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Activating transcription factor 3 (ATF3) responds to diverse cellular stresses, and regulates oncogenic activities (for example, proliferation, survival and migration) through direct transcriptional regulation or protein-protein interactions. Although aberrant ATF3 expression is frequently found in human cancers, the role of ATF3 in tumorigenesis is poorly understood. Here, we demonstrate that ATF3 suppresses the development of prostate cancer induced by knockout of the tumor suppressor Pten in mouse prostates. Whereas the oncogenic stress elicited by Pten loss induced ATF3 expression in prostate epithelium, we found that ATF3 deficiency increased cell proliferation and promoted cell survival, leading to early onset of mouse prostatic intraepithelial neoplasia and the progression of prostate lesions to invasive adenocarcinoma. Importantly, the loss of ATF3 promoted activation of the oncogenic AKT signaling evidenced by high levels of phosphorylated AKT and S6 proteins in ATF3-null prostate lesions. In line with these in vivo results, knockdown of ATF3 expression in human prostate cancer cells by single guided RNA-mediated targeting activated AKT and increased matrix metalloproteinase-9 expression. Our results thus link ATF3 to the AKT signaling, and suggest that ATF3 is a tumor suppressor for the major subset of prostate cancers harboring dysfunctional Pten. [ABSTRACT FROM AUTHOR]

Deleted in Breast Cancer 1 (DBC1), a negative regulator of deacetylase SIRT1, has been shown to act as an estrogen receptor α (ER) coactivator that has a key role in ER transcription complex assembly and estrogen-dependent breast cancer cell proliferation. However, little is known about its physiological role and mechanism of action in ER-negative breast cancer cells. Here we report that DBC1 functions as a coactivator for the oncogenic ETS transcription factor PEA3 to promote ER-negative breast cancer progression. DBC1 is required for the expression of PEA3 target genes and for recruitment of PEA3 and RNA polymerase II to PEA3 target promoters. We also demonstrated that acetylation of PEA3 stimulates its DNA binding and association with DBC1 by disrupting the intramolecular interaction of PEA3. The molecular mechanism underlying DBC1 function in PEA3-mediated transcription involves inhibition of SIRT1 interaction with PEA3 and of SIRT1-mediated deacetylation of PEA3. Moreover, DBC1 depletion inhibited the tumorigenic properties of ER-negative breast cancer cells in vitro and in vivo. Importantly, increased DBC1 expression correlated with shorter relapse-free survival of ER-negative breast cancer patients. Our results firmly established DBC1 as a critical coactivator of PEA3 and as a key player in PEA3-mediated breast cancer progression. [ABSTRACT FROM AUTHOR]

c-Myc transcription factor is a key protein involved in cellular growth, proliferation and metabolism. c-Myc is one of the most frequently activated oncogenes, highlighting the need to identify intracellular molecules that interact directly with c-Myc to suppress its function. Here we show that Hhex is able to interact with the basic region/helix-loop-helix/leucine zipper of c-Myc. Knockdown of Hhex increases proliferation rate in hepatocellular carcinoma cells, whereas Hhex expression cell-autonomously reduces cell proliferation rate in multiple cell lines by increasing G1 phase length through a c-Myc-dependent mechanism. Global transcriptomic analysis shows that Hhex counter-regulates multiple c-Myc targets involved in cell proliferation and metabolism. Concomitantly, Hhex expression leads to reduced cell size, lower levels of cellular RNA, downregulation of metabolism-related genes, decreased sensitivity to methotrexate and severe reduction in the ability to form tumours in nude mouse xenografts, all indicative of decreased c-Myc activity. Our data suggest that Hhex is a novel regulator of c-Myc function that limits c-Myc activity in transformed cells. [ABSTRACT FROM AUTHOR]

Basal-like breast tumors are aggressive cancers associated with high proliferation and metastasis. Chemotherapy is currently the only treatment option; however, resistance often occurs resulting in recurrence and patient death. Some extremely aggressive cancers are also associated with hypoxia, inflammation and high leukocyte infiltration. Herein, we discovered that the neural-specific transcription factor, Engrailed 1 (EN1), is exclusively overexpressed in these tumors. Short hairpin RNA (shRNA)-mediated knockdown of EN1 triggered potent and selective cell death. In contrast, ectopic overexpression of EN1 in normal cells activated survival pathways and conferred resistance to chemotherapeutic agents. Exogenous expression of EN1 cDNA reprogrammed the breast epithelial cells toward a long-lived, neural-like phenotype displaying dopaminergic markers. Gene expression microarrays demonstrated that the EN1 cDNA altered transcription of a high number of inflammatory molecules, notably chemokines and chemokine receptors, which could mediate prosurvival pathways. To block EN1 function, we engineered synthetic interference peptides (iPeps) comprising the EN1-specific sequences that mediate essential protein-protein interactions necessary for EN1 function and an N-terminal cell-penetrating peptide/nuclear localization sequence. These EN1-iPeps rapidly mediated a strong apoptotic response in tumor cells overexpressing EN1, with no toxicity to normal or non EN1-expressing cells. Delivery of EN1-iPeps into basal-like cancer cells significantly decreased the fifty percent inhibitory concentrations (IC50) of chemotherapeutic drugs routinely used to treat breast cancer. Lastly, matrix-assisted laser desorption/ionization-time of flight mass spectrometry and immunoprecipitation assays demonstrated that EN1-iPeps captured targets involved in transcriptional and post-transcriptional regulation. Importantly, the EN1-iPeps bound the glutamyl-prolyl tRNA synthetase (EPRS) target, which has been associated with the transcript-specific translational control of inflammatory proteins and activation of amino-acid stress pathways. This work unveils EN1 as an activator of intrinsic inflammatory pathways associated with prosurvival in basal-like breast cancer. We further build upon these results and describe the engineering of iPeps targeting EN1 (EN1-iPeps) as a novel and selective therapeutic strategy to combat these lethal forms of breast cancer. [ABSTRACT FROM AUTHOR]

P63, a p53 family member, is expressed as TA and ΔN isoforms. Interestingly, both TAp63 and ΔNp63 are transcription factors, and regulate both common and distinct sets of target genes. p63 is required for survival of some epithelial cell lineages, and lack of p63 leads to loss of epidermis and other epithelia in humans and mice. Here, we explored the role of p63 isoforms in cell proliferation, migration and tubulogenesis by using Madin-Darby Canine Kidney (MDCK) tubular epithelial cells in two- or three-dimensional (2-D or 3-D) culture. We found that like downregulation of p53, downregulation of p63 and TAp63 decreases expression of growth-suppressing genes, including p21, PUMA and MIC-1, and consequently promotes cell proliferation and migration in 2-D culture. However, in 3-D culture, downregulation of p63, especially TAp63, but not p53, decapacitates MDCK cells to form a cyst structure through enhanced epithelial-to-mesenchymal transition (EMT). In contrast, downregulation of ΔNp63 inhibits MDCK cell proliferation and migration in 2-D culture, and delays but does not block MDCK cell cyst formation and tubulogenesis in 3-D culture. Consistent with this, downregulation of ΔNp63 markedly upregulates growth-suppressing genes, including p21, PUMA and MIC-1. Taken together, these data suggest that TAp63 is the major isoform required for tubulogenesis by maintaining an appropriate level of EMT, whereas ΔNp63 fine-tunes the rate of cyst formation and tubulogenesis by maintaining an appropriate expression level of genes involved in cell cycle arrest and apoptosis. [ABSTRACT FROM AUTHOR]

Caudal-related homeobox 1 (CDX1), an intestinal-specific transcription factor, has been reported to have vital roles in gastric intestinal metaplasia (IM). Although IM is a high-risk factor for gastric cancer (GC), the specific role of CDX1 in GC is largely unknown. In this study, we investigated the expression of CDX1 and its functional roles in GC, and its upstream regulatory mechanisms at the microRNA (miRNA) level were further explored. We found that CDX1 is lost in GC when compared with adjacent IM tissues. Gain-of-function studies showed that CDX1 significantly inhibited GC cell growth by inducing cell cycle arrest and apoptosis. Interestingly, we identified and verified an onco-mir, miR-296-5p, as a direct upstream regulator of CDX1. miR-296-5p overexpression significantly promoted GC cell growth and attenuated the CDX1-induced anti-growth effects by recurring cell cycle distribution and apoptotic status, whereas knockdown of miR-296-5p decreased GC cell growth. Furthermore, we found that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and the subsequent downstream changes in protein levels related to cell cycle and apoptosis partly account for the miR-296-5p-CDX1-induced GC growth promotion. In addition, the detection of miR-296-5p and expression of CDX1 in primary GC tissues and adjacent IM tissues revealed that miR-296-5p is inversely correlated with CDX1, further supporting our in vitro results. Our results showed an anti-growth effect of CDX1 and identified its miRNA regulatory mechanism in GC. The identification of this novel miR-296-5p-CDX1-ERK1/2 axis sheds new light on the understanding of the process from IM to GC and may provide therapeutic targets for the treatment of GC. [ABSTRACT FROM AUTHOR]

Using microRNA (miRNA) expression array, we identified that miR-7 was deregulated in colorectal cancer (CRC). We studied the biological role and molecular target of miR-7 in CRC. miR-7 was downregulated in six out of seven colon cancer cell lines. Ectopic expression of miR-7 suppressed colon cancer cell proliferation (P<0.05), induced apoptosis (P<0.05) and caused cell-cycle arrest in G1 phase (P<0.05). The tumor suppressive function of miR-7 was further confirmed in nude mice (P<0.05). The 3′-untranslated region (3′UTR) of Yin Yang 1 (YY1) mRNA contains an evolutionarily conserved miR-7 binding site using in silico searches, luciferase reporter assay and western blot analysis confirmed that miR-7 directly bound to YY1 3′UTR to negatively regulate the protein expression of YY1 in colon cancer cell lines HCT116 and LOVO. Intriguingly, knock-down of YY1 in three colon cancer cell lines (HCT116, LOVO and DLD1) consistently suppressed cell proliferation (P<0.01) and induced apoptosis (P<0.01), indicating the opposite functions of miR-7 and YY1 in CRC. Consistent with these data, ectopic expression of YY1 promoted cell growth by increasing proliferation (P<0.01) and suppressing apoptosis (P<0.001). The tumorigenic ability of YY1 was further confirmed in vivo in xenograft-nude mouse model (P<0.01). In addition, pathway analyses revealed that the oncogenic effect by YY1 was associated with inhibiting p53 and modulating its downstream effectors p15, caspase cascades and C-Jun, and activating Wnt signaling pathway through activating β-catenin, anti-apoptotic survivin and fibroblast growth factor 4. Furthermore, multivariate analysis revealed that patients with YY1 protein high expression had a significant decrease in overall survival, and Kaplan-Meier survival curves showed that these patients had significantly shorter survival than others (P<0.0001). In conclusion, MiR-7 is a novel miRNA with tumor suppressive function in colon cancer by targeting oncogenic YY1. YY1 promotes colon cancer growth through inhibiting p53 and promoting Wnt signaling pathways and serves as an independent prognostic biomarker for CRC patients. [ABSTRACT FROM AUTHOR]

Involvement of the aryl hydrocarbon receptor (AHR) in carcinogenesis has been suggested in many studies. Upregulation of AHR has been reported in some cancer species, and an association between single-nucleotide polymorphisms (SNPs) of AHR and cancer risk or cancer development has also been reported. This evidence suggests the involvement of some specific SNPs in AHR transcriptional regulation in the process of carcinogenesis or cancer development, but there have been no studies to elucidate the mechanism involved. In this study, we identified the transcription factor Nuclear Factor 1-C (NF1C) as a candidate to regulate AHR transcription in a polymorphism-dependent manner. SNP rs10249788 was included in a consensus binding site for NF1C. Our results suggested that NF1C preferred the C allele to the T allele at rs10249788 for binding. Forced expression of NF1C suppressed the activity of the AHR promoter with C at rs10249788 stronger than that with T. Moreover, expression analysis of human uterine endometrial cancer (HEC) specimens showed greater upregulation of AHR and downregulation of NF1C than those of normal endometrium specimens. Sequence analysis showed HEC patients at advanced stages tended to possess T/T alleles more frequently than healthy women. We also demonstrated that NF1C suppressed proliferation, motility and invasion of HEC cells. This function was at least partially mediated by AHR. This study is the first to report that a polymorphism on the AHR regulatory region affected transcriptional regulation of the AHR gene in vitro. Because NF1C is a tumor suppressor, our new insights into AHR deregulation and its polymorphisms could reveal novel mechanisms of genetic susceptibility to cancer. [ABSTRACT FROM AUTHOR]

The Hedgehog-GLI (HH-GLI) signaling plays a critical role in controlling growth and tissue patterning during embryogenesis and is implicated in a variety of human malignancies, including those of the skin. Phosphorylation events have been shown to regulate the activity of the GLI transcription factors, the final effectors of the HH-GLI signaling pathway. Here, we show that WIP1 (or PPM1D), an oncogenic phosphatase amplified/overexpressed in several types of human cancer, is a positive modulator of the HH signaling. Mechanistically, WIP1 enhances the function of GLI1 by increasing its transcriptional activity, nuclear localization and protein stability, but not of GLI2 nor GLI3. We also find that WIP1 and GLI1 are in a complex. Modulation of the transcriptional activity of GLI1 by WIP1 depends on the latter's phosphatase activity and, remarkably, does not require p53, a known WIP1 target. Functionally, we find that WIP1 is required for melanoma and breast cancer cell proliferation and self-renewal in vitro and melanoma xenograft growth induced by activation of the HH signaling. Pharmacological blockade of the HH pathway with the SMOOTHENED antagonist cyclopamine acts synergistically with inhibition of WIP1 in reducing growth of melanoma and breast cancer cells in vitro. Overall, our data uncover a role for WIP1 in modulating the activity of GLI1 and in sustaining cancer cell growth and cancer stem cell self-renewal induced by activation of the HH pathway. These findings open a novel therapeutic approach for human melanomas and, possibly, other cancer types expressing WIP1 and with activated HH pathway. [ABSTRACT FROM AUTHOR]

KLF6, a ubiquitously expressed Krüppel-like transcription factor, is frequently inactivated in human cancer and has significant roles in cellular proliferation, apoptosis, differentiation and development. A key mechanism of KLF6-mediated growth suppression is through p53-independent transactivation of p21. Several cancer-derived KLF6 mutants lead to the loss of p21-mediated growth suppression through an unknown mechanism. Because several colorectal cancer and hepatocellular carcinoma-derived KLF6 mutations affect a glycogen synthase kinase 3β (GSK3β) phosphorylation consensus site, we investigated the role of GSK3β in the regulation of KLF6 function. Based on transient transfection, GSK3β augments the transactivation of a p21 promoter luciferase by KLF6. Reciprocal co-immunoprecipitation of hemagglutinin (HA)-GSK3β and Flag-KLF6 validated the interaction between these two proteins. KLF6 phosphorylation is augmented in the presence of GSK3β based on in vitro and in vivo 32P incorporation assays. Site-directed mutagenesis of the candidate phosphorylation sites to alanines ('KLF6-4A' phosphomutant) eliminated a higher molecular weight phosphorylated isoform of KLF6 based on western blot. GSK3β augmented the transactivation by wild-type KLF6, but not KLF6-4A, towards the p21 promoter, and increased p21 protein. Functionally, GSK3β enhanced KLF6-mediated growth suppression, which was abrogated by the KLF6-4A phosphomutant. These data establish that GSK3β directly phosphorylates KLF6, which augments its induction of p21 and resultant growth suppression. This interaction may account for the growth-promoting effects of cancer-derived KLF6 mutants that lack tumor suppressor activity. [ABSTRACT FROM AUTHOR]

The transcription factor E2F1 has pivotal roles in both cell proliferation and cell death, and is an important molecular target in cancer. Under proliferative conditions E2F1 induces the expression of genes that promote cell cycle progression, such as E2F2, whereas under proapoptotic conditions E2F1 induces expression of genes such as p73 that lead to apoptosis. The mechanism by which the apoptotic function of E2F1 is activated remains unclear, however. We now show that members of the E2F family are covalently conjugated with the ubiquitin-like modifier NEDD8. Overexpression of SENP8, a NEDD8-specific cysteine protease, resulted in deNEDDylation of E2F1 and promoted its transactivation activity at the p73 gene but not at the E2F2 gene. Knockdown of SENP8, on the other hand, attenuated p73 expression and apoptosis induced by E2F1 or by DNA damage. SENP8 also promoted the interaction between E2F1 and its cofactor Microcephalin 1, which is required for p73 induction. These results suggest that NEDDylation is a molecular trigger that modifies the target specificity of E2F1, and could have important implications for E2F1 regulation of apoptosis. [ABSTRACT FROM AUTHOR]

JunB, an activator protein-1 (AP-1) transcription factor component, acts either as a tumor suppressor or as an oncogene depending on the cell context. In particular, JunB is strongly upregulated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) where it enhances cell proliferation. Although its overexpression is linked to lymphomagenesis, the mechanisms whereby JunB promotes neoplastic growth are still largely obscure. Here, we show that JunB undergoes coordinated phosphorylation-dependent ubiquitylation during the G2 phase of the cell cycle. We characterized a critical consensus phospho-degron that controls JunB turnover and identified GSK3 and SCFFBXW7 as, respectively, the kinase and the E3 ubiquitin ligase responsible for its degradation in G2. Pharmacological or genetic inactivation of the GSK3-FBXW7-JunB axis induced accumulation of JunB in G2/M and entailed transcriptional repression of the DNA helicase DDX11, leading to premature sister chromatid separation. This abnormal phenotype due to dysregulation of the GSK3β/JunB/DDX11 pathway is phenocopied in ALK-positive ALCL. Thus, our results reveal a novel mechanism by which mitosis progression and chromatid cohesion are regulated through GSK3/SCFFBXW7-mediated proteolysis of JunB, and suggest that JunB proteolysis in G2 is an essential step in maintaining genetic fidelity during mitosis. [ABSTRACT FROM AUTHOR]

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and dasatinib. In the CML-derived K562 cell line, low concentrations of imatinib induce proliferative arrest and erythroid differentiation. We found that imatinib upregulated the cell cycle inhibitor p27KIP1 (p27) in a time- and -concentration dependent manner, and that the extent of imatinib-mediated differentiation was severely decreased in cells with depleted p27. MYC (c-Myc) is a transcription factor frequently deregulated in human cancer. MYC is overexpressed in untreated CML and is associated to poor response to imatinib. Using K562 sublines with conditional MYC expression (induced by Zn2+ or activated by 4-hydroxy-tamoxifen) we show that MYC prevented the erythroid differentiation induced by imatinib and dasatinib. The differentiation inhibition is not due to increased proliferation of MYC-expressing clones or enhanced apoptosis of differentiated cells. As p27 overexpression is reported to induce erythroid differentiation in K562, we explored the effect of MYC on imatinib-dependent induction of p27. We show that MYC abrogated the imatinib-induced upregulation of p27 concomitantly with the differentiation inhibition, suggesting that MYC inhibits differentiation by antagonizing the imatinib-mediated upregulation of p27. This effect occurs mainly by p27 protein destabilization. This was in part due to MYC-dependent induction of SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation. [ABSTRACT FROM AUTHOR]

The RE1 silencing transcription factor (REST) is a repressor of neuronal differentiation and its elevated expression in neural cells blocks neuronal differentiation. In this study, we demonstrate a role for REST in the control of proliferation of medulloblastoma cells. REST expression decreased the levels of cyclin-dependent kinase (CDK)NIB/p27, a CDK inhibitor and a brake of cell proliferation in these cells. The reciprocal relationship between REST and p27 was validated in human tumor samples. REST knockdown in medulloblastoma cells derepessed a novel REST target gene encoding the deubiquitylase ubiquitin (Ub)-specific peptidase 37 (USP37). Ectopically expressed wild-type USP37 formed a complex with p27, promoted its deubiquitination and stabilization and blocked cell proliferation. Knockdown of REST and USP37 prevented p27 stabilization and blocked the diminution in proliferative potential that normally accompanied REST loss. Unexpectedly, wild-type USP37 expression also induced the expression of REST-target neuronal differentiation genes even though REST levels were unaffected. In contrast, a mutant of USP37 carrying a site-directed change in a conserved cysteine failed to rescue REST-mediated p27 destabilization, maintenance of cell proliferation and blockade to neuronal differentiation. Consistent with these findings, a significant correlation between USP37 and p27 was observed in patient tumors. Collectively, these findings provide a novel connection between REST and the proteasomal machinery in the control of p27 and cell proliferation in medulloblastoma cells. [ABSTRACT FROM AUTHOR]

Macrophages have a key role in tumor-associated pulmonary inflammation that supports the proliferation of tumor cells and promotes lung tumor growth. Although increased numbers of tumor-associated macrophages are linked to poor prognosis in lung cancer patients, little is known regarding the transcriptional mechanisms control-ling recruitment of macrophages during lung tumorigen-esis. Forkhead Box ml (Foxml) transcription factor is induced in multiple cell types within tumor lesions and its increased expression is associated with poor prognosis in patients with lung adenocarcinomas. To determine the role of Foxml in recruitment of tumor-associated macro-phages, a mouse line with macrophage-specific Foxml deletion was generated (macFoxm1-/-). Lung tumorigen-esis was induced using a 3-methylcholanthrene/butylated hydroxytoluene (BHT; 3,5-di-t-butyl-4-hydroxytoluene) tumor initiation/promotion protocol. Ablation of Foxml in macrophages reduced the number and size of lung tumors in macFoxm1-/- mice. Decreased tumorigenesis was associated with diminished proliferation of tumor cells and decreased recruitment of macrophages during the early stages of tumor formation. The expression levels of the pro-inflammatory genes iNOS, Cox-2, interleukin-lb (IL-lb) and IL-6, as well as the migration-related genes macrophage inflammatory protein-1 (MIP-1α), MIP-2 and MMP-12, were decreased in macrophages isolated from macFoxm1-/- mice. Migration of Foxml-deficient macrophages was reduced in vitro. The chemokine receptors responsible for monocyte recruitment to the lung, CX3CR1 and CXCR4, were decreased in Foxml -deficient monocytes. In co-transfection experiments, Foxml directly bound to and transcriptionally activated the CX3CR1 promoter. Adoptive transfer of wild-type monocytes to macFoxm1-/- mice restored BHT-induced pulmonary inflammation to the levels observed in control mice. Expression of Foxml in macrophages is required for pulmonary inflammation, recruitment of macrophages into tumor sites and lung tumor growth. [ABSTRACT FROM AUTHOR]

MafB, a member of the large Maf transcription factor family, is essential for the embryonic and terminal differentiation of pancreatic α- and β-cells. However, the role of MafB in the control of adult islet-cell proliferation remains unknown. Considering its oncogenic potential in several other tissues, we investigated the possible alteration of its expression in adult mouse β-cells under different conditions of proliferation. We found that MafB, in general silenced in these cells, was reexpressed in ∼30% of adaptive β-cells both in gestational female mice and in mice fed with a high-fat diet. Importantly, reactivated MafB expression was also observed in the early β-cell lesions and insulinomas that developed in β-cell specific Men1 mutant mice, appearing in >80% of β-cells in hyperplasic or dysplastic islets from the mutant mice >4 months of age. Moreover, MafB expression could be induced by glucose stimulation in INS-1 rat insulinoma cells. The induction was further reinforced following Men1 knockdown by siRNA. Furthermore, MafB overexpression in cultured βTC3 cells enhanced cell foci formation both in culture medium and on soft agar, accompanied with the increased expression of Cyclin B1 and D2. Conversely, MafB downregulation by siRNA transfection reduced BrdU incorporation in INS-1E cells. Taken together, our data reveal that Men1 inactivation leads to MafB reexpression in mouse β-cells in vivo, and provides evidence that deregulated ectopic MafB expression may have a hitherto unknown role in adult β-cell proliferation and Men1-related tumorigenesis. [ABSTRACT FROM AUTHOR]

The NF-κB transcription factor has a central role in diverse processes, including inflammation, proliferation and cell survival, and its activity is dysregulated in diseases such as autoimmunity and cancer. We recently identified the TRE17/ubiquitin-specific protease 6 (USP6) oncogene as the first de-ubiquitinating enzyme to activate NF-κB. TRE17/USP6 is translocated and overexpressed in aneurysmal bone cyst (ABC), a pediatric tumor characterized by extensive bone degradation and inflammatory recruitment. In the current study, we explore the mechanism by which TRE17 induces activation of NF-κB, and find that it activates the classical NF-κB pathway through an atypical mechanism that does not involve IκB degradation. TRE17 co-precipitates with IκB kinase (IKK), and IKK activity is augmented in stable cell lines overexpressing TRE17, in a USP-dependent manner. Optimal activation of NF-κB by TRE17 requires both catalytic subunits of IKK, distinguishing its mechanism from the classical and non-canonical pathways, which require either IKKβ or IKKα, respectively. TRE17 stimulates phosphorylation of p65 at serine 536, a modification that has been associated with enhanced transcriptional activity and nuclear retention. Induction of S536 phosphorylation by TRE17 requires both IKKα and IKKβ, as well as the IKKγ/NEMO regulatory subunit of IKK. We further demonstrate that TRE17(long) is highly tumorigenic when overexpressed in NIH3T3 fibroblasts, and that inhibition of NF-κB significantly attenuates tumor formation. In summary, these studies uncover an unexpected signaling mechanism for activation of classical NF-κB by TRE17. They further reveal a critical role for NF-κB in TRE17-mediated tumorigenesis, and suggest that NF-κB inhibitors may function as effective therapeutic agents in the treatment of ABC. [ABSTRACT FROM AUTHOR]

Malignant glioma is the most common brain tumor with 16 000 new cases diagnosed annually in the United States. We performed a systematic large-scale transcriptomics data mining study of 9783 tissue samples from the GeneSapiens database to systematically identify genes that are most glioma-specific. We searched for genes that were highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared with 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (hairy and enhancer of split 6) emerged as the most glioma-specific gene. Immunostaining of a tissue microarray showed HES6 expression in 335 (98.8%) out of the 339 glioma samples. HES6 was expressed in endothelial cells of the normal brain and glioma tissue. Recurrent grade 2 astrocytomas and grade 2 or 3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. High HES6 mRNA expression correlated with the proneural subtype that generally has a favorable outcome but is prone to recur. Functional studies suggested an important role for HES6 in supporting survival of glioma cells, as evidenced by reduction of cancer cell proliferation and migration after HES6 silencing. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which implicated a role for HES6 in p53, c-myc and nuclear factor-κB transcriptional networks. We conclude that HES6 is important for glioma cell proliferation and migration, and may have a role in angiogenesis. [ABSTRACT FROM AUTHOR]

Expression of the breast cancer-associated gene 1 (BRCA1) in sporadic breast cancers is usually reduced, yet the underlying mechanisms remains elusive. To identify factors that are responsible for reduced BRCA1 expression, we screened 92 known transcription factors for their ability to regulate expression of BRCA1. Among several potential regulators, the Gli-Krueppel-related transcription factor Yin Yang 1 (YY1) showed the most dramatic transactivation of the BRCA1 promoter. YY1 binds to the promoter of BRCA1, and its overexpression resulted in increased expression of BRCA1 and a number of BRCA1 downstream genes. We further showed that overexpression of YY1 in cancer cells inhibited cell proliferation, foci formation and tumor growth in nude mice. To assess the clinical relevance between YY1 and BRCA1, we studied expression of YY1 and BRCA1 from human breast cancer samples and tissue arrays, and detected a significant positive correlation between the level of YY1 and BRCA1 expression in these cancers. Taken together, these findings suggest that YY1 is a key regulator of BRCA1 expression and may be causally linked to the molecular etiology of human breast cancer. [ABSTRACT FROM AUTHOR]

Breast cancer is genetically and clinically heterogeneous. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is usually associated with poor outcome and lack of benefit from targeted therapy. We used microarray analysis to perform a pathway analysis of TNBC compared with non-triple negative breast cancer (non-TNBC). Overexpression of several Wnt pathway genes, such as frizzled homolog 7 (FZD7), low density lipoprotein receptor-related protein 6 and transcription factor 7 (TCF7) was observed in TNBC, and we directed our focus to the Wnt pathway receptor, FZD7. To validate the function of FZD7, FZD7shRNA was used to knock down FZD7 expression. Notably, reduced cell proliferation and suppressed invasiveness and colony formation were observed in TNBC MDA-MB-231 and BT-20 cells. Study of the possible mechanism indicated that these effects occurred through silencing of the canonical Wnt signaling pathway, as evidenced by loss of nuclear accumulation of β-catenin and decreased transcriptional activity of TCF7. In vivo studies revealed that FZD7shRNA significantly suppressed tumor formation, through reduced cell proliferation, in mice bearing xenografts without FZD7 expression. Our findings suggest that FZD7-involved canonical Wnt signaling pathway is essential for tumorigenesis of TNBC, and thus, FZD7 shows promise as a biomarker and a potential therapeutic target for TNBC. [ABSTRACT FROM AUTHOR]

Deregulation of the pRb/E2F or Wnt/β-catenin pathway occurs frequently in human cancers, which is often associated with inappropriate cell proliferation. Although the oncogenic roles of pRb/E2F1 and Wnt/β-catenin pathways have been well studied, the functional interaction between the two pathways has only recently been characterized. In particular, E2F1 has been recently reported to negatively regulate Wnt/β-catenin activity in human colorectal cancers, though the mechanism underlying this regulation is not fully understood. Here we provide evidence that β-catenin interacting protein 1 (CTNNBIP1), also known as ICAT (inhibitor of β-catenin and TCF4), functions as a crucial node to mediate the cross talk between E2F1 and β-catenin signaling. We show that ICAT is a direct transcriptional target of E2F1, and that activation of ICAT by E2F1 is required for E2F1 to inhibit β-catenin activity. This study provides a mechanistic insight into the antagonistic interaction between E2F1 and β-catenin signaling. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) α factor (LITAF) is a multiple functional molecule whose sequence is identical to the small integral membrane protein of the lysosome/late endosome. LITAF was initially identified as a transcription factor that activates transcription of proinflammatory cytokine in macrophages in response to LPS. Mutations of the LITAF gene are associated with a genetic disease, called Charcot-Marie-Tooth syndrome. Recently, we have reported that mRNA levels of LITAF and TNF superfamily member 15 (TNFSF15) are upregulated by 5′ adenosine monophosphate (AMP)-activated protein kinase (AMPK). The present study further assesses their biological functions. Thus, we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a pharmacological activator of AMPK, increases the abundance of LITAF and TNFSF15 in LNCaP and C4-2 prostate cancer cells, which is abrogated by small hairpin RNA (shRNA) or the dominant-negative mutant of AMPK α1 subunit. Our data further demonstrate that AMPK activation upregulates the transcription of LITAF. Intriguingly, silencing LITAF by shRNA enhances proliferation, anchorage-independent growth of these cancer cells and tumor growth in the xenograft model. In addition, our study reveals that LITAF mediates the effect of AMPK by binding to a specific sequence in the promoter region. Furthermore, we show that TNFSF15 remarkably inhibits the growth of prostate cancer cells and bovine aortic endothelial cells in vitro, with a more potent effect toward the latter. In conjuncture, intratumoral injection of TNFSF15 significantly reduces the size of tumors and number of blood vessels and induces changes that are characteristic of tumor cell differentiation. Therefore, our studies for the first time establish the regulatory axis of AMPK-LITAF-TNFSF15 and also suggest that LITAF may function as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dubé syndrome. For this, we analysed a Birt-Hogg-Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dubé-associated renal lesions. [ABSTRACT FROM AUTHOR]

The Krüppel-like zinc-finger protein GLI1 functions as a downstream transcription factor of Hedgehog signaling and plays a pivotal role in the cellular proliferation of many types of tumors, including pancreatic ductal adenocarcinoma (PDA). PDA develops from dysplastic lesions called pancreatic intraepithelial neoplasia (PanIN) through a multistep carcinogenesis process that changes its cellular characteristics, including a mucin expression profile. Increased expression of a gel-forming mucin, MUC5AC, was previously revealed as a major biomarker for the poor prognosis of PDA patients, but the molecular mechanisms responsible for its expression and correlation with poor prognosis are not fully understood. Here we show that MUC5AC is a direct transcriptional target of GLI1 in PDA cells. Overexpression of GLI1 enhanced MUC5AC expression, and a double knockdown of GLI1 and GLI2 suppressed endogenous MUC5AC expression in PDA cells. Luciferase reporter assays revealed that GLI1 and GLI2 can activate the MUC5AC promoter through its conserved CACCC-box-like cis-regulatory elements. We also found that GLI1-upregulated MUC5AC was expressed in the intercellular junction between cultured PDA cells and interfered with the membrane localization of E-cadherin, leading to decreased E-cadherin-dependent cell-cell adhesion and promoting the migration and invasion of PDA cells. Consistently, GLI1 induced the nuclear accumulation and target gene expression of β-catenin in a MUC5AC-dependent manner. Finally, immunohistochemical analysis revealed that GLI1 expression statistically correlated with MUC5AC expression and also with altered subcellular localization of E-cadherin and β-catenin in PanIN lesions and PDA. This evidence revealed a new aspect of GLI1 function in modulating E-cadherin/β-catenin-regulated cancer cell properties through the expression of a gel-forming mucin. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway is an ubiquitous cell-cell interaction mechanism, which is essential in controlling processes like cell proliferation, cell fate decision, differentiation or stem cell maintenance. Recent data have shown that Notch signaling is RBP-Jκ-dependent in melanocytes, being required for survival of these pigment cells that are responsible for coloration of the skin and hairs in mammals. In addition, Notch is believed to function as an oncogene in melanoma, whereas it is a tumor suppressor in mouse epidermis. In this study, we addressed the implication of the Notch signaling in the development of another population of pigment cells forming the retinal pigment epithelium (RPE) in mammalian eyes. The constitutive activity of Notch in Tyrp1::NotchIC/° transgenic mice enhanced RPE cell proliferation, and the resulting RPE-derived pigmented tumor severely affected the overall eye structure. This RPE cell proliferation is dependent on the presence of the transcription factor RBP-Jκ, as it is rescued in mice lacking RBP-Jκ in the RPE. In conclusion, Notch signaling in the RPE uses the canonical pathway, which is dependent on the transcription factor RBP-Jκ. In addition, it is of importance for RPE development, and constitutive Notch activity leads to hyperproliferation and benign tumors of these pigment cells. [ABSTRACT FROM AUTHOR]

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V267NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check. [ABSTRACT FROM AUTHOR]

The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is a negative regulator of gene expression restricting the expression of neuronal genes to the nervous system. NRSF/REST is highly expressed in non-neuronal tissues like the lung. In previous work, we identified small-cell lung cancer (SCLC) cell lines with no detectable NRSF/REST expression that, as a consequence, expressed neuronal markers like L1-cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (NCAM). The loss of NRSF/REST expression was linked to malignant progression; however, its mechanistic role remained elusive. Here, we show that NRSF/REST itself, rather than one of its regulated genes, acts like a classic tumour suppressor, being in part regulated by methylation. In SCLCs, NRSF/REST is positively regulated by CREB, with an NRSF/REST promoter fragment showing cell type specificity. Downstream, NRSF/REST directly regulates AKT2, in which NRSF/REST loss leads to an epidermal growth factor-mediated de-regulation of AKT-Serine473 phosphorylation, important for cellular proliferation and survival. Assaying anchorage-independent growth, we observed that with reduced NRSF/REST expression, proliferation was significantly enhanced, whereas NRSF/REST rescue decreased the potential of cells to grow anchorage independently. Our observations support the fact that NRSF/REST may act as an important modulator of malignant progression in SCLC. [ABSTRACT FROM AUTHOR]

Zinc-finger (ZF) transcriptional repressor growth factor independence 1 (Gfi-1) has an important role in hematopoiesis and inner ear development, and also functions as an oncoprotein that cooperates with c-Myc in lymphomagenesis. Gfi-1 represses transcription by directly binding to conserved sequences in the promoters of its target genes. CDKN1A encoding p21Cip1 has been identified as a Gfi-1 target gene and has been shown to contain Gfi-1 binding sites in the upstream promoter region. We show here that Gfi-1 represses CDKN1A in a manner that is independent of its DNA binding activity. Gfi-1 interacts with POZ-ZF transcription factor Miz-1, originally shown to be a c-Myc-interacting partner, and through Miz-1 binds to the CDKN1A core promoter. Interestingly, Gfi-1 and c-Myc, through Miz-1, form a ternary complex on the CDKN1A promoter, and function in collaboration to repress CDKN1A. Gfi-1 knockdown results in enhanced levels of p21Cip1 and attenuated cell proliferation. Notably, similar to c-Myc, the expression of Gfi-1 is downregulated by transforming growth factor-β (TGFβ) and the level of Gfi-1 influences the response of cells to the cytostatic effect of TGFβ. Our data reveal an important mechanism by which Gfi-1 regulates cell proliferation and may also have implications for understanding the role of Gfi-1 in lymphomagenesis. [ABSTRACT FROM AUTHOR]

The adenomatous polyposis coli (APC) gene product is mutated in the vast majority of human colorectal cancers. APC negatively regulates the WNT pathway by aiding in the degradation of β-catenin, which is the transcription factor activated downstream of WNT signaling. APC mutations result in β-catenin stabilization and constitutive WNT pathway activation, leading to aberrant cellular proliferation. APC mutations associated with colorectal cancer commonly fall in a region of the gene termed the mutation cluster region and result in expression of an N-terminal fragment of the APC protein. Biochemical and molecular studies have revealed localization of APC/Apc to different sub-cellular compartments and various proteins outside of the WNT pathway that associate with truncated APC/Apc. These observations and genotype–phenotype correlations have led to the suggestion that truncated APC bears neomorphic and/or dominant-negative function that support tumor development. To analyze this possibility, we have generated a novel allele of Apc in the mouse that yields complete loss of Apc protein. Our studies reveal that whole-gene deletion of Apc results in more rapid tumor development than the APC multiple intestinal neoplasia (ApcMin) truncation. Furthermore, we found that adenomas bearing truncated Apc had increased β-catenin activity when compared with tumors lacking Apc protein, which could lead to context-dependent inhibition of tumorigenesis. [ABSTRACT FROM AUTHOR]

Nuclear expression of CCAAT enhancer binding protein-α (C/EBPα), which supports tissue differentiation through several antiproliferative protein–protein interactions, augurs terminal differentiation of prostate epithelial cells. C/EBPα is also a tumor suppressor, but in many tumors its antiproliferative interactions may be attenuated by de-phosphorylation. C/EBPα acts as a corepressor of the classical androgen response element (ARE)-mediated gene activation by the androgen receptor (AR), but this is paradoxical as the genotropic actions of AR are crucial not only for the growth of the prostate but also for its maintenance and function. We show that DNA-bound C/EPBα recruits AR to activate transcription. C/EBPα-dependent trans-activation by AR also overrode suppression of AREs by C/EBPα elsewhere in a promoter. This mechanism was remarkable in that its androgen dependence was apparently for nuclear translocation of AR; it was otherwise androgen independent, flutamide insensitive and tolerant to disruption of AR dimerization. Gene response profiles and global chromatin associations in situ supported the direct bimodal regulation of AR transcriptional signaling by C/EBPα. This unique mechanism explains the functional coordination between AR and C/EPBα in the prostate and also shows that hormone-refractory AR signaling in prostate cancer could occur through receptor tethering. [ABSTRACT FROM AUTHOR]

The nerve growth factor (NGF) receptor, trkA, the tumour suppressor p53 and the phosphatase SHP-1 are critical in cell proliferation and differentiation. SHP-1 is a trkA phosphatase that dephosphorylates trkA at tyrosines (Y) 674 and 675. p53 can induce trkA activation and tyrosine phosphorylation in the absence of NGF stimulation. In breast cancer tumours trkA expression is associated with increased patient survival. TrkA protein expression is higher in breast-cancer cell lines than in normal breast epithelia. In cell lines (but not in normal breast epithelia) trkA is functional and can be NGF-stimulated to promote cell proliferation. This study investigates the functional relationship between trkA, p53 and SHP-1 in breast-cancer, and reveals that in wild-type (wt) trkA expressing breast-cancer cells both endogenous wtp53, activated by therapeutic agents, and transfected wtp53 repress expression of SHP-1 through the proximal CCAAT sequence of the SHP-1-P1-promoter and the transcription factor NF-Y. In these cells trkA-Y674/Y675 phosphorylation is detected when SHP-1 protein levels decrease in a wtp53-dependent manner. Proliferation and cell-cycle assays, with cells expressing endogenous or transfected wt-trkA and a temperature-sensitive p53 grown at 32 °C (when p53 is in the wt configuration), show suppressed cell proliferation. Suppression is not detected when grown at 37 °C (when p53 is in the mutant configuration). A release from suppression is observed when these cells are transiently transfected with wt-SHP-1 and grown at 32 °C. Suppression is also detected when, as control, wt-trkA-expressing cells are transiently transfected with SHP-1-siRNA, but not when a dominant-negative (DN) mutant trkA is used to abolish wt-trkA activity. Importantly, suppression is not seen with control trkA-negative breast-cancer cells (expressing wtp53, wt-SHP-1 and undetectable trkA), transfected with Y674F/Y675F mutant-trkA. BrdU-incorporation experiments reveal lack of incorporation in cells expressing wt-trkA and wtp53, or wt-trkA and SHP-1-siRNA. However, BrdU is incorporated in the presence of Y674F/Y675F mutant trkA or DN mutant trkA. These results indicate that p53 repression of SHP-1 expression leads to trkA-Y674/Y675 phosphorylation and trkA-dependent suppression of breast-cancer cell proliferation. These data provide an explanation as to why high trkA levels are associated with favourable prognosis. [ABSTRACT FROM AUTHOR]

ErbB2/HER2/Neu-overexpressing breast cancers are characterized by poor survival due to high proliferation and metastasis rates and identifying downstream targets of ErbB2 should facilitate developing novel therapies for this disease. Gene expression profiling revealed the transcriptional regulator LIM-only protein 4 (LMO4) is upregulated during ErbB2-induced mouse mammary gland tumorigenesis. Although LMO4 is frequently overexpressed in breast cancer and LMO4-overexpressing mice develop mammary epithelial tumors, the mechanisms involved are unknown. In this study, we report that LMO4 is a downstream target of ErbB2 and PI3K in ErbB2-dependent breast cancer cells. Furthermore, LMO4 silencing reduces proliferation of these cells, inducing a G2/M arrest that was associated with decreased cullin-3, an E3-ubiquitin ligase component important for mitosis. Loss of LMO4 subsequently results in reduced Cyclin D1 and Cyclin E. Further supporting a role for LMO4 in modulating proliferation by regulating cullin-3 expression, we found that LMO4 expression oscillates throughout the cell cycle with maximum expression occurring during G2/M and these changes precede oscillations in cullin-3 levels. LMO4 levels are also highest in high-grade/less differentiated breast cancers, which are characteristically highly proliferative. We conclude that LMO4 is a novel cell cycle regulator with a key role in mediating ErbB2-induced proliferation, a hallmark of ErbB2-positive disease. [ABSTRACT FROM AUTHOR]

Sustained Hedgehog (HH) signaling is implicated in basal cell carcinoma of the skin and other types of cancer. Here we show that GLI1 and GLI2, the main transcriptional activators of the HH pathway, directly regulate expression of the activator protein 1 (AP-1) family member JUN, a transcription factor controlling keratinocyte proliferation and skin homeostasis. Activation of the JUN promoter by GLI is dependent on a GLI-binding site and the AP-1 sites known to be involved in self-activation of JUN. Transcription of JUN is greatly enhanced in the presence of GLI and requires activated JUN protein. GLI2act is a more potent activator than GLI1 in these experiments and physical interaction with phosphorylated JUN was only detected for GLI2act. The synergistic effect of GLI and JUN extends to the activation of further GLI target genes as shown by shRNA-mediated knockdown of JUN in human keratinocytes. Some of these cooperatively activated genes are involved in cell-cycle progression, which is consistent with a significant reduction of the proliferative potential of GLI in the absence of JUN. These results suggest a novel connection between HH/GLI pathway activity and JUN, which may contribute to the oncogenic activity of HH/GLI signaling in skin.Oncogene (2009) 28, 1639–1651; doi:10.1038/onc.2009.10; published online 16 February 2009 [ABSTRACT FROM AUTHOR]

A defining feature of basal-like breast cancer, a breast cancer subtype with poor clinical prognosis, is the high expression of ‘proliferation signature’ genes. We identified B-Myb, a MYB family transcription factor that is often amplified and overexpressed in many tumor types, as being highly expressed in the proliferation signature. However, the roles of B-Myb in disease progression, and its mammary-specific transcriptional targets, are poorly understood. Here, we showed that B-Myb expression is a significant predictor of survival and pathological complete response to neoadjuvant chemotherapy in breast cancer patients. We also identified a significant association between the G/G genotype of a nonsynonymous B-Myb germline variant (rs2070235, S427G) and an increased risk of basal-like breast cancer [OR 2.0, 95% CI (1.1–3.8)]. In immortalized, human mammary epithelial cell lines, but not in basal-like tumor lines, cells ectopically expressing wild-type B-Myb or the S427G variant showed increased sensitivity to two DNA topoisomerase IIα inhibitors, but not to other chemotherapeutics. In addition, microarray analyses identified many G2/M genes as being induced in B-Myb overexpressing cells. These results confirm that B-Myb is involved in cell cycle control, and that its dysregulation may contribute to increased sensitivity to a specific class of chemotherapeutic agents. These data provide insight into the influence of B-Myb in human breast cancer, which is of potential clinical importance for determining disease risk and for guiding treatment.Oncogene (2009) 28, 742–751; doi:10.1038/onc.2008.430; published online 1 December 2008 [ABSTRACT FROM AUTHOR]

The E2f transcription factors are key downstream targets of the retinoblastoma protein tumor suppressor that control cell proliferation. E2F3 has garnered particular attention because it is amplified in various human tumors. E2f3 mutant mice typically die around birth and E2f3-deficient cells have a proliferation defect that correlates with impaired E2f target gene activation and also induction of p19Arf and p53. The E2f3 locus encodes two isoforms, E2f3a and E2f3b, which differ in their N-termini. However, it is unclear how E2f3a versus E2f3b contributes to E2f3's requirement in either proliferation or development. To address this, we use E2f3a- and E2f3b-specific knockouts. We show that inactivation of E2f3a results in a low penetrance proliferation defect in vitro whereas loss of E2f3b has no effect. This proliferation defect appears insufficient to disrupt normal development as E2f3a and E2f3b mutant mice are both fully viable and have no detectable defects. However, when combined with E2f1 mutation, inactivation of E2f3a, but not E2f3b, causes significant proliferation defects in vitro, neonatal lethality and also a striking cartilage defect. Thus, we conclude that E2f3a and E2f3b have largely overlapping functions in vivo and that E2f3a can fully substitute for E2f1 and E2f3 in most murine tissues.Oncogene (2008) 27, 6561–6570; doi:10.1038/onc.2008.253; published online 28 July 2008 [ABSTRACT FROM AUTHOR]

The forkhead box m1 (Foxm1 or Foxm1b) protein (previously called HFH-11B, Trident, Win or MPP2) is abundantly expressed in human non-small cell lung cancers where it transcriptionally induces expression of genes essential for proliferation of tumor cells. In this study, we used Rosa26-Foxm1 transgenic mice, in which the Rosa26 promoter drives ubiquitous expression of Foxm1 transgene, to identify new signaling pathways regulated by Foxm1. Lung tumors were induced in Rosa26-Foxm1 mice using the 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT) lung tumor initiation/promotion protocol. Tumors from MCA/BHT-treated Rosa26-Foxm1 mice displayed a significant increase in the number, size and DNA replication compared to wild-type mice. Elevated tumor formation in Rosa26-Foxm1 transgenic lungs was associated with persistent pulmonary inflammation, macrophage infiltration and increased expression of cyclooxygenase-2 (Cox-2), Cdc25C phosphatase, cyclin E2, chemokine ligands CXCL5, CXCL1 and CCL3, cathepsins and matrix metalloprotease-12. Cell culture experiments with A549 human lung adenocarcinoma cells demonstrated that depletion of Foxm1 by either short interfering RNA transfection or treatment with Foxm1-inhibiting ARF 26-44 peptide significantly reduced Cox-2 expression. In co-transfection experiments, Foxm1 protein-induced Cox-2 promoter activity and directly bound to the −2566/−2580 bp region of human Cox-2 promoter.Oncogene (2008) 27, 4137–4149; doi:10.1038/onc.2008.60; published online 17 March 2008 [ABSTRACT FROM AUTHOR]

Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Primary ATL cells frequently express CCR4 at high levels. Since HTLV-1 Tax does not induce CCR4 expression, transcription factor(s) constitutively active in ATL may be responsible for its strong expression. We identified an activator protein-1 (AP-1) site in the CCR4 promoter as the major positive regulatory element in ATL cells. Among the AP-1 family members, Fra-2, JunB and JunD are highly expressed in fresh primary ATL cells. Consistently, the Fra-2/JunB and Fra-2/JunD heterodimers strongly activated the CCR4 promoter in Jurkat cells. Furthermore, Fra-2 small interfering RNA (siRNA) or JunD siRNA, but not JunB siRNA, effectively reduced CCR4 expression and cell growth in ATL cells. Conversely, Fra-2 or JunD overexpression promoted cell growth in Jurkat cells. We identified 49 genes, including c-Myb, BCL-6 and MDM2, which were downregulated by Fra-2 siRNA in ATL cells. c-Myb, BCL-6 and MDM2 were also downregulated by JunD siRNA. As Fra-2, these proto-oncogenes were highly expressed in primary ATL cells but not in normal CD4+ T cells. Collectively, aberrantly expressed Fra-2 in association with JunD may play a major role in CCR4 expression and oncogenesis in ATL.Oncogene (2008) 27, 3221–3232; doi:10.1038/sj.onc.1210984; published online 10 December 2007 [ABSTRACT FROM AUTHOR]

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation and survival in immune responses, hematopoiesis, neurogenesis and other biological processes. For example, STAT3 has been reported to be constitutively activated in numerous cancer cells. To clarify the molecular mechanisms underlying the STAT activation, we performed yeast two-hybrid screening and identified KAP1/TIF1β as a novel STAT-binding partner. KAP1 is a universal corepressor protein for the Kruppel-associated box zinc-finger protein superfamily of transcriptional repressors. We found endogenous KAP1 associated with endogenous STAT3 in vivo. Importantly, small-interfering RNA-mediated reduction of KAP1 expression enhanced interleukin (IL)-6-induced STAT3-dependent transcription and gene expression. Furthermore, reduction of KAP1 expression resulted in the marked accumulation of STAT3 phosphorylated on Ser727 in the nucleus, a modification that regulates its transcriptional activation. These results indicate that KAP1 may serve as a transcriptional regulator of the IL-6/STAT3 signaling pathway.Oncogene (2008) 27, 3054–3059; doi:10.1038/sj.onc.1210952; published online 26 November 2007 [ABSTRACT FROM AUTHOR]

Early growth response 3 (Egr3) is a member of a zinc-finger transcription factor subfamily, which we previously found to be strongly upregulated by vascular endothelial growth factor (VEGF)-A in an oligonucleotide microarray screen of endothelial cells. Here, we show that Egr3 is the predominant Egr family member upregulated by VEGF in endothelial cells at 45 min, and that VEGF induced a rapid increase in Egr-dependent transcriptional activation mediated via its major signalling receptor, VEGFR2/KDR, and the protein kinase C (PKC) pathway. VEGF-induced Egr3 gene expression was also mediated in part via a PKC-dependent activation of protein kinase D. Inhibition of Egr3 gene expression by RNA interference was effective in inhibiting basal and VEGF-induced Egr3 gene expression, and it also inhibited VEGF-mediated endothelial cell proliferation, migration and tubulogenesis. These findings indicate that Egr3 has an essential downstream role in VEGF-mediated endothelial functions leading to angiogenesis and may have particular relevance for adult angiogenic processes involved in vascular repair and neovascular disease.Oncogene (2008) 27, 2989–2998; doi:10.1038/sj.onc.1210959; published online 3 December 2007 [ABSTRACT FROM AUTHOR]

The FOXO subgroup of forkhead transcription factors plays a central role in cell-cycle control, differentiation, metabolism control, stress response and apoptosis. Therefore, the function of these important molecules is tightly controlled by a wide range of protein–protein interactions and posttranslational modifications including phosphorylation, acetylation and ubiquitination. The mechanisms by which these processes regulate FOXO activity are mostly elusive. This review focuses on recent advances in structural studies of forkhead transcription factors and the insights they provide into the mechanism of DNA recognition. On the basis of these data, we discuss structural aspects of protein–protein interactions and posttranslational modifications that target the forkhead domain and the nuclear localization signal of FOXO proteins.Oncogene (2008) 27, 2263–2275; doi:10.1038/onc.2008.20 [ABSTRACT FROM AUTHOR]

The FoxO family of Forkhead transcription factors plays an important role in longevity and tumor suppression by upregulating target genes involved in stress resistance, metabolism, cell cycle arrest and apoptosis. FoxO transcription factors translate a variety of environmental stimuli, including insulin, growth factors, nutrients and oxidative stress, into specific gene-expression programs. These environmental stimuli control FoxO activity primarily by regulating their subcellular localization, but also by affecting their protein levels, DNA-binding properties and transcriptional activity. The precise regulation of FoxO transcription factors is enacted by an intricate combination of post-translational modifications (PTMs), including phosphorylation, acetylation and ubiquitination, and binding protein partners. An intriguing possibility is that FoxO PTMs may act as a ‘molecular FoxO code’ read by selective protein partners to rapidly regulate gene-expression programs. The effective control of FoxO activity in response to environmental stimuli is likely to be critical to prevent aging and age-dependent diseases, including cancer, neurodegenerative diseases and diabetes.Oncogene (2008) 27, 2276–2288; doi:10.1038/onc.2008.21 [ABSTRACT FROM AUTHOR]

FoxO transcription factors are an evolutionary conserved subfamily of the forkhead transcription factors, characterized by the forkhead DNA-binding domain. FoxO factors regulate a number of cellular processes involved in cell-fate decisions in a cell-type- and environment-specific manner, including metabolism, differentiation, apoptosis and proliferation. A key mechanism by which FoxO determines cell fate is through regulation of the cell cycle machinery, and as such the cellular consequence of FoxO deregulation is often manifested through perturbation of the cell cycle. Consequently, the deregulation of FoxO factors is implicated in the development of numerous proliferative diseases, in particular cancer.Oncogene (2008) 27, 2300–2311; doi:10.1038/onc.2008.23 [ABSTRACT FROM AUTHOR]

Modulation FOXO transcription factor activities can lead to a variety of cellular outputs resulting in changes in proliferation, apoptosis, differentiation and metabolic responses. Although FOXO proteins all contain an identical DNA-binding domain their cellular functions appear to be distinct, as exemplified by differences in the phenotype of Foxo1, Foxo3 and Foxo4 null mutant mice. While some of these differences may be attributable to the differential expression patterns of these transcription factors, many cells and tissues express several FOXO isoforms. Recently it has become clear that FOXO proteins can regulate transcriptional responses independently of direct DNA-binding. It has been demonstrated that FOXOs can associate with a variety of unrelated transcription factors, regulating activation or repression of diverse target genes. The complement of transcription factors expressed in a particular cell type is thus critical in determining the functional end point of FOXO activity. These interactions greatly expand the possibilities for FOXO-mediated regulation of transcriptional programmes. This review details currently described FOXO-binding partners and examines the role of these interactions in regulating cell fate decisions.Oncogene (2008) 27, 2289–2299; doi:10.1038/onc.2008.22 [ABSTRACT FROM AUTHOR]

Members of the Forkhead box O (FOXO) class of transcription factors are key players in the regulation of cell-fate decisions, such as cell death, cell proliferation and cell metabolism. Furthermore, in model organisms, it has by now been demonstrated that FOXO function affects the life span of these organisms. Multiple signal transduction pathways regulate FOXO function, but most importantly, they are negatively regulated by protein kinase B (PKB/AKT)-mediated phosphorylation and constitute, therefore, an important downstream component of insulin signalling. This review issue provides a timely overview of our understanding of FOXO function and how signalling affects FOXO function. Taken together, the reviewed studies on FOXO function and regulation provide compelling evidence that FOXOs act at the crossroad between aging and age-related diseases including diabetes and cancer. With this perspective, further studies on FOXO function and regulation may shed light on how age impacts on the onset and progression of disease.Oncogene (2008) 27, 2258–2262; doi:10.1038/onc.2008.29 [ABSTRACT FROM AUTHOR]

FOXO (Forkhead box O) transcription factors are involved in cell-cycle arrest or apoptosis induction by transcripting cell-cycle inhibitor p27KIP1 or apoptosis-related genes, respectively. Akt/protein kinase B promotes cell proliferation and suppresses apoptosis, in part, by phosphorylating FOXOs. Phosphorylated FOXOs could not exhibit transcriptional activity because of their nuclear export. Here we show that p15INK4b and p19INK4d transcription is associated with FOXO-mediated G1 cell-cycle arrest. Inhibition of Akt signaling by PI3K inhibitors, a PDK1 inhibitor, or dominant-negative Akt transfection increased expression of p15INK4b and p19INK4d but not p16INK4a and p18INK4c. Ectopic expression of wild type or active FOXO but not inactive form also increased p15INK4b and p19INK4d levels. FOXOs bound to promoter regions and induced transcription of these genes. No increase in the G1-arrested cell population, mediated by PI3K inhibitor LY294002, was observed in INK4b−/− or INK4d−/− murine embryonic fibroblasts. In summary, FOXOs are involved in G1 arrest caused by Akt inactivation via p15INK4b and p19INK4d transcription.Oncogene (2008) 27, 1677–1686; doi:10.1038/sj.onc.1210813; published online 17 September 2007 [ABSTRACT FROM AUTHOR]

The FoxM1 transcription factor plays critical roles in the expression of genes that are essential for cell proliferation. FoxM1 null or depleted cells fail to progress through mitosis, as expression of several mitotic genes depends upon FoxM1. The transcriptional activity of FoxM1 is stimulated by cyclin-cdk-mediated phosphorylation at a site within the transcriptional activation domain. Here, we characterize the role of an N-terminal inhibitory domain in the transcriptional activity of FoxM1. Deletion of the N-terminal 232 amino-acid residues increases the transcriptional and transforming activities of FoxM1. Moreover, while the activity of the full-length FoxM1 is stimulated by growth factors, the activity of the N-terminal deletion mutant is constitutively high in all phases of the cell cycle. The N-terminal deletion also eliminates the requirement for cyclin-cdk to activate FoxM1. We provide evidence that the N-terminal domain interacts with the C-terminal half of the transcription factor to attenuate its transcriptional activity. Moreover, the N-terminal fragment inhibits the transcriptional activity of FoxM1 in G1/S cells, but not in G2/M cells. Our results suggest that cyclin-cdk phosphorylates FoxM1 to counteract the inhibition by the N-terminal domain to fully activate FoxM1 in G2/M phase.Oncogene (2008) 27, 1696–1704; doi:10.1038/sj.onc.1210814; published online 24 September 2007 [ABSTRACT FROM AUTHOR]

The paired/homeodomain transcription factor Pax4 is essential for islet β-cell generation during pancreas development and their survival in adulthood. High Pax4 expression was reported in human insulinomas indicating that deregulation of the gene may be associated with tumorigenesis. We report that rat insulinoma INS-1E cells express 25-fold higher Pax4 mRNA levels than rat islets. In contrast to primary β-cells, activin A but not betacellulin or glucose induced Pax4 mRNA levels indicating dissociation of Pax4 expression from insulinoma cell proliferation. Short hairpin RNA adenoviral constructs targeted to the paired domain or homeodomain (viPax4PD and viPax4HD) were generated. Pax4 mRNA levels were lowered by 73 and 50% in cells expressing either viPax4PD or viPax4HD. Transcript levels of the Pax4 target gene bcl-xl were reduced by 53 and 47%, whereas Pax6 and Pdx1 mRNA levels were unchanged. viPax4PD-infected cells displayed a twofold increase in spontaneous apoptosis and were more susceptible to cytokine-induced cell death. In contrast, proliferation was unaltered. RNA interference-mediated repression of insulin had no adverse effects on either Pax4 or Pdx1 expression as well as on cell replication or apoptosis. These results indicate that Pax4 is redundant for proliferation of insulinoma cells, whereas it is essential for survival through upregulation of the antiapoptotic gene bcl-xl.Oncogene (2007) 26, 4261–4271; doi:10.1038/sj.onc.1210205; published online 29 January 2007 [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are ∼22 nucleotide non-coding RNA molecules that regulate gene expression post-transcriptionally. Although aberrant expression of miRNAs in various human cancers suggests a role for miRNAs in tumorigenesis, it remains largely unclear as to whether knockdown of a specific miRNA affects tumor growth. In this study, we profiled miRNA expression in matched normal breast tissue and breast tumor tissues by TaqMan real-time polymerase chain reaction miRNA array methods. Consistent with previous findings, we found that miR-21 was highly overexpressed in breast tumors compared to the matched normal breast tissues among 157 human miRNAs analysed. To better evaluate the role of miR-21 in tumorigenesis, we transfected breast cancer MCF-7 cells with anti-miR-21 oligonucleotides and found that anti-miR-21 suppressed both cell growth in vitro and tumor growth in the xenograft mouse model. Furthermore, this anti-miR-21-mediated cell growth inhibition was associated with increased apoptosis and decreased cell proliferation, which could be in part owing to downregulation of the antiapoptotic Bcl-2 in anti-miR-21-treated tumor cells. Together, these results suggest that miR-21 functions as an oncogene and modulates tumorigenesis through regulation of genes such as bcl-2 and thus, it may serve as a novel therapeutic target.Oncogene (2007) 26, 2799–2803. doi:10.1038/sj.onc.1210083; published online 30 October 2006 [ABSTRACT FROM AUTHOR]

Constitutively activated nuclear factor-κB (NF-κB) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-κB in a series of HNSCC cell lines. We confirmed that NF-κB was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-κB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-κB (IκBα), the natural inhibitor of NF-κB. Constitutive NF-κB activation in HNSCC was found to be due to constitutive activation of IκBα kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IκBα and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-κB activation. The expression of constitutively active NF-κB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-κB reporter activity was inhibited by DN-TNF receptor-associated death domain (TRADD), DN-TNF receptor-associated factor (TRAF)2, DN-receptor-interacting protein (RIP), DN-transforming growth factor-β-activated kinase 1 (TAK1), DN-κ-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-κB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-κB and its ligand in HNSCC cells but not interleukin (IL)-1β. All HNSCC cell lines expressed IL-6, a NF-κB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-κB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-κB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.Oncogene (2007) 26, 1385–1397. doi:10.1038/sj.onc.1209945; published online 4 September 2006 [ABSTRACT FROM AUTHOR]

The path to the discovery of suberoylanilide hydroxamic acid (SAHA, vorinostat) began over three decades ago with our studies designed to understand why dimethylsulfoxide causes terminal differentiation of the virus-transformed cells, murine erythroleukemia cells. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations that have little or no toxic effects on normal cells. It was discovered that SAHA inhibits the activity of histone deacetylases (HDACs), including all 11 known human class I and class II HDACs. HDACs have many protein targets whose structure and function are altered by acetylation including histones and non-histone proteins component of transcription factors controlling gene expression and proteins that regulate cell proliferation, migration and death. SAHA is in clinical trials and has significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients. A new drug application has been approved for SAHA (vorinostat) treatment of cutaneous T-cell lymphoma.Oncogene (2007) 26, 1351–1356. doi:10.1038/sj.onc.1210204 [ABSTRACT FROM AUTHOR]