HIF-2α promotes the formation of vasculogenic mimicry in pancreatic cancer by regulating the binding of Twist1 to the VE-cadherin promoter

Abstract

Vasculogenic mimicry (VM) is a blood supply modality that occurs independently of endothelial cell angiogenesis. Hypoxia and the epithelial-mesenchymal transition (EMT) induce VM formation by remodeling the extracellular matrix. Our previous study demonstrated that hypoxia-inducible factor-2 alpha (HIF-2α) promotes the progress of EMT in pancreatic cancer; however, whether HIF-2α promotes VM formation in pancreatic cancer remains unknown. In this study, we investigated HIF-2α expression and VM by immunohistochemistry in 70 pancreatic cancer patients as well as the role of Twist1and Twist2 in HIF-2α-induced VM in vitro and in vivo. We found that the overexpression of HIF-2α and VM were correlated with poor tumor differentiation, late clinical stage and lymph node metastasis, and a poor prognosis in pancreatic cancer. Moreover, the upregulation of HIF-2α in SW1990 cells induced VM formation, whereas the opposite results were found after silencing HIF-2α in AsPC-1 cells. A mechanistic study indicated that HIF-2α might regulate the binding of twist1 to vascular endothelial cadherin (VE-cadherin) to promote VM formation in pancreatic cancer cells, and that the P1 (-421bp) and P4 (-2110bp) regions of the Twist1 binding sequences are positive regulatory elements for VE-cadherin. In addition, we confirmed that the overexpression of HIF-2α increased Twist1 expression and promoted tumor growth and VM formation in pancreatic cancer xenografts in nude mice. These findings indicated that HIF-2α might play a critical role in VM and that HIF-2α and the pathway of HIF-2α inducing VM formation are potential therapeutic targets for pancreatic cancer.

Keywords: HIF-2α; Twist; VE-cadherin; pancreatic cancer; vasculogenic mimicry (VM).

Figure 1

(A) Expression of HIF-2α (by IHC). (B) VM structure by CD34/PAS double-staining: the yellow arrow indicates endothelial-dependent vessels that are CD34-positive and PAS-positive; the red arrow indicates VM structures that wereCD34-negative and PAS-positive in pancreatic cancer and adjacent non-tumor tissues. Original magnification100× or 400×. The images are representative of three independent experiments. NT: normal tissue; PC: pancreatic cancer.

Figure 2. Correlation of HIF-2α, VE-cadherin, and VM in tissues, and the HIF-2α and VM expression survival curves in pancreatic cancer patients

(A) Representative immunohistochemical staining of HIF-2α and VE-cadherin in the two groups of pancreatic cancer tissues exhibiting either VM-positive or VM-negative expression. Original magnification100×. The images are representative of three independent experiments. (B) The survival ratesof70 pancreatic cancer patients that were positive and negative for HIF-2α and VM expression (log-rank test, P<0.001).

Figure 3. Expression of HIF-2α in pancreatic cell lines and regulation of HIF-2α in AsPC-1 and SW1990 cells

(A) The relative expression of HIF-2α in pancreatic cancer cell lines (SW1990, AsPC-1, CaPan-2, PaTu8988, BXPC-3, and CFPANC-1) was measured by Western blot. (B) Knockdown and ectopic expression of HIF-2α in AsPC-1 and SW1990 cells by Western blot. The images are representative of three independent experiments. * = P<0.05.

Figure 4. HIF-2α promotes cell migration, invasion, and VM formation in vitro

(A) Cell invasion was detected using a Transwell® assay. Representative images of cell invasion captured under an inverted microscope (original magnification, 200×). The data represent the means ± SD of five experiments. ** = P<0.01. (B) Cell migration was detected using a wound scrape assay. Representative images of cell migration in the wound scrape model at 0, 24, and 48 h are shown; original magnification, 100×. The data represent the means ± SD of three experiments. * = P<0.05. (C) Changes in VM formation in three-dimensional culture in vitro. Representative images captured under an inverted microscope (original magnification, 200×). (D) The effect of HIF-2α on expression of VM-related proteins (VE-cadherin, Twist1, Twist2, MMP2, and MMP9) was examined by Western blot. The images are representative of three independent experiments. * = P<0.05

Figure 5. The transcriptional factor of Twist1 binding to the promoter region of VE-cadherin

(A) ChIP assay of Twist1 and Twist2 interactions with the VE-cadherin promoter. Bands are PCR products targeting P1-P4 of the VE-cadherin promoter. Specific anti-Twist1, anti-Twist2, or control normal mouse IgG was used for immunoprecipitations, whereas genomic DNA was used as the input control. (B) The effect of Twist1 on the transcriptional activity of the VE-cadherin promoter. Mutated promoters that specifically delete P1 or P4 of the Twist1 binding elements were introduced into AsPC-1 cells. Firefly luciferase expression levels were normalized to the luciferase activity of the internal Renilla control. The results indicate that both P1 and P4 elements are positive regulatory elements. Separate experiments performed in triplicate for each group. One-way analysis of variance (ANOVA) was used, * = P<0.05.

Figure 6. The effect of Twist1 in the progress of HIF-2α promoting cell migration, invasion, and VM formation in vitro

(A) Expression of Twist1 after regulating HIF-2α or Twist1 by Western blot. The images are representative of three independent experiments. * = P<0.05. (B) Cell invasion was detected using a Transwell® assay. Representative images of cell invasion captured under an inverted microscope (original magnification, 200×). The data represent the means ± SD of five experiments. ** = P<0.01. (C) Cell migration was detected using a wound scrape assay. Representative images of cell migration in the wound scrape model at 0, 24 h are shown; original magnification, 100×. The data represent the means ± SD of three experiments. * = P<0.05. (D) Change in VM formation after regulating HIF-2α or Twist1 by three-dimensional cell culture in vitro. The images are representative of three independent experiments (original magnification, 200×). * = P<0.05.

Figure 7. HIF-2α promotes VM formation in vivo

(A) Size and tumor volume of the pancreatic cancer xenografts harvested two weeks after orthotopic inoculation. * = P<0.05. (B) Representative CD34/PAS staining (Red arrows indicate typical VM structures, whereas yellow arrows indicate endothelial vessels). Images of Twist1 and VE-cadherin expression in tumor sections from pancreatic cancer xenografts with silencing or overexpression of HIF-2α and their corresponding controls. Original magnification100× or 400×. The images are representative of three independent experiments