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1.  Centrosomal Localisation of the Cancer/Testis (CT) Antigens NY-ESO-1 and MAGE-C1 Is Regulated by Proteasome Activity in Tumour Cells 
PLoS ONE  2013;8(12):e83212.
The Cancer/Testis (CT) antigen family of genes are transcriptionally repressed in most human tissues but are atypically re-expressed in many malignant tumour types. Their restricted expression profile makes CT antigens ideal targets for cancer immunotherapy. As little is known about whether CT antigens may be regulated by post-translational processing, we investigated the mechanisms governing degradation of NY-ESO-1 and MAGE-C1 in selected cancer cell lines. Inhibitors of proteasome-mediated degradation induced the partitioning of NY-ESO-1 and MAGE-C1 into a detergent insoluble fraction. Moreover, this treatment also resulted in increased localisation of NY-ESO-1 and MAGE-C1 at the centrosome. Despite their interaction, relocation of either NY-ESO-1 or MAGE-C1 to the centrosome could occur independently of each other. Using a series of truncated fragments, the regions corresponding to NY-ESO-191-150 and MAGE-C1900-1116 were established as important for controlling both stability and localisation of these CT antigens. Our findings demonstrate that the steady state levels of NY-ESO-1 and MAGE-C1 are regulated by proteasomal degradation and that both behave as aggregation-prone proteins upon accumulation. With proteasome inhibitors being increasingly used as front-line treatment in cancer, these data raise issues about CT antigen processing for antigenic presentation and therefore immunogenicity in cancer patients.
doi:10.1371/journal.pone.0083212
PMCID: PMC3858345  PMID: 24340093
2.  Humanized Lewis-Y specific antibody based delivery of STAT3 siRNA 
ACS chemical biology  2011;6(9):10.1021/cb200176v.
The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Ley), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Ley expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Ley-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a non-covalent construct based on (D-Arginine)9 (9r) modified hu3S193. Ley-specific binding and internalization of both the covalent and non-covalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the non-covalent system led to efficient STAT3 silencing in Ley-positive cancer cells (A431), but not in Ley-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified non-covalent construct, induced ~70% STAT3 knockdown at sub-micromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ~50% inhibition of cell proliferation of Ley-positive cells. Non-covalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.
doi:10.1021/cb200176v
PMCID: PMC3831028  PMID: 21766840
3.  Collaborative research networks work 
Journal of Clinical Investigation  2003;112(4):468-471.
Brazil was heralded for completion of the first genome sequence of a plant pathogen following the development of a virtual research center — a collaborative network of laboratories throughout the state of São Paulo, drawing on the expertise of a dispersed and diverse scientific community and on investment from both the government and the private sector. Strategies key to the success of this model are discussed here in the context of continuing collaborative scientific endeavors in both developed and developing countries.
doi:10.1172/JCI200319520
PMCID: PMC171397  PMID: 12925684
4.  Alternative Spliced Transcripts as Cancer Markers 
Disease Markers  2002;17(2):67-75.
Eukaryotic mRNAs are transcribed as precursors containing their intronic sequences. These are subsequently excised and the exons are spliced together to form mature mRNAs. This process can lead to transcript diversification through the phenomenon of alternative splicing. Alternative splicing can take the form of one or more skipped exons, variable position of intron splicing or intron retention. The effect of alternative splicing in expanding protein repertoire might partially underlie the apparent discrepancy between gene number and the complexity of higher eukaryotes. It is likely that more than 50% form. Many cancer-associated genes, such as CD44 and WT1 are alternatively spliced. Variation of the splicing process occurs during tumor progression and may play a major role in tumorigenesis. Furthermore, alternatively spliced transcripts may be extremely useful as cancer markers, since it appears likely that there may be striking contrasts in usage of alternatively spliced transcript variants between normal and tumor tissue than in alterations in the general levels of gene expression.
doi:10.1155/2001/184856
PMCID: PMC3851395  PMID: 11673653
5.  Effects of CT-Xp Gene Knock down in Melanoma Cell Lines 
Oncotarget  2013;4(4):531-541.
Cancer/testis (CT) genes are encoded by genes that are normally expressed only in the human germ line but which are activated in various malignancies. CT proteins are frequently immunogenic in cancer patients and their expression is highly restricted to tumors. They are thus important targets for anticancer immunotherapy. In several different tumor types, the expression of CT-X genes is associated with advanced disease and poor outcome, indicating that their expression might contribute to tumorigenesis. CT-X genes encoding members of the MAGE protein family on Xq28 have been shown to potentially influence the tumorigenic phenotype. We used small interfering RNA (siRNA) to investigate whether CT-X mapping to the short arm of the X-chromosome might also have tumorigenic properties and therefore be potentially targeted by functional inhibitors in a therapeutic setting. siRNAs specific to GAGE, SSX and XAGE1 were used in cell proliferation, migration and cell survival assays using cell lines derived from melanoma, a tumor type known to present high frequencies of expression of CT antigens. We found that of these, those specific to GAGE and XAGE1 most significantly impeded melanoma cell migration and invasion and those specific to SSX4 and XAGE1 decreased the clonogenic survival of melanoma cells. Our results suggest that GAGE, XAGE1 and SSX4 might each have a role in tumor progression and are possible therapeutic targets for the treatment of melanoma and other malignancies.
PMCID: PMC3720601  PMID: 23625514
Cancer/testis genes; GAGE; XAGE1; SSX; siRNA; melanoma
6.  Differential Evolution of MAGE Genes Based on Expression Pattern and Selection Pressure 
PLoS ONE  2012;7(10):e48240.
Starting from publicly-accessible datasets, we have utilized comparative and phylogenetic genome analyses to characterize the evolution of the human MAGE gene family. Our characterization of genomic structures in representative genomes of primates, rodents, carnivora, and macroscelidea indicates that both Type I and Type II MAGE genes have undergone lineage-specific evolution. The restricted expression pattern in germ cells of Type I MAGE orthologs is observed throughout evolutionary history. Unlike Type II MAGEs that have conserved promoter sequences, Type I MAGEs lack promoter conservation, suggesting that epigenetic regulation is a central mechanism for controlling their expression. Codon analysis shows that Type I but not Type II MAGE genes have been under positive selection. The combination of genomic and expression analysis suggests that Type 1 MAGE promoters and genes continue to evolve in the hominin lineage, perhaps towards functional diversification or acquiring additional specific functions, and that selection pressure at codon level is associated with expression spectrum.
doi:10.1371/journal.pone.0048240
PMCID: PMC3484994  PMID: 23133577
7.  CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens 
Nucleic Acids Research  2008;37(Database issue):D816-D819.
The potency of the immune response has still to be harnessed effectively to combat human cancers. However, the discovery of T-cell targets in melanomas and other tumors has raised the possibility that cancer vaccines can be used to induce a therapeutically effective immune response against cancer. The targets, cancer-testis (CT) antigens, are immunogenic proteins preferentially expressed in normal gametogenic tissues and different histological types of tumors. Therapeutic cancer vaccines directed against CT antigens are currently in late-stage clinical trials testing whether they can delay or prevent recurrence of lung cancer and melanoma following surgical removal of primary tumors. CT antigens constitute a large, but ill-defined, family of proteins that exhibit a remarkably restricted expression. Currently, there is a considerable amount of information about these proteins, but the data are scattered through the literature and in several bioinformatic databases. The database presented here, CTdatabase (http://www.cta.lncc.br), unifies this knowledge to facilitate both the mining of the existing deluge of data, and the identification of proteins alleged to be CT antigens, but that do not have their characteristic restricted expression pattern. CTdatabase is more than a repository of CT antigen data, since all the available information was carefully curated and annotated with most data being specifically processed for CT antigens and stored locally. Starting from a compilation of known CT antigens, CTdatabase provides basic information including gene names and aliases, RefSeq accession numbers, genomic location, known splicing variants, gene duplications and additional family members. Gene expression at the mRNA level in normal and tumor tissues has been collated from publicly available data obtained by several different technologies. Manually curated data related to mRNA and protein expression, and antigen-specific immune responses in cancer patients are also available, together with links to PubMed for relevant CT antigen articles.
doi:10.1093/nar/gkn673
PMCID: PMC2686577  PMID: 18838390
8.  Cancer-testis (CT) antigen expression in medulloblastoma 
Medulloblastoma is the most common childhood malignant tumor of the central nervous system. Treatment of medulloblastoma requires harmful therapy and nevertheless carries a poor prognosis. Due to their presence in various cancers and their limited expression in normal tissues, CT antigens are ideal vaccine targets for tumor immunotherapy. CT antigens, such as MAGE and NY-ESO-1, have been employed in clinical trials in various malignancies but little is known about their presence in medulloblastoma. We analyzed 25 medulloblastomas for the expression of a panel of CT antigens by RT-PCR and immunohistochemistry. Messenger RNA expression in the samples was as follows: GAGE 64%, MAGEA3/6 56%, SYCP1 44%, SLCO6A1 32%, MAGEC1 28%, MAGEC2 28%, MAGEA4 28%, NY-ESO-1 20%, MAGEA1 16%, and TPTE 0%. All cases except one (96%) were positive for mRNA expression of at least one CT gene. However, CT antigen expression was scarce on a protein level. Immunoreaction to monoclonal antibody E978 (NY-ESO-1) was negative in all cases; MA454 (MAGEA1), 57B (MAGEA4), M3H67 (MAGEA3/6), CT10#5 (MAGEC2) and #23 (GAGE) were each positive in 1 case, while the highest incidence of positive immunostaining, albeit heterogeneous, was seen with CT7-33 (MAGEC1) in 3 out of the 25 cases. The absence of correlation between mRNA and protein expression in medulloblastoma has not been observed in other tumors and further studies addressing the biology of CT antigens are necessary to investigate the present discrepant results.
PMCID: PMC2935780  PMID: 18426187
human; medulloblastoma; CT antigens; RT-PCR; immunohistochemistry
9.  PLAC1, a trophoblast-specific cell surface protein, is expressed in a range of human tumors and elicits spontaneous antibody responses 
Identification of genes that are upregulated in tumors, and whose normal expression excludes adult somatic tissues but includes germline and/or embryonic tissues, has resulted in a rich variety of cancer antigens that are attractive targets for cancer vaccine and other therapeutic approaches. In the present study, we extended this approach to include genes strongly and restrictively expressed in the placenta by mining publicly available SAGE and EST databases. We identified a number of genes with high expression in placenta and different cancer types but with relatively restricted expression in normal tissues. The gene with the most distinctive expression pattern was found to be PLAC1, which encodes a putative cell surface protein that is highly expressed in placenta, testis, cancer cell lines and lung tumors. Hence we have designated it CT92. We found by ELISA that PLAC1 is immunogenic in a subset of cancer patients and healthy women. Its physical and expression characteristics render it a potential target for both active and passive cancer immunotherapeutic strategies.
PMCID: PMC2935750  PMID: 17983203
human; tumor antigens; PLAC1; mRNA; tissue distribution; humoral immunity
10.  Frequent and specific immunity to the embryonal stem cell–associated antigen SOX2 in patients with monoclonal gammopathy 
Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138− compartment in MGUS. SOX2 expression is also detected in a proportion of CD138+ cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.
doi:10.1084/jem.20062387
PMCID: PMC2118551  PMID: 17389240
11.  Cancer/testis (CT) antigens are expressed in fetal ovary 
Cancer/testis (CT) antigens are named after their expression pattern as they are typically present in various types of tumors and in the germ cells of normal adult testis. Adult ovarian tissue is usually reported to be CT antigen negative. Based on the differences in female versus male gonadal development, the ovarian counterpart of the most predominant CT antigen positive testicular germ cells are not prevalent in the adult ovary. Hence, we analyzed the protein expression of several CT antigens in fetal ovary by immunohistochemistry with various monoclonal antibodies (mAbs) previously generated by our group. The mAbs used were: MA454 (MAGE-A1), M3H67 (MAGE-A3), 57B (MAGE-A4), CT7-33 (CT7/MAGE-C1), and ES121 (NY-ESO-1). All mAbs showed some immunopositivity in fetal ovarian germ cells. The most intense staining was seen with mAbs M3H67, 57B, and CT7-33 during weeks 16 – 23 of gestation. The most prevalent cells stained were oogonia, with only focal staining of oocytes of the primordial follicle. We conclude that CT antigens are regularly expressed in fetal ovarian germ cells and might play an important role in male and female germ cell biology.
PMCID: PMC3077293  PMID: 17217256
human; fetal; ovary; CT antigens; immunohistochemistry
12.  HPtaa database-potential target genes for clinical diagnosis and immunotherapy of human carcinoma 
Nucleic Acids Research  2005;34(Database issue):D607-D612.
Tumor-associated antigens (TAAs) have been the most actively employed targets in the clinical diagnosis and treatment of human carcinoma, such as PSA in the diagnosis of prostate cancer and NY-ESO-1 in the immunotherapy of melanoma and other cancers. However, identification of TAAs has often been hampered by the complicated and laborsome laboratory procedures. In order to accelerate the process of tumor antigen discovery, and thereby improve diagnosis and treatment of human carcinoma, we have made an effort to establish a publicly available Human Potential Tumor Associated Antigen database (HPtaa) with potential TAAs identified by in silico computing (). Tumor specificity was chosen as the core of tumor antigen evaluation, together with other relevant clues. Various platforms of gene expression, including microarray, expressed sequence tag and SAGE data, were processed and integrated by several penalty algorithms. A total of 3518 potential TAAs have been included in the database, which is freely available to academic users. As far as we know, this database is the first one addressing human potential TAAs, and the first one integrating various kinds of expression platforms for one purpose.
doi:10.1093/nar/gkj082
PMCID: PMC1347445  PMID: 16381942
13.  Colorectal Cancer “Methylator Phenotype”: Fact or Artifact?1 
Neoplasia (New York, N.Y.)  2005;7(4):331-335.
Abstract
It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a “CpG island methylated phenotype (CIMP),” characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI). We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF) in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations.
PMCID: PMC1501152  PMID: 15967110
CpG methylation; phenotype; colorectal; cancer; microsatellite instability
14.  Definition of the Gene Content of the Human Genome: The Need for Deep Experimental Verification 
Based on the analysis of the drafts of the human genome sequence, it is being speculated that our species may possess an unexpectedly low number of genes. The quality of the drafts, the impossibility of accurate gene prediction and the lack of sufficient transcript sequence data, however, render such speculations very premature. The complexity of human gene structure requires additional and extensive experimental verification of transcripts that may result in major revisions of these early estimates of the number of human genes.
doi:10.1002/cfg.81
PMCID: PMC2447206  PMID: 18628909
15.  Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual 
Nucleic Acids Research  2011;39(14):6056-6068.
Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein–protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.
doi:10.1093/nar/gkr221
PMCID: PMC3152357  PMID: 21493686
16.  Network-Guided Analysis of Genes with Altered Somatic Copy Number and Gene Expression Reveals Pathways Commonly Perturbed in Metastatic Melanoma 
PLoS ONE  2011;6(4):e18369.
Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression (‘SCNA-genes’) in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.
doi:10.1371/journal.pone.0018369
PMCID: PMC3072964  PMID: 21494657

Results 1-16 (16)