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Human papillomaviruses (HPVs) cause more than 4.5% of all cancer in the world and more than half of these cases are attributed to human papillomavirus type 16 (HPV16). Prophylactic vaccines are available but antiviral drugs are not. Novel targets for therapy are urgently needed. Alternative RNA splicing is extensively used by HPVs to express all their genes and HPV16 is no exception. This process must function to perfection since mis-splicing could perturb the HPV gene expression program by altering mRNA levels or by generating dysfunctional mRNAs. Cis-acting RNA elements on the viral mRNAs and their cognate cellular trans-acting factors control papillomavirus RNA splicing. The precise but delicate nature of the splicing process renders splicing sensitive to interference. As such, papillomavirus RNA splicing is a potential target for therapy. Here we summarize our current understanding of cis-acting HPV16 RNA elements that control HPV16 mRNA splicing via cellular proteins and discuss how they may be exploited as targets for therapy to papillomavirus infections and cancer.

Circadian clock dysregulation has been implicated in various types of cancer and represents an area of growing research. However, the role of the circadian clock in prostate cancer has been relatively unexplored. This literature review will highlight the potential role of circadian clock dysregulation in prostate cancer by examining molecular, epidemiologic, and clinical data. The influence of melatonin, light, night shift work, chronotherapy, and androgen independence are discussed as they relate to the existing literature on their role in prostate cancer.

Introduction: Recent series of congenital anomaly (CA) rates (CARs) have showed the close and epidemiologically causal relationship of cannabis exposure to many CARs. We investigated these trends in Europe where similar trends have occurred. Methods: CARs from EUROCAT. Drug use from European Monitoring Centre for Drugs and Drug Addiction. Income data from World Bank. Results: CARs were higher in countries with increasing daily use overall (p = 9.99 × 10−14, minimum E-value (mEV) = 2.09) and especially for maternal infections, situs inversus, teratogenic syndromes and VACTERL syndrome (p = 1.49 × 10−15, mEV = 3.04). In inverse probability weighted panel regression models the series of anomalies: all anomalies, VACTERL, foetal alcohol syndrome, situs inversus (SI), lateralization (L), and teratogenic syndromes (TS; AAVFASSILTS) had cannabis metric p-values from: p < 2.2 × 10−16, 1.52 × 10−12, 1.44 × 10−13, 1.88 × 10−7, 7.39 × 10−6 and −16. In a series of spatiotemporal models this anomaly series had cannabis metric p-values from: 8.96 × 10−6, 6.56 × 10−6, 0.0004, 0.0019, 0.0006, 5.65 × 10−5. Considering E-values, the cannabis effect size order was VACTERL > situs inversus > teratogenic syndromes > FAS > lateralization syndromes > all anomalies. 50/64 (78.1%) E-value estimates and 42/64 (65.6%) mEVs > 9. Daily cannabis use was the strongest predictor for all anomalies. Conclusion: Data confirmed laboratory, preclinical and recent epidemiological studies from Canada, Australia, Hawaii, Colorado and USA for teratological links between cannabis exposure and AAVFASSILTS anomalies, fulfilled epidemiological criteria for causality and underscored importance of cannabis teratogenicity. VACTERL data are consistent with causation via cannabis-induced Sonic Hedgehog inhibition. TS data suggest cannabinoid contribution. SI&L data are consistent with results for cardiovascular CAs. Overall, these data show that cannabis is linked across space and time and in a manner which fulfill

Whilst the cannabis-cancer link has been traditionally described as controversial recent whole nation and whole continent studies have demonstrated that well documented laboratory-based multimodal cannabinoid genotoxicity is indeed reflected in numerous cancer types in larger epidemiological series. A recent longitudinal human sperm epigenome-wide DNA methylation screen in both cannabis dependence and cannabis withdrawal has revealed remarkable insights into the manner in which widespread perturbations of DNA methylation may lead to cancerogenic changes in both the exposed and subsequent generations as a result of both cannabis exposure and withdrawal. These results therefore powerfully strengthen and further robustify the causal nature of the relationship between cannabinoid exposure and cancerous outcomes well beyond the previously published extensive mechanistic literature on cannabinoid genotoxicity. The reported epigenomic results are strongly hypothesis generating and call powerfully for further work to investigate oncogenic mechanisms in many tissues, organs and preclinical models. These epigenomic results provide an extraordinarily close predictive account for the epidemiologically observed pattern of cannabis-related malignant disease and indicate that malignant and multigenerational cannabinoid epigenotoxicity is potentially a significant and major public health concern.

Introduction: Congenital anomalies (CA’s) of most of the gastrointestinal tract have been linked causally with prenatal or community cannabis exposure. Therefore, we studied this relationship in Europe. Methods: CA data were from Eurocat. Drug-use data were sourced from the European Monitoring Centre for Drugs and Drug Addiction. Income data were taken from the World Bank. Results: When countries with increasing rates of daily cannabis use were compared with those which were not, the overall rate of gastrointestinal CA’s (GCA’s) was higher in the former group (p = 0.0032). The five anomalies which were related to the metrics of cannabis exposure on bivariate analysis were bile duct atresia, Hirschsprungs, digestive disorders, annular pancreas and anorectal stenosis or atresia. The following sequence of GCA’s was significantly linked with cannabis metrics at inverse-probability-weighted-panel modelling, as indicated: esophageal stenosis or atresia, bile duct atresia, small intestinal stenosis or atresia, anorectal stenosis or atresia, Hirschsprungs disease: p = 1.83 × 10−5, 0.0046, 3.55 × 10−12, 7.35 × 10−6 and 2.00 × 10−12, respectively. When this GCA series was considered in geospatial modelling, the GCA’s were significantly cannabis-related from p = 0.0003, N.S., 0.0086, 6.652 × 10−5, 0.0002, 71.4% of 35 E-value estimates and 54.3% minimum E-values (mEVv’s) > 9 (high zone) and 100% and 97.1% > 1.25 (causality threshold). The order of cannabis sensitivity by median mEVv was Hirschsprungs > esophageal atresia > small intestinal atresia > anorectal atresia > bile duct atresia. Conclusions: Seven of eight GCA’s were related to cannabis exposure and fulfilled the quantitative criteria for epidemiologically causal relationships. Penetration of cannabinoids into the community should be carefully scrutinized and controlled to protect against exponential and multigenerational genotoxicity ensuing from multiple cannabinoids.

As global interest in the therapeutic potential of cannabis and its’ derivatives for the management of selected diseases increases, it is increasingly imperative that the toxic profile of cannabinoids be thoroughly understood in order to correctly assess the balance between the therapeutic risks and benefits. Modern studies across a number of jurisdictions, including Canada, Australia, the US and Europe have confirmed that some of the most worrying and severe historical reports of both congenital anomalies and cancer induction following cannabis exposure actually underestimate the multisystem thousand megabase-scale transgenerational genetic damage. These findings from teratogenic and carcinogenic literature are supported by recent data showing the accelerated patterns of chronic disease and the advanced DNA methylation epigenomic clock age in cannabis exposed patients. Together, the increased multisystem carcinogenesis, teratogenesis and accelerated aging point strongly to cannabinoid-related genotoxicity being much more clinically significant than it is widely supposed and, thus, of very considerable public health and multigenerational impact. Recently reported longitudinal epigenome-wide association studies elegantly explain many of these observed effects with considerable methodological sophistication, including multiple pathways for the inhibition of the normal chromosomal segregation and DNA repair, the inhibition of the basic epigenetic machinery for DNA methylation and the demethylation and telomerase acceleration of the epigenomic promoter hypermethylation characterizing aging. For cancer, 810 hits were also noted. The types of malignancy which were observed have all been documented epidemiologically. Detailed epigenomic explications of the brain, heart, face, uronephrological, gastrointestinal and limb development were provided, which amply explained the observed teratological patterns, including the inhibition of the key morphogenic gradients. Hence, thes

FOXO family of proteins are transcription factors involved in many physiological and pathological processes including cellular homeostasis, stem cell maintenance, cancer, metabolic, and cardiovascular diseases. Genetic evidence has been accumulating to suggest a prominent role of FOXOs in lifespan regulation in animal systems from hydra, C elegans, Drosophila, and mice. Together with the observation that FOXO3 is the second most replicated gene associated with extreme human longevity suggests that pharmacological targeting of FOXO proteins can be a promising approach to treat cancer and other age-related diseases and extend life and health span. However, due to the broad range of cellular functions of the FOXO family members FOXO1, 3, 4, and 6, isoform-specific targeting of FOXOs might lead to greater benefits and cause fewer side effects. Therefore, a deeper understanding of the common and specific features of these proteins as well as their redundant and specific functions in our cells represents the basis of specific targeting strategies. In this review, we provide an overview of the evolution, structure, function, and disease-relevance of each of the FOXO family members.

The use of ∆8THC is increasing at present across the USA in association with widespread cannabis legalization and the common notion that it is “legal weed”. As genotoxic actions have been described for many cannabinoids, we studied the cancer epidemiology of ∆8THC. Data on 34 cancer types was from the Centers for Disease Control Atlanta Georgia, substance abuse data from the Substance Abuse and Mental Health Services Administration, ethnicity and income data from the U.S. Census Bureau, and cannabinoid concentration data from the Drug Enforcement Agency, were combined and processed in R. Eight cancers (corpus uteri, liver, gastric cardia, breast and post-menopausal breast, anorectum, pancreas, and thyroid) were related to ∆8THC exposure on bivariate testing, and 18 (additionally, stomach, Hodgkins, and Non-Hodgkins lymphomas, ovary, cervix uteri, gall bladder, oropharynx, bladder, lung, esophagus, colorectal cancer, and all cancers (excluding non-melanoma skin cancer)) demonstrated positive average marginal effects on fully adjusted inverse probability weighted interactive panel regression. Many minimum E-Values (mEVs) were infinite. p-values rose from 8.04 × 10−78. Marginal effect calculations revealed that 18 ∆8THCrelated cancers are predicted to lead to a further 8.58 cases/100,000 compared to 7.93 for alcoholism and −8.48 for tobacco. Results indicate that between 8 and 20/34 cancer types were associated with ∆8THC exposure, with very high effect sizes (mEVs) and marginal effects after adjustment exceeding tobacco and alcohol, fulfilling the epidemiological criteria of causality and suggesting a cannabinoid class effect. The inclusion of pediatric leukemias and testicular cancer herein demonstrates heritable malignant teratogenesis.

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.

The p53 protein is an indispensable tumor suppressor that is involved in numerous cellular processes, such as regulation of the cell cycle and metabolism. About two decades ago, two proteins with significant homology to p53 were discovered, and termed p63 and p73. To date, these three proteins make up the p53 family of transcription factors. Each gene has the capacity to form multiple protein isoforms through the use of various promoters and alternative splicing, with p73 forming the most isoforms. At the 5` end of the p73 gene, transcription initiation from promoters 1 and 2 form the TAp73 and DNp73 N-terminal isoforms, respectively. At the 3` end, alternative splicing in p73 exons 11 through 13 give rise to seven known C-terminal isoforms (a, b, g, d, e, z, h). The function of a majority of the C-terminal isoforms is not well-studied. As such, the work in this dissertation focuses on characterizing the function of the p73 C-terminal isoforms. Chapter one serves as an introduction to the p53 family of proteins, while comprehensively compiling the literature that describes how these three proteins are involved in regulating lipid and iron metabolism. This chapter highlights the numerous ways in which p53 regulates lipid and iron metabolism, while revealing the limited literature on the role of p63 and p73 in these processes.Chapter two lays the foundation for the rest of the dissertation, which focuses on characterizing the function of a novel p73 C-terminal isoform that we discovered. This novel isoform, termed p73a1, results from the exclusion of p73 exon 12 (E12). By using CRIPSR to remove E12 from multiple cancer cell lines, the role of p73a1 in oncogenesis was studied. To study the physiological function of p73a1, E12 heterozygous (E12+/-) mice were generated and life-span, tumor burden, and histopathology were analyzed. It was found that p73a1 functions as a tumor suppressor, both in vitro and in vivo. Moreover, E12+/- mice exhibited widespread systemic inflam

TP73, a member of the p53 family, is expressed as TAp73 and DNp73 along with multiple C-terminal isoforms (a-n). DNp73 is primarily expressed in neuronal cells and necessary for neuronal development. Interestingly, while TAp73a is a tumor suppressor and predominantly expressed in normal cells, TAp73 is found to be frequently altered in human cancers, suggesting a role of TAp73 C-terminal isoforms in tumorigenesis. To test this, the TCGA SpliceSeq database was searched and showed that exon 11 (E11) exclusion occurs frequently in several human cancers. We also found that p73a to p73g isoform switch resulting from E11 skipping occurs frequently in human prostate cancers and dog lymphomas. To determine whether p73a to p73g isoform switch plays a role in tumorigenesis, CRISPR technology was used to generate multiple cancer cell lines and a mouse model in that Trp73 E11 is deleted. Surprisingly, we found that in E11-deificient cells, p73g becomes the predominant isoform and exerts oncogenic activities by promoting cell proliferation and migration. In line with this, E11-deficient mice were more prone to obesity and B-cell lymphomas, indicating a unique role of p73g in lipid metabolism and tumorigenesis. Additionally, we found that E11- deficient mice phenocopies Trp73-deficient mice with short lifespan, infertility, and chronic inflammation. Mechanistically, Mechanistically, we showed that Leptin, a pleiotropic adipocytokine involved in energy metabolism and oncogenesis, was highly induced by p73g, necessary for p73g-mediated oncogenic activity, and associated with p73a to g isoform switch in human prostate cancer and dog lymphoma. Finally, we showed that E11-knockout promoted, whereas knockdown of p73g or Leptin suppressed, xenograft growth in mice. Our study indicates that the p73g-Leptin pathway promotes tumorigenesis and alters lipid metabolism, which may be targeted for cancer management.

Felis catus gammaherpesvirus-1 (FcaGHV1), a novel candidate oncogenic virus, infects cats worldwide. Whether the oropharynx is a site of virus shedding and persistence, and whether oronasal carcinomas harbor FcaGHV1 nucleic acid were investigated. In a prospective molecular epidemiological study, FcaGHV1 DNA was detected by cPCR in oropharyngeal swabs from 26/155 (16.8%) of cats. Oropharyngeal shedding was less frequently detected in kittens ≤3 months of age (5/94, 5.3%) than in older animals; >3 months to ≤1 year: 8/26, 30.8%, (p = 0.001, OR 7.91, 95% CI (2.320, 26.979)); >1 year to ≤6 years: 10/20, 50%, (p < 0.001, OR 17.8 95% CI (5.065, 62.557)); >6 years: 3/15, 33% (p = 0.078). Provenance (shelter-owned/privately owned) was not associated with shedding. In situ hybridization (ISH) identified FcaGHV1-infected cells in salivary glandular epithelium but not in other oronasal tissues from two of three cats shedding viral DNA in the oropharynx. In a retrospective dataset of 11 oronasopharyngeal carcinomas, a single tumor tested positive for FcaGHV1 DNA by ISH, a papillary carcinoma, where scattered neoplastic cells showed discrete nuclear hybridization. These data support the oronasopharynx as a site of FcaGHV1 shedding, particularly after maternal antibodies are expected to decline. The salivary epithelium is identified as a potential site of FcaGHV1 persistence. No evidence supporting a role for FcaGHV1 in feline oronasal carcinomas was found in the examined tumours.

Introduction: Recent reports linking prenatal and community cannabis exposure to elevated uronephrological congenital anomaly (UCA) rates (UCAR’s) raise the question of its European epidemiology given recent increases in community cannabinoid penetration there. Methods: UCAR data from Eurocat. Drug use data from European Monitoring Centre for Drugs and Drug Addiction. Income from World bank. Results: UCAR increased across Spain, Netherlands, Poland and France. UCAR’s and cannabis resin THC increased simultaneously in France, Spain, Netherlands and Bulgaria. At bivariate analysis all UCA’s were related to cannabis herb and resin THC concentrations. All UCAR’s were bivariately related to cannabis metrics ordered by median minimum E-value (mEV) as hypospadias > multicystic renal disease > bilateral renal agenesis > UCA’s > hydronephrosis > posterior urethral valve > bladder exstrophy/epispadias. At inverse probability weighted multivariable analysis terms including cannabis were significant for the following series of anomalies: UCA’s, multicystic renal disease, bilateral renal agenesis, hydronephrosis, congenital posterior urethral valves from P = 1.91 × 10−5, 2.61 × 10−8, 4.60 × 10−15, 4.60 × 10−15 and 2.66 × 10−10. At geospatial analysis the same series of UCA’s were significantly related to cannabis from P = 7.84 × 10−15, 7.72 × 10−5, 0.0023, 6.95 × 10−5, and 8.82 × 10−5. 45/51 (88.2%) of E-value estimates and 31/51 (60.8%) of mEV’s >9. Conclusion: Analysis confirms a close relationship between cannabis metrics and all seven UCA’s and fulfill formal criteria for quantitative causal inference. Given the exponential cannabinoid genotoxicity dose–response relationship results provide a powerful stimulus to constrain community cannabinoid exposure including protection of the food chain to preserve the genome and epigenome of coming generations.

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.

Este número muestra la investigación del CSIC sobre el cáncer en las áreas de investigación básica: inicio del cáncer, metástasis, reacción sistémica, terapias, inmunoterapia, nuevos fármacos, detección y prevención. También aborda la transferencia del conocimiento (innovación), la cooperación internacional, formación, la cultura científica y los institutos de referencia del organismo en oncología. También cita los retos recogidos en el Libro Blanco del CSIC sobre Biomedicina, como la creación de un Instituto Virtual del Cáncer que coordine toda la investigación en oncología de la institución.

Copy neutral loss of heterozygosity (cnLOH) is a common event in several human malignancies—positing this as a mechanism of carcinogenesis. However, the role of cnLOH in synchronous colorectal cancer (SCRC), a unique CRC subtype, is not well understood. The aim of this study was to establish a cnLOH profile of SCRC using a single-nucleotide polymorphism array (SNP-A), and to explore associations between cnLOH and the genomic landscape of frequently mutated genes in SCRC. Among 74 paired SCRC cases, the most frequently altered regions were 16p11.2–p11.1 (59.5%) and 11p11.2–p11.12 (28.4%). Notably, the 6q11.21–q11.22 region altered by cnLOH was uniquely associated with polyclonal SCRCs (p = 0.038). Together, our analysis suggests that inactivation of tumor suppressor genes and cnLOH are rare events among SCRC cases. This study defines distinct patterns of cnLOH in SCRC, and provides initial evidence of a role for cnLOH in SCRC etiology.

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis., This study has been supported by the German Ministry of Science and Education (BMBF) in the framework of the ICGC MMML-Seq project (01KU1002A-J) the MMML-MYC-SYS project (036166B) and the project ICGC DE-MINING (01KU1505E), the European Union in the framework of the BLUEPRINT Project (HEALTH-F5-2011-282510) and the KinderKrebsInitiative Buchholz/Holm-Seppensen. This work was supported by the BMBF-funded Heidelberg Center for Human Bioinformatics (HD-HuB) within the German Network for Bioinformatics Infrastructure (de.NBI) (#031A537A, #031A537C). Former grant support of MMML by the Deutsche Krebshilfe (2003–2011) is gratefully acknowledged. We acknowledge COSMIC and use of Cancer Gene Census. Part of the work was performed in association with SFB1074 (particularly subproject B1) funded by DFG. We wish to thank Barbara Hutter, Ivo Buchhalter, Zuguang Gu, and Natalie Jäger for skillful technical assistance. We thank the High-Throughput Sequencing Unit of the Genome and Proteome Core Facility and the Omics IT and Data Management Core Facility of the German Cancer Research Center (DKFZ, Heidelberg) as well as the Institute of Clinical Molecular Biology (IKMB, Christian-Albrechts-University Kiel) for excellent technical support and expertise. DH is a member of the Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS) and of the MD/PhD-program of the University of Heidelberg. KK and UHT are funded by the Helmholtz International Graduate School for Cancer Research at the German Cancer Research Center. SHB, HK, and SH acknowledge support by LIFE (Leipzig Research Center for Civilization Diseases), Leipzig University. LIFE is funded by the European Union, the European Regional Development Fund (ERDF), the European Social Fund (ESF), and the Free State of Saxony. This work has been carried out with the help of the Interdisciplinary Bank of Biomaterials and Data of the University Hospital of Würzburg and the Julius Maximilian Universit, Peer Reviewed, "Article signat per 70 autors/es:Daniel Hübschmann, Kortine Kleinheinz, Rabea Wagener, Stephan H. Bernhart, Cristina López, Umut H. Toprak, Stephanie Sungalee, Naveed Ishaque, Helene Kretzmer, Markus Kreuz, Sebastian M. Waszak, Nagarajan Paramasivam, Ole Ammerpohl, Sietse M. Aukema, Renée Beekman, Anke K. Bergmann, Matthias Bieg, Hans Binder, Arndt Borkhardt, Christoph Borst, Benedikt Brors, Philipp Bruns, Enrique Carrillo de Santa Pau, Alexander Claviez, Gero Doose, Andrea Haake, Dennis Karsch, Siegfried Haas, Martin-Leo Hansmann, Jessica I. Hoell, Volker Hovestadt, Bingding Huang, Michael Hummel, Christina Jäger-Schmidt, Jules N. A. Kerssemakers, Jan O. Korbel, Dieter Kube, Chris Lawerenz, Dido Lenze, Joost H. A. Martens, German Ott, Bernhard Radlwimmer, Eva Reisinger, Julia Richter, Daniel Rico, Philip Rosenstiel, Andreas Rosenwald, Markus Schillhabel, Stephan Stilgenbauer, Peter F. Stadler, José I. Martín-Subero, Monika Szczepanowski, Gregor Warsow, Marc A. Weniger, Marc Zapatka, Alfonso Valencia, Hendrik G. Stunnenberg, Peter Lichter, Peter Möller, Markus Loeffler, Roland Eils, Wolfram Klapper, Steve Hoffmann, Lorenz Trümper, ICGC MMML-Seq consortium, ICGC DE-Mining consortium, BLUEPRINT consortium, Ralf Küppers, Matthias Schlesner & Reiner Siebert", Postprint (published version)

Economic and social development has essential repercussions on the health of populations, as it alters the morbidity and mortality profile and favors the increase in exposures and risks to human health, especially the risk of cancer. Cancer is considered a significant public health problem worldwide, and remains with high incidence rates, being considered a complex disease, with multifactorial causes. The permanent incidence rates of prostate cancer prove to be one of the most prevalent, considered the second most common cancer in men worldwide, and a leading cause of deaths from chronic noncommunicable diseases. Among the different types of cancer, prostate cancer has been the subject of great scientific interest. In this context, animal models are valuable for studying cancer-related aspects, the use of animal models has the potential to increase our understanding of carcinogenesis, tumor biology, and the impact of specific molecular events on tumor biology. Animal models with specific human cancer characteristics can be used to test cancer prevention and treatment strategies. In this review, we aim to show how the use of animal models as an essential tool in the study of the molecular mechanisms of carcinogenesis.

The development of cancer is driven by the accumulation of many oncogenesis-related genetic alterations and tumorigenesis is triggered by complex networks of involved genes rather than independent actions. To explore the epistasis existing among oncogenesis-related genes in lung cancer development, we conducted pairwise genetic interaction analyses among 35,031 SNPs from 2027 oncogenesis-related genes. The genotypes from three independent genome-wide association studies including a total of 24,037 lung cancer patients and 20,401 healthy controls with Caucasian ancestry were analyzed in the study. Using a two-stage study design including discovery and replication studies, and stringent Bonferroni correction for multiple statistical analysis, we identified significant genetic interactions between SNPs in RGL1:RAD51B (OR=0.44, p value=3.27x10-11 in overall lung cancer and OR=0.41, p value=9.71x10-11 in non-small cell lung cancer), SYNE1:RNF43 (OR=0.73, p value=1.01x10-12 in adenocarcinoma) and FHIT:TSPAN8 (OR=1.82, p value=7.62x10-11 in squamous cell carcinoma) in our analysis. None of these genes have been identified from previous main effect association studies in lung cancer. Further eQTL gene expression analysis in lung tissues provided information supporting the functional role of the identified epistasis in lung tumorigenesis. Gene set enrichment analysis revealed potential pathways and gene networks underlying molecular mechanisms in overall lung cancer as well as histology subtypes development. Our results provide evidence that genetic interactions between oncogenesis-related genes play an important role in lung tumorigenesis and epistasis analysis, combined with functional annotation, provides a valuable tool for uncovering functional novel susceptibility genes that contribute to lung cancer development by interacting with other modifier genes.

In 2015, over 850,000 people died from chronic hepatitis and hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV). A novel hepatitis B-like virus has recently been identified in domestic cats. The pathogenic potential of domestic cat hepadnavirus (DCH), for which 6.5% to 10.8% of pet cats are viremic, is unknown. We evaluated stored formalin-fixed, paraffin-embedded biopsies of diseased and normal feline liver for the presence of DCH using PCR and in situ hybridization (ISH). DCH was detected in 43% (6/14) of chronic hepatitis cases and 28% (8/29) of HCCs, whereas cholangitis (n = 6), biliary carcinoma (n = 18) and normal liver (n = 15) all tested negative for DCH. Furthermore, in DCH-associated cases, the histologic features of inflammation and neoplasia, and the viral distribution on ISH were strikingly similar to those seen with HBV-associated disease. Several histological features common in human HBV-associated hepatitis, including piecemeal necrosis and apoptotic bodies, were identified in DCH-positive cases of chronic hepatitis. In two cases of HCC examined, the proliferation index in regions that were ISH-positive was higher than in ISH-negative regions. The intracellular distribution of virus in both hepatitis and HCC demonstrated that viral nucleic acid is present in both nuclear and cytoplasmic forms. Collectively, these findings demonstrate a compelling association between DCH and some cases of chronic hepatitis and hepatocellular carcinoma in the cat that mirrors features of HBV-associated hepatopathies. Future investigations of viral epidemiology and natural history are needed to establish the impact of DCH on feline health.

Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic human T-cell lymphotropic virus 1 infection, triggered by the virally encoded oncoprotein Tax. The transforming activity and subcellular localization of Tax is strongly influenced by posttranslational modifications, among which ubiquitylation and SUMOylation have been identified as key regulators of the nuclear/cytoplasmic shuttling of Tax, as well as its ability to activate NF-κB signaling. Results: Adding to the complex posttranslational modification landscape of Tax, we here demonstrate that Tax also interacts with the ubiquitin-related modifier 1 (Urm1). Conjugation of Urm1 to Tax results in a redistribution of Tax to the cytoplasm and major increase in the transcription of the NF-ĸB targets Rantes and interleukin-6. Utilizing a tax-transgenic Drosophila model, we show that the Urm1-dependent subcellular targeting of Tax is evolutionary conserved, and that the presence of Urm1 is strongly correlated with the transcriptional output of Diptericin, an antimicrobial peptide and established downstream target of NF-κB in flies. Conclusions: These data put forward Urm1 as a novel Tax modifier that modulates its oncogenic activity and hence represents a potential novel target for developing new strategies for treating ATL., Originally included in thesis in manuscript form.

Circular RNA (circRNA) is a type of non-coding RNA molecule that affects the cellular regulatory network by sequestering microRNA (miRNA) like a sponge. This study was performed to identify differentially-expressed circRNA in oral squamous cell carcinomas (OSCCs). By high-throughput sequencing, microarray circRNA expression profiles were acquired from patients with OSCCs (n = 8) and controls (n = 8), which totaled 1921 existing circRNA molecules and 10021 novel circRNA molecules. Most of the circular RNA is from exons and distributed in the No. 1 and 2 chromosomes. Eight up-regulated and down-regulated circRNA molecules were identified as differentially-expressed in OSCCs. Among this, the expression of circ-000334, circ-006740, and circ-006371 are significantly down-regulated in 42 pairs of samples, which means that these circRNA molecules might be implicated in oncogenesis and development of OSCCs. © 2018 Frontiers Media S.A. All Rights Reserved.

Hedgehog signaling pathway is physiologically activated during embryogenesis, especially in lung development. It is also reactivated in many solid tumors. In lung cancer, Hedgehog pathway is closely associated with cancer stem cells (CSCs). Recent works have shown that CSCs produced a full-length Sonic Hedgehog (Shh) protein, with paracrine activity and induction of tumor development. Hedgehog pathway is also involved in tumor drug resistance in lung cancer, as cytotoxic chemotherapy, radiotherapy, and targeted therapies. This review proposes to describe the activation mechanisms of Hedgehog pathway in lung cancer, the clinical implications for overcoming drug resistance, and the perspectives for further research.

The PIM family of Ser/Thr kinase proteins has been implicated in tumorigenesis at different levels. PIM proteins are overexpressed in several tumor types and have been associated with chemoresistance. However, their role in hormone-dependent female tissues has not been explored, especially in the uterus, breast and ovary. We generated conditional transgenic mice with confined expression of human PIM1 or PIM2 genes in these tissues. We characterized the tumoral response to these genetic alterations corroborating their role as oncogenes since they induce hyperproliferation in all tissues and tumors in mammary gland and uterus. Furthermore, we observed a high degree of inflammatory infiltration in these tissues of transgenic mice accompanied by NFAT and mTOR activation and IL6 expression. Moreover, PIM1/2 were overexpressed in human breast, uterine and ovarian tumors, correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression provoke tissue alterations and a large IL6-dependent inflammatory response that may act synergistically during the process of tumorigenesis. The possible end-point is an increased percentage of cancer stem cells, which may be partly responsible for the therapy resistance found in tumors overexpressing PIM kinases.

Our hypothesis is that reversible alterations in histones are a determining factor for low FANCD2 expression in ovarian surface epithelial (OSE) cells in women with a familial risk for ovarian cancer, and that cells with reduced FANCD2 levels are hypersensitive to the genotoxic effects of estrogen, therefore predisposing OSE to malignant transformation. During the study we have screened a large number of normal (no familial risk), high risk (with familial history of this disease), and ovarian cancer cell lines, and determined levels of FANCD2 protein and mRNA. In the first set of experiments, aimed at determining whether histone modifications (i.e. acetylation and/or methylation) affect FANCD2 levels, we established that Trichostatin A (TSA;10nM for 24 hours) corrects FANCD2 levels but not consistently. In the second set of experiments, aimed at establishing whether the estrogen metabolite 4-OHE2 is genotoxic for cells with low FANCD2 levels we: 1) identified the minimal concentration of 4- OHE2 that is associated with DNA damage in human and murine OSE, and; 2) found that OSE cultures with low FANCD2 exhibit significantly increased DNA damage after exposure to 50 uM 4-OHE2, in comparison with OSE cultures with normal levels of FANCD2 protein expression., The original document contains color images.

Analysis of tumor adjacent tissue is assumed to reveal a temporal record of molecular pathways that define oncogenesis. The present study determines expression of the key transcription factor and potential marker of field cancerization early growth response 1 (EGR-1) in human prostate tissues derived from prostatectomies and biopsies. Expression was detected using immunofluorescence and quantified using ImageJ software. Accordingly, EGR-1 expression was similar in cancerous and in histologically normal adjacent tissues from prostatectomy and biopsy specimens. EGR-1 could be exploited as pre-surgical disease indicator in false negative biopsies, identify areas of repeat biopsy, and add molecular information to surgical margins.

The overarching goal of this grant award is to characterize differential splicing of oncongenes and tumor suppressor genes in prostate cancer disparities between African American (AA) and Caucasian American (CA) prostate cancer (PCa). In year 1 of this award, we have focused our efforts on two oncogenes, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) and fibroblast growth factor receptor 3 (FGFR3), undergoing population-dependent differential splicing where the AA-specific variants are engendered with a more aggressive oncogenic phenotype in vitro and in vivo. Full-length cloning of the AA and CA variants of both PIK3CD and FGFR3 have been accomplished. PCa cell lines genetically manipulated to predominantly express the AA-variant of PIK3CD or FGFR3 exhibit greater proliferative and invasive capacity. Detailed analysis of PCa cell lines over-expressing the AA-variant of PIK3CD revealed enhanced activation of the PI3K/AKT pathway compared to the same lines over-expressing the CA-variant. Moreover, proliferative capacity of the CA-variant lines was sensitive to inhibition by CAL-101, a small molecule inhibitor designed specifically against PIK3CD. In contrast, proliferative capacity of the AA-variant lines was resistant to CAL-101 inhibition. And these findings (CA variants sensitive and AA variants insensitive to CAL-101) were recapitulated in a xenograft mouse model of proliferation. We are currently testing a xenograft mouse model of metastasis. Year 2 will focus on in vitro and in vivo characterization of the AA and CA variants of FGFR3., The original document contains color images.

The goal of the project is to investigate the potential of inhibiting iron metabolism to inhibit prostate cancer growth. Specifically, we will study Deferiprone, an iron chelator, and focus on its effect on aconitase in prostate tumors. It has been shown that changes in citrate metabolism at the level of mitochondrial aconitase, is an early change in carcinogenesis in the prostate. This change in metabolism is detectable by magnetic resonance. The project includes both in vitro and in vivo studies to determine its potential utility for clinical translation. Our findings to date include 1) showing that Deferiprone inhibits tumor growth in 2 prostate cell lines, 2) demonstrated an effect on metabolism in perfused cells (one cell line), and 3) confirmed changes in metabolism by measuring changes in oxygen consumption rate. After 24 hours of incubation, deferiprone treated TRAMP C2 cells also showed lower oxygen consumption rates (OCR) and down regulation of mitochondrial aconitase expression, leading to lower enzyme activity as detected in both TRAMP C2 and Myc-Cap cell lines. The effects on both metabolism and proliferation are encouraging and we hope in Year 2 to begin in vivo studies and complete in vitro studies, The original document contains color images.

Long-wave ultraviolet A (UVA) is the major component of terrestrial UV radiation and is also the predominant constituent of indoor sunlamps, both of which have been shown to increase cutaneous melanoma risk. Using a 2-chamber model, we show that UVA-exposed target cells induce an intercellular oxidative signaling to non-irradiated bystander cells. This UVA-mediated bystander stress is observed between all three cutaneous cell types (i.e. keratinocytes, melanocytes and fibroblasts). Significantly, melanocytes appear to be more resistant to direct UVA effects compared to keratinocytes and fibroblasts although melanocytes are also more susceptible to bystander oxidative signaling. The extensive intercellular flux of oxidative species has not been previously appreciated and could possibly contribute to the observed cancer risk associated with prolonged UVA exposure, such as those associated with indoor tanning., The original document contains color images.

The Ron receptor tyrosine kinase is over-expressed and over-activated in a cohort of human cancers, with the most compelling data yet found in breast cancer. Specifically, Ron is overexpressed in approximately 50% of human breast cancers, and has been shown to be an independent predictor of both metastases and poor prognosis in women with this disease. While Ron overexpression appears to be an important factor in human breast cancer growth and metastasis, a significant gap exists in our knowledge about the signaling pathways that Ron activates in breast tumors, and about the importance of these pathways with respect to overall tumor growth and metastatic dissemination. Our laboratories have shown that mammary tumors from mice overexpressing Ron selectively in the mammary epithelium exhibit increased levels of the DEK proto-oncogene. In addition, we also show that ligand-induced Ron activation in human and murine breast cancer cell lines induces the accumulation of DEK protein. This accumulation of DEK is significant as DEK overexpression in breast cancer cell lines leads to increases in cell growth and migration while DEK depletion in breast cancer cells leads to dramatic reductions in cell growth and migration. Moreover, we also show that DEK deficient cells are more susceptible to DNA damage. Based on these data, our goal is to test the hypothesis that Ron-mediated DEK upregulation contributes functionally to breast cancer development, dissemination and resistance to clastogenic therapies and that targeting the Ron-DEK signaling axis may represent an important new therapeutic option for the treatment of breast cancer. To test this hypothesis, two Specific Aims were proposed. In Aim 1, we will determine the requirement of DEK in Ron overexpressing breast cancers utilizing a combination of DEK loss of function and Ron transgenic overexpression. Aim 2 will examine the therapeutic utility of targeting Ron and DEK on beta-catenin activation and breast cancer growth., The original document contains color images.

We previously generated a PB-Cre4/CAG-SMIL transgenic model allowing Cre-induced expression of c-Myc oncogene and Luc2 (for BLI imaging in vivo) in prostate epithelia. Once turned on, c-Myc and Luc2 expression will not rely on androgen, which allows studying castration response and CRPC. However, most Cre4/CAG-SMIL mice did not develop invasive prostate tumors up to 2-year of age, potentially due to c-Myc induced p53 activation. Hence, we proposed to generate PB-Cre4/CAG-SMIL/p53loxP/loxP mice to conditionally knock out p53 and turn on c-Myc expression in prostate for rapid onset of PCa, and use this model to study CRPC and chemoresistance of CRPC. In the initial year, we have carried out all the proposed studies as described in SOW on schedule, including performing extensive multi-rounds of crossing to generate the target PB-Cre4/CAG-SMIL/p53loxP/loxP mice, and the initial characterization of prostate histopathology to confirm early onset of mPIN in 8-week old PB-Cre4/CAG-SMIL/ p53loxP/loxP mice. We have also identified a potential technical problem and provided alternative approaches to address it., The original document contains color images.

Mutations in mitochondrial DNA (mtDNA) are frequent in prostate cancer and they seem to occur early during prostate malignant transformation. Depletion of mtDNA in prostate cancer cells has been linked to acquisition of androgenindependence, progression to an invasive phenotype that is resistant to conventional chemotherapies, as well as induction of epithelial-mesenchymal transition leading to cancer metastasis. Using long-range genomic polymerase chain reaction, large deletion of mtDNA can be detected in prostate cancer tissues but not benign or normal prostate tissues. Noticeably, our study excludes the germ-line origin of the mutant mtDNA pattern in prostate cancer patient through analysis of the blood of the corresponding patient. Our data conclude that mtDNA deletion is due to carcinogenesis process in somatic prostate cells. In addition, our data have unveiled the molecular alteration in prostate cancer cells resulted from mtDNA deletion. For example, Skp2 protein elevation is often associated in prostate cells with loss of mtDNA. Also, the presence of Skp2 expression can decrease the expression of BRCA2 protein as an early biomarker of prostate neoplastic transformation, which is due to BRCA2 proteolysis., The original document contains color images.

Our study is testing whether pregnancy affects the growth of breast cancer cells that expressed a mutant form of p53, which we found is responsible for the expression of PAPP-A. One of the hypothesis that was raised is that PAPP-A is a pregnancydependent oncogene. This year, we begun the characterization of the PAPP-A transgenic mice. The progress made led to a publication that is pending review. In short, PAPP-A was confirmed to be a pregnancy-dependent oncogene. We also made progress on the role of mutant p53. We found that the growth of genetically identical cells carrying a mutation in p53 is accelerated by the passage through pregnancy. The tumors volumes are bigger in the pregnancy group compared to the virgin group. We also found using microarray analysis that there are genes that are up-regulated specifically in the pregnancy group and not in the virgin group. We have begun the characterization of this pregnancy signature. We also obtained mice carrying a p53 mutant knockin in the p53 knockout mice background. We have established a colony and analyzed the effect of pregnancy on mammary tumor formation. So far we found only one mammary tumor in younger females. We are currently aging these mice. In addition, we have collected primary breast cancer sections and pregnancy history of breast cancer patients prior to their surgery. The analysis of this patient cohort is consistent with the data obtained in the PAPP-A transgenic mice and therefore the data is included in the same publication., The original document contains color images.

Prostate carcinogenesis is closely linked to aberrant activation of Ras or Ras signaling pathways (e.g., Raf-MEK, or PI3K pathways). The incidence of activating PI3K mutations in early and advanced prostate cancer, or loss of PTEN is very high. Increased expression of the Ras/Raf/MEK/ERK pathway has been associated with advanced prostate cancer, hormonal independence and a poor prognosis. We have demonstrated that, when aberrantly activated, Ras is lethal to the cell unless a survival pathway also initiated by Ras is active. This survival pathway requires PKC . Unlike the classical PKC isozymes, PKC is not required for cell survival, and its inhibition or down-regulation in normal cells and tissues has no significant adverse effects. Inhibition of PKC in human and murine cells containing an activated Ras protein, however, initiates rapid and profound apoptosis. In this work, we have validated the hypothesis that inhibition or down-regulation of PKC in human and murine models of prostate cancer with aberrant activation of Ras signaling will cause targeted cytotoxicity in these tumors. We have developed PKC specific small molecule inhibitors and demonstrated their anti-tumor activity against prostate cancer in vitro and in vivo., The original document contains color images.

Prostate cancer is a leading cause of cancer and cancer-related mortality in males in the United States. Acid ceramidase is frequently overexpressed in prostate cancer and catalyzes the deacylation of proapoptotic ceramide into sphingosine. Sphingosine is converted by sphingosine kinases into sphingosine 1-phosphate, which promotes numerous pro-cancer phenotypes. Due to the frequent overexpression of acid ceramidase in prostate cancer and its net cancer promoting signaling effects achieved by reducing ceramide and generating sphingosine 1-phosphate, the impact of acid ceramidase, and the lipid profiles it alters, on prostate cancer cell signaling were studied. Here we report the activation of the oncogenic PI3K/Akt signaling pathway when acid ceramidase is overexpressed both in human prostate tissue and in prostate cancer cell lines. This effect was found to depend on sphingosine kinase 1; potentially reflecting increased accessibility of sphingosine kinase 1 to lysosome-derived sphingosine. Surprisingly, we found that S1P receptor 2 mediates acid ceramidase-induced Akt activation, which challenges the prevailing view in the literature that suggests predominantly tumor suppressive functions for S1P receptor 2. Functionally, acid ceramidase overexpressing cells were found to be resistant to cytotoxic chemotherapies, but profoundly sensitive to inhibition of PI3K or Akt in proliferation assays and soft agar colony formation, suggesting acid ceramidase overexpressing cells become dependent on Akt signaling for their oncogenic phenotypes. The tumor suppressor PTEN classically antagonizes PI3K-mediated activation of Akt, however emerging evidence suggests that nuclear PTEN is also critically important in suppressing cancer. Thus, mechanisms mediating nucleocytoplasmic shuttling of PTEN are of great interest., The original document contains color images.

Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients. TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling. The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment wi

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown.

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown.

The aggressive behavior of malignant breast cancer is determined by a complex array of signaling pathways that regulate cell growth, survival and migration. The PI 3-K-Akt pathway has been linked to all of these responses. The current paradigm states that deregulated PI 3-K-Akt signaling promotes cancer progression. Many of the enzymes that regulate PI 3-K signaling are frequently mutated in human breast cancer, thereby up-regulating Akt activity and increasing tumor cell growth and survival. This is best illustrated by the finding that the catalytic subunit of PI 3-K, PIK3CA, is the most frequently mutated oncogene in breast cancer. However, recent studies have demonstrated that distinct Akt isoforms can either inhibit or enhance breast cancer cell invasive migration and metastasis in vitro and in vivo, probably by phosphorylating a different set of substrates in an isoform specific manner. Afadin is a Ras target gene that regulates cell-cell adhesion downstream of Ras. Afadin protein loss of expression is associated with poor outcome in breast cancer patients. The most significant finding of this research thus far is that Afadin isoform 3, which is the longer, ubiquitously expressed form of the protein, is a substrate of Akt, down stream of the PI-3K pathway., The original document contains color images.

It has been suggested that some inherited mtDNA variants could have an adverse effect by increasing the generation of reactive oxygen species (ROS). Besides the sequence variations in mtDNA, the mtDNA CNV might also affect cancer risk by disturbing crosstalk between the mitochondria and the nucleus, and consequently altering nuclear DNA stability. Variability in the mtDNA (sequence and copy number) might be extremely relevant to prostate cancer because oxidative stress has been suggested to play a significant role in prostate cancer carcinogenesis. Even more intriguingly, the geographic and racial polymorphisms of mtDNA might have implications in the racial disparity of prostate cancer because African Americans are at a disproportionately higher risk for many oxidative stress related medical conditions, including prostate cancer. In this current proposal, we plan to utilize the valuable biospecimens and data collected through North Carolina-Louisiana Prostate Cancer Project (PCaP) to comprehensively study the associations between mtDNA polymorphisms/haplogroups and prostate cancer tumor characteristics at baseline and progression in both CA and AA men. Our hypothesis is that genetic variations (sequence and copy number) in mtDNA are associated with prostate cancer aggressiveness at diagnosis and prostate cancer progression. So far, we have completed genotyping analyses for 75 genetic polymorphisms in all study subjects for Aim 1. In Aim 2, we have completed CNV analyses in about 500 study subjects. We have not started Aim 3 yet.

The incidence of human malignancies increases significantly with age, suggesting a mechanistic connection between aging (longevity) and carcinogenesis Breast malignancies that develop with increasing age very likely represent a spectrum of cancers arising from different types of hereditary and spontaneous tumors. Murine models for hereditary breast cancer in younger women have been established by altering the expression of BRCA genes. In contrast, there are no murine models for the spontaneous breast cancers that are more common in older women. The work supported by this DOD idea award has allows us to not only validate the first ER+ in vivo murine model for breast cancers but is has also allowed the discovery of several potential molecular targets that may potential be used for either chemoprevention or as anticancer agents., The original document contains color images.

Activating mutations in N-RAS are found in 33% of primary melanomas, and are correlated with sun exposure and nodular lesions. Potential therapeutic strategies have been devised in the past to directly target RAS. Unfortunately, these have shown minimal if any activity in melanoma in clinical trials, because wild-type Ras and its downstream effectors are required for many critical cellular functions in normal cells, the therapeutic window for inhibiting Ras directly may be too narrow to exploit. Our novel alternative strategy has the potential to circumvent this limitation. Aberrantly activated, K-RAS or Ha-RAS are lethal to a tumor cell unless a survival pathway requiring PKCd is also active. Inhibition of PKCd in human and murine cells containing an activated K- or Ha-RAS protein initiates rapid and profound apoptosis. The dependency of tumor cells upon the activity of a non-oncogenic protein is sometimes termed non-oncogene addiction. Hypothesis: inhibition or down-regulation of PKCd in human and murine models of melanoma with aberrant activation of N-RAS signaling will cause targeted cytotoxicity in these tumors. The Specific Aims/Study Design of this Discovery Proposal are: Test the hypothesis that inhibition or down-regulation of PKCd in human melanoma cell lines with NRAS mutations selectively induces apoptosis; 2) Determine whether aberrant activation of pathways downstream of RAS (in the setting of wild-type RAS alleles) will similarly sensitize human melanoma cells to PKCd inhibition; 3) Test this targeted approach in in vivo models of human melanoma. Impact: A novel therapeutic modality selectively targeting melanomas with activation of N-RAS would make a significant impact on the way melanoma is treated., The original document contains color images.

Our hypothesis is that reversible alterations in histones are a determining factor for low FANCD2 expression in ovarian surface epithelial (OSE) cells in women with a familial risk for ovarian cancer, and that cells with reduced FANCD2 levels are hypersensitive to the genotoxic effects of estrogen, therefore predisposing OSE to malignant transformation. During this year we have screened a large number of normal (no familial risk), high-risk (with familial history of this disease), and ovarian cancer cell lines, and determined levels of FANCD2 protein and mRNA. In the first set of experiments, aimed at determining whether histone modifications (i.e. acetylation and/or methylation) affect FANCD2 levels, we established that Trichostatin A (TSA;10nM for 24 hours) corrects FANCD2 levels.In the second set of experiments, aimed at establishing whether the estrogen metabolite 4-OHE2 is genotoxic for cells with low FANCD2 levels we: 1) identified the minimal concentration of 4-OHE2 that is associated with DNA damage in human and murine OSE, and; 2) found that OSE cultures that express low FANCD2 exhibit significantly increased DNA damage after exposure to 50 uM 4-OHE2, in comparison with OSE cultures with normal levels of FANCD2 protein expression., The original document contains color images.

During our first year of research, we demonstrated a localized field cancerization phenomenon on gene expression in the airway of patients with lung cancer, and we identified several pathways preferentially activated in the airway adjacent to tumors. In addition, we have identified markers of stem cells in the airway that may represent tumor-initiating cells of the airway and are evaluating profiles of these cells. We have identified Snail as a novel marker of stem cells in the airway that promote epithelial-mesenchymal transition. We have made a major technical advance in developing the methods required to use low quality and quantity laser capture microdissected material to sequence the transcriptome. This allows us to examine the gene expression profiles in premalignant lesions and compare it to the histologically normal airway epithelium and tumor. We have validated this approach and the data will allow us to identify novel pathways for lung carcinogenesis. All of these studies are identifying biomarkers that could be used for early lung cancer detection and pathways that are involved in field cancerization . Understanding this field cancerization and development of premalignant lesions is likely to shed light on novel pathways in lung carcinogenesis that could lead to diagnostic tests, therapies and cancer chemoprevention strategies for lung cancer., The original document contains color images.

The mammalian Sirtuin genes are homologs of the yeast Saccharomyces cerevisiae Sir2 gene that is implicated in the regulation of longevity (Haigis et al., 2012). There are seven Sirtuin proteins, with SIRT1, SIRT6, and SIRT7 localized in the nucleus, SIRT2 localized in the cytoplasm and SIRT3, SIRT4, and SIRT5 localized in the mitochondria (Finkel et al., 2009). The mitochondrial deacetylase SIRT3 is thought to act on numerous substrates to regulate seevral processes including fat and amino-acid metabolism as well as electron transport (Huang et al., 2010)., The original document contains color images.