Characterization of BRCAA1 and its novel antigen epitope identification.

Looking for novel breast cancer antigen epitopes is helpful for its treatment, diagnosis, and prevention. brcaa1 gene is mapped at 1q42.1-q43, its whole genome is 93.857 kb, including 18 exons and 17 introns. BRCAA1 protein is composed of 1,214 amino acids with 10 glycosylate sites, and shares 37% amino acid identity and an identical antigen epitope with Rb binding protein 1. The novel antigen epitope, SSKKQKRSHK, was predicted to locate in the region 610 to 619 sites, was synthesized, and its antibody was fabricated. Competent inhibition analysis showed that SSKKQKRSHK is the shortest effective peptide. The antigen epitope was mapped in the cytoplasm of MCF-7 cells. Immunohistochemistry analysis showed that the antigen epitope exhibited positive expression in 65% (39 of 60) breast cancer specimens and negative expression in 60 non-cancerous tissues. Statistical analysis shows that its expression is closely associated with status of ER and PR, with sensitivity of 100% and specificity of 81%, and confidence interval of 85.9% to 96.9%. ELISA analysis showed that the mean absorbance of sera antibody titers from breast cancer patients and healthy donors were 0401 +/- 0.163 SD and 0.137 +/- 0.121 SD, respectively. Sixty-four percent breast cancer patient sera and 13% healthy donor sera had higher titer than mean titer of healthy donors, and there exists significant difference between breast cancer patients and healthy donors (P < 0.001). In this study, a novel breast cancer antigen epitope, SSKKQKRSHK, is identified. Its expression is associated with characteristics that are themselves associated with prognosis of breast cancer, and its sera antibody level may be helpful for breast cancer diagnosis.     

Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability-positive endometrial cancer.

PURPOSE: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX). EXPERIMENTAL DESIGN: cDNA expression libraries made from testis or endometrial cancer cell lines were screened using sera from patients with endometrial cancer or melanoma patients immunized with dendritic cells pulsed with autologous tum or lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated. RESULTS: A cancer/testis antigen, CAGE, was isolated by two independently performed SEREX. CAGE was expressed in various cancer cell lines including endometrial cancer, colon cancer, and melanoma in 7 of 10 endometrial cancer tissues and in 1 of 3 atypical endometrial hyperplasia, but not in normal tissues including the endometrium and testis. The protein expression on cancer cells was confirmed by Western blot analysis with the recombinant CAGE protein, anti-CAGE IgG antibody was detected in sera from 5 of 45 endometrial cancer, 2 of 24 melanoma, and 2 of 33 colon cancer patients, but not in sera from healthy individuals. By ELISA analysis, anti-CAGE antibody was detected in 12 of 45 endometrial cancer, 2 of 20 melanoma, and 4 of 33 colon cancer patients. Intriguingly, anti-CAGE antibody was highly positive in 7 of the 13 (53.8%) microsatellite instability (MSI)-H patients with endometrial cancer, but negative in 20 non-MSI-H patients (P = 0.001). CONCLUSION: CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.     

Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer

A monoclonal antibody (323/A3) with a high degree of selectivity for binding to breast cancer cells was produced by immunization of mice with MCF-7 human breast cancer cells. The antigen recognized by 323/A3 on MCF-7 appears to be surface localized, and by enzyme-linked immunosorbent assay, the antibody was found to bind strongly with four of six breast cancer cell lines examined while no binding was detectable with nonbreast cancer cell lines. In vivo distribution of the 323/A3 antigen was screened by immunoperoxidase staining of formalin-fixed paraffin sections of normal human tissues and tumors. Among breast tissues, positive staining was detected with 75% (6 of 8) of metastatic lymph nodes, 59% (76 of 128) of primary breast tumors, 20% (13 of 63) of benign breast lesions, and 0% (0 of 10) of normal breast. No immunostaining was detected with a large variety and number of other normal human tissues with the exception of staining observed with epithelium of normal colon. Antigen distribution appears not to be disease specific, since positive staining was also observed with adenocarcinomas other than breast. The antigen recognized by the 323/A3 antibody was identified by Western blot analysis as a Mr 43,000 protein. The glycoprotein nature of the antigen was demonstrated by its binding to concanavalin A, specific elution with sugar, and immunoprecipitation of a Mr 43,000 radiolabeled protein from extracts of MCF-7 cells after pulse labeling with [3H]glucosamine. The 323/A3 antigen appears to be the same Mr 43,000 protein in cell lines as in breast tumors in vivo. Based on a comparison with the molecular weights of other known tumor-associated antigens and with their immunocytochemical tissue distribution, the Mr 43,000 glycoprotein described here represents a tumor-associated antigen previously undescribed in breast cancer or in other tumors. Since the Mr 43,000 glycoprotein is present on the surface of most breast cancer cells and is either absent or expressed at very low levels in most normal tissues including normal breast, the monoclonal antibody described here may have potential applications in diagnosis and management of breast cancer.     

A cDNA for the estradiol-regulated 24K protein: Control of mRNA levels in MCF-7 cells

We have previously demonstrated an estradiol-regulated 24 kDa (24K) protein in human breast cancer tissue culture cells and human tumor biopsies. The presence of 24K correlates well with the presence of steroid hormone receptors. In order to further study the hormonal regulation of the 24K protein and gene, we have isolated cDNA clones corresponding to the 24K mRNA.Poly(A)⁺ RNA isolated from the MCF-7 human breast cancer cell line was translated in a cell-free translation system containing [³⁵S]-methionine. The translation products were immunoprecipitated with a 24K monoclonal antibody, and thein vitro synthesis of 24K protein was confirmed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. The same poly(A)⁺ RNA was used to construct an oligo(dT)-primed cDNA library in thegt11 expression vector system. The library was screened with a highly specific polyclonal antibody raised against 24K protein purified by immunoaffinity chromatography. Four recombinant clones reacting with the antibody by virtue of antigen expression were isolated and three were used in hybridization-selected translation. Three clones were able to hybridize specifically to a messenger RNA (mRNA) that yielded a Mr 24,000 protein when translatedin vitro and analyzed by SDS/polyacrylamide gel electrophoresis. This protein was also immunoprecipitable by the 24K monoclonal antibody. MCF-7 mRNA size fractionated by formaldehyde-agarose gel electrophoresis was transferred to nitrocellulose paper and hybridized to a nick-translated 24K cDNA clone. A single band of hybridization corresponding to a mRNA size of approximately 0.9–1.0 kilobase (kb) was observed. Using this same technique, 24K cDNA was hybridized to mRNA extracted from MCF-7 cells that had been treated for varying periods with either estradiol, nafoxidine, or tamoxifen. The 24K mRNA was elevated by the addition of estradiol, and clearly diminished by nafoxidine and tamoxifen.These results demonstrate that we have isolated cDNA clones for the study of the hormonal regulation of the 24K gene in breast cancer cells, and have shown that the mRNA is regulated by estradiol.     

A human breast tissue-associated antigen detected by a monoclonal antibody

Two monoclonal antibodies were produced in mice immunized with the human breast carcinoma cell line MCF-7. One antibody (24-17.1) reacted with MCF-7 and other breast tumor cell lines and detected an antigen of Mr 95,000. This antigen was not breast-specific because other tumor cell lines were also reactive. The second antibody (24-17.2) detected an antigen of Mr 100,000 (initially appearing to be specific for breast tissue and possibly for breast carcinomas) which was present on 10 of 10 malignant breast lines and absent from 41 of 43 other cell lines of differing origins. The antigen could not be detected by absorption or a direct test on normal tissues (liver, kidney, heart, spleen) or on lymphocytes. In addition, the 24-17.2 antibody reacted absorptively with 12 of 13 fresh breast carcinoma samples but not with fresh colon carcinoma samples. The 100,000-Mr antigen detected by the 24-17.2 antibody appeared to be distinct from the other components of normal breast, such as casein, lactalbumin, or milk fat globulin protein. This evidence indicated that the 24-17.2 antibody detected a human breast carcinoma-associated antigen (HBCAA). However, further histologic studies were used to determine the cellular distribution of the HBCAA, which was found on malignant breast epithelium, the epithelium of gynecomastia, and in lesser amounts and differently distributed on normal breast epithelium. The antigen was also found in several other tissues; nonetheless, the anti-HBCAA could be detected in increased amounts in the sera of patients with breast cancer.     

Antitumor effectiveness of different amounts of electrical charge in Ehrlich and fibrosarcoma Sa-37 tumors

Background

Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX).

Methods

Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer.

Results

A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27).

Conclusions

Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.     

Human Mena protein, a serex-defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T-cell immune response

Screening of a cDNA expression library from a primary breast tumor with the autologous patient serum led to the isolation of 6 cDNA clones corresponding to 3 different genes, including a novel gene that maps to chromosome 1 and encodes the human homologue of mouse Mena (hMena, cDNA clone RMNY-BR-55), a protein of the Ena/VASP family involved in the regulation of cell motility and adhesion. A cancer-restricted antibody response against hMena was demonstrated, since 18/93 cancer patient sera, the majority (10/52) from breast cancer, showed anti-hMena-specific IgG, while no antibodies were present in healthy donors. When hMena protein expression was analyzed by Western blot and immunohistochemistry, the antigen was overexpressed in the majority of breast cancer cell lines and in 75% of primary breast tumor lesions evaluated. Furthermore, when HLA-A2-restricted peptides from the hMena sequence were used to stimulate CD8+ T cells, an hMena-specific response was found in 9 out of 12 HLA-A2+ breast cancer patients. In 4 patients, this cell-mediated immune response was concomitant with antibody response to hMena. Furthermore, an hMena-specific T-cell line was established from an HLA-A2+ breast cancer patient whose primary tumor lesion overexpressed the hMena protein. The present findings highlight the emerging role that overexpression of cytoskeleton regulatory components may have in the induction of a specific antitumor immune response.     

Identification of breast cancer-restricted antigens by antibody screening of SKBR3 cDNA library using a preselected patient's serum

Screening of a breast cancer cDNA library from SKBR3 human breast cancer cells by SEREX (serological analysis of cDNA expression library) using a preselected serum from a breast cancer patient revealed 13 genes, two of which, INT-MI-1 and INT-MI-2, encode novel gene products, while the remaining 11 genes and their products are identical with or highly homologous to known GenBank entries. Immunoscreening of the 13 clones using 20 allogeneic sera from breast cancer patients and 20 samples from age- and gender-matched healthy donors showed that lactate dehydrogenase-A (LDH-A), lactate dehydrogenase-B (LDH-B), fibulin-1, and thyroid hormone-binding protein (THBP) were recognized principally by the breast cancer patient sera, indicating the immunogenicity of these molecules in vivo. The other antigens were similarly recognized by normal and patients sera, and thus not tumor-restricted immunologically. RT-PCR analysis revealed strong expression of fibulin-1 in tumor cell lines and surgical specimen whereas in the same experimental conditions, normal tissues scored negative. Also THBP expression was found in various tumors whereas in normal tissues, its expression is restricted to the testis and, at lower levels, in ovary, liver, and spleen. In contrast, LDH-A and LDH-B were ubiquitously expressed in normal and tumor tissues, with LDH-B levels considerably lower and heterogeneous in normal samples compared to those expressed in tumor cell lines. The differential expression of fibulin-1 between the normal tissues and breast carcinoma cell lines (5/6) and surgical specimens (5/6) suggests the possible involvement of the overexpression of this extracellular matrix-associated glycoprotein in the pathogenesis of this neoplasm.     

Generation of monoclonal antibodies to human milk-fat globule membrane antigens, with special reference to a precipitable secretory product of breast and ovarian carcinomas

Monoclonal antibodies were generated against protein antigens extracted from human milk-fat globule (HMFG) membranes. Of more than 500 hybridoma clones, 16 secreted antibodies reacting with breast and ovarian carcinoma cells, but not with lymphocytes or macrophages. Five clones were further expanded and characterized; four reacted with plasma membranes of all breast and ovarian carcinomas rested, but also with normal breast. One of the monoclonal antibodies, III D 5, reacted mainly against the cytoplasmic components of breast cancer cells, whereas the reaction with normal or mastopathic breast was against the luminal plasma membranes. The antigen recognized by the antibody was secreted in ascites fluid or pleural effusions of patients with widely spread ovarian or breast carcinomas, and could be demonstrated by immunodiffusion. It is suggested that the antigen is composed of several glycoprotein subunits bearing the same repetitive epitope. This might be the physicochemical basis for its precipitability with a monoclonal antibody.     

LMAN2在HR阳性乳腺癌组织中的表达与患者预后的关系及其对MCF-7细胞增殖和迁移的影响

目的：探究甘露糖结合凝集素2(LMAN2)在激素受体（HR）阳性乳腺癌组织中的表达水平与乳腺癌患者预后的关系及其对MCF-7细胞增殖和迁移的影响。方法：通过TCGA、Bc-GenExMiner、GEPIA和Kaplan-Meier Plotter数据库分析LMAN2在乳腺癌组织和正常乳腺组织中的差异性表达及其与患者预后的关系。采用小RNA干扰技术将si-LMAN2#1、si-LMAN2#2及si-NC转染至MCF-7细胞,将过表达LMAN载体（pc-LMAN）及空载体pcDNA3.1阴性对照（pc-NC）转染至MCF-7细胞,实验分为si-LMAN2#1、si-LMAN2#2、si-NC、pc-LMAN2和pc-NC组。通过qPCR和WB实验检测各组细胞中LMAN2 mRNA和蛋白的表达水平,CCK-8、克隆形成、Transwell迁移、WB等实验检测敲低和过表达LMAN 2对MCF-7细胞增殖、克隆形成、迁移及AKT信号通路相关蛋白表达的影响。结果：LMAN2在乳腺癌组织中的表达水平显著高于正常乳腺组织（P<0.001）。HR阳性乳腺癌组织中LMAN2表达水平显著高于HR阴性乳...     

Neoplastic progression of breast epithelial cells--a molecular analysis.

Molecular changes associated with breast cancer progression were characterized using the MCF-10F cell series. MCF-10F was established from fibrous mastectomy tissue of a patient without detectable cancer. In vitro treatment of MCF-10F cells with benzo(a)pyrene resulted in a transformed subclone MCF-10F-BP1 (BP1). Transfection of clone BP1 with T24-Hras resulted in the tumorigenic line MCF-10F-BP1-Tras (BP1-Tras). Using flow cytometry, the expression of HLA I, ERBB-2 and MUC-1 was found to be comparable in ''normal'' MCF-10F, transformed BP1 and tumorigenic BP1-Tras cells. Glycosylated mucin is elevated in BP1 but reduced in BP1-Tras cells. Using mRNA differential display analysis, cDNA profiles of the ''normal'', transformed and tumorigenic cell lines were strikingly similar, yet distinct and elevated expression of several common cDNA fragments was detected in BP1 and BP1-Tras when compared with MCF-10F cells. These fragments were cloned and sequenced. The sequences of clones T1-360 and C4-310 are homologous to two reported EST cDNA clones from human fetal tissue and were further characterized. Elevated expression of the genes corresponding to clones T1-360 and C4-310 was verified using Northern blotting. High-level expression of these genes was also detected in the breast cancer cell line MCF-7 that was derived from the pleural effusion of a patient with advanced breast cancer. Therefore, specific molecular changes associated with breast cancer development were identified and may be indicators of neoplastic progression.     

Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer.

Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250-256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247-256) and KASIFLKTRV (250-259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml(-1) interleukin-2, 25 IU ml(-1) interleukin-7 and 500 IU ml(-1) granulocyte-macrophage colony-stimulating factor. Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2-, A3 CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer.     

Screening a Novel Human Breast Cancer- Associated Antigen from a cDNA Expression Library of Breast Cancer

OBJECTIVE The aim of this research was to clone and express the antigen of the previously prepared monoclonal antibody named M4G3.METHODS Western blots were used to screen a breast cancer cell line that overexpresses the M4G3-associated antigen. A λ zap cDNA expression library of breast cancer cells was constructed and screened using M4G3 as a probe to clone the antigen. The positive clones were subcloned and identified by homologous comparison using BLAST.RESULTS The λ zap cDNA expression library had 1.0x106 independent clones. Fifteen positive clones were isolated following 3 rounds of immunoscreening and identified as being from Mycoplasma pulmonis.CONCLUSION The specific antigen that matched the monoclonal M4G3 antibody is an unknown protein of M. pulmonis. This work is helpful for the further study of the association of M. pulmonis infection with breast cancer.     

Autoantibodies to Annexin XI-A and Other Autoantigens in the Diagnosis of Breast Cancer

We report on the identification of autoantigens commonly recognized by sera from patients with breast cancer. We selected ten sera from patients with invasive ductal carcinoma (IDC) of the breast with high titer IgG autoantibodies for biopanning of a T7 phage breast cancer cDNA display library. A high throughput method involved the assembly of 938 T7 phages encoding potential breast cancer autoantigens. Microarrays of positive phages were probed with sera from 90 patients with breast cancer [15 patients with ductal carcinoma in situ (DCIS) and 75 patients with IDC of the breast], with 51 non-cancer control sera and with sera from 21 patients with systemic autoimmune diseases. A 12-phage breast cancer predictor group was constructed with phage inserts recognized by sera from patients with breast cancer and not by non-cancer or autoimmune control sera (P < 0.0001). Several autoantigens including annexin XI-A, the p80 subunit of the Ku antigen, ribosomal protein S6, and other unknown autoantigens could significantly discriminate between breast cancer and non-cancer control sera. Biopanning with three different sera led to the cloning of partial cDNA sequences identical to annexin XI-A. IgG autoantibodies reacting with the amino acid 41-74 sequence of annexin XI-A were found in 19% of all women with breast cancer but in 60% of sera from women with DCIS of the breast. In addition, partial sequences identical to annexin XI-A, nucleolar protein interacting with the forkhead-associated (FHA) domain of pKi-67, the KIAA1671 gene product, ribosomal protein S6, cyclin K, elongation factor-2, Grb2-associated protein 2, and other unknown proteins could distinguish DCIS from IDC of the breast and appear to be potential biomarkers for the diagnosis of breast cancer.     

Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21

The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH). Six breast cancer patients immunized with this conjugate plus the immunological adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the beta turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells.