Malignant glioma is the most common and aggressive primary brain tumor and the overall prognosis for glioma patients remains poor. Clarification of the molecular mechanism responsible for glioma progression is critical for the effective treatment of glioma. Melanoma antigen gene (MAGE)-A2 (MAGEA2) is a member of the MAGE-A family proteins widely studied for cancer vaccine development and identification of tumor markers. However, MAGEA2 clinical significance  and biological function in glioma remain unclear, especially for the prognosis of glioma patients. This study investigates MAGEA2 expression in glioma tissue samples and its significance in predicting glioma patient prognosis. MAGEA2 protein expression in tissue samples was measured by immunohistochemistry and western blotting, and MAGEA2 mRNA expression was determined by real-time polymerase chain reaction. Our results confirmed that MAGEA2 mRNA and protein expression levels were upregulated in glioma tissues, compared with normal brain tissue. The high expression of MAGEA2 in glioma tissues significantly correlated with World Health Organization advanced grade. Univariate and multivariate analyses revealed that high MAGEA2 expression is an independent prognostic factor for glioma patient poor overall survival. The P53 mRNA expression levels were downregulated in glioma tissues compared to noncancerous brain tissue and MAGEA2 expression negatively correlated with P53 expression. Taken together, our results suggest that MAGEA2 plays an oncogenic role in glioma progression, and they provide insight into MAGEA2 application as a novel predictor of clinical outcomes and a potential glioma biomarker.

Cancer-testis antigens, effective markers of tissue malignant transformation, are characterized by heterogonous transcription depending on the pathological features of breast cancer. We performed screening of transcription profile of cancer-testis antigens specific for breast tumor tissues in female patients with and without regional metastasis. The relative expression of 16 genes (MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEB1, MAGEB2, GAGE1, GAGE3, GAGE4, MAGEC1, BAGE, XAGE3, NY-ESO1, SSX2, SYCP1, and PRAME1) was analyzed by RT-qPCR method in biopsy specimens of the mammary gland tissues obtained during surgery from 25 patients. Differential transcription activity of cancer-testis antigens genes was observed in patients with metastatic (enhanced expression of MAGEA2, MAGEB1, and XAGE3 genes) and non-metastatic (enhanced expression of GAGE3 and PRAME1 genes) breast cancer.

PURPOSE: To describe the histopathological characteristics and survival of female breast cancer (BC) patients in a rural setting with limited access to adjuvant treatment.
METHODS: A prospective study of 107 histologically confirmed BC patients treated with surgery from 2010 to 2016 from rural parts of western Ethiopia. Referral pathology was performed, and active follow-up was conducted. Adjusted cox regression analysis (hazard ratio [HR]) was performed.
RESULTS: The median age at diagnosis was 45 (16-83) years; 57% of the patients presented with cT3/4 tumors, 71% with clinically positive lymph nodes, 21% with HER2-overexpression (Dako3+) and 68% with grade 3 tumors. Estrogen and/or progesterone receptor expressions were present in 66% and triple-negative disease in 25%. The estimated 1- and 2-year overall survival probability rates were 78 and 53%, respectively. The 2-year survival for patients with clinically positive lymph nodes was 44% compared to 73% for patients with lymph node-negative disease (HR 2.44; 95% confidence interval [95% CI] 1.19-5.02). The corresponding 2-year survival for patients with cT4 tumors was 25% versus 68% for patients with cT1-2 tumors (cT1-3 vs. cT4 HR 3.86; 95% CI 1.82-13.63). The 2-year survival for patients with hormone receptor-negative disease was 40% compared to 59% for patients with hormone receptor-positive disease (HR 1.92; 95% CI 1.06-3.47).
CONCLUSION: The majority of breast cancer patients treated with surgery in rural parts of western Ethiopia are diagnosed at advanced stage and have hormone receptor-positive disease. Nearly half of the patients die within 2 years. These findings underscore the need for provision of adjuvant hormonal therapy and for the establishment of pathology service including hormone receptor testing.

BACKGROUND: MAGE-A genes belong to the cancer/testis antigens family. The prognostic significance of MAGE-A expression in the peripheral blood of patients with lung cancer is unknown. Therefore, this study evaluated the expression and possible prognostic significance of MAGE-A in the peripheral blood of patients with lung cancer.
METHODS: In this study, we detected MAGE-A gene expression in the peripheral blood of 150 patients with lung cancer and 30 healthy donors using multiplex semi-nested PCR and analyzed their correlation with clinicopathological risk factors.
RESULTS: MAGE-A expression was associated with factors indicating poor prognosis. The expression of MAGE-A and each individual MAGE-A gene were also associated with low overall survival in patients with lung cancer.
CONCLUSION: The expression of MAGE-A genes in peripheral blood may act as a poor prognostic marker in patients with lung cancer.

Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma-associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2-overexpressing iPS cells from hMAGEA2-overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self-renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self-renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells.

Melanoma-associated antigens (MAGE) are expressed in different type of cancers including lung cancer and have been shown to be functionally related to p53 tumor suppressor gene. Little is known about the relationship between MAGE genes and p53 aberrant expression in lung cancer. The aims of this study were to observe the expression of MAGEA2, examine the role of MAGEA2 in lung cancer survival, investigate its correlation between MAGEA2 and p53, and explore its clinicopathologic significance as a prognostic marker. Quantitative reverse transcription-polymerase chain reaction was performed to detect the expression of MAGEA2 using 36 primary tumors and 31 metastatic lymph nodes from patients with lung cancer. The role of MAGEA2 in cancer cell growth and in the regulation of p53 downstream genes were examined using small interfering RNA. The expression of MAGEA2 and p53 were analyzed immunohistochemically using tissue microarray from 353 resected lung specimens. High-level expression of MAGEA2 (High-MAGEA2) was confirmed in lung tumors with high frequency. Inhibiting MAGEA2 expression effectively suppressed cancer cell growth and decreased the expression of p53 downstream target genes in vitro. In adenocarcinoma, High-MAGEA2 was strongly associated with aberrant p53 expression (P<0.001) and was associated with worse clinical outcomes (5-year OS, 87.1% in low vs. 74.1% in high, P=0.014). Aberrant p53 expression was also significant worse prognostic factor (P=0.029). Among the adenocarcinoma patients with wild-type p53, High-MAGEA2 had poorer prognosis than low-level MAGEA2 groups (5-year OS, 90.1% vs. 72.1%, P=0.037), whereas had no difference in p53 aberrant tumors. On multivariate analysis, MAGEA2 was independently associated with survival (hazard ratio; 2.12, P=0.030). In conclusion, suppression of MAGEA2 in lung cancer cells significantly reduced the growth/survival of cancer cells. High-MAGEA2 was identified as an independent prognostic factor in lung adenocarcinoma. Specific inhibition of MAGEA2 may be a promising therapeutic strategy for patients with lung cancer.

Breast cancer is the most abundant cancer worldwide and a severe problem for women. Notably, breast cancer has a high mortality rate, mainly because of tumor progression and metastasis. Triple-negative breast cancer (TNBC) is highly progressive and lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Therefore, there are no established therapeutic targets against TNBC. In this study, we investigated whether the expression of human melanoma-associated antigen A2 (MAGEA2) is associated with TNBC. We found that hMAGEA2 is significantly overexpressed in human TNBC tissues; we also observed oncogenic properties using TNBC cell lines (MDA-MB-231 and MDA-MB-468). The overexpression of hMAGEA2 in MDA-MB-231 cell line showed dramatically increased cellular proliferation, colony formation, invasion, and xenograft tumor formation and growth. Conversely, knockdown of hMAEGA2 in MDA-MB-468 cell line suppressed cellular proliferation, colony formation, and xenograft tumor formation. Additionally, we showed that hMAGEA2 regulated the activation of Akt and Erk1/2 signaling pathways. These data indicate that hMAGEA2 is important for progression of TNBC and may serve as a novel molecular therapeutic target.

Global loss of DNA methylation is frequently observed in the genome of human tumors. Although this epigenetic alteration is clearly associated with cancer progression, the way it exerts its pro-tumoral effect remains incompletely understood. A remarkable consequence of DNA hypomethylation in tumors is the aberrant activation of "cancer-germline" genes (also known as "cancer-testis" genes), which comprise a diverse group of germline-specific genes that use DNA methylation as a primary mechanism for repression in normal somatic tissues. Here we review the evidence that such cancer-germline genes contribute to key processes of tumor development. Notably, several cancer-germline genes were found to stimulate oncogenic pathways involved in cell proliferation (SSX, DDX43, MAEL, PIWIL1), angiogenesis (DDX53), immortality (BORIS/CTCFL), and metastasis (CT-GABRA3). Others appear to inhibit tumor suppressor pathways, including those controlling growth inhibition signals (MAGEA11, MAGEB2), apoptosis (MAGEA2, MAGEC2), and genome integrity (HORMAD1, NXF2). Cancer-germline genes were also implicated in the regulation of tumor metabolism (MAGEA3/MAGEA6). Together, our survey substantiates the concept that DNA hypomethylation promotes tumorigenesis via transcriptional activation of oncogenes. Importantly, considering their highly restricted pattern of expression, cancer-germline genes may represent valuable targets for the development of anti-cancer therapies with limited side effects.

Bladder cancer has an unexplained, high recurrence rate. Causes of recurrence might include the presence of sporadic tumor micro-foci in the residual urothelial tissue after surgery associated with an inverted ratio between intratumoral effector and regulatory T cell subsets. Hence, surgical specimens of both tumors and autologous, macroscopically/histologically free-of-tumor tissues were collected from 28 and 20 patients affected by bladder or renal cancer, respectively. The frequencies of effector (IFNγ+ and IL17+ T cells) and regulatory (CD4+CD25hiCD127lo and CD8+CD28-CD127loCD39+ Treg) T cell subpopulations among tumor infiltrating lymphocytes were analyzed by immunofluorescence, while the gene expression of MAGE-A1 and MAGE-A2 tumor-associated antigens was studied by RT-PCR. The results show that both the T cell infiltrate and the frequency of MAGE-A1/A2 gene expression were comparable in tumors and in autologous free-of-tumor tissues in bladder cancer, while the autologous free-of-tumor renal tissues showed reduced T cell infiltrate and frequency of MAGE gene expression as compared to the autologous tumors. Importantly, the intra-tumor T effector/Treg cell ratio was consistently <1 in bladder cancer patients (n. 7) who relapsed within two years, while it was always >1 in patients (n. 6) without recurrence (regardless of tumor stage) (P = 0.0006, Odds ratio = 195). These unprecedented findings clarify the pathogenic mechanism of bladder cancer recurrence and suggest that microscopically undetectable micro-foci of tumor may predispose to recurrence when associated with an inverted intratumoral T effector/Treg cell ratio.

The antiestrogen tamoxifen is a well-tolerated, effective treatment for estrogen receptor-α-positive (ER+) breast cancer, but development of resistance eventually limits its use. Here we show that expression of MAGEA2, and related members of this cancer-testis antigen family, is upregulated in tamoxifen-resistant tumor cells. Expression of MAGEA2 in tumor lines grown in vitro or as xenografts led to continued proliferation in the presence of tamoxifen. At the molecular level, we demonstrate that MAGEA2 protein localizes to the nucleus and forms complexes with p53 and ERα, resulting in repression of the p53 pathway but increased ER-dependent signaling. In a series of ER+, tamoxifen-treated breast cancer patients, we show a highly significant (P=0.006) association between MAGEA (melanoma-associated antigen) expression and reduced overall survival, confirming the clinical significance of our observations.

Previous studies identified the frequent loss of adherens junction-associated protein 1 (AJAP1) expression in glioblastoma (GBM) and its correlation with worse survival. AJAP1 may suppress glioma cell migration, which plays an important role in tumor progression in malignant gliomas such as GBM. However, the role of AJAP1 in cell cycle arrest or apoptosis and resistance to chemotherapy remains unclear. Based on microarray screening results, quantitative PCR and luciferase plasmid reporter constructs were used to evaluate the possible regulatory role of AJAP1 on MAGEA2 expression and function. Cell death assays, TUNEL and other markers of apoptosis were utilized to detect cell apoptosis. Restoration of AJAP1 expression in glioma cells was analyzed after temozolomide exposure. AJAP1 suppressed the expression of MAGEA2 and inhibited the transcriptional activity of MAGEA2 in glioma cells. As AJAP1 expression decreased MAGEA2 protein expression apoptosis increased moderately. Consistent with increased cell death, the induced loss of MAGEA2 expression correlated with increased caspase 3/7 activity, BCL2/BAX ratio and TUNEL signal. AJAP1 expression enhanced cell death in the presence of temozolomide. This study suggests AJAP1 may also function as a pro-apoptotic factor and potentiate cell death by temozolomide in glioma cells. This effect may be partially explained by AJAP1-mediated gene regulation of MAGEA2.

In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas.

Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.

PURPOSE: Aim of this study was to determine whether BORIS (Brother of the Regulator of Imprinted Sites) is a regulator of MAGEA2, MAGEA3, and MAGEA4 genes in lung cancer.
EXPERIMENTAL DESIGN: Changes in expression of MAGEA genes upon BORIS induction/knockdown were studied. Recruitment of BORIS and changes in histone modifications at their promoters upon BORIS induction were analyzed. Luciferase assays were used to study their activation by BORIS. Changes in methylation at these promoters upon BORIS induction were evaluated.
RESULTS: Alteration of BORIS expression by induction/knockdown directly correlated with expression of MAGEA genes. BORIS was enriched at their promoters in H1299 cells, which show high expression of these cancer testis antigens (CTA), compared with normal human bronchial epithelial (NHBE) cells which show low expression of the target CTAs. BORIS induction in A549 cells resulted in increased amounts of BORIS and activating histone modifications at their promoters along with a corresponding increase in their expression. Similarly, BORIS binding at these promoters in H1299 correlates with enrichment of activating modifications, whereas absence of BORIS binding in NHBE is associated with enrichment of repressive marks. BORIS induction of MAGEA3 was associated with promoter demethylation, but no methylation changes were noted with activation of MAGEA2 and MAGEA4.
CONCLUSIONS: These data suggest that BORIS positively regulates these CTAs by binding and inducing a shift to a more open chromatin conformation with promoter demethylation for MAGEA3 or independent of promoter demethylation in case of MAGEA2 and MAGEA4 and may be a key effector involved in their derepression in lung cancer.

OBJECTIVE: To examine the role of MAGEA2 in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC).
DESIGN: Primary tissue microarray data and quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that MAGEA2 is differentially overexpressed in HNSCC. Functional analyses were then performed using MAGEA2 transfections and small-interfering RNA knockdowns with subsequent anchorage-dependent growth studies and cell cycle analyses. Quantitative RT-PCR was used to evaluate expression changes in p53 downstream targets after transfection of MAGEA2 into normal upper aerodigestive cell lines.
RESULTS: MAGEA2 is differentially overexpressed in HNSCC. In addition, MAGEA2 promotes growth in normal oral keratinocytes, whereas knockdown of MAGEA2 in HNSCC cells decreases growth. Using the HCT116 p53 wt and null cell line system, transfection of MAGEA2 induced growth in the p53 wt cell line while providing no growth advantage in the p53 mutant cells. Subsequently, transfection of MAGEA2 induced a decrease in messenger RNA expression of the p53 downstream targets CDKN1A and BAX and decreased G1 arrest in cells allowed to remain confluent for longer than 48 hours.
CONCLUSIONS: These data suggest that MAGEA2 is differentially expressed in HNSCC and functions, in part, through the p53 pathway by increasing cellular proliferation and abrogating cell cycle arrest. This improved understanding of MAGEA2 function and expression patterns will potentially allow for the improved ability to use MAGEA2 for detection, surveillance, and targeted therapeutics.

BACKGROUND: The cancer/testis antigens (CTAs) are a unique group of proteins normally expressed in germ cells but aberrantly expressed in several types of cancers including prostate cancer (PCa). However, their role in PCa has not been fully explored.
METHODS: CTA expression profiling in PCa samples and cell lines was done utilizing a custom microarray that contained probes for two-thirds of all CTAs. The data were validated by quantitative PCR (Q-PCR). Functional studies were carried out by silencing gene expression with siRNA. DNA methylation was determined by methylation-specific PCR.
RESULTS: A majority of CTAs expressed in PCa are located on the X chromosome (CT-X antigens). Several CT-X antigens from the MAGEA/CSAG subfamilies are coordinately upregulated in castrate-resistant prostate cancer (CRPC) but not in primary PCa. In contrast, PAGE4 is highly upregulated in primary PCa but is virtually silent in CRPC. Further, there was good correlation between the extent of promoter DNA methylation and CTA expression. Finally, silencing the expression of MAGEA2 the most highly upregulated member, significantly impaired proliferation of prostate cancer cells while increasing their chemosensitivity.
CONCLUSIONS: Considered together, the remarkable stage-specific expression patterns of the CT-X antigens strongly suggests that these CTAs may serve as unique biomarkers that could potentially be used to distinguish men with aggressive disease who need treatment from men with indolent disease not requiring immediate intervention. The data also suggest that the CT-X antigens may be novel therapeutic targets for CRPC for which there are currently no effective therapeutics.

This study aims to analyze the expression of 14 cancer/testis (CT) antigens in multiple myeloma (MM) to identify possible prognostic markers and therapeutic targets. The expression of MAGEA1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA10, MAGEA12, BAGE1, MAGEC1/CT7, the GAGE family, LAGE-1, PRAME, NY-ESO-1, SPA17 and SSX1 was studied by RT-PCR in 15 normal tissues, a pool of 10 normal bone marrow samples, 3 normal tonsils and bone marrow aspirates from 6 normal donors, 3 monoclonal gammopathies of undetermined significance (MGUS), 5 solitary plasmacytomas, 39 MM samples (95% advanced stage) and the MM cell line U266. MAGEC1/CT7 was expressed in bone marrow aspirates from one MGUS and one plasmacytoma. The frequencies at which CT antigen were found to be expressed in MM patients were MAGEC1/CT7 77%, LAGE-1 49%, MAGEA3/6 41%, MAGEA2 36%, GAGE family 33%, NY-ESO-1 33%, BAGE-1 28%, MAGEA1 26%, PRAME 23%, SSX-1 26%, MAGEA12 20.5%, MAGEA4 0%, and MAGEA10 0%. Cox's regression model showed that GAGE family expression and having >6 CT antigens expressed were independent prognostic factors when all patients were analyzed. However, MAGEC1/CT7 expression was the only independent prognostic factor when non-transplanted patients where analyzed. Based on our findings, MAGEC1/CT7, MAGEA3/6 and LAGE-1 are good candidates for immunotherapy, since together they cover 85% of our MM cases. Furthermore, expression of the GAGE family, >6 CT antigens and MAGEC1/CT7 seem to have impact on MM prognosis.

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.

Cancer-testis antigens expressed by different-histotype transformed cells are suitable targets for tumor immunotherapy. However, their heterogeneous expression in neoplastic lesions limits the eligibility of patients for cancer-testis antigen-directed vaccination, and low levels of cancer-testis antigens' expression may impair immune recognition of malignant cells. Because of the primary clinical relevance of cancer-testis antigens' expression in neoplastic tissues, 68 unrelated or sequential metastatic lesions from 56 patients were used to characterize the molecular mechanisms regulating the presence and levels of expression of different cancer-testis antigens of the MAGE family (i.e., MAGE2, 3 and 4) in cutaneous melanoma. Polymerase chain reaction-based methylation analyses showed that methylation status of specific cytosine-guanine dinucleotides in the promoters of investigated cancer-testis antigens correlated with their heterogeneous expression within unrelated metastatic melanoma lesions, and with their homogeneous expression among sequential metastases from three patients with melanoma. Unlike methylated promoters, unmethylated promoters of MAGE2, 3 and 4 genes drove the expression of reporter gene-enhanced green fluorescent protein after transient transfection of cancer-testis antigen-positive Mel 142 melanoma cells. Furthermore, de novo expression of MAGE3 gene induced by the treatment of Mel 195 melanoma cells with the DNA hypomethylating agent 5-aza-2'-deoxycytidine was associated with a 6%-12% demethylation of selected cytosine-guanine dinucleotides in its promoter. Finally, 5-aza-2'-deoxycytidine induced a 16-fold increase of MAGE3 expression in Mel 313 melanoma cells expressing constitutively low levels of the antigen, but did not affect that of Mel 275 melanoma cells expressing high baseline levels of MAGE3. Overall, these findings identify promoter methylation as a shared mechanism directly regulating the expression of therapeutic cancer-testis antigens in metastatic melanomas, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemoimmunotherapeutic strategies in patients with melanoma.