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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

Author information

Wang F#1,2, Xia X#1,3, Yang C#4, Shen J1, Mai J1, Kim HC1, Kirui D1, Kang Y5, Fleming JB5, Koay EJ6, Mitra S4, Ferrari M1,7, Shen H8,9,10.
1
Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas.
2
Department of Gastroenterology, The Tenth People's Hospital of Shanghai, Tongji University, Shanghai, China.
3
Department of Experimental Medicine, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China.
4
Department of Radiation Oncology, Houston Methodist Research Institute, Houston, Texas.
5
Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
6
Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
7
Department of Medicine, Weill Cornell Medical College, New York, New York.
8
Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas. hshen@houstonmethodist.org.
9
Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York.
10
Houston Methodist Cancer Center, Houston, Texas.
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Contributed equally