Abstract
BackgroundLymphatic metastasis, facilitated by lymphangiogenesis is a common occurrence in breast cancer, the molecular mechanisms remaining incompletely understood. We had earlier shown that cyclooxygenase (COX)-2 expression by human or murine breast cancer cells promoted lymphangiogenesis and lymphatic metastasis by upregulating VEGF-C/D production by tumor cells or tumor-associated macrophages primarily due to activation of the prostaglandin receptor EP4 by endogenous PGE2. It is not clear whether tumor or host-derived PGE2 has any direct effect on lymphangiogenesis, and if so, whether EP4 receptors on lymphatic endothelial cells (LEC) play any role.MethodsHere, we address these questions employing in vitro studies with a COX-2-expressing and VEGF-C/D-producing murine breast cancer cell line C3L5 and a rat mesenteric (RM) LEC line and in vivo studies in nude mice.ResultsRMLEC responded to PGE2, an EP4 agonist PGE1OH, or C3L5 cell-conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor reduced Matrigel. Native tube formation by RMLEC on Matrigel was abrogated in the presence of a selective COX-2 inhibitor or an EP4 antagonist. Addition of PGE2 or EP4 agonist, or C3L5-CM individually in the presence of COX-2 inhibitor, or EP4 antagonist, restored tube formation, reinforcing the role of EP4 on RMLEC in tubulogenesis. These results were partially duplicated with a human dermal LEC (HMVEC-dLyAd) and a COX-2 expressing human breast cancer cell line MDA-MB-231. Knocking down EP4 with shRNA in RMLEC abrogated their tube forming capacity on Matrigel in the absence or presence of PGE2, EP4 agonist, or C3L5-CM. RMLEC tubulogenesis following EP4 activation by agonist treatment was dependent on PI3K/Akt and Erk signaling pathways and VEGFR-3 stimulation. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) we demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4 agonist in vivo.Discussion/conclusionsThese results demonstrate the roles of tumor as well as host-derived PGE2 in inducing lymphangiogenesis, at least in part, by activating EP4 and VEGFR-3 on LEC. EP4 being a common target on both tumor and host cells contributing to tumor-associated lymphangiogenesis reaffirms the therapeutic value of EP4 antagonists in the intervention of lymphatic metastasis in breast cancer.
Highlights
Lymphatic metastasis, facilitated by lymphangiogenesis is a common occurrence in breast cancer, the molecular mechanisms remaining incompletely understood
We addressed whether: (a) the native tube forming capacity of the Lymphatic endothelial cells (LECs) on Matrigel is dependent on Cyclooxygenase 2 (COX-2) or EP4 activity; (b) soluble products of COX-2expressing breast cancer cells stimulate tube formation by the LEC; (c) proliferation, migration and tube forming capacity of the LEC are stimulated by exogenous Prostaglandin E2 (PGE2) or selective EP4 agonists and if so, what are the underlying signaling mechanisms
RNeasy Mini Kit was from Qiagen, qScriptTM, cDNA Synthesis Kit and PerfeCTa® Green SuperMix from Quanta Biosciences, Gaithersberg, MD, USA; Indomethacin from Sigma (Oakville, ON, Canada) and selective EP4 antagonist RQ15986 was a gift from RaQualia Pharma Inc (Ask/At), Japan
Summary
Lymphatic metastasis, facilitated by lymphangiogenesis is a common occurrence in breast cancer, the molecular mechanisms remaining incompletely understood. Lymphatic metastasis is a frequent occurrence in most epithelial cancers [1], including cancers of the breast [2], stomach [3], colon [4], pharynx and larynx [5], lungs [6], uterine cervix [7], prostate [8] and the ovary [9], negatively impacting patient survival This is the first route of spread for many cancers, which can subsequently metastasize from the lymph nodes to other organs via the blood stream. Development of new lymphatic capillaries (lymphangiogenesis) is an event common to inflammation and carcinogenesis suggesting a commonality of molecular players in both events Numerous ligands such as VEGF-C, VEGF–D, neuropilins, and certain chemokines have been shown to exert a direct growth-stimulatory effect on cancer-associated lymphatic endothelial cells (LEC) by activation of their respective receptors [12,13,14,15]. The roles of prostaglandin (PG) E2, another key inflammation-associated molecule, remain to be explored fully in breast cancer-associated lymphatic outgrowth
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