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1.  DDB2 Suppresses Epithelial to Mesenchymal Transition in Colon Cancer 
Cancer research  2013;73(12):3771-3782.
Colon cancer is one of the deadliest cancers worldwide because of its metastasis to other essential organs. Metastasis of colon cancer involves a complex set of events, including epithelial to mesenchymal transition (EMT) that increases invasiveness of the tumor cells. Here we show that the xeroderma pigmentosum group E (XPE) gene product DDB2 is down-regulated in high-grade colon cancers, and it plays a dominant role in the suppression of EMT of the colon cancer cells. Depletion of DDB2 promotes mesenchymal phenotype, whereas expression of DDB2 promotes epithelial phenotype. DDB2 constitutively represses genes that are the key activators of EMT, indicating that DDB2 is a master regulator of EMT of the colon cancer cells. Moreover, we observed evidence that DDB2 functions as a barrier for EMT induced by hypoxia and TGF-β. Also, we provide evidence that DDB2 inhibits metastasis of colon cancer. The results presented here identify a transcriptional regulatory pathway of DDB2 that is directly linked to the mechanisms that suppress metastasis of colon cancer.
PMCID: PMC3686976  PMID: 23610444
2.  Targeting FoxM1 effectively retards p53-null lymphoma and sarcoma 
Molecular cancer therapeutics  2013;12(5):759-767.
The forkhead box transcription factor FOXM1 is considered to be a promising target for cancer therapy. However, the significance of FOXM1 in tumors harboring mutation in p53, which is very common, is unclear. In this study, we investigated the efficacy of FoxM1-targeting in spontaneous p53-null tumors using genetic ablation as well as using a peptide-inhibitor of FOXM1. We show that conditional deletion of FoxM1 inhibits growth of the p53 null thymic lymphoma and sarcoma cells. In addition, deletion of FoxM1 induces apoptotic cell death of the p53 null tumors, accompanied by reduced expression of the FOXM1 target genes Survivin and Bmi1. An ARF-derived peptide that inhibits the activity of FOXM1, by targeting it to the nucleolus, also induces apoptosis in the p53 null sarcoma and lymphoma, leading to a strong inhibition of their metastatic colonization. Together, our observations suggest that FOXM1 is critical for survival and growth of the p53-null lymphoma and sarcoma, and provide proof-of-principle that FOXM1 is an effective therapeutic target for sarcoma and lymphoma carrying loss of function mutation in p53.
PMCID: PMC3651795  PMID: 23427295
3.  Tumor regression by phenethyl isothiocyanate involves DDB2 
Cancer Biology & Therapy  2013;14(2):108-116.
Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent commonly found in edible cruciferous vegetables. It has been implicated also for therapy, and is in clinical trial for lung cancer. Here, we provide evidence that the tumor suppressive effect of PEITC is related to its ability to induce expression of damaged DNA binding protein 2 (DDB2), a DNA repair protein involved also in apoptosis and premature senescence. DDB2 expression is attenuated in a wide variety of cancers including the aggressive colon cancers. We show that, in colon cancer cells, reactive oxygen species, which are induced by PEITC, augment expression of DDB2 through the p38MAPK/JNK pathway, independently of p53. PEITC-induced expression of DDB2 is critical for inhibition of tumor progression by PEITC. Tumors derived from DDB2-deficient colon cancer cells are refractory to PEITC-treatments, resulting from deficiencies in apoptosis and senescence. The DDB2-proficient tumors, on the other hand, respond effectively to PEITC. The results show that PEITC can be used to induce expression of DDB2, and that expression of DDB2 is critical for effective response of tumors to PEITC.
PMCID: PMC3571992  PMID: 23114715
DDB2; PEITC; ROS; apoptosis; colon cancer; drug resistance; senescence
4.  FoxM1 in Tumorigenicity of the Neuroblastoma Cells and Renewal of the Neural Progenitors 
Cancer research  2011;71(12):4292-4302.
Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state.
PMCID: PMC3771352  PMID: 21507930
5.  Cul4A is Essential for Spermatogenesis and Male Fertility 
Developmental biology  2011;352(2):278-287.
The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A-/- strain lacking exons 4-8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A-/- mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.
PMCID: PMC3065526  PMID: 21291880
Cul4A; spermatogenesis; homologous recombination
6.  Damaged DNA Binding Protein 2 in Reactive Oxygen Species (ROS) Regulation and Premature Senescence 
Premature senescence induced by DNA damage or oncogene is a critical mechanism of tumor suppression. Reactive oxygen species (ROS) have been implicated in the induction of premature senescence response. Several pathological disorders such as cancer, aging and age related neurological abnormalities have been linked to ROS deregulation. Here, we discuss how Damaged DNA binding Protein-2 (DDB2), a nucleotide excision repair protein, plays an important role in ROS regulation by epigenetically repressing the antioxidant genes MnSOD and Catalase. We further revisit a model in which DDB2 plays an instrumental role in DNA damage induced ROS accumulation, ROS induced premature senescence and inhibition of skin tumorigenesis.
PMCID: PMC3472727  PMID: 23109835
DDB2; senescence; reactive oxygen species; apoptosis; NER
7.  DDB2 (Damaged DNA binding protein 2) in nucleotide excision repair and DNA damage response 
Cell cycle (Georgetown, Tex.)  2009;8(24):4067-4071.
DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.
PMCID: PMC3107032  PMID: 19923893
DDB2; UV damage; Cul4A; E3 ligase; NER
8.  Destabilization of Rb by human papillomavirus E7 is cell cycle dependent: E2-25K is involved in the proteolysis 
Virology  2009;396(1):118-124.
The HPV-oncoprotein, E7 promotes proteasomal degradation of the tumor suppressor protein, Rb. In this study, we analyzed the regulation of E7-induced Rb proteolysis in HPV-containing Caski cervical cancer cells. We show that the Rb proteolysis is cell cycle dependent; in S phase Rb is stable while in post-mitotic early G1 phase cells and in differentiated cells, Rb is unstable. Similarly, the in vivo Rb/E7 interaction is not detected in S phase cells, but is readily detected in differentiating Caski cells. The ubiquitinating enzymes involved in Rb proteolysis have not been identified. We find that the E3 ligase MDM2 is not involved in the Rb proteolysis in Caski cells. An in vivo analysis using multiple catalytic-site mutant dominant negative E2-enzymes show that the C92A E2-25K most effectively blocks E7-induced Rb proteolysis. Taken together, these results show that E7 induces Rb proteolysis in growth-arrested cells and E2-25K is involved in the proteolysis.
PMCID: PMC2796474  PMID: 19906396
9.  DDB2, an Essential Mediator of Premature Senescence▿  
Molecular and Cellular Biology  2010;30(11):2681-2692.
Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.
PMCID: PMC2876515  PMID: 20351176
10.  Damaged-DNA Binding Protein-2 Drives Apoptosis Following DNA Damage 
Cell Division  2010;5:3.
Apoptosis induced by DNA damage is an important mechanism of tumor suppression and it is significant also in cancer chemotherapy. Mammalian cells activate the pathways of p53 to induce apoptosis of cells harboring irreparable DNA damages. While p53 induces expression of various pro-apoptotic genes and directly participates in the disruption of mitochondrial membrane polarization, it also increases expression of the cell cycle inhibitor p21 that is a dominant inhibitor of caspase-activation and apoptosis. Here we discuss how Damaged-DNA Binding Protein-2 (DDB2) subdues the level of p21 in cells harboring irreparable DNA damage to support activation of the caspases. We speculate a model in which DDB2 detects and couples the presence of un-repaired DNA damages to the proteolysis of p21, leading to the induction of apoptosis.
PMCID: PMC2822757  PMID: 20205757
11.  Proliferation Defects and Genome Instability in Cells Lacking Cul4A 
Oncogene  2009;28(26):2456-2465.
The Cul4A gene, which encodes a core component of a cullin-based E3 ubiquitin ligase complex, is over-expressed in breast and hepatocellular cancers. In breast cancers, over-expression of Cul4A strongly correlates with poor prognosis. Also, Cul4A is required for early embryonic development. Early lethality of mouse embryos prevented a detailed analysis of the functions of Cul4A. Here, we used a strain of mice carrying floxed alleles of Cul4A to study its role in cell division, in vitro and in vivo. Embryonic fibroblasts exhibit a severe deficiency in cell proliferation following deletion of Cul4A. We observed that the Cul4A protein is abundantly expressed in brain, liver and in the mammary tissue of pregnant mice. Deletion of Cul4A in liver impairs hepatocyte proliferation during regeneration following carbon tetrachloride induced injury. The Cul4A-deleted cells are slow in entering S phase, and are deficient in progressing through early M phase. Several cell cycle regulators, including p53 and p27Kip1, are de-regulated in the Cul4A-deleted cells. Expression of a dominant negative mutant of p53 causes significant reversal of the proliferation defects in Cul4A-deleted cells. The Cul4A-deleted cells exhibit aberrant number of centrosome, multipolar spindles and micronuclei formation. Furthermore, those cells are sensitive to UV irradiation and exhibit reduced levels of unscheduled DNA synthesis. Together, our observations indicate that Cul4A is required for efficient cell proliferation, control of the centrosome amplification and genome stability.
PMCID: PMC2705479  PMID: 19430492
12.  Deregulation of eIF4E: 4E-BP1 in Differentiated Human Papillomavirus-Containing Cells Leads to High Levels of Expression of the E7 Oncoprotein 
Journal of Virology  2006;80(14):7079-7088.
Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.
PMCID: PMC1489051  PMID: 16809313
13.  Cul4A and DDB1 Associate with Skp2 To Target p27Kip1 for Proteolysis Involving the COP9 Signalosome†  
Molecular and Cellular Biology  2006;26(7):2531-2539.
DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.
PMCID: PMC1430311  PMID: 16537899
14.  The Papillomavirus E7 Oncoprotein Is Ubiquitinated by UbcH7 and Cullin 1- and Skp2-Containing E3 Ligase 
Journal of Virology  2004;78(10):5338-5346.
Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2−/− mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.
PMCID: PMC400333  PMID: 15113913
15.  Stability of the Sp3–DNA complex is promoter-specific: Sp3 efficiently competes with Sp1 for binding to promoters containing multiple Sp-sites 
Nucleic Acids Research  2003;31(18):5368-5376.
The transcription regulatory protein Sp3 shares more than 90% sequence homology with Sp1 in the DNA-binding domain and they bind to the same cognate DNA-element. However, the transcriptional activities of these two Sp-family factors are not equivalent. While Sp1 functions strictly as a transcriptional activator, Sp3 has been shown to be transcriptionally inactive for promoters containing multiple Sp-binding sites. In the present study, we show that the DNA-binding property of Sp3 is promoter dependent and is different from Sp1. The 116 kDa Sp3 polypeptide binds as a monomer to a single Sp-binding site but readily forms slower migrating complexes with adjacent Sp-binding sites. The slower migrating Sp3–DNA complexes are significantly more stable than monomeric Sp3–DNA complexes or multimeric Sp1–DNA complexes. As a consequence, Sp3 can efficiently compete with Sp1 for binding to regions containing multiple Sp sites. The transcription regulatory function of Sp3 is also significantly different from Sp1. Unlike Sp1, Sp3 does not synergistically activate transcription of promoters containing multiple Sp-binding sites. Therefore, although Sp3 is a transcription activator, Sp3 reduces Sp1-dependent transcription of promoters containing adjacent Sp-binding sites by competing with Sp1 for promoter occupancy and thereby blocking the synergistic transactivation function of Sp1. Taken together, this study provides a possible mechanism of the promoter-specific transcription repression function of Sp3.
PMCID: PMC203306  PMID: 12954773

Results 1-15 (15)