The mammalian Retinoblastoma (RB) family including pRB, p107, and p130 represses E2F target genes through mechanisms that are not fully understood. In D. melanogaster, RB-dependent repression is mediated in part by the multisubunit protein complex Drosophila RBF, E2F, and Myb (dREAM) that contains homologs of the C. elegans synthetic multivulva class B (synMuvB) gene products. Using an integrated approach combining proteomics, genomics, and bioinformatic analyses, we identified a p130 complex termed DP, RB-like, E2F, and MuvB (DREAM) that contains mammalian homologs of synMuvB proteins LIN-9, LIN-37, LIN-52, LIN-54, and LIN-53/RBBP4. DREAM bound to more than 800 human promoters in G0 and was required for repression of E2F target genes. In S phase, MuvB proteins dissociated from p130 and formed a distinct submodule that bound MYB. This work reveals an evolutionarily conserved multisubunit protein complex that contains p130 and E2F4, but not pRB, and mediates the repression of cell cycle-dependent genes in quiescence.

Preface The dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM) complex provides a previously unsuspected unifying role in the cell cycle by directly linking p130, p107, E2F, BMYB and FOXM1. DREAM mediates gene repression during G0 and coordinates periodic gene expression with peaks during G1/S and G2/M. Perturbations in DREAM regulation shift the balance from quiescence towards proliferation and contribute to increased mitotic gene expression levels frequently observed in cancers with poor prognosis.

Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex-comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4-serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis.

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The tumor suppressor protein p53 regulates transcription of many genes that mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Here we show that Ipaf, a human CED-4 homologue and an activator of caspase-1, is induced by p53. Overexpression of p53 by transfection in U2OS and A549 cells increased Ipaf mRNA levels. Treatment of p53-positive cell lines U2OS and MCF-7 with the DNA damaging drug, doxorubicin, which increases p53 protein level, induced expression of Ipaf mRNA but similar treatment of MCF-7-mp53 (a clone of MCF-7 cells expressing mutant p53) and p53-negative K562 cells showed much less induction of Ipaf gene expression. Expression analysis for Ipaf mRNA in doxorubicin-treated human tumor cell lines suggests that p53-dependent as well as p53-independent mechanisms are involved in the regulation of Ipaf gene expression in a cell-type-specific manner. The Ipaf promoter was activated by normal p53 but not by His 273 mutant of p53. A functional p53-binding site was identified in the Ipaf promoter. A dominant-negative mutant of Ipaf inhibited p53-induced and doxorubicin-induced apoptosis by about 50%. Ipaf-directed small hairpin RNA downregulated p53-induced Ipaf gene expression and also reduced p53-induced apoptosis. Doxorubicin-induced apoptosis was also inhibited by Ipaf-directed small hairpin RNA. Our results show that p53 can directly induce Ipaf gene transcription, which contributes to p53-dependent apoptosis in at least some human cells.

PTP‐S2/TC45 is a nuclear protein tyrosine phosphatase that activates p53 and induces caspase 1‐dependent apoptosis. We analyzed the role of ICE protease‐activating factor (Ipaf), an activator of caspase 1 in p53‐dependent apoptosis. We also determined the sequence of events that lead to apoptosis upon caspase 1 activation by Ipaf. PTP‐S2 expression induced Ipaf mRNA in MCF‐7 cells which was dependent on p53. PTP‐S2‐induced apoptosis was inhibited by a dominant‐negative mutant of Ipaf and also by an Ipaf‐directed short‐hairpin RNA. Doxorubicin‐induced apoptosis was potentiated by the expression of caspase 1 (but not by a catalytic mutant of caspase 1) and required endogenous Ipaf. Doxorubicin treatment of MCF‐7 cells resulted in activation of exogenous caspase 1, which was partly dependent on endogenous Ipaf. An activated form of Ipaf induced caspase 1‐dependent apoptosis that was inhibited by Bcl2 and also by a dominant inhibitor of caspase 9 (caspase 9s). Caspase 1‐dependent apoptosis induced by doxorubicin was also inhibited by Bcl2 and caspase 9s, but caspase 1 activation by activated Ipaf was not inhibited by Bcl2. Mitochondrial membrane permeabilization was induced by caspase 1 and activated Ipaf, which was inhibited by Bcl2, but not by caspase 9s. Expression of caspase 1 with activated Ipaf resulted in the activation of Bax at mitochondria. Our results suggest that Ipaf is involved in PTP‐S2‐induced apoptosis and that caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby functioning as an initiator caspase.

BackgroundSequencing studies across multiple cancers continue to reveal mutations and genes involved in the pathobiology of these cancers. Exome sequencing of oral cancers, a subset of Head and Neck Squamous cell Carcinomas (HNSCs) common among tobacco-chewing populations, revealed that ∼34% of the affected patients harbor mutations in the CASP8 gene. Uterine Corpus Endometrial Carcinoma (UCEC) is another cancer where ∼10% cases harbor CASP8 mutations. Caspase-8, the protease encoded by CASP8 gene, plays a dual role in programmed cell death, which in turn has an important role in tumor cell death and drug resistance. CASP8 is a protease required for the extrinsic pathway of apoptosis and is also a negative regulator of necroptosis. Using multiple tools such as differential gene expression, gene set enrichment, gene ontology, in silico immune cell estimates, and survival analyses to mine data in The Cancer Genome Atlas, we compared the molecular features and survival of these carcinomas with and without CASP8 mutations.ResultsDifferential gene expression followed by gene set enrichment analysis showed that HNSCs with CASP8 mutations displayed a prominent signature of genes involved in immune response and inflammation. Analysis of abundance estimates of immune cells in these tumors further revealed that mutant-CASP8 HNSCs were rich in immune cell infiltrates. However, in contrast to Human Papilloma Virus-positive HNSCs that also exhibit high immune cell infiltration, which in turn is correlated with better overall survival, HNSC patients with mutant-CASP8 tumors did not display any survival advantage. Similar analyses of UCECs revealed that while UCECs with CASP8 mutations also displayed an immune signature, they had better overall survival, in contrast to the HNSC scenario. There was also a significant up-regulation of neutrophils (p-value = 0.0001638) as well as high levels of IL33 mRNA (p-value = 7.63747E−08) in mutant-CASP8 HNSCs, which were not observed in mutant-CASP8 UCECs.ConclusionsThese results suggested that carcinomas with mutant CASP8 have broadly similar immune signatures albeit with different effects on survival. We hypothesize that subtle tissue-dependent differences could influence survival by modifying the micro-environment of mutant-CASP8 carcinomas. High neutrophil numbers, a well-known negative prognosticator in HNSCs, and/or high IL33 levels may be some of the factors affecting survival of mutant-CASP8 cases.

Introduction 3. Oral cancer stem cells-identification and isolation 3.1. Isolation of CSCs from oral cancers using the cell surface marker CD44 3.2. Isolation of CSCs from oral cancers using other markers 4. Correlation between oral CSC numbers and clinical prognosis 5. Molecular features of oral CSCs 5.1. Expression of stem cell-and pluripotency-related genes, and inducers of cellular proliferation 5.2. Loss of cell adhesion and terminal differentiation markers 5.3. Duality-Presence of epithelial and epithelial-to-mesenchymal transition (EMT) features 6. Concluding remarks 7. Acknowledgments 8. References