Junctional Adhesion Molecule-A/F11 Receptor (JAM-A/F11R) Is a Novel Biomarker and a Potential Treatment Target In Multiple Myeloma Tumor and Its Microvascular Milieu

Multiple myeloma (MM) is a disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal immunoglobulin (IG) production, BM failure, osteolytic lesions, and kidney disease. Although initially treatable, tumor cells ultimately become resistant to drug-treatment, and the disease is invariably fatal. Therefore, novel treatment targets need to be identified. The tumor microenvironment, and vascular endothelial cells in particular, play a key role in the adhesion and migration of MM cells and thus govern tumor survival and growth, as well as the acquisition of drug-resistance. Hence, the adhesion/migration systems of MM cells are key potential therapeutic targets. The cell membrane protein JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the IG superfamily, and its expression is upregulated by TNF-a through NF-κB signaling. F11R also alters EC migration and paracellular permeability via stabilization of β1 integrin. We have previously shown that F11R gene expression and serum levels are upregulated in patients with MM compared to healthy controls. In this study, we further explored the functions of F11R within MM cells in order to gain insight into the potential role of this molecule in the progression and treatment of MM.

The MM cell line RPMI-8266 (RPMI) was examined for functional studies in vitro. Informed consent was obtained from all subjects. Primary BM tumor cells were enriched to > 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138– fraction was used for outgrowth of confluent EPCs (> 98% vWF/CD133/KDR+). Human umbilical vein endothelial cells (HUVECs) served as controls. F11R mRNA levels were assessed by Affymetrix GeneChip analysis and by F11R probe-based real-time PCR compared to a standard curve normalized to GAPDH mRNA levels. F11R protein levels were measured by immunofluorescence (IF) and flow cytometry. The role of F11R in MM cell migration and survival was quantified by examining these functions in RPMI cells in which F11R was knocked down by siRNA silencing and comparing them with control untransfected RPMI cells or cells transfected with a non-targeting siRNA or lipofectamine. Tumor migration and survival were determined by the Millipore QCM Chemotaxis assay (using a 5 micron pore size) and an Promega Cell Proliferation Assay, respectively. Each assay was performed in triplicate and replicated at least twice. Statistical analyses were performed using Student's t-test, two-tailed; P≤.05 was considered significant.

Inhibition of F11R gene expression by siRNA resulted in 70% cell death compared to control untransfected (P<.001), non-targeting siRNA (P=.04), or lipofectamine-treated (P=.003) MM cells (RPMI). Moreover, migration of MM cells was also inhibited by 23% after silencing of F11R expression compared to cells transfected with control siRNA (P=.008). Elevated F11R mRNA levels in MM cell lines and patient-derived tumor endothelial progenitor cells (EPCs) was confirmed by IF and flow cytometry using a specific monoclonal antibody, and showed increased expression of both membrane and cytoplasmic F11R compared to controls. Gene expression profiles from 20 patients' corresponding BM tumor cells and EPCs showed that F11R mRNA levels in tumor cells were higher than MM in EPCs by 12.62 fold, (P=1×10-4). However, F11R had a higher level of expression in MM EPCs compared to healthy control EPCs by 2.41 fold (P=.001), reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer cells (Naik et al., 2008).

We show, for the first time, that targeted inhibition of F11R/JAM-A expression bears key anti-myeloma consequences, defined by inhibition of tumor migration and survival. Taken together with elevated gene and protein expression of F11R/JAM-A expression, these results underscore the importance of this receptor as a tumor biomarker and a potential MM treatment target that warrants further validation.

Under investigation are the in vivo effect of F11R silencing in combination with other anti-myeloma strategies in a murine myeloma model; and also, whether F11R effects on MM cell migration involve stabilization of β1 integrin, as recently described in cardiovascular disease by Azari BM et al. 2010.

No relevant conflicts of interest to declare.