Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells.

Targeting tumor angiogenesis is an established strategy for cancer therapy. Because angiogenesis is not limited to pathological conditions such as cancer, molecular markers that can distinguish between physiological and pathological angiogenesis are required to develop more effective and safer approaches for cancer treatment. To identify such molecules, we determined the gene expression profiles of murine tumor endothelial cells (mTEC) and murine normal endothelial cells using DNA microarray analysis followed by quantitative reverse transcription-polymerase chain reaction analysis. We identified 131 genes that were differentially upregulated in mTEC. Functional analysis using siRNA-mediated gene silencing revealed five novel tumor endothelial cell markers that were involved in the proliferation or migration of mTEC. The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma. Comparative gene expression analysis revealed molecular differences between tumor endothelial cells and normal endothelial cells and identified novel tumor endothelial cell markers that may be exploited to target tumor angiogenesis for cancer treatment.

Expression and function of FERMT genes in colon carcinoma cells.

Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

Identification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from the lung carcinoma antigen, Lengsin.

Lengsin is an eye lens protein with a glutamine synthetase domain. We previously identified this protein as a lung carcinoma antigen through cDNA microarray analysis. Lengsin protein is overexpressed irrespective of the histological type of lung carcinoma, but not in normal tissues other than the lens. Therefore, to significantly extend the use of Lengsin-based T-cell immunotherapies for the treatment of patients with lung carcinoma, we searched for HLA-A*0201-restricted epitopes from this protein by screening predicted Lengsin-derived candidate peptides for the induction of tumor-reactive CTLs. Four Lengsin-derived peptides were selected by computerized algorithm based on a permissive HLA-A*0201 binding motif, and were used to immunize HLA-A*0201 transgenic (HHD) mice. Two of the immunizing peptides, Lengsin(206-215)(FIYDFCIFGV) and Lengsin(270-279)(FLPEFGISSA), induced peptide-specific cytotoxic T lymphocytes (CTLs) in HHD mice, and thus were used to stimulate human peripheral blood lymphocytes in vitro. Lengsin(206-215) and Lengsin (270-279) also induced human peptide-specific CTLs, and we were able to generate Lengsin(206-215)- and Lengsin(270-279)-specific CTL clones. The Lengsin(270-279)-specific CTL clone specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and Lengsin, and HLA-A*0201+/Lengsin+ lung carcinoma cells in an HLA-A*0201-restricted manner. On the other hand, the Lengsin(206-215)-specific CTL clone failed to recognize HLA-A*0201+/Lengsin+ target cells in the absence of cognate peptide. These results suggest that Lengsin(270-279) is naturally processed and presented by HLA-A*0201 molecules on the surface of lung carcinoma cells and may be a new target for antigen-specific T-cell immunotherapy against lung cancer.

Acquired resistance to miriplatin in rat hepatoma AH109A/MP10 is associated with increased Bcl-2 expression, leading to defects in inducing apoptosis.

Miriplatin, a novel lipophilic platinum complex approved to treat hepatocellular carcinoma, is administered into the hepatic artery after suspension in an oily contrast medium. Little is known concerning the mechanism of acquired resistance to miriplatin. In this study, we established and characterized a rat hepatoma cell subline, AH109A/MP10, which was about 10-fold more resistant to miriplatin than the parental cell line, AH109A. The established miriplatin-resistant cells showed clear cross-resistance to platinum complexes containing diaminocyclohexane as a carrier ligand, such as oxaliplatin and dichloro[(1R,2R)-1,2-cyclohexanediamine-N,N']platinum (DPC), while three human cancer cell lines selected for resistance to cisplatin (A2780cis, NCI-H69/CPR, MOR/CPR) did not show cross-resistance to miriplatin. There was no apparent difference in either intracellular platinum accumulation or platinum-DNA adducts in formation between resistant and parental cells after treatment with miriplatin or cisplatin, consisted with the unchanged expression of proteins involved in DNA repair, such as excision repair cross-complementing 1 (ERCC1) and mutL homolog 1 (MLH1). The increased expression of Bcl-2 was observed in AH109A/MP10 cells, in which apoptosis induced by miriplatin, but not cisplatin, was reduced. In addition, Bcl-2 inhibitor YC137 partially reversed the resistance of AH109A/MP10 cells to miriplatin. These findings suggested that the acquired resistance to miriplatin in AH109A/MP10 cells was associated in part with increased Bcl-2 expression, leading to defects in inducing apoptosis.

Cep55/c10orf3, a tumor antigen derived from a centrosome residing protein in breast carcinoma.

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.

Novel spliced form of a lens protein as a novel lung cancer antigen, Lengsin splicing variant 4.

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.

Identification of an immunogenic CTL epitope of HIFPH3 for immunotherapy of renal cell carcinoma.

PURPOSE: CD8(+) CTLs have an essential role in immune response against tumor. Although tumor-associated antigens have been identified in renal cell carcinoma (RCC), few of these are commonly shared and investigated as therapeutic targets in the clinical medicine. In this report, we show that HIFPH3, a member of prolyl hydroxylases that function as oxygen sensor, is a novel tumor antigen and HIFPH3-specific CTLs are induced from peripheral blood lymphocytes of RCC patients. EXPERIMENTAL DESIGN: Expression of HIFPH3 was examined by reverse transcription-PCR and immunostaining with anti-HIFPH3 antibody. To identify HLA-A24-restricted T-cell epitopes of HIFPH3, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. Peptide-specific CTLs were induced by stimulating peripheral blood lymphocytes of HLA-A24-positive RCC patients with these peptides in vitro. HLA-A24-restricted cytotoxicity of the CTLs against HIFPH3(+) RCC lines was assessed by chromium release assay. RESULTS: HIFPH3 was overexpressed in many RCC cell lines and primary RCC tissues, whereas it was not detectable in normal adult tissues by reverse transcription-PCR. Of the eight peptides that contained HLA-A24-binding motif, HIFPH3-8 peptide (amino acid sequence, RYAMTVWYF) could induce the peptide-specific CTLs from 3 of 6 patients with HIFPH3-positive RCC. Furthermore, HIFPH3-8 peptide-specific CTLs showed cytotoxicity against HIFPH3(+) RCC cell lines in a HLA-A24-restricted manner. CONCLUSIONS: HIFPH3 may be a target antigen in immunotherapy for RCC and HIFPH3-8 peptide could be used as a peptide vaccine for HLA-A*2402(+)/HIFPH3(+) RCC patients.

Improved generation of HLA class I/peptide tetramers.

As a tetrameric major histocompatibility complex (MHC) class I-peptide complex (tetramer) is capable of detecting antigen-specific cytotoxic T lymphocyte (CTL) by flow cytometry, significant information about the generation of in vivo immunity can be obtained. It is, however, difficult to make a soluble wild type of MHC class I heavy chain by the prokaryotic expression system. Therefore, we developed a new method for making soluble mutant HLA-A*2402 heavy chain. In this method, signal sequences were deleted, and the codon was changed to silent mutated nucleotide sequences that bacteria could use as preferable codon. When purified mutant HLA-A*2402 molecules were examined for the protein generation by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting using anti-HLA class I monoclonal antibody (mAb) as compared with wild type, a large amount of mutant heavy chain could be detected. In contrast, the expression of wild-type stable HLA-A*2402 heavy chain molecule was not detected in this system. Consequently, by using mutant HLA-A*2402/peptide tetramers, CTL precursors (CTLp) that specifically recognize antigenic peptide derived from the X;18 chromosomal translocations of synovial sarcoma were detected in patients' PBL.

Development of HLA-A2402/K(b) transgenic mice.

HLA-transgenic mice have been developed to facilitate studies of HLA-restricted cytotoxic responses, e.g., for the identification of immunodominant HLA-restricted CTL epitopes and the optimization of peptide or DNA vaccine constructs for human use. We have developed HLA-A2402/K(b)-transgenic mice expressing chimeric human (alpha1 and alpha2 domains of HLA-A2402) and mouse (alpha3, transmembrane and cytoplasmic domains of H-2K(b)) class I molecules. Immunization of these HLA-A2402/K(b)-transgenic mice with various known HLA-A24-restricted immunodominant cancer CTL epitope peptides derived from gp100, MAGE-1, MAGE-3, Her2/neu, CEA and TERT induced HLA-A24-restricted, peptide-specific CTLs. Using these transgenic mice, we identified a novel HLA-A24-restricted CTL epitope, PSA(152-160), encoded by human prostate-specific antigen. Staining with HLA tetramers showed that the cytotoxic activity induced by immunizing with PSA(152-160) in HLA-A2402/K(b) transgenic mice was HLA-A2402-restricted and CD8-dependent. Therefore, PSA(152-160) might be a candidate peptide for vaccination of HLA-A24(+) patients with prostate cancer. Our results suggest that HLA-A2402/K(b) transgenic mice might be useful in the search for HLA-A24-restricted CTL epitopes functioning as human cancer antigens and for the development of peptide-based cancer immunotherapy.

A simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in the circulation.

The detection and monitoring of peptide-specific cytotoxic T lymphocyte (CTL) precursors is essential for successful peptide-based immunotherapy against cancers. In contrast to the development of effective methods of detecting antigen-specific CTL, such as ELISpot and HLA-class I tetramer assay, stimulation with peptide-pulsed antigen-presenting cells (APC) has for some time been conventionally employed to induce peptide-specific CTL from peripheral blood mononuclear cells (PBMC). This culture protocol, however, needs a substantial number of PBMC to test the reactivity against a panel of peptides. In the present study, we established a simple culture protocol which has no need of additional APC. Addition of a corresponding peptide every 3 days was found to induce not only Epstein-Barr virus (EBV)-specific CTL from healthy donors, but also tumor antigen-derived peptide-specific CTL from cancer patients. A 10-ml blood sample was almost sufficient to test the presence of CTL precursors against 20 different peptides in triplicate assays. Overall, this culture protocol can be useful in detecting and monitoring peptide-specific CTL precursors in the circulation in peptide-based immunotherapy against cancer.