Inhibition of histone lysine methylation enhances cancer-testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer

Cancer-testis antigens (CTA), such as NY-ESO-1, MAGE-A1, and MAGE-A3, are immunogenic proteins encoded by genes, which are normally expressed only in male germ cells but are activated by ill-defined epigenetic mechanisms in human tumors, including lung cancers. Previously, we reported induction of these CTAs in cancer cells, but not normal cells, by DNA-demethylating agents and histone deacetylase inhibitors using clinically achievable exposure conditions. In the present study, we evaluated chromatin alterations associated with repression/activation of cancer-testis genes in lung cancer cells to further develop gene-induction regimens for cancer immunotherapy. Repression of NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment, and binding of polycomb-group proteins, and histone heterochromatin modifications within the promoters of these genes. Derepression coincided with DNA demethylation, dissociation of polycomb proteins, and presence of euchromatin marks within the respective promoters. Short hairpin RNAs were used to inhibit several histone methyltransferases (KMT) and histone demethylases (KDM) that mediate histone methylation and repress gene expression. Knockdown of KMT6, KDM1, or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated activation of these cancer-testis genes in lung cancer cells. DZNep, a pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6 knockdown. Following DAC-DZNep exposure, lung cancer cells were specifically recognized and lysed by allogeneic lymphocytes expressing recombinant T-cell receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA-demethylating agents with compounds, such as DZNep, that modulate histone lysine methylation may provide a novel epigenetic strategy to augment cancer-testis gene expression as an adjunct to adoptive cancer immunotherapy.     

Cancer/testis antigen expression and specific cytotoxic T lymphocyte responses in non small cell lung cancer

Non small cell lung cancers (NSCLC) express cancer/testis antigens (CTA) genes and MAGE-A expression correlates with poor prognosis in squamous cell carcinomas. We addressed cytotoxic T lymphocytes (CTL) responses to HLA class I restricted CTA epitopes in TIL from NSCLC in an unselected group of 33 patients consecutively undergoing surgery. Expression of MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1 CTA genes was tested by quantitative RT-PCR. Monoclonal antibodies (MAb) recognizing MAGE-A and NY-ESO-1 CTA were used to detect CTA by immunohistochemistry. CD8(+) TIL obtained from tumors upon culture with anti CD3 and anti CD28 mAb and IL-2 were stimulated with autologous mature DC (mDC) and HLA-A*0101 restricted MAGE-A1(161-169) or MAGE-A3(168-176) peptides or HLA-A*0201 restricted MAGE-A4(230-239), MAGE-A10(254-262), NY-ESO-1(157-165) or multi-MAGE-A (YLEYRQVPV) peptides or a recombinant vaccinia virus (rVV) encoding MAGE-A and NY-ESO-1 HLA-A*0201 restricted epitopes and CD80 co-stimulatory molecule. Specificity was assessed by (51)Cr release and multimer staining. At least one CTA gene was expressed in tumors from 15/33 patients. In 10 specimens, at least 4 CTA genes were concomitantly expressed. These data were largely confirmed by immunohistochemistry. TIL were expanded from 26/33 specimens and CTA-specific CTL activity was detectable in 7/26 TIL. In 6, however, specific cytotoxicity was weak, (<40% lysis at a 50:1 E:T ratio) and multimer staining was undetectable. In one case, high (>60% lysis at 50:1 E:T ratio) MAGE-A10(254-262) specific, HLA-A*0201 restricted response was observed. Supportive evidence was provided by corresponding multimer staining. Although CTA genes are frequently expressed in NSCLC, detection of CTL reactivity against CTA epitopes in TIL from nonimmunized NSCLC patients represents a rare event.     

Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens in serous ovarian neoplasms.

PURPOSE: The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms and is silent in normal tissues, except for the testis. Expression of two members of this family, MAGE-A4 and NY-ESO-1, has been described in melanomas, germ cell tumors, certain carcinomas and sarcomas, and more recently in uterine neoplasms. The objective of this study was to evaluate the extent and prognostic significance of CT antigen expression in ovarian serous neoplasms. EXPERIMENTAL DESIGN: Seventy-four patients with ovarian neoplasms, including 10 with serous cystadenomas, 11 with serous tumors of borderline malignancy, and 53 with serous carcinomas, were studied. Immunohistochemistry was performed with the 57B monoclonal antibody, which recognizes predominantly the MAGE-A4 antigen and the D8.38 antibody that recognizes NY-ESO-1. RESULTS: MAGE-A4 expression was found to be present in 57% of the serous carcinomas and only in 9% of the serous tumors of borderline malignancy. No staining was detected in serous cystadenomas or in the normal ovary. In 8 of 30 positively stained serous carcinomas, >50% of the tumor cells expressed MAGE-A4. NY-ESO-1 expression was seen in 19% of the serous carcinomas, whereas serous tumors of borderline malignancy and cystadenomas were negative. A significant inverse correlation was found between MAGE-A4 expression and patient survival (P = 0.016). Multivariate analysis revealed that both tumor stage and MAGE-A4 expression were independent predictors of patient survival (P = 0.022 and P = 0.013, respectively). CONCLUSIONS: Cancer-testis antigen expression in ovarian serous neoplasms correlates directly with their degree of malignancy. MAGE-A4 expression, and to a lesser degree NY-ESO-1 expression, is characteristic of the majority of serous carcinomas. Determining the degree of MAGE-A4 expression in these tumors may provide important prognostic information. Finally, MAGE-A4 may represent a novel target for immunotherapy in serous ovarian neoplasms.     

Expression of MAGE-A3, NY-ESO-1, LAGE-1 and PRAME in urothelial carcinoma

Background:

The potential for cancer-testis (CT) antigens as targets for immunotherapy in cancer patients has been heavily investigated, and currently cancer vaccine trials based on the CT antigens, MAGE-A3 and NY-ESO-1, are being carried out.

Methods:

We used specific q-RT-PCR assays to analyse the expression of the CT genes MAGE-A3, NY-ESO-1 (CTAG1B), LAGE-1 (CTAG2) and PRAME in a panel of bladder tumours from 350 patients with long-term follow-up and detailed treatment information.

Results:

Overall, 43% of the tumours expressed MAGE-A3, 35% expressed NY-ESO-1, 27% expressed LAGE-1 and 20% expressed PRAME. In all, 56% of the tumours expressed at least one of the CT genes analysed. Univariate Cox regression analysis of CT gene expression in non-muscle-invasive tumours showed that expression of MAGE-A3 (P=0.026), LAGE-1 (P=0.001) and NY-ESO-1 (P=0.040) was significantly associated with a shorter progression-free survival. In addition, we found that patients with tumours expressing PRAME responded poorly to chemotherapy (P=0.02, χ²-test).

Conclusion:

Cancer-testis genes are frequently expressed in bladder cancer and especially in tumours of high stage and grade. In addition, the CT gene expression may have both prognostic and predictive value. Development of specific immunotherapy against the CT antigens in bladder cancer may ultimately increase patient survival.     

Heterogeneous expression of GAGE, NY-ESO-1, MAGE-A and SSX proteins in esophageal cancer: Implications for immunotherapy

Cancer/testis antigens (CTAs) elicit immune response in cancer patients and are therefore targets of immunotherapy. Current information on CTA expression is primarily based on mRNA assays and little is known about their expression at the protein level. The objectives of our study are to analyze GAGE, NY-ESO-1, MAGE-A and SSX protein expression in esophageal cancer and to correlate their expression patterns with clinicopathologic parameters and survival. We examined CTA protein expression in 213 patients with esophageal cancer by immunohistochemistry. Antigen-positive tumors were evaluated once and antigen-negative tumors were evaluated 3 times by examining different parts of the cancer specimen. GAGE, NY-ESO-1 and MAGE-A were heterogeneously expressed in 42 (20%), 44 (21%) and 111 (52%) tumors, respectively, whereas SSX expression was not detected. Of the 126 (59%) patients expressing CTAs, 70 (33%) expressed 1, 41 (19%) expressed 2 and 15 (7%) expressed 3 antigens. The expression of MAGE-A was correlated with those of GAGE (p = 0.001) and NY-ESO-1 (p = 0.002), and the expression of GAGE was correlated with that of NY-ESO-1 (p = 0.002). One hundred fifty-six (79%) sections were positively stained in the first evaluation, whereas 37 (19%) and 4 (2%) positive sections were identified in the second and third evaluations, respectively. Particularly, MAGE and GAGE expression showed overlaps. GAGE, NY-ESO-1 and MAGE-A protein expression was not correlated with the disease progression, TNM factors or survival. The detection of immunonegative cells in every specimen suggests addition of other drugs such as 5-aza-2'-deoxycytidine to increase the therapeutic effect of CTA-specific cancer vaccines.     

Case-Control Study: Smoking History Affects the Production of Tumor Antigen–Specific Antibodies NY-ESO-1 in Patients with Lung Cancer in Comparison with Cancer Disease–Free Group

IntroductionLung cancer is the leading cause of cancer mortality worldwide; therefore, understanding the biological or clinical role of tumor-associated antigens and autoantibodies is of eminent interest for designing antitumor immunotherapeutic strategies.MethodsHere we prospectively analyzed the serum frequencies of New York esophageal squamous cell carcinoma 1 (NY-ESO-1), human epidermal growth factor 2/neu, and melanoma-associated antigen A4 (MAGE-A4) antibodies and expression of the corresponding antigens in tumors of 121 patients with NSCLC undergoing an operation without prior neoadjuvant chemotherapy and compared them with those in 57 control age-matched patients with no history of a malignant disease.ResultsWe found that only antibodies specific for NY-ESO-1 (19.8% [n = 24 of 121]) were significantly increased in the group of patients with NSCLC compared with in the controls. NY-ESO-1 seropositivity was significantly positively associated with an active smoking history in patients with NSCLC but not in smokers from the control group. In tumors, the frequency of NY-ESO-1 mRNA expression was 6.3% (in four of 64 patients), the frequency of human epidermal growth factor 2/neu (HER 2/neu) expression was 11.9% (five of 42), and the frequency of MAGE-A4 expression was 35.1% (20 of 57). MAGE-A4 expression in tumors correlated with smoking status and male sex in patients with NSCLC. Patients with squamous cell carcinoma displayed higher expression of NY-ESO-1 and MAGE-A4 in tumors than did patients with adenocarcinoma. On the other hand, 94.7% of nonsmoking patients in our study had adenocarcinoma (of whom 73.7% were women).ConclusionThese results confirm the reported high immunogenicity of NY-ESO-1 and suggest that a smoking-induced chronic inflammatory state might potentiate the development of NY-ESO-1–specific immune responses. Moreover, smoking might contribute to the expression of other cancer/testis antigens such as MAGE-A4 at early stages of NSCLC development.     

Decitabine facilitates immune recognition of sarcoma cells by upregulating CT antigens,MHC molecules,and ICAM-1

Rhabdomyosarcoma, osteosarcoma, and Ewing’s sarcoma are the most common types of sarcoma in children. Despite standard therapy, nearly one third of the patients with Ewing’s sarcoma relapse, and there are limited options with curative potential. Immunotherapy is a promising approach as it can target tumor-specific antigens that are specifically expressed on tumors while sparing non-malignant cells. We have demonstrated that a demethylating chemotherapeutic drug, 5-aza-2’-deoxycytidine (decitabine, DAC) can upregulate the expression of cancer-testis (CT) antigens, MHC molecules, and intracellular cell adhesion molecule-1 on pediatric sarcoma cell lines, resulting in enhanced killing of tumor cells by CT antigen-specific cytotoxic T lymphocytes derived from pediatric sarcoma patients. A significant increase in the mRNA expression levels of MAGE-A1 and MAGE-A3 were found in 70 %, and NY-ESO-1 in 80 % of the sarcoma lines following exposure to pharmacological levels of DAC. The high expression levels of MAGE-A1, MAGE-A3, and NY-ESO-1 were sustained in sarcoma lines and primary tumor lines over 30 days after the cessation of DAC. Furthermore, DAC treatment induced upregulation of MAGE-A1, MAGE-A3, or NY-ESO-1 protein expression in seven of nine lines studied. These studies show that demethylating chemotherapy could be combined with CT antigen-directed immunotherapy for treating pediatric sarcoma.     

Melanoma-associated antigen-A1 expression predicts resistance to docetaxel and paclitaxel in advanced and recurrent gastric cancer

Melanoma-associated antigen (MAGE) genes are cancer-testis antigen genes that serve as immunotherapy targets in several human cancers. Previous studies have revealed that the forced expression of MAGE genes induces a paclitaxel-resistant phenotype. In the present study, we examined whether the expression of MAGE-A1 could predict the response of advanced and recurrent gastric cancers (GCs) to taxan (doce-taxel or paclitaxel)-based chemotherapy. The expression of MAGE-A1 was analyzed by immunostaining in 41 primary GC samples. DNA demethylation was assessed by methylation-specific polymerase chain reaction and the effect of the forced expression of MAGE-A1 on drug resistance to taxan drugs was monitored by MTT assay. The expression of MAGE-A1 in primary GC was observed in 4 (9.8%) of 41 cases. All 4 patients with MAGE-A1-positive GC showed progressive disease, whereas MAGE-A1 expression was not detected in any of the 23 patients showing partial response (P=0.0302). There was no association between MAGE-A1 gene demethylation and response to chemotherapy (P=0.7245). The forced MAGE-A1 expression in the TMK-1 GC cell line increased the sensitivity to paclitaxel and docetaxel. These results suggest that although MAGE-A1 does not participate directly in the drug-resistant phenotype, the expression of MAGE-A1 could be a marker for the prediction of resistance to taxan-based chemotherapies in patients with GC.     

Expression and immunotherapeutic targeting of the SSX family of cancer-testis antigens in prostate cancer

Recent U.S. Food and Drug Administration approval of the first immunotherapy for prostate cancer encourages efforts to improve immune targeting of this disease. The synovial sarcoma X chromosome breakpoint (SSX) proteins comprise a set of cancer-testis antigens that are upregulated in MHC class I-deficient germline cells and in various types of advanced cancers with a poor prognosis. Humoral and cell-mediated immune responses to the SSX family member SSX2 can arise spontaneously in prostate cancer patients. Thus, SSX2 and other proteins of the SSX family may offer useful targets for tumor immunotherapy. In this study, we evaluated the expression of SSX family members in prostate cancer cell lines and tumor biopsies to identify which members might be most appropriate for immune targeting. We found that SSX2 was expressed most frequently in prostate cell lines, but that SSX1 and SSX5 were also expressed after treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. Immunohistochemical analysis of microarrayed tissue biopsies confirmed a differential level of SSX protein expression in human prostate cancers. Notably, SSX expression in patient tumor samples was restricted to metastatic lesions (5/22; 23%) and no expression was detected in primary prostate tumors examined (0/73; P < 0.001). We determined that cross-reactive immune responses to a dominant HLA-A2-specific SSX epitope (p103-111) could be elicited by immunization of A2/DR1 transgenic mice with SSX vaccines. Our findings suggest that multiple SSX family members are expressed in metastatic prostate cancers which are amenable to simultaneous targeting.     

Expression of multiple epigenetically regulated cancer/germline genes in nonsmall cell lung cancer

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized on the X-chromosome may provide effective treatment for an extended number of patients.