athymic mice, which was further reduced by IR with downregulated β1-integrin expression. Direct interactions between β1-integrin and NF-κB p65 were induced in nonmalignant breast epithelial cells, but not in malignant cells, indicating context-specific regulation. As β1-integrin also activates NF-κB, our findings reveal a novel forward feedback pathway that could be targeted to enhance therapy.

Figure 1

Inhibition of NF-κB or β1-integrin increased radiosensitivity in malignant breast cancer T4-2 cells. A and B, T4-2 malignant breast cells and its counterpart non-malignant S1 breast epithelial cells were left untreated (A) or T4-2 cells were treated with NF-κB activation inhibitor JSH-23 (5 µM) or β1-integrin inhibitory antibody AIIB2 (0.1 µg/µl) (B) before exposure to 1-, 2-, 4- or 8-Gy x-ray. Clonogenic survival was measured 14 days after ionizing radiation (IR). Colonies consisting of more than 50 cells were scored as surviving colonies and normalized against nonirradiated clones (n = 3, mean ± SD). C and D, Immunofluorescence analysis of NF-κB p65 in S1 and T4-2 cells exposed to sham or 4-Gy IR. Arrows indicate nuclear p65. Scale bar, 200 µm (C). Graphical representation of the p65 positive nuclei. A representative experiment from n = 3 is shown (D). E, NF-κB DNA binding assay using nuclear extracts of S1 and T4-2 cells exposed to sham or 4-Gy IR. Data are mean ± SD of pooled results from three independent experiments; p <0.01 (**). F, An NF-κB binding site is identified in the promoter region of β1-integrin gene.

Figure 2

Radiation-induced β1-integrin expression in 3D lrECM in human breast cancer T4-2 cells was mediated by NF-κB through increased transcriptional activity. A, Experimental schema. T4-2 colonies and S1 acini formed at day 4 and 6, respectively, were treated with NF-κB activation inhibitor JSH-23 or vehicle (DMSO), and then exposed to sham or 4-Gy x-ray at day 5 (T4-2) and 7 (S1). Whole cell lysates were prepared 48 h post-IR for Western and NF-κB DNA binding assays. B, Western blot analyses on the expression of phospho and total β1-integrins, and p65/p50 (major heterodimer of NF-κB) using whole lysates prepared from malignant breast cancer T4-2 and non-malignant S1 breast epithelial cells at day 7 and 9, respectively, in 3D lrECM. β-actin serves as an internal loading control. C and E, Western analyses of phospho and total β1-integrins (C), and p65 and p50 (E) in T4-2 cells treated with different concentrations of JSH-23 and exposed to sham or 4-Gy x-ray. The lysates were also blotted for β-actin as an internal loading control. Right panels: relative expression levels of phospho-β1-integrin (C) and NF-κB p65/p50 (E) normalized to the expression levels of β-actin. D, Quantitative RT-PCR analysis of β1-integrin mRNA expression in T4-2 cells treated as above. GAPDH serves as an internal control. F, NF-κB DNA binding assay using whole cell lysates of T4-2 and S1 cells exposed to sham or 4-Gy x-ray as shown in the schematic diagram in A. The 50-bp β1-integrin promoter region containing wild-type or mutated NF-κB binding sequence was synthesized and used as a DNA probe to assess the binding. Data are mean ± SD of pooled results from three independent experiments; p <0.01 (**).

Figure 3

Inhibition of NF-κB activity in T4-2 cells resulted in decreased colony size and cell proliferation, and increased apoptosis in 3D lrECM culture. A, Colony size, apoptosis (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; TUNEL) and proliferation (detected by indirect immunofluorescence of Ki-67 nuclear antigen) of T4-2 cells treated with JSH-23 (40 µM), and then exposed to sham or 4-Gy x-ray as shown in the schematic diagram in Fig. 2A. Phase-contrast photographs of the colonies were taken to measure the diameter of 100 colonies with or without IR using Spot Advanced software; Scale bar, 100 µm (upper panel). T4-2 cell smears fixed onto glass slides were prepared from the colonies at day 7 for apoptosis (middle panel) and proliferation (bottom panel) assays. Arrows indicate positive nuclei for Tunel (red) and Ki-67 (green), respectively. Scale bar, 50 µm. B, Graphical representation of the relative diameter of T4-2 colonies; p <0.01 (**). C and E, Graphical representation of the TUNEL (C) and Ki-67 (E) positive cells. Columns, mean (n = 3); bars, SD; p <0.001 (***); p <0.01 (**). D, The activation of caspase-3/7, considered a reliable marker for cells undergoing apoptosis, was highly induced by NF-κB inhibition post-IR. T4-2 cells treated with JSH-23, AIIB2 or JSH-23+AIIB2 with or without pan-caspase inhibitor Z-VAD-FMK were exposed to sham or 4-Gy x-ray (schematic diagram is shown in Fig. 2A), single cells were dissociated from lrECM and Caspase-3/7 activity was measured by Caspase-Glo® 3/7 Assay Kit. Asterisk indicates statistically significant difference (**, p <0.01) compared with DMSO-treated cells; columns, mean (n = 3); bars, SD.

Figure 4

Inhibition of NF-κB activity resulted in increased apoptosis and decreased proliferation in MDA-MB-231 and MCF-7 cells in 3D lrECM. A, Experimental schema. B-E, Apoptosis (B and D) and proliferation (C and E) levels of MDA-MB-231 and MCF-7 colonies in 3D lrECM using TUNEL and Ki-67 staining. MDA-MB-231 and MCF-7 colonies formed at day 3 and 4, respectively, were treated with NF-κB activation inhibitor JSH-23 or vehicle (DMSO), and then exposed to sham or 4-Gy x-ray at day 4 (MDA-MB-231) and 5 (MCF-7). Smears were fixed onto glass slides 48 h post-IR. Columns, mean (n = 3); bars, SD; p <0.05 (*); p <0.01 (**); p <0.001 (***).

Figure 5

NF-κB inhibition by 20 µM JSH-23 in T4-2 cells delayed tumor growth in nude mice via inhibition of β1-integrin expression. A, Tumor growth curves obtained following subcutaneous injection of T4-2 cells treated with vehicle (DMSO), or JSH-23 with sham (T4+JSH) or 4-Gy x-ray (T4+JSH+IR) in female NCR nude mice (nu/nu). Eight animals were used per group and the data represent the mean ± SE; p <0.01 (**); p <0.001 (***). B, Whole tumors excised from mice injected with T4+JSH, T4+JSH+IR or control cells. C, Immunohistochemical staining of the tumor sections from T4+JSH, T4+JSH+IR or control mice using NF-κB p65 or β1-integrin antibody. After the immunoreaction, sections were counterstained with hematoxylin. Nuclear p65 and β1-integrin are indicated by yellow and white arrows, respectively. Images were captured at 1000× magnifications; scale bar, 50 µm. D, Phospho and total β1-integrins and NF-κB p65/50 expressions in control, T4+JSH and T4+JSH+IR mouse tumors detected by Western blot analysis. Lamin A/C serves as an internal control.

Figure 6

Inhibition of β1-integrin reversed IR-induced NF-κB expression and DNA binding activity in 3D lrECM. A, Western blot analyses on the expression of NF-κB p65/p50 and β1-integrin using whole cell lysates prepared from malignant breast cancer T4-2 cells at day 7 in 3D lrECM as shown in Fig. 2A. β-actin serves as an internal loading control. Right panel: relative expression levels of NF-κB p65/p50 normalized to the expression levels of β-actin. B, NF-κB DNA binding assay using whole cell lysates prepared from T4-2 cells as above. Data are mean ± SD of pooled results from three independent experiments (n = 3); p <0.01 (**). C, Schematic representation of radiation-induced loop-like β1-integrin–NF-κB–β1-integrin pathway in radioresistance of malignant breast cells in 3D lrECM.