Ovarian cancer derived PKR1 positive exosomes promote angiogenesis by promoting migration and tube formation in vitro.

Cancer cell derived exosomes play important roles in cancer progression and modulation of the tumour microenvironment. This study aims to investigate the role of prokineticin receptor 1 (PKR1) positive exosomes on angiogenesis. In the present study, PKR1 expression in tumour samples from ovarian cancer patients were examined firstly. Then, two ovarian cancer cell lines, namely A2780 and HO-8910 cells, were used to isolate and obtain the PKR1 positive exosomes from the serum free medium. The function analysis of PKR1 positive exosomes on angiogenesis was conducted by cell proliferation and migration assay, tube formation analysis, and tumour volume assay. The results showed that PKR1 expression was down regulated in tumour samples of ovarian cancer patients compared with adjacent normal tissues. The intracellular expression of PKR1 could be detected in A2780 and HO-8910 cells. And, the isolated exosomes from the serum free medium were confirmed by transmission electron microscopic and NTA analysis, as well as the co-presence of PKR1 with exosome marker CD63. The function analysis of PKR1 positive exosomes on angiogenesis demonstrated the uptake of PKR1 positive exosomes by human umbilical vein endothelial cells through immunofluorescence staining. The angiogenesis assays in vitro indicated that PKR1 positive exosomes promoted migration and tube formation of HUVECs but not proliferation. The endogenous PKR1 was also verified to help to enhance migration and promote tube formation of vascular endothelial cells, which might involved in the phosphorylation of STAT3. Additionally, The tumour volume from exosomes treated A2780 tumour-bearing mice was significantly increased compared with the control group, accompanied with the induced PKR1 expression and phosphorylation of STAT3 level. SIGNIFICANCE OF THE STUDY: This study proved the important role of PKR1 positive exosomes released from ovarian cancer cells on promoting angiogenesis. The data indicated that PKR1 derived from ovarian cancer cells could act as an important tumour associated antigen and biomolecular factor for cellular communication in tumour microenvironment.

EG-VEGF silencing inhibits cell proliferation and promotes cell apoptosis in pancreatic carcinoma via PI3K/AKT/mTOR signaling pathway.

OBJECTIVE: Pancreatic carcinoma (PC), one of the most prevalent and malignant tumors, has a poor prognosis and a high mortality rate. EG-VEGF, a vascular endothelial growth factor from endocrine glands, also termed as PROK1, has a high positive expression rate in PC tissues and is involved in the pathogenesis of various tumors. However, the expression and potential role of EG-VEGF in PC has not been thoroughly explored. The aim of this study was to better clarify the expression and potential role of EG-VEGF in pancreatic carcinoma. METHODS: Immunohistochemical staining, western blotting, and RT-qPCR analysis were performed to detect the EG-VEGF level in PC tissues and cells. Subsequently, two short hairpin RNA (shRNA) lentiviral expression vector, shPROK1-1/shPROK1-2, were transfected into PANC-1 and BxPC-3 PC cell lines. MTT assay was used to determine cell proliferation. Meanwhile, flow cytometry assay was conducted to measure cell cycle and cell apoptosis. The protein levels of PI3K/AKT/mTOR pathway-related genes were also determined by western blotting. RESULTS: EG-VEGF was aberrantly expressed in PC samples, as compared with paracancerous samples. Knockdown of PROK1 notably decreased the protein level of EG-VEGF, indicating a successful downregulation model of EG-VEGF. EG-VEGF silencing remarkably attenuated cell proliferation, while also induced G0/G1 arrest and magnified the extent of cell apoptosis. Further, EG-VEGF knockdown significantly inhibited PI3K/AKT/mTOR signaling pathway by downregulating p-PI3K, p-AKT, and p-mTOR levels. CONCLUSION: This study identified the high-expression of EG-VEGF in pancreatic carcinoma tissues and cells, and demonstrated that EG-VEGF silencing inhibits the proliferation of PC cells and promotes apoptosis via regulating PI3K/AKT/mTOR pathway. Thus, EG-VEGF may become an essential target for the therapy of pancreatic cancer in the future.

Antagonism of EG-VEGF Receptors as Targeted Therapy for Choriocarcinoma Progression In Vitro and In Vivo.

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland-derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy.Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (n = 20) and in distinctive CHM (n = 38) and CC (n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice.Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions.Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130-40. ©2017 AACR.

Effects of antibodies to EG-VEGF on angiogenesis in the chick embryo chorioallantoic membrane.

BACKGROUND: Endocrine gland-related vascular endothelial growth factor (EG-VEGF), is an angiogenic factor specifically targeting endothelial cells derived from endocrine tissues. The inhibition of the EG-VEGF/prokineticin receptor pathway could represent a selective antiangiogenic and anticancer strategy. AIM: to evaluate the impact of an antibody to EG-VEGF on the rapidly growing capillary plexus of the chick embryo chorioallantoic membrane (CAM). MATERIALS AND METHODS: The in ovo CAM assay was performed for the humanized EG-VEGF antibody. RESULTS: Hemorrhagic damage was induced in the capillaries, which led to early death of the embryos. Upon morphological staining, there was evidence of vascular disruption and extravasation of red blood cells in the chorion. Signs of vacuolization of the covering epithelium were also observed. CONCLUSION: Blocking endogenous EG-VEGF might represent a valuable approach of impairing or inhibiting angiogenesis in steroidogenic-derived embryonic tissues.

Prokineticin 1 modulates IL-8 expression via the calcineurin/NFAT signaling pathway.

Prokineticins and their receptors are expressed in various cellular compartments in human endometrium, with prokineticin 1 (PROK1) showing a dynamic pattern of expression across the menstrual cycle and during pregnancy. Previous studies suggest that PROK1 can play an important role in implantation and early pregnancy by inducing vascular remodeling and increasing vascular permeability. Here we demonstrate that PROK1 induces the expression of IL-8, a chemokine with angiogenic properties, in endometrial epithelial Ishikawa cells stably expressing prokineticin receptor 1 and in human first trimester decidua. We also show that IL-8 promoter activity is induced by PROK1 and that this requires the presence of AP1 and NFAT motifs. The role of calcineurin/NFAT signaling pathway is confirmed by the use of specific chemical inhibitors. Additionally, PROK1 induces the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway. A modulatory role for RCAN1-4 is demonstrated by RCAN1-4 overexpression which results in the inhibition of PROK1-induced IL-8 expression whereas reduction in RCAN1-4 endogenous expression results in an increase in PROK1-induced IL-8 production. Our findings show that in endometrial cells PROK1 can activate the calcineurin/NFAT pathway to induce IL-8 expression and that this is negatively modulated by the induction of expression of RCAN1-4.