Promote Vasculogenic Mimicry Formation Research Articles

Gallbladder cancer-associated fibroblasts promote vasculogenic mimicry formation and tumor growth in gallbladder cancer via upregulating the expression of NOX4, a poor prognosis factor, through IL-6-JAK-STAT3 signal pathway

BackgroundCancer-associated fibroblasts (CAFs) and vasculogenic mimicry (VM) play important roles in the occurrence and development of tumors. However, the relationship between CAFs and VM formation, especially in gallbladder cancer (GBC) has not been clarified. In this study, we investigated whether gallbladder CAFs (GCAFs) can promote VM formation and tumor growth and explored the underlying molecular mechanism.MethodsA co-culture system of human GBC cells and fibroblasts or HUVECs was established. VM formation, proliferation, invasion, migration, tube formation assays, CD31-PAS double staining, optic/electron microscopy and tumor xenograft assay were used to detect VM formation and malignant phenotypes of 3-D co-culture matrices in vitro, as well as the VM formation and tumor growth of xenografts in vivo, respectively. Microarray analysis was used to analyze gene expression profile in GCAFs/NFs and VM (+)/VM (−) in vitro. QRT-PCR, western blotting, IHC and CIF were used to detected NOX4 expression in GCAFs/NFs, 3-D culture/co-culture matrices in vitro, the xenografts in vivo and human gallbladder tissue/stroma samples. The correlation between NOX4 expression and clinicopathological and prognostic factors of GBC patients was analyzed. And, the underlying molecular mechanism of GCAFs promoting VM formation and tumor growth in GBC was explored.ResultsGCAFs promote VM formation and tumor growth in GBC; and the finding was confirmed by facts that GCAFs induced proliferation, invasion, migration and tube formation of GBC cells in vitro, and promoted VM formation and tumor growth of xenografts in vivo. NOX4 is highly expressed in GBC and its stroma, which is the key gene for VM formation, and is correlated with tumor aggression and survival of GBC patients. The GBC patients with high NOX4 expression in tumor cells and stroma have a poor prognosis. The underlying molecular mechanism may be related to the upregulation of NOX4 expression through paracrine IL-6 mediated IL-6/JAK/STAT3 signaling pathway.ConclusionsGCAFs promote VM formation and tumor growth in GBC via upregulating NOX4 expression through the activation of IL-6-JAK-STAT3 signal pathway. NOX4, as a VM-related gene in GBC, is overexpressed in GBC cells and GCAFs, which is related to aggression and unfavorable prognosis of GBC patients.

CD276 Promotes Vasculogenic Mimicry Formation in Hepatocellular Carcinoma via the PI3K/AKT/MMPs Pathway.

PurposeCD276 protein expression and vasculogenic mimicry (VM) formation are associated with the poor prognosis of hepatocellular carcinoma (HCC) patients. Although both the effects of CD276 and VM formation involve the activation of matrix metalloproteinases, and their relationship has not yet been explored. The following study investigated the effect of CD276 expression on VM formation and the potential mechanisms.Materials and MethodsCD276 expression and VM were examined in commercial tissue microarrays by immunohistochemistry and CD31/PAS double staining. Tumor cell proliferation, invasion, migration and, tube formation were detected in vitro after transfecting HCC cell lines with an shRNA lentiviral vector against CD276. The expression of MMP14, MMP2, VE-cadherin, E-cadherin, and vimentin and MMPs activation was detected by Western blot, immunofluorescence and gelatin zymography assay. In addition, an orthotopic xenograft model of HCC cells was established in vivo, after which VM was detected, along with its marker molecules.ResultsCD276 expression was associated with VM and poor prognosis in HCC patients. RNA interference of CD276 reduced tumor cell proliferation, invasion, migration, and VM formation in vitro and in vivo. Furthermore, CD276 knockdown up-regulated the expression of E-cadherin but inhibited the phosphorylation of AKT, the expression of MMP14, MMP2, VE-cadherin, vimentin and the activation of MMP2 and MMP9 in HCC cell lines.ConclusionCD276 may promote VM formation by activating the PI3K/AKT/MMPs pathway and inducing the EMT process in HCC. CD276 may serve as a promising candidate for the anti-VM treatment of HCC.

VEGF Induce Vasculogenic Mimicry of Choroidal Melanoma through the PI3k Signal Pathway.

Purpose To explore the effect of VEGF (vascular endothelial growth factor) on the vasculogenic mimicry (VM) formation of Choroidal Melanoma (CM) through PI3k signal pathway, to find novel targets for CM therapy. Methods This research investigated the molecular mechanism of VEGF promoting VM formation of CM. First, we evaluated the expressions of VEGF in 20 CM specimens by immunohistochemical determination. Then we detected expressions of VEGF, AKT, MT1-MMP, MMP2, and MMP9 of OCM-1 in hypoxia. siRNA was used to inhibit the expression of VEGF, to realize the control of the VM formation. The VM formation was evaluated through wound healing assay, transwell assay, and apoptosis. And then we testify the correlation of the VM and the factors in protein and mRNA level preliminarily. Results VEGF protein was expressed in CM in all 20 cases of CM, especially along the VM. In hypoxia, the expression of VEGF in OCM-1 increased significantly. VEGF gene deletion reduced the proliferation, migration, and invasion of OCM-1. VEGF gene deletion impaired the expression of invasive associated genes like VEGF, p-AKT, AKT, MT1-MMP, MMP2, and MMP9. These results indicate that VEGF induce VM formation in CM by activating PI3K/AKT signaling pathway. Conclusions VEGF promoted VM formation by the PI3K signal transduction pathway, indicating a molecular mechanism which may be used to develop new therapeutic targets for the clinical treatment of CM.

Vasculogenic Mimicry Formation Is Associated with Erythropoietin Expression but Not with Erythropoietin Receptor Expression in Cervical Squamous Cell Carcinoma.

Background Vasculogenic mimicry (VM), as an endothelium-independent cancer microcirculation, has been observed in many malignancies including cervical cancer. Erythropoietin (EPO) and erythropoietin receptor (EPO-R) could produce an angiogenic effect to promote cervical squamous cell carcinoma (CSCC) progression. However, the association between VM formation and EPO/EPO-R expression in CSCC is poorly explored. Methods Seventy-six paraffin-embedded CSCC samples, 25 high-grade squamous intraepithelial lesion (HSIL) samples, 20 low-grade squamous intraepithelial lesion (LSIL) samples, and 20 normal cervix samples were collected. Immunohistochemistry SP method was performed to detect EPO/EPO-R expression and CD31/periodic acid-Schiff (PAS) double staining was performed to detect VM formation. The associations of EPO/EPO-R and VM with clinicopathological parameters of CSCC were analyzed. The associations between VM formation and EPO/EPO-R expression were also analyzed. Results The positive expression rates of EPO and EPO-R were gradually increasing along the progression of normal cervix-LSIL-HSIL-CSCC sequence (P<0.05). EPO and EPO-R expression were not significantly associated with clinicopathological parameters of CSCC patients (P>0.05). VM was significantly associated with FIGO stage, lymphovascular space involvement, and lymph node metastasis (P<0.05). VM was positively associated with EPO expression (r=0.284, P<0.05) but was not associated with EPO-R expression (P>0.05). Conclusion These data suggest that increased EPO/EPO-R expression may play an important role in cervical carcinogenesis. EPO overexpression may promote VM formation in CSCC.

Galectin-1 Promotes Vasculogenic Mimicry in Gastric Cancer by Upregulating EMT Signaling.

Background: Galectin-1 (Gal-1) expression was positively associated with vasculogenic mimicry (VM) in primary gastric cancer (GC) tissue, and that both Gal-1 expression and VM in GC tissue are indicators of poor prognosis. However, whether Gal-1 promotes VM, and by what mechanismsremains unknown.Methods: To investigate the underlying mechanisms,wound healing assay, proliferation assay, invasion assay, and three-dimensional culture were used to evaluate the invasion, metastasis and promoted VM formation effects of the Gal-1. We monitored the expression level of sociated proteins in GC tissues, cell lines in vitro and nude mice tumorigenicity in vivo by immunohistochemistry and western blot.Results: Gal-1 overexpression significantly promoted the proliferation, invasion, migration, and VM formation of MGC-803 cells. Gal-1 was associated with E-cadherin and vimentin in vitro and in clinical samples. The epithelial-to-mesenchymal transition (EMT) induced in MGC-803 cells by TGF-β1 was accompanied by Gal-1 activation and promotion of VM formation, while knockdown of Gal-1 reduced the response to TGF-β1, suggesting that Gal-1 promotes VM formation by activating EMT signaling. Overexpression of Gal-1 accelerated subcutaneous xenograft growth and facilitated pulmonary metastasis in athymic mice, enhanced the expression of EMT markers, and promoted VM formation in vivo.Conclusion: Our results indicated that Gal-1 promotes VM in GC by upregulating EMT signaling; thus, Gal-1 and this pathway are potential novel targets to treat VM in GC.

LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma.

Lysyl oxidase‐like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid‐Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.

The involvement of autophagy in melanoma vasculogenic mimicry

Introduction. Autophagy, a catabolic process of protein and organelle recycling by transferring defective cytoplasm and organelles into double-membraned vesicles to degrade and regenerate materials, plays a critical role in maintaining energy homeostasis. Autophagy also protects against stress and infection, participates at the development of autoimmune disease. In recent years, the existence of alternative blood circulation system in tumors, vasculogenic mimicry (VM), which can partially compensate the lack of nutrients and oxygen under the hypoxic conditions, has been described. Objective. To elucidate the relationship between autophagy and VM. Materials and methods. In this study we used 2D- and 3D-culturing of melanoma cells derived from surgical species of patients with disseminated melanoma, electrophoresis and western blot, knockdown of the genes by using small interfering RNA (siRNA), flow cytometry, fluorescence microscopy. Results. We detected the basal level autophagy by examining the expression of autophagy-specific protein (LC-3B) by flow cytometry and cellular immunofluorescence staining by monodancylcadaverine. Both assays are the markers of autophagy late stage. Here we show that the level of autophagy in melanoma cells mel P, participated in capillary-like structures (CLS) formation in matrigel, was considerably higher than in mel Me cells which do not involve in VM. To explore the function of autophagy in the ability of melanoma cells to form CLS 3-methyladenine (3-MA) or chloroquine - inhibitors of initiation and terminal stage of autophagy - were used. Both inhibitors reduced the ability of melanoma cells to engage in VM. The data obtained were confirmed by siRNA-mediated gene silencing of BECN1 involved in the initiation of autophagy and ATG5 gene which is considered to be a marker of late stage of autophagy. Knockdown of BECN1 or ATG5 in mel P melanoma cells reduced the level of protein Beclin-1 and Atg5 about 70-75 %, and suppressed CLS formation in matrigel. Melanoma cells with the ATG5 gene knockdown changed the shape but maintained the ability to migrate and recognize each other, the formation of CLS was not observed. Low molecular weight VM inhibitor LCS-1269, significantly reduced the basic level of autophagy. Conclusion. Our data indicate that autophagy participates in CLS formation, and inhibition of autophagy suppresses CLS formation. We suggest that autophagy plays a dual role in the survival and development of tumors: autophagy helps cancer cells against environment stress and provides a temporary survival pathway by promoting energy regeneration, autophagy also promotes VM formation which supplies nutrients and oxygen to less vascularized area of tumor.

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

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.

Nodal signaling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3 pathway.

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.

Dickkopf-1-promoted vasculogenic mimicry in non-small cell lung cancer is associated with EMT and development of a cancer stem-like cell phenotype.

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.

Effects of HCG on human epithelial ovarian cancer vasculogenic mimicry formation in vivo.

Ovarian cancer is the leading cause of mortality due to gynecological malignancy, and vasculogenic mimicry (VM) formation is correlated with poor prognosis. In a previous study, the present authors observed that human chorionic gonadotropin (HCG) could promote VM formation in three-dimensional OVCAR-3 cell cultures. In order to investigate whether HCG could promote VM formation in ovarian cancer in vivo, the role of OVCAR-3 cells overexpressing or depleted of chorionic gonadotropin, beta polypeptide 5 (CGB5, which is the fifth subunit of β-HCG and was identified as the key part of HCG) were injected into nude mice in the present study, while BeWo cells were used as a positive control. The results demonstrated that overexpressed CGB5 promoted xenografts tumor formation in nude mice, and the results of hematoxylin and eosin and cluster of differentiation (CD)34-periodic acid-Schiff dual staining revealed that CGB5 promoted VM formation. Furthermore, reverse transcription-polymerase chain reaction and immunochemistry staining demonstrated that the expression of the vascular markers CD31, vascular endothelial growth factor and factor VIII was also upregulated in the CGB5-overexpressing xenografts tumors. In addition, the expression of luteinizing hormone receptor (LHR), the receptor of CGB5, was increased in CGB5-overexpressing cells. In conclusion, CGB5 may promote tumor growth and VM formation via activation of the LHR signal transduction pathway, which may support a novel strategy for ovarian cancer therapy.

Epithelial-to-endothelial transition and cancer stem cells: two cornerstones of vasculogenic mimicry in malignant tumors.

Vasculogenic mimicry (VM) is a functional microcirculation pattern in malignant tumors accompanied by endothelium-dependent vessels and mosaic vessels. VM has been identified in more than 15 solid tumor types and is associated with poor differentiation, late clinical stage and poor prognosis. Classic anti-angiogenic agents do not target endothelium-dependent vessels and are not efficacious against tumors exhibiting VM. Further insight into the molecular signaling that triggers and promotes VM formation could improve anti-angiogenic therapeutics. Recent studies have shown that cancer stem cells (CSCs) and epithelium-to-endothelium transition (EET), a subtype of epithelial-to-mesenchymal transition (EMT), accelerate VM formation by stimulating tumor cell plasticity, remodeling the extracellular matrix (ECM) and connecting VM channels with host blood vessels. VM channel-lining cells originate from CSCs due to expression of EMT inducers such as Twist1, which promote EET and ECM remodeling. Hypoxia and high interstitial fluid pressure in the tumor microenvironment induce a specific type of cell death, linearly patterned programmed cell necrosis (LPPCN), which spatially guides VM and endothelium-dependent vessel networks. This review focuses on the roles of CSCs and EET in VM, and on possible novel anti-angiogenic strategies against alternative tumor vascularization.

Vasculogenic mimicry and hypoxia-inducible factor-1α expression in cervical squamous cell carcinoma.

In this study, the existence of vasculogenic mimicry (VM) in cervical squamous cell carcinoma was investigated. To this end, the relationship between hypoxia-inducible factor 1α (HIF-1α) and the development, infiltration, and metastasis of cervical squamous cell carcinoma was studied. Between January 2010 and December 2010, 67 human cervical squamous carcinoma tissue samples were collected and stained by CD34/periodic acid-Schiff double staining to detect the existence of VM. HIF-1α expression was analyzed by immunohistochemistry. The relationship between VM and HIF-1α was also analyzed. Normal cervical tissues (20 cases) from patients who had uterine surgeries in the same period were collected as controls. In the cervical squamous carcinoma tissues, positive rates of VM and HIF-1α were 38.81% (26/67) and 64.18% (43/67), respectively. This was significantly higher than those in the normal cervical tissues [0 (0/20); P < 0.05]. VM rates in cervical squamous carcinoma tissues from patients with different pathological grades, Federation of Gynecology and Obstetrics (FIGO) stages, and lymph node metastasis states were also significantly different (P < 0.05). In addition, significant differences in HIF-1α positivity rates were observed among patients with varying tumor sizes and lymph node metastasis states (P < 0.05). Positive correlation was found between VM and HIF-1α (r = 0.339, P < 0.05). To summarize, we found VM in cervical squamous carcinoma; high expression of HIF-1α may promote VM formation, as well as cervical squamous cell infiltration and metastasis.

Hypoxia induced epithelial–mesenchymal transition and vasculogenic mimicry formation by promoting Bcl-2/Twist1 cooperation

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.

Hypoxia promotes vasculogenic mimicry formation by the Twist1-Bmi1 connection in hepatocellular carcinoma.

Aggressive tumor cells can mimic embryonic vasculogenic networks and form vasculogenic mimicry (VM). Preliminary studies demonstrated that hypoxia can promote VM formation; however, the underlying mechanism remains unclear. The present study aimed to investigate the role of the Twist1‑Bmi1 connection in hypoxia‑induced VM formation and the underlying mechanism. In the in vitro experiments, western blot analysis demonstrated that hypoxia upregulated the expression of Twist1, Bmi1, epithelial‑mesenchymal transition (EMT) markers, stem cell markers and VM‑associated markers. The 3D culture assay showed that hypoxia promoted VM formation in hepatocellular carcinoma (HCC) cell lines. Using transfection and in vitro cell experiments, the Twist1‑Bmi1 connection was confirmed to have an important role in inducing EMT, cell stemness and VM formation. In the in vivo experiments, the murine hypoxia models were established via incomplete femoral artery ligation and the mechanism by which hypoxia promoted Twist1 and Bmi1 expression and led to VM formation was demonstrated by immunohistochemistry staining and endomucin/periodic acid Schiff double‑staining. In conclusion, hypoxia upregulate the expression of Twist1 and Bmi1, and these two proteins have an important role in inducing EMT and cancer cell stemness, which contributed to VM formation.

Vasculogenic mimicry is a major feature and novel predictor of poor prognosis in patients with orbital rhabdomyosarcoma.

Vasculogenic mimicry (VM) is a key developmental program, frequently activated during cancer invasion and metastasis. The aim of the present study was to evaluate the role of VM in orbital rhabdomyosarcoma (RMS), the correlation between VM and tumor differentiation, recurrence and survival duration, as well as the contribution of epithelial cell kinase (EphA2) and matrix metalloproteinase-2 (MMP-2) in VM initiation. A total of 32 patients were enrolled to investigate the associations between VM in orbital RMS tumors and clinical characteristics, as well as its impact on overall survival. VM was identified and confirmed by CD31/periodic acid-Schiff double staining, while the presence of EphA2 and MMP-2 were examined by immunohistochemical analysis. VM was identified in eleven patients, particularly those with poorly differentiated orbital RMS (P=0.001). Patients with VM exhibited significantly worse survival rates (P=0.001, log-rank test), a significantly increased risk of mortality (P=0.008) and EphA2 and MMP-2 expression levels were enhanced (P=0.005 and 0.001, respectively). The VM and mitotic rate were independent predictors of poor prognosis (P=0.001 and 0.004, respectively), indicated by multivariate Cox proportional hazards models. These results demonstrated that VM is present in orbital RMS and represents an independent prognostic factor for overall survival. In addition, overexpression of EphA2 and MMP-2 may promote VM formation in orbital RMS.

ZEB2 promotes vasculogenic mimicry by TGF-β1 induced epithelial-to-mesenchymal transition in hepatocellular carcinoma

AimsZinc 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 resultsParaffin-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. ConclusionZEB2 can promote VM formation through the EMT pathway. Our findings may represent a novel therapeutic target in HCC.

Dual effects of collagenase-3 on melanoma: metastasis promotion and disruption of vasculogenic mimicry.

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.

Expression and significance of Beclin-1 in vasculogenic mimicry formation of gastric cancer

To explore the effect and mechanism of autophagy specific gene Beclin-1 in gastric cancer cell SGC7901 on vasculogenic mimicry (VM) forming ability. Plasmid vectors with and without integrated shRNA were transfected respectively into SGC7901 cell line (Beclin1-inhibited group and negative control group). Simple SGC7901 cell line was used as blank group. RT-PCR and Western blot were performed to examine the expression of Beclin-1 in 3 groups. Culture was used to construct the VM model in vitro. Different VM forming ability was measured and genes (beclin-1, notch-1) expression of each group was detected before and after VM formation. Beclin-1 and notch-1 expression increased significantly in the process of VM forming. When beclin-1 was inhibited, the formation of VM was limited and VM formative genes expression decreased. As compared to cells of negative control group, those of Beclin1-inhibited group had less number of VM forming cellular tube-like construction (15.4±1.1 vs. 37.8±1.9, P<0.05), shorter length of such construction [(316.8±24.6) mm vs. (385.1±14.2) mm, P<0.05], and less crossing point (11.6±1.1 vs. 27.2±1.1, P<0.05). Beclin-1 can promote VM formation through maintaining stable expression of gastric cancer cell VM regulating genes. Beclin-1 inhibition may be a new target for gastric cancer gene therapy.

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

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.