SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.
SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.
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DOI：
10.1186/1471-2407-14-181
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发表时间：
2014-03-14
期刊：
影响因子：
3.8
通讯作者：
Cheng KH
中科院分区：
文献类型：
--
作者：
Chen YW;Hsiao PJ;Weng CC;Kuo KK;Kuo TL;Wu DC;Hung WC;Cheng KH
SMAD 4是一种胃肠道恶性肿瘤特异性肿瘤抑制基因，在三分之一的结直肠癌标本和一半的胰腺肿瘤中发现突变。SMAD 4基因缺失或基因内突变导致的失活主要发生在胰腺导管腺癌（PDAC）的晚期。各种研究已经提出了在人类恶性肿瘤中潜在的SMAD 4介导的抗肿瘤作用;然而，SMAD 4在PDAC分子表型中的相关性尚未完全表征。使用AsPC-1、CFPAC-1和PANC-1人PDAC细胞系。通过蛋白质印迹、荧光素酶报告基因和免疫荧光测定证实PDAC细胞中SMAD 4表达的恢复或敲低。使用SMAD 4过表达或敲低的PDAC细胞进行体外细胞增殖、异种移植、伤口愈合、定量逆转录酶-聚合酶链反应（qRT-PCR）、蛋白质印迹和免疫组织化学分析。在这里，我们报告SMAD 4在SMAD 4-null PDAC细胞中的重新表达不影响体外或体内肿瘤细胞生长，但显著增强体外细胞迁移。SMAD 4恢复转录激活TGF-β1/Nestin通路并诱导几种转录因子的表达。相比之下，PDAC中SMAD 4缺失导致E-钙粘蛋白、血管内皮生长因子（VEGF）、表皮生长因子受体（EGFR）和CD 133的表达增加。此外，SMAD 4缺失导致多个激酶途径（特别是磷酸化ERK/p38/Akt途径）的改变，并增加体外化学抗性。最后，具有完整SMAD 4的PDAC细胞对TGF-β1抑制剂处理更敏感，从而减少细胞迁移;缺乏SMAD 4的PDAC细胞显示出对EGFR抑制剂处理的响应降低的细胞运动性。本研究揭示了PDAC中SMAD 4依赖性差异的分子基础，旨在确定可能对靶向TGF-β或EGFR信号通路的治疗有反应的患者亚组，并确定SMAD 4缺陷PDAC患者的潜在治疗干预。
SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized. The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down. Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment. This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.
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