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.     

Exosomal LBH inhibits epithelial-mesenchymal transition and angiogenesis in nasopharyngeal carcinoma via downregulating VEGFA signaling

The limb-bud and heart (LBH) gene was reported to suppress nasopharyngeal carcinoma (NPC) progression in our previous study. Distant metastasis predominantly accounts for the unsatisfactory prognosis of NPC treatment, in which epithelial-mesenchymal transition (EMT) and tumor angiogenesis are of great significance. The roles of exosomes in mediating NPC progression have been highlighted in recent researches, and attempts have been made to explore the clinical application of NPC exosomes. Here we investigated the function of the LBH gene in NPC exosomes, and its potential mechanism. NPC xenografts were constructed, showing that vascular endothelial growth factor A (VEGFA) expression and neovascularity were attenuated by LBH overexpression, together with diminished EMT progression. NPC-derived exosomes were isolated, identified and applied for in vitro/in vivo experiments, and the exosomal distribution of LBH was elevated in exosomes derived from LBH-upregulated cells. Ectopic LBH, αB-crystallin (CRYAB) and VEGFA expression was induced by lentiviral infection or plasmid transfection to explore their functions in modulating EMT and angiogenesis in NPC. The addition of LBH+ NPC exosomes during a Matrigel plug assay in mice suppressed in vivo angiogenesis, and the treatment of human umbilical vein endothelial cells (HUVECs) with LBH+ NPC exosomes inhibited cellular proliferation, migration and tube formation. The interactions among LBH, CRYAB and VEGFA were confirmed by colocalization and fluorescence resonance energy transfer (FRET) assays, and extracellular VEGFA secretion from both HUVECs and NPC cells under the treatment with LBH+ NPC exosomes was diminished according to ELISA results. We concluded that exosomal LBH inhibits EMT progression and angiogenesis in the NPC microenvironment, and that its effects are partially implemented by modulation of VEGFA expression, secretion and related signaling. Thus, LBH could serve as a promising therapeutic target in VEGFA-focused NPC treatment.     

Exosome-mediated lncRNA SNHG11 regulates angiogenesis in pancreatic carcinoma through miR-324-3p/VEGFA axis

Pancreatic carcinoma (PC) is one of the most common and deadly human malignancies worldwide. LncRNAs play significant roles in the occurrence and development of various cancers. LncRNA SNHG11 (SNHG11) has been found to display high expression in serum of PC patients, which implies that dysregulated SNHG11 may be related to the development of PC. However, there is still a knowledge gap concerning the specific function and molecular mechanism of SNHG11 in PC. After conducting experiments with constructed models in vitro or in vivo, we found that exosomal SNHG11 promoted cell proliferation, migration, and angiogenesis but impeded cell apoptosis in PC in vitro, and additionally, it facilitated tumor growth in vivo. Exosome-mediated SNHG11 regulated the expression of VEGFA through sponging miR-324-3p. Rescue assays validated that the inhibitory effect of SNHG11 depletion on cell proliferation, migration, and angiogenesis could be reversed by miR-324-3p downregulation or VEGFA upregulation, and the promoting effect of SNHG11 silence on cell apoptosis could be rescued by transfection of miR-324-3p inhibitor or pcDNA3.1-VEGFA. To conclude, exosomal-mediated SNHG11 could regulate PC progression via miR-324-3p/VEGFA axis. Our findings may provide a novel insight for understanding PC, which might contribute to the development of potential PC biomarker.     

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.     

RASAL2 mediated the enhancement of YAP1/TIAM1 signaling promotes malignant phenotypes of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high incidence of metastasis and dismal prognosis. As a member of Gas-Gap gene, RASAL2 is involved in the hydrolysis of RAS-GTP to RAS-GDP and abnormal expression in human cancers. Here we firstly described the function of RASAL2 on PDAC to enrich the knowledge of RAS family.We interestingly observed that RASAL2 expression was upregulated in PDAC at both mRNA and protein levels, and high expression of RASAL2 predicted a poor prognosis in PDAC patients. Additionally, RASAL2 promoted malignant behaviors of PDAC in vitro and in vivo. To determine the mechanistic roles of RASAL2 signaling and its potential as a therapeutic target in PDAC, we clarified that RASAL2 could accumulate the TIAM1 expression in different level through inhibiting YAP1 phosphorylation, increased TIAM1 mRNA expression and suppressed ubiquitination of TIAM1 protein. In conclusion, RASAL2 enhances YAP1/TIAM1 signaling and promotes PDAC development and progression.     

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.     

Piwil 2 Expression Is Correlated with Disease-Specific and Progression-Free Survival of Chemotherapy-Treated Bladder Cancer Patients

Piwi-like 2 (Piwil 2) belongs to the family of Argonaute genes/proteins. The expression of Piwil 2 is associated with stem cells. A role in tumorigenesis and/or tumor progression is proposed for different cancers but not yet for bladder cancer (BCa). We investigated Piwil 2 expression by immunohistochemistry in a cohort of 202 BCa patients treated by cystectomy and adjuvant chemotherapy. The association between Piwil 2 expression and disease-specific (DSS) or progression-free survival (PFS) was calculated using Kaplan-Meier analyses and univariate/multivariate Cox regression hazard models. In a multivariate Cox regression analysis, Piwil 2 expression, either in the cytoplasm or the nucleus, was significantly associated with DSS and PFS. A weak cytoplasmic staining pattern was associated with poor DSS and tumor progression (relative risk [RR] = 2.7, P = 0.004, and RR = 2.4, P = 0.027). Likewise, absent nuclear Piwil 2 immunoreactivity was associated with poor DSS and tumor progression (RR = 2.3, P = 0.023, and RR = 2.2, P = 0.022). BCa patients whose tumors exhibited a combination of weak cytoplasmic and absent nuclear immunoreactivity had a 6-fold increased risk of tumor-related death (P = 0.005) compared with patients with strong expression. Considering only patients with high-grade G3 tumors, a 7.8-fold risk of tumor-associated death and a 3.6-fold risk of tumor progression were detected independently of the histologic tumor subtype or the chemotherapy regimen. In summary, a combination of weak cytoplasmic and absent nuclear expression of Piwil 2 is significantly associated with an increased risk of DSS and tumor progression. This indicates that Piwil 2 could be a valuable prognostic marker for high-risk BCa patients.     

Microarray expression profiles analysis revealed lncRNA OXCT1-AS1 promoted bladder cancer cell aggressiveness via miR-455-5p/JAK1 signaling

Bladder cancer (BCa) is one of the most prevalent cancers of the urinary system worldwide. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) perform a vital function in the pathogenesis and progression of BCa. In the current study, we identified a novel lncRNA OXCT1-AS1 and investigated its role and potential mechanisms in BCa. The microarray results showed the expression of lncRNAs, microRNAs, and messenger RNAs between BCa primary tumor tissues and metastatic lymph nodes were significantly different. The quantitative polymerase chain reaction verification was performed to ensure the reliability of the screening results. The Cell Counting Kit 8 and transwell assay were used to assess the tumor cell proliferation and invasion abilities in vitro, respectively. The dual-luciferase activity assay was performed to investigate the potential mechanism of competing endogenous RNA network. lncRNA OXCT1-AS1, which elevated in metastasis lymph node, was significantly upregulated in BCa cell lines compared with SVHUC-1. We demonstrated OXCT1-AS1 inhibited miR-455-5p to decrease its binding to the JAK1 3′-untranslated region, which could upregulate the expression of JAK1 at the protein level, thus promoting BCa proliferation and invasion. Therefore, lncRNA OXCT1-AS1 could act as a potential biomarker and therapeutic target for patients with BCa.     

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.     

Paracrine and autocrine mechanisms of apelin signaling govern embryonic and tumor angiogenesis

Apelin and its G protein-coupled receptor APJ play important roles in blood pressure regulation, body fluid homeostasis, and possibly the modulation of immune responses. Here, we report that apelin-APJ signaling is essential for embryonic angiogenesis and upregulated during tumor angiogenesis. A detailed expression analysis demonstrates that both paracrine and autocrine mechanisms mark areas of embryonic and tumor angiogenesis. Knockdown studies in Xenopus reveal that apelin-APJ signaling is required for intersomitic vessel angiogenesis. Moreover, ectopic expression of apelin but not vascular endothelial growth factor A (VEGFA) is sufficient to trigger premature angiogenesis. In vitro, apelin is non-mitogenic for primary human endothelial cells but promotes chemotaxis. Epistasis studies in Xenopus embryos suggest that apelin-APJ signaling functions downstream of VEGFA. Finally, we show that apelin and APJ expression is highly upregulated in microvascular proliferations of brain tumors such as malignant gliomas. Thus, our results define apelin and APJ as genes of potential diagnostic value and promising targets for the development of a new generation of anti-tumor angiogenic drugs.     

Long noncoding RNA HOXD-AS1 induces epithelial-mesenchymal transition in breast cancer by acting as a competing endogenous RNA of miR-421

Breast cancer (BCa) is the most common malignant tumor in females. Long noncoding RNAs (lncRNAs) are deregulated in many types of human cancers, including BCa. The purpose of the present study was to examine the expression profile and biological role of HOXD cluster antisense RNA 1 (HOXD-AS1) in BCa. Our results revealed that HOXD-AS1 was upregulated in BCa tissues and cell lines, and high HOXD-AS1 expression was correlated with aggressive clinicopathological characteristics of BCa patients. Further gain-of-function and loss-of-function analysis showed that HOXD-AS1 overexpression promoted, whereas HOXD-AS1 knockdown inhibited BCa cell proliferation, cell cycle progression, migration, and invasion, indicating that HOXD-AS1 may function as a novel oncogene in BCa. Mechanistically, HOXD-AS1 could activate epithelial-mesenchymal transition (EMT) in BCa cells. We further proved that HOXD-AS1 might serve as a competing endogenous RNA of miR-421 in BCa cells, and miR-421 was downregulated and negatively correlated with HOXD-AS1 expression in BCa tissues. Besides, we confirmed that SOX4, a master regulator of EMT, was a direct target gene of miR-421. Further, rescue experiments suggested that miR-421 overexpression partly abrogated the oncogenic role of HOXD-AS1 in BCa cells. Therefore, we shed light on that HOXD-AS1/miR-421/SOX4 axis may be considered as a novel therapeutic target for the treatment of BCa patients.     

A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis

The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr¹²¹⁴ and its downstream effectors Akt and ERK1/2, and importantly its association with β1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity.     

XSSJS inhibits hepatic fibrosis by promoting the miR-29b-3p/VEGFA axis in vitro and in vivo

Hepatic pathological angiogenesis (HPA) is the key event of hepatic fibrosis (HF). Xueshisanjia powder (XSSJS), a Chinese herbal compound, is beneficial for alleviating pathological angiogenesis of hepatic tissue. The present study attempts to reveal the effect and mechanism of XSSJS via regulating miR-29b-3p/VEGFA axis against pathological angiogenesis in HF. In in vitro model, human embryonic kidney 293T cells were transfected with miR-29b-3p mimics, whereby the expression of miR-29b-3p was tested by real-time quantitative polymerase chain reaction (RT-qPCR), ensued by Luciferase assay determining the relationship between miR-29b-3p and vascular endothelial cell growth factor A (VEGFA). In addition, miR-29b-3p mimic transfected into the activated hepatic stellate cell T6 (HSC-T6). The Cell-Counting-Kit 8 (CCK8) and 5-Bromodeoxyuridine (BrdU) staining were first utilized to detect the antiproliferative efficiency of XSSJS following the XSSJS compound serum intervention, and then used to observe the expression of transforming growth factor-β (TGF-β), VEGFA, platelet-derived growth factor (PDGF) via RT-PCR, Western blot (WB), and Immunofluorescence (IF) methods. During the in vivo model, XSSJS with boil-free granules were fed to Wistar rats with liver fibrosis caused by intraperitoneal injection of pig serum followed by the transfection of miR-29b-3p adeno-associated virus (AAV). Hematoxylin–Eosin (HE) staining was used for histopathology assessment. The expression of miR-29b-3p, VEGFA, PDGF, TGF-β have been investigated in liver tissue using RT-PCR, WB, IF. The results verified that XSSJS could up-regulate miR-29b-3p and suppress the expression of VEGFA, PDGA, and TGF-β. In mechanism, miR-29b-3p primarily targeted the 3′UTR of VEGFA. In conclusion, XSSJS could modulate miR-29b-3p/VEGFA axis to inhibit the pathological angiogenesis of HF.