NRP2 axis in tumor lymphangiogenesis and lymphatic metastasis

Clinica Chimica Acta 461 (2016) 165–171

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Review

Pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis in tumor lymphangiogenesis and lymphatic metastasis Jingwen Wang, Yuhong Huang, Jun Zhang, Yuanyi Wei, Salma Mahoud, Ahmed Musa Hago Bakheet, Li Wang, Shuting Zhou, Jianwu Tang ⁎ Department of Pathology, Dalian Medical University, Key Laboratory for Tumor Metastasis and Intervention of Liaoning Province, 9 West, Lvshun Southern Road, Dalian, Liaoning 116044, China

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Article history: Received 24 June 2016 Received in revised form 7 August 2016 Accepted 11 August 2016 Available online 12 August 2016 Keywords: VEGFC/D-VEGFR3/NRP2 axis Pathway-related molecules Lymphangiogenesis lymphatic metastasis Tumor

a b s t r a c t Precondition for tumor lymphatic metastasis is that tumor cells induce formation of original and newborn lymphatic vessels and invade surrounding lymphatic vessels in tumor stroma, while some pathway-related molecules play an important role in mechanisms associated with proliferation and migration of lymphatic endothelial cells (LECs) and tumor cells. In lymphangiogenesis and lymphatic metastasis, the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, such as Furin-like enzyme, CNTN1, Prox1, LYVE-1, Podoplanin, SOX18, SDF1 and CXCR4, are direct constitutors as a portion of VEGFC/D-VEGFR3/NRP2 axis, and their biological activities rely on this ligand-receptor system. These axis-related signal molecules could gradually produce waterfall-like cascading effects, mediate differentiation and maturation of LECs, remodel original and neonatal lymphatic vessels, as well as ultimately promote tumor cell chemotaxis, migration, invasion and metastasis to lymphoid tracts. This review summarizes the structure and function features of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, the expression changes of these molecules in different anatomic organs or histopathologic types or development stages of various tumors, the characteristics of transduction, implementation, integration of signal networks, the interactive effects on biological behaviors between tumor cells and lymphatic endothelial cells, and their molecular mechanisms and significances in tumor lymphangiogenesis and lymphatic metastasis. © 2016 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structures and functions of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis . . . . . . . . . . . . . . . . . . . . . . . . . The expression changes of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis in tumor lymphangiogenesis and lymphatic metastasis The mechanisms and significances of the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis on tumor lymphangiogenesis and lymphatic metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Abbreviations: LECs, lymphatic endothelial cells; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptors; NRP2, neuropilin-2; PlGF, Placenta Growth Factor; CNTN1, contactin-1; Prox1, Prospero homeobox protein1; LYVE-1, lymphatic endothelial hyaluronic acid (HA) recepror-1; SOX, sex-determining region Y chromosome related high mobility group box; SDF1, stromal cell derived factor1; CXCR4, chemokine CXC receptor4. ⁎ Corresponding author. E-mail address: [email protected] (J. Tang).

http://dx.doi.org/10.1016/j.cca.2016.08.008 0009-8981/© 2016 Elsevier B.V. All rights reserved.

Tumor lymphatic metastasis can be successfully realized though tumor cell proliferation, invasion and migration, which is closely related to the numbers, structures and functions of original and neonatal lymphatic endothelial cells (LECs) in tumor stroma, to the interactive recognition and connection between tumor cells and LECs, to the capacity of tumor cell movement and penetration through the endothelial cell

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barriers of lymphatic vessels, and also to the effections of signal transduction pathway molecules that are activated by the binding of specific ligands to corresponding receptors. The vascular endothelial growth factor (VEGF) is early referred to the vascular permeability factor which is a heparin-binding growth factor for blood vascular endothelial cells and LECs. The VEGF family has several subtypes, such as VEGFA, B, C, D, E, F and PlGF (Placenta Growth Factor), which specifically combine with vascular endothelial growth factor receptors (VEGFR), such as VEGFR1,2,3 and NRP2. In general, VEGFA and VEGFB mostly combine with VEGFR1 and VEGFR2 on the surface of blood vascular endothelial cells, while VEGFC and VEGFD predominantly bind to VEGR3 and NRP2 on the surface of LECs. In the process of tumor lymphangiogenesis and lymphatic metastasis, the typical pathway-related molecules as direct constitutors of VEGFC/D-VEGFR3/ NRP2 axis play an extremely important role in the complicated biological activities for tumor growth and progression. Therefore, understanding the exact influences of interactions between tumor cells and LECs, knowing the features of representative pathway-related molecules involved in tumor lymphatic chemotaxis, adhesion, invasion and metastasis, comprehending the network functions of these pathway-related molecules, revealing the expression alterations of these molecules in different tissues or types or stages of tumor development and progression, and particularly, clarifying the biological molecular mechanisms and significances of tumor lymphangiogenesis and lymphatic metastasis, are absolutely essential for us going forward an further intensive research in this field. 2. Structures and functions of pathway-related molecules of VEGFC/ D-VEGFR3/NRP2 axis VEGFC/D-VEGFR3/NRP2 axis is a lymphatic specific biochemical axis, and it is composed by three essential parts, e.g. extracellular VEGF-C/D as ligands, cell membrane VEGFR3/NRP2 as receptors, and extracellular or intracellular pathway-related molecules as executors. The commonalities of these pathway-related molecules of VEGFC/DVEGFR3/NRP2 axis are that their own activations are initially dependent on the combination of VEGFC/D and VEGFR3/NRP2, and that their group functions complete finally in the formation and reconstruction of preexisting and newborn lymphatic vessels. The representative molecules of VEGFC/D-VEGFR3/NRP2 axis include Furin-like Enzyme, CNTN1, Prox1, LYVE-1, Podoplanin, SOX18, SDF1 and CXCR4, etc. Among VEGF family members, only VEGFC/D contains the Furin-like enzyme that refers to some intracellular protein components having a similar function like Furin enzyme. Furin enzyme is a type I transmembrane protein composed of 794 amino acids, which belongs to the precursor protein converting enzyme family of the serine protease subtilisin superfamily with endoprotease activities. Furin enzyme is produced in the form of precursor protein which needs two self-cleavages in secretion pathway to become a functional mature molecular. The catalytic carboxy-terminal region of Furin enzyme has a domain consisted of about 140 amino acids, which is mainly involved in enzyme activity, pH maintenance and regulation on calcium requirements. Another conserved region is a carboxy-terminal propeptide of secretion signal peptides, which is basically associated with the enfoldment, activation, transportation and activity regulation of Furin enzyme. Furin enzyme also plays an important role in embryogenesis, homeostasis, bacterial toxin activation, viral package and glycoprotein hydrolysis physiologically and pathologically [1]. Contactin-1 (CNTN1) is a member of nervous contacting molecules of immunoglobulin superfamily, whose gene is located on the region of chromosome 12q11-q12 and molecular weight is 135 kD. In many human tumors, the region of 12q11-q12 is a breaking point area, therefore, the role of CNTN1 in tumors and their metastasis is being seriously concerned. CNTN1 is a direct downstream molecule of VEGFC/D-/ VEGFR3/NRP2 axis, which consists of six Ig-like regions, four fibronectin type III-like fragments and glycosyl phosphatidy linositols (GPI)

anchoring to cell membrane [2]. CNTN1 involves in nervous system growth and embodiment function, such as nerve cell differentiation, migration, neurite outgrowth, synapse formation, myelination and nerve impulse conduction [3–5]. CNTN1 also has been shown to correlate with a variety of cell surface proteins and plays roles in various cellular signaling pathways and molecular biologic functions [6]. Prospero homeobox protein1 (Prox1) is the homeobox transcription factor of Drosophila homeobox gene prospero in mammals. Prox1 gene is located on chromosome lq32.2-lq32.3, with a length of about 40 kb. Prox1 gene contains at least five exons and four introns, encodes 83 kD of Prox1 protein. Prox1 is the primary regulator of primitive endothelial cell differentiation and induce the embryonic lymphatic bud growth, extension and phenotypic changes of LECs, as well as plays a decisive role in both normal and tumor lymphatic assembling, formation and remodeling [7,8]. Prox1 also promotes the expression of certain cell adhesion molecules and upregulates the metalloproteinase activity which are required for LEC sprouting and branching, and maintains signal transduction pathway activation in migration of LECs [9]. Prox1 is an excellent marker with a high specificity and sensitivity to LECs, mostly situated in the nucleus and easily double stained with other cytoplasmic or membrane markers for the research of the relationship between lymphangiogenesis and metastasis on morphological inspections [10]. LYVE-1 is the lymphatic endothelial hyaluronic acid (HA) recepror-1 and is the homolog compound of glycoprotein CD44. Human LYVE-1 gene is located on chromosome 11p15 with a total length of 2313 bp, which can be translated as a transmembrane glycoprotein with 322 amino acid residues. LYVE-1 expresses in the LECs and lies on the surface of lymph tube cavity, which is a relatively specific marker of lymphatic endothelium [11]. LYVE-1 can uptake and transport HA and take the catabolism in endothelial cells of lymphatic vessels, hepatic and splenic sinus, and transfer HA transmembranely from the surrounding tissue to lymphatic chamber, or be internalized subsequently by macrophage-like cells and fibroblasts, whereas HA may then cross the endothelium of lymph nodes, liver and spleen sinus [12,13]. LYVE-1 not only could be used alone as a marker for lymphatic endothelium, but also might be used in combination with Prox1 to further enhance the specific determination of LECs [14,15]. Podoplanin is a type I myxoid transmembrane glycoprotein, which has been first found on glomerular podocyte surface in puromycin-induced nephrotic rats. The size of this gene is 34.2 kb having 8 exons. Podoplanin molecular weight is 38kD and Podoplanin is composed of 162 amino acids, which contains a highly conserved transmembrane domain, an intracellular domain with 9 amino acids and two potential phosphorylation sites on C terminal in cytoplasma. Extracellular domain is changeable greatly in vary species, but is constantly rich in highly glycosylated serine and threonine residues. In addition, Podoplanin also has a region to provoke aggregation of platelets [16]. D2-40 is an excellent commercialized antibody that can against and combine to the segments of Podoplanin FC and carcinoembryonic antigen M2A, and therefore can recognize LECs as a sensitive marker. Podoplanin expresses abundantly in lumen wall of growing and maturating lymphatic tracts, very little in outer wall and sidewall of LEC chambers and organelles, and absently in blood capillary endothelial cells. Apart from preventing cell adhesion, preserving glomerular podocyte forms and maintaining glomerular permeability, Podoplanin is principally associated with generation and differentiation of LECs [17,18]. An animal experiment showed that Podoplanin knockout mice were death after the birth, with lymphatic dilation, dysfunction and lymphatic edema. Sex-determining region Y chromosome related high mobility group box (SOX) is a developmental gene that encodes a transcription factor, which is different from other genes with high mobility group sequence. SOX is able to binding to DNA in a sequence-specific manner, by which it recognizes the sequence of 5′ (a/T) (a/T) CAA (a/T) G3′. According to the sequence homology of high mobility group box, SOX gene family is divided into 10 (A-J) subfamilies, and SOX18 serves as a member of SOXF subfamily. SOX18 gene is located on chromosome 20q13.33,

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with a total length of 1901 bp. Human SOX18 protein is consisted of 384 amino acids, the molecular weight is about 41 kD. The binding of SOX18 to the region of Prox1 promoter mentioned above is crucial for generation of LECs [19,20]. Duong et al. has confirmed that SOX18 plays a key role as a “switch” in mice embryonic lymphangiogenesis [21]. Mutations of human SOX18 gene are usually caused by a dominant negative effect. Stromal cell derived factor1 (SDF1) is a member of chemokine CXC family, also is known as CXC ligand 12, while CXC receptor4 (CXCR4) is the specific receptor of SDF1. SDF1 gene is located on chromosome 10q11.1 and encodes 89 and 93 amino acid polypeptides. CXCR4 gene is located on the region of human chromosome 2q21, encoding 352 amino acid residues with highly conserved coding sequence, whose α-helix has been transmembraned by 7 times, and there are an extracellular N-terminal, three extracellular loops, three intracellular loops and an intracellular C-terminal. Only by the way of combining with Nterminal and interacting with the second extracellular loop of CXCR4, SDF1 can provoke downstream signaling pathways. SDF1 has the promoting functions for cell proliferation, migration and immune responses, as well as it has an important role in differentiation of endothelial progenitor cells and LECs [22,23]. 3. The expression changes of pathway-related molecules of VEGFC/ D-VEGFR3/NRP2 axis in tumor lymphangiogenesis and lymphatic metastasis In recent years, many studies have shown that pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis have varying changes in different anatomic organs, different histo-pathologic types and different clinicopathologic stages of tumor development, progression and lymphatic metastasis, both at genetic level and protein level, both in original lymphatic vessels and newborn lymphatic vessels, and both in primary tumors and secondary tumors. The upstream and downstream pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis not only have regular expression patterns linked to tumor lymphangiogenesis and lymphatic metastasis, but also demonstrate a lot of expression changes on different pathways and up or downstream sites in different tumor cells. As a precursor protein converting enzyme in cells, Furin-like enzyme could shear the precursors of VEGFC/D during cell secretion of VEGFC and VEGFD. Previous research data showed that the capacities of mature VEGFD combining to VEGFR2 and VEGFR3 were increased by approximately 290 and 400 times compared to immature precursors of VEGFD. Just because of such a huge difference, the mature VEGFC/D could regulate the selective signal activation that was related to VEGFR2/3, through the same two-ways feedback mechanism and thus played a decisive role on angiogenesis or lymphangiogenesis during tumor hematogenous or lymphatic metastasis [1]. The effections of Furin-like enzyme were also associated with malignant phenotypes, proliferation, adhesion, transference and immune response of tumor cells [24]. The activation of many growth factors and receptors depended on Furin splicing. There was high expression of Furin enzyme in non-small cell lung cancer, head and neck squamous cell carcinoma, cervical squamous cell carcinoma, pancreatic ductal adenocarcinoma, malignant glioma and other kinds of tumor cells [25–29]. Furin enzyme also enhanced the activity of membrane-type 1 matrix metalloproteinase, which was conducive to migration and lymphangio-genesis of tumor cells [30]. The former peptide fragments of Furin enzyme could be used as antagonists against Furin enzyme, which depressed the efficiency of cutting mature VEGFC, further more inhibited proliferation and invasion of malignant tumors. It was observed that Furin-like enzyme might improve the biological effects by modifying and processing VEGFC/D precursors [1,24]. It should be noted that Furin-like enzyme belongs to extracellular upstream molecules of VEGF-C/D-VEGFR-3/ NRP2 axis and is different in action sites from the following intracellular downstream molecules of this axis.

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CNTN1 was a GPI anchored nerve adhesion molecule. Lung cancer cell lines with different invasive ability had been tested at mRNA and protein levels, and data revealed that the higher the invasive ability of lung cancer cells was, the higher the expression of CNTN1 on these cancer cells was [31,32]. Filamentous actin (F-actin) was a cell migration related protein, the expression of F-actin, the mobility and adhesion of lung cancer cells were all significantly depressed when CNTN1 gene was interfered by short hairpin RNA (shRNA) in lung cancer metastasis of nude mice [33]. If CNTN1 was interfered by above RNA technique in oral squamous cells, the invasion ability of tumor cells was also distinctly decreased [34,35]. CNTN1 was involved in the development of astrocytic glioma and concerned with the malignancy degree of this tumor [36]. At the mRNA tests in lung cancer, the expression of CNTN1 in primary tumors with lymph node metastasis was more abundantly than in the cases without lymph node metastasis [37,38]. Some authors found that the expression of CNTN1 protein was distinctly higher in gastric cancer tissue than in adjacent normal gastric tissue, which is associated to higher lymphatic invasion and lymph node metastasis [39,40]. As detecting VEGFR3 in gastric cancer, the density of lymphatic vessels with CNTN1 protein positive cases was apparently increased compared to the CNTN1 protein negative cases, and the expression of CNTN1 was positively correlated with VEGFC and VEGFR3 expressions, which revealed the abnormal expression of CNTN1 may be the molecular basis of tumor lymphangiogenesis and metastasis [41]. CNTN1 had been proved as a key molecule in directly downstream pathways of VEGFC/VEGFR3 axis in some researches, and the interfered expression of VEGFR3 in gastric cancer cell line MKN45 could result in reduction of CNTN1. The expression of CNTN1 protein in primary tumors had accordance with clinicopathologic stages, lymphatic invasion and lymph node metastasis in esophageal squamous cell carcinoma. VEGFC also promoted cancer cell invasion by regulating the expression of CNTN1 [32]. The studies have found that the mutation and abnormal expression of Prox1 gene, as an important downstream pathway-related molecule of VEGFC/D-VEGFR3/NRP2 axis, were closely associated with lymphangiogenesis during the development and progression of many tumors. Lymphatic progenitor cells derived from embryos veins could be induced by Prox1, which regulated the differentiation of LECs [5]. Single Prox1 gene as a radical factor in lymphangiogenesis might differentiate blood vascular endothelial cells to lymphatic endothelial phenotypes, which was confirmed by Hong etc. in a gene transfection experiment [42,43]. Petrova found that even stable blood endothelial cells could also be converted to LECs under the role of over-expressed Prox1 [44]. An immunohistochemical study demonstrated that overexpressed Prox1 mainly located in the nucleus of LECs and had an increased expression in some LECs in tumor stroma [43,45]. The expression of Prox1 gene in colorectal cancer tissue was obviously higher, and correlated with high lymphatic vessel density, indicating that Prox1 overexpression was tightly associated with lymphangiogenesis of colorectal cancer compared with normal colon tissue. The expression of Prox1 gene not only evidently enhanced colorectal cancer growth, but also stimulated new lymphatic hyperplasia in tumor stroma while tested at mRNA level [46,47]. A research on breast cancer found that, the expression of Prox1 in lymph node-positive tumor was significantly higher than that in normal breast and lymph node-negative breast cancer tissues [45,48]. A study on cervical cancer investigated that the Prox1 protein in tumor tissue was gradually increased with occurrence of lymph node metastasis [49]. In animal models, the expression of Prox1 could induce more local aggression and invasion in mice xenografts and might increase the lymphatic invasion rate of tumor cells in vitro and in vivo [50,51]. The expression of LYVE-1 in advanced neuroblastoma with lymph node metastasis was significantly increased than that in peripheral lymph nodes without metastasis [52]. The content of LYVE-1 in breast cancer patients with lymph node metastasis was apparently higher than those without lymph node metastasis [45]. The overexpression of LYVE-1 was related to local recurrence and poor prognosis in head

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and neck squamous cell carcinoma [53]. Hou and his colleagues have found that the structure and function of the lymphatic vessels in the LYVE-1 gene knockout mice were significant abnormal, showing the expanded lymph tube cavity and the enhanced fluid circulation from interstitial to lymphatic tracts [54]. Therefore LYVE-1 might regulate the permeability of tumor associated lymphatic vessels to facilitate lymphatic metastasis. The high expression of LYVE-1 in lymphatic vessels of colorectal cancer expedited more invasion and lymph node metastasis [55]. The overexpression of LYVE-1 was a risk factor of the advanced staging and distant metastasis in gastric cancer [56]. As also, the increased LYVE-1 level and the pathway signal activation in cellular microenvironment could promote lymphangiogenesis and lymphatic metastasis of tumors. The reasons of LYVE-1 involved in tumor lymphatic metastasis might have three items: 1. LYVE-1 could bind growth factors such as VEGFC/D and platelet-derived growth factor-BB, which contained cell-surface retention sequence, made these growth factors to be persisted in the surrounding cell matrix and involved in the autocrine and paracrine regulation of growth and migration of tumor associated LECs. 2. LYVE-1 could damage cell connection mediated by vascular endothelial cadherin, to open the lymphatic endothelial gaps, to stimulate lymphatic endothelial cell contraction and to adjust lymphatic duct wall permeability [57]. 3. LYVE-1 could also interact with HA on the surface of tumor cells within the lymphatic vessels, to provoke adhesion and migration of tumor cells to LECs [58]. By Podoplanin/Ki67 double labeled staining, the LEC proliferation was observed in many tumors such as head and neck squamous cell carcinoma [59,60], melanoma [61], colorectal cancer [62] and non-small cell lung cancer [63], moreover Podoplanin was rich in lymphatic vessels around the tumor compared with the distant region [64]. Similarly, Podoplanin highly expressed at tumor edge in lung cancer, laryngeal cancer, cervical cancer, skin cancer and esophageal cancer [61,63,65– 67]. The study on skin squamous cell carcinoma showed that Podoplanin expressed in 79% of tumors, not expressed in well-differentiated carcinoma, over-expressed in tumor margin in middle differentiated carcinoma, and was more in undifferentiated carcinoma [68]. Podoplanin primarily expressed in lung squamous cell carcinoma but not in lung adenocarcinoma. The expression of Podoplanin in esophageal carcinoma with lymph node invasion was significantly higher than in that without lymphatic invasion, and the expression of podoplanin in astrocytoma was related to the degree of tumor malignancy [67,69]. It was also showed that Podoplanin was closely associated with the lymphatic invasion, metastasis and survival rate of cervical cancer and oral squamous cell carcinoma [66,70]. In cervical cone biopsy and radical hysterectomy specimens, 71% of cases expressed podoplanin, in which 59% on the tumor edge and focal expression was closely linked to lymphatic invasion, metastasis, recurrence and short-term survival rate of the tumors [71]. By gene transfection in breast cancer cell MCF7, the over-expression of Podoplanin had been found and lead to major changes of cellular morphology, such as cell elasticity reduction, filopodia-like membrane protrusion, strong adhesion and fibronectin diffusion in extracellular stroma, thereby facilitated the migration of MCF-7 cells and strengthened cancer cells to penetrate into the matrix [72]. In animal models of squamous cell carcinoma and insulin producing tumor, there were evidences that on the one hand, Podoplanin could accelerate the single cell invasion and migration of oral squamous cell carcinoma through the independent way of Epithelial-Mesenchymal Transition (EMT) and on the other hand, might be helpful to cell mobility mediated by the dependent way of EMT [72,73]. The SOX18-COUP-TF11-Prox1 pathway, in particular SOX18, only worked in the early stages of lymphangiogenesis, however, some other molecules like Foxc2, nuclear factor of activated T 1 (NFATc1), T homeobox gene 1 (Tbx1) and Ang-2 might act on the later stages of remodeling and maturing of lymphatic vessels [74]. Knocking out of SOX18 binding sites or dominant negative mutation of homozygote SOX18 gene in mice would lead to embryonic lethality and lymphedema [75]. Duong has shown that SOX18 was a key gene in tumor-induced

lymphangiogenesis, while inhibition of SOX18 could constrain tumor metastasis [19,76]. In an vivo study by bioluminescence imaging in B16-F10 melanoma cells with expression of firefly luciferase implanted in SOX18-defected mice, the probability of tumor cells who transferred to lymph nodes was reduced, indicating a positive relation to the reduction of diameter and density of tumor lymphatic vessels [76,77]. Saitoh reported that the expression of SOX18 mRNA was increased in gastric cancer cell lines MKN45 and TMK1 and could encourage cell growth and lymphangiogenesis in tumor LEC development [78]. The two proteins of SDF1 and CXCR4 occurred on the surfaces of lymphatic vessels and tumor cells. SDF1 which is secreted by LECs could promote lymphatic invasion of melanoma cells expressed CXCR4, while hypoxia would respectively induce the upregulation of SDF1 and CXCR4 in those two kinds of cells [79]. In a model of breast cancer in mice, SDF1 inhibitors might apparently reduce the numbers of breast cancer cells transferring to lymph node. Some studies found that the expression of CXCR4 would be increased by elevation of VEGFC and VEGFR3 levels, namely VEGFC/VEGFR3 together with SDF1/CXCR4 participated in the mechanism of lymphatic formation and both of these pathways may have some additive effects [80]. 4. The mechanisms and significances of the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis on tumor lymphangiogenesis and lymphatic metastasis A series of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, whose biological activities mainly rely on this reaction axis, eventually plays a major role in regulation of tumor lymphangiogenesis and lymphatic metastasis. It is through that the participation of these shaft pathway-related molecules could become a functional complex of VEGFC/D-VEGFR3/NRP2 axis, which has a complete, efficient and specific biochemical effect, and serve as a practitioner in introduction and generation of tumor lymphatic vessels and lymphatic metastasis. The recent studies show that the alterations in micro-environment both inside and outside of tumor cells and LECs can make more tumor cell populations have stronger abilities of proliferation, adhesion and invasion, and enter the existing or nascent tumor lymphatic vessels, however, the molecular mechanisms and significances of expression changes in pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis on tumor lymphangiogenesis and lymphatic metastasis are still unclear. According to the existing data, we speculate the following factors may be involved. First, the functional status of VEGFC/D-VEGFR3/NRP2 axis is impacted individually by the ligand proteolytic processes and the up or downstream pathway-related molecular activities. As a specific protein converting enzyme of VEGFC/D, Furin-like enzyme can splice VEGFC/D precursors, which has been reported being positively related to malignant phenotype, proliferation, adhesion of neoplasm cells [1,24]. The activation of VEGFC/D-VEGFR3/NRP2 signal pathway is an important molecular event in stimulating lymphangiogenesis and lymphatic metastasis. For instances, VEGFR3 could be phosphorylated and activate the knot together protein so that phosphotyrosine binding domain region of VEGFC/D can combine to Src homology 2 (SH2) domain in knot together protein and occur to the autophosphorylation. The knot together protein might also combine to SH2 domain of regulatory growth factor receptor bound protein 2, and improve this region to combine with purine nucleotide releasing factor SOS forming a complex. It could induce the further activation of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, including Prox1, Podoplanin, LYVE-1, SOX18, SDF1 and CXCR4, etc. and the other molecules such as phosphatidylcholine 3 kinase, protein kinase C-dependent p42/p44, mitogen activated protein kinase (MAPK) and AKT signaling pathways, which results in lymphangiogenesis and thereby promotes tumor cell migration to regional lymph nodes. Second, the up and downstream pathways-related molecules of VEGFC/D-VEGFR3/NRP2 axis form a complicated biochemical network and synergy in tumor lymphangiogenesis and lymphatic metastasis.

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For example, VEGFC/VEGFR3 axis upregulates the expression of CNTN1 through activating the Src-p38/MAPK-C/EBP dependent signaling pathway, whereas CNTN1 can also reduce E-cadherin expression in gene transcription, by activating Notch/AKT pathway and inhibiting transcription factor SNAI2 [81]. Again, Prox1 can adjust a9 integrin pathway to induce the differentiation of LECs under the effect of VEGFC [82]. Meanwhile Prox1 can procure lymphatic metastasis of liver cancer by regulating expression of hypoxia inducible factor-1α [5]. And also again, Podoplanin can bind to ERM (Ezrin, Radixin and Moesin) proteins by the conserved regions consisted with three amino acids in cytoplasm while adjusted by the prox1, which significantly increases the phosphorylation of ERM proteins and make Podoplanin has a linkage to actin recombinant and even to tumor cell migration [83]. Podoplanin also can enhance the tumor cell mobility by adjusting GTP activity of Rho family, especially that of RhoA [83,84]. Others, the mutation of heterozygous SOX18 gene can lead to upregulation of SOX7 and SOX17, restarting the reexpression of Prox1 gene in LEC precursor cells [85,86]. Third, the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis are specifically present in LECs, although it is not unique to LECs. The so-called specificity is to show a strength expression of those molecules in LECs rather than in blood vascular endothelial cells, and therefore these pathway-related molecules are often regarded as excellent markers of LECs to distinguish LECs from blood vascular endothelial cells. The so-called nonexclusion is reflected by the less presentation that these molecules could also be found in other types of cells, such as CNTN1, Prox1, LYVE-1 and Podoplanin might appear in macrophages, lymphatic sinus reticular cells, liver and spleen sinus endothelial cells, and in blood vascular endothelial cells. Meanwhile, some other pathway molecules can also be involved in mediation of LECs generation and differentiation, in which desmoplakin (I and II), 5′-nucleotidase, cyclooxygenase (COX) and β-chemokine D6 are often mentioned [87–90]. Fourth, the final functions of the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis would represent in promoting tumor lymphangiogenesis and lymphatic metastasis. They not only influence the process that tumor cells get into the lymph tracts through invading the original lymphatics, but also affect the process that tumor cells enter the lymph tracts through invading the neonatal lymphatics. It is much clear now that the formations of lymphangiogenesis and angiogenesis are in a very similar manner. The newborn lymphatic vessels proliferate and differentiate in tumor stroma, which are possibly produced by the way of “budding” of pre-existing lymphatic vessels, also may be derived from the migration of endothelial progenitor cells in bone marrow, and even may come from the direct conversion of certain mesenchymal cells. The number of the newborn lymphatic vessels has been increased in sentinel lymph nodes before the tumor cells have not yet reached the sentinel lymph nodes [48]. The significances of newborn lymphatics involved in tumor metastasis are predominantly embodied in following aspects: First, the number and density of lymphatic vessels in tumor stroma have risen, to increase contacting chances between tumor cells and LECs. Second, the nascent tumor LECs may exhibit phenotypes which are more conducive to tumor cell metastasis, such as the upregulated expression of adhesion molecules. Third, the wall of monolayer endothelial cells in lymphangiogenesis is thinner with less intact basement membrane, widen cell gaps, and low intraluminal pressure, which are more vulnerable to be squeezed, invaded and entered by cancer cells. Fourth, the original lymphatic channels are easily blocked due to compression by near large cancer cell nests so that the corresponding distant lymphatic tracts are deposited and expanded, and the certain endothelial cells are damaged and the gaps between LECs are opened, which conducively promotes tumor cells into the lymphatic tracts during tumor metastasis [91,92]. 5. Conclusion In recent years, the studies on pathway-related molecules of VEGFC/ D-VEGFR3/NRP2 axis have made quite a progress, and meanwhile, the

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knowledge of molecular significances on tumor lymphangiogenesis and lymphatic metastasis are distinctly generated. The specific pathway-related molecules, which are dependent on VEGFC/D-VEGFR3/ NRP2 ligand-receptor system and are a portion of VEGFC/D-VEGFR3/ NRP2 axis, could produce waterfall-like cascading effects, mediate differentiation and maturation of LECs, remodel original and neonatal lymphatic vessels, as well as promote tumor cell chemotaxis, migration, invasion and metastasis to lymphoid tracts. These pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis include Furin-like enzyme, CNTN1, Prox1, LYVE-1, Podoplanin, SOX18, SDF1 and CXCR etc. The expression changes of these molecules in different organs or types or stages of various tumors, the interactive effects on biological behaviors between tumor cells and lymphatic endothelial cells, and especially, the molecular significances in proliferation and migration of lymphatic endothelial cells and tumor cells, play an important role in mechanisms associated with lymphangiogenesis and lymphatic metastasis of malignances. It is believable that with the deepening of research in this field, such progresses will possibly provide new early evaluable indexes for tumorigenesis, lymphangiogenesis and lymphatic metastasis, and may ultimately offer a potential application value in guiding clinical diagnosis, treatment and prognosis of cancer in future. Conflict of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81071725) and the Financial Department of Liaoning Province (No. 20121203). References [1] G. Siegfried, A.M. Khatib, Processing of VEGF-C and-D by the proprotein convertases: importance in angiogenesis, lymphangiogenesis and tumorgenesis, Colloq. Ser. Protein Activation Cancer 2 (2) (2013) 1–66. [2] P. Liu, J. Zhou, H. Zhu, VEGF-C promotes the development of esophageal cancer via regulating CNTN-1 expression, Cytokine 55 (1) (2011) 8–17. [3] A. Bizzoca, P. Corsi, A. Polizzi, et al., F3/Contactin acts as a modulator of neurogenesis during cerebral cortex development, Dev. Biol. 365 (1) (2012) 133–151. [4] G. Çolakoğlu, U. Bergstrom-Tyrberg, E.O. Berglund, et al., Contactin-1 regulates myelination and nodal/paranodal domain organization in the central nervous system, Proc. Natl. Acad. Sci. U. S. A. 111 (3) (2014) E394–E403. [5] D. Puzzo, A. Bizzoca, L. Privitera, et al., Contactin promotes hippocampal neurogenesis, synaptic plasticity, and memory in adult mice, Hippocampus 23 (12) (2013) 1367–1382. [6] G. Yang, J.G. Song, Y. Li, Under hypoxia conditions contactin-1 regulates the migration of Mkn45 cells through the RhoA pathway, Mol. Biol. 49 (1) (2015) 112–119. [7] H.M. Teng, Y.Z. Yang, H.Y. Wei, et al., Fucoidan suppresses hypoxia induced lymphangiogenesis and lymphatic metastasis in mouse hepatocarcinoma, Mar. Drugs 13 (6) (2015) 3514–3530. [8] K. Koltowska, A.K. Lagendijk, C. Pichol-Thievend, et al., Vegfc regulates bipotential precursor division and prox1 expression to promote lymphatic identity in zebrafish, Cell Rep. 13 (9) (2015) 1828–1841. [9] R.S. Srinivasa, X. Geng, Y. Yang, The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of prox1 expression in lymphatic endothelial cells, Genes Dev. 24 (7) (2010) 696–707. [10] A. Kaser-Eichberger, F. Schroedl, L. Bieler, et al., Expression of lymphatic markers in the adult rat spinal cord, Front. Cell. Neurosci. (2016), http://dx.doi.org/10.3389/ fncel.2016.00023. [11] K. Nunomiya, Y. Shibata, S. Abe, Relationship between serum level of lymphatic vessel endothelial hyaluronan receptor-1 and prognosis in patients with lung cancer, J. Cancer 5 (3) (2014) 242–247. [12] M. Wu, Y. Du, Y.W. Liu, Low molecular weight hyaluronan induces lymphangiogenesis through LYVE-1 mediated signaling pathway, PLoS One 9 (3) (2014), e92857. [13] L. William, B.J. Suneale, A.J. Day, Binding of hyaluronan to the native lymphatic vessel endothelial receptor LYVE-1 is critically dependent on receptor clustering and hyaluronan organization, J. Biol. Chem. 291 (15) (2016) 8014–8030. [14] R. Tang, C.Y. Zhao, The correlation between expression of LYVE-1 and PROX-1 in breast cancer associated lymphatic vessel and lymphatic metastases, Label. Immunoass. Clin. Med. 22 (11) (2015) 1086–1089.

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