Cancer-testis Gene Expression Research Articles

Expression of cancer testis genes in gastric neoplasms — a preliminary study

Expression of cancer testis genes in gastric neoplasms — a preliminary study

BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites

BackgroundPervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters.ResultsHere we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements.ConclusionsThus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

Differential expression and regulation of ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12 gene profiles in colon cancer tissues and their in vitro response to epigenetic drugs.

Colon cancer (CC) is a significant cause of death worldwide, particularly in Saudi Arabia. To increase the accuracy of diagnosis and treatment, it is important to discover new specific biomarkers for CC. The main objectives of this research are to identify potential specific biomarkers for the early diagnosis of CC by analyzing the expressions of eight cancer testis (CT) genes, as well as to analyze how epigenetic mechanisms control the expression of these genes in CC cell lines. Tissue samples were collected from 15 male patients with CC tissues and matched NC tissues for gene expression analysis. The expression levels of specific CT genes, including ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12, were assessed using quantitative techniques. To validate the gene expression patterns, we used publicly available CC statistics. To investigate the effect of inhibition of DNA methylation and histone deacetylation on CT gene expression, in vitro experiments were performed using HCT116 and Caco-2 cell lines. There was no detected expression of the genes neither in the patient samples nor in NC tissues, except for TEX48, which exhibited upregulation in CC samples compared to NC tissues in online datasets. Notably, CT genes showed expression in testis samples. In vitro, experiments demonstrated significant enhancement in mRNA expression levels of ADAD1, DMRTC2, PRSS54, SYCE1, SYCP1, TEX101, TEX48, and TMPRSS12 following treatment with 5-aza-2'-deoxycytidine and trichostatin A in HCT116 and Caco-2 cell lines. Epigenetic treatments modify the expression of CT genes, indicating that these genes can potentially be used as biomarkers for CC. The importance of conducting further research to understand and target epigenetic mechanisms to improve CC treatment cannot be overemphasized.

The Expression Patterns of Human Cancer-Testis Genes Are Induced through Epigenetic Drugs in Colon Cancer Cells.

The expression of human germline genes is restricted to the germ cells of the gonads, which produce sperm and eggs. The germline genes involved in testis development and potentially activated in cancer cells are known as cancer-testis (CT) genes. These genes are potential therapeutic targets and biomarkers, as well as drivers of the oncogenic process. CT genes can be reactivated by treatment with drugs that demethylate DNA. The majority of the existing literature on CT gene activation focuses on X-chromosome-produced CT genes. We tested the hypothesis that epigenetic landscape changes, such as DNA methylation, can alter several CT gene expression profiles in cancer and germ cells. Colon cancer (CC) cell lines were treated with the DNA methyltransferase inhibitor (DNMTi) 5-aza-2'-deoxycytidine, or with the histone deacetylase inhibitor (HDACi) trichostatin A (TSA). The effects of these epigenetic treatments on the transcriptional activation of previously published CT genes (CTAG1A, SCP2D1, TKTL2, LYZL6, TEX33, and ACTRT1) and testis-specific genes (NUTM1, ASB17, ZSWIM2, ADAM2, and C10orf82) were investigated. We found that treatment of CC cell lines with 5-aza-2'-deoxycytidine or TSA correlated with activation of X-encoded CT genes and non-X-encoded CT genes in somatic (non-germline) cells. These findings confirm that a subset of CT genes can be regulated by hypomethylating drugs and subsequently provide a potential therapeutic target for cancer.

Cancer-Testis Gene Biomarkers Discovered in Colon Cancer Patients.

In Saudi Arabia, colon cancer (CC) is the most prevalent cancer in men and the third most common cancer in women. Rather than being detected through screening programs, most CC cases are diagnosed mainly during clinical exams. Because of the slow growth of CC and its ability to be treated at an early stage, screening for CC can reduce the incidence of death and mortality. Consequently, there is an urgent need to identify a potential new cancer-specific biomarker for detecting early illness. Much research has been conducted on distinct antigen classes as potential new cancer-specific biomarkers for the early identification of malignancy. The cancer-testis antigens (CTAs) are one such category of antigens, with protein presence largely normally confined to human germ line cells in the testis and aberrantly produced in some cancer cells. CTAs are potentially valuable for use as cancer biomarkers and in cancer therapeutics due to their distinctive expression pattern. The aim of this current study was to identify potential cancer-testis (CT) gene biomarkers in Saudi Arabian CC patients. In this study, a total of 20 matching CC and normal colon (NC) tissues were obtained from the Saudi population. Any genes that showed expression in CC tissues but not in matching NC tissues were subsequently verified for mRNA expression in eight breast and eight leukemia malignancies using RT-PCR to determine the specificity of any CC biomarkers. CTAG1A, SPZ1, LYZL6, SCP2D1, TEX33, and TKTL2 genes were expressed in varying numbers of CC tissues compared to no measurable expressions in all NC tissue specimens, making these genes suitable potential candidates for CC markers. The most frequently expressed CT genes in CC patients were CTAG1A (35%) and SCP2D1 (35%), followed by TKTL2 (25%), SPZ1 (20%), LYZL6 (15%), and TEX33 (5%). The LYZL6 gene shows a weak RT-PCR product in 25% of breast cancer (BC) patients but not in leukemia patients. The SCP2D1 gene appears to display expression in all leukemia patients but not in the BC patients. TKTL2 expression was also observed in 50% of leukemia samples but not in the BC samples. More experiments at the protein level and with a larger cohort of patients are required to evaluate this finding.

Cancer-Testis Gene Expression in Hepatocellular Carcinoma: Identification of Prognostic Markers and Potential Targets for Immunotherapy.

Background:Cancer-testis genes can serve as prognostic biomarkers and valuable targets for immunotherapy in multiple tumors because of their restricted expression in testis and cancer. However, their expression pattern in hepatocellular carcinoma is still not well understood. The purpose is to comprehensively characterize the cancer-testis gene expression in hepatocellular carcinoma as well as identify prognostic markers and potential targets for immunotherapy.Methods:Cancer-testis database and publicly available data sets reporting new cancer-testis genes were integrated, and then restricted them in a testis and hepatocellular carcinoma expression pattern. Pathway enrichment analysis and survival analysis were conducted to evaluate the biological function and prognostic effect of cancer-testis genes. Clustering analysis and coexpression analysis were performed to illustrate cancer-testis gene expression patterns in hepatocellular carcinoma. The association of gene expression of each cancer-testis gene to the corresponding methylation status was detected. Finally, we explored the associations between cancer-testis genes and CD8+ T-cell infiltration in hepatocellular carcinoma by TISIDB, and then validated it in an independent hepatocellular carcinoma cohort with 72 patients.Results:A total of 59 testis-specific genes were identified highly expressed in hepatocellular carcinoma. Pathway enrichment analysis revealed that cancer-testis genes in hepatocellular carcinoma significantly involves in the process of cell cycle regulation. Most of the cancer-testis genes were coexpressed, and cluster analysis suggested that cancer-testis gene expressed in hepatocellular carcinoma is independent of sex, hepatitis status, and histology type. We also found that demethylation might be a regulatory mechanism of cancer-testis gene expression in hepatocellular carcinoma. Survival analysis indicated that cancer-testis genes could predict the prognosis of patients with hepatocellular carcinoma. Furthermore, BUB1B was identified contributing to the resistance of CD8+ T-cell infiltration in hepatocellular carcinoma and was an independent prognostic factor both for overall survival and disease-free survival.Conclusions:Our analysis enables better understanding of cancer-testis genes in hepatocellular carcinoma and provides potential targets for hepatocellular carcinoma treatment. Experimental and clinical studies are needed for further validations.

Systematic cancer-testis gene expression analysis identified CDCA5 as a potential therapeutic target in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with poor prognosis. Cancer-testis genes (CTGs) have been vigorously pursued as targets for cancer immunotherapy, but the expressive patterns and functional roles of CTGs remain unclear in ESCC. MethodsA systematic screening strategy was adopted to screen CTGs in ESCC by integrating multiple public databases and RNA expression microarray data from 119 ESCC subjects. For the newly identified ESCC prognosis-associated CTGs, an independent cohort of 118 patients with ESCC was recruited to validate the relationship via immunohistochemistry. Furthermore, functional assays were performed to determine the underlying mechanisms. Findings21 genes were recognized as CTGs, in particular, CDCA5 was aberrantly upregulated in ESCC tissues and significantly associated with poor prognosis (HR = 1.85, 95%CI: 1.14–3.01, P = .013). Immunohistochemical staining confirmed that positive CDCA5 expression was associated with advanced TNM staging and a shorter overall survival rate (45.59% vs 28.00% for CDCA5−/+ subjects, P = 1.86 × 10−3). H3K27 acetylation in CDCA5 promoter might lead to the activation of CDCA5 during ESCC tumorigenesis. Functionally, in vitro assay of gain- and loss-of-function of CDCA5 suggested that CDCA5 could promote ESCC cells proliferation, invasion, migration, apoptosis resistance and reduce chemosensitivity to cisplatin. Moreover, in vivo assay showed that silenced CDCA5 could inhibit tumor growth. Mechanistically, CDCA5 knockdown led to an arrest in G2/M phase and changes in the expression of factors that played fundamental roles in the cell cycle pathway. InterpretationCDCA5 contributed to ESCC progression and might serve as an attractive target for ESCC immunotherapy. FundThis work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20181083 and BK20181496), Jiangsu Top Expert Program in Six Professions (No. WSW-003 and WSW-007), Major Program of Science and Technology Foundation of Jiangsu Province (No. BE2016790 and BE2018746), Jiangsu Medical Young Talent Project (No. QNRC2016566), the Program of Jiangsu Medical Innovation Team (No. CXTDA2017006), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1487) and Jiangsu Province 333 Talents Project (No. BRA2017545).

Some characteristics of cancer-testis gene expression regulation in colorectal cancer.

e14233 Background: Cancer-testis antigens (CTA) can be used in immunotherapy and for early detection of cancer. Despite numerous studies of the CTA expression in different tumors, their transcriptional activity and its regulation in colorectal cancer (CRC) remain poorly understood. The purpose of the study was to analyze the expression of cancer-testis genes (CT-genes) and mechanisms of its regulation in CRC. Methods: Tumor and intact tissues of the colon were studied in 60 patients. RNAs were isolated using the method described by Chomczynski and Sacchi (2006). The REVERTA-L reagent kit was used for the cDNA synthesis. Expression of 16 CT-genes (MAGE-A1, -A2, -A3, -A4, MAGE-B1, -B2, GAGE-1, -3, -4, MAGEC1, BAGE, XAGE3, NYESO1, SSX2, SCP1, PRAME1) and expression and copy number of 3 DNA methyltransferase genes (DNMT1, DNMT3A, DNMT3B) were determined by Real-Time qPCR (GAPDH and GUSB - reference genes). For the cluster analysis, we used our R scripts. Differences were assessed by the Mann-Whitney test, and correlations – by the Spearman's rank correlation coefficient (r). Results: The expression of 2 CT genes, SSX2 and PRAME1, was increased (p < 0.05) by 1.8 and 2.9 times, respectively, and the BAGE expression was decreased by 2.4 times in tumor tissues, compared to the normal tissues. The expression and copy number of DNMT3A in tumor tissues was 1.5 and 2 times higher (p < 0.05), and that of DNMT3B – 2 times lower (p < 0.005), compared to normal tissues. The copy number and expression of the DNMT1 gene did not change. A strong positive correlation (r = 0.996) between the expression and copy number of DNA methyltransferase genes was observed. Using cluster analysis (Hierarchical Clustering, Euclidean distance), we detected two clusters of CRC samples different in the expression of CT-genes and methyltransferases. Cluster 1 showed increased expression of DNMT1, DNMT3A and DNMT3B and decreased expression of BAGE, SSX2 and PRAME1; cluster 2 – decreased expression of DNMT1, DNMT3A and DNMT3B and increased expression of BAGE, SSX2 and PRAME1. Conclusions: The detected aberrant expression of the CT-genes BAGE, SSX2, PRAME1 in CRC depends on the expression of DNMT1, DNMT3A, DNMT3B, which in its turn depends on the copy number of the corresponding DNA methyltransferase genes.

Comprehensive characterization of cancer-testis genes in testicular germ cell tumor.

Cancer‐testis (CT) genes are a group of genes restrictedly expressed in testis and multiple cancers and can serve as candidate driver genes participating in the development of cancers. Our previous study identified a number of CT genes in nongerm cell tumors, but their expression pattern in testicular germ cell tumor (TGCT), a cancer type characterized by less genomic alterations, remained largely unknown. In this study, we systematically investigated the expression pattern of CT genes in TGCT samples and evaluated the transcriptome difference between TGCT and normal testis tissues, using datasets from the UCSC Xena platform, The Cancer Genome Atlas (TCGA) and the Genotype‐Tissue Expression (GTEx) project. Pathway enrichment analysis and survival analysis were conducted to evaluate the biological function and prognostic effect of expressed CT genes. We identified that 1036 testis‐specific expressed protein‐coding genes and 863 testis‐specific expressed long noncoding RNAs (lncRNAs) were expressed in TGCT samples, including 883 CT protein‐coding genes and 710 CT lncRNAs defined previously. The number of expressed CT genes was significantly higher in seminomas (P = 3.48 × 10−13) which were characterized by frequent mutations in driver genes (KIT, KRAS and NRAS). In contrast, the number of expressed CT genes showed a moderate negative correlation with the fraction of copy number altered genomes (cor = −0.28, P = 1.20 × 10−3). Unlike other cancers, our analysis revealed that 96.16% of the CT genes were down‐regulated in TGCT samples, while CT genes in stem cell maintenance related pathways were up‐regulated. Further survival analysis provided evidence that CT genes could also predict the prognosis of TGCT patients with both disease‐free interval and progression‐free interval as clinical endpoints. Taken together, our study provided a global view of CT genes in TGCT and provided evidence that CT genes played important roles in the progression and maintenance of TGCT.

Exploring a potential link between cell cycle regulation and Cancer Testis gene misregulation

Oncogenesis and misexpression of germline genes like Cancer Testis (CT) genes, called the soma‐to‐germline transformation, are commonly linked, but the mechanisms that drive the phenomenon remain underexplored. In the model system Caenorhabditis elegans, a soma‐to‐germline transformation occurs when the function of the highly conserved DREAM complex (for Dp, Retinoblastoma(Rb)‐like, E2F, and MuvB) is attenuated. In mammalian cells, DREAM's activity overlaps with the Retinoblastoma(Rb) protein to mediate establishment of cell cycle exit into quiescence, or G0. We hypothesize that CT gene misexpression in cancer cells occurs following loss of DREAM and Rb activity, similar to the soma‐to‐germline transformations that occur in C. elegans. Therefore, we expect that cancer cell lines that express CT genes will be unable to maintain an effective cell cycle exit. To test our hypothesis, cell cycle distributions of 8 cancer cell lines were analyzed using propidium iodide (PI) staining and flow cytometry before and after serum starvation. We observed that only 3 of the cell lines failed to arrest under growth limiting conditions, with the other 5 exhibiting some enrichment in G0/G1, indicating that these cell lines that did not arrest might exhibit upregulated expression of CT genes, and DREAM complex and Rb are deregulated. However, the cell lines that we observe G0arrest may still be dysfunctional in either DREAM complex or Rb activity. Therefore, we will next test expression analysis will test whether CT genes are differentially regulated between these groups and whether DREAM and/or Rb regulation is attenuated. This study is the first step towards understanding whether cell cycle regulation and the soma‐to‐germline transformation are linked in mammalian cells.Support or Funding InformationThe Saudi Arabian Cultural Mission (SACM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decitabine enhances tumor recognition by T cells through upregulating the MAGE-A3 expression in esophageal carcinoma

Cancer testis (CT) antigens are expressed in various types of tumors and represent the potential targets for T cell-based immunotherapy. Analysis of CT gene expression and DNA methylation have indicated that certain CT genes are epigenetically regulated and studies have confirmed that certain CT antigens are regulated by DNA methylation. In this study, we explored the epigenetic regulation of MAGE-A3 and improved the clinical outcome of MAGE-A3-specific T cell therapy in esophageal squamous cell carcinoma (ESCC).We used molecular profiling datasets in The Cancer Genome Atlas to analyze CT gene expression in ESCC and its regulation by DNA methylation. We performed quantitative reverse transcription PCR (qRT-PCR), immunohistochemistry and bisulfite sequencing in ESCC cell lines and ESCC tissues. Functional assays, such as flow cytometry, cytotoxicity assays and ELISA, were performed to determine the demethylation agent, decitabine (5-aza-2′-deoxycytidine, DAC)-treated cancer cell improved antigen specific T cells response. ESCC tumor cell-xenograft mouse model and enzyme-linked immunospot (ELISPOT) assays were used to determine the function of DAC treatment in enhancing anti-MAGE-3 T cell responses in ESCC. Furthermore, we performed qRT-PCR and flow cytometry in the peripheral blood mononuclear cells (PBMC) of myelodysplastic syndromes (MDS) patients.MAGE-A3, one of the CT antigens, expressed at various levels in ESCC and was interfered by DNA methylation. We observed an efficient increase in MAGE-A3 expression in tumor cells and tissues after the treatment of decitabine and the expression of MAGE-A3 was affected by DNA methylation. Functional assays showed enhanced secretion of IFN-γ and cytolysis of MAGE-A3 antigen-specific T cells by DAC-treated target cells. In the tumor cell-xenograft mouse model and ELISPOT assays, DAC increased the expression of MAGE-A3 and T cell mediated tumor clearance in ESCC as well. Notably, the proportions of MAGE-A3-responsive T cells were elevated in DAC-treated patients with MDS, indicating DAC dismissed the epigenetic inhibition of MAGE-A3.DAC would probably improve the clinical outcome of MAGE-A3-specific T cell therapy by augmenting the expression of target gene.

EXPRESSION OF CANCER-TESTIS GENES PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 IN PATIENTS WITH MULTIPLE MYELOMA, THEIR INFLUENCE ON OVERALL SURVIVAL AND RELAPSE RATE

Objective:to study the prognostic significance of the expression of cancer-testis (CT) genes PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 in patients with multiple myeloma (MM) and their influence on overall survival and relapse rate. To determine their effect on suсh clinical parameters as levels of lactate dehydrogenase, leucocytes, hemoglobin, calcium, albumen, creatinine, beta-2-microglobulin.Materials and methods.Real-time polymerase chain reaction was performed on complementary DNA obtained from bone marrow of 77 patients with MM. The statistical analysis was performed using the Statistica 10.0 software package. To estimate prognostic values of the CT gene expression data were analyzed by the Kaplan – Meier method.Results.The study was conducted to determine the level of expression of CT genes PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 in a group of patients with MM. The group included primary and receiving cancer treatment in MM patients. According to the log-rank criterion expression of any of the CT genes PRAME, NY-ESO1, GAGE1, MAGE A3, MAGE A6, MAGE A12, SSX1, SLLP1, PASD1 exerts a significant influence on overall survival and progression-free survival/relapse. It was also determined that providing expression of some CT genes, the levels of creatinine, calcium, beta-2-microglobulin were much higher to compare with patients without expression.

Genome-wide identification of cancer/testis genes and their association with prognosis in a pan-cancer analysis.

Cancer/testis (CT) genes are excellent candidates for cancer immunotherapies because of their restrict expression in normal tissues and the capacity to elicit an immune response when expressed in tumor cells. In this study, we provide a genome-wide screen for CT genes with the identification of 745 putative CT genes. Comparison with a set of known CT genes shows that 201 new CT genes were identified. Integration of gene expression and clinical data led us to identify dozens of CT genes associated with either good or poor prognosis. For the CT genes related to good prognosis, we show that there is a direct relationship between CT gene expression and a signal for CD8+ cells infiltration for some tumor types, especially melanoma.

A cis-eQTL genetic variant of the cancer-testis gene CCDC116 is associated with risk of multiple cancers.

Recent studies have found that cancer-testis (CT) genes, which are expressed predominantly in germ and cancer cells, may be candidate cancer drivers. Because of their crucial roles, genetic variants in these genes may contribute to the development of cancer. Here, we systematically evaluated associations of common variants in CT genes and their promoters for the risk of lung cancer in our initial GWAS (2331 cases and 3077 controls), followed by in silico replication using additional 10,512 lung cancer cases and 9562 controls. We found a significant association between rs3747093 located in the CCDC116 promoter and lung cancer risk (OR=0.91, P meta=7.81×10-6). Although CCDC116 was expressed at lower levels in somatic tissues compared to the testis, the protective allele A of rs3747093 was associated with decreased CCDC116 expression in many normal tissues, including the lung (P=8.1×10-13). We subsequently genotyped this variant in another four commonly diagnosed cancers (gastric, esophageal, colorectal, and breast cancers), as we found expression quantitative trait locus (eQTL) signals for rs3747093 and CCDC116 in their corresponding normal tissues. Interestingly, we observed consistent associations between rs3747093 and multiple cancers (gastric cancer: OR=0.85, P=2.21×10-4; esophageal cancer: OR=0.91, P=2.57×10-2; colorectal cancer: OR=0.80, P=1.85×10-6; and breast cancer: OR=0.87, P=1.55×10-3). Taken together, the A allele of rs3747093 showed significant protective effects on cancer risk (OR=0.88, P pool=6.52×10-13) in an Asian population. Moreover, our findings suggest that low abundance expression of CT genes in normal tissues may also contribute to tumorigenesis, providing a new mechanism of CT genes in the development of cancer.

Pulmonary Metastases Exhibit Epigenetic Clonality: Implications for Precision Cancer Therapy

Development of effective cancer therapies may be limited by intratumoral heterogeneity, which facilitates outgrowth and organ-specific dissemination of treatment resistant clones. At present, limited information is available regarding epigenetic landscapes of pulmonary metastases. This study was undertaken to characterize epigenetic signatures of pulmonary metastases and to identify potential therapeutic targets. RNA and DNA were extracted from 65 pulmonary metastases resected from 12 patients (5 with sarcoma, 7 with adrenocortical carcinoma). Quantitative reverse transcription polymerase chain reaction techniques were used to evaluate expression levels of cancer-testis (CT) genes (NY-ESO-1, MAGE-A3, MAGE-A9, MAGE-A12, GAGE1, CT-45, SSX-1, and SSX-2), tumor suppressor (TS) genes (p16 and RASSF1A), and genes encoding epigenetic modifiers (DNMT1, DNMT3A, DNMT3B, EZH2, EED, and SUZ12), aberrantly expressed in human malignant diseases. Pyrosequencing techniques were used to quantitate DNA methylation levels in LINE1, NBL2, and D4Z4 repetitive sequences and promoter methylation status of differentially regulated genes. Results of these analyses were compared with a standardized panel of normal lung tissues. Pulmonary metastases exhibited histologically related and patient-specific global DNA demethylation. Significant interpatient heterogeneity of gene expression was observed even among patients with similar tumor histologic features. Epigenetic signatures appeared consistent among metastases from the same patient, irrespective of the time of resection (synchronous/metachronous) or the anatomic location. EZH2, EED, and SUZ12 (core components of Polycomb repressive complex-2 [PRC-2]) were upregulated in the majority of metastases. Pulmonary metastases exhibit patient-specific epigenetic clonality, which may be exploited for precision therapies targeting aberrant CT or TS gene expression. PRC-2 may be a shared target for epigenetic therapy of pulmonary metastases.

Epigenetic mechanisms underlying the dynamic expression of cancer-testis genes, PAGE2, -2B and SPANX-B, during mesenchymal-to-epithelial transition.

Cancer-testis (CT) genes are expressed in various cancers but not in normal tissues other than in cells of the germline. Although DNA demethylation of promoter-proximal CpGs of CT genes is linked to their expression in cancer, the mechanisms leading to demethylation are unknown. To elucidate such mechanisms we chose to study the Caco-2 colorectal cancer cell line during the course of its spontaneous differentiation in vitro, as we found CT genes, in particular PAGE2, -2B and SPANX-B, to be up-regulated during this process. Differentiation of these cells resulted in a mesenchymal-to-epithelial transition (MET) as evidenced by the gain of epithelial markers CDX2, Claudin-4 and E-cadherin, and a concomitant loss of mesenchymal markers Vimentin, Fibronectin-1 and Transgelin. PAGE2 and SPAN-X up-regulation was accompanied by an increase in Ten-eleven translocation-2 (TET2) expression and cytosine 5-hydroxymethylation as well as the disassociation of heterochromatin protein 1 and the polycomb repressive complex 2 protein EZH2 from promoter-proximal regions of these genes. Reversal of differentiation resulted in down-regulation of PAGE2, -2B and SPANX-B, and induction of epithelial-to-mesenchymal transition (EMT) markers, demonstrating the dynamic nature of CT gene regulation in this model.

Ectopic Expression of Cancer–Testis Antigens in Cutaneous T-cell Lymphoma Patients

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains only partially understood. A number of recent studies attempted to identify novel diagnostic markers and future therapeutic targets. One group of antigens, cancer-testis (CT) antigens, normally present solely in testicular germ cells, can be ectopically expressed in a variety of cancers. Currently, only a few studies attempted to investigate the expression of CT antigens in CTCL. In the present work, we test the expression of CT genes in a cohort of patients with CTCL, normal skin samples, skin from benign inflammatory dermatoses, and in patient-derived CTCL cells. We correlate such expression with the p53 status and explore molecular mechanisms behind their ectopic expression in these cells. Our findings demonstrate that SYCP1, SYCP3, REC8, SPO11, and GTSF1 genes are heterogeneously expressed in patients with CTCL and patient-derived cell lines, whereas cTAGE1 (cutaneous T-cell lymphoma-associated antigen 1) was found to be robustly expressed in both. Mutated p53 status did not appear to be a requirement for the ectopic expression of CT antigens. While T-cell stimulation resulted in a significant upregulation of STAT3 and JUNB expression, it did not significantly alter the expression of CT antigens. Treatment of CTCL cells in vitro with vorinostat or romidepsin histone deacetylase inhibitors resulted in a significant dose-dependent upregulation of mRNA but not protein. Further expression analysis demonstrated that SYCP1, cTAGE1, and GTSF1 were expressed in CTCL, but not in normal skin or benign inflammatory dermatoses. A number of CT genes are ectopically expressed in patients with CTCL and can be used as biomarkers or novel targets for immunotherapy.

Cancer–Testis Genes as Candidates for Immunotherapy in Breast Cancer

Cancer-testis (CT) antigens are tumor-associated antigens attracting immunologists for their possible application in the immunotherapy of cancer. Several clinical trials have assessed their therapeutic potentials in cancer patients. Breast cancers, especially triple-negative cancers are among those with significant expression of CT genes. Identification of CT genes with high expression in cancer patients is the prerequisite for any immunotherapeutic approach. CT genes have gained attention not only for immunotherapy of cancer patients, but also for immunoprevention in high-risk individuals. Many CT genes have proved to be immunogenic in breast cancer patients suggesting the basis for the development of polyvalent vaccines.

Expression of Cancer/Testis genes in ductal carcinoma in situ and benign lesions of the breast.

Cancer/testis (CT) genes represent a unique class of genes, which are expressed by germ cells, normally silenced in somatic cells, but activated in various cancers. CT proteins can elicit spontaneous immune responses in cancer patients and this feature makes them attractive targets for immunotherapy-based approaches. We have previously reported that CTs are relatively commonly expressed in estrogen receptor (ER) negative, high risk carcinomas. In this study, we examined the expression of selected CT genes in ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS) and benign proliferative lesions of the breast. ER negative DCIS were found to be associated with significant CT gene expression together with HER2 positivity and a marked stromal immune response.

NY-ESO-1 expression in meningioma suggests a rationale for new immunotherapeutic approaches.

Meningiomas are the most common primary intracranial tumors. Surgical resection remains the treatment of choice for these tumors. However, a significant number of tumors are not surgically accessible, recur, or become malignant, necessitating the repetition of surgery and sometimes radiation. Chemotherapy is rarely used and is generally not recognized as an effective treatment. Cancer/testis (CT) genes represent a unique class of genes, which are expressed by germ cells, normally silenced in somatic cells, but activated in various cancers. CT proteins can elicit spontaneous immune responses in patients with cancer and this feature makes them attractive targets for immunotherapy-based approaches. We analyzed mRNA expression of 37 testis-restricted CT genes in a discovery set of 18 meningiomas by reverse transcription PCR. The overall frequency of expression of CT genes ranged from 5.6% to 27.8%. The most frequently expressed was NY-ESO-1, in 5 patients (27.8%). We subsequently analyzed NY-ESO-1 protein expression in a larger set of meningiomas by immunohistochemistry and found expression in 108 of 110 cases. In some cases, NY-ESO-1 expression was diffused and homogenous, but in most instances it was heterogeneous. Importantly, NY-ESO-1 expression was positively correlated with higher grade and patients presenting with higher levels of NY-ESO-1 staining had significantly worse disease-free and overall survival. We have also shown that NY-ESO-1 expression may lead to humoral immune response in patients with meningioma. Considering the limited treatment options for patients with meningioma, the potential of NY-ESO-1-based immunotherapy should be explored.