The Mechanism Of VEGF Induced Hyper-permeability Of Vascluar In The Lung Pre-metastatic Niche

Background and AimsMetastasis is the main reason leading to the death of cancer patients.Therefore,it is of vital importanc to reveal the mechanism of cancer metastasis and to develop new strategies to prevent and control tumor metastasis.During metastasis,cancer cells intravasate from the primary tumour,migrate to a secondary site via the blood vessels,extravasate into the surrounding tissue and form metastatic nodules.In addition,primary tumors can induce the formation of pre-metastatic niches in secondary organs,which are suitable for the colonization and outgrowth of cancer cells.Targeting the pre-metastatic niche may be an effective way to intervene in cancer metastasis.Therefore,it is of great value to identify molecules participating in the formation of pre-metastatic niche that could be used for the intervention of cancer metastasis.Six characteristics have been summarized to define the pre-metastatic niche,including reprogramming,inflammation,lymphangiogenesis,angiogenesis/vascular permeability,organotropism,and immunosuppression.Among these characteristics,the hyper-permeability of vessels in the pre-metastatic niche is the first step in the development of metastasis.A significant study found that metastatic cancer cells preferentially homed to the hyperpermeable foci in LLC-or E0771-mammary carcinoma-bearing-lungs.Therefore,it is crucial to find the mechanism of increased vascular permeability.VEGF is a family of vasoactive compounds with different isoforms,some of which play a key role in the formation of pre-metastatic niche.Recent evidence has suggested that vascular endothelial growth factor(VEGF)secreted by primary tumor cells induces vascular hyper-permeability in the pre-metastatic niche and promotes tumor metastasis.But little is known about the mechanism of vascular hyperpermeability under the VEGF induction in the formation of pre-metastatic niche.Several studies have identified that alterations of tight junction(TJ)complex proteins may lead to damage to the blood-retinal barrier(BRB)and vascular hyper-permeability during retinal diseases.Incubating the endothelial cells with VEGF alters occludin content and distribution in endothelial cell barriers.Occludin is involved in many pathological and physiological processes in vivo,including barrier permeability,cell growth,cell differentiation,epithelial-mesenchymal transition and cancer invasiveness.A recent study suggested that exosomal miR-25-3p regulates the expression of occludin in endothelial cells,consequently promoting vascular permeability and angiogenesis in the pre-metastatic niche.Therefore,we hypothesized that occludin may participate in the formation of pre-metastatic niche,as a downstream molecule of VEGF.David A.Antonetti et al.found that phosphorylation at Ser-490 and ubiquitination were related to the occludin decrease in response to VEGF in endothelial cells.In this study,we tested whether occludin phosphorylation and ubiquitination contribute to VEGF-induced occludin down regulation and hyper-permeability in the pre-metastatic niche.In the current study,we used the 4T1 breast cancer model to test the hypothesis that primary tumor secreted VEGF reduces occludin expression coordinately with increased vascular permeability in the lung pre-metastatic niche.Furthermore,we evaluated the mechanism of VEGF induced occludin reduction.We aimed to identify an underlying molecule for the intervention of cancer metastasis and to provide a potential strategy to prevent the formation of the lung pre-metastatic niche.Part 1 Primary Tumor Secreted VEGF Induce Vascular Hyper-permeability in Lung Pre-metastatic Niche by Down-regulating OccludinObjectiveTo verify that primary tumor increased the pulmonary vascular permeability in pre-metastatic phase by down-regulating the tight junction protein occludin.Methods1.Establishment of 4T1 breast cancer model:Female BALB/c mice,aged 6-8 weeks(20-22 g)were used to establish the 4T1 breast cancer model.In the 4T1 group,1×1054T1 tumor cells were inoculated into the coupled abdominal mammary glands to establish a tumor model.The control group was inoculated with isometric amounts of PBS.2.Calculation of wet/dry ratios and histological studies:The mice were randomly divided into two groups,the control group and the 4T1 group.On day 0,7,14 and 21,5 mice in the control and 4T1 groups were sacrificed.The left lung of the mouse was used for wet/dry analysis,the superior lobe of the right lung was used for H&E staining.3.ELISA assay:Cytokines in mouse serum and supernatants of 4T1 cell culture medium were measured using the Enzyme-Linked Immune Sorbent Assay(ELISA)kit.4.Permeability assay:Two weeks after establishing the 4T1 breast tumor model,rhodamine-conjugated dextran was intravenously injected into the mice.Four hours later,fluorescein isothiocyanate(FITC)-lectin was intravenously injected into mice.After thirty minutes,the mice were anesthetized and perfused with saline.Lung tissues were cryosectioned and examined under an LSM 700 confocal microscope.The effect of primary breast cancer on the expression of tight junction protein occludin in lung tissue was observed by immunofluorescence.5.Western Blotting and immunofluorescence were used to observe the expression of tight junction protein occludin.6.Transfection:Wild type occludin was transfected into the HUVECs to testify the effect of occludin on the permeability of HUVECs.Results1.The lung wet/dry-weight ratios were increased in the tumor-bearing mice,which suggested increased pulmonary vascular permeability.HE staining showed that,inflammatory cell was infiltrated in the lung tissues of 4T1 tumor-bearing mice.2.The permeability assay showed that:the blood vessels was destabilized and that the permeability of the vasculature was increased in the pre-metastatic lung.3.The ELISA assay indicated that the levels of IL-6,VEGF and GM-CSF were elevated in the 4T1-tumor-bearing mice.However,only the level of VEGF was markedly increased in the 4T1 cell culture medium,indicating that VEGF secreted by 4T1 cells may be critical in triggering the formation of the pre-metastatic niche.4.The pulmonary permeability was blocked by bevacizumab,which further verified the influence of VEGF in the induction of vascular hyper-permeability.5.Primary tumor induced the decrease of tight junction protein occludin in the pre-metastatic lung.6.In vitro studies demonstrated that VEGF increased the permeability of dextran and decreased the levels of occludin in HUVECs.7.Moreover,the hyper-permeability of vessels and the degradation of occludin was blocked by bevacizumab.8.Overexpression of occludin alleviated the VEGF induced hyper-permeability.Conclusions1.VEGF secreted by primary tumors was elevated in the pre-metastatic lung.2.Primary tumor secreted VEGF may induce the hyper-permeability of vessels by regulating the TJ proteins in the pre-metastatic lung.Part 2 Primary Tumor Secreted VEGF Induces the Down-regulatin of Occludin via the Phosphorylation/ubiquitination PathwayObjectiveTo verify that VEGF induce the down-regulation of occludin via the phosphorylation/ubiquitination pathway.Methods1.Western Blotting was used to detect the phosphorylation of occludin and after VEGF induction.2.Co-IP was used to detect the ubiquitination of occludin after VEGF induction.3.Flag-tagged occludin mutants in which Ser-490 was substituted with aspartic acid(S490DOcc)was transfected into HUVECs,ubiquitination was determined by Co-IP with an anti-Flag antibody followed by Western Blotting.4.Flag-tagged wild type occludin and occludin mutants in which Ser-490 was substituted to alanine(S490AOcc)were transfected into HUVECs,the ubiquitination of wild type occludin and S490AOcc were determined by Co-IP.5.After incubated with cycloheximide,VEGF and MG 132,degradation rates of occludin in HUVECs were evaluated by Western Blotting.Results1.Western Blotting showed that the Ser-490-phosphorylated occludin(pS490)level was increased by approximately fourfold at 15 minutes after VEGF induction.2.The ubiquitinated occludin at 59 kDa and poly-ubiquitinated occludin levels increased after VEGF treatment.3.Flag-tagged occludin mutants in which Ser-490 was substituted with aspartic acid(S490DOcc)was transfected into HUVECs by plasmid.Co-IP showed that VEGF increased ubiquitination of the Ser-490 phosphorylated form of occludin.4.Flag-tagged WtOcc and occludin mutants in which Ser-490 was substituted to alanine(S490AOcc)were transfected into HUVECs by plasmid.After treatment with VEGF,WtOcc was ubiquitinated and the ubiquitinated-S490AOcc was minimal.Our results indicated that phosphorylation at Ser-490 is indispensable for VEGF-induced occludin ubiquitination.5.VEGF promotes phosphorylation-ubiquitination of occludin by activating ERK signaling pathway.6.VEGF quicked the process of occludin degradation.The increased rate of occludin degradation was prevented by the proteasome inhibitor MG132,indicating that the VEGF-induced occludin degradation can be completely prevented by the proteasome inhibitor.Our results suggested that VEGF accelerated the process of occludin degradation,through the ubiquitin-proteasome system.ConclusionIn the pre-metastatic lung,VEGF induced the down-regulation of occludin via the phosphorylation/ubiquitination pathway.