Vascular Endothelial Growth Factor Receptor-3 Directly Interacts with Phosphatidylinositol 3-Kinase to Regulate Lymphangiogenesis

Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCc1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis

Molecular mechanisms regulating lymphangiogenesis

Lymphangiogenesis, described as growth of nascent lymphatic vessels in vivo, and proliferation, migration, tube formation of lymphatic endothelial cells (LECs) in vitro, plays an important physiological role in homeostasis, metabolism and immunity. Lymphatic vessel formation has also been implicated in a number of pathological conditions including cancer metastasis, lymphedema and impaired wound healing. Elucidating molecular mechanisms that regulate LEC motility and its relationship to lymphangiogenesis will play an important part in understanding lymphatic biology and in studying disease associated with altered lymphatic vessel architecture or function. Cancer is the most commonly diagnosed malignancy and the leading cause of mortality around the globe. A key turning point in the course of cancer progression is the development of metastatic potential as most cancer related deaths are not due to the primary tumor, but rather to metastases. A better understanding of molecular mechanisms that underlie the process of cancer metastasis to lymph nodes will most likely result in improved therapeutic options for patients and thus help reduce the burden of the disease. This project focuses on discovering the molecular mechanisms that regulate the spread of cancer to lymph nodes and lymphangiogenesis, and will identify potential new targets for therapy. Lymph node metastasis is the first route of cancer cell dissemination, and may provide a bridgehead that subsequently results in tumor cell seeding into the lymphatic system to distant organs, which in turn leads to one of the major causes of morbidity associated with cancer progression. Currently, there are no effective therapies against these metastases. The delineation of the processes that result in lymphangiogenesis, including the roles of LECs in vitro, and the interaction between cancer cells and lymphatic environment in vivo is anticipated to lead to novel therapeutic strategies. The first aim of this study was to identify signaling pathways regulating lymph node metastasis via vascular endothelial growth factor receptor (VEGFR)-3. This was investigated in vitro using human LECs (hLECs), and in vivo using clinical material. The direct association of VEGFR-3/ phosphatidylinositol 3-kinase (PI3K) was shown for the first time in the metastatic small cell lung carcinoma samples. Second aim of the study was to isolate and characterise murine LECs (mLECs) from dermis and prostate. We have shown that regulation of lymphangiogenesis by VEGF family ligands is conserved in mLECs and hLECs. This finding was an important discovery into how mLECs are regulated and will provide bridgehead into using these mLECs as tools for studying lymphatic system related diseases such as cancer. NADPH oxidases (Nox) family are emerging as novel regulators of tumor angiogenesis and were previously shown to regulate blood vascular endothelial signaling via reactive oxygen species (ROS). Nox2 was described as a major regulator of migration and proliferation of vascular endothelial cells. In the third aim of the study, using the techniques described in Aim 2, mLECs were derived from Nox2-/- mice and wild-type mice in order to define the role of Nox2. Key signaling pathways regulated by Nox2/VEGFR-2 that modulate murine and human LEC migration and tube formation were identified. This work provides an important insight into the role of Nox2 as a potential regulator of lymphangiogenesis. These findings add to the current knowledge of VEGF receptor signaling pathways, and introduce a potentially important new player, Nox2. Hence, targeting VEGF receptor family/Nox2 may provide new emerging therapeutic targets and could have beneficial clinical effects in treatment of lymphatic associated disorders

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function

NADPH oxidases as regulators of tumor angiogenesis: current and emerging concepts

Significance Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and peroxynitrite are generated ubiquitously by all mammalian cells and have been understood for many decades as inflicting cell damage and as causing cancer by oxidation and nitration of macromolecules, including DNA, RNA, proteins, and lipids. Recent Advances A current concept suggests that ROS can also promote cell signaling pathways triggered by growth factors and transcription factors that ultimately regulate cell proliferation, differentiation, and apoptosis, all of which are important hallmarks of tumor cell proliferation and angiogenesis. Moreover, an emerging concept indicates that ROS regulate the functions of immune cells that infiltrate the tumor environment and stimulate angiogenesis, such as macrophages and specific regulatory T cells. Critical Issues In this article, we highlight that the NADPH oxidase family of ROS-generating enzymes are the key sources of ROS and, thus, play an important role in redox signaling within tumor, endothelial, and immune cells thereby promoting tumor angiogenesis. Future Directions Knowledge of these intricate ROS signaling pathways and identification of the culprit NADPH oxidases is likely to reveal novel therapeutic opportunities to prevent angiogenesis that occurs during cancer and which is responsible for the revascularization after current antiangiogenic treatment

VEGF-C induces LEC tube formation.

<p><i>A</i>, Prostatic LEC tube length at 4.5 hours post VEGF-C treatment was quantified using ImageJ. VEGF-C significantly increased the number of tubes formed compared to vehicle control. Data expressed as mean±s.e.m., n = 3, ***<i>P</i>&lt;0.001 using One-way ANOVA, Bonferroni post-analysis. <i>B</i>, Western blotting analysis of VEGFR-2 and VEGFR-3 expression in lung, neonatal dermis and prostate LECs. β-tubulin was used as a loading control.</p

Direct interaction between PI3K p85 and VEGFR-3 in metastatic small cell lung carcinoma.

<p>VEGFR-3/PI3K complexes (<i>red</i>) detected by <i>in situ</i> PLA in lung tumor area and lymphatic vessels (<i>green,</i> podoplanin). Lymphatic vessel VEGFR-3/PI3K signals (<i>red</i>) and their representative co-staining with podoplanin (<i>green</i>) in metastatic (<i>A, top and middle panel,</i> respectively) and non-metastatic (<i>B, top and middle panel,</i> respectively) in small cell lung cancer tissue. VEGFR-3/PI3K signals were detected in cancer cells surrounding the lymphatic vessels in the metastatic samples (<i>A</i>, <i>top and bottom panel</i>) as well as the lymphatic vessels; whereas the low signal in non-metastatic samples was detected mostly away from the lymphatic vessels (<i>B</i>, <i>bottom panel</i>), and not in tumor cells inside or surrounding the non-metastatic lymphatic vessels (<i>B</i>, <i>top and middle panels</i>). Bar, 25 µm. DAPI (<i>blue</i>); <i>C.</i> Quantification of PLA signals (at least 5 different regions in each sample) in the lymphatic vessels (LV) (<i>C</i>, <i>left</i>) in lymph node negative (LN-; n = 7; two samples were excluded because there were no lymphatic vessels present in the sections examined) and lymph node positive (LN+; n = 10) samples using Olink Imaging Software. Plotted are mean values for individual patients. Line represents median value. *<i>P</i>&lt;0.05.</p

Direct interaction between PI3K p85 and VEGFR-3 in metastatic melanoma (A), breast (B) and colon (C) cancers.

<p>PI3K p85/VEGFR-3 complexes (<i>red</i>) detected by <i>in situ</i> PLA in tumor cells and lymphatic vessels (<i>green,</i> podoplanin). Bar, 50 µm. Nuclei stained with DAPI (<i>blue</i>).</p

Blocking PI3K inhibits VEGF-C-induced LEC tube formation and migration.

<p>Effect of anti-VEGFR-3 antibody (hF4-3C5, 20 µg/ml), selective PI3Kγ inhibitor (AS252424, 5 µM), Raf/MEK inhibitor (PD98059, 5 µM) and PLCγ1 inhibitor (U-73122, 1 µM) on VEGF-C-induced (200 ng/ml) LEC tube formation (<i>A</i>) and migration (<i>B</i>, <i>left panel</i>). Effect of PLCγ1 siRNA or non-targeting control (NTC) on LEC VEGF-C-induced migration (<i>B</i>, <i>right panel</i>). Control serum-free (with DMSO as appropriate) LEC is indicated by ‘0′. *<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01, ***<i>P</i>&lt;0.001, compared to VEGF-C treated cells/NTC. n = 3. Data shown as mean±s.e.m.</p

VEGF-C induces PI3Kγ-dependent P70S6K (A), eNOS (B), and PLCγ1 (C) phosphorylation via VEGFR-3 in LECs.

<p>Western blotting analysis of phosphorylated P70S6K (<i>A, top left</i>) and eNOS (S1177) (<i>B, top left</i>) in prostatic LECs following 15 minute stimulation with ligand: VEGF-C (100 ng/ml), VEGF-A (100 ng/ml), VEGF-C156S (250 ng/ml), VEGF-D (250 ng/ml) and VEGF-E (100 ng/ml). Time- and concentration-dependent phosphorylation of P70S6K (<i>A, top right</i>), eNOS (S1177) (<i>B, top right</i>), and PLCγ1 (Tyr783) (<i>C, top left</i>) in LECs in response to VEGF-C. The effect of inhibition of VEGFR-3 (hF4-3C5), VEGFR-2 (IMC-1121b) or VEGFR-1 (IMC-18F1) on phosphorylation of P70S6K (<i>A, bottom left</i>), eNOS (S1177) (<i>B, bottom left</i>); and VEGFR-3 (hF4-3C5) on PLCγ1 (Tyr783) (<i>C, bottom right</i>) on LEC response to VEGF-C (100 ng/ml). The effect of AS252424, LY294002, PD98059, and U-73122 on phosphorylation of P70S6K (<i>A, top right</i>), eNOS (S1177) (<i>B, bottom right</i>), and PLCγ1 (Tyr783) (<i>C, bottom right</i>) on LECs in response to VEGF-C. The effect of VEGF-C on phosphorylation of PLCγ2 (Tyr759, Tyr1217) in LECs (<i>C, bottom left</i>). Control serum-free vehicle treated LEC lysate is indicated by ‘0′ in all blots. n = 3. Densitometry analysis is shown under each blot in italics; where integrated intensity of phosphorylated molecules was firstly compared to that of the total target protein for each sample, and then expressed as fold change in integrated density compared to either serum-free (A, <i>top left and right panels</i>; B, <i>top left and right panels</i>; C, <i>top and bottom left panels</i>) or VEGF-C treated control samples (A, <i>bottom left and right panels</i>; B, <i>bottom left and right panels</i>; C, <i>bottom and bottom left panels</i>).</p

Differential expression of VEGF ligands and receptors in prostate cancer

BACKGROUND\ud Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer.\ud \ud METHODS\ud The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer.\ud \ud RESULTS\ud Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue.\ud \ud CONCLUSIONS\ud These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer