Gene Expression Profiling of Bone Marrow Endothelial Cells in Patients with Multiple Myeloma

Abstract

Purpose: To determine a "gene/molecular fingerprint" of multiple myeloma endothelial cells and identify vascular mechanisms governing the malignant progression from quiescent monoclonal gammopathy of undetermined significance. Experimental Design: Comparative gene expression profiling of multiple myeloma endothelial cells and monoclonal gammopathy of undetermined significance endothelial cells with the Affymetrix U133A Arrays was carried out in patients at diagnosis; expression and function of selective vascular markers was validated by real-time reverse transcriptase-PCR, Western blot, and small interfering RNA analyses. Results: Twenty-two genes were found differentially expressed (14 down-regulated and eight up-regulated) at relatively high stringency in multiple myeloma endothelial cells compared with monoclonal gammopathy of undetermined significance endothelial cells. Functional annotation revealed a role of these genes in the regulation of extracellular matrix formation and bone remodeling, cell adhesion, chemotaxis, angiogenesis, resistance to apoptosis, and cell-cycle regulation. Validation was focused on six genes (DIRAS3, SERPINF1, SRPX, BNIP3, IER3, and SEPW1) not previously found to be functionally correlated to the overangiogenic phenotype of multiple myeloma endothelial cells in active disease. The small interfering RNA knockdown of BNIP3, IER3, and SEPW1 genes affected critical multiple myeloma endothelial cell functions correlated with the overangiogenic phenotype. Conclusions: The distinct endothelial cell gene expression profiles and vascular phenotypes detected in this study may influence remodeling of the bone marrow microenvironment in patients with active multiple myeloma. A better understanding of the linkage between plasma cells and endothelial cells in multiple myeloma could contribute to the molecular classification of the disease and thus pinpoint selective gene targets for more effective antiangiogenic treatments. (Clin Cancer Res 2009;15(17):5369-78) The unique markers expressed by tumor vasculature distinguish it from normal endothelium. These abnormalities reflect the pathologic nature of its induction and attempts to discover tumor endothelial cell markers have always been hampered by technical difficulties in isolating functionally intact and phenotypically stable endothelial cells from patient samples. St. Croix et al. (1) were the first to show that colorectal cancer endothelial cells overexpress specific transcripts as a result of qualitative

Microvesicles secreted from human multiple myeloma cells promote angiogenesis

“…These results support the role of MM-MVs in angiogenesis, consistent with findings in other cancers [12,14,21] . Of note, the origin of MMendothelial cells is actually unknown [42,43] . The present study supports the idea that these cells are malignantly transformed endothelial cells, perhaps partly due to MM-MVs; this idea is highly consistent with the observation that TMVs can form niches that favor tumor development [22,44] .…”

Four proteins governing overangiogenic endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets

“…This proteomic signature renders MMECs very similar to transformed (such as tumor) cells than normal ECs. The MMECs' similarity to tumor cells has been also hypothesized in studies at the genomic level (Ria et al, 2009). As overexpression of FLNA, VIM, CRYAB and YWHAZ in MMECs has a key role in determining their overangiogenic phenotype, and as their silencing is antiangiogenic, these proteins could be considered new targets for molecular therapy in MM patients.…”

“…Real time reverse transcriptase PCR was performed as described (Ria et al, 2009) using primers (Invitrogen, Paisley, UK; Supplementary Table 2) and the Applied Biosystems methodology. Relative quantification of the mRNA level was performed using the comparative Ct method with GAPDH as the reference gene and with the 2 ÀDDCT formula (Livak and Schmittgen, 2001).…”

“…Knowledge of global changes in MMECs' proteome in active disease may be critical to elucidate these molecular mechanisms. Although gene microarray-based profiling of MMECs versus MGECs and normal ECs revealed a role of the identified genes in the regulation of extracellular matrix formation, cell adhesion, chemotaxis, angiogenesis and resistance to apoptosis (Ria et al, 2009), the relationships between mRNA production and protein synthesis is not closely correlated. Here, we compare MMEC with MGEC protein expression profiles, and identify four constantly overexpressed proteins in MMECs: filamin A (FLNA), vimentin (VIM), a-crystallin B (CRYAB) and 14-3-3z/d protein (YWHAZ) not yet previously correlated with overangiogenic activity.…”

“…Activated MMECs are endowed not only with enhanced angiogenesis but also with proliferative advantage over normal ECs, which implies resistance to apoptosis and genome instability (Vacca et al, 2003;Ria et al, 2009). Accordingly, several essential pathways regulating cytoskeletal organization, cell mitosis and apoptosis are altered in these cells.…”