Understanding the molecular underpinnings of cancer is of critical importance to developing targeted intervention strategies. Identification of such targets, however, is notoriously difficult and unpredictable. Malignant cell transformation requires the cooperation of a few oncogenic mutations that cause substantial reorganization of many cell features 1 and induce complex changes in gene expression patterns 2-6 . Genes critical to this multi-faceted cellular phenotype thus only have been identified following signaling pathway analysis 7-10 or on an ad hoc basis 4, 11-14 . Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signaling circuitry 15-17 suggest that malignant transformation is a highly cooperative process, involving synergy at multiple levels of regulation, including gene expression. Here we show that a large proportion of genes controlled synergistically by loss-of-function p53 and Ras activation are critical to the malignant state. Remarkably, 14 among 24 such 'cooperation response genes' (CRGs) were found to contribute to tumor formation in gene perturbation experiments. In contrast, only one in 14 perturbations of genes responding in a non-synergistic manner had a similar effect. Synergistic control of gene expression by oncogenic mutations thus emerges as an underlying key to malignancy and provides an attractive rationale for identifying intervention targets in gene networks downstream of oncogenic gain and loss-of-function mutations.To identify genes regulated synergistically by cooperating oncogenic mutations at genomic scale, we compared mRNA expression profiles of young adult murine colon (YAMC) cells

Human papillomaviruses (HPVs) cause squamous cancers of epithelial surfaces, of which genital cancers are the most common. In this article we have attempted to describe the properties and functions of the viral proteins of HPV type 16, a common cause of genital cancers, and have tried to suggest how their expression may lead to a dysregulated cell which may become malignant. These viruses are attempting to replicate in terminally differentiating keratinocytes and must stimulate G1 to S-phase progression for the replication of their genome. As part of the successful completion of replication and assembly of infectious virus particles, the virus needs at least partial differentiation to occur. Therefore, at the same time as differentiation is occurring, the nuclei of infected cells are in S-phase. While the mechanisms of action of the viral proteins are not completely understood, researchers are making progress and this article strives to bring together the conclusions from some of this work.

Motivation: Quantitative real-time PCR (qPCR) is one of the most widely used methods to measure gene expression. Despite extensive research in qPCR laboratory protocols, normalization and statistical analysis, little attention has been given to qPCR non-detects—those reactions failing to produce a minimum amount of signal.Results: We show that the common methods of handling qPCR non-detects lead to biased inference. Furthermore, we show that non-detects do not represent data missing completely at random and likely represent missing data occurring not at random. We propose a model of the missing data mechanism and develop a method to directly model non-detects as missing data. Finally, we show that our approach results in a sizeable reduction in bias when estimating both absolute and differential gene expression.Availability and implementation: The proposed algorithm is implemented in the R package, . This package also contains the raw data for the three example datasets used in this manuscript. The package is freely available at http://mnmccall.com/software and as part of the Bioconductor project.Contact: mccallm@gmail.com

Human papillomavirus type 16 (HPV-16), a DNA tumor virus, has a causal role in cervical cancer, and the viral oncoproteins E6 and E7 contribute to oncogenesis in multiple ways. E6 increases telomerase activity in keratinocytes through increased transcription of the telomerase catalytic subunit gene (TERT), but the factors involved in this have been elusive. We have found that mutation of the proximal E box in the TERT promoter has an activating effect in luciferase assays. This suggested that a repressive complex might be present at this site. HPV-16 E6 activated the TERT promoter predominantly through the proximal E box, and thus, might be acting on this repressive complex. This site is specific for the Myc/Mad/Max transcription factors as well as USF1 and USF2. Addition of exogenous USF1 or USF2 repressed activation of the TERT promoter by E6, dependent on the proximal E box. Using siRNA against USF1 or USF2 allowed for greater activation of the TERT promoter by E6. Conversely, loss of c-Myc function, through a dominant-negative Myc molecule, reduced activation by E6. Chromatin immunoprecipitations showed that in the presence of E6, there was a reduction in binding of USF1 and USF2 at the TERT promoter proximal E box, and a concomitant increase in c-Myc bound to this site. This shows that a repressive complex containing USF1 and USF2 is present in normal cells with little or no telomerase activity. In E6 keratinocytes, this repressive complex is replaced by c-Myc, which corresponds to higher levels of TERT transcription and consequently, telomerase activity.

Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.

Summary Leukemia stem cells (LSCs) represent a biologically distinct subpopulation of myeloid leukemias, with reduced cell cycle activity and increased resistance to therapeutic challenge. To better characterize key properties of LSCs, we employed a strategy based on identification of genes synergistically dysregulated by cooperating oncogenes. We hypothesized that such genes, termed “cooperation response genes” (CRGs), would represent regulators of LSC growth and survival. Using both a primary mouse model and human leukemia specimens, we show that CRGs are comprised of genes previously undescribed in leukemia pathogenesis in which multiple pathways modulate the biology of LSCs. In addition, our findings demonstrate that the CRG expression profile can be used as a drug discovery tool for identification of compounds that selectively target the LSC population. We conclude that CRG-based analyses provide a powerful means to characterize the basic biology of LSCs as well as to identify improved methods for therapeutic targeting.

Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable.

Fetal alcohol spectrum disorder (FASD), caused by gestational ethanol (EtOH) exposure, is one of the most common causes of non-heritable and life-long mental disability worldwide, with no standard treatment or therapy available. While EtOH exposure can alter the function of both neurons and glia, it is still unclear how EtOH influences brain development to cause deficits in sensory and cognitive processing later in life. Microglia play an important role in shaping synaptic function and plasticity during neural circuit development and have been shown to mount an acute immunological response to EtOH exposure in certain brain regions. Therefore, we hypothesized that microglial roles in the healthy brain could be permanently altered by early EtOH exposure leading to deficits in experience-dependent plasticity. We used a mouse model of human third trimester high binge EtOH exposure, administering EtOH twice daily by subcutaneous injections from postnatal day 4 through postnatal day 9 (P4-:P9). Using a monocular deprivation model to assess ocular dominance plasticity, we found an EtOH-induced deficit in this type of visually driven experience-dependent plasticity. However, using a combination of immunohistochemistry, confocal microscopy, and in vivo two-photon microscopy to assay microglial morphology and dynamics, as well as fluorescence activated cell sorting (FACS) and RNA-seq to examine the microglial transcriptome, we found no evidence of microglial dysfunction in early adolescence. We also found no evidence of microglial activation in visual cortex acutely after early ethanol exposure, possibly because we also did not observe EtOH-induced neuronal cell death in this brain region. We conclude that early EtOH exposure caused a deficit in experience-dependent synaptic plasticity in the visual cortex that was independent of changes in microglial phenotype or function. This demonstrates that neural plasticity can remain impaired by developmental ethanol exposure even in a brain region where microglia do not acutely assume nor maintain an activated phenotype.