Our study adds another piece of information in the complex interaction between the bone marrow microenvironment and cancer cells by introducing the role of cancer secreted microvesicles.
Our previous
in vitro and
in vivo work demonstrated that LAMA84 cells release exosomes able to induce in endothelial cells an angiogenic phenotype through the stimulation of an IL-8 dependent autocrine loop [
5‐
7]. In this study we show that LAMA84 cells may modulate
in vitro gene expression in endothelial cells through the release of miRNAs contained within exosomes. miRNAs profiling evidenced a similarity in the miRNA species found in exosomes and parental cells, however in line with other reports [
25] we show that not all miRNAs can be incorporated into exosomes thus suggesting a sorting mechanism, that is still unclear. Our experiments showed in Figure
1 indicate that exosomes, containing miRNAs, are incorporated and released through an energy and ceramide-dependent pathway given that incubation of LAMA84 cells at 4°C or with GW4869 inhibited the transfer of labelled miR-126 into HUVECs. Of the 124 miRNAs we identified in exosomes, we focused on miR-126. miR-126 is considered an angioMiR with an abundant level in highly vascularized tissues, and is known to regulate many aspects of endothelial cell biology including cell motility, vasculature integrity, cell survival and cytoskeletal organization [
11]. The involvement of miR-126 in cancer biology is not limited to modulation of angiogenesis and data in literature indicate that this miRNA plays a role in cancers of the gastrointestinal tract, breast, lung and other organs by altering several cellular mechanisms of cancer pathogenesis [
26]. In myeloid leukemia miR-126 was found to down-regulate HOXA9, an oncogene encoding a transcription factor that regulates hematopoietic development [
27], while Cammarata and colleagues found that miR-126, upregulated in acute myeloid leukemia, induced cell proliferation via the inhibition of PLK, one member of the Polo-like kinase that regulates the cell cycle [
28]. Our study suggests a different mechanism by which miR-126 may affect CML development, the alteration of the bone marrow microenvironment due to inappropriate cancer cell retention, adhesion and motility. We provide evidence that CXCL12 and VCAM1, critical components of the bone marrow niche are in part regulated, by miR-126 contained in LAMA84 exosomes. CXCL12 or SDF-1 is a chemokine that binds specifically to the G-protein coupled receptor, CXCR4. Sipkins and colleagues have demonstrated that disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to the BM [
29]. Our data indicate that exosomal delivery of miR-126 to endothelial cells decreases CXCL12 release from HUVECs and concomitantly reduces the motility of LAMA84 cells towards HUVEC conditioned medium. To further study the role of miR-126 in the modulation of CXCL12 secretion, we used the inhibitor of miR-126 that can bind and inhibit miR-126 molecules, or its negative control scramble oligomer, to transfect HUVECs. MiR-126 inhibitor decreased by 45% the miR-126 expression in the cells and concomitantly augmented CXCL12 protein level. Consistently with these data, the overexpression of miR-126 mimic caused a decrease of SDF-1 level and lower migration tendency of LAMA84 cells toward EC conditioned medium. Another target of miR-126 that may be relevant to CML disease progression is VCAM1, a cell-cell adhesion molecule. Experimental studies have demonstrated that miR-126 downregulates VCAM1 expression in endothelial cells through a post-trascriptional mechanism acting on mRNA translation [
13]. Fish et al. also reported that VCAM1 mRNA levels were elevated upon miR-126 inhibition, but were not decreased in the presence of miR-126 mimic thus supporting the hypothesis of a regulative mechanism at translational level [
12]. Moreover, it was demonstrated that forced expression of miR-126 in the Lin
− bone marrow cells induced minimal change in the relative levels of VCAM1 mRNA but caused a decrease in the proportion of surface VCAM1-positive Sca-1
hi cKit
hi cells within this population [
30]. From a functional point of view, a recent report from Salvucci and colleagues reported that miR-126 contained in G-CSF-mobilized vesicles in the bone marrow induced hematopoietic stem/progenitor cell (HSPC) mobilization by reducing the expression of VCAM1 in HSPC endothelial cells and other non-hematopoietic cells [
30]. We found that VCAM1 expression is decreased following incubation of endothelial cells with LAMA84 exosomes and that this effect was due to miR-126 contained in the nanovesicles (Figure
5). This effect was partially reduced by the introduction of miR-126 inhibitor in HUVECs (Figure
5). As a functional consequence of the diminished amount of VCAM1 in EC, the adhesion of LAMA84 to HUVECs was reduced after exosome treatment. In our previous study, we demonstrated that the treatment of endothelial cells with exosomes for short times (6 h), induced VCAM1 at both the mRNA and protein level and increased adhesion of LAMA84 cells on the HUVECs monolayer [
7].
In this new study, we found that the treatment of endothelial cells with exosomes for 24 hours downregulated VCAM1 mRNA and protein expression and caused a decrease of LAMA84 adhesion cells on the HUVECs monolayer. In order to explain these apparently contrasting results, we hypothesize that in the first 6 hours, the exosomes treatment of HUVECs induces the expression of VCAM1 to allow the adhesion of the cancer cells on the endothelium, as the first step of cells migration. After a longer time of HUVECs exposure to exosomes, LAMA84 cells lose the ability to adhere on the endothelial cells and increase their capacity to migrate towards a richer source of chemoattractants.