Abstract
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. © 2003 Wiley‐Liss, Inc.