Autophagy promotes radiation-induced senescence but inhibits bystander effects in human breast cancer cells

Abstract

Ionizing radiation induces cellular senescence to suppress cancer cell proliferation. However, it also induces deleterious bystander effects in the unirradiated neighboring cells through the release of senescence-associated secretory phenotypes (SASPs) that promote tumor progression. Although autophagy has been reported to promote senescence, its role is still unclear. We previously showed that radiation induces senescence in PTTG1-depleted cancer cells. In this study, we found that autophagy was required for the radiation-induced senescence in PTTG1-depleted breast cancer cells. Inhibition of autophagy caused the cells to switch from radiation-induced senescence to apoptosis. Senescent cancer cells exerted bystander effects by promoting the invasion and migration of unirradiated cells through the release of CSF2 and the subsequently activation of the JAK2-STAT3 and AKT pathways. However, the radiation-induced bystander effects were correlated with the inhibition of endogenous autophagy in bystander cells, which also resulted from the activation of the CSF2-JAK2 pathway. The induction of autophagy by rapamycin reduced the radiation-induced bystander effects. This study reveals, for the first time, the dual role of autophagy in radiation-induced senescence and bystander effects.

Keywords: angiogenesis; autophagy; invasion; migration; radiation; senescence-associated secretory phenotypes.

Figure 1. Radiation induced autophagy in MDA-MB-231-2A cells. (A) The levels of PTTG1 in MDA-MB-231, MDA-MB-231-2A, and MCF-7 cells were examined by western blot analysis. (B) MDA-MB-231-2A cells were exposed to different doses of radiation followed by 24 h of recovery time. The ratio of MAP1LC3-II/MAP1LC3-I was analyzed by western blot analysis. (C) EGFP-MAP1LC3-transfected MDA-MB-231-2A cells were exposed to 6-Gy radiation or serum starvation. EGFP intensity was measured by flow cytometry. (D) EGFP-MAP1LC3-transfected MDA-MB-231-2A cells were exposed to 6-Gy radiation or serum starvation. EGFP-MAP1LC3 puncta were measured using fluorescence microscopy. Scale bar: 500 µm. In (C and D), ** indicates significant differences (P < 0.01) between the control and serum starved cells. ## indicates significant differences (P < 0.01) between the control and irradiated cells. (E) MDA-MB-231-2A cells were exposed to 6-Gy radiation, and autophagosome-like structures (arrows) were observed by TEM.

Figure 2. Effect of the autophagic inhibition on radiation-induced autophagy in MDA-MB-231-2A and MCF-7 cells. (A) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. The MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis. (B) MDA-MB-231-2A cells were recovered for 18 h after exposure to 6-Gy radiation and were treated with or without bafilomycin A₁ for 6 h. The MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis. (C) MDA-MB-231-2A cells were exposed to different doses of radiation followed by 24 h of recovery time. SQSTM1 expression was measured by western blot analysis. (D) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. After 24 h of recovery time, SQSTM1 expression was measured by western blot analysis. (E and F) MCF-7 cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. After 24 h of recovery time, the MAP1LC3-II/MAP1LC3-I ratio (E) and SQSTM1 expression (F) were measured by western blot analysis. (G) MDA-MB-231-2A and MCF-7 cells were transfected with ATG5 or non-targeting siRNAs for 48 h before exposure to 6-Gy radiation. After 24 h of recovery time, the MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis.

Figure 3. Autophagy promoted radiation-induced senescence in MDA-MB-231-2A cells. (A) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. Bright-field microscopy observation and SA-β-gal staining were performed 2 d after irradiation. (B) MDA-MB-231-2A cells were recovered for 18 h after irradiation and were treated with or without bafilomycin A₁. The cells were then observed by bright-field microscopy and stained with SA-β-gal 2 d after irradiation. ** indicates significant differences (P < 0.01) between the control and irradiated cells. ## indicates significant differences (P < 0.01) between the inhibitor-treated and untreated cells. (C) MDA-MB-231-2A cells were transfected with ATG5 or nontargeting siRNAs for 48 h before exposure to 6-Gy radiation. Bright-field microscopy observation and SA-β-gal staining were performed 2 d after irradiation. ** indicates significant differences (P < 0.01) compared with si-Cont cells. n.s. indicates no significant differences between the unirradiated and irradiated si-ATG5 cells. (D) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. Cells were stained with C₁₂FDG, and the fluorescence was analyzed by flow cytometry.

Figure 4. Inhibition of autophagy enhanced the radiosensitivity of MDA-MB-231-2A cells. (A) Left part: MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. Right part: MDA-MB-231-2A cells were recovered for 18 h after irradiation and treated with bafilomycin A₁ for 6 h. Then, the cells were washed with PBS, and clonogenic survival assays were performed. ** indicates significant differences (P < 0.01) between the control and irradiated cells. ## indicates significant differences (P < 0.01) between the inhibitor-treated and untreated cells. (B) MDA-MB-231-2A cells were transfected with ATG5 or non-targeting siRNAs for 48 h before exposure to 4-Gy radiation. Then, clonogenic survival assays were performed. ** indicates significant differences (P < 0.01) between the si-Cont and irradiated si-Cont cells. ## indicates significant differences (P < 0.01) between the si-Cont and si-ATG5 cells. (C) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. Apoptotic cell death was measured by ANXA5-PI double staining 2 d after irradiation. ANXA5+ and PI-, as well as ANXA5+ and PI+ cells were quantified in (D). ## indicates significant differences (P < 0.01) between the inhibitor-treated and untreated cells. n.s. indicates no significant differences between the control and irradiated cells. (E) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation. Cell viability was measured by the MTT assay 2 d after irradiation. ** indicates significant differences (P < 0.01) between the control and irradiated cells. ## indicates significant differences (P < 0.01) between the inhibitor-treated and untreated cells.

Figure 5. CSF2 contributed to the radiation-induced bystander effects by promoting the invasion and migration of MDA-MB-231 cells. (A) MDA-MB-231-2A cells were pretreated with or without 3-MA before exposure to 6-Gy radiation, and the levels of CSF2 in 2A-CM were measured by ELISA. (B and C), MDA-MB-231 cells were treated with serum-free, 10% serum, 2A-CM, or 2A-CM from MDA-MB-231-2A cells pretreated with 3-MA before exposure to irradiation. In (D and E), CSF2-neutralizing antibody (5 µg/mL) was added to the 2A-CM for 1 h and then incubated with MDA-MB-231 cells. The invasion and migration of MDA-MB-231 cancer cells were measured using a Boyden chamber and a wound-healing assay, respectively. The numbers of the invaded and migrated cells were quantified. In (B and C), ** indicates significant differences (P < 0.01) between the control (10% serum) and CM-treated cells. ## indicates significant differences (P < 0.01) between the CM-treated and CM (3-MA)-treated cells. In (D and E), ** indicates significant differences (P < 0.01) between the control (10% serum) and CM-treated cells. ## indicates significant differences (P < 0.01) between the CSF2 antibody-treated and untreated cells.

Figure 6. CSF2 contributed to the radiation-induced bystander effects via the JAK2-STAT3 and AKT pathways. (A) MDA-MB-231 cells were treated with 2A-CM for the indicated time intervals. The levels of phospho-JAK2, phospho-STAT3, and phospho-AKT were examined by western blot analysis. (B) CSF2-neutralizing antibody (5 µg/mL) was added to the 2A-CM for 1 h and then incubated with MDA-MB-231 cells. phospho-JAK2, phospho-STAT3, and phospho-AKT were examined by western blot analysis. (C–E) MDA-MB-231 cells were treated with or without the JAK2 inhibitor AZD1480 before incubation with 2A-CM. The levels of phospho-JAK2, phospho-STAT3, and phospho-AKT were examined by western blot analysis (C). The invasion (D) and migration (E) of cells were measured using Boyden chamber and wound healing assays, respectively. In (D and E), ** indicates significant differences (P < 0.01) between the control (10% serum) and CM-treated cells. ## indicates significant differences (P < 0.01) between the inhibitor-treated and untreated cells.

Figure 7. Endogenous autophagy inhibited the radiation-induced bystander effects by promoting the invasion and migration of unirradiated MDA-MB-231 cells. (A) MDA-MB-231 cells were exposed to 2A-CM for the indicated time intervals, and the MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis and quantified at each time point. ** indicates significant differences (P < 0.01) compared with the CM-treated cells at day 1. ## indicates significant differences (P < 0.01) between the control and CM-treated cells at each time points. n.s. indicates no significant differences between the control and CM-treated cells. (B) CSF2-neutralizing antibody (5 µg/mL) was added to the 2A-CM for 1 h and then incubated with MDA-MB-231 cells for 1 h. The MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis. (C) MDA-MB-231 cells were treated with or without AZD1480 before incubation with 2A-CM. The MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis. (D) Upper part: MDA-MB-231 cells were treated with rapamycin for 3 h. Lower part: MDA-MB-231 cells were exposed to 2A-CM with or without rapamycin for 4 h. Western blot analysis was performed to examine the MAP1LC3-II/MAP1LC3-I ratio. (E and F), MDA-MB-231 cells were pretreated with or without rapamycin for 3 h before exposure to 2A-CM. The invasion (E) and the migration (F) of the cells were measured using a Boyden chamber and wound healing assays, respectively. ** indicates significant differences (P < 0.01) between the control (10% serum) and CM-treated cells. ## indicates significant differences (P < 0.01) between the rapamycin-treated and untreated cells.

Figure 8. Endogenous autophagy inhibited the radiation-induced bystander effects by promoting the invasion and migration of HUVECs. (A) Observation window of the fertilized chicken eggs was prepared (a) and incubated with control medium (b), 2A-CM (c) or CM from MDA-MB-231-2A cells pretreated with 3-MA before irradiation (d). Microvessels were observed under a dissecting scope (arrowhead). The method for counting the numbers of microvessels was described in Materials and Methods. Scale bars: (a) 5 mm; (b–d) 2 mm. (B) HUVECs were incubated with CM from MDA-MB-231-2A cells pretreated with or without 3-MA before irradiation. The effects on cell invasion and migration were examined using a Boyden chamber and wound healing assays, respectively. ** indicates significant differences (P < 0.01) between the control (10% serum) and CM-treated cells. ## indicates a significant differences (P < 0.01) between the CM- and CM (3-MA)-treated cells. (C) MDA-MB-231-2A cells were pretreated with 3-MA before irradiation. Then, the HUVECs were treated with 2A-CM. The levels of phospho-JAK2, phospho-STAT3 and phospho-AKT were examined by western blot analysis. (D) HUVECs were exposed to 2A-CM, and the MAP1LC3-II/MAP1LC3-I ratio was measured by western blot analysis and quantified at each time point. (E) HUVECs were pretreated with or without rapamycin (5 µM) for 3 h and incubated with 2A-CM. The effects on cell invasion and migration were examined using a Boyden chamber and wound healing assays, respectively. ** indicates significant differences (P < 0.01) between the control (10% serum) and CM-treated cells. ## indicates significant differences (P < 0.01) between the rapamycin-treated and untreated cells.

Figure 9. A hypothetical model for the role of autophagy in radiation-induced senescence and bystander effects. Radiation induces autophagy and senescence in PTTG1-depleted breast cancer cells (MDA-MB-231-2A and MCF-7). Inhibition of autophagy by 3-MA and bafilomycin A₁ blocks radiation-induced senescence. In contrast, induction of autophagy by rapamycin induces senescence. Conditioned medium (CM) from radiation-induced senescent MDA-MB-231-2A and MCF-7 cells promote the invasion and migration of unirradiated neighboring cancer (MDA-MB-231) and normal endothelial (HUVEC) cells (bystander effects). CSF2 in the CM from the radiation-induced senescent cancer cells acts as senescence-associated secretory phenotype (SASP) to exert the bystander effects. CSF2 promotes cell invasion and migration through either the activation of the JAK2-STAT3 and JAK-AKT pathways or the downregulation of endogenous autophagy in bystander cells.