Identification of sphingosine 1-phosphate receptors in exosomes released from breast cancer cells

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive lipid that promotes cell survival, proliferation, migration, angiogenesis and immune response, which are critical processes in cancer progression. Although some essential roles of intracellular S1P have recently been revealed, the majority of its biological effects are recognised to be mediated through activation of its cognate S1P1-5 receptors. This work demonstrates for the first time that the sphingosine 1-phosphate receptor 2 (S1P2, Mr = 42kDa) is shed in Hsp70 and CD63 containing exosomes from MDA-MB-231 breast cancer cells.;These exosomes can be taken up by fibroblasts where exosomal S1P2 receptor is proteolysed to a constitutively active shorter form of S1P2 (S1P2, Mr=38 kDa) which activates ERK-1/2 and stimulates DNA synthesis in fibroblasts. Therefore, conditioned medium and an exosomal preparation containing shed S1P2 and isolated from MDA-MB-231 cells was able to stimulate the ERK-1/2 pathway and DNA synthesis in fibroblasts.;Moreover, S1P2 siRNA knockdown in MDA-MB-231 cells reduced the ability of conditioned medium and exosomal preparations containing S1P2 to induce ERK-1/2 activation in fibroblasts. This finding links the constitutively active shorter form of S1P2 released from cancer cells with the proliferation of fibroblasts required for metastatic spread. In addition, it was demonstrated that S1P4 is released from MDA-MB-453 cells breast cancer cells.;However, conditioned medium containing S1P4 failed to activate ERK-1/2 in fibroblasts, suggesting that ERK-1/2 signalling is specifically regulated by the short form S1P2. Future work requires in vivo study of the truncated version of S1P2 receptor in an orthotopic graft mouse model to examine its impact on cancer metastasis.;In addition, S1P and inhibitors of sphingosine kinase (SK) increase the release of S1P4 from MDA-MB-453 cells. These findings, suggest that exogenous S1P might enhance S1P4 internalisation and its subsequent release into CM. SK inhibitors might increase intracellular ceramide, which has been demonstrated to be enriched in lipid raft microdomains and regulate MVBs formation and cargo sorting into ILVs (exosomes).;S1P and SK inhibitors had no effect on S1P2 release, suggesting that the effect on S1P4 is cell type-specific. In addition, it was demonstrated that the combination of the ceramide kinase (CERK) inhibitor, NVP-231 and the SK1 inhibitor, PF-543 produced less than additive effect on DNA synthesis, suggesting a functional interaction or overlapping regulation of CERK and SK1 in MDA-MB-453 and MDA-MB-231 breast cancer cells survival.;Therefore, modulating the sphingolipid rheostat using CERK and SK1 inhibitor failed to sensitise breast cancer cells to cell death. Further investigation is required to examine the effect of CERK inhibitor alone and in combination with SK1 inhibitors on S1P4 release.Taken together; the findings of this study, suggest a novel regulation of cancer progression by the S1P signalling pathway.Sphingosine-1-phosphate (S1P) is a bioactive lipid that promotes cell survival, proliferation, migration, angiogenesis and immune response, which are critical processes in cancer progression. Although some essential roles of intracellular S1P have recently been revealed, the majority of its biological effects are recognised to be mediated through activation of its cognate S1P1-5 receptors. This work demonstrates for the first time that the sphingosine 1-phosphate receptor 2 (S1P2, Mr = 42kDa) is shed in Hsp70 and CD63 containing exosomes from MDA-MB-231 breast cancer cells.;These exosomes can be taken up by fibroblasts where exosomal S1P2 receptor is proteolysed to a constitutively active shorter form of S1P2 (S1P2, Mr=38 kDa) which activates ERK-1/2 and stimulates DNA synthesis in fibroblasts. Therefore, conditioned medium and an exosomal preparation containing shed S1P2 and isolated from MDA-MB-231 cells was able to stimulate the ERK-1/2 pathway and DNA synthesis in fibroblasts.;Moreover, S1P2 siRNA knockdown in MDA-MB-231 cells reduced the ability of conditioned medium and exosomal preparations containing S1P2 to induce ERK-1/2 activation in fibroblasts. This finding links the constitutively active shorter form of S1P2 released from cancer cells with the proliferation of fibroblasts required for metastatic spread. In addition, it was demonstrated that S1P4 is released from MDA-MB-453 cells breast cancer cells.;However, conditioned medium containing S1P4 failed to activate ERK-1/2 in fibroblasts, suggesting that ERK-1/2 signalling is specifically regulated by the short form S1P2. Future work requires in vivo study of the truncated version of S1P2 receptor in an orthotopic graft mouse model to examine its impact on cancer metastasis.;In addition, S1P and inhibitors of sphingosine kinase (SK) increase the release of S1P4 from MDA-MB-453 cells. These findings, suggest that exogenous S1P might enhance S1P4 internalisation and its subsequent release into CM. SK inhibitors might increase intracellular ceramide, which has been demonstrated to be enriched in lipid raft microdomains and regulate MVBs formation and cargo sorting into ILVs (exosomes).;S1P and SK inhibitors had no effect on S1P2 release, suggesting that the effect on S1P4 is cell type-specific. In addition, it was demonstrated that the combination of the ceramide kinase (CERK) inhibitor, NVP-231 and the SK1 inhibitor, PF-543 produced less than additive effect on DNA synthesis, suggesting a functional interaction or overlapping regulation of CERK and SK1 in MDA-MB-453 and MDA-MB-231 breast cancer cells survival.;Therefore, modulating the sphingolipid rheostat using CERK and SK1 inhibitor failed to sensitise breast cancer cells to cell death. Further investigation is required to examine the effect of CERK inhibitor alone and in combination with SK1 inhibitors on S1P4 release.Taken together; the findings of this study, suggest a novel regulation of cancer progression by the S1P signalling pathway