ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFN gamma, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.

ADAM17 Promotes Motility, Invasion, and Sprouting of Lymphatic Endothelial Cells

Witold Waldemar Kilarski, Renata Katarzyna Mezyk-Kopec, Melody Swartz

Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGF beta 2 in ADAM17-silenced LEC. The antilym-phangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy.

Public Library Science2015

In vitro lymphatic endothelial morphogenesis

Carolyn Yong Pullin

Cell organization into functional multicellular three-dimensional structures is a long-standing challenge. In particular, engineering of vascularized tissues requires both blood and lymphatic neovascular network formation in a simultaneous and coordinated fashion. While extensive in vitro investigations in blood and lymphangiogenesis have identified a vast array of cellular phenomena and key cellular and extracellular bioactive substances, the dynamic natural cellular environment and its regulatory role in governing cellular response and interactions remain largely unclear. Furthermore, the wealth of existing knowledge describing the molecular regulation of vessel morphogenesis is heavily biased towards the angiogenic growth of blood capillaries, despite the importance of the lymphatic system. This thesis first addresses the differential gene regulation of isolated microvascular lymphatic endothelial cells (LECs) in response to the lymphatic growth factor VEGF-C and to fluid shear stress using gene arrays and QRT-PCR. First, characteristic of a response to a growth factor stimulus, the largest numbers of differentially expressed genes in response to VEGF-C stimulation were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C. The ability of lymphatic endothelium to sense and actively regulate drainage function is poorly understood, and shear stress is likely a key indicator of lymphatic drainage function. Thus, LECs were exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. Important adaptive responses to both low and high shear stress conditions that were correlated to lymphatic function, including changes in gene expression patterns related to water and solute transport, cell-cell and cell-matrix adhesion, inflammatory cytokines and cell cycle were found. These changes were consistent with increased transport function, both active and passive, in high shear conditions and indicate that during lymphedema, lymphatic endothelium shuts down transport functions. These data demonstrate a functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their drainage function. It has been previously shown in our lab that in vitro blood and lymphatic morphogenesis are differentially enhanced by interstitial flow. Using a three-dimensional tissue culture model, the molecular mechanisms orchestrating endothelial cell morphogenesis, specifically gene regulation of the lymphatic microvascular system was investigated, focusing on genes implicated in both cellular morphogenesis and lymphatic development. Finally, concurrent angiogenesis and lymphangiogenesis were studied in vitro to examine the influences and crosstalk between blood and lymphatic microvascular development. The data suggest that both blood endothelial cell (BEC) and LEC in vitro capillary morphogenesis are enhanced by the presence of BEC-secreted factors, and these effects are, in part, mediated through proliferative and migratory responses of which the latter was induced by VEGFR-2 and VEGFR-3 signaling for BECs and LECs, respectively. When maintained in mixed three-dimensional cultures, both homotypic and heterotypic interactions between LECs and BECs were observed. Furthermore, capillary morphogenesis and interactions were enhanced and distinct when under the additional influence of interstitial flow. In conclusion, the novelties of the work presented in this dissertation include studies of 1) overall LEC gene response to its primary growth factor VEGF-C and biophysical factor, fluid flow and 2) the genetic regulation underlying the morphogenetic processes of LECs under interstitial flow in vitro, by using unique approaches which combine bioengineering principles to address fundamental biological questions and thereby contribute to this novel niche which has so far been neglected. Furthermore, the importance of biophysical processes along with crucial biochemical signal regulation is demonstrated in the concurrent induction of angiogenesis and lymphangiogenesis. Such are the requisites for the understanding of microvascular development and the determination of useful design principles for the in vitro vascularization of engineered tissues.

EPFL2011

Microenvironmental regulation of tumour angiogenesis

Daniela Biziato, Michele De Palma

Tumours display considerable variation in the patterning and properties of angiogenic blood vessels, as well as in their responses to anti-angiogenic therapy. Angiogenic programming of neoplastic tissue is a multidimensional process regulated by cancer cells in concert with a variety of tumour-associated stromal cells and their bioactive products, which encompass cytokines and growth factors, the extracellular matrix and secreted microvesicles. In this Review, we discuss the extrinsic regulation of angiogenesis by the tumour microenvironment, highlighting potential vulnerabilities that could be targeted to improve the applicability and reach of anti-angiogenic cancer therapies.