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The aryl hydrocarbon receptor (AhR) is a nuclear transcription factor and xenobiotic sensor reported to mediate diet-induced obesity and is upregulated in high-grade prostate cancer (PCa). Previously published data from our lab shows AhR protein is overexpressed and constitutively active in castration-resistant PCa cells. AhR signaling is promoted by high fat diet-induced obesity, when circulating leptin levels are also high. Physiologically relevant leptin concentration is proportional to BMI and is secreted by adipose cells to inhibit hunger under normal circumstances. Our preliminary results suggest that both AhR and leptin independently desensitize PCa cells to clinically-relevant chemotherapeutics however, the underlying mechanisms must be elucidated and may require sustained AhR signaling. We also conducted an integrative bioinformatics analysis to explore the role of AHR and LEPR in PCa, which revealed AHR and LEPR mRNA expression positively correlates in prostate cancer (P Citation Format: Kofi K. Khamit-Kush, Joann B. Powell, Jeffrey A. Handy, Nathan J. Bowen, Valerie Odero-Marah, Daqing Wu. Aryl hydrocarbon receptor and leptin promote prostate cancer progression and may be associated with chemoresistance [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C023.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of "luminal committed basal cells", identifying a unique prostate developmental pathway regulated by Id4.

Patients with advanced epithelial ovarian cancer often experience disease recurrence after standard therapies, a critical factor in determining their five-year survival rate. Recent reports indicated that long-term or short-term survival is associated with varied gene expression of cancer cells. Thus, identification of novel prognostic biomarkers should be considered. Since the mouse genome is similar to the human genome, we explored potential prognostic biomarkers using two groups of mouse ovarian cancer cell lines (group 1: IG-10, IG-10pw, and IG-10pw/agar; group 2: IG-10 clones 2, 3, and 11) which display highly and moderately aggressive phenotypes in vivo. Mice injected with these cell lines have different survival time and rates, capacities of tumor, and ascites formations, reflecting different prognostic potentials. Using an Affymetrix Mouse Genome 430 2.0 Array, a total of 181 genes were differentially expressed (P

JunD, a member of the AP-1 family, is essential for cell proliferation in prostate cancer (PCa) cells. We recently demonstrated that JunD knock-down (KD) in PCa cells results in cell cycle arrest in G1-phase concomitant with a decrease in cyclin D1, Ki67, and c-MYC, but an increase in p21 levels. Furthermore, the over-expression of JunD significantly increased proliferation suggesting JunD regulation of genes required for cell cycle progression. Here, employing gene expression profiling, quantitative proteomics, and validation approaches, we demonstrate that JunD KD is associated with distinct gene and protein expression patterns. Comparative integrative analysis by Ingenuity Pathway Analysis (IPA) identified 1) cell cycle control/regulation as the top canonical pathway whose members exhibited a significant decrease in their expression following JunD KD including PRDX3, PEA15, KIF2C, and CDK2, and 2) JunD dependent genes are associated with cell proliferation, with MYC as the critical downstream regulator. Conversely, JunD over-expression induced the expression of the above genes including c-MYC. We conclude that JunD is a crucial regulator of cell cycle progression and inhibiting its target genes may be an effective approach to block prostate carcinogenesis.

Triple-Negative Breast Cancers (TNBCs) are the most difficult to treat subtype of breast cancer and are often associated with high nuclear expression of Snail and Cathepsin L (Cat L) protease. We have previously shown that Snail can increase Cat L expression/activity in prostate and breast cancer cells. This study investigated the role of CUX1 (a downstream substrate of Cat L) in TNBC. We showed that Cat L and CUX1 were highly expressed in TNBC patient tissue/cell lines, as compared to ER-positive samples, using cBioportal data and western blot/zymography analyses. Additionally, luciferase reporter and chromatin immunoprecipitation assays showed that CUX1 directly bound to estrogen receptor-alpha (ER-α) promoter in MDA-MB-468, a representative TNBC cell line, and that CUX1 siRNA could restore ER-α transcription and protein expression. Furthermore, Snail and CUX1 expression in various TNBC cell lines was inhibited by muscadine grape skin extract (MSKE, a natural grape product rich in anthocyanins) or Cat L inhibitor (Z-FY-CHO) leading to decreased cell invasion and migration. MSKE decreased cell viability and increased expression of apoptotic markers in MDA-MB-468 cells, with no effect on non-tumorigenic MCF10A cells. MSKE also decreased CUX1 binding to ER-α promoter and restored ER-α expression in TNBC cells, while both MSKE and CUX1 siRNA restored sensitivity to estradiol and 4-hydoxytamoxifen as shown by increased cell viability. Therefore, CUX1 activated by Snail-Cat L signaling may contribute to TNBC via ER-α repression, and may be a viable target for TNBC using natural products such as MSKE that targets cancer and not normal cells.

African Americans (AA) have higher death rates due to prostate and breast cancer as compared to Caucasian Americans (CA), and few biomarkers have been associated with this disparity. In our study we investigated whether epithelial-mesenchymal transition (EMT) with a focus on Snail and Cathepsin L (Cat L), could potentially be two markers associated with prostate and breast health disparities. We have previously shown that Snail can increase Cat L protein and activity in prostate and breast cancer. Western blot and real-time PCR analyses showed that mesenchymal protein expression (Snail, vimentin, Cat L) and Cat L activity (shown by zymography) was higher in AA prostate cancer cells as compared to CA normal transformed RWPE-1 prostate epithelial cells, and androgen-dependent cells, and comparable to metastatic CA cell lines. With respect to breast cancer, mesenchymal markers were higher in TNBC compared to non-TNBC cells. The higher mesenchymal marker expression was functionally associated with higher proliferative and migratory rates. Immunohistochemistry showed that both nuclear Snail and Cat L expression was significantly higher in cancer compared to normal for CA and Bahamas prostate patient tissue. Interestingly, AA normal tissue stained higher for nuclear Snail and Cat L that was not significantly different to cancer tissue for both prostate and breast tissue, but was significantly higher than CA normal tissue. AA TNBC tissue also displayed significantly higher nuclear Snail expression compared to CA TNBC, while no significant differences were observed with Luminal A cancer tissue. Therefore, increased EMT in AA compared to CA that may contribute to the more aggressive disease.

JunD, a member of the AP-1 transcription factor family, is involved in a variety of biological processes including cell proliferation, inflammation, differentiation, apoptosis, and carcinogenesis. We previously showed that JunD is essential for cell proliferation and demonstrated that the knock-down (KD) of JunD in PCa cells resulted in cell cycle arrest in G1-phase concomitant with a decrease in the levels of Id1, c-MYC, and Ki67, but an increased in p21 protein levels. Furthermore, the over-expression of JunD significantly increased proliferation in these cells suggesting that JunD regulates the expression of genes which are required for cell cycle progression. In this present study, employing gene expression profiling, quantitative proteomics, and validation approaches, we demonstrated that JunD KD is associated with distinct gene and protein expression patterns. Comparison integrative analysis by Ingenuity Pathway Analysis (IPA) identified 1) cell cycle control/regulation as the top canonical pathway whose members exhibited a significant decrease in their expression following JunD KD including PRDX3, PEA15, KIF2C, and CDK2, and 2) JunD target genes are involved in cell proliferation, with MYC as the key downstream regulator. Conversely, JunD over-expression induced cell proliferation and the expression of the above JunD target genes including c-MYC. When treated with JQ1, a c-MYC inhibitor, these cells exhibited a significant reduction in cell proliferation and a decrease in JunD target genes. Our data suggest that JunD-mediated cell proliferation involves MYC signaling and inhibiting its target genes may be an effective approach to blocking prostate carcinogenesis Citation Format: Bethtrice Thompson (Elliott), Ana Cecilia Millena, Lilya Matyunina, Mengnan Zhang, Jin Zou, Guangdi Wang, Qiang Zhang, Nathan Bowen, Vanessa Eaton, Gabrielle Webb, Shadyra Thompson, John McDonald, Shafiq Khan. JunD-induced cell proliferation requires MYC signaling in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4306.

Peroxidasin (PXDN), a human homolog of Drosophila PXDN, belongs to the family of heme peroxidases and has been found to promote oxidative stress in cardiovascular tissue, however, its role in prostate cancer has not been previously elucidated. We hypothesized that PXDN promotes prostate cancer progression via regulation of metabolic and oxidative stress pathways. We analyzed PXDN expression in prostate tissue by immunohistochemistry and found increased PXDN expression with prostate cancer progression as compared to normal tissue or cells. PXDN knockdown followed by proteomic analysis revealed an increase in oxidative stress, mitochondrial dysfunction and gluconeogenesis pathways. Additionally, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics confirmed that PXDN knockdown induced global reprogramming associated with increased oxidative stress and decreased nucleotide biosynthesis. We further demonstrated that PXDN knockdown led to an increase in reactive oxygen species (ROS) associated with decreased cell viability and increased apoptosis. Finally, PXDN knockdown decreased colony formation on soft agar. Overall, the data suggest that PXDN promotes progression of prostate cancer by regulating the metabolome, more specifically, by inhibiting oxidative stress leading to decreased apoptosis. Therefore, PXDN may be a biomarker associated with prostate cancer and a potential therapeutic target.

Background GLI pathogenesis-related 1 (GLIPR1) was originally identified in glioblastomas and its expression was also found to be down-regulated in prostate cancer. Functional studies revealed both growth suppression and proapoptotic activities for GLIPR1 in multiple cancer cell lines. GLIPR1’s role in lung cancer has not been investigated. Protein arginine methyltransferase 5 (PRMT5) is a protein arginine methyltransferase and forms a stoichiometric complex with the WD repeat domain 77 (WDR77) protein. Both PRMT5 and WDR77 are essential for growth of lung epithelial and cancer cells. But additional gene products that interact genetically or biochemichally with PRMT5 and WDR77 in the control of lung cancer cell growth are not characterized. Methods DNA microarray and immunostaining were used to detect GLIPR1 expression during lung development and lung tumorigenesis. GLIPR1 expression was also analyzed in the TCGA lung cancer cohort. The consequence of GLIPR1 on growth of lung cancer cells in the tissue culture and lung tumor xenografts in the nude mice was observed. Results We found that GLIPR1 expression is negatively associated with PRMT5/WDR77. GLIPR1 is absent in growing epithelial cells at the early stages of mouse lung development and highly expressed in the adult lung. Expression of GLIPR1 was down-regulated during lung tumorigenesis and its expression suppressed growth of lung cancer cells in the tissue culture and lung tumor xenografts in mice. GLIPR1 regulates lung cancer growth through the V-Erb-B avian erythroblastic leukemia viral oncogene homolog 3 (ErbB3). Conclusions This study reveals a novel pathway that PRMT5/WDR77 regulates GLIPR1 expression to control lung cancer cell growth and GLIPR1 as a potential therapeutic agent for lung cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0508-4) contains supplementary material, which is available to authorized users.

Heterogeneous bioeffects have been reported in previous studies of ultrasound-mediated gene delivery. The goal of this study is to identify the differences between cells that take up plasmid DNA (pDNA) after sonication but are not transfected and cells that similarly take up pDNA but are transfected. We used these findings to select drugs that regulate intracellular processes expected to enhance gene transfection in combination with US.Gene expression among DU145 human prostate cancer cells after ultrasound-mediated transfection was analyzed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Drug treatments suggested by the microarray analysis were combined with US exposure to regulate the corresponding intracellular processes. Cell viability and transfection efficiency were determined by flow cytometry to analyze the effects of US combined with drug treatment.Genes such as GADD45α (growth arrest and DNA-damage inducible, alpha) and Topoisomerase IIα were found to be associated with successful transfection. Drugs that regulate GADD45α and Topoisomerase IIα (e.g., ethyl methanesulfomate, amsacrine and chloroquine) were shown to increase ultrasound-mediated transfection efficiency by up to 2 fold.Among cells with pDNA uptake after sonication, we found that genes are differentially expressed among transfected cells versus non-transfected cells. Regulation of the expression level of GADD45α and TOP2α and other intracellular processes can yield higher efficiency of ultrasound-mediated gene transfection. This suggests that a strategy to increase gene transfection efficiency involving the combination of sonication and regulation of intracellular processes using drugs could further enhance US-mediated gene transfection.

It was previously thought that epigenetic histone modifications of mammalian transposable elements (TEs) serve primarily to defend the genome against deleterious effects associated with their activity. However, we recently showed that, genome-wide, human TEs can also be epigenetically modified in a manner consistent with their ability to regulate host genes. Here, we explore the ability of TE sequences to epigenetically regulate individual human genes by focusing on the histone modifications of promoter sequences derived from TEs. We found 1520 human genes that initiate transcription from within TE-derived promoter sequences. We evaluated the distributions of eight histone modifications across these TE-promoters, within and between the GM12878 and K562 cell lines, and related their modification status with the cell-type specific expression patterns of the genes that they regulate. TE-derived promoters are significantly enriched for active histone modifications, and depleted for repressive modifications, relative to the genomic background. Active histone modifications of TE-promoters peak at transcription start sites and are positively correlated with increasing expression within cell lines. Furthermore, differential modification of TE-derived promoters between cell lines is significantly correlated with differential gene expression. LTR-retrotransposon derived promoters in particular play a prominent role in mediating cell-type specific gene regulation, and a number of these LTR-promoter genes are implicated in lineage-specific cellular functions. The regulation of human genes mediated by histone modifications targeted to TE-derived promoters is consistent with the ability of TEs to contribute to the epigenomic landscape in a way that provides functional utility to the host genome.

Cell specification and tissue formation during embryonic development are precisely controlled by the local concentration and temporal presentation of morphogenic factors. Similarly, pluripotent embryonic stem cells can be induced to differentiate in vitro into specific phenotypes in response to morphogen treatment. Embryonic stem cells (ESCs) are commonly differentiated as 3D spheroids referred to as embryoid bodies (EBs); however, differentiation of cells within EBs is typically heterogeneous and disordered. In this study, we demonstrate that in contrast to soluble morphogen treatment, delivery of morphogenic factors directly within EB microenvironments in a spatiotemporally controlled manner using polymer microspheres yields homogeneous, synchronous and organized ESC differentiation. Degradable PLGA microspheres releasing retinoic acid were incorporated directly within EBs and induced the formation of cystic spheroids uniquely resembling the phenotype and structure of early streak mouse embryos (E6.75), with an exterior of FOXA2+ visceral endoderm enveloping an epiblast-like layer of OCT4+ cells. These results demonstrate that controlled morphogen presentation to stem cells using degradable microspheres more efficiently directs cell differentiation and tissue formation than simple soluble delivery methods and presents a unique route to study the spatiotemporal effects of morphogenic factors on embryonic developmental processes in vitro.

Objectives. Epithelial ovarian carcinomas develop from ovarian surface epithelia that undergo complex differentiation to form distinguishable phenotypes resembling those of the epithelia of the female urogenital regions. While previous studies have implicated regulatory developmental homeobox (HOX) genes in this process, other factors responsible for this differentiation are largely unknown. Aberrant transcriptional expression of PAX8 has been reported in epithelial ovarian cancer, prompting us to initiate the molecular characterization of this master regulatory gene in ovarian cancer development. Methods. Immunohistochemistry, immunoblotting and RT-PCR were used to investigate the presence of PAX8 and its protein products in epithelial ovarian cancer subtypes, normal ovarian surface epithelia, ovarian inclusion cysts and normal endosalpingeal epithelia. Results. In this report, we confirm microarray results indicating that the transcription factor, PAX8, is highly expressed in epithelial ovarian cancer but absent from the precursor ovarian surface epithelia of healthy individuals. Furthermore, we report that PAX8 is localized to the nucleus of non-ciliated epithelia in simple ovarian epithelial inclusion cysts and in three epithelial ovarian cancer subtypes (serous, endometrioid and clear cell). We also determined that PAX8 is expressed in the non-ciliated, secretory cells of healthy fallopian tube mucosal linings but not in the adjacent ciliated epithelia. Conclusion. These findings support the hypothesis that PAX8 plays parallel roles in the development of epithelial ovarian cancer and in the developmental differentiation of coelomic epithelia into endosalpingeal epithelia.

Prostate cancer is a major health burden within the ever-increasingly aging US population. The molecular mechanisms involved in prostate cancer are diverse and heterogeneous. In this context, epigenetic changes, both global and gene specific, are now an emerging alternate mechanism in disease initiation and progression. The three major risk factors in prostate cancer: age, geographic ancestry, and environment are all influenced by epigenetics and additional significant insight is required to gain an understanding of the underlying mechanisms. The androgen receptor and its downstream effector pathways, central to prostate cancer initiation and progression, are subject to a multitude of epigenetic alterations. In this review we focus on the global perspective of epigenetics and the use of recent next-generation sequencing platforms to interrogate epigenetic changes in the prostate cancer genome.

Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target.

The vertebrate Mi-2/NuRD complex is a multi-subunit protein complex containing both histone deacetylase and nucleosome-dependent ATPase subunits. Current models predict that this complex functions primarily in transcriptional repression. Surprisingly, every subunit of this complex presents heterogeneity at the protein and gene level. This raises the intriguing possibility of functional specialization resulting from incorporation of unique gene products into the complex. The MTA (metastasis-associated) proteins represent one class of alternative subunits of the human Mi-2/NuRD complex. The members of this family in human cells are differentially expressed depending on cell type and on physiologic parameters. We summarize evidence supporting the view that the alternative subunits of the complex that have arisen during vertebrate evolution endow unique functional properties.

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.

Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the Western world. The transition of androgen-dependent PCa to castration-resistant (CRPC) is a major clinical manifestation during disease progression and presents a therapeutic challenge. Our studies have shown that genetic ablation of inhibitor of differentiation 4 (Id4), a dominant-negative helix loop helix protein, in mice results in prostatic intraepithelial neoplasia lesions and decreased Nkx3.1 expression without the loss of androgen receptor (Ar) expression. ID4 is also epigenetically silenced in the majority of PCa. However, the clinical relevance and molecular pathways altered by ID4 inactivation in PCa are not known. This study investigates the effect of loss of ID4 in PCa cell lines on tumorigenicity and addresses the underlying mechanism. Stable silencing of ID4 in LNCaP cells (L-ID4) resulted in increased proliferation, migration, invasion, and anchorage-independent growth. An increase in the rate of tumor growth, weight, and volume was observed in L-ID4 xenografts compared with that in the LNCaP cells transfected with nonspecific short hairpin RNA (L+ns) in noncastrated mice. Interestingly, tumors were also observed in castrated mice, suggesting that loss of ID4 promotes CRPC. RNA sequence analysis revealed a gene signature mimicking that of constitutively active AR in L-ID4, which was consistent with gain of de novo steroidogenesis. Prostate-specific antigen expression as a result of persistent AR activation was observed in L-ID4 cells but not in L+ns cells. The results demonstrate that ID4 acts as a tumor suppressor in PCa, and its loss, frequently observed in PCa, promotes CRPC through constitutive AR activation.

Background: Amplified centrosomes in cancers are recently garnering a lot of attention as an emerging hub of diagnostic, prognostic and therapeutic targets. Ovarian adenocarcinomas commonly harbor supernumerary centrosomes that drive chromosomal instability. A centrosome clustering molecule, KIFC1, is indispensable for the viability of extra centrosome-bearing cancer cells, and may underlie progression of ovarian cancers. Methods: Centrosome amplification in low- and high- grade serous ovarian adenocarcinomas was quantitated employing confocal imaging. KIFC1 expression was analyzed in ovarian tumors using publically-available databases. Associated grade, stage and clinical information from these databases were plotted for KIFC1 gene expression values. Furthermore, interactions and functional annotation of KIFC1 and its highly correlated genes were studied using DAVID and STRING 9.1. Results: Clinical specimens of ovarian cancers display robust centrosome amplification and deploy centrosome clustering to execute an error-prone mitosis to enable karyotypic heterogeneity that fosters tumor progression and aggressiveness. Our in silico analyses showed KIFC1 overexpression in human ovarian tumors (n = 1090) and its upregulation associated with tumor aggressiveness utilizing publically-available gene expression databases. KIFC1 expression correlated with advanced tumor grade and stage. Dichotomization of KIFC1 levels revealed a significantly lower overall survival time for patients in high KIFC1 group. Intriguingly, in a matched-cohort of primary (n = 7) and metastatic (n = 7) ovarian samples, no significant differences in KIFC1 expression were detectable, suggesting that high KIFC1 expression may serve as a marker of metastases onset. Nonetheless, KIFC1 levels in both primary and matched metastatic sites were significantly higher compared to normal tissue . Ingenuity based network prediction algorithms combined with pre-established protein interaction networks uncovered several novel cell-cycle related partner genes on the basis of interconnectivity, illuminating the centrosome clustering independent agenda of KIFC1 in ovarian tumor progression. Conclusions: Ovarian cancers display amplified centrosomes, a feature of aggressive tumors. To cope up with the abnormal centrosomal load, ovarian cancer cells upregulate genes like KIFC1 that are known to induce centrosome clustering. Our data underscore KIFC1 as a putative biomarker that predicts worse prognosis, poor overall survival and may serve as a potential marker of onset of metastatic dissemination in ovarian cancer patients.

Retrotransposons are the most abundant and widespread class of eukaryotic transposable elements. The recent genome sequencing ofCaenorhabditis elegans has provided an unprecedented opportunity to analyze the evolutionary relationships among the entire complement of retrotransposons within a multicellular eukaryotic organism. In this article we report the results of an analysis of retroviral-like long terminal repeat retrotransposons in C. elegans that indicate that this class of elements may be even more abundant and divergent than previously expected. The unexpected presence, in C. elegans, of an element displaying a number of characteristics previously thought to be unique to vertebrate retroviruses suggests an ancient lineage for this important class of infectious agents.

Since LTR retrotransposons and retroviruses are especially prone to regional duplications and recombination events, these viral-like systems may be especially conducive to the evolution of closely spaced combinatorial regulatory motifs. Using the Drosophila copia LTR retrotransposon as a model, we show that a regulatory region contained within the element's untranslated leader region (ULR) consists of multiple copies of an 8 bp motif (TTGTGAAA) with similarity to the core sequence of the SV40 enhancer. Naturally occurring variation in the number of these motifs is correlated with the enhancer strength of the ULR. Our results indicate that inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and perhaps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to play a role in regulatory evolution.

Increasing evidence supports the existence of a subpopulation of cancer cells capable of self-renewal and differentiation into diverse cell lineages. These cancer stem-like or cancer-initiating cells (CICs) also demonstrate resistance to chemo- and radiotherapy and may function as a primary source of cancer recurrence. We report here on the isolation and in vitro propagation of multicellular ovarian cancer spheroids from a well-established ovarian cancer cell line (OVCAR-3). The spheroid-derived cells (SDCs) display self-renewal potential, the ability to produce differentiated progeny, and increased expression of genes previously associated with CICs. SDCs also demonstrate higher invasiveness, migration potential, and enhanced resistance to standard anticancer agents relative to parental OVCAR-3 cells. Furthermore, SDCs display up-regulation of genes associated with epithelial-to-mesenchymal transition (EMT), anticancer drug resistance and/or decreased susceptibility to apoptosis, as well as, down-regulation of genes typically associated with the epithelial cell phenotype and pro-apoptotic genes. Pathway and biological process enrichment analyses indicate significant differences between the SDCs and precursor OVCAR-3 cells in TGF-beta-dependent induction of EMT, regulation of lipid metabolism, NOTCH and Hedgehog signaling. Collectively, our results indicate that these SDCs will be a useful model for the study of ovarian CICs and for the development of novel CIC-targeted therapies.

Experimentally characterized enhancer regions have previously been shown to display specific patterns of enrichment for several different histone modifications. We modelled these enhancer chromatin profiles in the human genome and used them to guide the search for novel enhancers derived from transposable element (TE) sequences. To do this, a computational approach was taken to analyze the genome-wide histone modification landscape characterized by the ENCODE project in two human hematopoietic cell types, GM12878 and K562. We predicted the locations of 2,107 and 1,448 TE-derived enhancers in the GM12878 and K562 cell lines respectively. A vast majority of these putative enhancers are unique to each cell line; only 3.5% of the TE-derived enhancers are shared between the two. We evaluated the functional effect of TE-derived enhancers by associating them with the cell-type specific expression of nearby genes, and found that the number of TE-derived enhancers is strongly positively correlated with the expression of nearby genes in each cell line. Furthermore, genes that are differentially expressed between the two cell lines also possess a divergent number of TE-derived enhancers in their vicinity. As such, genes that are up-regulated in the GM12878 cell line and down-regulated in K562 have significantly more TE-derived enhancers in their vicinity in the GM12878 cell line and vice versa. These data indicate that human TE-derived sequences are likely to be involved in regulating cell-type specific gene expression on a broad scale and suggest that the enhancer activity of TE-derived sequences is mediated by epigenetic regulatory mechanisms.

Long terminal repeat (LTR) retrotransposons, the most abundant genomic components in flowering plants, are classifiable into autonomous and nonautonomous elements based on their structural completeness and transposition capacity. It has been proposed that selection is the major force for maintaining sequence (e.g., LTR) conservation between nonautonomous elements and their autonomous counterparts. Here, we report the structural, evolutionary, and expression characterization of a giant retrovirus-like soybean (Glycine max) LTR retrotransposon family, SNARE. This family contains two autonomous subfamilies, SAREA and SAREB, that appear to have evolved independently since the soybean genome tetraploidization event ∼13 million years ago, and a nonautonomous subfamily, SNRE, that originated from SAREA. Unexpectedly, a subset of the SNRE elements, which amplified from a single founding SNRE element within the last ∼3 million years, have been dramatically homogenized with either SAREA or SAREB primarily in the LTR regions and bifurcated into distinct subgroups corresponding to the two autonomous subfamilies. We uncovered evidence of region-specific swapping of nonautonomous elements with autonomous elements that primarily generated various nonautonomous recombinants with LTR sequences from autonomous elements of different evolutionary lineages, thus revealing a molecular mechanism for the enhancement of preexisting partnership and the establishment of new partnership between autonomous and nonautonomous elements.

BackgroundAccumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. The serous subtype of ovarian adenocarcinoma in humans has been hypothesized to arise from at least two possible classes of progenitor cells: the ovarian surface epithelia (OSE) and/or an as yet undefined class of progenitor cells residing in the distal end of the fallopian tube.MethodsComparative gene expression profiling analyses were carried out on OSE removed from the surface of normal human ovaries and ovarian cancer epithelial cells (CEPI) isolated by laser capture micro-dissection (LCM) from human serous papillary ovarian adenocarcinomas. The results of the gene expression analyses were randomly confirmed in paraffin embedded tissues from ovarian adenocarcinoma of serous subtype and non-neoplastic ovarian tissues using immunohistochemistry. Differentially expressed genes were analyzed using gene ontology, molecular pathway, and gene set enrichment analysis algorithms.ResultsConsistent with multipotent capacity, genes in pathways previously associated with adult stem cell maintenance are highly expressed in ovarian surface epithelia and are not expressed or expressed at very low levels in serous ovarian adenocarcinoma. Among the over 2000 genes that are significantly differentially expressed, a number of pathways and novel pathway interactions are identified that may contribute to ovarian adenocarcinoma development.ConclusionsOur results are consistent with the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as the origin of ovarian adenocarcinoma. While our findings do not rule out the possibility that ovarian cancers may also arise from other sources, they areinconsistentwith claims that ovarian surface epithelia cannot serve as the origin of ovarian cancer initiating cells.

Transposable elements (TEs) can donate regulatory sequences that help to control the expression of human genes. The oncogene c-Myc is a promiscuous transcription factor that is thought to regulate the expression of hundreds of genes. We evaluated the contribution of TEs to the c-Myc regulatory network by searching for c-Myc binding sites derived from TEs and by analyzing the expression and function of target genes with nearby TE-derived c-Myc binding sites. There are thousands of TE sequences in the human genome that are bound by c-Myc. A conservative analysis indicated that 816-4564 of these TEs contain canonical c-Myc binding site motifs. c-Myc binding sites are over-represented among sequences derived from the ancient TE families L2 and MIR, consistent with their preservation by purifying selection. Genes associated with TE-derived c-Myc binding sites are co-expressed with each other and with c-Myc. A number of these putative TE-derived c-Myc target genes are differentially expressed between Burkitt's lymphoma cells versus normal B cells and encode proteins with cancer-related functions. Despite several lines of evidence pointing to their regulation by c-Myc and relevance to cancer, the set of genes identified as TE-derived c-Myc targets does not significantly overlap with two previously characterized c-Myc target gene sets. These data point to a substantial contribution of TEs to the regulation of human genes by c-Myc. Genes that are regulated by TE-derived c-Myc binding sites appear to form a distinct c-Myc regulatory subnetwork.

Background Although it has long been appreciated that ovarian carcinoma subtypes (serous, clear cell, endometrioid, and mucinous) are associated with different natural histories, most ovarian carcinoma biomarker studies and current treatment protocols for women with this disease are not subtype specific. With the emergence of high-throughput molecular techniques, distinct pathogenetic pathways have been identified in these subtypes. We examined variation in biomarker expression rates between subtypes, and how this influences correlations between biomarker expression and stage at diagnosis or prognosis. Methods and Findings In this retrospective study we assessed the protein expression of 21 candidate tissue-based biomarkers (CA125, CRABP-II, EpCam, ER, F-Spondin, HE4, IGF2, K-Cadherin, Ki-67, KISS1, Matriptase, Mesothelin, MIF, MMP7, p21, p53, PAX8, PR, SLPI, TROP2, WT1) in a population-based cohort of 500 ovarian carcinomas that was collected over the period from 1984 to 2000. The expression of 20 of the 21 biomarkers differs significantly between subtypes, but does not vary across stage within each subtype. Survival analyses show that nine of the 21 biomarkers are prognostic indicators in the entire cohort but when analyzed by subtype only three remain prognostic indicators in the high-grade serous and none in the clear cell subtype. For example, tumor proliferation, as assessed by Ki-67 staining, varies markedly between different subtypes and is an unfavourable prognostic marker in the entire cohort (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.2%–2.4%) but is not of prognostic significance within any subtype. Prognostic associations can even show an inverse correlation within the entire cohort, when compared to a specific subtype. For example, WT1 is more frequently expressed in high-grade serous carcinomas, an aggressive subtype, and is an unfavourable prognostic marker within the entire cohort of ovarian carcinomas (RR 1.7, 95% CI 1.2%–2.3%), but is a favourable prognostic marker within the high-grade serous subtype (RR 0.5, 95% CI 0.3%–0.8%). Conclusions The association of biomarker expression with survival varies substantially between subtypes, and can easily be overlooked in whole cohort analyses. To avoid this effect, each subtype within a cohort should be analyzed discretely. Ovarian carcinoma subtypes are different diseases, and these differences should be reflected in clinical research study design and ultimately in the management of ovarian carcinoma., David Huntsman and colleagues describe the associations between biomarker expression patterns and survival in different ovarian cancer subtypes. They suggest that the management of ovarian cancer should reflect differences between these subtypes., Editors' Summary Background. Every year, about 200,000 women develop ovarian cancer and more than 100,000 die from the disease. Ovarian epithelial cancer (carcinoma) occurs when epithelial cells from the ovary or fallopian tube acquire mutations or equivalent changes that allow them to grow uncontrollably within one of the ovaries (two small organs in the pelvis that produce eggs) and acquire the potential to spread around the body (metastasize). While the cancer is confined to the ovaries, cancer specialists call this stage I disease; 70%–80% of women diagnosed with stage I ovarian cancer survive for at least 5 y. However, only a fifth of ovarian cancers are diagnosed at this stage; in the majority of patients the cancer has spread into the pelvis (stage II disease), into the peritoneal cavity (the space around the gut, stomach, and liver; stage III disease), or metastasized to distant organs such as brain (stage IV disease). This peritoneal spread might be associated with often only vague abdominal pain and mild digestive disturbances. Patients with advanced-stage ovarian carcinoma are treated with a combination of surgery and chemotherapy but, despite recent advances in treatment, only 15% of women diagnosed with stage IV disease survive for 5 y. Why Was This Study Done? Although it is usually regarded as a single disease, there are actually several distinct subtypes of ovarian carcinoma. These are classified according to their microscopic appearance as high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous ovarian carcinomas. These subtypes develop differently and respond differently to chemotherapy. Yet scientists studying ovarian carcinoma usually regard this cancer as a single entity, and current treatment protocols for the disease are not subtype specific. Might better progress be made toward understanding ovarian carcinoma and toward improving its treatment if each subtype were treated as a separate disease? Why are some tumors confined to the ovary, whereas the majority spread beyond the ovary at time of diagnosis? In this study, the researchers address these questions by asking whether correlations between the expression of “biomarkers” (molecules made by cancer cells that can be used to detect tumors and to monitor treatment effectiveness) and the stage at diagnosis or length of survival can be explained by differential biomarker expression between different subtypes of ovarian carcinoma. They also address the question of whether early stage and late stage ovarian carcinomas are fundamentally different. What Did the Researchers Do and Find? The researchers measured the expression of 21 candidate protein biomarkers in 500 ovarian carcinoma samples collected in British Columbia, Canada, between 1984 and 2000. For 20 of the biomarkers, the fraction of tumors expressing the biomarker varied significantly between ovarian carcinoma subtypes. Considering all the tumors together, ten biomarkers had different expression levels in early and late stage tumors. However, when each subtype was considered separately, the expression of none of the biomarkers varied with stage. When the researchers asked whether the expression of any of the biomarkers correlated with survival times, they found that nine biomarkers were unfavorable indicators of outcome when all the tumors were considered together. That is, women whose tumors expressed any of these biomarkers had a higher risk of dying from ovarian cancer than women whose tumors did not express these biomarkers. However, only three biomarkers were unfavorable indicators for high-grade serous carcinomas considered alone and the expression of a biomarker called WT1 in this subtype of ovarian carcinoma is associated with a lower risk of dying. Similarly, expression of the biomarker Ki-67 was an unfavorable prognostic indicator when all the tumors were considered, but was not a prognostic indicator for any individual subtype. What Do These Findings Mean? These and other findings indicate that biomarker expression is more strongly associated with ovarian carcinoma subtype than with stage. In other words, biomarker expression is constant from early to late stage, but only within a given subtype. Second, the association of biomarker expression with survival varies between subtypes, hence lumping all subtypes together can yield misleading results. Although these findings need confirming in more tumor samples, they support the view that ovarian carcinoma subtypes are different diseases. In practical terms, therefore, these findings suggest that better ways to detect and treat ovarian cancer are more likely to be found if future biomarker studies and clinical research studies investigate each subtype of ovarian carcinoma separately rather than grouping them all together. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050232. The US National Cancer Institute provides a brief description of what cancer is and how it develops and information on all aspects of ovarian cancer for patients and professionals. It also provides a fact sheet on tumor markers (in English and Spanish) The UK charity Cancerbackup provides general information about cancer and more specific information about ovarian cancer, including tumor staging

Background Certain endogenous metabolites can influence the rate of cancer cell growth. For example, diacylglycerol, ceramides and sphingosine, NAD+ and arginine exert this effect by acting as signaling molecules, while carrying out other important cellular functions. Metabolites can also be involved in the control of cell proliferation by directly regulating gene expression in ways that are signaling pathway-independent, e.g. by direct activation of transcription factors or by inducing epigenetic processes. The fact that metabolites can affect the cancer process on so many levels suggests that the change in concentration of some metabolites that occurs in cancer cells could have an active role in the progress of the disease. Results CoMet, a fully automated Computational Metabolomics method to predict changes in metabolite levels in cancer cells compared to normal references has been developed and applied to Jurkat T leukemia cells with the goal of testing the following hypothesis: Up or down regulation in cancer cells of the expression of genes encoding for metabolic enzymes leads to changes in intracellular metabolite concentrations that contribute to disease progression. All nine metabolites predicted to be lowered in Jurkat cells with respect to lymphoblasts that were examined (riboflavin, tryptamine, 3-sulfino-L-alanine, menaquinone, dehydroepiandrosterone, α-hydroxystearic acid, hydroxyacetone, seleno-L-methionine and 5,6-dimethylbenzimidazole), exhibited antiproliferative activity that has not been reported before, while only two (bilirubin and androsterone) of the eleven tested metabolites predicted to be increased or unchanged in Jurkat cells displayed significant antiproliferative activity. Conclusion These results: a) demonstrate that CoMet is a valuable method to identify potential compounds for experimental validation, b) indicate that cancer cell metabolism may be regulated to reduce the intracellular concentration of certain antiproliferative metabolites, leading to uninhibited cellular growth and c) suggest that many other endogenous metabolites with important roles in carcinogenesis are awaiting discovery.

Gene expression profiles of malignant tumors surgically removed from ovarian cancer patients pre-treated with chemotherapy (neo-adjuvant) prior to surgery group into two distinct clusters. One group clusters with carcinomas from patients not pre-treated with chemotherapy prior to surgery (C-L), while the other clusters with non-malignant adenomas (A-L). We show here that although the C-L cluster is preferentially associated with p53 loss-of-function (LOF) mutations, the C-L cluster cancer patients display a more favorable clinical response to chemotherapy as evidenced by enhanced long-term survivorships. Our results support a model whereby p53 mediated cell-cycle-arrest/DNA repair serves as a barrier to optimal chemotherapeutic treatment of ovarian and perhaps other carcinomas and suggest that inhibition of p53 during chemotherapy may enhance clinical outcome.

Background Aberrant methylation of gene promoter regions has been linked to changes in gene expression in cancer development and progression. Genes associated with CpG islands (CGIs) are especially prone to methylation, but not all CGI-associated genes display changes in methylation patterns in cancers. Results In order to identify genes subject to regulation by methylation, we conducted gene expression profile analyses of an ovarian cancer cell line (OVCAR-3) before and after treatment with the demethylating agent 5-aza-deoxycytidine (5-aza-dC). An overlapping subset of these genes was found to display significant differences in gene expression between normal ovarian surface epithelial cells and malignant cells isolated from ovarian carcinomas. While 40% of all human genes are associated with CGIs, > 94% of the overlapping subset of genes is associated with CGIs. The predicted change in methylation status of genes randomly selected from the overlapping subset was experimentally verified. Conclusion We conclude that correlating genes that are upregulated in response to 5-aza-dC treatment of cancer cell lines with genes that are down-regulated in cancer cells may be a useful method to identify genes experiencing epigenetic-mediated changes in expression over cancer development.

The activity of transposable elements (TEs) has had a profound impact on the evolution of eukaryotic genomes. Once thought to be purely selfish genomic entities, TEs are now recognized to occupy a continuum of relationships, ranging from parasitic to mutualistic, with their host genomes. One of the many ways that TEs contribute to the function and evolution of the genomes in which they reside is through the donation of host protein coding sequences (CDSs). In this chapter, we will describe several notable examples of eukaryotic host CDSs that are derived from TEs. Despite the existence of a number of such well-established cases, the overall extent and significance of this phenomenon remains a matter of controversy. Genome-scale computational analyses have yielded vastly different estimates for the fraction of host CDSs that are derived from TEs. We explain how these seemingly contradictory findings are the result of specific ascertainment biases introduced by the different methods used to detect TE-related sequences. In light of this problem, we propose a comprehensive and systematic framework for definitively characterizing the contribution of TEs to eukaryotic CDSs.

Human endogenous retroviruses (HERVs) make up approximately 8.3% of the human genome (12). HERVs have previously been classified into 31 distinct families based upon sequence alignment of reverse transcriptase (RT) and envelope domains and subsequent phylogenetic analyses (1, 9, 16). Using the data mining program LTR_STRUC (13) in conjunction with conventional sequence homology techniques, we recently completed an analysis of chimpanzee long terminal repeat (LTR) retrotransposon families (unpublished data). Since LTR_STRUC searches for LTR retrotransposons based on structure (e.g., the presence of LTRs, target site duplications, tRNA binding sites, etc.) rather than homology, elements can be identified that go undetected in traditional BLAST searches. We identified nine chimpanzee LTR retrotransposon families that are orthologous to HERV families not previously identified. These nine newly discovered HERV families are described and characterized in this letter. LTR retrotransposons and retroviruses are grouped into three major classes (14). Class I contains elements related to gammaretroviruses, class II elements are related to betaretroviruses, and class III elements are related to spumaviruses. The RT-based phylogeny indicates that all the newly identified HERVs described here are class I elements (Fig. ​(Fig.1).1). The detailed characteristics of each of the newly discovered HERV families are presented in Table ​Table11 and Table ​Table2.2. All are low-abundance families, being composed of only one to seven full-length members with low homology to previously identified HERVs. This may, in part, explain why they have not been previously identified. The newly discovered full-length elements are of standard HERV length (7,198 to 10,675 bp with 359- to 682-bp LTRs) and display typically sized target site duplications (4 or 5 bp). With the exception of a few mutated copies, the newly identified elements have the same canonical dinucleotides terminating the LTRs as previously characterized HERVs (TG/CA). Since LTR_STRUC can only identify elements having two LTRs, we conducted BLAST searches by using identified full-length elements as query sequences to identify solo LTRs and other fragmented elements. Consistent with what has been reported previously for other HERV families (15), we have found that each of the newly identified families is represented by significantly more solo LTRs and fragmented sequences than full-length elements (Table ​(Table11). FIG. 1. Unrooted RT-based neighbor-joining tree of human LTR retrotransposon families. The phylogenetic tree is built from the DNA sequence (using MEGA3 software [11]) of the RTs taken from all the members of the newly identified HERV families together with representative ... TABLE 1. Representative elements of human LTR retrotransposon families characterized in this study TABLE 2. Characteristics of human LTR retrotransposon families identified in this study Because HERV LTRs are synthesized from the same RNA template during reverse transcription, they are identical in sequence at the time of integration (2). Using a primate pseudogene nucleotide substitution rate of 0.16% divergence/million years (5, 8, 10), the relative integration time or age of any full-length HERV can be estimated from the level of sequence divergence existing between the element's 5′ and 3′ LTRs. Using this method, the estimated ages of the new families of HERVs described here range from 18.0 to 49.5 million years, indicating that members of these families have not been transpositionally active in the primate lineage since well before chimpanzees and humans diverged from a common ancestor (6 million years ago) (4). Although caution must be taken when using LTR divergence to estimate the ages of individual elements because of confounding processes such as recombination and conversion (e.g., see references 6 and 7), the method is able to provide useful age estimates, at least to a first approximation (e.g., see reference 3). Our estimated ages of the newly identified human elements fall within the median range of previously described families of HERVs (16). The possible contribution of these newly identified LTR retrotransposons to primate gene or genome evolution is currently under investigation.

The unique properties of eukaryotic DNA modified via methylation of cytosine residues are believed to result from the action of a conserved family of proteins, the MBD family. The prototype member of this family, MeCP2, was isolated independently in two laboratories. One group isolated MeCP2 as a methylated DNA-binding protein, the second as a sequence-specific DNA-binding protein. Multiple lines of evidence suggest that MeCP2 functions in assembly of specialized chromatin architecture. While initial findings pointed to an enzymatic mechanism involving histone modification for transcriptional repression mediated by MeCP2, emerging studies clearly provide exceptions to this model. In a recent study, highly compacted, unique chromatin structures were generated by stoichiometric binding of MeCP2 to model chromatin fibers. These findings support the likelihood that MeCP2 can utilize two independent, but not mutually exclusive, mechanisms to repress transcription: enzymatic and structural mechanisms.

This chapter provides a simple guide for the computational analysis of transposable element (TE) sequences. Web links are provided for a number of sequence analysis applications, and their potential use in the analysis of TE sequences is briefly described. The level of detail provided is intended to be sufficient for a naive user to begin to analyze TE sequences in silico. The emphasis is placed on the identification, retrieval and manipulation of TE sequences. Information is also provided on the evolutionary study of TE sequences including the use phylogenetics programs.

This chapter provides a simple guide for the computational analysis of transposable element (TE) sequences. Web links are provided for a number of sequence analysis applications, and their potential use in the analysis of TE sequences is briefly described. The level of detail provided is intended to be sufficient for a naive user to begin to analyze TE sequences in silico. The emphasis is placed on the identification, retrieval and manipulation of TE sequences. Information is also provided on the evolutionary study of TE sequences including the use phylogenetics programs.

The p24 transmembrane proteins, also known as EMP24/GP25 (endomembrane protein precursor of 24kD (Schimmoller et al., 1995)) proteins, are components of coat protein (COP)-coated vesicles and are present in species as diverse as fungi, plants, flies, worms, and mammals, indicating that they have important conserved functions. Genetic, molecular, and biochemical characterization of these proteins and the loci that encode them has provided insights into their potential cellular roles, including postulated functions in vesicle cargo protein selection and sorting, COPI and COPII vesicle formation and budding, and quality control of proteins that mature through the secretory pathway. Recently, the first mutations in a Drosophila melanogaster p24 gene have been isolated and characterized. These alleles produce an interesting behavioral phenotype in females, affecting their ability to oviposit. This identification and mutant characterization of a p24 locus in Drosophila will pave the way for a better understanding of cell-type-specific functions and interactions among p24 proteins.

Eukaryotic transposable elements are ubiquitous and widespread mobile genetic entities. These elements often make up a substantial fraction of the host genomes in which they reside. For example, approximately 1/2 of the human genome was recently shown to consist of transposable element sequences. There is a growing body of evidence that demonstrates that transposable elements have been major players in genome evolution. A sample of this evidence is reviewed here with an emphasis on the role that transposable elements may have played in driving the evolution of eukaryotic complexity. A number of specific scenarios are presented that implicate transposable elements in the evolution of the complex molecular and cellular machinery that are characteristic of the eukaryotic domain of life.

The recent release of the complete euchromatic genome sequence ofDrosophila melanogaster offers a unique opportunity to explore the evolutionary history of transposable elements (TEs) within the genome of a higher eukaryote. In this report, we describe the annotation and phylogenetic comparison of 178 full-length long terminal repeat (LTR) retrotransposons from the sequenced component of theD. melanogaster genome. We report the characterization of 17 LTR retrotransposon families described previously and five newly discovered element families. Phylogenetically, these families can be divided into three distinct lineages that consist of members from the canonical Copia and Gypsy groups as well as a newly discovered third group containing BEL, mazi, and rooelements. Each family consists of members with average pairwise identities ≥99% at the nucleotide level, indicating they may be the products of recent transposition events. Consistent with the recent transposition hypothesis, we found that 70% (125/178) of the elements (across all families) have identical intra-element LTRs. Using the synonymous substitution rate that has been calculated previously forDrosophila (.016 substitutions per site per million years) and the intra-element LTR divergence calculated here, the average age of the remaining 30% (53/178) of the elements was found to be 137,000 ±89,000 yr. Collectively, these results indicate that many full-length LTR retrotransposons present in the D. melanogaster genome have transposed well after this species diverged from its closest relative Drosophila simulans, 2.3 ± .3 million years ago.

Accumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. The serous subtype of ovarian adenocarcinoma in humans has been hypothesized to arise from at least two possible classes of progenitor cells: the ovarian surface epithelia (OSE) and/or an as yet undefined class of progenitor cells residing in the distal end of the fallopian tube. Comparative gene expression profiling analyses were carried out on OSE removed from the surface of normal human ovaries and ovarian cancer epithelial cells (CEPI) isolated by laser capture microdissection (LCM) from human serous papillary ovarian adenocarcinomas. Differentially expressed genes were analyzed using gene ontology, molecular pathway, and gene set enrichment analysis algorithms. Consistent with pluripotent capacity, genes in pathways previously associated with adult stem cell maintenance are highly expressed in ovarian surface epithelia and are not expressed or expressed at very low levels in serous ovarian adenomcarcinoma. The results of the gene expression analyses were selectively confirmed using immunohistochemistry. Among the over 2000 genes that are significantly differentially expressed, a number of pathways and novel pathway interactions are identified that may contribute to ovarian adenocarcinoma development. Our results are consistent with the hypothesis that human ovarian surface epithelia are pluripotent and capable of serving as the origin of ovarian adenocarcinoma. While our findings do not rule out the possibility that ovarian cancers may also arise from other sources, they are inconsistent with claims that ovarian surface epithelia cannot serve as the origin of ovarian cancer initiating cells. Citation Information: Cancer Res 2009;69(23 Suppl):C3.

The identification and characterization of 42 families of chimpanzee endogenous retroviruses and a comparison to their human orthologs is described., Background Retrotransposons, the most abundant and widespread class of eukaryotic transposable elements, are believed to play a significant role in mutation and disease and to have contributed significantly to the evolution of genome structure and function. The recent sequencing of the chimpanzee genome is providing an unprecedented opportunity to study the functional significance of these elements in two closely related primate species and to better evaluate their role in primate evolution. Results We report here that the chimpanzee genome contains at least 42 separate families of endogenous retroviruses, nine of which were not previously identified. All but two (CERV 1/PTERV1 and CERV 2) of the 42 families of chimpanzee endogenous retroviruses were found to have orthologs in humans. Molecular analysis (PCR and Southern hybridization) of CERV 2 elements demonstrates that this family is present in chimpanzee, bonobo, gorilla and old-world monkeys but absent in human, orangutan and new-world monkeys. A survey of endogenous retroviral positional variation between chimpanzees and humans determined that approximately 7% of all chimpanzee-human INDEL variation is associated with endogenous retroviral sequences. Conclusion Nine families of chimpanzee endogenous retroviruses have been transpositionally active since chimpanzees and humans diverged from a common ancestor. Seven of these transpositionally active families have orthologs in humans, one of which has also been transpositionally active in humans since the human-chimpanzee divergence about six million years ago. Comparative analyses of orthologous regions of the human and chimpanzee genomes have revealed that a significant portion of INDEL variation between chimpanzees and humans is attributable to endogenous retroviruses and may be of evolutionary significance.

Background Dosage compensation in Drosophila is the epigenetic process by which the expression of genes located on the single X-chromosome of males is elevated to equal the expression of X-linked genes in females where there are two copies of the X-chromosome. While epigenetic mechanisms are hypothesized to have evolved originally to silence transposable elements, a connection between transposable elements and the evolution of dosage compensation has yet to be demonstrated. Results We show that transcription of the Drosophila melanogaster copia LTR (long terminal repeat) retrotransposon is significantly down regulated when in the hemizygous state. DNA digestion and chromatin immunoprecipitation (ChIP) analyses demonstrate that this down regulation is associated with changes in chromatin structure mediated by the histone acetyltransferase, MOF. MOF has previously been shown to play a central role in the Drosophila dosage compensation complex by binding to the hemizygous X-chromosome in males. Conclusion Our results are consistent with the hypothesis that MOF originally functioned to silence retrotransposons and, over evolutionary time, was co-opted to play an essential role in dosage compensation in Drosophila.