Crosstalk between chronic myelogenous leukemia (CML)and bone marrow stromal cells: role of exosomes in the IL8- dependent signalling mediated by EGFR activation

Abstract

Introduction: CML is a myeloproliferative disorder characterized by Bcr-Abl oncoprotein with a constitutive tyr-kinase activity. Exosomes (exo) shed by cancer cells potentially affect tumor-stroma interaction through the establishment of a bi-directional crosstalk. Interleukin-8 (IL8) is a proinflammatory chemokine that regulate proliferation and survival of cancer cells. We previously demonstrated that CML-derived exo modulate bone marrow microenvironment through the IL8 secretion from stromal cells. EGFR, as well as IL8, regulate cell proliferation and survival; it has been recently demonstrated that EGFR ligands can signal via exosomes shed by cancer cells. We hypothesized that the effects induced by IL8 are EGFR mediated and exosomes are involved in this pathway. Methods: Human cell lines used are LAMA84 (CML cells) and HS5 (stromal cells); gene expression analysis was performed by RT-PCR and western blot with antibodies for EGFR, pEGFR, AREG. For in vivo experiments, LAMA84 cells were inoculated in NOD/SCID mice and treated with IL8, vehicle (PBS), SB (IL8 receptors inhibitor) or IL8+SB. After 50 days, tumors were removed to calculate their weights and RNA was extracted from biopsies. Results: On the basis of the in vitro effects of IL8 on LAMA84 cells, we studied the in vivo role of this cytokine. Mice treated with IL8 develop larger tumours than control groups; co-treatment with SB resulted in a slower tumor growth compared with mice treated with IL8 alone. The expression of pro- and anti-apoptotic genes confirmed a pro-survival role of IL8 in vivo. LAMA84 cells and their exo showed different EGFR ligands. Exo treatment of stromal cells increase EGFR expression possibly inducing activation of survival pathways mediated by IL8. Conclusions: Our data show that IL8 promotes tumour growth and survival in vivo. Exo, carrying EGFR ligands, modulate bone marrow microenvironment through activation of EGFR signaling on stromal cells, demonstrating a new extracrine signaling mediated by EGFR ligands.