HIF-2α promotes epithelial-mesenchymal transition through regulating Twist2 binding to the promoter of E-cadherin in pancreatic cancer

Abstract

Background: 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.

Method: 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.

Results: 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.

Conclusion: 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.

Fig. 1

Expression of HIF-2α and E-cadherin in pancreatic cancer and adjacent non-tumor tissues. Original magnification × 100 or 400. Images are representative of three independent experiments

Fig. 2

Expression of HIF-2α in pancreatic cell lines and regulating HIF-2α in AsPC-1 and SW1990 cells. a Relative expression of HIF-2α protein in pancreatic cancer cell lines (BXPC-3, CaPan-2, PaTu8988, SW1990 and AsPC-1) was measured by Western blot. b Expression of ectopic expression and knockdown of HIF-2α in SW1990 or AsPC-1 cells by Western blot. Images are representative of three independent experiments. *P < 0.05

Fig. 3

HIF-2α promoted cell migration and invasion. a Cell invasion was detected by the Transwell assay. Representative images of cell invasion captured under an inverted microscope (original magnifiation, ×200). The data represent the means ± SD of 5 experiments. **P < 0.01. b Cell migration was detected by the wound scrape assay. Representative images of cell migration in the wound scrape model at 0, 24 h and 48 h are shown; original magnifiation, ×100. The data represent the means ± SD of three experiments. *P < 0.05

Fig. 4

Effect of HIF-2α on expression of EMT-related proteins. E-cadherin, β-catenin, MMP2 and MMP9 was examined in AsPC-1 cells silencing of HIF-2α expression and SW1990 cells transfected with HIF-2α cDNA by Western blot. Images are representative of three independent experiments. *P < 0.05

Fig. 5

HIF-2α promotes EMT through Twist binding to the promoter region of E-cadherin. a Twist1 and Twist2 expressions in response to up-regulation or silencing of HIF-2α in pancreatic cancer cells were detected by Western blot. b ChIP of Twist1 and Twist2 interactions with the E-cadherin promoter. Bands are PCR products targeting P1 and P2 of the E-cadherin promoter. The specific anti-Twist1, anti-Twist2 or control normal mouse IgG was used for immunoprecipitations, whereas genomic DNA was used as the input control

Fig. 6

Representative immunohistochemical staining of Twist1 and Twist2 in the two groups of pancreatic cancer tissues with E-cadherin positive or E-cadherin negative expression. Original magnification × 200. Images are representative of three independent experiments