We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients.
colorectal cancer; targeted therapy; cell surface proteins; somatic mutations
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.
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.
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.
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.
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.
Cancer/testis genes; GAGE; XAGE1; SSX; siRNA; melanoma
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.
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.
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.
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.
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
human; medulloblastoma; CT antigens; RT-PCR; immunohistochemistry
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.
human; tumor antigens; PLAC1; mRNA; tissue distribution; humoral
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.
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.
human; fetal; ovary; CT antigens; immunohistochemistry
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.
This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.
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.
CpG methylation; phenotype; colorectal; cancer; microsatellite instability
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.