Myung-Ha Song

Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon cancer patients by SEREX

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.

Pattern of cancer/testis antigen expression in lung cancer patients

Cancer/testis (CT) antigens represent promising targets for immunotherapy. We investigated the composite expression of 13 CT antigens by RT-PCR in 79 lung cancer tissues and by immunohistochemistry in 22 lung cancer tissues. In the 79 lung cancer tissues, MAGE-3 (42%) was expressed most frequently and followed by NY-SAR-35 (33%), NY-ESO-1 (30%), MAGE-1 (27%), CT-7 (20%), MAGE-4 (19%), LAGE-1 (16%), and MAGE-10 (14%). Twenty-one tissues did not express any of the CT antigens tested, 58 (73%) expressed at least one, 36 (46%) co-expressed two, 24 (30%) co-expressed three, 17 (22%) co-expressed four, 14 (18%) co-expressed five, 8 (10%) co-expressed six, 4 (6%) co-expressed seven and 2 tissues expressed 9 of the 13 examined CT antigens. Expression of CT antigens was significantly associated with age (P<0.001), smoking history (P=0.009), and gender (P=0.001) of patients, whereas no correlation was found between the expression of CT antigens and other clinical factors, such as pT status, pN status, tumor stage, and histology history. The present results show that CT antigens are potential candidates in lung cancer patients for polyvalent immunotherapy.

Identification of the cancer/testis antigens AKAP3 and CTp11 by SEREX in hepatocellular carcinoma

Cancer/testis (CT) antigens are considered promising target molecules for immunotherapy. To efficiently identify potential CT antigens, a testis cDNA library was immunoscreened with sera from hepatocellular carcinoma (HCC) patients. We isolated 3 different antigens, AKAP3, CTp11, and UBQLN3. Although AKAP3 and CTp11 have been previously reported as CT antigens, this is the first time that these 2 antigens have been isolated from HCC patients by SEREX. Conventional RT-PCR analysis showed that AKAP3 was frequently present in HCC cell lines (5/7) and HCC tissues (5/10), and the gene was broadly expressed in several cancer types, including breast cancer cell lines (3/6), breast cancer tissues (6/9), colon cancer cell lines (3/10), colon cancer tissues (5/6), ovary cancer cell lines (6/8), ovary cancer tissues (11/16), lung cancer cell lines (4/7) and lung cancer tissues (6/13). By phage plaque analysis, anti-AKAP3 antibody was detected in sera from 15 of 27 HCC patients and 8 of 27 healthy donors. These data suggest that AKAP3 may be useful for diagnosis and immunotherapy in HCC patients.

Expression of the human cancer/testis antigen NY-SAR-35 is activated by CpG island hypomethylation

The novel cancer/testis antigen gene, NY-SAR-35, is expressed exclusively in normal testis and in various histological types of tumor. However, the NY-SAR-35 gene expression is observed to be aberrant in several cancer cell lines and tissues. The analysis of methylation status of the NY-SAR-35 gene promoter in various cancer cell lines showed that its expression was related to methylation of the promoter region. Treatment of human cancer cell lines with the demethylating agent 5-aza-2′-deoxycytidine activated the expression of the NY-SAR-35 gene. In addition, transfection experiments on various fragments of the CpG-rich gene promoter indicate that in vitro methylation of the NY-SAR-35 gene promoter results in the loss of promoter activity. The expression of NY-SAR-35 is therefore activated by hypomethylation of the CpG island in the gene promoter.

A novel cancer/testis antigen KP-OVA-52 identified by SEREX in human ovarian cancer is regulated by DNA methylation

SEREX has proven to be a powerful method that takes advantage of the presence of spontaneous humoral immune response in some cancer patients. In this study, immunoscreening of normal testis and two ovarian cancer cell line cDNA expression libraries with sera from ovarian cancer patients led to the isolation of 75 independent antigens, designated KP-OVA-1 through KP-OVA-75. Of these, RT-PCR showed KP-OVA-52 to be expressed strongly in normal testis, in ovarian cancer cell lines (3/9) and in ovarian cancer tissues (1/17). The expression of KP-OVA-52 in cancer cells is also induced by the demethylating agent 5-aza-2′-deoxycytidine (ADC). To test immunogenicity, we used the Serum Antibody Detection Assay (SADA) to analyze anti-IgG antibodies against the 75 antigens that were initially isolated by SEREX. Four of the 75 antigens (KP-OVA-25, KP-OVA-35, KP-OVA-68 and KP-OVA-73) reacted exclusively with sera from cancer patients. However, KP-OVA-52 reacted with 1 of 20 ovarian cancer sera. These data suggest that the KP-OVA-52 can be considered a novel CT antigen that is regulated by DNA methylation.

KP-CoT-23 (CCDC83) is a novel immunogenic cancer/testis antigen in colon cancer

Cancer/testis (CT) antigens are considered target molecules for cancer immunotherapy. To identify novel CT antigens, immunoscreening of a testicular cDNA library was performed using serum obtained from a colon cancer patient who was immunized with a new dendritic cell vaccine. We isolated 64 positive cDNA clones comprised of 40 different genes, designated KP-CoT-1 through KP-CoT-40. Three of these putative antigens, including KP-CoT-23 (CCDC83), had testis-specific expression profiles in the Unigene database. RT-PCR analysis showed that the expression of 2 KP-Cot-23 variants was restricted to the testis in normal adult tissues. In addition, KP-CoT-23 variants were frequently expressed in a variety of tumors and cancer cell lines, including colon cancer. A serological western blot assay showed IgG antibodies to the KP-CoT-23 protein in 26 of 37 colon cancer patients and in 4 of 21 healthy patients. These data suggest that KP-CoT-23 is a novel CT antigen that may be useful for the diagnosis and immunotherapy of cancer.

Cancer/testis antigen NY-SAR-35 enhances cell proliferation, migration, and invasion

The cancer/testis antigen NY-SAR-35 is aberrantly expressed in various cancer tissues and cancer cell lines but not in normal tissues except for the testis. A previous study demonstrated that the expression of NY-SAR-35 is activated by hypomethylation in cancer cells. However, the functions of this antigen remain unexplored. In the present study, we investigated the role of NY-SAR‑35 in human embryonic kidney (HEK) 293 cells using exogenous expression system of the gene. NY-SAR‑35 was predominantly expressed at the cytoplasm and was mainly observed in spermatogonia and spermatocytes. Expression of NY-SAR-35 in stable HEK293 transfectant clones was 2-fold higher than the control cells promoting cell growth and proliferation. NY-SAR-35 overexpression also enhanced cell migration and invasion ~2-fold and 4-fold more than the control, respectively. In contrast, small interfering RNA-mediated knockdown of NY-SAR-35 suppressed cell proliferation, migration, and invasion in HEK293 stable transfectants. We concluded that NY-SAR-35 as a cancer/testis antigen enhanced cell proliferation and invasion.

A cancer/testis antigen, NY-SAR-35, induces EpCAM, CD44, and CD133, and activates ERK in HEK293 cells

The cancer/testis (CT) antigen NY-SAR-35 gene is located on the X chromosome and is aberrantly expressed in various cancers but not in normal tissues, other than testes. Previously, we reported the expression of NY-SAR-35 enhanced cell growth, proliferation, and invasion in HEK293 and cancer cells. To extend understanding of the NY-SAR-35 gene, we used a next generation sequencing (NGS) approach. NY-SAR-35 expression induced growth, proliferation, metastasis, and stemness genes, as indicated by the up-regulations of CXCR4, EpCAM, CD133, and CD44, at the mRNA and protein levels. The expression of NY-SAR-35 in HEK293 cells significantly increased ERK phosphorylation, but not the phosphorylation of AKT. In HEK293/NY-SAR-35 cells, the expressions of pro-apoptotic proteins, including p53, Bax, and p21, were reduced and that of cyclin E was increased. Also, NY-SAR-35 increased the expressions of pluripotency genes (Nanog, Oct-4, and Sox2) and the ability of HEK293 cells to form colonies. Taken together, the present study indicates NY-SAR-35 functions as a CT antigen that triggers oncogenesis and self-renewal.

Review of Cancer-Testis (CT) Genes

Cancer-testis (CT) antigen은 다양한 종양과 정상 고환에서 제한적으로 발현되고, 암환자에서 체액성 면역과 세포성 면역반응을 일으키는 종양항원이다. 지금까지 MAGE, NY-ESO-1, GAGE, BAGE, LAGE, SSX2, NY-SAR-35를 포함하여 100개 이상의 CT antigen들이 동정되었고, 이러한 CT antigen들은 백신을 이용한 면역치료에 있어서 중요한 요소로 작용할 것으로 사료된다. CT antigen은 여러 가지 방법들을 통해 확인할 수 있고, X 염색체에 암호화 되어있는 CT-X gene과 X 염색체에 암호화 되어있지 않은 non-X CT gene으로 나눌 수 있다. 또한 몇몇의 종양에서 비정상적으로 활성화되지 않거나 발현되지 않는 CT antigen도 존재한다. 생식세포 조직과 종양에서 CT-X gene의 생물학적 역할이 아직 명확하게 규명되지 않았지만, 현재 여러 종류의 CT antigen을 이용한 암백신 치료법이 시도되고 있다. 본 논문은 암의 면역치료를 위한 CT antigen의 최근 연구동향과 앞으로의 연구방향에 대해 서술하고자 한다. 【Cancer-testis (CT) antigens are immunogenic protein antigens with restricted expression in the testes and a wide range of human tumor types, eliciting both humoral and cellular immune responses in cancer patients. They are considered to be ideal targets for vaccine-based immunotherapy, and more than 100 CT antigens, including MAGE, NY-ESO-1, GAGE, BAGE, LAGE, SSX2 and NY-SAR-35 have been identified to date. The CT antigens were identified through various techniques and can be divided in those that are encoded on the X chromosome, the CT-X genes, and those that are not, the non-X-CT genes. CT genes are aberrantly activated and expressed in a proportion of various types of human cancers. The biological role of CT-X in both germ line tissues and tumors remains poorly understood. Cancer vaccine trials based on several CT antigens are currently ongoing. This paper reviews recent advances in and future trends of CT antigens for cancer immunotherapy.】

A Comprehensive Analysis of a Large Panel of Cancer/Testis (CT) Antigens in Korea Breast Cancer

Cancer/testis (CT) antigens are immunogenic in cancer patients, exhibit highly tissue-restricted expression, and are considered promising target molecules for cancer vaccines. We investigated the expression of 13 CT genes in 29 Korean patients with primary breast carcinoma. The most frequently expressed CT genes were MAGE-3 (66%) and MAGE-1 (57%), followed by LAGE-1 (55%), NY-SAR-35 (49%), MAGE-4 (41%), NY-ESO-1 (38%), CT-7 (24%) and SSX-4 (24%), whereas SSX-1, SSX-2, MAGE-10 and NY-TLU-57 were found to be expressed significantly less often (3-7%) and SCP-1 not all. Expression of at least one antigen was observed in 28 breast cancer samples. Immunohistochemistry was performed for NY-ESO-1 and MAGE-3 protein expression in breast tumor samples. NY-ESO-1 and MAGE-3 proteins were expressed in 11 of 29 (38%) and 12 of 29 (41%) breast tumors. Our results suggest that CT antigens may be potential candidates for polyvalent immunotherapy in Korean breast cancer patients.