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.

Establishment of a human induced pluripotent stem cell derived alveolar organoid for toxicity assessment.

Alveolar epithelial cells (AECs) are vulnerable to injury, which can result in epithelial hyperplasia, apoptosis, and chronic inflammation. In this study, we developed human induced pluripotent stem cell (hiPS) cell-derived AECs (iAECs) and the iAECs based organoids (AOs) for testing AEC toxicity after chemical exposure. HiPS cells were cultured for 14 days with differentiation medium corresponding to each step, and the iAECs-based AOs were maintained for another 14 days. SFTPC and AQP5 were expressed in the AOs, and mRNA levels of SOX9, NKX2.1, GATA6, HOPX, and ID2 were increased. The AOs were exposed for 24 h to nine chemical substances, and IC50 values of the nine chemicals were determined using MTT assay. When the correlations between iAECs 2D culture and AOs 3D culture were calculated using Pearson's correlation coefficient r value, the nine chemicals that caused a significant decrease of cell viability in 3D culture were found to be highly correlated in 2D culture. The cytotoxicity and nitric oxide release in AO cultured with macrophages were then investigated. When AOs with macrophages were exposed to sodium chromate for 24 h, the IC50 value and nitric oxide production were higher than when the AOs were exposed alone. Taken together, the AO-based 3D culture system provides a useful platform for understanding biological characteristics of AECs and modeling chemical exposures.

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.

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.

Effect of cancer/testis antigen NY-SAR-35 on the proliferation, migration and invasion of cancer cells.

NY-SAR-35 is a cancer/testis (CT) antigen that was identified by serological analysis of recombinant complementary DNA expression libraries. The gene encoding NY-SAR-35 is located on the × chromosome and is aberrantly expressed in a number of cancer types and germ cells, such as those in the testes, but not in normal tissue. It has been reported that treatment with a demethylating agent induced the expression of NY-SAR-35 in several types of cancer cells. However, the function of NY-SAR-35 in cancer remains undetermined. In present study, the role of NY-SAR-35 in human lung adenocarcinoma (SK-LC-14) and hepatocellular carcinoma (SNU-449) cells was investigated following stable transfection of the NY-SAR-35 gene. NY-SAR-35 was observed to be expressed in the cytoplasm of the cells. In addition, the bromodeoxyuridine incorporation assay and immunofluorescence staining for proliferating cell nuclear antigen and Ki-67 demonstrated that proliferation was increased in cells transfected with NY-SAR-35. In addition, the trypan blue exclusion assay indicated that NY-SAR-35 increased cancer cell viability. Furthermore, NY-SAR-35 increased the migration and invasion of the cells. These results indicate that NY-SAR-35 increases cancer cell viability, proliferation, migration and invasion.

Identification of tumor antigens in malignant mesothelioma.

Serological analysis of recombinant tumor cDNA expression library (SEREX) is a powerful and widely used method to explore the cancer immune environment. In the present study, immunoscreening of normal testicular tissues and malignant mesothelioma (MM) cancer MSTO-211H cell line cDNA libraries with sera from 5 MM patients led to the isolation of 16 independent antigens, which were designated 'Korea Pusan-Malignant Mesothelioma' (KP-MM)-1 to -16. In total, 3/16 antigens were identified using the results of previous SEREX analyses, and 13 were newly identified. Of these, KP-MM-8, which was subsequently identified as amyotrophic lateral sclerosis 2 chromosome region candidate 11, was shown to be tissue-restricted. Reverse transcription-polymerase chain reaction demonstrated KP-MM-8 to be expressed strongly only in the normal testis, and weakly in the spleen, prostate, ovary, heart and skeletal muscle. In addition, KP-MM-8 mRNA was identified in MM cell lines, and in various other cancer cell lines, including MM (3/4), lung cancer (5/7), melanoma (5/7) and liver cancer (5/5) cell lines. Additionally, 2/16 antigens (KP-MM-2 and KP-MM-6) exclusively reacted with sera from cancer patients. However, KP-MM-8 reacted with 1 of 8 MM sera. Notably, 8/8 patients with MM and 8/8 normal individuals exhibited antibodies reactive to KP-MM-5, which was identified as cell division cycle 25B, a known oncogene. Overall, this data suggests that KP-MM-8 may be considered as a cancer/testis-like antigen and KP-MM-5 as an immunogenic tumor antigen in MM patients.

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-SAR­35 in human embryonic kidney (HEK) 293 cells using exogenous expression system of the gene. NY-SAR­35 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 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 5­aza­2'­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 (KP­OVA­25, KP­OVA­35, KP­OVA­68 and KP­OVA­73) reacted exclusively with sera from cancer patients. However, KP­OVA­52 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.

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.

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.