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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.

SUBMITTER: Wang F

PROVIDER: S-EPMC6345154 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Wang Feng F Xia Xiaojun X Yang Chunying C Shen Jianliang J Mai Junhua J Kim Han-Cheon HC Kirui Dickson D Kang Ya'an Y Fleming Jason B JB Koay Eugene J EJ Mitra Sankar S Ferrari Mauro M Shen Haifa H

Clinical cancer research : an official journal of the American Association for Cancer Research 20180330 13

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 ...[more]

PMID: 29602802

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Project description:Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide and effective therapy remains a challenge. IFIT3 is an interferon-stimulated gene with antiviral and pro-inflammatory functions. Our previous work has shown that high expression of IFIT3 is correlated with poor survival in PDAC patients who receive chemotherapy suggesting a link between IFIT3 and chemotherapy resistance in PDAC. However, the exact role and molecular mechanism of IFIT3 in chemotherapy resistance in PDAC has been unclear. Methods: A group of transcriptome datasets were downloaded and analyzed for the characterization of IFIT3 in PDAC. Highly metastatic PDAC cell line L3.6pl and patient-derived primary cell TBO368 were used and IFIT3 knockdown and the corresponding knockin cells were established for in vitro studies. Chemotherapy-induced apoptosis, ROS production, confocal immunofluorescence, subcellular fractionation, chromatin-immunoprecipitation, co-immunoprecipitation and mass spectrometry analysis were determined to further explore the biological role of IFIT3 in chemotherapy resistance of PDAC. Results: Based on PDAC transcriptome data, we show that IFIT3 expression is associated with the squamous molecular subtype of PDAC and an increase in inflammatory response and apoptosis pathways. We further identify a crucial role for IFIT3 in the regulation of mitochondria-associated apoptosis during chemotherapy. Knockdown of IFIT3 attenuates the chemotherapy resistance of PDAC cells to gemcitabine, paclitaxel, and FOLFIRINOX regimen treatments, independent of individual chemotherapy regimens. While IFIT3 overexpression was found to promote drug resistance. Co-immunoprecipitation identified a direct interaction between IFIT3 and the mitochondrial channel protein VDAC2, an important regulator of mitochondria-associated apoptosis. It was subsequently found that IFIT3 regulates the post-translational modification-O-GlcNAcylation of VDAC2 by stabilizing the interaction of VDAC2 with O-GlcNAc transferase. Increased O-GlcNAcylation of VDAC2 protected PDAC cells from chemotherapy induced apoptosis. Conclusions: These results effectively demonstrate a central mechanism by which IFIT3 expression can affect chemotherapy resistance in PDAC. Targeting IFIT3/VDAC2 may represent a novel strategy to sensitize aggressive forms of pancreatic cancer to conventional chemotherapy regimens.

Project description:Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies. Given that macroautophagy/autophagy activation is prevalent in PDAC, the dual roles of autophagy could be involved in PDAC heterogeneity. In this work, we demonstrated that TGFB1 induced autophagic flux through SMAD4-dependent or SMAD4-independent pathways based on a distinct genetic context. In SMAD4-positive PDAC cells, TGFB1-induced autophagy promoted proliferation and inhibited migration by decreasing the nuclear translocation of SMAD4. Conversely, TGFB1-induced autophagy inhibited proliferation and promoted migration in SMAD4-negative cells through the regulation of MAPK/ERK activation. TGFB1 expression also positively correlated with LC3B expression in PDAC specimens. A high level of LC3B was associated with unfavorable overall survival (OS) and disease-free survival (DFS) in SMAD4-negative PDAC patients, although LC3B could not predict OS and DFS for the 110 PDAC patients. Thus, TGFB1-induced autophagy contributed to the different patterns of PDAC progression. This knowledge can aid in improving our understanding of the molecular classification of PDAC and might guide the development of therapeutic strategies for PDAC, especially for SMAD4-negative PDAC.Abbreviations: CDH1: cadherin 1; CDH2: cadherin 2; CI: combination index; CQ: chloroquine; DFS: disease-free survival; EMT: epithelial-to-mesenchymal transition; ERK: extracellular signal-regulated protein kinase; GFP: green fluorescent protein; IHC: immunohistochemistry; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; OS: overall survival; PBS: phosphate-buffered saline; PDAC: pancreatic ductal adenocarcinoma; RAP: rapamycin; RFP: red fluorescent protein; RT: room temperature; shRNA: short-hairpin RNA; SQSTM1: sequestosome 1; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; TGFB1: transforming growth factor beta 1; TMA: tissue microarray.

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

Publications

<i>SMAD4</i> Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy.

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