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APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G(1) phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE : This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

Introduction: The Nuclear Receptor Coactivator 4 (NCOA4) gene is frequently targeted by chromosomal rearrangements in thyroid carcinoma. These events join the 5’-ter of NCOA4 to the DNA sequence encoding the tyrosine kinase (TK) domain of the receptor tyrosine kinase RET, generating the RET/PTC3 chimeric gene, whose protein product displays oncogenic activity. The NCOA4 N-ter mediates homodimerization of the RET TK domain, followed by RET kinase activation and, in turn, gain of transforming activity. Matherials: We used Xenopus laevis egg extracts to study NCOA4 role in DNA replication by performing in vitro DNA replication assays, chromatin pull down and DNA fiber stretching; Mouse embryo fibroblasts (MEFs) from NCOA4+/+ and -/- embryos to study population doublings accumulation in culture, DNA damage and DNA damage response (DDR) activation by immufluorescence staining for γH2AX and 53BP1 and DNA fiber stretching to visualize replication stalling lesions, fork speed and inter-origin distance. Results: Here we show that NCOA4 protein binds MCM7 protein, a component of the MCM2-7 complex involved in DNA replication origin licensing and functioning as the major helicase of the DNA replication fork. In Xenopus laevis egg extracts, both exogenously expressed and endogenous NCOA4 protein inhibit DNA replication by interacting with the MCM2-7 complex and restraining replication origin activation, as shown by in vitro DNA replication assays, chromatin pull-down, and DNA fiber stretching experiments. NCOA4 -/- mouse embryonic fibroblasts (MEFs) undergo premature senescence, characterized by block of cell proliferation and SA-β galactosidase positive staining. This phenotype is characterized by replication stress-associated activation of DDR. Thus, using a DNA fiber stretching assay, we show that NCOA4 depleted cells accumulate replication stalling lesions and feature increased DNA replication origin activation. Conclusions: We conclude that NCOA4 protein is involved in the maintenance of genome integrity by controlling DNA replication , possibly acting as a new genome “caretaker”. Thus RET/PTC3 rearrangement in cancer may act as genetic double-hit by causing simultaneously a gain of oncogenic RET function and a loss of care-taker NCOA4 function.

The Nuclear Receptor Coactivator 4 (NCOA4) gene (also known as RFG/ELE1/ARA70) is frequently targeted by chromosomal rearrangements in papillary thyroid carcinoma (PTC). These events join the N-ter (exons 1-5, encoding amino acids 1-238) of the NCOA4 gene to the DNA sequence encoding the tyrosine kinase (TK) domain of the receptor tyrosine kinase RET, generating the RET/PTC3 chimeric gene, whose protein product displays oncogenic activity. The NCOA4 N-ter mediates homodimerization of the RET TK domain, followed by RET kinase activation and, in turn, gain of transforming activity. Disruption of NCOA4 gene might contribute to the neoplastic phenotype as well, however to date no cellular phenotype has been associated with NCOA4 deficiency. To gain insight into the physiological function of NCOA4, we disrupted NCOA4 in mice. NCOA4-deficient mice did not exhibit any apparent phenotypic alteration. However, when grown at 20% O2, NCOA4-/- mouse embryonic fibroblasts (MEFs) undergo premature senescence, characterized by block of cell proliferation and BrdU incorporation, as well as SA(senescence-associated)-β-galactosidase positive staining. This phenotype is associated with accumulation of DNA damage and activation of a DNA damage response (DDR). Using a fiber stretching assay, that monitors DNA replication fork progression, we show that NCOA4-depleted cells accumulate replication fork stalling lesions and feature increased DNA replication origin activation. In addition, NCOA4-/- MEFs, grown at 3% O2 concentration, feature increased sensitivity to genotoxic stress, particularly that induced by S-phase specific DNA damaging agents. In conclusion, these findings highlight a novel role for NCOA4 in the prevention of replication stress, DNA damage, and premature senescence and, thus, in the maintenance of genome stability. Loss of such NCOA4 function may contribute to thyroid cancer formation.

Iron is essential for deoxyribonucleotides production and for enzymes containing an Fe-S cluster involved in DNA replication and repair. How iron bioavailability and DNA metabolism are coordinated remains poorly understood. NCOA4 protein mediates autophagic degradation of ferritin to maintain iron homeostasis and inhibits DNA replication origin activation via hindrance of the MCM2-7 DNA helicase. Here, we show that iron deficiency inhibits DNA replication, parallel to nuclear NCOA4 stabilization. In iron-depleted cells, NCOA4 knockdown leads to unscheduled DNA synthesis, with replication stress, genome instability, and cell death. In mice, NCOA4 genetic inactivation causes defective intestinal regeneration upon dextran sulfate sodium-mediated injury, with DNA damage, defective cell proliferation, and cell death; in intestinal organoids, this is fostered by iron depletion. In summary, we describe a NCOA4-dependent mechanism that coordinates iron bioavailability and DNA replication. This function prevents replication stress, maintains genome integrity, and sustains high rates of cell proliferation during tissue regeneration.

NCOA4 has been recently identified as a cargo receptor mediating autophagic ferritin degradation. Here, we show that NCOA4 deficiency in a knock out mouse model leads to iron accumulation in liver and spleen, pointing to a systemic alteration of iron homeostasis. Null mice show increased levels of transferrin saturation, serum ferritin and liver hepcidin with decreased duodenal ferroportin. Although NCOA4 null mice display iron overload signs, they suffer from a mild microcytic hypochromic anaemia. Under an iron-deprived diet regimen (2-3 mg/kg), they fail to release iron from ferritin storages and rapidly develop a severe microcytic hypochromic anaemia, with ineffective erythropoiesis associated with increased erythropoietin levels. On the contrary, when fed with an iron-enriched diet (2 gr/kg), knock out mice die prematurely, featuring signs of liver damage. In primary embryonic fibroblasts from NCOA4 null mice, the absence of NCOA4 protein induced accumulation of ferritin impeding its targeting to autophagosomes; the adoptive expression of NCOA4 COOH-terminal portion (aa 239-614) was sufficient to restore this function. All together these data suggest that NCOA4 plays a central role in balancing iron levels in vivo by controlling ferritin turnover to prevent excessive iron accumulation and ensure an efficient erythropoiesis.

PMID:22919068.-- et al., Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer., This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Ministero dell'Università e della Ricerca (MiUR), and by the grant MERIT of MIUR. G Garcia-Rostan is supported by Programa Ramón y Cajal, Ministerio de Ciencia e Innovación, Social EU Funds, Universidad de Valladolid, Spain. C Nucera (Principal Investigator, Program: Human Thyroid Cancers Preclinical and Translational Research) was funded by the NIHR21CA165039-01A1 and the American Thyroid Association for Thyroid Cancer Research.

Here we describe a conditional doxycycline-dependent mouse model of RET/PTC3 (NCOA4-RET) oncogene-induced thyroid tumorigenesis. In these mice, after 10 days of doxycycline (dox) administration, RET/PTC3 expression induced mitogen activated protein kinase (MAPK) stimulation and a proliferative response which resulted in the formation of hyperplastic thyroid lesions. This was followed, after 2 months, by growth arrest accompanied by typical features of oncogene-induced senescence (OIS), including upregulation of p16INK4A and p21CIP, positivity at the Sudan black B, activation of the DNA damage response (DDR) markers γH2AX and pChk2 T68, and induction of p53 and p19ARF. After 5 months, about half of thyroid lesions escaped OIS and formed tumors that remained dependent on RET/ PTC3 expression. This progression was accompanied by activation of AKT-FOXO1/3a pathway and increased serum TSH levels.

RET/papillary thyroid carcinoma (RET/PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase domain with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC stimulates the β-catenin pathway. By stimulating PI3K/AKT and Ras/extracellular signal–regulated kinase (ERK), RET/PTC promotes glycogen synthase kinase 3β (GSK3β) phosphorylation, thereby reducing GSK3β-mediated NH2-terminal β-catenin (Ser33/Ser37/Thr41) phosphorylation. In addition, RET/PTC physically interacts with β-catenin and increases its phosphotyrosine content. The increased free pool of S/T(nonphospho)/Y(phospho)β-catenin is stabilized as a result of the reduced binding affinity for the Axin/GSK3β complex and activates the transcription factor T-cell factor/lymphoid enhancer factor. Moreover, through the ERK pathway, RET/PTC stimulates cyclic AMP–responsive element binding protein (CREB) phosphorylation and promotes the formation of a β-catenin-CREB-CREB-binding protein/p300 transcriptional complex. Transcriptional complexes containing β-catenin are recruited to the cyclin D1 promoter and a cyclin D1 gene promoter reporter is active in RET/PTC–expressing cells. Silencing of β-catenin by small interfering RNA inhibits proliferation of RET/PTC–transformed PC Cl3 thyrocytes, whereas a constitutively active form of β-catenin stimulates autonomous proliferation of thyroid cells. Thus, multiple signaling events downstream from RET/PTC converge on β-catenin to stimulate cell proliferation. [Cancer Res 2009;69(5):1867–76]

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream G?q and G?12/13 GTPases, and consistently, other G?q and G?13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active G?qQL and G?12/13QL mutants stimulated Gli. By using cells null for G?q and G?12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, G?q- and G?12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/G?q-12/13/Rho mediated activation of nuclear factor ?B (NF?B), which can stimulate a NF-?B response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.Oncogene advance online publication, 21 April 2014; doi:10.1038/onc.2014.104.

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.