Abstract
BackgroundTumor-induced lymphangiogenesis facilitates breast cancer progression by generating new lymphatic vessels that serve as conduits for tumor dissemination to lymph nodes and beyond. Given the recent evidence suggesting the implication of C-C chemokine ligand 21/chemokine receptor 7 (CCL21/CCR7) in lymph node metastasis, the aim of our study was to define the role of this chemokine pair in breast cancer-associated lymphangiogenesis.MethodsThe expression analysis of CCL21/CCR7 pair and lymphatic endothelial cell (LEC) markers in breast cancer specimens was performed by means of quantitative real-time PCR. By utilizing CCR7 and CCL21 gene manipulated breast cancer cell implants into orthotopic sites of nude mice, lymphatic vessel formation was assessed through quantitative real-time PCR, immunohistochemistry and immunofluorescence assays. Finally, the lymphangiogenic potential of CCL21/CCR7 was assessed in vitro with primary LECs through separate functional assays, each attempting to mimic different stages of the lymphangiogenic process.ResultsWe found that CCR7 mRNA expression in human breast cancer tissues positively correlates with the expression of lymphatic endothelial markers LYVE-1, podoplanin, Prox-1, and vascular endothelial growth factor-C (VEGF-C). We demonstrated that the expression of CCL21/CCR7 by breast cancer cells has the ability to promote tumor-induced lymph-vascular recruitment in vivo. In vitro, CCL21/CCR7 chemokine axis regulates the expression and secretion of lymphangiogenic factor VEGF-C and thereby promotes proliferation, migration, as well as tube formation of the primary human LECs. Finally, we showed that protein kinase B (AKT) signaling pathway is the intracellular mechanism of CCR7-mediated VEGF-C secretion by human breast cancer cells.ConclusionsThese results reveal that CCR7 and VEGF-C display a significant crosstalk and suggest a novel role of the CCL21/CCR7 chemokine axis in the promotion of breast cancer-induced lymphangiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0306-4) contains supplementary material, which is available to authorized users.
Highlights
Tumor-induced lymphangiogenesis facilitates breast cancer progression by generating new lymphatic vessels that serve as conduits for tumor dissemination to lymph nodes and beyond
We had earlier reported that overexpression of cyclooxygenase-2 (COX-2) in breast cancer cells – resulting in increased prostaglandin E2 (PGE2) levels in the tumor milieu – promotes metastasis by multiple mechanisms including stimulation of tumor cell migration [29,30], invasiveness [31], tumor-associated angiogenesis [29], and lymphangiogenesis [32,33,34] caused by an upregulation of vascular endothelial growth factor C (VEGF-C) secretion via prostaglandin EP1/EP4 receptors [27,32,33]
The proposed molecular mechanism responsible for the regulation of VEGF-C was analyzed in MCF-7 mock and MCF-7-CCL21-knocked in (KI) breast cancer cells and the efficacy of nucleotransfection is presented in Figure 1H and I
Summary
Tumor-induced lymphangiogenesis facilitates breast cancer progression by generating new lymphatic vessels that serve as conduits for tumor dissemination to lymph nodes and beyond. Given the recent evidence suggesting the implication of C-C chemokine ligand 21/chemokine receptor 7 (CCL21/CCR7) in lymph node metastasis, the aim of our study was to define the role of this chemokine pair in breast cancer-associated lymphangiogenesis. The objective of the present study was to investigate whether CCL21/ CCR7 signaling promotes breast cancer-associated lymphangiogenesis through CCR7-dependent stimulation of VEGF-C secretion followed by LECs activation towards the development of new lymphatic vessels. This objective was achieved by a combination of in situ, in vivo, and in vitro approaches
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