SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy

Abstract

Purpose: Understanding the mechanism of radioresistance could help develop strategies to improve therapeutic response of patients with PDAC. The SMAD4 gene is frequently mutated in pancreatic cancer. In this study, we investigated the role of SMAD4 deficiency in pancreatic cancer cells' response to radiotherapy.Experimental Design: We downregulated SMAD4 expression with SMAD4 siRNA or SMAD4 shRNA and overexpressed SMAD4 in SMAD4 mutant pancreatic cancer cells followed by clonogenic survival assay to evaluate their effects on cell radioresistance. To study the mechanism of radioresistance, the effects of SMAD4 loss on reactive oxygen species (ROS) and autophagy were determined by flow cytometry and immunoblot analysis, respectively. Furthermore, we measured radioresistance by clonogenic survival assay after treatment with autophagy inhibitor (Chloroquine) and ROS inhibitor (N-acetyl-l-cysteine) in SMAD4-depleted pancreatic cancer cells. Finally, the effects of SMAD4 on radioresistance were also confirmed in an orthotopic tumor model derived from SMAD4-depleted Panc-1 cells.Results:SMAD4-depleted pancreatic cancer cells were more resistant to radiotherapy based on clonogenic survival assay. Overexpression of wild-type SMAD4 in SMAD4-mutant cells rescued their radiosensitivity. Radioresistance mediated by SMAD4 depletion was associated with persistently higher levels of ROS and radiation-induced autophagy. Finally, SMAD4 depletion induced in vivo radioresistance in Panc-1-derived orthotopic tumor model (P = 0.038). More interestingly, we observed that the protein level of SMAD4 is inversely correlated with autophagy in orthotopic tumor tissue samples.Conclusions: Our results demonstrate that defective SMAD4 is responsible for radioresistance in pancreatic cancer through induction of ROS and increased level of radiation-induced autophagy. Clin Cancer Res; 24(13); 3176-85. ©2018 AACR.

Figure 1.. SMAD4 knockdown resulted in irradiation resistance in PDAC cells.

(A-C) Panc-1 cells were transfected with a control shRNA retroviral vector or two independent shRNA retroviral vector against SMAD4 (A), Panc-1 cells were transfected with scrambled siRNA or four SMAD4-specific siRNA for 72h (B), Panc-1, MIA PaCa-2 cells were transfected with scrambled siRNA or pool of SMAD4-specific siRNA for 72h (C), and total cell lysates were harvested followed by immunoblotting with the indicated antibodies. (D-F) Panc-1 cells were transfected with a control shRNA retroviral vector or two independent shRNA retroviral vector targeting SMAD4 (D) or pool of SMAD4-specific siRNA (E), MIA PaCa-2 cells were transfected with scrambled siRNA or SMAD4-specific siRNA pool (F), and then treated with indicated doses of IR. Colony formation assays were conducted and more than 50 cells were counted. (G) Colony formation assays were conducted in two SMAD4 mutated cell lines (Bxpc-3, Capan-1) and two SMAD4 WT cell lines (Panc-1, MIA PaCa-2). The differences between SMAD4 WT and mutated cells selected to be compared were detected using repeated measures ANOVA. * , P < 0.05. (H) Bxpc-3 cells were transfected with Flag-SMAD4 plasmid or empty vector, followed by colony formation assays. Shown are the averages of triplicate samples. Standard errors are shown by error bars. The differences in the two groups selected to be compared were detected using repeated measures ANOVA. * , P < 0.05. (si-SMAD4-1=si-SMAD4-a+si-SMAD4-b; si-SMAD4-2=si-SMAD4-c+si-SMAD4-d)

Figure 2.. SMAD4 depletion impaired DNA Double-Strand Break Signaling in Panc-1 cells in response to ionizing radiation.

(A) Panc-1 cells were transfected with scrambled siRNA, or four independent SMAD4-specific siRNAs for 72 h, and then total cell lysates were harvested followed by immunoblotting with the indicated antibodies. (B, C) Panc-1 cells were transfected with scrambled siRNA or pool of SMAD4-specific siRNAs and then exposed to IR (6 Gy). Laser confocal microscopy images of pancreatic cancer cell lines labeled with fluorescent antibodies to γ-H2AX (red channel), SMAD4 (green channel) and DAPI (blue channel) at 2 h post irradiation or mock treatment. Focal g-H2AX staining signals were quantified in cells. (C) The typical staining of γ-H2AX in Panc-1 cells before and after the treatment with irradiation. The average data point was calculated from three independent experiments. At least 1000 cells were counted for each cell line. The differences in those two groups were detected using t-test. **, P <0.01.

Figure 3.. SMAD4 depletion induced high level of autophagy in response to ionizing radiation.

(A-C) Panc-1 and MIA PaCa-2 were transfected with scrambled siRNA or SMAD4-specific siRNA for 48 h and then were treated with or without IR (6 Gy). Cell lysates were harvested at the indicated time points after IR. (A) Immunoblot of LC3 isoforms were measured using antibody against LC3. Quantification of the LC3-II/LC3-I ratio in Panc-1 cells (B) and MIA PaCa-2 cells (C). The protein level of the β-actin was used as the internal standard. Shown are the averages of triplicate experiments. (D) Representative confocal images of endogenous LC3 staining in Panc-1 transfected with scramble or SMAD4-specific siRNA for 48 h, and treated with or without IR (6 Gy). Cells were stained with antibody to LC3 (red) and DAPI (blue). Scale bars, 10 μm. (E) Quantification of LC3 puncta in Panc-1 cell lines.

Figure 4.. SMAD4 depletion increased reactive oxygen species generation in PDAC cells.

(A, B) Panc-1 and MIA PaCa-2 cells were transfected with scrambled siRNA or SMAD4-specific siRNA for 48 h and then treated with IR (6 Gy). SMAD4 depletion was confirmed by Western blot. (C, D) Cells were then incubated with ROS indicator (CM-H2DCFDA), and fluorescence intensity was assessed by flow cytometry at indicated time points. Labeled tert-butyl hydrogen peroxide (TBHP, 50μM) serve as a positive control. The differences in those two groups were detected using t-test. * , P < 0.05, **, P < 0.01.

Figure 5.. SMAD4-induced IR-resistance is partially rescued by pretreatment of autophagy inhibitor or ROS scavenger.

(A) Panc-1 cells were transfected with scrambled siRNA or SMAD4-specific siRNA for 72h. SMAD4 depletion was confirmed by Western blot. (B, C) Cells were pretreated with 10μM chloroquine (CQ) (B) or 3mM N-acetyl-L-cysteine (NAC) (C) for 1 h, and then treated with indicated doses of IR. Colony formation assays were conducted and more than 50 cells counted. Shown are the averages of triplicate samples. Standard errors are shown by error bars. The differences between the two groups selected to be compared were detected using repeated measures ANOVA. *, P < 0.05.

Figure 6.. SMAD4 depletion induced radio-resistance in an orthotopic pancreatic cancer mouse model.

(A) Schemes for the establishment and treatment of the pancreatic cancer orthotopic model. (B, C) Nude mice were injected with 1×106 Panc-1 cells infected with control retroviral vector, or shSMAD4 vector with 5% Matrigel into the pancreas. After 21 days, tumors were irradiated thrice with 4-Gy dose of IR every other day. The mice were sacrificed at day 42, and the tumors were isolated, weighed, and compared between the groups using ANOVA. (D) Immunohistochemistry (IHC) was performed on serial sections using antibodies against SMAD4, LC3 and g-H2AX in tumor tissues.