Tumor‐derived prostaglandins (PGs) have been implicated in the progression of murine and human breast cancer. Chronic treatment with a non‐selective PG inhibitor indomethacin was shown in this laboratory to retard the development and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The present study examined the role of endogenous prostaglandins in the proliferation/survival, the migratory and invasive behavior and angiogenic ability of a highly metastatic murine mammary tumor cell line, C3L5, originally derived from a C3H/HeJ spontaneous mammary tumor. This cell line was shown to express high levels of cyclooxygenase (COX) –2 mRNA and protein as detected by Northern and Western blotting as well as immunostaining. PGE₂ production by C3L5 cells was primarily owing to COX‐2, since this was blocked similarly with non‐selective COX inhibitor indomethacin and selective COX‐2 inhibitor NS‐398, but unaffected with the selective COX‐1 inhibitor valeryl salicylate. C3L5 cell proliferation/survival in vitro was not influenced by PGs, since their cellularity remained unaffected in the presence of PGE₂ or NS‐398 or PG‐receptor (EP1/EP2) antagonist AH6809; a marginal decline was noted only at high doses of indomethacin, which was not abrogated by addition of exogenous PGE₂. Migratory and invasive abilities of C3L5 cells, as quantitated with in vitro transwell migration/invasion assays, were inhibited with indomethacin or NS‐398 or AH6809 in a dose‐dependent manner; the indomethacin and NS‐398‐mediated inhibition was partially reversed upon addition of exogenous PGE₂. An in vivo angiogenesis assay that used subcutaneous implants of growth factor‐reduced matrigel inclusive of tumor cells showed a significant inhibition of blood vessel formation in these implants in animals treated with indomethacin compared with animals receiving vehicle alone. These studies show that selective and nonselective COX‐2 inhibitors retarded tumor progression in this COX‐2‐expressing murine mammary tumor model by inhibiting tumor cell migration, invasiveness and tumor‐induced angiogenesis. The inhibitory effects were not entirely PG dependent; some PG‐independent effects were also noted.

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE₂. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors.

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 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. We 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. These 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.

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.

Using clonal derivatives of spontaneous mammary tumours in C3H/HeJ mice, we had earlier shown that tumour‐derived nitric oxide (NO), resulting from endothelial type (e) NO synthase (NOS) expression by tumour cells, promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell invasiveness, migration and angiogenesis. Our present study examined the signaling mechanisms underlying NO‐mediated promotion of tumour cell migration in a highly metastatic and high eNOS‐expressing C3H/HeJ mammary tumour cell line, C3L5. C3L5 cell migration was reduced in the presence of N^G‐nitro‐L‐arginine methyl ester (L‐NAME, NOS inhibitor) in a concentration‐dependent manner and restored in the additional presence of excess L‐arginine (NOS substrate), confirming a migration‐promoting role of endogenous NO. Migratory capacity of C3L5 cells was reduced after treatment with the guanylate cyclase (GC) inhibitor 1‐H‐[1,2,4]oxadiaxolo[4,3‐a]quinolalin‐1‐one (ODQ) and restored in the additional presence of 8‐bromoguanosine 3′5′‐cyclic monophosphate (8‐Br cGMP, cGMP analogue), demonstrating a pivotal role for GC in C3L5 cell migration. Mitogen‐activated protein kinase kinase (MAPKK; MEK) inhibitor, UO126, blocked migration, demonstrating MEK involvement in C3L5 cell migration. Furthermore, both ODQ and UO126 blocked migration‐restoring effects of L‐arginine in L‐NAME‐treated cells, indicating that GC and MAPK pathways are required for endogenous NO‐mediated migratory responses. Similarly, L‐NAME reduced and additional treatment with excess L‐arginine or sodium nitroprusside (SNP, NO donor) stimulated phosphorylation of extracellular signal‐regulated kinases (ERK_1/2), demonstrating a role for endogenous and exogenous NO in ERK_1/2 activation. ODQ inhibited ERK_1/2 activation, whereas 8‐Br cGMP stimulated ERK_1/2 phosphorylation in L‐NAME‐treated cells, indicating that cGMP is a downstream effector of NOS for ERK_1/2 activation. Finally, both ODQ and UO126 blocked the capacity of L‐arginine to restore ERK_1/2 phosphorylation in L‐NAME‐treated cells, demonstrating that GC and MEK are both required for endogenous NO‐mediated MAPK activation. Together, these results indicate sequential activation of NOS, GC and MAPK pathways in mediating signals for C3L5 cell migration, an essential step in invasion and metastasis. Since NOS activity is positively associated with human breast cancer progression, the present results are relevant for development of therapeutic modalities for this disease.