Paraneoplastic Antigen Research Articles

Impaired male fertility and atrophy of seminiferous tubules caused by haploinsufficiency for Foxa3

Foxa1, 2 and 3 (formerly HNF-3α, -β and -γ) constitute a sub-family of winged helix transcription factors with multiple roles in mammalian organ development. While all three Foxa mRNAs are present in endoderm derivatives including liver and pancreas, only Foxa3 is expressed in the testis. Here we demonstrate by genetic lineage tracing that Foxa3 is expressed in postmeiotic germ and interstitial Leydig cells. The germinal epithelium of Foxa3-deficient testes is characterized by a loss of germ cells secondary to an increase in germ cell apoptosis that ultimately leads to a Sertoli cell-only syndrome. Remarkably, not only the Foxa3 −/− mice but also Foxa3 +/− mice exhibited loss of germ cells. This cellular phenotype caused significantly reduced fertility and testis weight of both Foxa3 −/− and Foxa3 +/− mice. Using microarray analysis, we found a dramatic downregulation of the zinc finger protein 93 and the testicular tumor-associated paraneoplastic Ma antigen (PNMA) and increased expression of a number of genes including zinc finger protein 94 and several kallikrein 1-related peptidases which could account for at least part of the observed phenotype. In summary, we have identified Foxa3 as a transcriptional regulator with a dominant phenotype in germ cell maintenance and suggest FOXA3 as a potential candidate gene for subfertility in man.

A differential gene expression profiles by cDNA microarrays in endometrioid endometrial carcinoma: a preliminary study

Objective : Endometrial carcinoma is the most common gynecological malignant disease in industrialized countries. However, the molecular bases for endometrial tumoriogenesis are not clearly elucidated. Our hypothesis is that there may be some difference in gene expression patterns between normal endometrium and endometrial cancer lesion. In this study, we analyzed the difference of gene expression profile with cDNA microarray. Methods : Normal endometrial tissues and cancer lesions were gathered from three patient with endometrioid endometrial cancer. cDNA microarray technique (KNU 4.8K chip) was applied to screen the different gene expression profiles. Results : Many genes such as interleukin-1 receptor-associated kinase 1 (IRAK1), bifunctional apoptosis regulator (BFAR), paraneoplastic antigen MA2 (PNMA2), zinc finger protein 257 (ZNF257), ras homolog gene family, member F (in filopodia) (ARHF), cell division cycle 27 (CDC27) were over-expressed in the endometrial cancer tissue. The genes were down-regulated in the endometrial cancer samples included fibronectin 1 (FN1), meiotic checkpoint regulator (MCPR), transforming growth factor beta-stimulated protein TSC-22 (TSC22), programmed cell death 4 (neoplastic transformation inhibitor) (PDCD4), transcript variant 2, matrix metalloproteinase 2 (MMP2), insulin-like growth factor binding protein 4 (IGFBP4), retinoblastoma binding protein 7 (RBBP7), insulin-like growth factor binding protein 3 (IGFBP3), downregulated in ovarian cancer 1 (DOC1). Conclusion : The result of this analysis supports the hypothesis that the endometrial cancer tissue has distinct gene expression profile from normal endometium. But, the vaildation of gene expression with RT-PCR and the further study are needed.

A Functional –1 Ribosomal Frameshift Signal in the Human Paraneoplastic Ma3 Gene

A bioinformatics approach to finding new cases of -1 frameshifting in the expression of human genes revealed a classical retrovirus-like heptanucleotide shift site followed by a potential structural stimulator in the paraneoplastic antigen Ma3 and Ma5 genes. Analysis of the sequence 3' of the shift site demonstrated that an RNA pseudoknot in Ma3 is important for promoting efficient -1 frame-shifting. Ma3 is a member of a family of six genes in humans whose protein products contain homology to retroviral Gag proteins. The -1 frameshift site and pseudoknot structure are conserved in other mammals, but there are some sequence differences. Although the functions of the Ma genes are unknown, the serious neurological effects of ectopic expression in tumor cells indicate their importance in the brain.

Recoverin as a cancer-retina antigen.

In photoreceptor cells the Ca(2+) -binding protein recoverin controls phosphorylation of the visual receptor rhodopsin by inhibiting rhodopsin kinase (GRK-1). It can also serve as a paraneoplastic antigen in the development of retinal degeneration in some patients with cancer. The aberrant expression of recoverin in cancer cells and the presence of autoantibodies against recoverin are essential for the occurrence of cancer-associated retinopathy, which finally results in the apoptosis of photoreceptor cells. Noteworthy in cancer patients, the aberrant recoverin expression and the appearance of autoantibodies against recoverin are more frequent than paraneoplastic syndromes. We suggest the term "cancer-retina antigens" for this kind of proteins like recoverin that are solely expressed in retina and tumor tissues and evoke antibodies and/or T cells in patients with cancer. The rare development of a paraneoplastic syndrome is possibly caused by this immune response and probably depends on further events allowing to overcome the blood-retina barrier and the immune privileged status of the retina. It is still unknown whether aberrantly expressed recoverin could have a specific function in cancer cells, though it is suggested that it can be functionally associated with G-protein-coupled receptor kinases. This paper reviews the present knowledge on paraneoplastic syndromes associated with the aberrant expression of recoverin. A possible application of recoverin as a potential target for immunotherapy of cancer is discussed.

Induction of humoral responses specific for paraneoplastic cerebellar degeneration-associated antigen by whole recombinant yeast immunization

Paraneoplastic cerebellar degeneration (PCD) is a potent autoimmune disorder in which antigen-driven responses toward the onconeural antigen are assumed to occur in patients. Yeast cell wall has adjuvant capacity and provides immunostimulatory effects of the antigen expressing in viable cells. The recombinant yeast expressing the PCD-associated antigen may become an immunogen for inducing PCD-associated autoimmunity in mice. We attempted to induce autoimmune responses with whole recombinant yeast expressing PCD-associated antigen. SJL/J strain of mouse is found to be a responder to the major epitope on the antigen for anti-Purkinje cell antibodies, and whole recombinant yeast could induce cellular and humoral autoimmune responses in vivo ion SJL/J mice. The immunization technique based on the recombinant yeast expressing a PCD-associated antigen provides a new tool for analyzing the underlying immunological pathomechanisms of PCD.

Stimulation of the aberrant expression of a paraneoplastic antigen, recoverin, in small cell lung cancer cell lines

Recoverin, a retina-specific Ca 2+-binding protein, is one of the paraneoplastic antigens (PNAs) which are normally present in neurons, but can also be aberrantly expressed in malignant tumors localized outside the nervous system. In this study, we have analyzed 16 small cell lung carcinoma (SCLC) and 12 non-small cell lung carcinoma cell lines and found that none of them is capable of expressing recoverin in vitro. However, two small cell lung carcinoma lines, NCI-H69 and NCI-H82, became recoverin-positive after cultivation in the presence of butyrate. Recoverin expression in the butyrate-treated cells has been detected by immunoblotting with polyclonal (monospecific) antibodies against recoverin and confirmed by the analysis of recoverin mRNA expression. To our knowledge, this work is the first to demonstrate stimulation of the aberrant expression of recoverin in cancer cell lines in vitro. This result opens the way to investigation of the mechanisms underlying the aberrant expression of recoverin, as well as other paraneoplastic antigens, in tumor cells.

Stimulation of the aberrant expression of a paraneoplastic antigen, recoverin, in small cell lung cancer cell lines

Recoverin, a retina-specific Ca2+-binding protein, is one of the paraneoplastic antigens (PNAs) which are normally present in neurons, but can also be aberrantly expressed in malignant tumors localized outside the nervous system. In this study, we have analyzed 16 small cell lung carcinoma (SCLC) and 12 non-small cell lung carcinoma cell lines and found that none of them is capable of expressing recoverin in vitro. However, two small cell lung carcinoma lines, NCI-H69 and NCI-H82, became recoverin-positive after cultivation in the presence of butyrate. Recoverin expression in the butyrate-treated cells has been detected by immunoblotting with polyclonal (monospecific) antibodies against recoverin and confirmed by the analysis of recoverin mRNA expression. To our knowledge, this work is the first to demonstrate stimulation of the aberrant expression of recoverin in cancer cell lines in vitro. This result opens the way to investigation of the mechanisms underlying the aberrant expression of recoverin, as well as other paraneoplastic antigens, in tumor cells.

Recoverin as a paraneoplastic antigen in lung cancer: the occurrence of anti-recoverin autoantibodies in sera and recoverin in tumors

Using immunoblotting with recombinant recoverin as an antigen, we have examined 279 serum samples from individuals with small cell lung carcinoma (SCLC, 99 patients), non-small cell lung carcinoma (NSCLC, 44 patients), and non-malignant pulmonary disorders (86 patients) as well as sera from 50 healthy donors. Autoantibodies against recoverin (anti-Rc) were detected in sera from 15 patients with SCLC (15% of cases) and from 9 patients with NSCLC (about 20% of cases). Only two anti-Rc positive cases were detected in patients with non-malignant pulmonary disorders, while no such cases were found in healthy individuals. Immunohistochemical investigation of paraffin sections of 44 SCLC and 40 NSCLC tumors revealed recoverin-positive reaction in 30 SCLC (68%) and 34 NSCLC (85%) sections. Despite the high specificity (98%), the low sensitivity (less than 20%) does not allow serum anti-Rc to be considered as a valuable marker of lung cancer. However, taking into account the high occurrence of aberrant expression of recoverin in lung tumors, this PNA could be considered as a potential target for immunotherapy of lung cancer.

P-137 Stimulation of the aberrant expression of the paraneoplastic antigen recoverin in small-cell lung cancer cell lines

P-137 Stimulation of the aberrant expression of the paraneoplastic antigen recoverin in small-cell lung cancer cell lines

Interaction of a Paraneoplastic Cerebellar Degeneration-Associated Neuronal Protein with the Nuclear Helix-Loop-Helix Leucine Zipper Protein MRG X

Paraneoplastic cerebellar degeneration-associated antigen PCD17/cdr2 is a neuronal protein expressed predominantly in the cytoplasm of cerebellar Purkinje cells. The biological activities of this protein are not known; however, the presence of a leucine zipper motif in its amino acid sequence suggests that this protein might interact with other proteins harboring a leucine zipper motif. In this study we found by means of a yeast two-hybrid system, ligand overlay assay, and co-immunoprecipitation assay that PCD17 interacts with a nuclear helix-loop-helix leucine zipper protein, MRG X. Overexpression of MRG X in T98G glioblastoma cells by transfection caused abnormal morphological changes in the nucleus and induced cell death. On the other hand, coexpression of PCD17 with MRG X prevented nuclear morphological changes and cell death in T98G cells. MRG X, which is thought to be functionally related to the cell cycle and cell growth, may be regulated by PCD17/cdr2 in Purkinje cells.

Induction of Cytotoxic T Lymphocytes Specific for Paraneoplastic Cerebellar Degeneration-associated Antigen in vivo by DNA Immunization

Paraneoplastic cerebellar degeneration associated with gynecological and breast malignancies (PCD) is known to develop autoantibodies and autoreactive cytotoxic T lymphocytes (CTLs) specific for a cytoplasmic protein of Purkinje cells PCD17/cdr2, in the blood of patients. The functional roles of these antibodies and CTLs in the pathogenesis of PCD are unknown. Induction of immune response to this antigen in experimental animals should be useful to clarify the immune mechanisms in PCD patients. We immunized Balb/c mice with naked DNA, which is encoded human PCD17 neural protein in an eukaryotic expression plasmid under control of CMV promoter, and explored whether or noth humoral and cell-mediated immune responses against PCD17 could be induced in vivo. We show that DNA immunization with nake pcd17 cDNA could induce autoantibodies against the cytoplasmic protein of Purkinje cells and CTLs could lyse syngenic myeloma cells pulsed with H-2K-restricted PCD17 peptide. In spite of the generation of anti-Purkinje cell antibodies and PCD17-specific CTLs in vivo, neither clinical nor pathological changes consistent with significant cerebellar degeneration have been detected.

Suppression of the transcriptional activity and DNA binding of nuclear factor-kappa B by a paraneoplastic cerebellar degeneration-associated antigen

Paraneoplastic cerebellar degeneration (PCD) associated with gynecological malignancies is a disorder in which an autoimmune mechanism has been suggested, and both antibody- and cell-mediated immune responses exist against pcd17/cdr2, a neural protein expressed in cerebellar Purkinje neurons and brainstem neurons. In this report, we describe that pcd17 can suppress the basal or activated NF-κB-dependent transcriptional activity in a co-transfection study. The DNA binding of constitutive NF-κB complexes decreased in the nucleus of TNF-α-stimulated neuroblastoma cells, though pcd17 does not bind to classical NF-κB consensus site. These data indicate that pcd17 is a potential repressor for NF-κB-dependent gene transcription in neurons.

Retinal degeneration under the effect of antibodies to recoverin.

Serious changes in the retina, diagnosed as retinal degeneration and uveitis, are observed only in the presence of high titers of antibodies to recoverin (Ca2+-binding protein, a paraneoplastic antigen) in the blood of rabbits. Negligible changes in the retina of rabbits with low antibody titers are detected only by cytohistochemical analysis of the retina. No changes in the retina develop in rabbits intravenously injected with antibodies to recoverin.

Paraneoplastic syndromes of the nervous system.

Paraneoplastic syndromes affecting the nervous system are unique among immune-mediated disorders in that the trigger of the immune response is known: tumor expression of proteins normally restricted to neurons (or other immunoprivileged sites, such as testis) but ectopically expressed in some cancers results in an immunological response characterized by high titers of antibodies targeting the "onconeuronal" antigen. A T-cell response is also elicited in some paraneoplastic syndromes and may be the cause of neuronal destruction. In some instances genes that code for the antigens recognized by the autoantibodies have been identified, cloned and sequenced. Some of the proteins so identified are RNA binding proteins but their specific function has not been identified. In some individuals with cancer but no paraneoplastic syndrome, low titers of antibody can be identified in the serum. Low titers of antibody are associated with a better prognosis of the cancer. Experimental animals immunized against a paraneoplastic antigen are partially protected against tumors that express that antigen.

DNA vaccination against HuD antigen elicits antitumor activity in a small-cell lung cancer murine model.

There is a clinically significant correlation between the presence of an antibody against the paraneoplastic encephalomyelitis antigen HuD and the limitation of tumor spread in patients with small-cell lung cancer (SCLC). This suggests that HuD is a possible target molecule for antitumor immunotherapy against SCLC. We have hypothesized that anti-HuD immunity suppresses in vivo growth of HuD-expressing tumor cells. In this study, Colon 26, a murine adenocarcinoma cell line, stably transfected with the HuD gene (Colon 26/HuD cell) was used as a target cell, and the immunity against HuD was evoked by intramuscular injection of a HuD-expressing plasmid, a technique of DNA vaccination previously used in BALB/c mice. Colon 26/HuD cells were injected subcutaneously and tumor size was calculated as a product of width and length. Antitumor activity was investigated by using two different lots of Colon26/HuD cells in two protocols: Protocol 1, in which either Colon 26/HuD or Colon 26 cells were injected in each side, and Protocol 2, in which Colon 26/HuD cells alone were injected. The size of Colon 26/HuD tumors obtained from mice vaccinated with HuD-expressing plasmid was significantly smaller than those from negative control plasmid-vaccinated mice (86.6 +/- 29.9 versus 195.3 +/- 48.1 mm2, P < 0.05 in Protocol 1; 107.7 +/- 12.8 versus 156.6 +/- 22.8 mm2, P < 0.05 in Protocol 2). Moreover, the de novo DNA synthesis of spleen cells obtained from HuD-vaccinated mice was significantly enhanced. In addition, anti-HuD antibody was found in individual sera obtained from HuD-vaccinated mice. DNA vaccination with mouse HuD antigen suppressed HuD-expressing tumor growth in a murine SCLC model.

PKN Interacts with a Paraneoplastic Cerebellar Degeneration-Associated Antigen, Which Is a Potential Transcription Factor

PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and α-actinin. To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed. Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library. The interaction between PKN and PCD17 was also determined by thein vitrobinding analysis. PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17. PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 μM arachidonic acid. The amino-terminal region of PCD17 could form a homodimerin vitro,and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells. These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus.

POP66, a paraneoplastic encephalomyelitis-related antigen, is a marker of adult oligodendrocytes.

Paraneoplastic neurological diseases are disorders of the central nervous system, associated with neuronal degeneration in patients with systemic cancer, but are not a direct result of the tumor mass or metastasis. The biological diagnosis of these syndromes is based mainly on the detection, in the patient's serum and cerebrospinal fluid, of autoantibodies (anti-Hu, anti-Yo, for example), suggesting an autoimmune origin for these disorders. Recently, we described novel autoantibodies (anti-CV2 autoantibodies) associated with paraneoplastic neurological disease, which recognize a 66 kDa brain protein. We named this antigen POP66, for Paraneoplastic Oligodendrocyte Protein of 66 kDa molecular weight, as in the adult human, rat, and mouse brain, it is specifically expressed by a subpopulation of oligodendrocytes. This cell type specificity was surprising given the fact that the cell loss in the brains of patients with anti-CV2 autoantibodies is neuronal. POP66-expressing oligodendrocytes are distributed along the longitudinal axis of the brain according to an increasing rostro-caudal gradient, with no positive oligodendrocytes being found in the forebrain and the greatest number found in the spinal cord. In addition, in transverse sections of the spinal cord, the distribution of POP66-positive oligodendrocytes follows an increasing dorsal-to-ventral gradient, which may be related to different oligodendrocyte precursor pools. In addition, the neuronal loss without demyelination seen in the brains of patients with anti-CV2 autoantibodies, together with the exclusive oligodendroglial expression of POP66 in the adult brain, raises the question of the possible involvement of POP66 in neuron survival via neuron/oligodendrocyte interactions.

Human periplakin: cDNA and genomic cloning, chromosomal mapping, mRNA expression, and a paraneoplastic antigen

Human periplakin: cDNA and genomic cloning, chromosomal mapping, mRNA expression, and a paraneoplastic antigen

A Post-Transcriptional Regulatory Mechanism Restricts Expression of the Paraneoplastic Cerebellar Degeneration Antigen cdr2 to Immune Privileged Tissues

Paraneoplastic cerebellar degeneration (PCD) is believed to be an autoimmune disorder initiated by the ectopic expression of a neuron-specific protein in breast and ovarian tumors. PCD antisera was used previously to identify several cerebellar degeneration-related (cdr) genes encoding putative PCD antigens. We have found that the cdr2 gene, which encodes a cytoplasmic leucine zipper protein of unknown function, is expressed in PCD-associated tumors, whereas other cdr genes are not; thus, cdr2 encodes the PCD tumor antigen. To determine whether the expression pattern of cdr2 is consistent with its proposed role in PCD, we have isolated the mouse homolog and examined both the mRNA and protein distribution in adult tissues. We have found that cdr2 mRNA is expressed in almost all tissues, whereas the protein is expressed only in the brain and testis. Within the brain, both the cdr2 mRNA and immunoreactivity are confined primarily to neurons in the cerebellum and brainstem, the regions most affected in PCD. These results suggest first that the tissue-specific expression of cdr2 is regulated at a post-transcriptional level. Moreover, because the brain and testis are considered to be immune-privileged sites, the expression pattern of cdr2 is compatible with the autoimmune model of PCD pathogenesis.

The HuD paraneoplastic protein shares immunogenic regions between PEM/PSN patients and several strains and species of experimental animals

Patients with small-cell carcinoma of the lung and paraneoplastic encephalomyelitis develop antibodies (anti-Hu) against HuD, a member of a family of neuronal specific RNA-binding proteins, which are also expressed by the tumor. In order to determine if the human HuD paraneoplastic antigen shares immunogenic regions between patients and several strains and species of animals, we immunized animals with HuD and several deletion constructs of this protein. All immunized animals developed high titers of anti-HuD antibodies, comparable to the antibody titers found in paraneoplastic patients. Using immunohistochemistry and Western blot analysis of human and mouse cerebral cortex, the pattern of reactivity of these antibodies could not be differentiated from the human paraneoplastic anti-Hu antibodies. None of the immunized animals developed neurologic symptoms. Western blot analysis of HuD deletion constructs demonstrated that the first and second RNA binding domains were the main immunodominant regions, and that the animal immune response was both strain and species dependent.