Autoantibodies in breast cancer sera are not epiphenomena and may participate in carcinogenesis.

BACKGROUND: The objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases. METHODS: We performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens. RESULTS: Autoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative. CONCLUSIONS: The distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women. Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.

Anti-centrosome antibodies in breast cancer are the expression of autoimmunity.

Centrosome abnormalities have been observed in nearly all human solid tumors, but their role in tumorigenesis is unclear. We have demonstrated that autoantibodies reacting with antigens in centrosomes are frequently found in BC sera. In this work, we attempted to characterize the centrosome antigens associated with BC. We immunoscreened a T7 cDNA library of BC proteins with BC sera, and the autoantigens identified were printed as a microarray and hybridized with BC and control sera. We used immunohistochemistry (IHC) to investigate the expression of the cloned autoantigens in BC tissue. Immunoscreening with BC sera led to the cloning of autoantibodies recognizing epitopes developing in a family of proteins located on centrosomes such as peri-centriolar material-1, isomorph CRA, stathmin1, HS actin gamma1, SUMO/sentrin peptidase 2, and ubiquitin-conjugating enzyme E2 variant 1. Antibody reactivity to these proteins that are associated with centrosome assembly and/or microtubule function was highly associated with the diagnosis of BC. IHC staining of formalin-fixed paraffin-embedded sections with specific antibodies showed that aurora and stathmin are expressed in BC. The discovery of autoantibodies to important centrosome antigens associated with BC suggests that this immune reactivity could be related to autoimmunity developing in BC. Our finding that some of these antibodies are also present in a group of healthy women suggests that breakdown of tolerance to centrosome proteins may occur early in breast carcinogenesis and that autoantibodies to centrosome antigens might be biomarkers of early BC.

Anti-DFS70/LEDGF antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases.

OBJECTIVE: Antinuclear antibodies (ANA) are a serological hallmark of systemic autoimmune rheumatic diseases (SARD) such as systemic lupus erythematosus (SLE). While a number of ANA patterns detected by indirect immunofluorescence (IIF) have diagnostic significance, autoantibodies producing the dense fine speckled (DFS) pattern have been reported to be more prevalent in healthy individuals than in SARD. METHODS: Sequential samples submitted for ANA testing were screened for anti-DFS antibodies by IIF (n = 3263). Samples with the DFS pattern were tested for anti-DFS70/lens epithelium-derived growth factor (LEDGF) antibodies by ELISA and by a novel chemiluminescence assay (CIA, Quanta Flash DFS70). Sera from patients with various diseases and healthy individuals were tested for anti-DFS70/LEDGF antibodies by CIA. A cohort of 251 patients with SLE was used to analyze serological and clinical associations of anti-DFS70 antibodies. RESULTS: The frequency of anti-DFS antibodies by IIF was 1.62%. The prevalence of anti-DFS70/LEDGF antibodies as detected by CIA in the different cohorts was 8.9% in healthy individuals, 2.8% in SLE, 2.6% in rheumatoid arthritis, 4.0% in asthma, 5.0% in interstitial cystitis, 1.7% in Graves' disease, and 6.0% in Hashimoto's thyroiditis. Of note, the prevalence of anti-DFS70/LEDGF antibodies was significantly higher in healthy individuals compared to patients with SARD (p = 0.00085). In SLE results, anti-DFS70/LEDGF antibodies were not significantly associated with clinical features or other autoantibodies typically found in SLE. Only 1/7 SLE sera showed anti-DFS70/LEDGF, but no other autoantibody reactivity. CONCLUSION: "Monospecific" anti-DFS70/LEDGF antibodies may represent a biomarker for differentiating SARD from non-SARD individuals, but there is a need for a reliable assay to ensure reactivity to DFS70.

The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA).

Autoantibodies targeting the proliferating cell nuclear antigen (PCNA) were first described over 30 years ago and are historically most commonly associated with systemic lupus erythematosus (SLE). The primary antigenic target is a 34 kDa protein that is part of the DNA polymerase delta multi-protein complex. A number of diagnostic platforms have incorporated PCNA into their diagnostic assays and algorithms. However, little is known about the clinical utility of autoantibodies to PCNA, especially with novel detection systems. This review will focus on the history of the discovery of the PCNA autoantigen and the current status of the diagnostic significance of anti-PCNA and suggest future studies that are required to strengthen our understanding of their clinical utility.

Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F.

Autoantibodies to the centromere proteins (CENP), which are major constituents of the primary constriction of metaphase chromosomes, were first described in 1980. In those seminal publications and 30 years of research that have followed, a number of CENP have been identified as autoantibody targets in human diseases. Historically, autoantibodies directed to CENP-A, -B and -C have been considered relatively specific biomarkers for limited cutaneous systemic sclerosis (lcSSc) or the calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) syndrome. These autoantibodies, found in up to 40% of SSc sera, can be identified by indirect immunofluorescence (IIF) on a variety of tissue culture cell lines as a discrete speckled staining pattern of both interphase nuclei and metaphase chromatin. Early in the investigation of anti-CENP, it became apparent that some autoantibodies had a similar IIF pattern wherein as cells entered into the cell cycle, speckled staining of the metaphase chromatin could be observed but, unlike conventional CENP staining, interphase nuclei were not stained. Subsequent studies identified one of the targets of these autoantibodies to be CENP-F, a kinesin binding protein essential for completion of the cell cycle. Early clinical studies found that, unlike antibodies to the earlier described CENP, lcSSc rarely expressed anti-CENP-F and approximately 50% of these patients had a malignancy. This review provides a historical perspective of CENP autoantibodies and focuses on an update of the information on CENP-F and their clinical associations.

Arc regulates spine morphology and maintains network stability in vivo.

Long-term memory relies on modulation of synaptic connections in response to experience. This plasticity involves trafficking of AMPA receptors (AMPAR) and alteration of spine morphology. Arc, a gene induced by synaptic activity, mediates the endocytosis of AMPA receptors and is required for both long-term and homeostatic plasticity. We found that Arc increases spine density and regulates spine morphology by increasing the proportion of thin spines. Furthermore, Arc specifically reduces surface GluR1 internalization at thin spines, and Arc mutants that fail to facilitate AMPAR endocytosis do not increase the proportion of thin spines, suggesting that Arc-mediated AMPAR endocytosis facilitates alterations in spine morphology. Thus, by linking spine morphology with AMPAR endocytosis, Arc balances synaptic downscaling with increased structural plasticity. Supporting this, loss of Arc in vivo leads to a significant decrease in the proportion of thin spines and an epileptic-like network hyperexcitability.

The serum response factor and a putative novel transcription factor regulate expression of the immediate-early gene Arc/Arg3.1 in neurons.

The immediate-early effector gene Arc/Arg3.1 is robustly upregulated by synaptic activity associated with learning and memory. Here we show in primary cortical neuron culture that diverse stimuli induce Arc expression through new transcription. Searching for regulatory regions important for Arc transcription, we found nine DNaseI-sensitive nucleosome-depleted sites at this genomic locus. A reporter gene encompassing these sites responded to synaptic activity in an NMDA receptor-dependent manner, consistent with endogenous Arc mRNA. Responsiveness mapped to two enhancer regions approximately 6.5 kb and approximately 1.4 kb upstream of Arc. We dissected these regions further and found that the proximal enhancer contains a functional and conserved "Zeste-like" response element that binds a putative novel nuclear protein in neurons. Therefore, activity regulates Arc transcription partly by a novel signaling pathway. We also found that the distal enhancer has a functional and highly conserved serum response element. This element binds serum response factor, which is recruited by synaptic activity to regulate Arc. Thus, Arc is the first target of serum response factor that functions at synapses to mediate plasticity.

AMPA receptors regulate transcription of the plasticity-related immediate-early gene Arc.

Learning and memory depend critically on long-term synaptic plasticity, which requires neuronal gene expression. In the prevailing view, AMPA receptors mediate fast excitatory synaptic transmission and effect short-term plasticity, but they do not directly regulate neuronal gene expression. By studying regulation of Arc, a gene required for long-term plasticity, we uncovered a new role for AMPA receptors in neuronal gene expression. Spontaneous synaptic activity or activity induced by brain-derived neurotrophic factor (BDNF) elicited Arc expression in cultures of rat cortical neurons and in organotypic brain slices. Notably, inhibiting AMPA receptors strongly potentiated activity-dependent Arc expression. We found that AMPA receptors negatively regulate Arc transcription, but not translation or stability, through a mechanism involving a pertussis toxin-sensitive G protein. These results provide insights into the activity-dependent mechanisms of Arc expression and suggest that, in addition to effecting short-term plasticity, AMPA receptors regulate genes involved in long-term plasticity.

Autoantibody to hLSm4 and the heptameric LSm complex in anti-Sm sera.

OBJECTIVE: To characterize the 15-kd human SmD-like autoantigen and its associated proteins previously shown to be recognized by IgM antibodies in patients with Epstein-Barr virus (EBV)-induced infectious mononucleosis. METHODS: The full-length complementary DNA for the 15-kd protein was expressed as recombinant protein and analyzed for reactivity using biochemical analysis and immunoprecipitation (IP). RESULTS: The 15-kd protein was determined to be the human like-Sm protein LSm4 (hLSm4). Rabbit antibody raised against the C-terminal polypeptide immunoprecipitated a 68-kd complex composed of LSm4 together with a group of smaller proteins ranging in size from 6.5 to 14 kd, consistent with the reported heptameric LSm complexes involved in U4/U6 duplex formation and messenger RNA (mRNA) decapping/degradation. About 80% of all anti-Sm sera from patients with systemic lupus erythematosus (SLE) recognized the hLSm4 in vitro translated product, while 6.7% (29 of 434) immunoprecipitated from cell extracts hLSm4 together with the other members of the hLSm complex. Four sera (0.92%) showed apparently exclusive reactivity to the hLSm complex in the absence of reactivity to Sm core proteins in the IP assay. CONCLUSION: These findings document that while IgM, but not IgG, autoantibodies to LSm4 were found in sera from patients with EBV infection, IgG autoantibodies to hLSm4 are detected in a large number of anti-Sm-positive sera from patients with SLE. Importantly, in a small number of anti-Sm sera the LSm complex can be recognized independently of the Sm core protein antigens. Our data introduce the concept that "Sm" autoantigens include Sm as well as LSm complexes involved in the maturation and degradation of mRNA.