Elevated APOBEC3B Correlates with Poor Outcomes for Estrogen-Receptor-Positive Breast Cancers

Recent observations connected DNA cytosine deaminase APOBEC3B to the genetic evolution of breast cancer. We addressed whether APOBEC3B is associated with breast cancer clinical outcomes. APOBEC3B messenger RNA (mRNA) levels were related in 1,491 primary breast cancers to disease-free (DFS), metastasis-free (MFS), and overall survival (OS). For independent validation, APOBEC3B mRNA expression was associated with patient outcome data in five additional cohorts (over 3,500 breast cancer cases). In univariate Cox regression analysis, increasing APOBEC3B expression as a continuous variable was associated with worse DFS, MFS, and OS (hazard ratio [HR] = 1.20, 1.21, and 1.24, respectively; all P < .001). Also, in untreated ER-positive (ER+), but not in ER−, lymph-node-negative patients, high APOBEC3B levels were associated with a poor DFS (continuous variable: HR = 1.29, P = .001; dichotomized at the median level, HR = 1.66, P = .0002). This implies that APOBEC3B is a marker of pure prognosis in ER + disease. These findings were confirmed in the analyses of five independent patient sets. In these analyses, APOBEC3B expression dichotomized at the median level was associated with adverse outcomes (METABRIC discovery and validation, 788 and 706 ER + cases, disease-specific survival (DSS), HR = 1.77 and HR = 1.77, respectively, both P < .001; Affymetrix dataset, 754 ER + cases, DFS, HR = 1.57, P = 2.46E-04; NKI295, 181 ER + cases, DFS, HR = 1.72, P = .054; and BIG 1-98, 1,219 ER + cases, breast-cancer-free interval (BCFI), HR = 1.42, P = 0.0079). APOBEC3B is a marker of pure prognosis and poor outcomes for ER + breast cancer, which strongly suggests that genetic aberrations induced by APOBEC3B contribute to breast cancer progression. Electronic supplementary material The online version of this article (doi: 10.1007/s12672-014-0196-8) contains supplementary material, which is available to authorized users

The (I/Y)XGG Motif of Adenovirus DNA Polymerase Affects Template DNA Binding and the Transition from Initiation to Elongation

Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increasedK m(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.This work was supported in part by the Netherlands Organization for scientific research (to P. C. v. d. V.), by National Institutes of Health Grant 2R01 GM27242-21 and Dirección General de Investigación Cientı́fica y Técnica grant PB98–0645 (to M. S.), by an institutional grant from the Fundación Ramón Areces (to the Centro de Biologı́a Molecular “Severo Ochoa”), and by European Union Contract FMRX-CT97-0125 (to P. C. v. d. V. and M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Peer reviewe

Differential Programming of B Cells in AID Deficient Mice

<div><p>The <i>Aicda</i> locus encodes the activation induced cytidine deaminase (AID) and is highly expressed in germinal center (GC) B cells to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. Besides these Ig specific activities in B cells, AID has been implicated in active DNA demethylation in non-B cell systems. We here determined a potential role of AID as an epigenetic eraser and transcriptional regulator in B cells. RNA-Seq on different B cell subsets revealed that <i>Aicda^−/−</i> B cells are developmentally affected. However as shown by RNA-Seq, MethylCap-Seq, and SNP analysis these transcriptome alterations may not relate to AID, but alternatively to a CBA mouse strain derived region around the targeted <i>Aicda</i> locus. These unexpected confounding parameters provide alternative, AID-independent interpretations on genotype-phenotype correlations previously reported in numerous studies on AID using the <i>Aicda^−/−</i> mouse strain.</p></div

Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages

The extracellular serine protease inhibitor serpinE2 is overexpressed in breast cancer and has been shown to foster metastatic spread. Here, we investigated the hypothesis that serpinE2 creates tumor-promoting conditions in the tumor microenvironment (TME) by affecting extracellular matrix remodeling. Using two different breast cancer models, we show that blocking serpinE2, either by knock-down (KD) in tumor cells or in response to a serpinE2 binding antibody, decreases metastatic dissemination from primary tumors to the lungs. We demonstrate that in response to serpinE2 KD or antibody treatment there are dramatic changes in the TME. Multiphoton intravital imaging revealed deposition of a dense extracellular collagen I matrix encapsulating serpinE2 KD or antibody-treated tumors. This is accompanied by a reduction in the population of tumor-promoting macrophages, as well as a decrease in chemokine ligand 2, which is known to affect macrophage abundance and polarization. In addition, TIMP-1 secretion is increased, which may directly inhibit matrix metalloproteases critical for collagen degradation in the tumor. In summary, our findings suggest that serpinE2 is required in the extracellular milieu of tumors where it acts in multiple ways to regulate tumor matrix deposition, thereby controlling tumor cell dissemination

Transcriptome comparisons of GC B cells and <i>in vitro</i> activated B cells from <i>Aicda^+/+</i> and <i>Aicda^−/−</i> mice.

<p>A) Box-plot of previously defined gene groups which are differentially expressed between <i>Aicda^+/+</i> and <i>Aicda^−/−</i> GC (left panel) and activated (right panel) B cells: CON, control group; LZS light zone signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Victora1" target="_blank">[29]</a>, DZS, dark zone signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Victora1" target="_blank">[29]</a>; NVS, naïve B cell signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Klein2" target="_blank">[28]</a>, CBS, centroblast signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Klein2" target="_blank">[28]</a>. For statistical analysis the sign test was applied. B) Volcano-plot of genes differentially expressed between <i>Aicda^+/+</i> and <i>Aicda^−/−</i> GC (left panel) and activated (right panel) B cells. The <i>Ighv</i> genes are shown in red. For statistical analysis the sign test was applied.</p

Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G1 cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21Cip. Notably, concomitant genetic inactivation of p53 or p21Cip indicates that Hdac1 and Hdac2 regulate p53–p21Cip-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors

Assessment of AID-dependent DNA demethylation.

<p>A) Comparisons of CpG methylation load between <i>Aicda^+/+</i> and <i>Aicda^−/−</i> GC B cells in defined genomic elements. TSS, transcription start sides (TSS); EXN, exons; INT, introns; ING, intergenic regions are shown. Various statistical tests revealed no differences. B) Correlation plot of MethylCap data obtained from <i>Aicda^+/+</i> and <i>Aicda^−/−</i> GC B cells. The correlation coefficient, <i>r</i> was determined applying the Persons’ test.</p

Naïve B cells are pre-activated.

<p>A) Box-plot of previously defined gene groups differentially expressed between <i>Aicda^+/+</i> and <i>Aicda^−/−</i> naive B cells: CON, control group; LZS light zone signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Victora1" target="_blank">[29]</a>, DZS, dark zone signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Victora1" target="_blank">[29]</a>; NVS, naïve B cell signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Klein2" target="_blank">[28]</a>, CBS, centroblast signature genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069815#pone.0069815-Klein2" target="_blank">[28]</a>. B) Volcano-plot of genes differentially expressed between <i>Aicda^+/+</i> and <i>Aicda^−/−</i> naïve B cells. The DZS genes are shown in red.</p