SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy.
SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy.
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SMAD4 基因突变通过促进自噬使胰腺癌对放疗产生抵抗力
DOI：
10.1158/1078-0432.ccr-17-3435
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发表时间：
2018-07-01
期刊：
影响因子：
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通讯作者：
Shen H
中科院分区：
文献类型：
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作者：
Wang F;Xia X;Yang C;Shen J;Mai J;Kim HC;Kirui D;Kang Y;Fleming JB;Koay EJ;Mitra S;Ferrari M;Shen H
目的：了解PDAC的放射抵抗机制有助于制定改善PDAC治疗反应的策略。SMAD 4基因在胰腺癌中经常发生突变。在这项研究中，我们研究了SMAD 4缺陷在胰腺癌细胞对放射治疗反应中的作用。实验设计：我们用SMAD 4 siRNA或SMAD 4 shRNA下调SMAD 4的表达，并在SMAD 4突变的胰腺癌细胞中过表达SMAD 4，然后通过克隆形成存活试验来评估它们对细胞辐射抗性的影响。为了研究辐射抗性的机制，SMAD 4损失对活性氧簇（ROS）和自噬的影响分别通过流式细胞术和免疫印迹分析来确定。此外，我们在SMAD 4耗尽的胰腺癌细胞中用自噬抑制剂（氯喹）和ROS抑制剂（N-乙酰基-L-半胱氨酸）处理后，通过克隆形成存活测定来测量辐射抗性。最后，SMAD 4对辐射抗性的作用也在源自SMAD 4耗尽的Panc-1细胞的原位肿瘤模型中得到证实。结果：SMAD 4缺失的胰腺癌细胞对放射治疗具有更强的抵抗性。野生型SMAD 4在SMAD 4突变细胞中的过表达挽救了它们的放射敏感性。SMAD 4耗竭介导的辐射抗性与持续较高水平的ROS和辐射诱导的自噬相关。最后，SMAD 4耗尽在Panc-1衍生的原位肿瘤模型中诱导体内放射抗性（P = 0.038）。更有趣的是，我们观察到SMAD 4的蛋白水平与原位肿瘤组织样品中的自噬呈负相关。结论：我们的研究结果表明，SMAD 4缺陷是负责通过诱导ROS和辐射诱导的自噬水平增加在胰腺癌的放射抗性。临床癌症研究; 24（13）; 3176-85。©2018 AACR.
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.