The micros in harmony: microRNA and microenvironment

Multiple myeloma (MM) is a B-cell neoplasm characterized by the aberrant expansion of plasma cells within the bone marrow. Previously, glucocorticoids and alkylating agents were the mainstream chemotherapeutic agents for this neoplasm [1], whereas proteasome inhibitors and immunomodulatory agents have now become the standard of care. A few other inhibitors of heat shock protein-90 (HSP-90), cyclin-dependent kinases (CDKs) and cell surface antigens are being tested as experimental drugs [2]. Bortezomib (PS-341; Velcade) is an inhibitor of proteasome, a ubiquitous complex that degrades cell cycle regulating proteins and induces proteolysis of IκB, a specific inhibitor of the transcription factor NF-κB (nuclear factor-κB). Mechanistically, bortezomib induces caspase-dependent apoptosis by promoting up-regulation of NOXA and down-regulation of apoptosis inhibitors via NF-κB blockade. Despite the progress in target discoveries and their potential inhibitors, long-term disease-free survival is rare in MM. This is because MM is not a single disease entity, but rather is a complex malignancy that is in network with the bone marrow (BM) microenvironment, a bona fide candidate for chemoresistance [3]. Therefore, moderating treatment options for myeloma is not limited to merely eliminating plasma cells, but to ultimately disrupting stromal cell-mediated signaling pathways.

Plasma cells in myeloma home to the BM microenvironment, comprising adhesion molecules, integrins (intracellular adhesion molcule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], very late antigen-4 [VLA-4], VLA-5), growth factors (hepatocyte growth factor [HGF], vascular endothelial growth factor [VEGF], basic fibroblast growth factor [bFGF], platelet derived growth factor [PDGF], epidermal growth factor [EGF], insulin-like growth factor [IGF]), growth factor receptors (c-Met, VEGFR, FGFR, PDGFR, EGFR, IGFR, human epidermal growth factor receptor 2 [HER2], insulin receptor [InsR]), cytokines (interleukin-6 [IL-6], IL-8, IL-3, tumor necrosis factor α [TNFα]), chemokines (macrophage inflammatory protein 3α [MIP3α], CCL20, CXCR6, MIP1 (CCL3), monocyte chemotactic protein-1 [MCP-1], stromal cell derived factor-1 [SDF-1]) and transcription factors [4]. Adhesion of MM cells to extracellular matrixproteins confers cell adhesion-mediated drug resistance (CAM-DR), whereas binding of myeloma cells to bone marrow stromal cells (BMSCs) triggers the transcription and secretion of cytokines, including IL-6, VEGF and SDF-1α, which mediate MM cell proliferation, survival, drug resistance, migration and angiogenesis [Figure 1(a)]. In addition, BAFF (B-cell activating factor), a cytokine that belongs to the TNF ligand family, highly secreted by MM-BMSCs, binds to three receptors specifically expressed on B cells, TACI (TRAF interacting receptor), BCMA (B cell maturation), and BAFF-R [5]. These receptors signal by recruiting TNF receptor-associated factors (TRAFs) to their cytoplasmic tails and activate the degradation of IκB. The subsequent nuclear translocation of NF-κB transcriptionally activates genes involved in B-cell proliferation, differentiation and survival. Taken together, this network of survival signals creates an environment affording MM cells resistance to chemotherapy.

Figure 1. (a) Previously established interplay between myeloma plasma cell and the microenvironment in response to bortezomib mediated apoptosis [2–4]. There are several mechanisms through which a myeloma cell can receive signals for angiogenesis, proliferation and survival (purple circles): (1) epigenetic silencing of miR-15a/16, (2) NF-κB activation and (3) VEGF or IL-6 mediated induction of Bcl-2 family anti-apoptotic proteins. While the first two pathways can be potentially targeted via miR therapy or bortezomib, the ever-growing challenge is to disrupt the interactions of BMSC-MM plasma cell and the consequent downstream signaling molecules. BMSC, bone marrow stromal cell; VEGF, vascular endothelial growth factor; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intracellular adhesion molecule-1; SDF-1, stromal cell derived factor-1; BAFF, B-cell activating factor; BCMA, B cell maturation; TACI, TRAF interacting receptor; BAFF-R, B-cell activating factor receptor, IL-6, interleukin-6; IAPs, inhibitory apoptotic proteins; AA, anti-apoptotic proteins; AM, adhesion molecules. (b) Current evidence on inhibitory role of MM-BMSC in bortezomib-induced apoptosis [8]. BMSC induced VEGF, Bcl-2 and cyclin D production is inhibited by bortezomib and miR-15a (purple circles 1 and 2). Bortezomib-mediated miR-15a up-regulation and concurrent repression of its respective targets (VEGF, cyclin D, Bcl-2) are blocked by BMSCs, whose mechanism is unknown (purple circle 3).

While myeloma plasma cell survival is hugely driven by microenvironment, there are reports evidencing epigenetic silencing of microRNAs (miRs), and deregulations of miR signature contribute to MM pathogenesis. First, overexpression of miR-21, miR-106b˜25 cluster and miR-181a and b is reported in myeloma plasma cells compared to healthy plasma cells (PCs). Second, selective up-regulation of miR-32 and miR-17˜92 cluster was identified in myeloma PCs but not in healthy PCs. Third, two distinct miRNAs, miR-19a and 19b, that are part of the miR-17˜92 cluster, were shown to down-regulate expression of SOCS-1, a gene frequently silenced in MM, which plays a critical role as an inhibitor of IL-6 growth signaling. Fourth, p300-CBP-associated factor, a gene involved in p53 regulation in MM, has been identified as a bona fide target of the miR-106b˜25 cluster, miR-181a and b and miR-32. Fifth, xenograft studies using human myeloma cell lines treated with miR-19a and b and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. Sixth, microRNA 192, 194 and 215 genes, that are activated by transcription factor p53, are silent in newly diagnosed MM. Seventh, human MDM2 mRNA is a direct target of three miRs: 192, 194 and 215, which can substantially enhance the pharmacological activity of MDM2 inhibitors. In addition to MDM2 (murine double minute 2), miRs 192 and 215 target the IGF pathway, preventing enhanced migration of plasma cells into the bone marrow. Eighth, as in chronic lymphocytic leukemia, 13q14 deletion is also seen in the early stages of MM, suggesting loss of miR-15a/16-1 in this neoplasm. In addition to Bcl-2 and Mcl-1, BM components such as FGFR1, phosphatidylinositol 3-kinase (PI3K), MDM4 and VEGFα are additional targets of miR-16. Ninth, myeloma cells express germinal center B-cell associated miRs such as 93 and 181b, 30a and 223, constituting the CD19(−)/miR-223(+) phenotype. In summary, the MM microRNA signature modulates the expression of proteins critical to myeloma pathogenesis, underscoring the role of miRNA in myeloma biology [6].

While microenvironment and miR signature and their interaction with malignant PCs have been focused individually, their functional role as a team is still unclear. Raccaro et al. studied the regulatory role of miRs in MM progression in context with BM milieu. They reported a myeloma-specific miR signature that is characterized by the repression of miR-15a/16-1 and overexpression of miR-222/-221/-382/-181a/-181b in the presence of stroma [7]. They also revealed the functional role of miR-15a/16-1 in regulating the proliferation of MM cells by inhibiting PI3 kinases. Following this observation, Hao et al. [8] in this issue of Leukemia and Lymphoma demonstrate that bortezomib as a single agent sensitized myeloma cells (U266), whose apoptosis was abrogated when co-cultured in the presence of MM-BMSCs, but not when co-cultured with normal BMSCs. Although bortezomib is a classic proteasomal inhibitor, these results confirm its lack of ability to overcome MM-BMSC-associated NF-κB activation. Their attempt to investigate the mechanism behind the protection associating regulatory microRNAs revealed the interaction between the miRs and BMSCs. BMSC-induced apoptosis blockade was mediated through Bcl-2 up-regulation, but not Mcl-1. Though Mcl-1 is a direct target of miR-15a, why it remains unaffected is not answered. Because Bcl-2 is a direct target of miR-15a, they tested miR-15a expression and confirmed this miR to be repressed in cells incubated with BMSCs. However, mechanistic studies to elucidate how bortezomib can mediate up-regulation of miR-15a or which factor upstream in BMSCs is associated with miR-15a repression are lacking. Transfection studies with miR-15a confirmed decreases in VEGF, cyclin D and Bcl-2, the direct targets of miR-15a. Transwell experiments substantiate that direct cell–cell contact and adhesion of MM cells to BMSCs are required to attenuate the BMSC-featured biological events via an increase in adhesion molecules VCAM-1 and ICAM-1 [Figure 1(b)].

Although the work described here provides a mechanism of resistance, it also raises several questions. The focus was on a single miR (miR-15a) and its downstream targets (VEGF, Bcl-2 and cyclin D), while there are other miRs (miR-146) in the vicinity whose downstream targets could be essential components of BMSCs (TNFα, CXCR4, EGFR, IL-2 and NF-κB) [9]. Pichiorri et al. highlighted several other miRs (besides miR-15a/16-1) repressed in MM [10]. The insight into putative targets of these miRs or indirect targets of miR-15a/16-1 whose expression is perhaps repressed by another direct target of miR-15a may reveal a different mechanism of association between these factors. The data obtained in the current study is from two myeloma cell lines with 13q14 del, and it would be interesting to study other lines without 13q14 deletion and the mechanism of resistance to bortezomib in the presence of BMSCs, or perhaps the implication for primary myeloma cells with the same genetic lesion. Because 13q14 deletion also holds true for chronic lymphocytic leukemia, would the mechanism of chemoresistance be the same in this B-cell leukemia? Finally, while microRNA regulates the expression of several target genes, it may prove useful to investigate the role of bone marrow constituents in the regulation of miR biogenesis or bio-machinery. New investigations focusing on these questions may provide a novel insight into the harmony between miRNAs and the microenvironment.

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