Overexpression of HIF-1α in primary gallbladder carcinoma and its relation to vasculogenic mimicry and unfavourable prognosis

Department of Surgery, Tongji Hospital, Tongji University School of Medicine, Shanghai, PR China.
Oncology Reports (Impact Factor: 2.3). 06/2012; 27(6):1990-2002. DOI: 10.3892/or.2012.1746
Source: PubMed


As a novel mode of tumor neovascularization, vasculogenic mimicry (VM) has been reported to increase tumor-related mortality in many different solid tumors. In the present study, two established human gallbladder carcinoma (GBC) cell lines (highly aggressive GBC-SD and poorly aggressive SGC-996) cultured on a three-dimensional matrix were assessed for the ability of VM channel formation under normoxic or hypoxic conditions. In addition, the relationship between HIF-1α gene expression and VM channel formation of GBC cells in vitro was measured using the small interfering RNA (siRNA) technique, western blotting and real-time reverse transcription (RT)-PCR analysis. Furthermore, H&E and CD31/periodic acid-Schiff (PAS) staining were used to observe VM in GBC tissue samples. Additionally, all seventy-one specimens with VM and non-VM were stained for hypoxia inducible factor-1 α (HIF-1α) and its correlation with clinicopathological features and prognosis was analyzed simultaneously. We found that hypoxia could induce more VM channel formation and elevated HIF-1α expression in highly aggressive GBC-SD cells. HIF-1α siRNA efficiently knocked down HIF-1α expression and GBC VM networks under either normoxic or hypoxic conditions. VM was present in human primary GBC and overexpression of HIF-1α was significantly correlated with depth of invasion and perineural involvement in the non-VM group. Moreover, VM and HIF-1α were independent factors for the overall survival of GBC patients and correlated with decreased survival. In conclusion, VM was present in human GBC. As a critical mediator in VM formation, high expression of HIF-1α was associated with VM and tumor progression in GBC patients.

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    • "Because VM is an alternative pathway for highly aggressive tumors to guarantee their blood supply, whereas not an angiogenic event arising from pre-existing vessels, it is necessary to find potential therapeutic approaches for targeting VM or in addition to antiangiogenic therapies [23]–[25]. We reported that VM existed in human gallbladder cancers, highly aggressive gallbladder cancer GBC-SD cells and xenografts; whereas poorly aggressive SGC-996 cells did not form the VM networks under the same conditions [13], [17], [18], [42]. In this study, the results showed that NCTD, similar to TIMP-2, inhibited tumor growth and VM formation of GBC-SD xenografts, and prolonged survival time of the xenograft mice. "
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    ABSTRACT: Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2)/focal adhesion kinase (FAK)/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.
    PLoS ONE 05/2014; 9(5):e96982. DOI:10.1371/journal.pone.0096982 · 3.23 Impact Factor
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    ABSTRACT: Vasculogenic mimicry (VM) is a new tumor blood supply in some highly aggressive malignant tumors. We previously reported VM in human gallbladder carcinomas, 3-D matrices in vitro and nude mouse xenografts in vivo of highly aggressive GBC-SD cells and its clinical significance. In this study, we further studied the underlying mechanisms of VM in gallbladder carcinomas via the 3-D matrix in vitro, the nude mouse xenografts in vivo of GBC-SD or SGC-996 cells, immunohistochemistry (H&E staining and CD31-PAS double staining), electron microscopy, expression of MMP-2, MT1-MMP, PI3K, Ln-5γ2, EphA2, FAK and Paxillin-P proteins/mRNAs determined by SABC, ELISA, immunofluorescence, western blotting and qRT-PCR, respectively. It was shown that all of untreated highly aggressive GBC-SD cells and xenografts formed vasculogenic-like structures within 2 weeks of seeding and injecting, and facilitated the growth of tumor cells or xenografts; whereas poorly aggressive SGC-996 cells or GBC-SD cells treated by TIMP-2 were unable to form the vasculogenic-like structures with the same conditions; and tumor xenograft growth was inhibited. Expression of MMP-2, MT1-MMP proteins/mRNAs from sections and supernates of 3-D matrix in vitro, expression of PI3K, MMP-2, MT1-MMP, Ln-5γ2, EphA2, FAK and Paxillin-P proteins/mRNAs from sections of xenografts in vivo in untreated GBC-SD group was upregulated significantly (all P<0.001); however, expression of these VM signal-related proteins/mRNAs in the SGC-996 group and GBC-SD treated by the TIMP-2 group was significantly downregulated (all P<0.001). Thus, we identified for the first time that highly aggressive GBC-SD cells formed VM in vitro and in vivo through the upregulation of PI3K/MMPs/Ln-5γ2 and/or EphA2/FAK/Paxillin signaling. PI3K/MMPs/Ln-5γ2 and EphA2/FAK/Paxillin as key signaling pathways in a coordinated manner contributed to tumor growth and VM of gallbladder carcinomas and provided novel targets that could be potentially exploited for therapeutic intervention of human gallbladder carcinomas.
    International Journal of Oncology 04/2013; 42(6). DOI:10.3892/ijo.2013.1897 · 3.03 Impact Factor
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    ABSTRACT: This study aims to find good markers for predicting the prognosis of patients with esophageal squamous cell carcinoma (ESCC). Vasculogenic mimicry (VM) and the expression of hypoxia inducible factor-lα (HIF-lα)/E-cad protein in ESCC were investigated by immunostaining. The association between VM, HIF-lα/E-cad and clinicopathologic characteristics and 5-year-survival rate of patients with ESCC was analyzed. A total of 160 ESCC specimens were involved in this study and 28 specimens of normal esophageal mucosa served as controls. VM channels were identified in 78 (48.75%) of the 160 ESCC specimens and none of the normal esophageal mucosa was found to have VM. The rates of high-expression of HIF-lα and E-cad in ESCC were 43.75% and 38.75%, while the rates in control were 17.86% and 71.43%, respectively (P<0.05 for all). VM and the expression levels of HIF-lα and E-cad were significantly related to lymph node metastasis, serosa infiltration, PTNM staging and 5-year-survival rates of patients with ESCC (P<0.05 for all). VM was positively correlated with HIF-lα but negatively with E-cad, and HIF-lα was negatively correlated with E-cad (P<0.001 for all). The 5-year-survival rate of patients with ESCC was 6.41% (5/78) in VM group and 65% (52/82) in non-VM group, 7.14% (5/70) in high HIF-lα expression group and 57.78% (52/90) in low HIF-lα expression group. Oppositely, the 5-year-survival rate in high E-cad expression group was 80.65% (50/62) and that in low E-cad expression group was 7.37% (7/98) (P<0.05 for all). Cox multifactor regression analysis indicated that lymph node metastasis, PTNM stage, VM and expression levels of HIF-lα and E-cad were independent risk factors of patients with ESCC (P<0.05 for all). Combined detection of VM, HIF-lα and E-cad plays an important role in predicting the invasion, metastasis and prognosis of patients with ESCC.
    Journal of Huazhong University of Science and Technology 06/2013; 33(3):385-91. DOI:10.1007/s11596-013-1129-4 · 0.83 Impact Factor
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