Communication between vascular (endothelial) and blood (haematopoietic) cells in adult bone marrow (BM) is essential for maintaining normal haematopoiesis. Micro RNAs (miRNAs) are key regulators of gene expression, involved in the regulation of numerous cellular processes. miRNA signatures (profiles) of BM disorders may allow the prediction of disease prognosis and responses to treatments. In this article we suggest that miRNA profiling of BM endothelial cells may provide specific clues to aid in the understanding of normal and malignant BM function.
by Dr Ana Costa and Sérgio Dias
The BM vascular niche, mostly comprised of endothelial cells, plays a crucial homeostatic role in the maintenance of adequate BM function haematopoiesis. The BM endothelial cells convey signals to haematopoietic elements, favouring cell proliferation, differentiation and ultimately migration into the peripheral circulation. Consequently, interfering with the integrity of the BM vascular niche impairs BM recovery following radiation or chemotherapy-induced injury [1]. It is now increasingly accepted that abnormal (malignant) BM dysfunction is accompanied by a “malignant” (ie abnormal) vascular niche; BM endothelial cells have, for instance been found to express the molecular traits of the corresponding BM disorders in myeloma and chronic leukaemias. Therefore, molecular characterisation of a malignant BM vascular niche may reveal novel targets suitable for therapeutic intervention.
miRNAs are small RNAs (usually 19-25 nt) processed from long hairpin-shaped transcript precursors (primary miRNAs). Generally, miRNAs are known to regulate gene expression at the post-transcriptional level by promoting the degradation or inhibition of translation of a target mRNA. However, miRNAs are also able to induce epigenetic modifications, leading to transcriptional gene silencing. Furthermore, each miRNA is able to regulate hundreds of different target genes while a single target mRNA can be regulated by dozens of different miRNAs.
Deregulation of miRNAs expression has been shown in cancer, where they are thought to act as oncogenes (oncomirs) or as tumour suppressors. Although aberrant expression of miRNAs can induce tumourigenesis, miRNAs are also involved in the tumour progression and in the metastatic process [2]. Recently it was shown that specific patterns of miRNAs regulate tumour blood vessel formation (angiogenesis) and consequently may affect cancer progression [3].
Importantly, miRNA expression profiles are now recognised to be more accurate and informative than the mRNA expression profiles, allowing the classification of poorly differentiated tumours that were not discriminated by mRNA profiles [4].
In the case of BM disorders, it has been shown that miR-15a and miR-16-1 are frequently deleted or down-regulated in chronic lymphocytic leukaemia (CLL) [5]. These miRNAs target the anti-apoptotic BCL2 transcript, acting as tumour suppressors; down-regulation or deletion results in inhibition of apoptosis, allowing the tumour cells to grow [6]. The authors of this pivotal study further showed that deregulated miRNAs are often located in fragile chromosomal sites deleted in certain cancers, such as miR15a and miR-16-1, which are found in 13q14.3 region, frequently deleted in CLL [5, 7].
Moreover, the progression of CLL is usually associated with the presence of mutations in the immunoglobulin (Ig) heavy-chain variable-region gene (VH) or expression level of protein ZAP-70 (70-kDa z-chain-associated protein kinase 70): CLL with an aggressive course is characterised by few mutations in the IgVH gene and high-levels of ZAP-70 protein. miRNA expression profiling provided a unique signature of 13 miRNAs, which could discriminate the CLL patients with poor prognosis (unmutated IgVH/ZAP-70 positive patients), and was therefore associated with CLL progression, showing significant prognostic value [8].
Studies of other BM disorders showed that acute myeloid leukaemia (AML) could be distinguished from acute lymphoblastic leukaemia (ALL) based on miRNA expression profiles [9]. From 27 miRNAs differentially expressed, it was shown that as few as two of these were sufficient to discriminate between AML and ALL. Importantly, in ALL, the miRNAs were associated with cytogenetics and disease subtypes. A detailed study defined a miRNA signature associated with cytoplasmic mutated nucleophosmin, one of the commonest cytogenetic defects in AML [10].
Myelodysplastic syndromes (MDS) represent a distinct group of BM disorders whose incidence has increased significantly over the last decade, which has been largely attributed to the secondary effects of conventional anti-cancer treatments including chemotherapy and radiotherapy. Importantly, a subset of MDS patients eventually develops fatal, and clinically aggressive, acute leukaemias. The progression from MDS to acute leukaemia has been recognised as involving activation of endothelial cells in the BM microenvironment, favouring malignant transformation and the progression of leukaemia. MDS patients with poor prognosis are at higher risk of acute leukaemia progression, are characterised by cytogenetic aberrations such as deletions in chromosome 5 or 7, and are unresponsive to aggressive treatments. Recently, miR-145 and miR-146a were shown to be mediators of the MDS patients shown to have deletions in the long arm of chromosome 5; interestingly, these miRNAs
localise in the 5q chromosome [11].
In our lab, we have recently developed a murine model of therapy-induced MDS, which involves sequential whole body irradiations; under this scheme, 60% of irradiated mice develop an MDS-like BM disorder which rapidly progresses into a fatal acute leukaemia, similar to patients with MDS (Cachaco et al, PlosOne, in press). Using this model, we have identified miRNAs differentially expressed in MDS BM compared to control BM. These miRNAs are being investigated to try to understand how their deregulation contributes to BM dysfunction and malignant transformation. Moreover, to understand the role of the BM endothelium in acute leukaemia onset and progression, we also identified miRNA that was differentially expressed on MDS BM endothelial cells versus control BM endothelial cells, and we have shown that the miRNA profiles of BM endothelial cells strongly correlate with the onset of BM disease. Our results show that miRNA regulation of BM endothelial cells contributes towards BM dysfunction, is associated with augmented BM cell apoptosis, and leads to disease onset and progression. Furthermore, our data strongly suggest that the reduced heterogeneity of the BM endothelial cell population allows a better definition of miRNA profiles specifically associated with BM disorders (Costa A et al, manuscript in preparation) [Figure 1 and Figure 2].
Given their therapeutic potential, strategies are being developed to efficiently down-regulate miRNAs for the treatment of different cancers. As miRNAs can regulate different targets and one mRNA target can be regulated by families of related miRNAs, a new therapeutic concept has been put forward, where the management of a single miRNA could be directed to multiple targets. This could be carried out by modifying levels of expression of endogenous miRNAs or by designing artificial miRNAs to induce down-regulation of multiple targets [12]. Generally, therapeutic strategies aim at reducing the level of oncomirs and/or increasing the level of tumour suppressor miRNAs. Strategies to silence miRNA expression have also been developed. Given the importance of BM endothelial cells in the regulation of normal BM function and also for the onset of BM diseases, we suggest that the targeted manipulation of miRNA levels on BM endothelial cells shows enormous potential.
The use of miRNA profiling to classify BM disorders has not only created significant excitement in the field but has also proven to be clinically relevant. Our proposed approach in this field of biomedical research is selectively to isolate a crucial component of the BM microenvironment (endothelial cells), which mirrors the molecular complexity of the malignancy, and to characterise its miRNA profile, resulting in greater specificity and the possible identification of suitable targets for therapeutic intervention.
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The authors
Dr Ana Costa*^º and Sérgio Dias*^º
*Angiogenesis Lab,
CIPM
Instituto Portugues de Oncologia de
Francisco Gentil,
Centro de Lisboa,
EPE, Lisboa, Portugal;
^Instituto Gulbenkian de Ciencia,
Oeiras, Portugal;
ºCEDOC,
Faculdade de Ciencias Médicas da
Universidade Nova de Lisboa,
Lisboa, Portugal
