Search

Cancer stem–like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC. Cancer Res; 72(11); 2844–54. ©2012 AACR.

In our previous study, we demonstrated that a peptide derived from the novel centrosome residing protein Cep55/c10orf3 can be targeted by the cytotoxic T lymphocytes (CTLs) in peripheral blood mononuclear cells (PBMCs) of breast carcinoma patients. In this report, we evaluated the feasibility of cancer immunotherapy using Cep55/c10orf3 peptide for colorectal carcinoma (CRC). To evaluate the expression of Cep55/c10orf3 in CRC tissues, we performed immunohistochemical staining of using anti-Cep55/c10orf3 monoclonal antibody. Sixty-three percent cases showed weak positive for Cep55/c10orf3 in total 70 CRC cases. The Cep55/c10orf3 expression intention was collated with high histological grade of CRC. Thus, we hypothesized that Cep55/c10orf3 can also be the target of CTLs in CRC cases. We generated CTLs from PBMCs of human leukocyte antigen (HLA)-A24-positive colorectal carcinoma patients using HLA-A24-restricted Cep55/c10orf3 peptides. Two of 6 colorectal cancer patients were reactive for the Cep55/c10orf3_193(10) peptide, which was the only immunogenic peptide in breast carcinoma patients. CTL clone specific for Cep55/c10orf3_193(10) recognized and lysed HLA-A24 (+) and Cep55/c10orf3 (+) colorectal carcinoma cell lines. In addition, 1 of 6 colorectal carcinoma patients was reactive for the Cep55/c10orf3_402(11) and Cep55/c10orf3_283(12) peptides, but not for Cep55/c10orf3_193(10) with the ELISPOT assay. These observations suggest that the antigenic peptide repertoire presented by HLA-A24 in colorectal carcinoma might be different from that in breast carcinoma. Thus, these peptide vaccination peptide mixture of Cep55/c10orf3_193(10), Cep55/c10orf3_402(11) and Cep55/c10orf3_283(12) might be more effective than a single peptide in the treatment of colorectal carcinoma patients.

Pancreatic cancer is associated with the poorest prognosis of any digestive cancer due to the high incidence of liver metastasis. This study evaluated the possibility that osteopontin (OPN) RNA interference (RNAi) and anti-OPN antibody (Ab) could have antimetastatic effects. The differential gene expression was measured in a parental cell line, HPC-3, and an established highly liver metastatic cell line, HPC-3H4. This study investigated the effect of OPN RNAi and anti-OPN Ab on the metastatic ability of HPC-3H4 to the liver. An OPN RNAi-expressing vector was introduced into HPC-3H4 cells (HPC-3H4/miOPN), in which OPN production was reduced to the level of the parental HPC-3 cells. Finally, the ability of anti-OPN Ab to suppress liver metastasis was investigated. Osteopontin was upregulated 11.1-fold in HPC-3H4 in comparison to HPC-3. The metastatic rate of HPC-3H4/miOPN was significantly reduced to 25% in comparison to the 100% metastatic rate of HPC-3H4 and control HPC-3H4/miNeg cells (P < 0.01). The metastatic rate of the group given anti-OPN Ab was 50%. OPN RNAi and anti-OPN Ab had remarkable inhibitory effects against liver metastasis by the pancreatic cancer cell line.

A glutamine synthetase I family protein, Lengsin, was previously identified as a novel lens-specific transcript in the vertebrate eye. In this report, we show for the first time that Lengsin is a novel tumor-associated antigen expressed ectopically in lung cancer. Interestingly, a novel spliced form of human Lengsin termed 'splicing variant 4', gaining exon 3 that codes extra 63 amino acids, is the dominant transcript form in lung cancer cells. Lengsin mRNA could be detected in 7 of 12 (58%) lung cancer cell lines and 7 of 7 (100%) surgically resected lung cancer tissues. On the other hand, Lengsin transcripts could not be detected in normal major tissues or in other cancer cell lines, including melanoma, colorectal carcinoma, breast carcinoma and hepatocellular carcinoma. In addition, knockdown of Lengsin mRNA with RNAi caused cell death and a decrease of cell viability, suggesting that Lengsin has some essential role in cell survival. Since the lens is an immune-privileged site, we regard Lengsin as a highly immunogenic cancer antigen. Anti-Lengsin autoantibodies were detectable in sera of lung cancer patients, although these patients did not show any lens-related disturbances. Hence, Lengsin splicing variant 4 might be an immunogenic lung cancer-specific antigen that is suitable as a diagnostic marker and for molecular targeting therapy, including immunotherapy.

Several mechanisms have been described by which malignant cells escape from the immune system and/or chemotherapy. Recently we isolated TUCAN-54 gene that is over-expressed in a wide variety of epithelial cancers but not in normal counterparts. TUCAN-54 inhibited both death receptor-mediated and mitochondria/cytochrome C-mediated apoptosis. Moreover, down-regulation of TUCAN-54 in colon cancer cells by siRNA treatment could increase the sensitivity of the cells to an anti-cancer drug. Finally, we revealed that Fas-associated death domain protein (FADD) was physically associated with TUCAN-54, but not with TUCAN-48. Thus, TUCAN-54 might be a novel tumor-specific anti-apoptotic molecule, which might aggravate malignant potential of cancer cells, such as immunoresistance and chemoresistance.

Survivin, a member of the inhibitor of apoptosis protein family, is widely expressed in a variety of human cancer tissues. Survivin inhibits activation of caspases, and its overexpression can lead to resistance to apoptotic stimuli. In this study, survivin protein expression was assessed by immunohistochemical staining of 195 invasive breast cancer specimens. Overall, 79.5% of the tumors were positive for survivin. The expression of epidermal growth factor receptor (EGFR) family, human epidermal growth factor receptor 2 (HER2) and EGFR, was also examined in 53 cases, and consequently, it was indicated that survivin positivity might be correlated with the coexpression of HER2 and EGFR. To clarify the regulatory mechanism of survivin expression in breast cancer cells, the effect of HER2 and/or EGFR expression on the survivin levels was examined. It was revealed that the survivin protein level was up-regulated by the coexpression of HER2 and EGFR, leading to the increased resistance against etoposide-induced apoptosis in breast cancer cells. Conversely, survivin levels and apoptosis resistance were decreased when cells were treated with HER2-specific inhibitor, Herceptin. Although Herceptin could down-regulate both phosphatidylinositol 3-kinase (PI3K)/AKT signal and mitogen-activated protein/extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signal in HER2-positive breast cancer cells, PI3K-specific inhibitor but not MEK1-specific inhibitor could decrease the survivin levels. The present study clarified the regulatory mechanism of HER2 in the expression of survivin protein in breast cancer cells.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA–mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16–induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.

CD8+ CTLs have an essential role in immune response against tumor. Although an increasing number of tumor-associated antigens that can be recognized by CTLs have been identified from human tumors, a limited number of tumor-associated antigens is known in lung cancer. In addition, because some of them are expressed in noncancerous tissues, there exist limitations in their application to tumor immunotherapy. Livin/ML-IAP is one of recently identified inhibitor of apoptosis protein (IAP) family, which is overexpressed in melanoma cells. In this report, we show that Livin/ML-IAP is aberrantly expressed in many lung cancer cell lines and primary lung cancer tissues, whereas it is not detectable in normal tissues, including lung by reverse transcription-PCR methods. To identify HLA-A24-restricted T-cell epitopes of Livin/ML-IAP, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. It was revealed that Livin7 peptide (amino acid sequence, KWFPSCQFLL) had the highest affinity to HLA-A24. By stimulating peripheral blood lymphocytes of HLA-A24-positive lung cancer patients with Livin7 peptide in vitro, the peptide-specific CTLs were successfully induced from four of five patients with Livin/ML-IAP-positive lung cancer but not from any of four patients without Livin/ML-IAP expression in their cancer tissues. Furthermore, the CTLs induced by Livin7 peptide showed cytotoxicity against Livin/ML-IAP+ lung cancer cell lines in an HLA-A24-restricted manner. Our data suggest that Livin/ML-IAP may be an excellent target antigen in immunotherapy for lung cancer and Livin7 peptide may serve as a potent peptide vaccine for HLA-A*2402+/Livin+ lung cancer patients.

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer.

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125FAK) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.

We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

We present a unique case of crisis type chronic adult T-cell leukemia (ATL), in which the majority of leukemic cells had abundant periodic acid-Schiff (PAS)-positive cytoplasmic inclusions. These inclusions were found to be composed of glycogen because the PAS-positivity completely disappeared after digestion with amylase or human saliva. Electron microscopy also revealed that the inclusions consisted of aggregated beta particles of glycogen. The mechanism of glycogen accumulation in leukemic cells remains unknown; however, the presence of such inclusions in leukemic cells may be helpful diagnostically in T-lymphocyte malignancies.

Identification of tumor-associated antigens may facilitate vaccination strategies to treat patients with malignant diseases. We have found that the centrosomal protein, Cep55/c10orf3 acts as a novel breast carcinoma-associated tumor-associated antigen. Cep55/c10orf3 mRNA was detectable in a wide variety of tumor cell lines. Expression was barely detectable in normal tissues except for testis and thymus. Moreover, Cep55/c10orf3 protein could be detected by a monoclonal anti-Cep55/c10orf3 antibody (# 11-55) in 69.8% of breast carcinoma, 25% of colorectal carcinoma, and 57.8% of lung carcinoma tissues. The expression of Cep55/c10orf3 protein did not show any relationship with the hormone receptors such as estrogen receptor and progesterone receptor or expression patterns of p185 HER2/neu. We designed 11 peptides which displayed a human leukocyte antigen-A24 binding motif. One Cep55/c10orf3-peptide, Cep55/c10orf3_193(10) (VYVKGLLAKI), induced cytotoxic T lymphocytes (CTLs) in 3 of 3 patients with Cep55/c10orf3 (# 11-55)-positive breast carcinoma. A Cep55/c10orf3_193(10)-specific CTL clone could also recognize Cep55/c10orf3 (+) displayed on human leukocyte antigen-A24 (+) cancer cell lines. These data indicate that Cep55/c10orf3 peptides were naturally presented by breast cancer cells and can cause CTL clonal expansion in vivo. Monoclonal antibody # 11-55 and the Cep55/c10orf3_193(10) peptides may be useful as part of a therapeutic strategy for hormonal therapy or anti-p185 HER2/neu monoclonal antibody therapy-resistant breast carcinoma patients.

Introduction We have generated HLA-A*2601-restricted CD8+ CTL clones against an autologous pancreatic cancer cell line. Aims To characterize the antigen expressed on the cancer cells. Methodology We assessed cytotoxic activities and cytokine production of these CTL clones reacting against cancer cell lines that stably or transiently expressed the HLA-A*2601 gene. Results These CTL clones recognized 4 of 10 allogeneic pancreatic cancer cell lines and a gallbladder cancer cell line in the context of HLA-A*2601. However, the CTL clones did not recognize three hepatocellular carcinoma cell lines, two esophageal squamous cell carcinoma cell lines, or a lung adenocarcinoma cell line. Conclusions Thus, the CTL clones may recognize a shared, but not ubiquitously expressed, tumor antigen on pancreatic and gallbladder cancer cells.

Purpose: Pancreatic cancer is associated with the poorest prognosis of any digestive cancer due to the high incidence of peritoneal dissemination, which is the cause of death in most cases. To determine the mechanisms of peritoneal dissemination in pancreatic cancer, we established a mouse model of high peritoneal dissemination. Methods: A novel highly peritoneal-disseminating cell line was established from the human pancreatic cancer cell line; CAPAN-1. The new cell line, CAPAN-1P4a, was established from CAPAN-1 by repeated in vivo selection (four times) of the tumor cell line. To clarify the candidate genes implicated in peritoneal dissemination of pancreatic cancer, global gene expression screening was done using a cDNA macroarray. Results: CAPAN-1P4a cells showed 100% metastasis 3 weeks after injection and high reproducibility in the inoculated mice. Twenty-seven genes were upregulated and 14 genes were downregulated in CAPAN-1P4a cells compared with CAPAN-1 cells. The genes differentially expressed in the two cell lines were included as tumor suppressor/apoptosis genes, regulatory transcription factor, membrane receptors, cell adhesion protein, membrane receptors, and so on. Conclusions: Our established CAPAN-1P4a model offers a new means of conducting global gene expression analysis of pancreatic cancer cells with peritoneal dissemination and it has the potential to provide new insights into the mechanism of peritoneal dissemination in human pancreatic cancer.

Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4 T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4 T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30-40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human alpha-enolase, suggesting that it was derived from the processed parental alpha-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.