MAGE Gene Family Research Articles

Cancer/testis antigen expression and co-expression patterns in gastroesophageal adenocarcinoma

Gastroesophageal adenocarcinoma (GEAC) poses a significant challenge due to its poor prognosis and limited treatment options. Recently, Cancer/testis antigens (CTAs) have emerged as potential therapy targets due to their high expression in tumor cells and their immunogenic nature. We aimed to explore the expression and co-expression of CTAs in GEAC. We analyzed 63 GEAC patients initially and validated our findings in 329 patients from The Cancer Genome Atlas (TCGA) database. CTA expression was measured after RNA sequencing, while clinical information, including survival outcomes and treatment details, was collected from an institutional database. Co-expression patterns among CTAs were determined using Spearman correlation analysis. The majority of the study cohort were male (87%), Caucasian (94%), and had stage IV disease (64%). CTAs were highly prevalent, ranging from 58 to 19%. The MAGE gene family showed the highest expression, consistent across both cohorts. The correlation matrix revealed a distinct cluster of significantly co-expressed genes, including MAGEA3, NY-ESO-1, and others (0.27 ≤ r ≤ 0.73). Survival analysis revealed that individual CTAs were associated with poorer survival outcomes in patients not receiving immunotherapy while showing potential for improved survival in those undergoing immunotherapy, although these findings lacked robust reliability. Our study provides a comprehensive characterization of CTA expression and co-expression in GEAC. The strong correlation among CTAs like MAGE, NY-ESO-1, and GAGE suggests a potential for therapies targeting multiple CTAs simultaneously. Further research, including prospective trials, is warranted to assess the prognostic value of CTAs and their suitability as therapeutic targets.

Cancer/testis antigen expression and co-expression patterns in Gastroesophageal Adenocarcinoma.

Gastroesophageal adenocarcinoma (GEAC) poses a significant challenge due to its poor prognosis and limited treatment options. Recently, Cancer/testis antigens (CTAs) have emerged as potential therapy targets due to their high expression in tumor cells and their immunogenic nature. We aimed to explore the expression and co-expression of CTAs in GEAC. We analyzed 63 GEAC patients initially and validated our findings in 329 patients from The Cancer Genome Atlas (TCGA) database. CTA expression was measured after RNA sequencing, while clinical information, including survival outcomes and treatment details, was collected from an institutional database. Co-expression patterns among CTAs were determined using Pearson correlation analysis. The majority of the study cohort were male (87%), Caucasian (94%), and had stage IV disease (64%). CTAs were highly prevalent, ranging from 58-19%. The MAGE gene family showed the highest expression, consistent across both cohorts. The correlation matrix revealed a distinct cluster of significantly co-expressed genes, including MAGEA3, NY-ESO-1, and others (0.27 ≤ r ≤ 0.73). Survival analysis revealed that individual CTAs were associated with poorer survival outcomes in patients not receiving immunotherapy while showing potential for improved survival in those undergoing immunotherapy, although these findings lacked robust reliability. Our study provides a comprehensive characterization of CTA expression and co-expression in GEAC. The strong correlation among CTAs like MAGE, NY-ESO-1, and GAGE suggests a potential for therapies targeting multiple CTAs simultaneously. Further research, including prospective trials, is warranted to assess the prognostic value of CTAs and their suitability as therapeutic targets.

Abstract 4880: MAGE gene family expression in pediatric medulloblastoma: frequency and possible therapeutic target

Abstract Background: The melanoma antigen gene family (MAGE) is a group of related genes that were first discovered in malignant melanoma. Among the approximately 40 MAGE genes in the human genome, two thirds are classified as Type I (MAGE-A, MAGE-B, and MAGE-C), referred to also as cancer testis antigens (CTAs), proteins that are restricted to testis but often aberrantly expressed in many adult cancers. Despite their common expression in adult cancers and their oncogenic potential, little is known about their expression and role in pediatric cancers. To determine the frequency of MAGE expression in pediatric medulloblastoma, we measured expression levels of Type I MAGEs in patient tumors. In addition, we examined how MAGE expression in medulloblastoma cells affects their growth and oncogenic potential. Methods: We searched the Children’s Health pathology database for pediatric medulloblastoma between 2008 - 2015 and were able to recover an adequate amount of RNA from formalin-fixed paraffin-embedded tissue samples of 34 patients. The RNA was converted to cDNA using reverse transcriptase, and the relative expression of 23 Type I MAGE genes was measured by qRT-PCR. To determine if MAGE expression is critical for medulloblastoma cell survival we knocked down several MAGEs using siRNAs in two MAGE-expressing medulloblastoma cell lines (D283 and DAOY) and evaluated the effect by cell viability (Alamar Blue and Cell titer Glo), apoptosis assays (Annexin V), and colony formation in soft agar. Results: In 34 medulloblastoma patient samples we found that 10 (29.4%) expressed at least one MAGE gene, and one case had high levels of expression of 10 of the 23 Type I MAGEs. MAGE expression was associated with the non-WNT/SHH (G3/G4) molecular subgroup of tumors in our study. There was no correlation between MAGE expression and histologic subtype. Furthermore, using MAGE-A/-B2 positive medulloblastoma cell lines, we showed that siRNA knock down of multiple MAGE-As and -B2 decreased cell viability by 15 - 80%, increased apoptosis (-B2), and decreased growth of cells in soft agar. Conclusion: Type I MAGE genes are expressed in 30% of pediatric medulloblastoma in our study. In MAGE positive medulloblastoma cells, MAGE genes are important for cell survival and clonogenic growth. The decreased cell survival after MAGE knockdown appears to be related, at least in part, to increased apoptosis. Our results indicate that targeting of MAGEs in medulloblastoma may be a potential therapeutic option. Citation Format: Rebecca R. Collins, Klementina Fon Tacer, Dinesh Rakheja, Patrick Ryan Potts. MAGE gene family expression in pediatric medulloblastoma: frequency and possible therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4880. doi:10.1158/1538-7445.AM2017-4880

MAGEA1 interacts with FBXW7 and regulates ubiquitin ligase-mediated turnover of NICD1 in breast and ovarian cancer cells

Melanoma-associated antigen A1 (MAGEA1) is member of the MAGE gene family that is expressed in male germ line cells and placenta under normal physiological conditions. Although MAGEA1's expression levels have been evaluated as one of the cancer testis (CT) antigens for immunotherapy in melanoma and several other cancers, its functional role and signaling mechanisms are largely unknown. In this study, we examined the functional involvement and signaling mechanisms of MAGEA1 in breast and ovarian cancer cells. Inhibitory effects of MAGEA1 on cell proliferation and migration were observed using both gain-of- (MAGEA1 overexpression) and loss-of- (siRNA interference) function approaches in breast (MCF-7 and MDA-MB-231) and ovarian (SKOV3 and SKOV3ip) cancer cell lines. We revealed a novel interaction between MAGEA1 and the intracellular segment of NOTCH1 receptor (NICD1). MAGEA1 reduced NICD1's stability by promoting the ubiquitin modification of NICD1. MAGEA1 also interacted with FBXW7, subunit of E3 ubiquitin protein ligase complex SCF, and the latter was functionally involved in NICD1 ubiquitination and degradation. In addition, siRNA interference of FBXW7 reversed the inhibitory effect of MAGEA1 on migration and proliferation of MCF-7 and MDA-MB-231 cells. These newly discovered MAGEA1-NICD1 and MAGEA1-FBXW7 interactions have potential clinical implications in breast and ovarian cancer treatment.

Cancer–testis antigen HCA587/MAGE-C2 interacts with BS69 and promotes its degradation in the ubiquitin–proteasome pathway

HCA587, also known as MAGE-C2, belonging to the MAGE gene family which is characterized by a conserved MAGE Homology Domain, is active in various types of tumors and silent in normal tissues except in male germ-line cells. The biological function of HCA587 is largely unknown. To analyze it, we attempted to identify protein partners of HCA587. We immunopurified HCA587-containing complex from HEK293 cells and identified BS69, a potential tumor suppressor, as an associated protein by mass spectrometry, and the following Immunoprecipitation and GST pull-down assays confirmed HCA587 interaction with BS69. Interestingly, overexpression of HCA587 promoted ubiquitination and the proteasomal degradation of BS69 whereas knockdown of endogenous HCA587 increased the protein level of BS69. Consistent with a functional role for BS69 in negatively regulating LMP1-induced NF-κB activation, overexpression of HCA587 resulted in a significant enhancement of LMP1-induced IL-6 production. These data indicate that HCA587 is a new negative regulator of BS69.

Differential Evolution of MAGE Genes Based on Expression Pattern and Selection Pressure

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.

Loss of Maged1 results in obesity, deficits of social interactions, impaired sexual behavior and severe alteration of mature oxytocin production in the hypothalamus

MAGED1, NECDIN and MAGEL2 are members of the MAGE gene family. The latter two of these genes have been involved in Prader-Willi syndrome (PWS), which includes hyperphagia, repetitive and compulsive behaviors, and cognitive impairment. Here, we show that Maged1-deficient mice develop progressive obesity associated with hyperphagia and reduced motor activity. Loss of Maged1 also results in a complex behavioral syndrome that includes reduced social interactions and memory, deficient sexual behavior, as well as increased anxiety and self-grooming. Oxytocin (OT), which is produced in the hypothalamus, can act as a neurotransmitter that reduces anxiety, promotes social behaviors and regulates food intake. Growing evidences indicate that OT is involved in autism. We found that Maged1 mutants showed a severe reduction in the levels of mature OT, but not of its precursors, in the hypothalamus. Moreover, the administration of OT rescued the deficit in social memory of these mice. We conclude that Maged1 is required for OT processing or stability. A decrease in mature OT levels in Maged1 mutants affects social interactions and possibly other behavioral processes. Our observations suggest that, in human, MAGED1 could play a role in autism or cause a neurodevelopmental condition that is reminiscent of the PWS.

Prediction and analysis of HLA-A2/A24-restricted cytotoxic T-lymphocyte epitopes of the tumor antigen MAGE-n using the artificial neural networks method on NetCTL1.2 Server

Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n(+)/HLA-A2(+) tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.

Frequent MAGE Mutations in Human Melanoma

BackgroundCancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype.Methodology/Principal FindingsWe undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1.SignificanceThese results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Androgen receptor molecular biology and potential targets in prostate cancer

The androgen receptor (AR) is a key transcriptional regulator and therapeutic target in prostate cancer. During androgen deprivation therapy to treat metastatic prostate cancer, surviving cells acquire increased AR signaling through a variety of mechanisms, one of which is enhanced interactions with AR coactivators. One recently identified AR-specific coregulator expressed only in human and nonhuman primates is the melanoma antigen gene protein-A11 (MAGE-11). MAGE-11 increases AR transcriptional activity through direct interactions with AR and other coactivators, and its levels increase during prostate cancer progression to castration-recurrent growth. The MAGE-11 gene is located at Xq28 on the human X chromosome as part of an X-linked MAGE gene family of cancer-testis antigens. MAGE-11 stabilizes AR when androgen levels are low, and functions in a transcriptional hub to promote AR-mediated gene activation. The evolutionary development and organization of the MAGE-11 gene within the cancer-testis antigen family suggests that MAGE-11 provides a gain-of-function to AR among primates in both normal physiology and cancer, and may serve as a therapeutic target in the treatment of advanced prostate cancer.

Melanoma Antigen-11 Inhibits the Hypoxia-Inducible Factor Prolyl Hydroxylase 2 and Activates Hypoxic Response

Activation of hypoxia-inducible factors (HIF), responsible for tumor angiogenesis and glycolytic switch, is regulated by reduced oxygen availability. Normally, HIF-alpha proteins are maintained at low levels, controlled by site-specific hydroxylation carried out by HIF prolyl hydroxylases (PHD) and subsequent proteasomal degradation via the von Hippel-Lindau ubiquitin ligase. Using a yeast two-hybrid screen, we identified an interaction between melanoma antigen-11 (MAGE-11) cancer-testis antigen and the major HIF-alpha hydroxylating enzyme PHD2. The interaction was confirmed by a pull-down assay, coimmunoprecipitation, and colocalization in both normoxic and hypoxic conditions. Furthermore, MAGE-9, the closest homologue of MAGE-11, was also found to interact with PHD2. MAGE-11 inhibited PHD activity without affecting protein levels. This inhibition was accompanied by stabilization of ectopic or endogenous HIF-1alpha protein. Knockdown of MAGE-11 by small interfering RNA results in decreased hypoxic induction of HIF-1alpha and its target genes. Inhibition of PHD by MAGE-11, and following activation of HIFs, is a novel tumor-associated HIF regulatory mechanism. This finding provides new insights into the significance of MAGE expression in tumors and may provide valuable tools for therapeutic intervention because of the restricted expression of the MAGE gene family in cancers, but not in normal tissues.

NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.

The anti-tumor immune response induced by a combination of MAGE-3/MAGE-n-derived peptides

Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). MAGE gene products are of particular interest owing to their wide expression in many tumors and their potential to induce tumor-specific CTL responses. Antigen-specific CTLs induced by MAGE gene-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumors. MAGE-3 has been used as a target for tumor immunotherapy. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma (HCC). However, the majority of previous investigations focused on the single MAGE antigen-derived peptides as a cancer vaccine which has many limitations. The tumor antigen expression is known to be heterogeneous and tumor cells can express multiple tumor antigens. Thus, vaccines incorporating single antigen-derived epitopes may be inadequate in generating a complete immune response against the tumor. Instead, a polyvalent vaccine incorporating epitopes derived from several tumor antigens may be more effective. Our study combined the MAGE-3 and MAGE-n-derived peptides as a cancer vaccine. The results showed that the combination of MAGE-3 and MAGE-n epitopes induced more effective anti-tumor immune responses than either of the peptides alone. In addition, the peptide-specific activity was observed to be in an MHC-restricted manner. Our study indicated that the combination of several tumor antigen-derived peptides may present a better peptide-based cancer immunotherapy.

Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor

The androgen receptor (AR) is a ligand-activated transcription factor that interacts with coregulatory proteins during androgen-dependent gene regulation. Melanoma antigen gene protein 11 (MAGE-11) is an AR coregulator that specifically binds the AR NH(2)-terminal FXXLF motif and modulates the AR NH(2)- and carboxyl-terminal N/C interaction to increase AR transcriptional activity. Here we demonstrate that epidermal growth factor (EGF) signaling increases androgen-dependent AR transcriptional activity through the posttranslational modification of MAGE-11. EGF in the presence of dihydrotestosterone stabilizes the AR-MAGE complex through the site-specific phosphorylation of MAGE-11 at Thr-360 and ubiquitinylation at Lys-240 and Lys-245. The time-dependent EGF-induced increase in AR transcriptional activity by MAGE-11 is mediated through AR activation functions 1 and 2 in association with the increased turnover of AR and MAGE-11. The results reveal a dynamic mechanism whereby growth factor signaling increases AR transcriptional activity through the covalent modification of an AR-specific coregulatory protein. Sequence conservation of the MAGE-11 phosphorylation and ubiquitinylation sites throughout the MAGE gene family suggests common regulatory mechanisms for this group of cancer-testis antigens.

Expression of MAGE-A1 and MAGE-A3 in Bone Marrow from Monoclonal Gammapathy to Myeloma Patients.

Multiple myeloma (MM) is an incurable tumor characterized by multifocal clonal expansion of malignant plasma cells within bone marrow (BM), with sometimes extramedullary location, i.e. mainly in peripheral blood (PB), pleural effusion or ascites. Cancer/testis antigens (CTA) are a category of tumor antigens with normal expression restricted to a male germ cells in the testis but not in adult somatic tissues. CTA are promising candidates for cancer immunotherapy. Presently, 44 distinct CT-antigen families have been identified. Presence of RNA transcripts encoding members of the MAGE gene family, as well as NY-ESO-1, CT7/MAGE-C1 in myeloma tumor cells and cell lines has been documented. In MM, cancer germ-line specific genes are preferentially expressed in advanced disease (up to 50%) and in almost all cell lines. Most of the genes of the MAGE-A family were found to be expressed in myeloma cells, although with various incidences. The aim of this study was to evaluate the posibility of using these genes as molecular markers of the progression MGUS to MM and the early relapse of the MM. Due to rare extramedullary location of malignant plasma cells we tried to undercover if expression is detecable in PB simultaneously with expression in the BM. Total of 151 samples from BM were evaluated: 83 samples from advanced myeloma, 12 samples of patients with early stage of MM who did not required treatment (smoldering MM 4x, stage IA 8x), 41 samples of MGUS patiens and 15 samples of normal healthy donors served as control group. In 15 advanced myeloma patients we simultaneously assessed BM sample and PB sample. Total RNA was evaluated by RT-PCR and then by real-time PCR using FRET probes. For relative quantification we used G6PDH housekeeping gene as external standard. As positive control we used myeloma cell line U266. Only 4,8% (2 /41) samples from MGUS patient showed expression of MAGE-A1 or MAGE-A3. 50% (6/12) patients with early stage of MM (IA and smoldering) showed expression of MAGE-A1 or MAGE-A3. 39,7% (33/83) of the samples from the advanced myeloma patients expressed at least 1 of these genes, or both (20 cases). We tested 15 BM from normal donors. All were negative. In 15 advanced myeloma BM samples we have observed expression in 8/15 cases but no expression in PB sample. We have confirmed that expression of MAGE is not present in samples of healthy donors. There is an obvious correlation between expression of the MAGE genes and early-late stage of the disease. Our evaluation confirmed the detection of low expression levels of MAGE-type mRNA in BM from patients with MGUS and early stage of MM. It is possible that MAGE antigen monitoring may predict the evolution towards more advanced disease. But due to need of separated myeloma cells or BM samples this metod can not be used for monitoring minimal residual disease in patients with MM.

MAGE-A tumor antigens target p53 transactivation function through histone deacetylase recruitment and confer resistance to chemotherapeutic agents

The MAGE gene family is characterized by a conserved domain (MAGE Homology Domain). A subset of highly homologous MAGE genes (group A; MAGE-A) belong to the chromosome X-clustered cancer/testis antigens. MAGE-A genes are normally expressed in the human germ line and overexpressed in various tumor types; however, their biological function is largely unknown. Here we present evidence indicating that MageA2 protein, belonging to the MAGE-A subfamily, confers wild-type-p53-sensitive resistance to etoposide (ET) by inducing a novel p53 inhibitory loop involving recruitment of histone deacetylase 3 (HDAC3) to MageA2/p53 complex, thus strongly down-regulating p53 transactivation function. In fact, enhanced MageA2 protein levels, in addition to ET resistance, correlate with impaired acetylation of both p53 and histones surrounding p53-binding sites. Association between MAGE-A expression levels and resistance to ET treatment is clearly shown in short-term cell lines obtained from melanoma biopsies harboring wild-type-p53, whereas cells naturally, or siRNA-mediated expressing low MAGE-A levels, correlate with enhanced p53-dependent sensitivity to ET. In addition, combined trichostatin A/ET treatment in melanoma cells expressing high MAGE-A levels reestablishes p53 response and reverts the chemoresistance.

Expression of the TRAG-3 gene in human esophageal cancer: The frequent synchronous expression of MAGE-3 gene

We previously reported some cancer testis antigens, especially for MAGE genes, to be expressed in a relatively high population of gastro-intestinal and breast cancers. TRAG-3 (Taxol resistant associated gene-3) may be another cancer testis antigen; however, its expression has still not been fully studied. The TRAG-3 expression was evaluated in a total of 57 cancer cell lines and 322 cancer samples of gastrointestinal and breast cancers by RT-PCR. TRAG-3 was expressed in 23/57 (40%) of the cell lines: the highest expression was found in gastric cancer (6/9: 67%), followed by esophageal (13/28: 46%), colon (3/11: 27%) and liver (1/4: 25%) cancers. In clinical samples, the expression was the highest in esophageal cancer (32/58: 55%), followed by liver (13/50: 26%), bile duct (5/27: 19%), gastric (5/50: 10%), breast (5/50: 10%) and colon (2/87: 2.3%) cancers. The TRAG-3 expression significantly correlated with the expression of MAGE-3 in esophageal cancer (p<0.05). As the TRAG-3 gene is located on Xq28, which is the same locus as the MAGE gene family, we found a frequent synchronous expression pattern with TRAG-3 and MAGE-3 in esophageal cancer.

Promoter Demethylation and Histone Acetylation Mediate Gene Expression of MAGE-A1, -A2, -A3, and -A12 in Human Cancer Cells

The broad range of expression of cancer-testis antigens in various tumor types makes the proteins encoded by human MAGE gene family promising targets for anticancer immunotherapy. However, a major drawback is their heterogeneous expression. In the current study, we have examined the influence of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) together with the histone deacetylase inhibitor trichostatin A on the expression of MAGE-A1, -A2, -A3, and -A12 genes in different cell lines. Reverse transcription-PCR, Western blot analyses, and immunocytochemical staining show that trichostatin A was able to significantly up-regulate 5-aza-CdR-induced MAGE gene expression. Transient transfection assays with methylated reporter plasmids containing promoter fragments of the different MAGE genes show that trichostatin A was able to overcome gene silencing. In addition, the methylation status of the MAGE promoters was assessed by sodium bisulfite mapping in the various cell lines before and after stimulation with 5-aza-CdR and/or trichostatin A. In contrast to the methylation patterns, which clearly correlated with the basal MAGE RNA transcripts, up-regulation of the MAGE-A mediated by both agents only resulted in a reduction in promoter methylation ranging between 1% and 19%. In conclusion, our data show for the first time that not only hypermethylation but also histone deacetylation is responsible for the mechanism underlying MAGE gene silencing.

Expression of MAGE Tumor-Associated Antigen in Thyroid Carcinomas

The 12 members of the MAGE gene family encode tumor-specific antigens that are recognized by autologous cytotoxic T lymphocytes. The MAGE genes are expressed not only in melanoma but in other malignant tumors as well. There is, however, little information on their expression in thyroid carcinomas. We studied the expression of the MAGE-3 antigen in human thyroid carcinomas to explore the possibility of specific immunotherapy using MAGE peptides. Tumor tissue samples of thyroid carcinomas were obtained from 60 patients. Standard pathohistologic analysis followed by immunohistochemistry analysis of MAGE-3 expression was performed in all patients. The overall expression rate of MAGE-3 antigen in thyroid carcinomas was 65%. According to histological types of thyroid carcinomas, expression rate of MAGE-3 antigen was as follows: 0% in anaplastic, 20% in medullary, 29% in follicular, and 80% in papillary thyroid carcinomas (p < 0.01). On the other hand, significantly higher expression of MAGE-3 antigen was observed in classical subtypes of papillary thyroid carcinomas and in small papillary tumors sized to 1 cm in diameter. These findings demonstrated that MAGE-3 antigen expression seems to be particularly high in the small, typical papillary carcinomas, thus suggesting that MAGE-3 gene abnormality is an early step in thyroid cancer progression.

BRCA2 Suppresses Cell Proliferation via Stabilizing MAGE-D1

Germ line mutations in BRCA2 gene predispose women to early-onset familial breast and ovarian cancer. BRCA2 is a protein of multiple functions. In addition to its role in DNA double-strand break repair, BRCA2 also plays a role in stabilization of stalled DNA replication forks, cytokinesis, transcription regulation, mammalian gametogenesis, centrosome duplication, and suppression of cell proliferation. However, how BRCA2 mutations predispose women specifically to breast and ovarian cancer remains undefined. Here we found that BRCA2 binds and stabilizes MAGE-D1, a member of the MAGE gene family of proteins. Expression of BRCA2 and MAGE-D1 synergistically suppresses cell proliferation independently of the p53 pathway. Using two MAGE-D1 RNA interferences and two cell lines expressing low or undetectable levels of MAGE-D1, we further showed that the expression of MAGE-D1 is required for BRCA2-mediated suppression of cell proliferation, indicating that MAGE-D1 is a downstream target of BRCA2 and that BRCA2 suppresses cell proliferation via stabilizing MAGE-D1. Importantly, MAGE-D1 protein expression was reduced in 6 of 16 breast carcinoma cell lines tested as compared with untransformed immortal mammary epithelial cell lines, suggesting that suppression of MAGE-D1 expression may be involved in the tumorigenesis of a subset of sporadic breast cancers.