TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK

<p>Abstract</p> <p>Background</p> <p>Cytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.</p> <p>Results</p> <p>TGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.</p> <p>Conclusions</p> <p>We suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.</p

Mutational dynamics and immune evasion in diffuse large B-cell lymphoma explored in a relapse-enriched patient series

Peer reviewe

A Gene Panel, Including <i>LRP12</i>, Is Frequently Hypermethylated in Major Types of B-Cell Lymphoma

<div><p>Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt's lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (<i>BMP7, BMPER</i>, <i>CDH1</i>, <i>DUSP4</i> and <i>LRP12</i>), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes <i>LRP12</i> and <i>CDH1</i> showed the highest methylation frequencies (94% and 92%, respectively). <i>BMPER</i> (58%), <i>DUSP4</i> (32%) and <i>BMP7</i> (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of <i>LRP12</i> and <i>CDH1</i> methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma.</p></div

Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma

<div><p>Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of <i>DSP</i>, <i>FZD8</i>, <i>KCNH2,</i> and <i>PPP1R14A</i> were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of <i>DSP</i>, <i>FZD8</i>, <i>KCNH2,</i> and <i>PPP1R14A</i> was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.</p></div

BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5

<div><p>Selection and maturation of B cells into plasma cells producing high-affinity antibodies occur in germinal centers (GC). GCs form transiently in secondary lymphoid organs upon antigen challenge, and the GC reaction is a highly regulated process. TGF-β is a potent negative regulator, but the influence of other family members including bone morphogenetic proteins (BMPs) is less known. Studies of human peripheral blood B lymphocytes showed that BMP-6 suppressed plasmablast differentiation, whereas BMP-7 induced apoptosis. Here, we show that human naïve and GC B cells had a strikingly different receptor expression pattern. GC B cells expressed high levels of BMP type I receptor but low levels of type II receptors, whereas naïve B cells had the opposite pattern. Furthermore, GC B cells had elevated levels of downstream signaling components <i>SMAD1</i> and <i>SMAD5</i>, but reduced levels of the inhibitory <i>SMAD7</i>. Functional assays of GC B cells revealed that BMP-7 suppressed the viability-promoting effect of CD40L and IL-21, but had no effect on CD40L- and IL-21-induced differentiation into plasmablasts. BMP-7-induced apoptosis was counteracted by a selective TGF-β type I receptor (ALK4/5/7) inhibitor, but not by a selective BMP receptor type I inhibitor. Furthermore, overexpression of truncated ALK5 in a B-cell line counteracted BMP-7-induced apoptosis, whereas overexpression of truncated ALK4 had no effect. BMP-7 mRNA and protein was readily detected in tonsillar B cells, indicating a physiological relevance of the study. Altogether, we identified BMP-7 as a negative regulator of GC B-cell survival. The effect was counteracted by truncated ALK5, suggesting greater complexity in regulating BMP-7 signaling than previously believed.</p></div

Receiver Operating Characteristics (ROC) curves for individual and combined markers in lymphoma patients versus healthy donors.

<p>The area under the ROC curve (AUC) represents how accurate the individual and combined biomarkers can discriminate between lymphoma and control samples. A) Lymphoma patients versus controls for individual genes. B) Lymphoma patients versus controls for the combined gene panel.</p

Receiver Operating Characteristics (ROC) curves for individual and combined markers in lymphoma patients versus healthy donors.

<p>The area under the ROC curve (AUC) represents how accurate the individual and combined biomarkers can discriminate between lymphomas and normal controls. A) Individual genes. B) The combined gene panel.</p

Methylation frequencies and range of PMR values (brackets) assessed by qMSP in the clinical test and validation sets.

<p>The various healthy controls were unmethylated in all analyzed markers. Genes in bold (<i>DSP</i>, <i>FZD8</i>, <i>KCNH2</i>, <i>PPP1R14A</i>) are included in the combined biomarker panel. Abbreviations: Burkitt`s lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) activated B-cell type (ABC), germinal center B-cell type (GCB), follicular lymphoma (FL), primary mediastinal B-cell lymphoma (PMBL) and non-Hodgkin lymphoma (NHL).</p