TP53INP1 inhibits hypoxia‐induced vasculogenic mimicry formation via the ROS/snail signalling axis in breast cancer

Tumour protein p53‐inducible nuclear protein 1 (TP53INP1) is a tumour suppressor associated with malignant tumour metastasis. Vasculogenic mimicry (VM) is a new tumour vascular supply pattern that significantly influences tumour metastasis and contributes to a poor prognosis. However, the molecular mechanism of the relationship between TP53INP1 and breast cancer VM formation is unknown. Here, we explored the underlying mechanism by which TP53INP1 regulates VM formation in vitro and in vivo. High TP53INP1 expression was not only negatively correlated with a poor prognosis but also had a negative relationship with VE‐cadherin, HIF‐1α and Snail expression. TP53INP1 overexpression inhibited breast cancer invasion, migration, epithelial‐mesenchymal transition (EMT) and VM formation; conversely, TP53INP1 down‐regulation promoted these processes in vitro by functional experiments and Western blot analysis. We established a hypoxia model induced by CoCl₂ and assessed the effects of TP53INP1 on hypoxia‐induced EMT and VM formation. In addition, we confirmed that a reactive oxygen species (ROS)‐mediated signalling pathway participated in TP53INP1‐mediated VM formation. Together, our results show that TP53INP1 inhibits hypoxia‐induced EMT and VM formation via the ROS/GSK‐3β/Snail pathway in breast cancer, which offers new insights into breast cancer clinical therapy.     

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.     

Slug promoted vasculogenic mimicry in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.     

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix‐rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20–30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double‐staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three‐dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF‐7 cells induced vessel‐like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up‐regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.     

Advanced research on vasculogenic mimicry in cancer

Vasculogenic mimicry (VM) is a brand‐new tumour vascular paradigm independent of angiogenesis that describes the specific capacity of aggressive cancer cells to form vessel‐like networks that provide adequate blood supply for tumour growth. A variety of molecule mechanisms and signal pathways participate in VM induction. Additionally, cancer stem cell and epithelial‐mesenchymal transitions are also shown to be implicated in VM formation. As a unique perfusion way, VM is associated with tumour invasion, metastasis and poor cancer patient prognosis. Due to VM's important effects on tumour progression, more VM‐related strategies are being utilized for anticancer treatment. Here, with regard to the above aspects, we make a review of advanced research on VM in cancer.     

Exploring stemness gene expression and vasculogenic mimicry capacity in well- and poorly-differentiated hepatocellular carcinoma cell lines

Vasculogenic mimicry (VM) is the phenomenon where cancer cells mimic endothelial cells by forming blood vessels. A stem cell-like phenotype has been proposed to be involved in this tumor plasticity. VM seems to correlate with metastasis rate, but there have been no reports on the effects of pro-metastatic and pro-angiogenic factors or hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) on VM formation of hepatocellular carcinoma (HCC) cells. Here, we determine VM capacity and expression of stemness genes (Oct4, Sox2, Nanog and CD133) in well- and poorly-differentiated HCC cell lines. The poorly-differentiated cell line SK-Hep-1 with mesenchymal features (high invasiveness and expressing Vimentin, with no E-cadherin) could form VM in vitro, while the well-differentiated cell line HepG2 did not form VM. There was no correlation between expression of stemness genes and intrinsic VM capacity. However, HGF but not VEGF, could induce VM formation in HepG2, concomitant with epithelial-mesenchymal transition (EMT), de-differentiation and increased expression of stemness genes. Our results show that the role of stemness genes in VM capacity of HCC cells is likely to depend on differentiation status.     

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple‐negative breast cancer

The up‐regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple‐negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down‐regulated in human TNBC, and Claudin15 down‐regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down‐regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non‐TNBC MCF‐7 cells. Conversely, an up‐regulation of Claudin15 remarkably reduced TNBC MDA‐MB‐231 cell migration, invasion and VM formation. We also showed that down‐regulation of Claudin15 was Twist1‐dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15‐deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1‐Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.     

Silencing of Prrx1b suppresses cellular proliferation,migration, invasion and epithelial–mesenchymal transition in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired‐related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial–mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up‐regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal‐like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β‐catenin signaling pathway and induced the mesenchymal‐epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.     

MMP‐2 and MMP‐13 affect vasculogenic mimicry formation in large cell lung cancer

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.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

Long non‐coding RNA H19 is responsible for the progression of lung adenocarcinoma by mediating methylation‐dependent repression of CDH1 promoter

Lung adenocarcinoma is a common histologic type of lung cancer with a high death rate globally. Increasing evidence shows that long non‐coding RNA H19 (lncRNA H19) and CDH1 methylation are involved in multiple tumours. Here, we tried to investigate whether lncRNA H19 or CDH1 methylation could affect the development of lung adenocarcinoma. First, lung adenocarcinoma tissues were collected to detect CDH1 methylation. Then, the regulatory mechanisms of lncRNA H19 were detected mainly in concert with the treatment of overexpression of lncRNA H19, siRNA against lncRNA H19, overexpression of CDH1 and demethylating agent A‐5az in lung adenocarcinoma A549 cell. The expression of lncRNA H19 and epithelial‐mesenchymal transition (EMT)‐related factors as well as cell proliferation, sphere‐forming ability, apoptosis, migration and invasion were detected. Finally, we observed xenograft tumour in nude mice so as to ascertain tumorigenicity of lung adenocarcinoma cells. LncRNA H19 and methylation of CDH1 were highly expressed in lung adenocarcinoma tissues. A549 cells with silencing of lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation by demethylating agent 5‐Az had suppressed cell proliferation, sphere‐forming ability, apoptosis, migration and invasion, in addition to inhibited EMT process. Silencing lncRNA H19 could reduce methylation level of CDH1. In vivo, A549 cells with silencing lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation exhibited low tumorigenicity, reflected by the smaller tumour size and lighter tumour weight. Taken together, this study demonstrates that silencing of lncRNA H19 inhibits EMT and proliferation while promoting apoptosis of lung adenocarcinoma cells by inhibiting methylation of CDH1 promoter.     

Traditional Chinese medicine as targeted treatment for epithelial-mesenchymal transition-induced cancer progression

The epithelial-mesenchymal transition (EMT) program, which loosens cell-cell adhesion complexes, endows cells with enhanced migratory and invasive properties. Furthermore, this process facilitates both the development of drug resistance and immunosuppression by tumor cells, which preclude the successful treatment of cancer. Recent research has demonstrated that many signaling pathways are involved in EMT progression. In addition, cancer stem cells (CSCs), vasculogenic mimicry (VM) and the tumor-related immune microenvironment all play important roles in tumor formation. However, there are few reports on the relationships between EMT and these factors. In addition, in recent years, traditional Chinese medicine (TCM) has developed a unique system for treating cancer. In this review, we summarize the crucial signaling pathways associated with the EMT process in cancer patients and discuss the interconnections between EMT and other molecular factors (such as CSCs, VM, and the tumor-related immune microenvironment). We attempt to identify common regulators that might be potential therapeutic targets to thereby optimize tumor treatment. In addition, we outline recent research on TCM approaches that target EMT and thereby provide a foundation for further research on the exact mechanisms by which TCMs affect EMT in cancer.     

Comparative mRNA/micro-RNA co-expression network drives melanomagenesis by promoting epithelial–mesenchymal transition and vasculogenic mimicry signaling

This study aimed to identify a novel disease-associated differentially co-expressed mRNA-microRNA (miRNA) that is associated with vasculogenic mimicry (VM) and epithelial-to-mesenchymal transition (EMT) network at different stages of melanoma. By applying weighted gene co-expression network analysis, we constructed a VM+EMT biological network with the available microarray dataset downloaded from a public database. Quantitative real-time PCR, immunohistochemical staining, and CD31-periodic acid solution dual staining were performed to confirm the expression of genes associated with EMT and VM formation in subjects with malignant melanoma (n = 18) and primary melanoma (n = 13) and in healthy subjects (n = 10). Our findings suggested that phosphatidylserine-specific phospholipase A1-alpha (PLA1A) and dermokine (DMKN) genes function as oncogenes that trigger VM and EMT processes during melanomagenesis on interaction with miR-370, miR-563, and miR-770–5p. PLA1A and DMKN genes can be considered potential VM+EMT network-based diagnostic biomarkers for distinguishing between melanoma patients. We postulate that a network with altered PLA1A/miR-563 and DMNK/miR-770–5p/miR-370 may contribute to melanomagenesis by triggering the EMT signaling pathway and VM formation. This study provides a potentially valuable approach for the early diagnosis and prognosis of melanoma progression.     

Comparative mRNA/micro-RNA co-expression network drives melanomagenesis by promoting epithelial–mesenchymal transition and vasculogenic mimicry signaling

This study aimed to identify a novel disease-associated differentially co-expressed mRNA-microRNA (miRNA) that is associated with vasculogenic mimicry (VM) and epithelial-to-mesenchymal transition (EMT) network at different stages of melanoma. By applying weighted gene co-expression network analysis, we constructed a VM+EMT biological network with the available microarray dataset downloaded from a public database. Quantitative real-time PCR, immunohistochemical staining, and CD31-periodic acid solution dual staining were performed to confirm the expression of genes associated with EMT and VM formation in subjects with malignant melanoma (n = 18) and primary melanoma (n = 13) and in healthy subjects (n = 10). Our findings suggested that phosphatidylserine-specific phospholipase A1-alpha (PLA1A) and dermokine (DMKN) genes function as oncogenes that trigger VM and EMT processes during melanomagenesis on interaction with miR-370, miR-563, and miR-770–5p. PLA1A and DMKN genes can be considered potential VM+EMT network-based diagnostic biomarkers for distinguishing between melanoma patients. We postulate that a network with altered PLA1A/miR-563 and DMNK/miR-770–5p/miR-370 may contribute to melanomagenesis by triggering the EMT signaling pathway and VM formation. This study provides a potentially valuable approach for the early diagnosis and prognosis of melanoma progression.     

Comparative mRNA/micro-RNA co-expression network drives melanomagenesis by promoting epithelial–mesenchymal transition and vasculogenic mimicry signaling

This study aimed to identify a novel disease-associated differentially co-expressed mRNA-microRNA (miRNA) that is associated with vasculogenic mimicry (VM) and epithelial-to-mesenchymal transition (EMT) network at different stages of melanoma. By applying weighted gene co-expression network analysis, we constructed a VM+EMT biological network with the available microarray dataset downloaded from a public database. Quantitative real-time PCR, immunohistochemical staining, and CD31-periodic acid solution dual staining were performed to confirm the expression of genes associated with EMT and VM formation in subjects with malignant melanoma (n = 18) and primary melanoma (n = 13) and in healthy subjects (n = 10). Our findings suggested that phosphatidylserine-specific phospholipase A1-alpha (PLA1A) and dermokine (DMKN) genes function as oncogenes that trigger VM and EMT processes during melanomagenesis on interaction with miR-370, miR-563, and miR-770–5p. PLA1A and DMKN genes can be considered potential VM+EMT network-based diagnostic biomarkers for distinguishing between melanoma patients. We postulate that a network with altered PLA1A/miR-563 and DMNK/miR-770–5p/miR-370 may contribute to melanomagenesis by triggering the EMT signaling pathway and VM formation. This study provides a potentially valuable approach for the early diagnosis and prognosis of melanoma progression.     

Down‐regulating Myoferlin inhibits the vasculogenic mimicry of melanoma via decreasing MMP‐2 and inducing mesenchymal‐to‐epithelial transition

Vasculogenic mimicry (VM) constitutes a novel approach for tumour blood supply and contributes to tumour metastasis and poor prognosis in patients with melanoma. Myoferlin (MYOF), a type II membrane protein involved in membrane regeneration and repair, is elevated in several malignant tumours, especially in advanced melanomas. This study aims to investigate the role and mechanism of MYOF in the regulation of VM. VM structures were found in 14 of 52 tested melanoma samples, and high MYOF expression correlated with VM structures. According to Kaplan–Meier survival curves, VM channels and elevated MYOF expression both correlated with poor prognosis in melanoma patients. Down‐regulation of MYOF by siRNA severely impaired the capability of A375 cells to form VM structures in vitro. Further studies demonstrated MYOF knockdown inhibited cell migration and invasion, which is required for VM formation, via decreasing MMP‐2 expression as evidenced by Western blotting, RT‐RCP and ELISA results. SB‐3CT, a specific inhibitor of MMP‐2, showed similar inhibiting effects with siMYOF, further supporting that MYOF down‐regulation inhibits MMP‐2 expression to affect VM formation. Moreover, MYOF knockdown suppress VM formation by A375 cells by inducing mesenchymal‐to‐epithelial transition (MET). After down‐regulating MYOF, focal adhesions were enlarged and A375 cells developed into a clear epithelial morphology. Such cells acquired the expression of E‐cadherin at adherens junctions along with a loss of mesenchymal markers, such as Vimentin and Twist1. In conclusion, MYOF plays an important role in VM and knockdown of MYOF suppresses VM formation via decreasing MMP‐2 and inducing MET in A375 melanoma cells.     

Mesenchymal stem cells promote cell invasion and migration and autophagy‐induced epithelial‐mesenchymal transition in A549 lung adenocarcinoma cells

Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment‐induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial‐mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E‐cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture‐mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture‐mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell‐containing microenvironments and MSC‐induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion.     

Signalling pathways in vasculogenic mimicry

Solid tumour growth is dependent on the development of an adequate blood supply. For years, sprouting angiogenesis has been considered an exclusive mechanism of tumour vascularization. However, over the last years, several other mechanisms have been identified, including vessel-co-option, intussusception, recruitment of endothelial precursor cells (EPCs) and even mechanisms that do not involve endothelial cells, a process called vasculogenic mimicry (VM). The latter describes a mechanism by which highly aggressive tumour cells can form vessel-like structures themselves, by virtue of their high plasticity. VM has been observed in several tumour types and its occurrence is strongly associated with a poor prognosis. This review will focus on signalling molecules and cascades involved in VM. In addition, we will discuss the presence of VM in relation to ongoing cancer research. Finally, we describe the clinical significance of VM regarding anti-angiogenesis treatment modalities.