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Background Quantitative real-time PCR (qPCR) is one of the most widely used methods to measure gene expression. An important aspect of qPCR data that has been largely ignored is the presence of non-detects: reactions failing to exceed the quantification threshold and therefore lacking a measurement of expression. While most current software replaces these non-detects with a value representing the limit of detection, this introduces substantial bias in the estimation of both absolute and differential expression. Single imputation procedures, while an improvement on previously used methods, underestimate residual variance, which can lead to anti-conservative inference. Results We propose to treat non-detects as non-random missing data, model the missing data mechanism, and use this model to impute missing values or obtain direct estimates of model parameters. To account for the uncertainty inherent in the imputation, we propose a multiple imputation procedure, which provides a set of plausible values for each non-detect. We assess the proposed methods via simulation studies and demonstrate the applicability of these methods to three experimental data sets. We compare our methods to mean imputation, single imputation, and a penalized EM algorithm incorporating non-random missingness (PEMM). The developed methods are implemented in the R/Bioconductor package . Conclusions The statistical methods introduced here reduce discrepancies in gene expression values derived from qPCR experiments in the presence of non-detects, providing increased confidence in downstream analyses.

Malignant cell transformation and the underlying genomic scale reprogramming of gene expression require cooperation of multiple oncogenic mutations. Notably, this cooperation is reflected in the synergistic regulation of downstream genes, so-called cooperation response genes (CRGs). CRGs impact diverse hallmark features of cancer cells and are not known to be functionally connected. Yet, they act as critical mediators of the cancer phenotype at an unexpectedly high frequency of >50%, as indicated by genetic perturbations. Here we demonstrate that CRGs function within a network of strong genetic interdependencies that are critical to the robustness of the malignant state. Our approach, termed TopNet, utilizes attractor-based ternary network modeling that takes the novel approach of incorporating uncertainty in the underlying gene perturbation data and is capable of identifying non-linear gene interactions. TopNet reveals topological gene network architecture that effectively predicts previously unknown, functionally relevant epistatic gene interactions, and thus, among a broad range of applications, has utility for identification of non-mutant targets for cancer intervention.

// Justin Komisarof 1 , Matthew McCall 2 , Laurel Newman 1 , Wiam Bshara 3 , James L. Mohler 4 , Carl Morrison 3 , Hartmut Land 1, 5 1 Departments of Biomedical Genetics, University of Rochester Medical Center, Rochester NY, 14642, USA 2 Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester NY, 14642, USA 3 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, 14623, USA 4 Department of Urology, Roswell Park Cancer Institute, Buffalo, NY, 14623, USA 5 Wilmot Cancer Institute, University of Rochester Medical Center, Rochester NY, 14642, USA Correspondence to: Hartmut Land, email: Land@urmc.rochester.edu Keywords: prostate cancer, biochemical recurrence, cooperation response genes, radical prostatectomy, algorithmic prediction Received: January 19, 2016 Accepted: November 21, 2016 Published: December 09, 2016 ABSTRACT Prostate cancer is the most common form of non-dermatological cancer among US men, with an increasing incidence due to the aging population. Patients diagnosed with clinically localized disease identified as intermediate or high-risk are often treated by radical prostatectomy. Approximately 33% of these patients will suffer recurrence after surgery. Identifying patients likely to experience recurrence after radical prostatectomy would lead to improved clinical outcomes, as these patients could receive adjuvant radiotherapy. Here, we report a new tool for prediction of prostate cancer recurrence based on the expression pattern of a small set of cooperation response genes (CRGs). CRGs are a group of genes downstream of cooperating oncogenic mutations previously identified in a colon cancer model that are critical to the cancer phenotype. We show that systemic dysregulation of CRGs is also found in prostate cancer, including a 4-gene signature (HBEGF, HOXC13, IGFBP2, and SATB1) capable of differentiating recurrent from non-recurrent prostate cancer. To develop a suitable diagnostic tool to predict disease outcomes in individual patients, multiple algorithms and data handling strategies were evaluated on a training set using leave-one-out cross-validation (LOOCV). The best-performing algorithm, when used in combination with a predictive nomogram based on clinical staging, predicted recurrent and non-recurrent disease outcomes in a blinded validation set with 83% accuracy, outperforming previous methods. Disease-free survival times between the cohort of prostate cancers predicted to recur and predicted not to recur differed significantly (p = 1.38x10 -6 ). Therefore, this test allows us to accurately identify prostate cancer patients likely to experience future recurrent disease immediately following removal of the primary tumor.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

SummaryMutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA) and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

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, nondetects. 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

Birt-Hogg-Dubesyndrome (BHD) is a human cancer disorder caused by mutations in the tumor suppressor gene Folliculin (FLCN) with unknown biological functions. Here, we show that the Drosophila homolog of FLCN, dFLCN (a.k.a. dBHD) localizes to the nucleolus and physically interacts with the 19S proteasomal ATPase, Rpt4, a nucleolar resident and known regulator of rRNA transcription. Downregulation of dFLCN resulted in an increase in nucleolar volume and upregulation of rRNA synthesis, whereas dFLCN overexpres- sion reduced rRNA transcription and counteracted the effects of Rpt4 on rRNA production by preventing the association of Rpt4 with the rDNA locus. We further show that human FLCN exhibited evolutionarily con- served function and that Rpt4 knockdown inhibits the growth of FLCN-deficient human renal cancer cells in mouse xenografts. Our study suggests that FLCN functions as a tumor suppressor by negatively regulat- ing rRNA synthesis.

Malignant cell transformation commonly results in the deregulation of thousands of cellular genes, an observation that suggests a complex biological process and an inherently challenging scenario for the development of effective cancer interventions. To better define the genes/pathways essential to regulating the malignant phenotype, we recently described a novel strategy based on the cooperative nature of carcinogenesis that focuses on genes synergistically deregulated in response to cooperating oncogenic mutations. These so-called “cooperation response genes” (CRGs) are highly enriched for genes critical for the cancer phenotype, thereby suggesting their causal role in the malignant state. Here we show that CRGs play an essential role in drug-mediated anti-cancer activity and that anti-cancer agents can be identified through their ability to antagonize the CRG expression profile. These findings provide proof-of-concept for the use of the CRG signature as a novel means of drug discovery with relevance to underlying anti-cancer drug mechanisms.

SummaryLeukemia 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 comprise 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.

We introduce a nonparametric test intended for large-scale simultaneous inference in situations where the utility of distribution-free tests is limited because of their discrete nature. Such situations are frequently dealt with in microarray analysis where the number of tests is much larger than the sample size. The proposed test statistic is based on a certain distance between the distributions from which the samples under study are drawn. In a simulation study, the proposed permutation test is compared with permutation counterparts of the t-test and the Kolmogorov-Smirnov test. The usefulness of the proposed test is discussed in the context of microarray gene expression data and illustrated with an application to real datasets.

Mit der Anwendung von DIN 1045-1, Ausgabe Juli 2001 sind eingreifende Anderungen bei der Bemessung und Konstruktion von Teilfertigdecken verbunden. Das betrifft insbesondere den Nachweis der Schubkraftaufnahme in der Fuge Fertigplatte/Ortbeton. Fur diesen aufwendigen Nachweis werden die Grundlagen zusammengestellt und ein Beispiel gegeben. Detailliert werden Regelungen fur die Anordnung von Gittertragern als Verbund-/Querkraftbewehrung sowie fur die Betondeckungen aufgefuhrt. Auflagerausbildungen sind zukunftig einfacher ausfuhrbar. Partly pre-fabricated reinforced concrete Floors/Ceilings according to DIN 1045-1. Important Points of Design and Construction There are invasive changes in design and construction of partly pre-fabricated reinforced concrete floors/ceilings connected with the application of DIN 1045-1, July 2001 issue. This applies specifically to the verification of acceptance of thrust force in the joint between pre-cast slab and the on-site concrete. The basic elements will be gathered for this extensive verification and an example is given. Regulations for the arrangement of lattice girders as bond/shear force reinforcement as well as concrete covers will be shown in detail. Structures of support are easier to implement in the future.

In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53, but the molecular basis for this co-operation remains unresolved. Here we show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a p53 and p21Cip1-dependent cycle arrest and to terminal differentiation. Raf activation in keratinocytes lacking p53 or p21Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21Cip1 function is necessary to disable growth-inhibitory Raf/MAP kinase signalling. Activation of oncogenes, including Ras, has been reported to stabilize and activate p53 via induction of the tumour suppressor p19ARF. However, the response to Raf in p19ARFI-/- keratinocytes was indistinguishable from wild-type controls. Thus, p19ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms.

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G(1) cell cycle inhibitors p27(Kip1) and p21(Cip1) (Ckis) via a Myc-induced protein. We provide biochemical and genetic evidence to show that this sequestration is mediated via induction of cyclin D1 and/or cyclin D2 protein synthesis rates. Consistent with this conclusion, primary cells from cyclin D1(-/-) and cyclin D2(-/-) mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1(-/-) cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant forming kinase-defective, Cki-binding cyclin-cdk complexes. The sequestration function of D cyclins thus appears essential for Myc-induced cell cycle progression but dispensable for apoptosis.

Induction of the Myc-oestrogen receptor fusion protein (MycER) by 4-OH-tamoxifen (OHT) leads to the activation of Cyclin E/Cyclin-dependent kinase 2 (CycE/Cdk2) complexes followed by the induction of DNA synthesis. As CycE/Cdk2 activity is essential for G1/S transition, we have investigated the mechanism by which Myc can activiate CycE/Cdk2. Our results suggest that this activation may involve at least two Myc-dependent steps: the induction of cyclin E gene transcription followed by accumulation of cyclin E mRNA in a protein synthesis-independent manner and the inhibition of p27(Kip1) association with CycE/Cdk2 complexes containing newly synthesised CycE. As a consequence phosphorylation of CycE-bound Cdk2 by cyclin activating kinase (CAK) is accelerated. We propose a model in which the active newly synthesised CycE/Cdk2 complexes trigger a positive feed-back mechanism to activate preexisting complexes through phosphorylation-dependent p27(Kip1) release.

The cooperation of oncogenes in the transformation of primary rat Schwann cells is a strikingly synergistic process. We have explored the molecular mechanisms involved. Activation of an inducible Raf kinase results in morphologically transformed cells that are arrested in G1 via the induction of p21(CiP1) and subsequent inhibition of cyclin/cdk activity. In contrast, coexpression of SV40 large T (LT) or a dominant-negative mutant of p53 abolishes p21(CiP1) induction and alleviates the growth arrest. Moreover in this scenario, Raf activation results in an increase in the specific activity of cyclin/cdk complexes with Raf and LT cooperating to superinduce cyclin A/cdk2 activity and stimulate proliferation in the absence of mitogens. Thus, signaling by Raf and its cooperating partners converges at the regulation of cyclin/cdk complexes, with the cellular responses to Raf modulated by p53.

Tumor suppressors known to date impede cancer growth by arresting the cell cycle or promoting apoptosis. Here we show that unphosphorylated human STAT5A functions as a tumor suppressor capable of repressing multiple oncogenes via heterochromatin formation. Unphosphorylated STAT5A binds to heterochromatin protein 1α (HP1α) and stabilizes heterochromatin. Expressing unphosphorylated STAT5A or HP1α inhibits colon cancer growth in mouse xenograft models. Transcriptome profiling shows that expressing an unphosphorylatable STAT5A has similar effects to overexpressing HP1α in global gene expression. Notably, the majority of the genes commonly repressed by unphosphorylated STAT5A and HP1α have been implicated in cancer development. Finally, down-regulation, somatic mutations, and deletions of STAT5 genes are found in certain human cancers. These results suggest that unphosphorylated STAT5A may epigenetically suppress tumor growth by promoting heterochromatin formation.

c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ). Using a genetic approach, we demonstrate that binding to Max is essential for Myc transforming activity and that Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-LZ domains or reciprocally modifying LZ dimerization specificities. While transformation defective on their own, complementary mutants restore Myc transforming activity when coexpressed in cells. The HLH-LZ exchange mutants also have dominant negative activity on wild-type Myc function. In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and activator of Myc. A general model for the role of Myc and Max in growth control is discussed.

The transcription factor c-Myc and its dimerisation partner Max are members of the basic/helix-loop-helix/leucine-zipper (bHLH-Z) family and bind to the DNA core sequence CACGTG. Using a site-selection protocol, we determined the complete 12 base pair consensus binding sites of c-Myc/Max (RACCACGTGGTY) and Max/Max (RANCACGTGNTY) dimers. We find that the c-Myc/Max dimer fails to bind the core when it is flanked by a 5'T or a 3'A, while the Max/Max dimer readily binds such sequences. Furthermore we show that inappropriate flanking sequences preclude transactivation by c-Myc in vivo. In conclusion, Max/Max dimers are less discriminatory than c-Myc/Max and may regulate other genes in addition to c-Myc/Max targets.

Understanding the molecular underpinnings of cancer is of critical importance to the development of 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 and induce complex changes in gene expression patterns. Genes critical to this multifaceted cellular phenotype have therefore only been identified after signalling pathway analysis or on an ad hoc basis. Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signalling circuitry 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 of murine and human colon cells. Notably, 14 out of 24 'cooperation response genes' were found to contribute to tumour formation in gene perturbation experiments. In contrast, only 1 in 14 perturbations of the 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.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

The introduction of activated c-myc and v-myc genes into a variety of non-established and established cells results in the suppression of endogenous c-myc expression. As measured in Rat-1 fibroblasts, the suppression occurs at the level of transcriptional initiation. Moreover, the extent of the down-regulation is proportional to the cellular concentration of c-myc protein, and the critical concentration range in which the endogenous c-myc RNA is effectively suppressed corresponds to that found in non-transformed cells. In addition, the autoregulatory mechanism is not only dependent on c-myc protein, but also requires additional trans-acting factors. These results support a role for c-myc in the regulation of cellular gene transcription and suggest that a negative feedback mechanism can act as a homeostatic regulator of c-myc expression in vivo.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve. These progenitors are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. When exposed to platelet-derived growth factor (PDGF), O-2A progenitors divide a limited number of times before clonally related cells differentiate into oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. O-2A progenitors can become immortalized through at least two different mechanisms. First, when O-2A progenitors are exposed to a combination of PDGF and basic fibroblast growth factor (bFGF) these cells undergo continuous self-renewal in the absence of differentiation. In contrast, the application of bFGF alone is associated with premature oligodendrocytic differentiation of dividing O-2A lineage cells. Thus, cooperation between growth factors can modulate O-2A progenitor self-renewal in a defined chemical environment by eliciting a novel programme of division and differentiation which cannot be predicted from the effects of either factor examined in isolation. A further mechanism which allows prolonged self-renewal in the O-2A lineage is the generation of a stem cell. O-2A progenitors isolated from optic nerves of perinatal rats also have the capacity to give rise to a population of cells called O-2Aadult progenitors, which differ from their perinatal counterparts in many characteristics. Most importantly, O-2Aadult progenitors have a slow cell cycle, divide and differentiate asymmetrically and appear to have the capacity for prolonged self-renewal. Thus, immortalization in this lineage can also be achieved by the generation of a cell with stem cell-like characteristics from a rapidly dividing progenitor population.

A) Pancreatic ductal adenocarcinoma (PDA) depends on a marked reprogramming of metabolic pathways, including the acquisition of autophagy dependence, for survival and growth. How common mutations in PDA cause autophagy dependence as well as the timing of autophagy activation in the course of cancer progression, have not been established. Here we show how Plac8, a gene synergistically up-regulated in response to the common cooperating oncogenic mutations found in PDA (RAS activation and functional loss of p53), is critical to the growth of PDA by sustaining autophagy via facititating autophagosome-lysosome fusion. Furthermore, we delineate that Kras and p53 mutations cooperate to induce autophagic flux. B) To establish Plac8’s role in PDA growth and autophagy we use murine cell lines and human PDA cell lines to determine 1) the lysosomal localization of Plac8, 2) its role in regulating autophagy using both loss of function and gain of function approaches, 3) the impact on lysosomal biology, and 4) the relationship between Plac8 and other genetic pathways governing autophagy. Using genetically engineered models of PDA we determine the timing of autophagy activation in PDA progression and the impact of Plac8 mutation. C) We identify Plac8 as a novel regulator of autophagosome-lysosome fusion required for PDA growth, thus providing a mechanistic link between oncogenic mutations and the activation of autophagy in cancer. Plac8 expression is required for growth of human PDA cells as xenografts in mice, as well as activation of autophagy. We find that concurrent mutation of KRAS and p53 is critical for maximal induction of autophagy in vitro. Correspondingly, using genetically engineered mouse models of PDA (Pdx1-Cre; LSL-KrasG12D; p53L/+), in which loss of p53 function occurs in a step-wise manner relying on the spontaneous loss of a heterozygous WT p53 allele, we see a step-wise incremental increase in LC3 puncta in vivo with each histological stage through the course of PDA progression. Thus, we find that the cooperative effects of KRAS and p53 drive activation of autophagy rather than either mutation alone. The overall survival of a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8null murine cohort (OS 27.9 wks) was significantly longer than a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8wt cohort (OS 17.0 wks, p=0.0006) demonstrating in vivo that genetic inactivation of Plac8 impedes cancer progression and resulting death. Our data suggest that the role of Plac8 in facilitating autophagy is critical to cancer, as the requirement of Plac8 for both tumorigenicity and autophagy can be compensated by over-expression of Atg12, a gene critical for autophagosome formation or by constitutively activated Rab7, a gene encoding a GTP-binding protein stimulating autophagosome-lysosome fusion. D) We conclude that Plac8 may offer a potential therapeutic window and point of intervention, as Plac8 mutation in the engineered PDA model inhibits cancer progression and significantly improves survival while having a minimal impact on the overall fitness of the animals. In fact, Plac8, and regulation of autophagosome-lysosome fusion, has specific relevance to regulation of autophagy during malignant cell transformation as Plac8 and the processes it regulates, appear to be largely dispensable to many normal physiologic processes. This abstract is also presented as Poster B36. Citation Format: Vijaya Balakrishnan, Kinsey Conan, Michael O'Dell, Jing Li Huang, Laurel Newman, Christa Whitney-Miller, Hartmut Land, Aram Hezel. Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR09.

ABSTACT: During the past few years, retroviral vectors have become a very important and widely used means of gene transfer. In the laboratory, their use has expanded the capabilities of investigators to perform important experiments that have solved previously unanswerable biological questions. Retroviral vectors exploit the inherent capacity of retrovkuses to transfer genetic material stably into a cell's genome and subsequently express it in a manner that is generally not detrimental to the host-cell. Initial entry of genetic material into cells via retroviral vectors is so efficient that it permits successful transfer of genes into limiting numbers of cells, such as hematopoietic stem cells in explanted bone marrow, and transfer approaching 100% in tissue-culture experiments. In comparison, chemical and electrical means of gene transfer require large initial numbers of cells and, under optimal circumstances, function at efficiencies several orders of magnitude lower than retroviral vectors, and only in a limited number of specific cell types.

TRRAP links Myc with histone acetylases and appears to be an important mediator of its oncogenic function. Here we show that interaction with TRRAP is required for cellular transformation not only by Myc, but also by the adenovirus E1A protein. Substitution of the 262 N-terminal residues of Myc with a small domain of E1A (residues 12-54) restores Myc transforming function. E1A(12-54) contains a TRRAP-interaction domain, that recruits TRRAP to either E1A-Myc chimeras, or the native 12S E1A protein. Overexpression of a competing TRRAP fragment in vivo blocks interaction of cellular TRRAP with either E1A-Myc or E1A, and suppresses cellular transformation by both oncoproteins. Moreover, E1A(Delta26-35) that fails to bind TRRAP but is capable of binding the Retinoblastoma (Rb)-family and p300/CBP proteins is defective in cellular immortalization, transformation and cell cycle deregulation. Thus in addition to disrupting Rb and p300/CBP functions, E1A must recruit TRRAP to transform cells.

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.

Publisher Summary This chapter focuses on the transformation of primary rat embryo cells. Carcinogenesis is a multistep process involving the activation of oncogenes and the loss or inactivation of tumor suppressor genes. The transfection of certain combinations of distinct cellular oncogenes (for example, ras and myc) or cellular and viral oncogenes (for example, ras and adenovirus E1A) is required to induce tumorigenic conversion. The rat embryo cell cotransformation assay provides an invaluable tool to score the biological activity of these genes easily. The preparation of rat embryo cells and the cotransformation assay are described and materials and reagents required are also highlighted.

The Myc oncoprotein dimerizes with its partner, Max, to bind DNA, activate transcription, and promote cell proliferation, as well as programmed cell death. Max also forms homodimers or heterodimers with its alternative partners, Mad and Mxi-1. These complexes behave as antagonists of Myc/Max through competition for common DNA targets, and perhaps permit cellular differentiation.

Abstract 553 In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures: No relevant conflicts of interest to declare.

Oncogenesis induces profound changes to the cellular metabolic network. A number of these cancer-cell metabolic phenotypes have been found to be important for in vivo tumorigenesis. We have utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to globally profile the metabolic network of a genetically-defined experimental model of oncogenesis in an attempt to identify novel metabolic activities induced by transformation. Furthermore, we sought to delineate how common oncogenic mutations contribute to specific metabolic phenotypes. We have analyzed how dominant negative p53 and constitutively active Ras expression, either independently or in concert, impacts the metabolic activity of non-transformed parental colon crypt epithelial cells. In addition to measuring steady-state metabolite concentrations, we have employed isotope tracer analysis to measure the impact of oncogenic mutation on metabolic pathway kinetics. We find that combined p53 and Ras oncogenic mutation synergistically activates numerous aspects of central carbon metabolism including glycolysis, glutamine catabolism, and pyrimidine and purine biosynthesis. Our data indicate that many well-defined oncogenic metabolic phenotypes are the result of a cooperative effect of combinatorial mutation highlighting the importance of identifying the interaction between oncogenic signal transduction and metabolic regulation for understanding the etiology of the cancer-cell metabolic state. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1249. doi:10.1158/1538-7445.AM2011-1249

The c-Myc protein (Myc) is involved in cellular transformation and mitogenesis, but is also a potent inducer of programmed cell death, or apoptosis. Whether these apparently opposite functions are mediated through common or distinct molecular mechanisms remains unclear. Myc and its partner protein, Max, dimerize and bind DNA in vitro and in vivo through basic/helix-loop-helix/leucine zipper motifs (bHLH-LZ). By using complementary leucine zipper mutants (termed MycEG and MaxEG), which dimerize efficiently with each other but not with their wild-type partners, we demonstrate that both cell cycle progression and apoptosis in nontransformed rodent fibroblasts are induced by Myc-Max dimers. MycEG or MaxEG alone are inactive, but co-expression restores ability to prevent withdrawal from the cell cycle and to induce cell death upon removal of growth factors. Thus, Myc can control two alternative cell fates through dimerization with a single partner, Max.

THE c-myc protein (Myc) contains an amino-terminal transcriptional activation domain1 and a carboxy-terminal basic helix–loop–helix–leucine zipper (bHLH-Z) domain2–5 that directs dimerization of Myc with its partner, the max protein (Max), and promotes DNA binding to sites containing a CACGTG core consensus sequence6–9. Despite these characteristics and the observation that Myc can modulate gene expression4,5,10, a direct role for Myc or Max as transcription factors has never been demonstrated. Here we use Saccharomyces cerevisiae as an in vivo model system to show that the Myc protein is a sequence-specific transcriptional activator whose DNA binding is strictly dependent on dimerization with Max. Transactivation is mediated by the amino-terminal domain of Myc. We find that Max homodimers bind to the same DNA sequence as Myc + Max but that they fail to transactivate and thus can antagonize Myc + Max function. We also show that the Max HLH-Z domain has a higher affinity for the Myc HLH-Z domain than for itself, and suggest that the heterodimeric Myc + Max activator forms preferentially at equilibrium.

Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.

Bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve, are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. Previous studies have shown that O-2A progenitors exposed to platelet-derived growth factor (PDGF) divide as migratory bipolar cells a limited number of times, with a cell cycle time of 18 hr, before clonally related progenitors differentiate into nondividing oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but instead differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. We now have found that O-2A progenitors can be induced to undergo continuous self-renewal in the absence of oligodendrocytic differentiation by exposure to a combination of PDGF and basic fibroblast growth factor (bFGF). With the exception of the inhibition of differentiation, the O-2A progenitors exposed to PDGF and bFGF behaved similarly to those exposed to PDGF alone. In contrast, progenitors exposed to basic bFGF alone were multipolar, had a cell-cycle length of 45 hr, showed little migratory behavior, underwent premature oligodendrocytic differentiation, and did not cease division upon expression of oligodendrocyte marker antigens. Thus, inhibition of differentiation required the presence of both mitogens. Our results demonstrate that PDGF and bFGF act on O-2A progenitors as both inducers of division and as regulators of differentiation that modulate multiple aspects of O-2A progenitor development and, additionally, reveal a previously unrecognized means of regulating self-renewal processes, wherein cooperation between growth factors promotes continuous division in the absence of differentiation.

We report the development of an advanced system for transfer and expression of exogenous genes in mammalian cells based on Moloney murine leukemia virus (Mo MuLV). Extensive deletion/mutagenesis analysis to identify cis-acting signals involved in virus transmission has led to the design of a family of novel, highly efficient retroviral vectors and a partner helper-free packaging cell line. The pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR). Each of these vectors has been constructed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The high titre ecotropic helper free packaging cell line, omega E, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo MuLV via homologous recombination events. The omega E cell line was generated with separate gagpol and ecotropic env expression constructs with minimal sequence overlap and decreased sequence homology achieved by 'codon wobbling'. Homologous env coding sequences were deleted from the pBabe vectors without diminishing recombinant vector titre. Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.

Messenger RNA activities for tyrosine aminotransferase and tryptophan oxygenase have been determined in two Morris hepatomas, 5123C and 9618A and in HTC cells by translation in vitro of poly(A)-containing RNA in a wheat germ system, followed by immunoprecipitation of the synthesized enzyme subunits. The amount of specific translatable mRNA was correlated to the respective enzyme activity in the tumors and in the host livers of untreated and hydrocortisone-treated rats. In hepatoma 5123C, the activities of tyrosine aminotransferase mRNA parallel the corresponding enzyme activities. The basal levels of enzyme activity and of mRNA activity are several-fold higher than in the host liver and can be induced by hydrocortisone, reaching levels somewhat higher than in maximally induced liver. In hepatoma 9618A, tyrosine aminotransferase activity and the respective mRNA activity is virtually absent in non-induced animals. Enzyme activity is only slightly induced by treatment with hydrocortisone, whereas translatable tyrosine aminotransferase mRNA accumulates after hormone administration, reaching levels of approximately one third of the maximum liver values. Tryptophan oxygenase activity and the corresponding mRNA activity is virtually absent in both Morris hepatomas tested and cannot be unequivocally detected after administration of hydrocortisone. Dexamethasone-treated HTC cells accumulate translatable tyrosine aminotransferase mRNA in direct correlation with the increased activity of the respective enzyme, whereas no tryptophan oxygenase or its respective mRNA could be detected either in untreated or dexamethasone-treated cells. The patterns of the major proteins synthesized in the wheat germ system under the direction of poly(A)-containing RNA from liver, hepatoma 5123C, hepatoma 9618A and HTC cells show striking differences. A mRNA coding for a protein of Mr approximately 40 000 is induced by dexamethasone solely in HTC cells.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.

In HTC cells dexamethasone induces the accumulation of tyrosine aminotransferase mRNA activity in addition to a mRNA activity coding for a protein of a yet unknown function (DIP). The kinetics of decay of these two mRNA activities were followed in induced cells under the influence of cordycepin and in cells withdrawn from the hormone. For tyrosine amino-transferase mRNA activity a relative half life of approximately 1 hour was estimated in both cases. However, DIP mRNA activity decayed to 50 % of the fully induced level within 3 to 4 hours following withdrawal of the hormone, whereas after application of cordycepin to induced cells only a small and delayed decay could be observed. Application of cordycepin two hours after withdrawal of hormone caused a stabilization of DIP mRNA activity.

Transfection of embryo fibroblasts by a human ras oncogene does not convert them into tumour cells unless the fibroblasts are established and immortalized before transfection. The embryo fibroblasts become tumorigenic if a second oncogene such as a viral or cellular myc gene or the gene for the polyoma large-T antigen is introduced together with the ras gene.

In order to study the deployment of cells during gastrulation and early organogenesis, it is necessary to have an in situ cell marker which can be used to follow cell fate. To create such a marker a transgenic mouse strain, designated Tg(Act-lac Z)-1, which carries 6 copies of the Escherichia coli lac Z gene under the control of the rat β-actin promoter, was made by pronuclear injection of DNA. Staining early postimplantation hemizygous mouse conceptuses, during gastrulation and early organogenesis, for β-galactosidase activity shows that lac Z expression is ubiquitous and constitutive in all epiblast derivatives of the 10th day conceptus. No activity is seen in trophectoderm and primitive endoderm derivatives. Postimplantation grafts of [3H]thymidine-labelled transgenic cells establish the cell autonomy of this transgenic marker. Preliminary observations on the distribution of inner cell mass (ICM) descendant clones, identified in situ in midgestation conceptuses, confirm the pluripotency of individual ICM cells. The implications regarding patterns of cell growth in nascent fetal primordia are discussed.

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.

The ABCA1 protein mediates the transfer of cellular cholesterol across the plasma membrane to apolipoprotein A-I. Loss-of-function mutations in the ABCA1 gene induce Tangier disease and familial hypoalphalipoproteinemia, both cardio-vascular conditions characterized by abnormally low levels of serum cholesterol, increased cholesterol in macrophages and subsequent formation of vascular plaque. Increased intra-cellular cholesterol levels are also frequently found in cancer cells. Here we demonstrate anti-cancer activity of ABCA1 efflux function, which is compromised following inhibition of ABCA1 gene expression by oncogenic mutations or cancer-specific ABCA1 loss-of-function mutations. In concert with elevated cholesterol synthesis found in cancer cells, ABCA1 deficiency allows for increased mitochondrial cholesterol, inhibits release of mitochondrial cell death-promoting molecules and thus facilitates cancer cell survival, overall suggesting that elevated mitochondrial cholesterol is essential to the cancer phenotype.