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Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self-renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high-resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)-cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Matrix metalloproteinases (MMPs) have critical functions in tumour vasculogenic mimicry (VM). This study explored the mechanisms underlying MMP-13 and MMP-2 regulation of tumour VM formation in large cell lung cancer (LCLC). In our study, laminin5 (Ln-5) fragments cleaved by MMP-2 promoted tubular structure formation by the LCLC cell lines H460 and H661 in three-dimensional (3D) cultures. Transient up-regulation of MMP-13 or treatment with recombinant MMP-13 protein abrogated tubular structure formation of H460 cells in 3D culture. Treated cells with Ln-5 fragments cleaved by MMP-2 stimulated EGFR and F-actin expression. Ln-5 fragments cleaved by MMP-13 decreased EGFR/F-actin expression and disrupted VM formation. MMP-13 expression was negatively correlated with VM, Ln-5 and EGFR in LCLC tissues and xenograft. In vivo experiments revealed that VM was decreased when the number of endothelium-dependent vessels (EDVs) increased during xenograft tumour growth, whereas MMP-13 expression was progressively increased. In conclusion, MMP-2 promoted and MMP-13 disrupted VM formation in LCLC by cleaving Ln-5 to influence EGFR signal activation. MMP-13 may regulate VM and EDV formation., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Vasculogenic mimicry (VM) is a nonangiogenesis-dependent pathway that promotes tumor growth and disease progression. Nodal signaling has several vital roles in both embryo development and cancer progression. However, the effects of Nodal signaling on VM formation in breast cancer and its underlying mechanisms are ill-defined. We analyzed the relationship between Nodal signaling and VM formation in one hundred human breast cancer cases and the results showed that the expression of Nodal was significantly correlated with VM formation, tumor metastasis, differentiation grade, TNM stage and poor prognosis. Furthermore, up-regulation of Nodal expression promoted VM formation of breast cancer cells in vitro and in vivo. Knockdown of Nodal expression restrained VM formation. In addition, Nodal induced EMT and up-regulated the expression of Slug, Snail and c-Myc. We found that blocking the Smad2/3 pathway by administering SB431542 inhibited VM formation in breast cancer cell lines and xenografts. Taken together, Nodal signaling through the Smad2/3 pathway up-regulated Slug, Snail and c-Myc to induce EMT, thereby promoting VM formation. Our study suggests that the Nodal signaling pathway may serve as a therapeutic target to inhibit VM formation and improve prognosis in breast cancer patients.

MicroRNAs (miRNAs) play vital roles in cell proliferation, differentiation and apoptosis in hepatocellular carcinoma (HCC). miR-26b has been confirmed as an important regulator in carcinogenesis and other pathological processes. miR-26b-5p is one member of the mature miR-26 family, and its functional role in proliferation, angiogenesis and apoptosis in HCC remains unknown. Here, we demonstrate that miR-26b-5p expression was significantly decreased in HCC tissues and HCC cell lines compared with normal liver tissues and liver cells by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between miR-26b-5p and the clinical characteristics of HCC patients were further analysed, and miR-26b-5p was positively correlated with the differentiation of HCC cells. Computational searches were further used to identify the downstream targets and signalling pathways of miR-26b-5p in HCC cells. Cell viability, proliferation and tube formation abilities were assessed by scrape, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and three-dimensional culture assays to confirm that miR-26b-5p inhibited HCC cell growth and impaired the tube formation ability of the HCC cells. Both in vitro and in vivo studies showed that miR-26b-5p could suppress vascular mimicry (VM) and angiogenesis by down-regulating the expression of VE-cadherin, Snail and MMP2 and could inhibit the apoptosis of HCC cells. Using mouse models, we revealed that tumours derived from miR-26b-5p-expressing HCC cells displayed a significant decrease in microvessel density compared with those derived from control cells. Therefore, our data provide further insight into the role of miR-26b-5p as a negative regulator of proliferation, angiogenesis, and apoptosis in HCC.

Hypoxia plays a pivotal role in tumor progression. The functions of hypoxia and subsequent Bcl-2/Twist1 activation in epithelial-mesenchymal transition (EMT) and vasculogenic mimicry (VM) formation are currently unclear. This study aimed to investigate the role of Bcl-2/Twist1 cooperation in hypoxia-induced EMT and VM formation. In in vitro experiments, we found that hypoxia resulted in co-overexpression of Bcl-2 and Twist1, facilitated Twist1 nuclear translocation and promoted EMT and VM formation. Co-overexpression of Bcl-2 and Twist1 under normoxia could also induce EMT and promote VM formation. Furthermore, blocking Bcl-2 or Twist1 attenuated the effects of hypoxia on EMT progress and VM formation in hepatocellular carcinoma cells. In in vivo experiments, the mechanism by which hypoxia promoted Bcl-2 and Twist1 co-overexpression and induced EMT process and VM formation was demonstrated using murine xenograft models. These results above suggest that hypoxia could activate the cooperation of Bcl-2 and Twist1, Bcl-2 plays an important role in assisting Twist1 nuclear translocation which could change the expression of a wide range of genes and lead to the induction of EMT and VM formation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vasculogenic mimicry (VM), a newly defined pattern of tumor blood supply, describes the functional plasticity of aggressive cancer cells that form vascular networks. In our previous study, breast cancer stem cells (CSC) were shown to potentially participate in VM formation. In this study, breast CSCs presented centrosome amplification (CA) phenotype and ubiquitin-specific protease 44 (USP44) upregulation. USP44 expression contributed to the establishment of bipolar spindles in breast CSCs with supernumerary centrosomes by localizing at pole-associated centrosomes. The bipolar spindle patterns of breast CSCs with CA, including planar-like and apico-basal-like, functioned differently during the VM process of CSCs. Moreover, the ability of transendothelial migration in VM-forming cells was increased. In vivo experiment results showed that CSC xenografts presented linearly patterned programmed cell necrosis, which provided a spatial foundation for VM formation as well as angiogenesis. Breast CSCs further showed increased levels of IL6 and IL8. However, USP44 silencing induced spindle multipolarity, abated VM, reduced transendothelial migration, and consequently decreased IL6 and IL8 levels in breast CSCs. Finally, USP44(+) CSC subclones (ALDH1(+)/USP44(+)/IL6(+)/IL8(+)) were identified in breast cancer specimens through consecutive sections scanning. The subclones were related not only to CA, but also to VM. Statistical analysis suggested that USP44(+) CSC subclones could be used as an independent prognostic biomarker of poor clinical outcomes in patients with breast cancer. Collectively, the identification of USP44(+) CSC subclones may contribute to the prediction of VM formation and aggressive behavior. This study provides novel insights into the therapy for advanced breast cancer., (©2015 American Association for Cancer Research.)

Aims: Zinc finger E-box binding homeobox 2 (ZEB2), an epithelial-mesenchymal transition (EMT) regulator, has been involved in invasion and metastasis of human tumor. Although EMT may be involved in vasculogenic mimicry (VM) formation, no reports describing the relation between ZEB2 and VM are available. We hypothesize that ZEB2 may promote VM formation in hepatocellular carcinoma (HCC)., Methods and Results: Paraffin-embedded tumor tissue samples from 92 patients were immunostained with anti-ZEB2 antibody. We found that the ZEB2 nuclear expression was significantly associated with VM formation and metastasis. Patients with VM and ZEB2 nuclear expression had a shorter survival period than those without expression. In vitro, ZEB2 overexpression significantly enhanced cell motility, invasiveness, and VM formation of HepG2 cells. ZEB2 upregulation also increased VE-cadherin, Flt-1, and Flk-1 expression and activated MMPs. ZEB2 knockdown inhibited cell motility, invasiveness, and VM formation in Bel7402 cells. ZEB2 knockdown also decreased VE-cadherin, Flt-1, and Flk-1 expression and MMP activity. In addition, EMT in HepG2 cells was induced by TGF-β1 treatment, and the kinetics of expression of EMT markers and regulators were assessed by Western blot analysis. The expression of ZEB2 increased significantly, and VM formation was promoted., Conclusion: ZEB2 can promote VM formation through the EMT pathway. Our findings may represent a novel therapeutic target in HCC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vasculogenic mimicry (VM) is a functional microcirculation formed by tumor cells. Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, promote VM formation. Another specific MMP, collagenase-3 (MMP-13), has broad substrate specificity and potentially affects tumor metastasis and invasion. Here we found that MMP-13 was associated with metastasis and poor survival in 79 patients with melanoma. MMP-13 expression was inversely correlated with VM. These results were confirmed in human and mouse melanoma cell lines. We found that MMP-13 cleaves laminin-5 (Ln-5) into small fragments to accelerate tumor metastasis. Degradation of Ln-5 and VE-cadherin by MMP-13 inhibited VM formation. In conclusion, MMP-13 has a dual effect in melanoma, as it promotes invasion and metastasis but disrupts VM formation.

The urokinase plasminogen activator receptor (uPAR) is closely associated with poor prognosis in various aggressive cancers including large-cell lung cancer (LCLC). Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks involving the blood supply in early tumor formation. We demonstrate the statistically positive correlation of uPAR expression with VM formation, metastasis, and poor prognosis of LCLC patients. uPAR(+) cells sorted from the LCLC H460 cell line show higher invasion, migration capacity, and tube structure formation capability on Matrigel compared with uPAR(-) cells. uPAR(+) tumor cells highly expressed vimentin and VE-cadherin; the epithelial marker E-cadherin was low expressed. Higher EMT-regulated protein twist and snail expressions were also observed in these cells. uPAR(+) cells injected subcutaneously into nude mice markedly increased tumor growth, induced VM formation and liver metastasis; by contrast, uPAR(-) cells did not. The data suggest that uPAR expression may predict VM formation, tumor metastasis and poorer prognosis of LCLC patients. The uPAR gene may be used as a novel therapeutic target for inhibiting angiogenesis and metastasis in LCLC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To evaluate the prognostic value of OCT4 expression and vasculogenic mimicry (VM) in human breast cancer, we examined OCT4 expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-schiff) double staining on 90 breast cancer specimens. All patients were followed up for five-149 months following surgery. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between OCT4 expression and VM formation (p<0.05). Both OCT4 expression and VM were also positively correlated with lymph node metastasis, higher histological grade, and Nottingham prognostic index (p<0.05). Patients with OCT4 expression or VM formation exhibited poorer overall survival (OS) and disease-free survival (DFS) than OCT4-negative or VM-negative patients (p<0.05). OCT4-positive/VM-positive patients also had the worst OS and DFS (p<0.05). In multivariate survival analysis, VM, Nottingham prognostic index (NPI), and Her2 were independent prognostic factors related to OS and OCT4-positive/VM-positive patients, whereas NPI and Her2 were independent predictors of DFS. These results suggest that a combined OCT4 expression/VM could improve the prognostic judgment for breast cancer patients.