HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer

Background: HAGE protein is a known immunogenic cancer-specific antigen. Methods: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. Results: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGEþ) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGEþexpression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+ did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+ and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+ residual disease (P=0.0003). Conclusions: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC

Genetic Screening of New Genes Responsible for Cellular Adaptation to Hypoxia Using a Genome-Wide shRNA Library

Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress

Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients

FATE and TPTE genes were originally reported to be specifically expressed in the adult testis. We searched for the databases of Unigene and serial analysis of gene expression ( SAGE) implying that these two gene transcripts might also be expressed in tumours. Herein, we demonstrated that FATE and TPTE mRNA transcripts were expressed in different histological types of tumours and normal testis. Both are cancer-testis (CT) antigens and renamed as FATE/BJ-HCC-2 and TPTE/BJ-HCC-5, respectively. Comparison at nucleotide sequence, the FATE/BJ-HCC-2 cDNA, was identical to that of FATE, whereas the TPTE/BJ-HCC-5 was found to have two isoforms in both cancers and testis: one was identical in cDNA sequence to TPTE, encoding a protein of 551 amino acids, and the other variant lacked an exon of 54 bp, encoding a protein of 533 amino acids. The mRNA expression was analysed by RT-PCR and real-time PCR. FATE/BJ-HCC-2 mRNA was detected in 66% ( 41 out of 62) in hepatocellular carcinoma (HCC) samples and 21% ( three out of 14) in colon cancer samples, whereas the TPTE/BJ-HCC-5 mRNA was detected in 39% ( 24 out of 62) and 36% ( five out of 14) in HCC and non-small lung cancer samples, respectively. The recombinant proteins were prepared and the reactivity of allogenic sera to these two antigens was screened. The frequency of antibody response against FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 proteins was 7.3% ( three out of 41) and 25.0% ( six out of 24), respectively, in HCC patients bearing respective gene transcripts. Therefore, FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 are the novel CT antigens capable of eliciting antibody response in cancer patients.OncologySCI(E)PubMed22ARTICLE2291-2978

High Expression of Testes-Specific Protease 50 Is Associated with Poor Prognosis in Colorectal Carcinoma

Testes-specific protease 50 (TSP50) is normally expressed in testes and abnormally expressed in breast cancer, but whether TSP50 is expressed in colorectal carcinoma (CRC) and its clinical significance is unclear. We aimed to detect TSP50 expression in CRC, correlate it with clinicopathological factors, and assess its potential diagnostic and prognostic value. = 0.009).Our data demonstrate that TSP50 is a potential effective indicator of poor survival for CRC patients, especially for those with early-stage tumors

Dead-box proteins: a family affair—active and passive players in RNP-remodeling

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family

Hage et sage : deux nouveaux gènes exprimés préférentiellement dans les tumeurs

Genes MAGE, BAGE, GAGE and LAGE/NY-ESO-1 encode surface antigens that are recognized on tumor cells by autologous cytolytic T lymphocytes. Since these genes are expressed in a significant proportion of tumors from various histological types but nor in normal tissues, the antigens they encode may serve as targets for cancer immunotherapy. Clinical trials are going on melanoma patients and the results, although preliminary, are rather encouraging. In order to identify new genes presenting the same pattern of expression as the MAGE-type genes, i.e. expressed in tumors but not in normal tissues except testis, we applied the Representational Difference Analysis method, that couples subtractive hybridization and PCR amplification of cDNA. We have performed a testis cDNA enrichment in order to test whether genes expressed in testis were also activated in tumors. We observed that most of the cDNA fragments that we obtained were derived from gene expressed only or preferentially in testis. Those genes were not activated in tumors. This suggests that the MAGE-type genes belong to a minor subset of testis-specific genes that are expressed in tumors of various types. We have also enriched cDNA from the sarcoma cell line. LB23-SAR. This allowed us to identify three genes presenting an interesting pattern of expression. Gene SAR35 is highly expressed in a high percentage of tumors from various histological types but the expression of this gene in some normal tissues may prevent its use in immunotherapy. On the other hand, genes SAGE and HAGE present a pattern of expression similar to that of MAGE-type genes: they are not expressed in normal tissues except testis and they are activated in tumors. Because of this tumor-specific expression, genes SAGE en HAGE ought to encode antigens that could be useful for anti-tumoral therapeutic vaccination. The function of these three genes is unknown. However preliminary results and the features common to the protein encoded by gen SAR35 and protein HEC suggest that this gene could be involved in cell proliferation. Gene HAGE encodes a protein that presents all the motifs of the DEAD box proteins, that are putative ATP-dependent RNA helicasesLes gènes MAGE, BAGE, GAGE, et LAGE/NY-ESO-1 codent des antigènes qui sont reconnus à la surface des cellules tumorales par des lymphocytes T cytolytiques autologues. Etant donné que ces gènes sont exprimés dans une proportion significative de tumeurs de différents types histologiques, mais pas dans les tissus sains, les antigènes qu’ils codent peuvent servir de cibles pour une immunothérapie du cancer. Des essais cliniques sont actuellement en cours sur des patients atteints de mélanome et les résultats, bien que préliminaires, sont plutôt encourageants. Afin d’identifier de nouveau gènes présentant le même profil d’expression que les gènes de type MAGE, c’est-à-dire exprimés dans les tumeurs, mais pas dans les tissus normaux à l’exception du testicule, nous avons appliqué la technique de Representational Difference Analysis qui couple hybridation soustractive et amplification d’ADNc afin d’isoler des gènes exprimés de façon différentielle. Nous avons ainsi réalisé un enrichissement d’ADNc de testicule afin de savoir si beacuoup de gènes exprimés dans le testicule étaient également activés dans les tumeurs. Nous avons observé que la plupart des ADNc obtenus provenaient de gènes exprimés uniquement ou préférentiellement dans le testicule. Ces gènes n’étaient pas activés dans les tumeurs. Les gènes de type MAGE semble donc appartenir à un groupe minoriatire de gènes qui sont exprimés à la fois dans le testicule et dans les tumeurs. Nus avons également enrichi l’ADNc de la lignée de sarcome LB23-SAR, ce qui nous a permis d’identifier trosi gènes présentant un profil d’expression intéressant. Le gène SAR35 est fortement exprimé dans un pourcentage élevé de tumeurs de différents types histologiques mais il est également faiblement exprimé dans certains tissus normaux. Par contre, les gènes SAGE et HAGE présentent un profil d’expression similaire à celui des gènes de type MAGE : ils ne sont pas exprimés dans les tissus normaux à l’exception du testicule et ils sont activés dans les tumeurs. Etant donné ce profil d’expression restreint aux tumeurs, les gènes SAGE et HAGE pourraient donc coder des antigènes utiles dans le cadre d’une immunothérapie du cancer. La fonction de ces trois gènes n’est pas connue. Des caractéristiques communes à la protéine codée par le gène SAR35 et à la protéine HEC, ainsi que quelques résultats préliminaires, nous suggèrent que le gène SAR35 pourrait être impliqué dans la prolifération cellulaire. Le gène HAGE code une protéine qui a tous les motifs des protéines DEAD-box, dont certaines sont connues pour avoir une fonction d’ARN hélicase ATP-dépendanteThèse de doctorat en sciences biomédicales -- UCL, 200

Identification of human testis-specific transcripts and analysis of their expression in tumor cells

Tumor-specific antigens recognized by autologous T lymphocytes are encoded by genes, including those of the MAGE, BAGE, and GAGE gene families, that are expressed in a significant fraction of tumors of various types, but not in normal adult tissues, except for testis where they appear to be expressed in germ cells. Because male germ cells are known to express many genes that are not expressed in other normal adult tissues, we wished to determine whether most of these genes are occasionally activated in tumor cells. Representational difference analysis was used to obtain testis-specific transcripts. The expression of 15 testis-specific cDNA sequences was tested by RT-PCR in a series of tumor cell lines. Only one cDNA sequence showed a significant level of expression in some tumor cell lines. Remarkably, this cDNA clone proved to be a new gene of the MAGE family. These results suggest that MAGE, BAGE, and GAGE genes belong to a minor subset of testis-specific genes that is often activated in tumors of various types, whereas most testis-specific genes are either never or very rarely activated in tumors. (C) 1997 Academic Press

DNA Methylation Is the Primary Silencing Mechanism for a Set of Germ Line- and Tumor-Specific Genes with a CpG-Rich Promoter

A subset of male germ line-specific genes, the MAGE-type genes, are activated in many human tumors, where they produce tumor-specific antigens recognized by cytolytic T lymphocytes. Previous studies on gene MAGE-A1 indicated that transcription factors regulating its expression are present in all tumor cell lines whether or not they express the gene. The analysis of two CpG sites located in the promoter showed a strong correlation between expression and demethylation. It was also shown that MAGE-A1 transcription was induced in cell cultures treated with demethylating agent 5′-aza-2′-deoxycytidine. We have now analyzed all of the CpG sites within the 5′ region of MAGE-A1 and show that for all of them, demethylation correlates with the transcription of the gene. We also show that the induction of MAGE-A1 with 5′-aza-2′-deoxycytidine is stable and that in all the cell clones it correlates with demethylation, indicating that demethylation is necessary and sufficient to produce expression. Conversely, transfection experiments with in vitro-methylated MAGE-A1 sequences indicated that heavy methylation suffices to stably repress the gene in cells containing the transcription factors required for expression. Most MAGE-type genes were found to have promoters with a high CpG content. Remarkably, although CpG-rich promoters are classically unmethylated in all normal tissues, those of MAGE-A1 and LAGE-1 were highly methylated in somatic tissues. In contrast, they were largely unmethylated in male germ cells. We conclude that MAGE-type genes belong to a unique subset of germ line-specific genes that use DNA methylation as a primary silencing mechanism