Zinc finger E-box binding homeobox 1 promotes vasculogenic mimicry in colorectal cancer through induction of epithelial-to-mesenchymal transition

Abstract

Our previous studies have shown that epithelial-mesenchymal transition (EMT) may be involved in the vasculogenic mimicry (VM) formation in hepatocellular carcinoma. Here, we hypothesize that zinc finger E-box binding homeobox 1 (ZEB1) promotes VM formation in colorectal carcinoma (CRC) by inducing EMT. We identified VM in 39 (19.2%) out of 203 CRC patients. The presence of VM was associated with aggressive biological behavior and was an unfavorable prognostic indicator. By immunohistochemical analysis, we found that the VM-positive CRC samples showed increased ZEB1 expression compared with the VM-negative samples and the ZEB1 expression occurred concomitantly with features of EMT. In vitro, knockdown of ZEB1 in poorly differentiated HCT116 CRC cells destroyed the vessel-like structures in the 3-D culture, a property associated with VM formation. Knockdown of ZEB1 resulted in restoration of epithelial phenotypes and significantly inhibited the ability to migrate and invade. In addition, ZEB1 underexpression decreased the expression of vascular endothelial (VE)-cadherin and Flk-1, which are characteristics of endothelial cells. Taken together, our results suggest that ZEB1 can promote VM formation by inducing EMT in CRC and might represent an important target in CRC.

Figure 1

(A) Vasculogenic mimicry (VM) channel (black arrow) lined with tumor cells and containing red blood cells (HE staining, ×400). (B) Endothelial‐dependent vessel lined with shuttle‐like endothelium cells (red arrow) (HE staining, ×400). (C) The VM channel formed by tumor cells was negative for CD34 and the base membrane‐like structure between red blood cells and tumor cells was positive for periodic acid Schiff (PAS) (black arrow) (CD34/PAS double‐staining, ×400). (D) The endothelial‐dependent vessel was both positive for CD34 and PAS (red arrows) (CD34/PAS double‐staining, ×400). (E) Kaplan–Meier survival analysis showing that the VM‐positive patients have shorter survival time than VM‐negative patients. (F) Kaplan–Meier survival analysis showing the zinc finger E‐box binding homeobox 1 (ZEB1)‐positive patients have shorter survival time than ZEB1‐negative patients.

Figure 2

Expression of zinc finger E‐box binding homeobox 1 (ZEB1) and epithelial–mesenchymal transition (EMT) markers in colorectal carcinoma (CRC) samples. (A) The ZEB1 expression (black arrows) in the vasculogenic mimicry (VM)‐positive group was higher than in the VM‐negative group. In contrast, except for some stromal cells (red arrows), tumor cells in the VM‐negative group showed almost no ZEB1 expression (immunohistochemical staining, ×400). (B) E‐cadherin expression was lower in the ZEB1‐positive CRC tissue samples than in the ZEB1‐negative ones. Some tumor cells showed vimentin expression in ZEB1‐positive CRC samples (black arrow), whereas the tumor cells showed no vimentin expression and only some stromal cells showed vimentin expression (red arrow) in ZEB1‐negative CRC samples (IHC staining, ×400).

Figure 3

Effect of zinc finger E‐box binding homeobox 1 (ZEB1) on the vasculogenic mimicry (VM) formation of three colorectal carcinoma (CRC) cell lines in Matrigel. (A) HCT116 cells can form typical tube‐like structures in the 3‐D culture (red arrow), whereas HT‐29 and SW480 cannot. The typical tube‐like structures formed by human umbilical vein endothelial cells (HUVEC) were regarded as positive control. After ZEB1 siRNA treatment, VM formation was significantly reduced in HCT116 cells. (B) Quantitative analysis of the mean number of tube‐like structures formed by HUVEC, control HCT116 and siZEB1 HCT116 cells in 3‐D culture. (C) Western blot analysis showed that the ZEB1 knockdown in HCT116 cells results in reduced VE‐cadherin and Flk‐1 expression. No significant change in Flt‐1 expression was observed.

Figure 4

The zinc finger E‐box binding homeobox 1 (ZEB1) knockdown resulted in the restoration of epithelial phenotype in HCT116 cells. (A) The ZEB1 knockdown in HCT116 cells resulted in a morphological change from a fibroblastic to an epithelial appearance. (B) Immunofluorescence and (C) western blot analysis revealed upregulated epithelial marker (E‐cadherin, claudin‐4) and downregulated mesenchymal marker (vimentin, fibronectin) expression, respectively, after ZEB1 siRNA treatment.

Figure 5

The zinc finger E‐box binding homeobox 1 (ZEB1) knockdown in HCT116 cells resulted in reduced migratory and invasive capabilities. (A) Scratch wounds were made at 0 and 24 h after wounds (×200). (B) Quantitative measurement of wound gaps showed a larger reduction in the cellular motility of ZEB1 siRNA‐transfected HCT116 cells than control cells. (C,D) There were much fewer ZEB1 knockdown HCT116 cells that invaded through Matrigel compared to the control cells (×200).