Frequent Down Regulation of the Tumor Suppressor Gene A20 in Multiple Myeloma

<div><p>Multiple myeloma (MM) is a malignant clonal expansion of plasma cells in the bone marrow and belongs to the mature B-cell neoplams. The pathogenesis of MM is associated with constitutive NF-κB activation. However, genetic alterations causing constitutive NF-κB activation are still incompletely understood. Since A20 (<i>TNFAIP3</i>) is a suppressor of the NF-κB pathway and is frequently inactivated in various lymphoid malignancies, we investigated the genetic and epigenetic properties of A20 in MM. In total, of 46 patient specimens analyzed, 3 single base pair exchanges, 2 synonymous mutations and one missense mutation were detected by direct sequencing. Gene copy number analysis revealed a reduced A20 gene copy number in 8 of 45 (17.7%) patients. Furthermore, immunohistochemical staining confirmed that A20 expression correlates with the reduction of A20 gene copy number. These data suggest that A20 contributes to tumor formation in a significant fraction of myeloma patients.</p></div

MSPCs are responsible for oxygen sensing and induction of neo-vasculogenesis.

<p>(<b>A</b>) The experimental strategy to delineate the hypoxia response used pre-treatment of either ECFCs or MSPCs before transplantation with YC-1 (inhibiting both HIF-1α as well as HIF-2α) or specific knockdown of HIF-1α RNA (by sh-RNA;). The surprising outcome of vessel formation despite the inhibition of ECFC hypoxia response and collapse of neo-vasculogenesis after MSPC inhibition is graphically illustrated. (<b>B</b>) Based on our current insight a model is proposed describing three temporarily distinct phases of adult vasculogenesis.</p

Mural cells are more sensitive to hypoxia than endothelial cells.

<p>(<b>A</b>) Immune histochemical staining of Matrigel plugs 1 day (d) after implantation of either ECFCs only (left picture), ECFCs/MSPCs together (middle picture) or MSPCs only (right picture). Plugs were explanted and sections were stained with anti-HIF-1α (DAB, brown) and co-stained with hematoxylin (blue; scale bar 50 µm). Staining revealed that 100% of MSPCs display positive signals for HIF-1α, while ECFCs alone show no HIF-1α stabilization and in plugs of ECFCs together with MSPCs the number of positive cells corresponds to the 20% MSPCs. (<b>B</b>) To measure the hypoxia response at the single cell level <i>in vitro</i>, ECFCs and MSPCs were cultured for 4 h at 1%, 5% and 20% O₂. Phase contrast microphotographs show an unchanged appearance. Hypoxyprobe (pimonidazole, green; 4′,6-Diamidin-2-phenylindol, DAPI nuclear stain in blue) revealed that MSPCs sense hypoxia at 5% O₂ and both ECFCs and MSPCs sense hypoxia at 1% O₂. Accordingly, nuclear HIF-1α protein accumulation was detected in MSPCs at 5% and in both progenitor cell types at 1% O₂ (scale bar 100 µm for phase contrast and pimonidazole and 20 µm for HIF-1α pictures). (<b>C</b>) Equal amounts of protein from cell lysates corresponding to the analysis in (B) were tested in western blots showing higher amounts of HIF-1α in MSPCs. MSPCs stabilize HIF-1α at 5% and display a minute baseline signal at ambient air levels of 20% O₂ (n = 3; representative blot regions are shown; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044468#pone.0044468.s001" target="_blank">Figure S1E</a> for complete blot scans).</p

Specific knock-down of HIF-1α in MSPCs but not in ECFCs inhibits vessel formation <i>in vivo</i>.

<p>(<b>A</b>) Total cell lysates of untreated control (<b>−</b>) ECFCs and MSPCs or after infection with either pGIPZ-HIF1α-shRNA (shHIF-1α) or non-specific pGIPZ-scramble-shRNA (NS) were separated by SDS-PAGE after 6 hours of incubation at 1% O₂. Blots were stained with HIF-1α, HIF-2α, HIF-1β or β-actin (β-Act). Full blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044468#pone.0044468.s002" target="_blank">Figure S2</a>. (<b>B</b>) To delineate the impact of genetic ablation of HIF-1α in either ECFCs or MSPCs before co-transplantation, vessel formation was determined one week after co-transplantation of HIF-1α-silenced (ECFC^shHIF-1α, n = 3; MSPC^shHIF-1α, n = 7) or mock-transfected non-silenced cells (ECFC^NS, n = 3; MSPC^NS, n = 7) with genetically un-manipulated corresponding (+ECFC and +MSPC) partner cells as indicated above the columns. Analysis was performed as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044468#pone-0044468-g003" target="_blank">Figure 3A</a> and is identified at the left side of the picture. Scale bars indicate magnification (first and second row, 200 µm; third row, 50 µm; fourth row, 20 µm). (<b>C</b>) Microvessel density was quantified in 200x magnifications of HE stained Matrigel plug sections by ImageJ counting red blood cell filled vessel structures. A significantly decreased capacity to form perfused vessels was found in implants containing shHIF-1α transfected MSPCs compared to untreated co-transplants. (n = 3, 5 high power fields 200x, mean ± SEM; ***p<0.0001).</p

HIF-competent MSPCs are required to avoid premature ECFC apoptosis <i>in vivo</i>.

<p>(<b>A</b>) Apoptosis assay using TdT-mediated dUTP nick end labeling (TUNEL) of ECFC-only or MSPC-only transplants and treated or untreated ECFC+MSPC co-transplants, as indicated above each photograph. All plugs were explanted 24 h after implantation <i>in vivo</i>. DNA strand breaks of apoptotic cells were detected by a TUNEL assay kit (Promega) and nuclei were counterstained with propidium iodide (PI, red). Green fluorescence is due to FITC-labeled nucleotide binding to DNA strand breaks of apoptotic cells. Representative pictures from one experiment are shown (scale bar 100 µm, from at least three different donors performed per transplantation type as specified in the legends to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044468#pone-0044468-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044468#pone-0044468-g004" target="_blank">4</a>). (<b>B</b>) Apoptotic cells depicted as percentage of total cells ± SD with groups corresponding to the representative pictures in (A); five high power fields 200x; ***p<0.0001, **p<0.01).</p

Angiogenic factors can partially substitute MSPC HIF-1α deficiency <i>in vivo</i>.

<p>ECFCs alone or in combination with MSPCs (either un-manipulated or after shHIF-1α knockdown) were (co)-transplanted in either Matrigel or pooled human platelet lysate (pHPL) gel with or without additional growth factors (GF) as specified. The GF-Mix comprised VEGF, EGF, IGF, FGF-2, hydrocortisone and ascorbic acid. Mean ± SD results of the number of perfused vessels counted in five high power fields (200x, at least two independent plugs and two independent animals). Microphotographs correspond to the treatment group (scale bar 50 µm; ***p<0.0001, *p<0.05).</p

Genetic aberrations of A20 in multiple myeloma.

<p><b>a: Electropherogram of rs368271377 in exon 7:</b> Arrows indicate the single base pair substitution. <b>b: Electropherogram of rs143002189 in exon 9:</b> The arrow indicates the single base pair substitution. <b>c: Gene copy number analysis of A20 of selected cases:</b> For the gene copy number assays two technical replicates of each samples were used. The blue bar represents the data for exon 4 and the red one for exon 6. Each bar represents the mean values of expression levels ± standard deviation (SD). Cut off for deletion—depicted as red line—was set at 0.7 through the fact that samples exhibited up to 40% non-neoplastic surrounding tissue. <b>d: Representative immunohistochemical A20 staining of multiple myeloma samples.</b> i and ii: multiple myeloma samples with reduced A20 gene copy number. iii and iv: multiple myeloma samples with normal A20 gene copy number.</p

mRNA expression analysis of A20 and 7 NF-κB target genes (BCL2, Cyclin D1, CCR7, CD44, CXCR2, cFlip, IRF4) of MM cases with (n = 6) and without (n = 14) monoallelic A20 deletions and of non-neoplastic bone marrow biopsies (BM; n = 6).

<p>mRNA expression levels were calculated as relative expression in comparison with peripheral mononucleated cells serving as a calibrator. Each bar represents the mean values of expression levels ± standard deviation (SD). The comparison of the expression levels was performed by using the Mann-Whitney U test; all significant associations were corrected for multiple testing by applying a Bonferroni correction.</p