PGE2 promotes breast cancer-associated lymphangiogenesis by activation of EP4 receptor on lymphatic endothelial cellsReport as inadecuate

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BMC Cancer

, 17:11

Cell and molecular biology


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.

KeywordsPGE2 Cyclooxygenase COX-2 Lymphangiogenesis Angiognesis EP4 receptors EP4 antagonist Breast cancer Metastasis Lymphatic endothelial cells Directed in vivo lymphangiogenesis assay DIVLA AbbreviationsCCL21-6CkineC-C chemokine ligand 21

CCR7C-C chemokine receptor 7

CD31Cluster of differentiation 31

COX-2Cyclooxygenase 2

DAPI4′, 6-diamidino-2-phenylindole

DIVLADirected in vivo lymphangiogenic assay

DNADeoxyribonucleic acid

ELISAEnzyme-linked immunosorbent assay

EPProstanoid receptor

ERK1-2Extracellular regulated kinase 1-2

GAPDHGlyceraldehyde 3-phosphate dehydrogenase

HMVEC-dLyAdPrimary adult human dermal lymphatic microvascular endothelial cells

Lyve-1Lymphatic vessel endothelial receptor-1

LECsLymphatic endothelial cells

PI3KPhosphatidylinositol 3-kinase

Prox-1Prospero homebox protein-1

AKT-PKBProtein kinase B

PGE2Prostaglandin E2

RMLECRat mesenteric lymphatic endothelial cells

RT-PCRReverse transcriptase polymerase chain reaction

SLCStem like cell

VEGFVascular endothelial growth factor

VEGFR-3Vascular endothelial growth factor receptor-3

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Author: Pinki Nandi - Gannareddy V. Girish - Mousumi Majumder - Xiping Xin - Elena Tutunea-Fatan - Peeyush K. Lala


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