Luminal breast epithelial cells from wildtype and BRCA mutation carriers harbor copy number alterations commonly associated with breast cancer.

Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNAs in >42,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Accumulation of individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) is detectable in almost all breast tissues and, in those from BRCA1 or BRCA2 mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells not basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from BRCA1 or BRCA2 mutation carriers contain a small percentage of cells with extreme aneuploidy, featuring loss of TP53 , LOH of BRCA1 or BRCA2 , and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs are not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium partially mirror those of established cancer genomes and are chromosome- and cell lineage-specific.

Statistical distribution of micro and macro pores in acrylic bone cement- effect of amount of antibiotic content.

Aseptic loosening due to mechanical failure of bone cement is considered to be a leading cause of revision of joint replacement systems. Detailed quantified information on the number, size and distribution pattern of pores can help to obtain a deeper understanding of the bone cement's fatigue behavior. The objective of this study was to provide statistical descriptions for the pore distribution characteristics of laboratory bone cement specimens with different amounts of antibiotic contents. For four groups of bone cement (Palacos) specimens, containing 0.3, 0.6, 1.2 and 2.4 wt/wt% of telavancin antibiotic, seven samples per group were micro computed tomography scanned (38.97 µm voxel size). The images were first preprocessed in Mimics and then analyzed in Dragonfly, with the level of threshold being set such that single-pixel pores become visible. The normalized pore volume data of the specimens were then used to extract the logarithmic histograms of the pore densities for antibiotic groups, as well as their three-parameter Weibull probability density functions. Statistical comparison of the pore distribution data of the antibiotic groups using the Mann-Whitney non-parametric test revealed a significantly larger porosity (p < 0.05) in groups with larger added antibiotic contents (2.4 and 0.6 wt/wt% vs 0.3 wt/wt%). Further analysis revealed that this effect was associated with the significantly larger frequency of micropores of 0.1-0.5 mm diameter (p < 0.05) in groups with larger antibiotic content (2.4 wt/wt% vs and 0.6 and 0.3 wt/wt%), implying that the elution of the added antibiotic produces micropores in this diameter range mainly. Based on this observation and the fatigue test results in the literature, it was suggested that micropore clusters have a detrimental effect on the mechanical properties of bone cement and play a major role in initiating fatigue cracks in highly antibiotic added specimens.

Influence of Porosity on Fracture Toughness and Fracture Behavior of Antibiotic-Loaded PMMA Bone Cement.

Aseptic loosening is the most common reason for the long-term revision of cemented arthroplasties with fracture of the cement being a postulated cause or contributing factor. In our previous studies we showed that adding an antibiotic to a polymethylmethacrylate (PMMA) bone cement led to detrimental effects on various mechanical properties of the cement such as bending strength, compressive strength and fracture toughness (KIC). This finding implied that the mechanical failure of antibiotic-loaded PMMA bone cement was influenced by its pore volume fraction. Up to now this aspect has not been studied. Hence the purposes of this study were to determine (1) the influence of antibiotic (telavancin) loading on the KIC of a widely used PMMA bone cement brand (Palacos®R) and (2) the influence of pore size and pore distribution on the fracture behavior of the KIC specimens. For (2) both experimental and numerical methods (extended finite element method [XFEM]) were used allowing a comparison between the two sets of results. We found that: (1) KIC decreased with increased porosity with the drop (relative to the value for the control cement) being significant when the telavancin loading was 4.8 wt/wt % (2 g of telavancin added to 40 g of control cement powder); (2) there was a critical pore size above which there was a significant decrease in KIC and is 1 mm; (3) crack propagation was strongly influenced by pore size and pore locations (pore-pore interactions); and, (4) there was good agreement between the experimental and XFEM results. The implications of these findings for the use of a telavancin-loaded PMMA bone cement in cemented total joint arthroplasties are commented upon.

A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene.

Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.

Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1.

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1.

Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila.

Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.

Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1.

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity.

A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing.

The exon junction complex controls the splicing of MAPK and other long intron-containing transcripts in Drosophila.

Signaling pathways are controlled by a vast array of posttranslational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. We conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a key element of this pathway. The EJC binds the exon-exon junctions of mRNAs and thus far, has been linked exclusively to postsplicing events. Here, we report that the EJC is required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC.

The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

Alphabet, a Ser/Thr phosphatase of the protein phosphatase 2C family, negatively regulates RAS/MAPK signaling in Drosophila.

Signal transduction through the RAS/mitogen-activated protein kinase (MAPK) pathway depends on a diverse collection of proteins regulating positively and negatively signaling flow. We previously conducted a genetic screen in Drosophila to identify novel components of this signaling pathway. Here, we present the identification and characterization of a new gene, alphabet (alph), whose activity negatively regulates RAS/MAPK-dependent developmental processes in Drosophila and this, at a step downstream or in parallel to RAS. alph encodes a protein phosphatase 2C (PP2C) family member closely related to the mammalian PP2C alpha and beta isoforms. Interestingly, although alph gene product does not appear to be essential for viability, its elimination leads to weak but significant developmental defects reminiscent of an overactivated RAS/MAPK pathway. Consistent with this interpretation, strong genetic interactions are observed between alph alleles and mutations in bona fide components of the pathway. Together, this work identifies a PP2C of the alpha/beta subfamily as a novel negative regulator of the RAS/MAPK pathway and suggests that these evolutionarily conserved enzymes play a similar role in other metazoans. Finally, despite the relatively large size of the PP2C gene family in metazoans, this study represents only the second genetic characterization of a PP2C in these organisms.