Evidence That Selenium Binding Protein 1 Is a Tumor Suppressor in Prostate Cancer

Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56) is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA) recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.     

The RING Finger Protein RNF8 Ubiquitinates Nbs1 to Promote DNA Double-strand Break Repair by Homologous Recombination

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.     

HOXB1 Is a Tumor Suppressor Gene Regulated by miR-3175 in Glioma

The HOXB1 gene plays a critical role as an oncogene in diverse tumors. However, the functional role of HOXB1 and the mechanism regulating HOXB1 expression in glioma are not fully understood. A preliminary bioinformatics analysis showed that HOXB1 is ectopically expressed in glioma, and that HOXB1 is a possible target of miR-3175. In this study, we investigated the function of HOXB1 and the relationship between HOXB1 and miR-3175 in glioma. We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy. Reduced HOXB1 expression promoted the proliferation and invasion of glioma cells, and inhibited their apoptosis in vitro, and the downregulation of HOXB1 was also associated with worse survival in glioma patients. More importantly, HOXB1 was shown experimentally to be a direct target of miR-3175 in this study. The downregulated expression of miR-3175 inhibited cell proliferation and invasion, and promoted apoptosis in glioma. The oncogenicity induced by low HOXB1 expression was prevented by an miR-3175 inhibitor in glioma cells. Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma. These results clarify the pathogenesis of glioma and offer a potential target for its treatment.     

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.     

Tumor Cell Death Mediated by Peptides That Recognize Branched Intermediates of DNA Replication and Repair

Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50–100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.     

Replication Protein A (RPA1a) Is Required for Meiotic and Somatic DNA Repair But Is Dispensable for DNA Replication and Homologous Recombination in Rice

Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.In a population of organisms, it is crucial to maintain the integrity of genome among individuals as well as shuffle genetic information at the population level. To maintain such genetic integrity, cells have evolved elaborate mechanisms such as base excision repair (BER; Hegde et al., 2008), nucleotide excision repair (NER; Shuck et al., 2008), homologous recombination (HR; Li and Heyer, 2008) repair, and nonhomologous end joining (Weterings and Chen, 2008) pathways to repair diverse types of DNA damage. To allow for variation, however, organisms utilize meiosis to shuffle genetic material so as to increase genetic diversity in populations and in the species.DNA double-strand break (DSB) repair is particularly important in maintaining the integrity of genome among individuals and shuffling genetic information among population, because DSBs are generated not only in meiotic cells but also from the action of certain endogenous or exogenous DNA-damaging agents and during repair of other kinds of DNA lesions by NER or BER (West et al., 2004; Bleuyard et al., 2006). The past decade has witnessed an explosion in understanding of this complex process by using yeast (Saccharomyces cerevisiae) as a model organism (Aylon and Kupiec, 2004). Cells can repair DSBs by the relatively inaccurate process of rejoining the two broken ends directly (i.e. nonhomologous end joining) or much more accurately by HR (Bleuyard et al., 2006; Wyman and Kanaar, 2006). These two pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type (Shrivastav et al., 2008). According to the current general model for meiotic DSB repair (Bishop and Zickler, 2004; Ma, 2006; San Filippo et al., 2008), when DSBs occur the MRN complex (composed of Mre11, Rad50, and NBS1) resects the DSBs to generate 5′→3′ single-stranded DNA (ssDNA) ends. Subsequently, the replication protein A (RPA) protein complex binds to the ssDNA ends to protect them from attack by endogenous exonucleases; then, in concert with catalysis by Rad52, Rad55, and Rad57, the recombinase Rad51 displaces RPA, resulting in the generation of a Rad51 nucleoprotein filament that in turn catalyzes the search and invasion into the recombination partner with the help of proteins belonging to the RAD52 epistasis group to form a D loop that accompanies DNA synthesis. Thereafter, at least two competing mechanisms may come into play. One is the DSB repair pathway, in which the capture of the second DSB end and additional DNA synthesis result in an intermediate that harbors two Holliday junctions. The subsequent resolution of Holliday junctions results in the formation of crossovers. Alternatively, in the synthesis-dependent strand annealing pathway, the D loop dissociates and the invading single strand with newly synthesized DNA reanneals with the other DSB end, followed by gap-filling DNA synthesis and ligation, forming only noncrossover products (Ma, 2006; San Filippo et al., 2008).RPA is comprised of three subunits of RPA1, 2, and 3, alternatively termed as RPA70, 32, and 14, respectively, according to their apparent M_rs (Wold, 1997; Iftode et al., 1999). RPA is an essential protein in various DNA metabolism pathways such as DNA replication, repair, and HR (Wold, 1997; Iftode et al., 1999). In these pathways, the most basic function of RPA is binding to ssDNA to protect it from exonucleases, and its general roles in DNA metabolism depend on its interactions with other proteins in various pathways (Wold, 1997; Iftode et al., 1999). For example, in human NER pathway, RPA binds to damaged DNA and interacts with xeroderma pigmentosum damage-recognition protein, XPA, in the damage recognition step, and then the endonucleases XPG and ERCC1/XPF are recruited to the RPA-XPA-damaged DNA complex in the excision step (He et al., 1995). Interactions of RPA with those proteins are critical in this process (Wold, 1997; Iftode et al., 1999). A great deal of protein dynamics research has indicated that the interactions between RPA and other DNA-metabolism proteins are choreographed on the ssDNA to recruit the required protein present at the proper time (Fanning et al., 2006).Human, animals, and fungi have single copy for each subunit of RPA (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, have multiple genes for most RPA subunits (Ishibashi et al., 2006; Shultz et al., 2007). Most of them have not unveiled exact function up to now. To elucidate the molecular basis of meiosis in rice, we performed a large-scale screen for sterile mutants using our T-DNA insertion mutant library (Wu et al., 2003). Previously, we reported the cloning of OsPAIR3, a novel gene required for homologous chromosome pairing and synapsis in rice (Yuan et al., 2009). Here we report the characterization of another sterile mutant with a T-DNA insertion in OsRPA1a. Our results indicate that OsRPA1a is essential for DNA repair but may play redundant roles in DNA replication and recombination in rice.     

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.     

DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.     

Bacillus subtilis hlpB Encodes a Conserved Stand-Alone HNH Nuclease-Like Protein That Is Essential for Viability Unless the hlpB Deletion Is Accompanied by the Deletion of Genes Encoding the AddAB DNA Repair Complex

The HNH domain is found in many different proteins in all phylogenetic kingdoms and in many cases confers nuclease activity. We have found that the Bacillus subtilis hlpB (yisB) gene encodes a stand-alone HNH domain, homologs of which are present in several bacterial genomes. We show that the protein we term HlpB is essential for viability. The depletion of HlpB leads to growth arrest and to the generation of cells containing a single, decondensed nucleoid. This apparent condensation-segregation defect was cured by additional hlpB copies in trans. Purified HlpB showed cooperative binding to a variety of double-stranded and single-stranded DNA sequences, depending on the presence of zinc, nickel, or cobalt ions. Binding of HlpB was also influenced by pH and different metals, reminiscent of HNH domains. Lethality of the hlpB deletion was relieved in the absence of addA and of addAB, two genes encoding proteins forming a RecBCD-like end resection complex, but not of recJ, which is responsible for a second end-resectioning avenue. Like AddA-green fluorescent protein (AddA-GFP), functional HlpB-YFP or HlpB-FlAsH fusions were present throughout the cytosol in growing B. subtilis cells. Upon induction of DNA damage, HlpB-FlAsH formed a single focus on the nucleoid in a subset of cells, many of which colocalized with the replication machinery. Our data suggest that HlpB plays a role in DNA repair by rescuing AddAB-mediated recombination intermediates in B. subtilis and possibly also in many other bacteria.     

MicroRNA-219-2-3p Functions as a Tumor Suppressor in Gastric Cancer and Is Regulated by DNA Methylation

Background & Aims

Gastric cancer is the most frequent gastrointestinal tumor in adults and is the most lethal form of human cancer. Despite of the improvements in treatments, the underlying mechanism of gastric carcinogenesis is not well known. To define novel modulators that regulate susceptibility to tumorgenesis, we focused on miR-219-2-3p.

Methods

Quantitative RT-PCR was employed to investigate the level of miR-219-2-3p in gastric cancer (GC) tissues (n = 113) and their matched adjacent normal tissues (n = 113). In vitro cell proliferation, apoptosis assays, cell migration, and invasion assays were performed to elucidate biological effects of miR-219-2-3p. Since silencing of miRNA by promoter CpG island methylation may be an important mechanism in tumorgenesis, GC cells were treated with 5-aza-2′-deoxycytidine and trichostatin A, and expression changes of miR-219-2-3p were subsequently examined by quantitative RT-PCR. Finally, the methylation status of CpG island upstream of miR-219-2-3p was analyzed by methylation-specific PCR in GC tissues (n = 22).

Results

miR-219-2-3p was down-regulated in GC and cell lines. In addition, the experiments documented the lower expression of miR-219-2-3p in GC specimens with higher grade and later stage tumors. Meanwhile, miR-219-2-3p exerted antiproliferative, proapoptotic, and antimetastatic roles and reduced levels of p-ERK1/2 in GC cells. Furthermore, 5-aza-2′-deoxycytidine and trichostatin A increased the expression (∼2 fold) of miR-219-2-3p in GC cells. By methylation-specific PCR, DNA methylation in the upstream region of miR-219-2-3p was detected in both adjacent normal tissues and cancer tissues. As expected, the methylation level was considerably higher in the miR-219-2-3p down-regulated group than up-regulated group.

Conclusions

miR-219-2-3p is potentially involved in gastric cancer progression and metastasis by regulating ERK1/2-related signal pathways, which may provide a novel therapeutic strategy for treatment of gastric cancer. Methylation mechanism may be involved in modulating the expression level of miR-219-2-3p in gastric cancer.     

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.