High nuclear MSK1 is associated with longer survival in breast cancer patients

Purpose: Mitogen- and stress- activated kinases (MSKs) are important substrates of the mitogen-activated protein kinase (MAPK)-activated protein kinase family. MSK1 and MSK2 are both nuclear serine/threonine protein kinases, with MSK1 being suggested to potentially play a role in breast cancer cell proliferation, cell cycle progression, cell migration, invasion and tumour growth. The aim of the current study was to assess MSK1 protein expression in breast cancer tumour specimens, evaluating its prognostic significance. Methods: A large cohort of 1902 early stage invasive breast cancer patients was used to explore the expression of MSK1. Protein expression was examined using standard immunohistochemistry on tissue microarrays. Results: Low MSK1 protein expression was associated with younger age (P=0.004), higher tumour grade (P<0.001), higher Nottingham Prognostic Index scores (P=0.007), negative ER (P<0.001) and PR (P<0.001) status, and with triple-negative (P<0.001) and basal-like (P<0.001) phenotypes. Low MSK1 protein expression was significantly associated with shorter time to distant metastasis (P<0.001), and recurrence (P=0.013) and early death due to breast cancer (P=0.01). This association between high MSK1 expression and improved breast cancer-specific survival was observed in the whole cohort (P=0.009) and in the HER2 negative and non-basal like tumours (P=0.006 and P=0.024, respectively). Multivariate analysis including other prognostic variables indicated that MSK1 is not an independent marker of outcome. Conclusions: High MSK1 is associated with improved breast cancer-specific survival in early stage invasive breast cancer patients, and has additional prognostic value in HER2 negative and non-basal like disease. Although not an independent marker of outcome we believe such findings, and significant associations with well-established negative prognostic factors (age, grade, Nottingham Prognostic Index, hormone receptor status, time to distant metastasi

Suppression of Lung Adenocarcinoma Progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras[superscript LSL-G12D/+];p53[superscript flox/flox] mice initiates lung adenocarcinoma development4. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limitsmetastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions ofNkx2-1 in the sametumourNational Institutes of Health (U.S.) (grant U01-CA84306 )National Institutes of Health (U.S.) (grant K99-CA151968)Howard Hughes Medical InstituteLudwig Center for Molecular OncologyNational Cancer Institute (U.S.) (Cancer Center Support (core) grant P30-CA14051

Reconstruction of primary vertices at the ATLAS experiment in Run 1 proton–proton collisions at the LHC

This paper presents the method and performance of primary vertex reconstruction in proton–proton collision data recorded by the ATLAS experiment during Run 1 of the LHC. The studies presented focus on data taken during 2012 at a centre-of-mass energy of √s=8 TeV. The performance has been measured as a function of the number of interactions per bunch crossing over a wide range, from one to seventy. The measurement of the position and size of the luminous region and its use as a constraint to improve the primary vertex resolution are discussed. A longitudinal vertex position resolution of about 30μm is achieved for events with high multiplicity of reconstructed tracks. The transverse position resolution is better than 20μm and is dominated by the precision on the size of the luminous region. An analytical model is proposed to describe the primary vertex reconstruction efficiency as a function of the number of interactions per bunch crossing and of the longitudinal size of the luminous region. Agreement between the data and the predictions of this model is better than 3% up to seventy interactions per bunch crossing

Absence of Inhibin Alpha and Retinoblastoma Protein Leads to Early Sertoli Cell Dysfunction

Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin α knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10–14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice

Polyoma Virus-Induced Osteosarcomas in Inbred Strains of Mice: Host Determinants of Metastasis

The mouse polyoma virus induces a broad array of solid tumors in mice of many inbred strains. In most strains tumors grow rapidly but fail to metastasize. An exception has been found in the Czech-II/Ei mouse in which bone tumors metastasize regularly to the lung. These tumors resemble human osteosarcoma in their propensity for pulmonary metastasis. Cell lines established from these metastatic tumors have been compared with ones from non-metastatic osteosarcomas arising in C3H/BiDa mice. Osteopontin, a chemokine implicated in migration and metastasis, is known to be transcriptionally induced by the viral middle T antigen. Czech-II/Ei and C3H/BiDa tumor cells expressed middle T and secreted osteopontin at comparable levels as the major chemoattractant. The tumor cell lines migrated equally well in response to recombinant osteopontin as the sole attractant. An important difference emerged in assays for invasion in which tumor cells from Czech-II/Ei mice were able to invade across an extracellular matrix barrier while those from C3H/BiDa mice were unable to invade. Invasive behavior was linked to elevated levels of the metalloproteinase MMP-2 and of the transcription factor NFAT. Inhibition of either MMP-2 or NFAT inhibited invasion by Czech-II/Ei osteosarcoma cells. The metastatic phenotype is dominant in F1 mice. Osteosarcoma cell lines from F1 mice expressed intermediate levels of MMP-2 and NFAT and were invasive. Osteosarcomas in Czech-II/Ei mice retain functional p53. This virus-host model of metastasis differs from engineered models targeting p53 or pRb and provides a system for investigating the genetic and molecular basis of bone tumor metastasis in the absence of p53 loss

The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

Background: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. Methodology/Principal Findings: We have generated conditional knockout mice to obtain NFAT5−/− T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5−/− cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5−/− cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5−/− lymphocytes. Conclusions/Significance: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.Molecular and Cellular Biolog

Knockout and transgenic mice of Trp53: what have we learned about p53 in breast cancer?

The human p53 tumor suppressor gene TP53 is mutated at a high frequency in sporadic breast cancer, and Li-Fraumeni syndrome patients who carry germline mutations in one TP53 allele have a high incidence of breast cancer. In the 10 years since the first knockout of the mouse p53 tumor suppressor gene (designated Trp53) was published, much has been learned about the contribution of p53 to biology and tumor suppression in the breast through the use of p53 transgenic and knockout mice. The original mice deficient in p53 showed no mammary gland phenotype. However, studies using BALB/c-Trp53-deficient mice have demonstrated a delayed involution phenotype and a mammary tumor phenotype. Together with other studies of mutant p53 transgenes and p53 bitransgenics, a greater understanding has been gained of the role of p53 in involution, of the regulation of p53 activity by hormones, of the effect of mouse strain and modifier genes on tumor phenotype, and of the cooperation between p53 and other oncogenic pathways, chemical carcinogens and hormonal stimulation in mammary tumorigenesis. Both p53 transgenic and knockout mice are important in vivo tools for understanding breast cancer, and are yet to be exploited for developing therapeutic strategies in breast cancer

Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-κB-dependent Genes

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells

Overcoming barriers in evaluating outbreaks of diarrheal disease in resource poor settings: assessment of recurrent outbreaks in Chobe District, Botswana

BACKGROUND: Diarrheal illness remains a leading cause of global morbidity and mortality, with the majority of deaths occurring in children <5 years of age. Lack of resources often prohibits the evaluation of outbreak characteristics and limits progress in managing this important disease syndrome, particularly in Africa. Relying only on existing medical staff and hospital resources, we assess the use of a questionnaire survey tool to identify baseline outbreak characteristics during recurrent diarrheal outbreaks in Chobe, Botswana. METHODS: Using historical surveillance data (2006–2009), the temporal pattern of recurrent diarrheal outbreaks was evaluated among patients <5 years of age presenting to health facilities in Chobe District. Using a questionnaire survey tool, medical staff from selected health facilities assessed patients (all ages) presenting with diarrheal disease during two diarrheal outbreaks (2011–2012). Cluster analysis and classification and regression trees (CART) were used to evaluate patient attributes by outbreak. RESULTS: We identified a bimodal, annual pattern of acute diarrhea in children <5 years of age across years (Wilcox test, W = 456.5, p = 0.052). Historical outbreak periods appeared to coincide with major hydrological phenomena (rainfall/flood recession). Across health facilities, a significant percent of patients in the prospective study were in the ≥5 age class (44%, n = 515 and 35%, n = 333 in the dry and wet season outbreaks, respectively). Cluster analysis of questionnaire data identified two main branches associated with patient age (<5 and ≥5 years of age). Patients did not cluster by outbreak or village. CART examination identified sex and hospitalization as being most predictive of patients <5 years and household diarrhea in patients ≥5 years. Water shortages and water quality deficiencies were identified in both outbreaks. CONCLUSIONS: Diarrhea is a persistent, seasonally occurring disease in Chobe District, Botswana. Lack of variation in outbreak variables suggests the possibility of environmental drivers influencing outbreak dynamics and the potential importance of human-environmental linkages in this region. Public health strategy should be directed at securing improved water service and correcting water quality deficiencies. Public health education should include increased emphasis on sanitation practices when providing care to household members with diarrhea. While global diarrheal disease surveillance is directed at the under-5 age group, this may not be appropriate in areas of high HIV prevalence such as that found in our study area where a large immune-compromised population may warrant increased surveillance across age groups. The approach used in this study provided the first detailed characterization of diarrheal disease outbreaks in the area, an important starting point for immediate intervention and development of working hypotheses for future disease investigations. While data derived from this approach are necessarily limited, they identify critical information on outbreak characteristics in resource poor settings where data gaps continue and disease incidence is high