Xueyi Dong

CD133 + cells with cancer stem cell characteristics associates with vasculogenic mimicry in triple-negative breast cancer

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. In the study we demonstrated that CD133 expression was the highest in triple-negative (TN) breast cancer specimens. Importantly, VM showed statistical correlation with CD133+ expression. The presence of the close relationship between VM and CD133+ expression might be central for TN tumor relapse and progression. The TN breast cancer cell line, MDA-MB-231 cells developed a range of colony morphologies paralleling the holoclone, meroclone and paraclone morphologies produced by normal keratinocytes and other epithelial cancer cell lines when plated at clonal densities. Holoclone cells were capable of forming more colonies on soft agar than meroclone cells and paraclone cells, suggesting that holoclone cells had higher self-renew potential and might harbors cancer stem cells (CSCs) subpopulation. Strikingly, it was holoclone that displayed CD133+ phenotype and formed VM. In addition, holoclone acquired endothelial cell marker vascular endothelial-cadherin expression and upregulated VM mediators matrix metalloproteinase (MMP)-2 and MMP-9 expression. The subpopulation with holoclone morphology, CD133+ phenotype and CSCs characteristics might have the capacity of transdifferentiation and contributed to VM in TN breast cancer. The related molecular pathways may be used as novel therapeutic targets for the inhibition of angiogenesis and metastasis in TN breast carcinoma.

Hypoxia promotes vasculogenic mimicry formation by inducing epithelial-mesenchymal transition in ovarian carcinoma.

OBJECTIVES The functions of hypoxia and subsequent hypoxia-inducible factor-1α (HIF-1α) activation in vasculogenic mimicry (VM) are currently unclear. This study aimed to investigate the effects of hypoxia on VM formation in ovarian cancer, and explore the possible mechanism involved. METHODS The expression levels of HIF-1α, E-cadherin, vimentin, Twist1, Slug, and VE-cadherin proteins were analyzed by immunohistochemistry in 71 specimens of epithelial ovarian cancer. The results were correlated with VM and survival analysis. We used a well-established in vitro model of a three-dimensional culture to compare VM formation under hypoxia and normoxia in ovarian cancer cell lines SKOV3 and OVCAR3. To explore the potential mechanism, we examined the effects of hypoxia on the mRNA and protein expression levels of both E-cadherin and vimentin. RESULTS HIF-1α expression was correlated with loss of E-cadherin expression and up-regulated vimentin expression in 11 of the 18 VM-positive patients. Ovarian cancer with evidence of VM was significantly more likely to have high Twist1, Slug, and VE-cadherin expression levels. VM was observed in vitro under hypoxia. The ovarian cancer cells presented morphological epithelial-mesenchymal transition (EMT)-like changes (more fibroblastoid morphology and loss of cellular cohesiveness) under hypoxic conditions. The mRNA and protein levels demonstrated the induction of EMT after hypoxia. Clinicopathological analysis revealed that both VM and HIF-1α expression levels presented shorter survival durations. CONCLUSIONS Hypoxia contributed to VM formation by inducing EMT. These results may offer new insights for consideration in ovarian cancer treatment strategies.

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.

Twist1 expression induced by sunitinib accelerates tumor cell vasculogenic mimicry by increasing the population of CD133+ cells in triple-negative breast cancer

BackgroundHypoxia induced by antiangiogenic agents is linked to the generation of cancer stem cells (CSCs) and treatment failure through unknown mechanisms. The generation of endothelial cell-independent microcirculation in malignant tumors is defined as tumor cell vasculogenic mimicry (VM). In the present study, we analyzed the effects of an antiangiogenic agent on VM in triple-negative breast cancer (TNBC).MethodsMicrocirculation patterns were detected in patients with TNBC and non-TNBC. Tientsin Albino 2 (TA2) mice engrafted with mouse TNBC cells and nude mice engrafted with human breast cancer cell lines with TNBC or non-TNBC phenotypes were administered sunitinib and analyzed to determine tumor progression, survival, microcirculation, and oxygen concentration. Further, we evaluated the effects of hypoxia induced with CoCl2 and the expression levels of the transcription factor Twist1, in the presence or absence of a Twist siRNA, on the population of CD133+ cells and VM in TNBC and non-TNBC cells.ResultsVM was detected in 35.8 and 17.8% of patients with TNBC or with non-TNBC, respectively. The growth of tumors in TNBC and non-TNBC-bearing mice was inhibited by sunitinib. The tumors in TA2 mice engrafted with mouse TNBCs and in mice engrafted a human TNBC cell line (MDA-MB-231) regrew after terminating sunitinib administration. However, this effect was not observed in mice engrafted with a non-TNBC tumor cell line. Tumor metastases in sunitinib-treated TA2 mice was accelerated, and the survival of these mice decreased when sunitinib was withdrawn. VM was the major component of the microcirculation in sunitinib-treated mice with TNBC tumors, and the population of CD133+ cells increased in hypoxic areas. Hypoxia also induced MDA-MB-231 cells to express Twist1, and CD133+ cells present in the MDA-MB-231 cell population induced VM after reoxygenation. Moreover, hypoxia did not induce MDA-MB-231 cells transfected with an sh-Twist1 siRNA cell to form VM and generate CD133+ cells. Conversely, hypoxia induced MCF-7 cells transfected with Twist to form VM and generate CD133+ cells.ConclusionsSunitinib induced hypoxia in TNBCs, and Twist1 expression induced by hypoxia accelerated VM by increasing population of CD133+ cells. VM was responsible for the regrowth of TNBCs sunitinib administration was terminated.

Clinical significances and prognostic value of cancer stem-like cells markers and vasculogenic mimicry in renal cell carcinoma

The aim of this study was to investigate the clinical significances and prognostic value of CD133 and CD44 (markers of cancer stem‐like cells, CSCs), and vasculogenic mimicry (VM) in renal cell carcinoma (RCC).

Wnt5a promotes vasculogenic mimicry and epithelial-mesenchymal transition via protein kinase Cα in epithelial ovarian cancer.

Epithelial ovarian cancer is one of the most common causes of cancer-related death in women. The majority of epithelial ovarian cancer patients present with metastasis at the time of initial diagnosis. Studies have demonstrated that vasculogenic mimicry (VM) is highly correlated with metastasis and invasiveness, and epithelial-mesenchymal transition (EMT) is pivotal in VM formation. Wnt5a, a member of the Wnt protein family, can activate the non-canonical Wnt signaling pathway mediating cancer initiation and progression. Thus, the present study aimed to investigate the relationship between Wnt5a and VM and its mechanism in epithelial ovarian cancer. The present results showed that Wnt5a staining was significantly correlated with metastasis in epithelial ovarian cancer. The correlation between the expression of Wnt5a and VM or protein kinase Cα (PKCα) indicated that Wnt5a was associated with VM and may be linked to the PKC pathway. In vitro experiments revealed that Wnt5a enhanced the vasculogenic capacity, motility and invasiveness of ovarian cancer cells; however, the PKCα inhibitor blocked these effects. Western blot analysis showed that changes in Wnt5a expression coincided with changes in PKC expression and that PI3K and Snail expression increased along with Wnt5a upregulation. However, no change was observed in β-catenin levels, indicating that Wnt5a may mediate EMT and VM in ovarian cancer cells via the PKCα pathway.

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.

miR-27a-3p suppresses tumor metastasis and VM by down-regulating VE-cadherin expression and inhibiting EMT: an essential role for Twist-1 in HCC

Twist-1 and miRNAs have been reported to be associated with tumor metastasis and angiogenesis. However, the relationship between Twist-1 and miRNAs and the function of miRNAs remain largely undefined. We aimed to reveal the Twist-1-related miRNA expression profile and to determine whether Twist-1 functions in tumor metastasis and vasculogenic mimicry (VM) by regulating miRNA expression in hepatocellular carcinoma (HCC). Results showed that the expression of miR-27a-3p was consistently down-regulated in HCC cell lines and tissue samples displaying high expression of Twist-1. Both loss- and gain-of-function assays revealed suppressive effects of miR-27a-3p. Low miR-27a-3p expression was significantly associated with early metastasis in HCC. Subsequent investigations revealed that miR-27a-3p mediated the inhibition of epithelial–mesenchymal transition (EMT). Additional experiments showed that VE-cadherin is a direct target of miR-27a-3p and further demonstrated the critical role of miR-27a-3p in suppressing tumor metastasis and VM. Conclusions: Twist-1 up-regulation in HepG2 cells resulted in the differential expression of 18 miRNAs. Among them, miR-27a-3p deregulation contributed to VM and metastasis. The miR-27a-3p-mediated down-regulation of VE-cadherin and inhibition of EMT may be essential for Twist-1 to induce tumor metastasis and VM. Our findings highlight the importance of miR-27a-3p and suggest a promising new strategy for anti-HCC therapy.

Doxycycline as an Inhibitor of the Epithelial-to-Mesenchymal Transition and Vasculogenic Mimicry in Hepatocellular Carcinoma

This study was conducted to examine the effects of doxycycline on the survival time and proliferation of hepatocellular carcinoma (HCC) in vivo and on the biologic functions of HCC in vitro. This study was also designed to evaluate the effects of doxycycline on epithelial-to-mesenchymal transition (EMT)– and vasculogenic mimicry (VM)–related protein expression and on matrix metalloproteinase (MMP) and DNA methyltransferase (DNMT) activity in vitro. Human MHCC97H cells were injected into BALB/c mice, which were divided into treatment and control groups. Doxycycline treatment prolonged the mouse survival time and partly suppressed the growth of engrafted HCC tumor cells, with an inhibition rate of 43.39%. Higher amounts of VM and endothelium-dependent vessels were found in the control group than the treatment group. IHC indicated that epithelial (E)-cadherin expression was increased in the doxycycline-treated mice compared with the control group. In in vitro experiments, doxycycline promoted HCC cell adhesion but inhibited HCC cell viability, proliferation, migration, and invasion. Western blot analysis, semiquantitative RT-PCR, qRT-PCR, and immunofluorescence demonstrated that doxycycline inhibited the degradation of the epithelial marker E-cadherin and downregulated the expression levels of EMT promoters, the mesenchymal marker vimentin, and the VM-associated marker vascular endothelial (VE)-cadherin. Furthermore, the activities of MMPs and DNMTs were examined in different groups via gelatin zymography and a DNMT activity assay kit. A methylation-specific PCR was performed to assess the promoter methylation of CDH1 (the gene encoding E-cadherin). Doxycycline prolonged the mouse survival time by inhibiting EMT progression and VM formation. Mol Cancer Ther; 13(12); 3107–22. ©2014 AACR.

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

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.