The up-regulation and nuclear relocation of epithelial-mesenchymal transition (EMT) regulator Twist1 have been implicated in the tumor invasion and metastasis of human hepatocellular carcinoma (HCC). The term vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. However, the relationship between Twist1 and VM formation is not clear. In this study, we explored HCC as a VM and EMT model in order to investigate the role of Twist1 in VM formation. We first examined the expression of Twist1 in human HCC samples and cell lines and found that Twist1 was frequently overexpressed in the nuclear relocation occurring in VM-positive HCCs (13/18 [72%]). Twist1 nuclear expression was likewise significantly associated with VM formation. Clinicopathological analysis revealed that both VM and Twist1 nuclear expressions present shorter survival durations than those without expression. We consistently demonstrated that an overexpression of Twist1 significantly enhanced cell motility, invasiveness, and VM formation in an HepG2 cell. Conversely, a knockdown of Twist1 by the short hairpin RNA approach remarkably reduced Bel7402 cell migration, invasion, and VM formation. Using chromatin immunoprecipitation, we also showed that Twist1 binds to the vascular endothelial (VE)-cadherin promoter and enhances its activity in a transactivation assay. Conclusion: The results of this study indicate that Twist1 induces HCC cell plasticity in VM cells more through the suppression of E-cadherin expression and the induction of VE-cadherin up-regulation than through the VM pattern in vivo and in a three-dimensional in vitro system. Our findings also demonstrate a novel cogitation in cancer stem-like cell differentiation and that related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis. (HEPATOLOGY 2010;51:545-556.)

Background Hyperglycaemia associated with myocardial oxidative stress and fibrosis is the main cause of diabetic cardiomyopathy. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor has recently been reported to improve glycaemic control in patients with type 2 diabetes in an insulin-independent manner. The aim of this study was to investigate the effect of empagliflozin on myocardium injury and the potential mechanism in type 2 diabetic KK-Ay mice. Methods Thirty diabetic KK-Ay mice were administered empagliflozin (10 mg/kg/day) by oral gavage daily for 8 weeks. After 8 weeks, heart structure and function were evaluated by echocardiography. Oxidants and antioxidants were measured and cardiac fibrosis was analysed using immunohistochemistry, Masson’s trichrome stain and Western blot. Results Results showed that empagliflozin improved diabetic myocardial structure and function, decreased myocardial oxidative stress and ameliorated myocardial fibrosis. Further study indicated that empagliflozin suppressed oxidative stress and fibrosis through inhibition of the transforming growth factor β/Smad pathway and activation of Nrf2/ARE signaling. Conclusions Glycaemic control with empagliflozin significantly ameliorated myocardial oxidative stress injury and cardiac fibrosis in diabetic mice. Taken together, these results indicate that the empagliflozin is a promising agent for the prevention and treatment of diabetic cardiomyopathy.

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.

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent extrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effects of ATPase inhibitory factor 1 (IF1), an inhibitor of the mitochondrial H(1)-adenosine triphosphate (ATP) synthase, on HCC angiogenesis and metastasis. We found that increased expression of IF1 in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and metastasis, expressed high levels of IF1. Invasive tumors overexpressing IF1 were featured by active epithelial-mesenchymal transition (EMT) and increased angiogenesis, whereas silencing IF1 expression attenuated EMT and invasion of HCC cells. Mechanistically, IF1 promoted Snai1 and vascular endothelial growth factor (VEGF) expression by way of activating nuclear factor kappa B (NFjB) signaling, which depended on the binding of tumor necrosis factor (TNF) receptorassociated factor 1 (TRAF1) to NF-jB-inducing kinase (NIK) and the disruption of NIK association with the TRAF2-cIAP2 complex. Suppression of the NF-jB pathway interfered with IF1-mediated EMT and invasion. Chromatin immunoprecipitation assay showed that NF-jB can bind to the Snai1 promoter and trigger its transcription. IF1 was directly transcribed by NF-jB, thus forming a positive feedback signaling loop. There was a significant correlation between IF1 expression and pp65 levels in a cohort of HCC biopsies, and the combination of these two parameters was a more powerful predictor of poor prognosis. Conclusion: IF1 promotes HCC angiogenesis and metastasis by up-regulation of Snai1 and VEGF transcription, thereby providing new insight into HCC progression and IF1 function. (HEPATOLOGY 2014;60:1659-1673

The antiapoptotic protein Bcl-2 plays multiple roles in apoptosis, immunity, and autophagy. Its expression in tumors correlates with tumor grade and malignancy. The recapitulation of the normal developmental process of epithelial-mesenchymal transition (EMT) contributes to tumor cell plasticity. This process is also a characteristic of metastatic cells and vasculogenic mimicry. In the present study we report functional and structural interactions between Bcl-2 and the EMT-regulating transcription factor Twist1 and the relationship with metastasis and vascular mimicry. Bcl-2 and Twist1 are coexpressed under hypoxia conditions. The Bcl-2 can bind to Twist1 in vivo and in vitro. This interaction involves basic helix-loop-helix DNA binding domain within Twist1 and through two separate domains within Bcl-2 protein. Formation of the Bcl-2/Twist1 complex facilitates the nuclear transport of Twist1 and leads to transcriptional activation of wide ranges of genes that can increase the tumor cell plasticity, metastasis, and vasculogenic mimicry. Finally, nuclear expression of Bcl-2 and Twist1 is correlated with poor survival of these patients in a cohort of 97 cases of human hepatocellular carcinoma. Conclusion: The results describe a novel function of Bcl-2 in EMT induction, provide insight into tumor progression, and implicate the Bcl-2/Twist1 complex as a potential target for developing chemotherapeutics.

Abstract. Vasculogenic mimicry (VM) has increasingly been recognized as a form of angiogenesis. In VM, epithelial cells are integrated into the malignant tumor vasculature. An association has been observed between VM and poor clinical prognosis in some malignant tumors. However, whether VM is present and clinically significant in hepatocellular carcinoma (HCC) is unknown. In this study, we determined whether VM was present in HCC and whether it was associated with tumor grade, invasion and metastasis, and survival duration. We collected paraffin-embedded HCC tumor samples, along with complete clinical and pathologic data for all the cases, and performed immunohistochemical staining for CD31, CD105 (endoglin), hepatocyte, vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9. The VM status was compared with the clinical and pathological data using statistical tests. Kaplan-Meier survival analysis and log-rank test were used to compare survival durations between patients with and without VM. The VM vessel cells were CD31 and CD105-negative and hepatocyte and vascular endothelial growth factor-positive, showing that they were not derived from endothelial cells but were HCC tumor cells. Patients with VM had a higher metastasis rate than did those without VM (P=0.003). Consistent with this finding, MMP-2 and MMP-9 were present in all the VM cases but were found less frequently in non-VM cases (P<0.05). The Kaplan-Meier survival analysis showed that patients in the VM group had a significantly shorter survival duration than did those in the non-VM group. In conclusion, VM is a marker of poor clinical prognosis in HCC: Its presence may be associated with a high tumor grade, invasion and metastasis, and short survival.

Systemic therapy with anti-VEGF drugs such as bevacizumab is widely used for treatment of human patients with various solid tumors. However, systemic impacts of such drugs in host healthy vasculatures remain poorly understood. Here, we show that, in mice, systemic delivery of an anti-VEGF or an anti–VEGF receptor (VEGFR)-2 neutralizing antibody caused global vascular regression. Among all examined tissues, vasculatures in endocrine glands, intestinal villi, and uterus are the most affected in response to VEGF or VEGFR-2 blockades. Thyroid vascular fenestrations were virtually completely blocked by VEGF blockade, leading to marked accumulation of intraendothelial caveolae vesicles. VEGF blockade markedly increased thyroid endothelial cell apoptosis, and withdrawal of anti-VEGF resulted in full recovery of vascular density and architecture after 14 d. Prolonged anti-VEGF treatment resulted in a significant decrease of the circulating level of the predominant thyroid hormone free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid functions. Conversely, VEGFR-1–specific blockade produced virtually no obvious phenotypes. These findings provide structural and functional bases of anti-VEGF–specific drug-induced side effects in relation to vascular changes in healthy tissues. Understanding anti-VEGF drug-induced vascular alterations in healthy tissues is crucial to minimize and even to avoid adverse effects produced by currently used anti-VEGF–specific drugs.

Yes-associated protein (YAP), a transcriptional co-activator, has important regulatory roles in cell signaling and is dysregulated in a number of cancers. However, the role of YAP in cholangiocarcinoma (CCA) progression remains unclear. Here, we demonstrated that YAP was overexpressed in CCA cells and human specimens. High levels of nuclear YAP (nYAP) correlated with histological differentiation, TNM stage, metastasis and poor prognosis in CCA. Silencing YAP increased tumor sensitivity to chemotherapy and inhibited CCA tumorigenesis and metastasis both in vivo and in vitro. YAP overexpression in vivo and in vitro promoted CCA tumorigenesis and metastasis. Additionally, we found that YAP induced epithelial-mesenchymal transition (EMT) and formed a regulatory circuit with miR-29c, IGF1, AKT and gankyrin to promote the progression of CCA. Results of CCA tissue microarray showed positive correlations between nYAP and gankyrin or p-AKT expression. Combination of nYAP and gankyrin or p-AKT exhibited improved prognostic accuracy for CCA patients. In conclusion, YAP promotes carcinogenesis and metastasis by up-regulating gankyrin through activation of the AKT pathway.