American Association for Cancer Research
10780432ccr173435-sup-192574_3_supp_4629274_p60tdp.pdf (3.78 MB)

Figure S2 from SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy

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journal contribution
posted on 2023-03-31, 20:46 authored by Feng Wang, Xiaojun Xia, Chunying Yang, Jianliang Shen, Junhua Mai, Han-Cheon Kim, Dickson Kirui, Ya'an Kang, Jason B. Fleming, Eugene J. Koay, Sankar Mitra, Mauro Ferrari, Haifa Shen

Figure S2. SMAD4 depletion induced high level of autophagy in response to ionizing radiation in Panc-1 cells using shRNA lentiviral vector. Immunoblot of LC3 isoforms were measured using antibody against LC3 in Panc-1-CTL, Panc-1-sh-SMAD4-#2 Cells. (A) The protein level of the β-actin was used as the internal standard.(B) Representative confocal images of endogenous LC3 staining in Panc-1 transfected with control shRNA lentiviral or shRNA lentiviral vector against SMAD4, and treated with or without IR (6 Gy). Cells were stained with antibody to LC3 (red) and DAPI (blue).


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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.

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