Yang Jian, Zhang Xu, Zhang Yi, Zhu Dongming, Zhang Lifeng, Li Ye, Zhu Yanbo, Li Dechun, Zhou Jian
Department of General Surgery, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, China.
Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China.
J Exp Clin Cancer Res. 2016 Feb 3;35:26. doi: 10.1186/s13046-016-0298-y.
Epithelial-mesenchymal transition (EMT) is a dedifferentiation process that mainly involves in mesenchymal marker upregulation, epithelial maker downregulation and cell polarity loss. Related hypoxia factors play a crucial role in EMT, however, it remains few evidence to clarify the role of HIF-2α in EMT in pancreatic cancer.
In this study, we investigated the expression of HIF-2α and E-cadherin by immunohistochemistry in 70 pancreatic cancer patients, as well as the correlation to the clinicopathologic characteristics. Then we regulated the expression of HIF-2α in pancreatic cancer cells to examine the role of HIF-2α on invasion and migration in vitro. Finally, we tested the relation of HIF-2α and EMT related proteins by Western blot and determined whether HIF-2α regulated EMT through Twist regulating the expression of E-cadherin by Chromatin immunoprecipitation (ChIP) assay.
We found that HIF-2α protein was expressed positively in 67.1% (47/70) of pancreatic cancer tissues and 11.4% (8/70) of adjacent non-tumor pancreatic tissues, and there was a significant difference in the positive rate of HIF-2α protein between two groups (χ2 = 45.549, P < 0.05). In addition, the staining for HIF-2α was correlated with tumor differentiation (P < 0.05), clinical stage (P < 0.05) and lymph node metastasis (P < 0.05), while E-cadherin expression was only correlated with lymph node metastasis (P < 0.05). HIF-2α promoted cell migration, invasion in vitro, and regulated the expression of E-cadherin and MMPs, which are critical to EMT. Our further ChIP assay suggested that only Twist2 could bind to the promoter of E-cadherin in -714 bp region site, but there is no positive binding capacity in -295 bp promoter region site of E-cadherin. Clinical tissues IHC staining showed that Twist2 and E-cadherin expression had an obviously negative correlation in pancreatic cancer. Nevertheless, it had no obvious correlation between Twist1 and E-cadherin.
These findings indicated that HIF-2α promotes EMT in pancreatic cancer by regulating Twist2 binding to the promoter of E-cadherin, which meant that HIF-2α and this pathway may be effective therapeutic targets for pancreatic cancer.
上皮-间质转化（EMT）是一种去分化过程，主要涉及间充质标志物上调、上皮标志物下调和细胞极性丧失。相关的缺氧因子在EMT中起关键作用，然而，关于缺氧诱导因子-2α（HIF-2α）在胰腺癌EMT中的作用仍缺乏证据。
在本研究中，我们通过免疫组织化学检测了70例胰腺癌患者中HIF-2α和E-钙黏蛋白的表达，并分析其与临床病理特征的相关性。然后我们调节胰腺癌细胞中HIF-2α的表达，以研究HIF-2α在体外对侵袭和迁移的作用。最后，我们通过蛋白质免疫印迹法检测HIF-2α与EMT相关蛋白的关系，并通过染色质免疫沉淀（ChIP）试验确定HIF-2α是否通过Twist调节E-钙黏蛋白的表达来调控EMT。
我们发现HIF-2α蛋白在67.1%（47/70）的胰腺癌组织中呈阳性表达，在11.4%（8/70）的癌旁非肿瘤胰腺组织中呈阳性表达，两组之间HIF-2α蛋白阳性率有显著差异（χ2 = 45.549，P < 0.05）。此外，HIF-2α染色与肿瘤分化（P < 0.05）、临床分期（P < 0.05）和淋巴结转移（P < 0.05）相关，而E-钙黏蛋白表达仅与淋巴结转移相关（P < 0.05）。HIF-2α促进体外细胞迁移、侵袭，并调节对EMT至关重要的E-钙黏蛋白和基质金属蛋白酶的表达。我们进一步的ChIP试验表明，只有Twist2能在E-钙黏蛋白启动子-714 bp区域位点与之结合，但在E-钙黏蛋白启动子-295 bp区域位点没有阳性结合能力。临床组织免疫组化染色显示，胰腺癌中Twist2和E-钙黏蛋白表达呈明显负相关。然而，Twist1与E-钙黏蛋白之间无明显相关性。
这些发现表明，HIF-2α通过调节Twist2与E-钙黏蛋白启动子的结合促进胰腺癌中的EMT，这意味着HIF-2α及其相关通路可能是胰腺癌有效的治疗靶点。
