Molecular analysis of the human cytoglobin mRNA isoforms.

Multiple functions have been proposed for the ubiquitously expressed vertebrate globin cytoglobin (Cygb), including nitric oxide (NO) metabolism, lipid peroxidation/signalling, superoxide dismutase activity, reactive oxygen/nitrogen species (RONS) scavenging, regulation of blood pressure, antifibrosis, and both tumour suppressor and oncogenic effects. Since alternative splicing can expand the biological roles of a gene, we investigated whether this mechanism contributes to the functional diversity of Cygb. By mining of cDNA data and molecular analysis, we identified five alternative mRNA isoforms for the human CYGB gene (V-1 to V-5). Comprehensive RNA-seq analyses of public datasets from human tissues and cells confirmed that the canonical CYGB V-1 isoform is the primary CYGB transcript in the majority of analysed datasets. Interestingly, we revealed that isoform V-3 represented the predominant CYGB variant in hepatoblastoma (HB) cell lines and in the majority of analysed normal and HB liver tissues. CYGB V-3 mRNA is transcribed from an alternate upstream promoter and hypothetically encodes a N-terminally truncated CYGB protein, which is not recognized by some antibodies used in published studies. Little to no transcriptional evidence was found for the other CYGB isoforms. Comparative transcriptomics and flow cytometry on CYGB+/+ and gene-edited CYGB-/- HepG2 HB cells did not unveil a knockout phenotype and, thus, a potential function for CYGB V-3. Our study reveals that the CYGB gene is transcriptionally more complex than previously described as it expresses alternative mRNA isoforms of unknown function. Additional experimental data are needed to clarify the biological meaning of those alternative CYGB transcripts.

Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors.

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

NOXA Accentuates Apoptosis Induction by a Novel Histone Deacetylase Inhibitor.

Epigenetic modifiers of the histone deacetylase (HDAC) family are often dysregulated in cancer cells. Experiments with small molecule HDAC inhibitors (HDACi) have proven that HDACs are a vulnerability of transformed cells. We evaluated a novel hydroxamic acid-based HDACi (KH16; termed yanostat) in human pancreatic ductal adenocarcinoma (PDAC) cells, short- and long-term cultured colorectal cancer (CRC) cells, and retinal pigment epithelial cells. We show that KH16 induces cell cycle arrest and apoptosis, both time and dose dependently in PDAC and CRC cells. This is associated with altered expression of BCL2 family members controlling intrinsic apoptosis. Recent data illustrate that PDAC cells frequently have an altered expression of the pro-apoptotic BH3-only protein NOXA and that HDACi induce an accumulation of NOXA. Using PDAC cells with a deletion of NOXA by CRISPR-Cas9, we found that a lack of NOXA delayed apoptosis induction by KH16. These results suggest that KH16 is a new chemotype of hydroxamic acid HDACi with superior activity against solid tumor-derived cells. Thus, KH16 is a scaffold for future research on compounds with nanomolar activity against HDACs.

Rapid translocation of intracellular toll-like receptors depends on endosomal NADPH oxidase.

Endosomal toll-like receptors (TLRs) must be translocated from the endoplasmic reticulum (ER) to the endosome and proteolytically cleaved within the endosome before they can induce cellular signals. As ligands for these TLRs are also liberated from apoptotic or necrotic cells, this process is controlled by several mechanisms which shall ensure that there is no inadvertent activation. We have shown previously that antiphospholipid antibodies induce endosomal NADPH-oxidase (NOX) followed by the translocation of TLR7/8 to the endosome. We show now that endosomal NOX is required for the rapid translocation of TLR3, TLR7/8, and TLR9. Deficiency of gp91phox, the catalytic subunit of NOX2, or inhibition of endosomal NOX by the chloride channel blocker niflumic acid both prevent immediate (i.e., within 30 min) translocation of these TLRs as shown by confocal laser scanning microscopy. Under these conditions, the induction of mRNA synthesis for TNF-α and secretion of TNF-α is delayed by approx. 6-9 h. However, maximal expression of TNF-α mRNA or secretion of TNF-α is not significantly reduced. In conclusion, these data add NOX2 as another component involved in the orchestration of cellular responses to ligands of endosomal TLRs.

A complex epigenome-splicing crosstalk governs epithelial-to-mesenchymal transition in metastasis and brain development.

Epithelial-to-mesenchymal transition (EMT) renders epithelial cells migratory properties. While epigenetic and splicing changes have been implicated in EMT, the mechanisms governing their crosstalk remain poorly understood. Here we discovered that a C2H2 zinc finger protein, ZNF827, is strongly induced during various contexts of EMT, including in brain development and breast cancer metastasis, and is required for the molecular and phenotypic changes underlying EMT in these processes. Mechanistically, ZNF827 mediated these responses by orchestrating a large-scale remodelling of the splicing landscape by recruiting HDAC1 for epigenetic modulation of distinct genomic loci, thereby slowing RNA polymerase II progression and altering the splicing of genes encoding key EMT regulators in cis. Our findings reveal an unprecedented complexity of crosstalk between epigenetic landscape and splicing programme in governing EMT and identify ZNF827 as a master regulator coupling these processes during EMT in brain development and breast cancer metastasis.

Increased stability of the TM helix oligomer abrogates the apoptotic activity of the human Fas receptor.

Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is coupled with a decreased ligand-sensitivity of cells expressing the Fas receptor, and thus in a decreased number of apoptotic events. Mutation of Pro183 likely results in a substantial rearrangement of the self-associated Fas receptor transmembrane trimer, which likely abolishes further signaling of the apoptotic signal but may activate other signaling pathways. Our study shows that formation of a stable Fas receptor transmembrane helix oligomer does not per se result in receptor activation.

Application of patient-derived liver cancer cells for phenotypic characterization and therapeutic target identification.

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were performed using next-generation sequencing (NGS). Key oncogenic alterations were identified by targeted NGS and cell lines carrying potentially actionable mutations were treated with corresponding specific inhibitors. PDCL fully resembled morphological features of the primary cancers in vitro and in vivo over extended period in culture. Genomic alterations as well as transcriptome profiles showed high similarity with primary tumors and remained stable during long-term culturing. Targeted-NGS confirmed that key oncogenic mutations such as TP53, KRAS, CTNNB1 as well as actionable mutations (e.g. MET, cKIT, KDR) were highly conserved in PDCL and amenable for individualized therapeutic approaches. Integrative genomic and transcriptomic approaches further demonstrated that PDCL more closely resemble molecular and prognostic features of PLC than established cell lines and are valuable tool for direct target evaluation. Our integrative analysis demonstrates that PDCL represents refined model for discovery of relevant molecular subgroups and exploration of precision medicine approaches for the treatment of this deadly disease.

Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver.

Ginkgo biloba (EGb761) is a widely used botanical drug. Several reports indicate that EGb761 confers preventive as well as anti-tumorigenic properties in a variety of tumors, including hepatocellular carcinoma (HCC). We here evaluate functional effects and molecular alterations induced by EGb761 in hepatoma cells and non-malignant hepatocytes. Hepatoma cell lines, primary human HCC cells and immortalized human hepatocytes (IH) were exposed to various concentrations (0-1000 µg/ml) of EGb761. Apoptosis and proliferation were evaluated after 72h of EGb761 exposure. Response to oxidative stress, tumorigenic properties and molecular changes were further investigated. While anti-oxidant effects were detected in all cell lines, EGb761 promoted anti-proliferative and pro-apoptotic effects mainly in hepatoma cells. Consistently, EGb761 treatment caused a significant reduction in colony and sphere forming ability in hepatoma cells and no mentionable changes in IH. Transcriptomic changes involved oxidative stress response as well as key oncogenic pathways resembling Nrf2- and mTOR signaling pathway. Taken together, EGb761 induces differential effects in non-transformed and cancer cells. While treatment confers protective effects in non-malignant cells, EGb761 significantly impairs tumorigenic properties in cancer cells by affecting key oncogenic pathways. Results provide the rational for clinical testing of EGb761 in preventive and therapeutic strategies in human liver diseases.

The histone deacetylases HDAC1 and HDAC2 are required for the growth and survival of renal carcinoma cells.

Novel therapies are required for the treatment of metastatic renal cell carcinoma (RCC), which is associated with inoperable disease and patient death. Histone deacetylases (HDACs) are epigenetic modifiers and potential drug targets. Additional information on molecular pathways that are altered by histone deacetylase inhibitors (HDACi) in RCC cells is warranted. It should equally be delineated further which individual members of the 18 mammalian HDACs determine the survival and tumor-associated gene expression programs of such cells. Most importantly, an ongoing dispute whether HDACi promote or suppress metastasis-associated epithelial-to-mesenchymal transition (EMT) has to be resolved before HDACi are considered further as clinically relevant drugs. Here we show how HDACi affect murine and primary human RCC cells. We find that these agents induce morphological alterations resembling the metastasis-associated EMT. However, individual and proteomics-based analyses of epithelial and mesenchymal marker proteins and of EMT-associated transcription factors (EMT-TFs) reveal that HDACi do not trigger EMT. Pathway deconvolution analysis identifies reduced proliferation and apoptosis induction as key effects of HDACi. Furthermore, these drugs lead to a reduction of the cell adhesion molecule E-cadherin and of the platelet-derived growth factor receptor-ß (PDGFRß), which is a key driver of RCC metastasis formation. Accordingly, HDACi reduce the pulmonary spread of syngeneic transplanted renal carcinoma cells in mice. Specific genetic elimination of the histone deacetylases HDAC1/HDAC2 reflects the effects of pharmacological HDAC inhibition regarding growth suppression, apoptosis, and the downregulation of E-cadherin and PDGFRß. Thus, these epigenetic modifiers are non-redundant gatekeepers of cell fate and precise pharmacological targets.

Molecular endoscopic imaging in cancer.

Cancer is one of the major causes of death in both the USA and Europe. Molecular imaging is a novel field that is revolutionizing cancer management. It is based on the molecular signature of cells in order to study the human body both in normal and diseased conditions. The emergence of molecular imaging has been driven by the difficulties associated with cancer detection, particularly early-stage premalignant lesions which are often unnoticed as a result of minimal or no structural changes. Endoscopic surveillance is the standard method for early-stage cancer detection. In addition to recent major advancements in endoscopic instruments, significant progress has been achieved in the exploration of highly specific molecular probes and the combination of both will permit significant improvement of patient care. In this review, we provide an outline of the current status of endoscopic imaging and focus on recent applications of molecular imaging in gastrointestinal, hepatic and other cancers in the context of detection, targeted therapy and personalized medicine. As new imaging agents have the potential to broadly expand our cancer diagnostic capability, we will also present an overview of the main types of optical molecular probes with their pros and cons. We conclude by discussing the challenges and future prospects of the field.

FBXO32 promotes microenvironment underlying epithelial-mesenchymal transition via CtBP1 during tumour metastasis and brain development.

The set of events that convert adherent epithelial cells into migratory cells are collectively known as epithelial-mesenchymal transition (EMT). EMT is involved during development, for example, in triggering neural crest migration, and in pathogenesis such as metastasis. Here we discover FBXO32, an E3 ubiquitin ligase, to be critical for hallmark gene expression and phenotypic changes underlying EMT. Interestingly, FBXO32 directly ubiquitinates CtBP1, which is required for its stability and nuclear retention. This is essential for epigenetic remodeling and transcriptional induction of CtBP1 target genes, which create a suitable microenvironment for EMT progression. FBXO32 is also amplified in metastatic cancers and its depletion in a NSG mouse xenograft model inhibits tumor growth and metastasis. In addition, FBXO32 is essential for neuronal EMT during brain development. Together, these findings establish that FBXO32 acts as an upstream regulator of EMT by governing the gene expression program underlying this process during development and disease.

Adverse genomic alterations and stemness features are induced by field cancerization in the microenvironment of hepatocellular carcinomas.

Hepatocellular Carcinoma (HCC) commonly develops in chronically damaged liver tissues. The resulting regenerative and inflammatory processes create an adverse milieu that promotes tumor-initiation and progression. A better understanding of the hepatic tumor-microenvironment interaction might infer profound therapeutic implications.Integrative whole genome and transcriptome analyses of different tumor regions, the invasive tumor border and tumor-surrounding liver (SL) were performed to identify associated molecular alterations and integrated with our existing HCC database. Expression levels and localization of established CSC markers were assessed in pre-neoplastic lesions and confirmed in two independent patient cohorts using qRT-PCR, immunohistochemistry and immunofluorescence.Our results indicate that genomic and transcriptomic profiles between SL and different tumor regions are quite distinct. Progressive increase in genetic alterations and activation of pathways related to proliferation as well as apoptosis were observed in the tumor tissue, while activation of stemness markers was present in cirrhotic SL and continuously decreased from pre-neoplastic lesions to HCC. Interestingly, the invasive tumor border was characterized by inflammatory and EMT-related gene sets as well as activation of pro-survival signaling. Consistently, integration of gene expression signatures with two independent HCC databases containing 300 HCCs revealed that border signatures are predictive of HCC patient survival.Prognostic significance of the permissive liver microenvironment might be a consequence of a pro-oncogenic field effect that is caused by chronic regenerative processes. Activation of key oncogenic features and immune-response signaling indicates that the cross-talk between tumor and microenvironment might be a promising therapeutic and/or preventive target.

Glycine-functionalized copper(ii) hydroxide nanoparticles with high intrinsic superoxide dismutase activity.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O2-) radicals into molecular oxygen (O2) and H2O2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(ii) hydroxide nanoparticles (Gly-Cu(OH)2 NPs) are functional SOD mimics, whereas bulk Cu(OH)2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH)2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH)2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concentrations of toxic reactive oxygen species (ROS, >1016 molecules per puff) including superoxide and reactive nitrogen species which lead to the development of chronic and degenerative diseases via oxidative damage and subsequent cell death. Embedded in cigarette filters Gly-Cu(OH)2 NPs efficiently removed ROS from smoke, thereby protecting lung cancer cell lines from cytotoxic effects. Their stability, ease of production and versatility make them a powerful tool for a wide range of applications in environmental chemistry, biotechnology and medicine.

Activation of silent mating type information regulation 2 homolog 1 by human chorionic gonadotropin exerts a therapeutic effect on hepatic injury and inflammation.

Incidence and prevalence of inflammatory liver diseases has increased over the last years, but therapeutic options are limited. Pregnancy induces a state of immune tolerance, which can result in spontaneous improvement of clinical symptoms of certain autoimmune diseases including autoimmune hepatitis (AIH). We investigated the immune-suppressive mechanisms of the human pregnancy hormone, chorionic gonadotropin (hCG), in the liver. hCG signaling activates silent mating type information regulation 2 homolog 1 (SIRT1), which deacetylates forkhead box o3 (FOXO3a), leading to repression of proapoptotic gene expression, because the immunosuppressive consequence attributed to the absence of caspase-3 activity of hepatocellular interleukin 16 (IL-16) is no longer processed and released. Thus, serum levels of IL-16, a key chemotactic factor for CD4+ lymphocytes, were reduced and migration to injured hepatocytes prevented. Furthermore, elevated IL-16 levels are found in the sera from patients with AIH, hepatitis B virus, hepatitis C virus, and nonalcoholic steatohepatitis. CONCLUSION: Here, we report that hCG regulates the SIRT1/FOXO3a axis in hepatocytes, resulting in immune suppression by attenuating caspase-3-dependent IL-16 processing and release, which concomitantly prevents autoaggressive T-cell infiltration of the liver. Considering the low toxicity profile of hCG in humans, interrupting the inflammatory cycle by hCG opens new perspectives for therapeutic intervention of inflammatory liver diseases. (Hepatology 2017;65:2074-2089).

Glucagon-like peptide-1 receptor signalling reduces microvascular thrombosis, nitro-oxidative stress and platelet activation in endotoxaemic mice.

BACKGROUND AND PURPOSE: Excessive inflammation in sepsis causes microvascular thrombosis and thrombocytopenia associated with organ dysfunction and high mortality. The present studies aimed to investigate whether inhibition of dipeptidyl peptidase-4 (DPP-4) and supplementation with glucagon-like peptide-1 (GLP-1) receptor agonists improved endotoxaemia-associated microvascular thrombosis via immunomodulatory effects. EXPERIMENTAL APPROACH: Endotoxaemia was induced in C57BL/6J mice by a single injection of LPS (17.5 mg kg-1 for survival and 10 mg kg-1 for all other studies). For survival studies, treatment was started 6 h after LPS injection. For all other studies, drugs were injected 48 h before LPS treatment. KEY RESULTS: Mice treated with LPS alone showed severe thrombocytopenia, microvascular thrombosis in the pulmonary circulation (fluorescence imaging), increased LDH activity, endothelial dysfunction and increased markers of inflammation in aorta and whole blood (leukocyte-dependent oxidative burst, nitrosyl-iron haemoglobin, a marker of nitrosative stress, and expression of inducible NOS). Treatment with the DPP-4 inhibitor linagliptin or the GLP-1 receptor agonist liraglutide, as well as genetic deletion of DPP-4 (DPP4-/- mice) improved all these parameters. In GLP-1 receptor-deficient mice, both linagliptin and liraglutide lost their beneficial effects and improvement of prognosis. Incubation of platelets and cultured monocytes (containing GLP-1 receptor protein) with GLP-1 receptor agonists inhibited the monocytic oxidative burst and platelet activation, with a GLP-1 receptor-dependent elevation of cAMP levels and PKA activation. CONCLUSIONS AND IMPLICATIONS: GLP-1 receptor activation in platelets by linagliptin and liraglutide strongly attenuated endotoxaemia-induced microvascular thrombosis and mortality by a cAMP/PKA-dependent mechanism, preventing systemic inflammation, vascular dysfunction and end organ damage. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Assessing the Histone Deacetylase Activity of SIRT6 in Primary Murine Hepatocytes via Proximity Ligation Assay.

Generation of primary cell culture of hepatocytes by mouse liver perfusion (MLP) combines the advantages of in vivo and in vitro models. It provides hepatocytes that grow under physiological conditions in mice, with the genotype of the whole organism or a specific gene knockout. In contrast to immortalized cell cultures, primary murine hepatocytes (pmHep) are non-cancerous cells with a limited lifespan but still amenable to classical in vitro methods such as treatment with drugs, small molecule inhibitors, and agonistic/antagonistic antibodies of surface receptors as well as transfection. One technique, which has gained popularity recently, is the analysis of protein-protein interactions by the proximity ligation assay (PLA). Here, we describe a liver perfusion protocol and the detection of the histone deacetylase function of Sirtuin 6 (SIRT6) using PLA in pmHep.

Identification of a classic nuclear localization signal at the N terminus that regulates the subcellular localization of Rbfox2 isoforms during differentiation of NMuMG and P19 cells.

Nuclear localization of the alternative splicing factor Rbfox2 is achieved by a C-terminal nuclear localization signal (NLS) which can be excluded from some Rbfox2 isoforms by alternative splicing. While this predicts nuclear and cytoplasmic localization, Rbfox2 is exclusively nuclear in some cell types. Here, we identify a second NLS in the N terminus of Rbfox2 isoform 1A that is not included in Rbfox2 isoform 1F. Rbfox2 1A isoforms lacking the C-terminal NLS are nuclear, whereas equivalent 1F isoforms are cytoplasmic. A shift in Rbfox2 expression toward cytoplasmic 1F isoforms occurs during epithelial to mesenchymal transition (EMT) and could be important in regulating the activity and function of Rbfox2.

CAF-like state in primary skin fibroblasts with constitutional BRCA1 epimutation sheds new light on tumor suppressor deficiency-related changes in healthy tissue.

Constitutive epimutations of tumor suppressor genes are increasingly considered as cancer predisposing factors equally to sequence mutations. In light of the emerging role of the microenvironment for cancer predisposition, initiation, and progression, we aimed to characterize the consequences of a BRCA1 epimutation in cells of mesenchymal origin. We performed a comprehensive molecular and cellular comparison of primary dermal fibroblasts taken from a monozygous twin pair discordant for recurrent cancers and BRCA1 epimutation, whose exceptional clinical case we previously reported in this journal. Comparative transcriptome analysis identified differential expression of extracellular matrix-related genes and pro-tumorigenic growth factors, such as collagens and CXC chemokines. Moreover, genes known to be key markers of so called cancer-associated fibroblasts (CAFs), such as ACTA2, FAP, PDPN, and TNC, were upregulated in fibroblasts of the affected twin (BRCA1(mosMe)) in comparison to those of the healthy twin (BRCA1(wt)). Further analyses detected CAF-typical cellular features, including an elevated growth rate, enhanced migration, altered actin architecture and increased production of ketone bodies in BRCA1(mosMe) fibroblasts compared to BRCA1(wt) fibroblasts. In addition, conditioned medium of BRCA1(mosMe) fibroblasts was more potent than conditioned medium of BRCA1(wt) fibroblasts to promote cell proliferation in an epithelial and a cancer cell line. Our data demonstrate, that a CAF-like state is not an exclusive feature of tumor-associated tissue but also exists in healthy tissue with tumor suppressor deficiency. The naturally occurring phenomenon of twin fibroblasts differing in their BRCA1 methylation status revealed to be a unique powerful tool for exploring tumor suppressor deficiency-related changes in healthy tissue, reinforcing their significance for cancer predisposition.

Translating bioinformatics in oncology: guilt-by-profiling analysis and identification of KIF18B and CDCA3 as novel driver genes in carcinogenesis.

MOTIVATION: Co-regulated genes are not identified in traditional microarray analyses, but may theoretically be closely functionally linked [guilt-by-association (GBA), guilt-by-profiling]. Thus, bioinformatics procedures for guilt-by-profiling/association analysis have yet to be applied to large-scale cancer biology. We analyzed 2158 full cancer transcriptomes from 163 diverse cancer entities in regard of their similarity of gene expression, using Pearson's correlation coefficient (CC). Subsequently, 428 highly co-regulated genes (|CC| ≥ 0.8) were clustered unsupervised to obtain small co-regulated networks. A major subnetwork containing 61 closely co-regulated genes showed highly significant enrichment of cancer bio-functions. All genes except kinesin family member 18B (KIF18B) and cell division cycle associated 3 (CDCA3) were of confirmed relevance for tumor biology. Therefore, we independently analyzed their differential regulation in multiple tumors and found severe deregulation in liver, breast, lung, ovarian and kidney cancers, thus proving our GBA hypothesis. Overexpression of KIF18B and CDCA3 in hepatoma cells and subsequent microarray analysis revealed significant deregulation of central cell cycle regulatory genes. Consistently, RT-PCR and proliferation assay confirmed the role of both genes in cell cycle progression. Finally, the prognostic significance of the identified KIF18B- and CDCA3-dependent predictors (P = 0.01, P = 0.04) was demonstrated in three independent HCC cohorts and several other tumors. In summary, we proved the efficacy of large-scale guilt-by-profiling/association strategies in oncology. We identified two novel oncogenes and functionally characterized them. The strong prognostic importance of downstream predictors for HCC and many other tumors indicates the clinical relevance of our findings. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SIRT1 prevents genotoxic stress-induced p53 activation in acute myeloid leukemia.

SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)(+)-cells protein, expression of SIRT1 is regulated by FLT3 kinase activity. In addition, SIRT1 function is modulated via the ATM-DBC1-SIRT1 axis in a FLT3-ITD-dependent manner. In murine leukemia models driven by MLL-AF9 or AML1-ETO coexpressing FLT3-ITD, SIRT1 acts as a safeguard to counteract oncogene-induced stress, and leukemic blasts become dependent on SIRT1 activity. Pharmacologic targeting or RNAi-mediated knockdown of SIRT1 inhibited cell growth and sensitized AML cells to tyrosine kinase inhibitor treatment and chemotherapy. This effect was a result of the restoration of p53 activity. Our data suggest that targeting SIRT1 represents an attractive therapeutic strategy to overcome primary resistance in defined subsets of patients with AML.