EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis

Eye absent homolog 4 (EYA4) has been demonstrated to be down‐regulated in hepatocellular carcinoma (HCC), but its biological function and the mechanism in HCC angiogenesis and metastasis remain largely unknown. Herein, we showed that EYA4 expression was frequently low in HCC tissue samples compared with matched adjacent non‐tumourous tissues. In the analysis of 302 HCC specimens, we revealed that decreased expression of EYA4 correlated with tumour differentiation status. Univariate and multivariate analyses identified EYA4 as an independent risk factor for recurrence‐free survival (RFS) and overall survival (OS) among the 302 patients. Functional assays showed that forced expression of EYA4 suppressed HCC cell migration, invasion and capillary tube formation of endothelial cells in vitro, as well as in vivo tumour angiogenesis and metastasis in a mouse model. Furthermore, mechanism study exhibited that EYA4 could inhibit HCC angiogenesis and metastasis by inhibiting c‐JUN/VEGFA pathway. Together, we provide proof that EYA4 is a novel tumour suppressor in HCC and a new prognostic biomarker and therapeutic target in HCC.     

Clinical Relevance of Increased Endothelial and Mesothelial Expression of Proangiogenic Proteases and VEGFA in the Omentum of Patients with Metastatic Ovarian High-Grade Serous Carcinoma

Epithelial ovarian cancer (EOC) metastasis to the omentum requires implantation and angiogenesis. We propose that prometastatic changes in the omental endothelium (for angiogenesis) and mesothelium (for implantation) are critical. We investigated the expression of angiogenic proteases [cathepsin D (CD), cathepsin L (CL), and matrix metalloproteinase 2 (MMP2) and MMP9] and vascular endothelial growth factor A (VEGFA) in the mesothelium and endothelium of omentum from patients with EOC with omental metastases and control patients with benign ovarian tumors. Endothelial expression of CL, VEGFA, and MMP9 and mesothelial expression of VEGFA, MMP9, and CD were significantly increased in patients with metastasized EOC. High expression of MMP9 and VEGFA in endothelium and mesothelium and CD in mesothelium was positively associated with poor disease-specific survival (DSS). High MMP9 expression in either endothelium or mesothelium and presence of ascites prospectively showed the greatest risk of shorter DSS [hazard ratio (HR)= 6.16, 95% confidence interval (CI) = 1.76-21.6, P = .0045; HR = 11.42, 95% CI = 2.59-50.35, P = .0013; and HR = 6.35, 95% CI = 2.01-20.1, P = .002, respectively]. High endothelial MMP9 expression and ascites were independent predictors of reduced DSS and overall survival, together resulting in worst patient prognosis. Our data show that omental metastasis of EOC is associated with increased proangiogenic protein expression in the omental endothelium and mesothelium.     

Vascular endothelial growth factor A is a potential prognostic biomarker and correlates with immune cell infiltration in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths among cancer patients. Vascular endothelial growth factor A (VEGFA) is involved in regulating biological processes, such as angiogenesis and vascular permeability, and is very closely related to the pathogenesis of various tumours, especially vascular-rich, solid tumours. Clinical data of patients with HCC and other tumours were analysed through public databases, such as the TCGA database, Gene Expression Omnibus database, Human Protein Atlas database, STRING, Tumour Immune Estimation Resource and Kaplan–Meier Plotter. The tumour tissues and adjacent normal tissues of patients with HCC from Hunan Provincial People's Hospital were collected to verify the expression of VEGFA by immunohistochemistry, immunofluorescence, Western blotting and qPCR. VEGFA expression is elevated in multiple tumour types and correlates with the prognosis of tumour patients. VEGFA is involved in regulating the tumour microenvironment and immune cell function in tumour development. Inhibition of VEGFA reduces proliferation, invasion, and migration and promotes apoptosis in HCC cells. VEGFA is a potential predictive biomarker for the diagnosis and prognosis of HCC.     

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.     

MicroRNA-26a Inhibits Angiogenesis by Down-Regulating VEGFA through the PIK3C2α/Akt/HIF-1α Pathway in Hepatocellular Carcinoma

Background & Aims

microRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC).

Methods

The expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings.

Results

Ectopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2α as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients.

Conclusions

miR-26a modulated angiogenesis of HCC through the PIK3C2α/Akt/HIF-1α/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.     

Murine pregnancy-specific glycoprotein 23 induces the proangiogenic factors transforming-growth factor beta 1 and vascular endothelial growth factor a in cell types involved in vascular remodeling in pregnancy

Haemochorial placentation is a unique physiological process in which the fetal trophoblast cells remodel the maternal decidual spiral arteries to establish the fetoplacental blood supply. Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen family. PSGs are produced by the placenta of rodents and primates and are secreted into the bloodstream. PSG23 is one of 17 members of the murine PSG family (designated PSG16 to PSG32). Previous studies determined that PSGs have immunoregulatory functions due to their ability to modulate macrophage cytokine secretion. Here we show that recombinant PSG23 induces transforming growth factor (TGF) beta1, TGFB1, and vascular endothelial growth factor A (VEGFA) in primary murine macrophages and the macrophage cell line RAW 264.7 cells. In addition, we identified new cell types that responded to PSG23 treatment. Dendritic cells, endothelial cells, and trophoblasts, which are involved in maternal vasculature remodeling during pregnancy, secreted TGFB1 and VEGFA in response to PSG23. PSG23 showed cross-reactivity with human cells, including human monocytes and the trophoblast cell line, HTR-8/SVneo cells. We analyzed the binding of PSG23 to the tetraspanin CD9, the receptor for PSG17, and found that CD9 is not essential for PSG23 binding and activity in macrophages. Overall these studies show that PSGs can modulate the secretion of important proangiogenic factors, TGFB1 and VEGFA, by different cell types involved in the development of the placenta.     

The Decrease of Mineralcorticoid Receptor Drives Angiogenic Pathways in Colorectal Cancer

Angiogenesis plays a crucial role in tumor growth and progression. Low expression of mineralocorticoid receptor (MR) in several malignant tumors correlates with disease recurrence and overall survival. Previous studies have shown that MR expression is decreased in colorectal cancer (CRC). Here we hypothesize that decreased MR expression can contribute to angiogenesis and poor patient survival in colorectal malignancies. In a cohort of CRC patients, we analyzed tumor MR expression, its correlation with tumor microvascular density and its impact on survival. Subsequently, we interrogated the role of MR in angiogenesis in an in vitro model, based on the colon cancer cell line HCT116, ingenierized to re-express a physiologically controlled MR. In CRC, decreased MR expression was associated with increased microvascular density and poor patient survival. In pchMR transfected HCT116, aldosterone or natural serum steroids largely inhibited mRNA expression levels of both VEGFA and its receptor 2/KDR. In CRC, MR activation may significantly decrease angiogenesis by directly inhibiting dysregulated VEGFA and hypoxia-induced VEGFA mRNA expression. In addition, MR activation attenuates the expression of the VEGF receptor 2/KDR, possibly dampening the activation of a VEGFA/KDR dependent signaling pathway important for the survival of tumor cells under hypoxic conditions.     

VEGFA Expression Is Inhibited by Arsenic Trioxide in HUVECs through the Upregulation of Ets-2 and miRNA-126

Arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia. Recently, studies have shown that ATO can induce apoptosis in leukemic cells and blood vessel endothelial cells in a time- and dose-dependent manner through the inhibition of vascular endothelial growth factor A (VEGFA) production. VEGFA is a key factor in angiogenesis initiation. Targeted inhibition of VEGF or VEGFA expression can suppress angiogenesis; however, little is known about the mechanism by which ATO inhibits VEGFA expression. In this study, we investigated the role of miRNA-126 in the mechanism of action of ATO in human umbilical vein endothelial cells (HUVECs). ATO significantly decreased the viability and proliferation of HUVECs and decreased their migration at 48 h. Cell proliferation was inhibited by 50% (IC50) when 5.0 μmol/L ATO was used. ATO treatment induced miR-126 upregulation and HUVEC apoptosis. Transfection with a miR-126 mimic significantly downregulated VEGFA mRNA levels, and transfection with a miR-126 inhibitor significantly upregulated VEGFA mRNA levels. Finally, we showed that ATO treatment upregulated Ets-2 and miR-126 expression in HUVECs. These results demonstrate that ATO inhibits the growth of HUVECs and induces apoptosis by downregulating VEGFA. One mechanism by which this occurs is Ets-2 upregulation, which results in an increase in miR-126 levels and downregulation of VEGFA expression.     

The balance of proangiogenic and antiangiogenic VEGFA isoforms regulate follicle development

Vascular endothelial growth factor A (VEGFA) has been extensively studied because of its role in follicular development and is a principal angiogenic factor essential for angiogenesis. Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, VEGFA might influence follicle growth via the regulation of angiogenesis. However, VEGFA might also influence follicular development through nonangiogenic mechanisms, since its expression has been localized in nonvascular follicles and cells. Alternative mRNA splicing of eight exons from the VEGFA gene results in the formation of various VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms (VEGFA_XXX) are encoded by exon 8a, whereas a sister set of isoforms (VEGFA_XXXB) with antiangiogenic properties is encoded by exon 8b. The antiangiogenic VEGFA_XXXB isoforms comprise the majority of VEGFA expressed in most tissues, whereas expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. Although proangiogenic and antiangiogenic isoforms can now be distinguished from one another, many studies evaluating VEGFA in ovarian and follicular development up to now have not differentiated proangiogenic VEGFA from antiangiogenic VEGFA. Experiments from our laboratory indicate that proangiogenic VEGFA promotes follicle recruitment and early follicular development and antiangiogenic VEGFA inhibits these processes. The balance of proangiogenic versus antiangiognic VEGFA isoforms is thus of importance during follicle development. Further studies are warranted to elucidate the way that this balance regulates follicular formation and progression.     

Tregs are involved in VEGFA/ VASH1-related angiogenesis pathway in ovarian cancer

Vasohibin1 (VASH1) is a kind of vasopressor, produced by negative feedback from vascular endothelial growth factor A (VEGFA). Anti-angiogenic therapy targeting VEGFA is currently the first-line treatment for advanced ovarian cancer (OC), but there are still many adverse effects. Regulatory T cells (Tregs) are the main lymphocytes mediating immune escape function in the tumor microenvironment (TME) and have been reported to influence the function of VEGFA. However, whether Tregs are associated with VASH1 and angiogenesis in TME in OC is unclear. We aimed to explore the relationship between angiogenesis and immunosuppression in the TME of OC. We validated the relationship between VEGFA, VASH1, and angiogenesis in ovarian cancer and their prognostic implications. The infiltration level of Tregs and its marker forkhead box protein 3 (FOXP3) were explored in relation to angiogenesis-related molecules. The results showed that VEGFA and VASH1 were associated with clinicopathological stage, microvessel density and poor prognosis of ovarian cancer. Both VEGFA and VASH1 expression were associated with angiogenic pathways and there was a positive correlation between VEGFA and VASH1 expression. Tregs correlated with angiogenesis-related molecules and indicated that high FOXP3 expression is harmful to the prognosis. Gene set enrichment analysis (GSEA) predicted that angiogenesis, IL6/JAK/STAT3 signaling, PI3K/AKT/mTOR signaling, TGF-β signaling, and TNF-α signaling via NF-κB may be common pathways for VEGFA, VASH1, and Tregs to be involved in the development of OC. These findings suggest that Tregs may be involved in the regulation of tumor angiogenesis through VEGFA and VASH1, providing new ideas for synergistic anti-angiogenic therapy and immunotherapy in OC.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Upregulated miR-182 increases drug resistance in cisplatin-treated HCC cell by regulating TP53INP1

Chemotherapy plays a crucial role in hepatocellular carcinoma (HCC) treatment especially for patients with advanced HCC. Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of HCC. However, acquisition of cisplatin resistance is common in patients with HCC, and the underlying mechanism of such resistance is not fully understood. In the study, we focused on identifying the role of miRNAs in chemotherapy resistance after cisplatin-based combination chemotherapy. We assayed the expression level of miR-182 after cisplatin-based chemotherapy in patients with advanced HCC, and defined the biological functions by real-time PCR analysis and CCK-8 assay. We found that miR-182 levels were significantly increased in HCC patients treated with cisplatin-based chemotherapy. miR-182 levels were also higher in cisplatin-resistant HepG2 (HepG2-R) cells than in HepG2 cells. Upregulated miR-182 significantly increased the cell viability, whereas miR-182 knockdown reduced the cell viability during cisplatin treatment. miR-182 inhibition also partially overcame cisplatin resistance in HepG2-R cell. Furthermore, we found that upregulated miR-182 inhibited the expression of tumor suppressor gene TP53INP1 (tumor protein 53-induced nuclear protein1) in vitro. In vivo, miR-182 and TP53INP1 expression was negatively correlated. We finally demonstrated that miR-182 increased cisplatin resistance of HCC cell, partly by targeting TP53INP1. These data suggest that miR-182/TP53INP1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.     

Plasma from pre‐eclamptic patients induces the expression of the anti‐angiogenic miR‐195‐5p in endothelial cells

We examined the effect of plasma incubation from preeclampsia pregnant on the antiangiogenic miR‐195‐5p expression. Higher miR‐195‐5p expression was found in cultures incubated with preeclampsia plasma compared to those incubated with healthy pregnant plasma. Next, as VEGF is a target of miR‐195‐5p we have quantified its expression by real‐time qPCR and ELISA. We found reduced VEGF levels in culture incubated with preeclampsia plasma. Therefore, we have concluded that the higher expression of miR‐195‐5p in endothelial cell cultures incubated with preeclampsia plasma may contribute to decreased expression of VEGFA (gene and protein) and increased antiangiogenic status in preeclampsia. Therefore, this miR may be an important target in preeclampsia.     

LINC00628 suppresses migration and invasion of hepatocellular carcinoma by its conserved region interacting with the promoter of VEGFA

Accumulated evidence revealed that numerous long noncoding RNAs (lncRNAs) have been found to be involved in the development and progression of hepatocellular carcinoma (HCC). LINC00628, a member of lncRNAs, has been reported to act as a tumor suppressor in gastric cancer and breast cancer. However, its potential role in HCC still remains unknown. Herein, we characterized the function of LINC00628 in HCC. Our investigation has revealed that LINC00628 were dramatically decreased in HCC tissues and cells, and inhibited the migration and invasion of HCC cells in vitro and in vivo. Moreover, LINC00628 exerted its tumor suppressive function by repressing the vascular endothelial growth factor A (VEGFA) promoter activity. A highly conserved region element in LINC00628 was identified by a cross-species comparative analysis, which is required for LINC00628 exerted its function. Dual-luciferase reporter assay showed that the conserved sequence mediated the interaction with a specific region of VEGFA promoter, resulting in a decrease of VEGFA expression. In conclusion, our results demonstrated that LINC00628 could function as a tumor suppressor in HCC via its conserved sequence elements interacting with a particular region of VEGFA promoter, suggesting that LINC00628 may serve as a novel promising target for diagnosis and therapy in HCC.     

VEGFA and VEGFR2 RNAscope determination in gastric cancer

Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.