Non-coding regions of nuclear-DNA-encoded mitochondrial genes and intergenic sequences are targeted by autoantibodies in breast cancer.

Autoantibodies against mitochondrial-derived antigens play a key role in chronic tissue inflammation in autoimmune disorders and cancers. Here, we identify autoreactive nuclear genomic DNA (nDNA)-encoded mitochondrial gene products (GAPDH, PKM2, GSTP1, SPATA5, MFF, TSPOAP1, PHB2, COA4, and HAGH) recognized by breast cancer (BC) patients' sera as nonself, supporting a direct relationship of mitochondrial autoimmunity to breast carcinogenesis. Autoreactivity of multiple nDNA-encoded mitochondrial gene products was mapped to protein-coding regions, 3' untranslated regions (UTRs), as well as introns. In addition, autoantibodies in BC sera targeted intergenic sequences that may be parts of long non-coding RNA (lncRNA) genes, including LINC02381 and other putative lncRNA neighbors of the protein-coding genes ERCC4, CXCL13, SOX3, PCDH1, EDDM3B, and GRB2. Increasing evidence indicates that lncRNAs play a key role in carcinogenesis. Consistent with this, our findings suggest that lncRNAs, as well as mRNAs of nDNA-encoded mitochondrial genes, mechanistically contribute to BC progression. This work supports a new paradigm of breast carcinogenesis based on a globally dysfunctional genome with altered function of multiple mitochondrial and non-mitochondrial oncogenic pathways caused by the effects of autoreactivity-induced dysregulation of multiple genes and their products. This autoimmunity-based model of carcinogenesis will open novel avenues for BC treatment.

Mitochondrial autoimmunity and MNRR1 in breast carcinogenesis.

BACKGROUND: Autoantibodies function as markers of tumorigenesis and have been proposed to enhance early detection of malignancies. We recently reported, using immunoscreening of a T7 complementary DNA (cDNA) library of breast cancer (BC) proteins with sera from patients with BC, the presence of autoantibodies targeting several mitochondrial DNA (mtDNA)-encoded subunits of the electron transport chain (ETC) in complexes I, IV, and V. METHODS: In this study, we have characterized the role of Mitochondrial-Nuclear Retrograde Regulator 1 (MNRR1, also known as CHCHD2), identified on immunoscreening, in breast carcinogenesis. We assessed the protein as well as transcript levels of MNRR1 in BC tissues and in derived cell lines representing tumors of graded aggressiveness. Mitochondrial function was also assayed and correlated with the levels of MNRR1. We studied the invasiveness of BC derived cells and the effect of MNRR1 levels on expression of genes associated with cell proliferation and migration such as Rictor and PGC-1α. Finally, we manipulated levels of MNRR1 to assess its effect on mitochondria and on some properties linked to a metastatic phenotype. RESULTS: We identified a nuclear DNA (nDNA)-encoded mitochondrial protein, MNRR1, that was significantly associated with the diagnosis of invasive ductal carcinoma (IDC) of the breast by autoantigen microarray analysis. In focusing on the mechanism of action of MNRR1 we found that its level was nearly twice as high in malignant versus benign breast tissue and up to 18 times as high in BC cell lines compared to MCF10A control cells, suggesting a relationship to aggressive potential. Furthermore, MNRR1 affected levels of multiple genes previously associated with cancer metastasis. CONCLUSIONS: MNRR1 regulates multiple genes that function in cell migration and cancer metastasis and is higher in cell lines derived from aggressive tumors. Since MNRR1 was identified as an autoantigen in breast carcinogenesis, the present data support our proposal that both mitochondrial autoimmunity and MNRR1 activity in particular are involved in breast carcinogenesis. Virtually all other nuclear encoded genes identified on immunoscreening of invasive BC harbor an MNRR1 binding site in their promoters, thereby placing MNRR1 upstream and potentially making it a novel marker for BC metastasis.

Autoantibodies in breast cancer.

In addition to their historical role, autoantibodies appear promising as biomarkers to facilitate diagnosis, improve patient outcome and decrease mortality in cancer. Autoantibodies may also be useful in the identification of subjects at risk for cancer, that is, those bearing premalignant changes. Numerous studies have demonstrated that cancer serum contains a variety of autoantibodies that react with autologous cellular antigens, that is, tumor-associated antigens. Interestingly, some of these antigens are involved in signal transduction, cell cycle regulation, cell proliferation, and apoptosis. As such, identification of these molecules has additional importance for development of novel anticancer drugs and vaccines. This review focuses on the use of autoantibodies in breast cancer, a major public health problem. We also address the need for additional research to validate this approach in cancer diagnostics and therapeutics in general.

New insight into the molecular mechanisms of the biological effects of DNA minor groove binders.

BACKGROUND: Bisbenzimides, or Hoechst 33258 (H258), and its derivative Hoechst 33342 (H342) are archetypal molecules for designing minor groove binders, and widely used as tools for staining DNA and analyzing side population cells. They are supravital DNA minor groove binders with AT selectivity. H342 and H258 share similar biological effects based on the similarity of their chemical structures, but also have their unique biological effects. For example, H342, but not H258, is a potent apoptotic inducer and both H342 and H258 can induce transgene overexpression in in vitro studies. However, the molecular mechanisms by which Hoechst dyes induce apoptosis and enhance transgene overexpression are unclear. METHODOLOGY/PRINCIPAL FINDINGS: To determine the molecular mechanisms underlying different biological effects between H342 and H258, microarray technique coupled with bioinformatics analyses and multiple other techniques has been utilized to detect differential global gene expression profiles, Hoechst dye-specific gene expression signatures, and changes in cell morphology and levels of apoptosis-associated proteins in malignant mesothelioma cells. H342-induced apoptosis occurs in a dose-dependent fashion and is associated with morphological changes, caspase-3 activation, cytochrome c mitochondrial translocation, and cleavage of apoptosis-associated proteins. The antagonistic effect of H258 on H342-induced apoptosis indicates a pharmacokinetic basis for the two dyes' different biological effects. Differential global gene expression profiles induced by H258 and H342 are accompanied by unique gene expression signatures determined by DNA microarray and bioinformatics software, indicating a genetic basis for their different biological effects. CONCLUSIONS/SIGNIFICANCE: A unique gene expression signature associated with H342-induced apoptosis provides a new avenue to predict and classify the therapeutic class of minor groove binders in the drug development process. Further analysis of H258-upregulated genes of transcription regulation may identify the genes that enhance transgene overexpression in gene therapy and promote recombinant protein products in biopharmaceutical companies. DATA DEPOSITION: The microarray data reported in this article have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.GSE28616).

Rare cause of thoracic myelopathy: ossified ligamentum flavum.

Myelopathy due to ossification of ligamentum flavum was first described in 1938 by Anzai (J Jpn Orthop Assosc. 1938;13:305-316). This is a rare condition that has been described mostly in the far east, most commonly in Japan (Miyasaka et al, Am J Neuroradiol. 1983;4:629-632). Cases reported from the western hemisphere are rare, particularly in the African-American population (Wiseman et al, J Spinal Disord Tech. 2002;15:542-545). We report an African-American patient who presented with progressive back and chest pain of recent onset, numbness, tingling, and gait disturbance due to T3 paraplegia secondary to ossified ligamentum flavum resulting in thoracic spinal stenosis. On cross-sectional imaging, there was cord effacement at multiple levels, most pronounced at T10. The patient required emergency laminectomies, which allowed him to recover significant function. This case presentation should heighten physician awareness to this unusual clinical entity, since early diagnosis and timely surgical intervention can prevent permanent neurologic deficits.

Autoantibodies in breast cancer sera: candidate biomarkers and reporters of tumorigenesis.

A plethora of promising breast cancer-associated autoantigens have been cloned by immunoscreening cDNA expression libraries with breast cancer sera or identified using proteomics, yet no biomarkers, whether individual autoantigens or panels of antigens developed using antibody-based methods have been validated and incorporated to routine oncologic practice for the early diagnosis of breast cancer. Recently, the addition of genomics, proteomics and high throughput technology to traditional immunological techniques has revived the interest in this field, and some of the most promising breast cancer autoantigens are in the process of being validated prospectively in large cohorts of patients with breast cancer. In addition, some of the identified breast cancer-associated autoantigens are recognized by T-cells and may prove to have a role in the treatment of breast cancer in the future. Autoantibodies found in breast cancer patient sera provide important clues about their significance. The discovery of breast cancer-associated antigens has provocative implications beyond the quest for novel diagnostic biomarkers, because autoantibodies target molecules involved in signal transduction, cell cycle regulation, cell proliferation and apoptosis, all of them key processes in carcinogenesis. Molecular components of the DNA double-strand break repair machinery as well as several members of the rapamycin-sensitive pathway elicit an autoantibody response in breast cancer. Data obtained by screening cDNA expression libraries of breast cancer antigens with autoantibodies present in breast cancer sera suggest that autoantibodies in cancer sera may be linked to the process of apoptosis. The studies reviewed here, clearly demonstrate the participation of autoimmunity in breast cancer to an extent previously unsuspected, which may have broad implications for the discovery of molecular targets for drug therapy and cancer biomarkers in general.

Improved approach to identify cancer-associated autoantigens.

The failure to identify biomarkers of clinical significance for cancer diagnosis and prognosis generated a great deal of skepticism in regard to the usefulness of autoantibody-based methods. SEREX was a major advancement in immunoscreening that resulted in the identification of a large group of autoantigens recognized by cancer sera. However, few SEREX-defined autoantigens have proven to have definitive diagnostic value in clinical practice. Often, the identified antigens are patient-specific rather than tumor-specific and many tumor-associated antigens are rare in expression libraries made from non-autologous cells. Since autoantibodies are part of the normal immune response, it can be difficult to single out tumor-associated antibodies from the scores of irrelevant patient-specific responses. In our view, any practical approach for identifying cancer-related autoantigens must include an integral strategy for demonstrating tumor relevance early in the screening process. Care must also be taken not to exclude potentially important autoantibodies by pre-screening manipulations to patient sera. We have introduced substantial modifications in SEREX, designed to minimize confounding effects of unrelated autoantibodies and to eliminate steps that preclude the identification of cancer-related autoantigens commonly recognized by cancer sera. In addition, we incorporate methodology to identify candidate antigens that have potential diagnostic or prognostic value prior to their molecular cloning and characterization.

Spectra of antinuclear antibodies in patients with squamous cell carcinoma of the lung and of the head and neck.

Squamous cell carcinoma of the head and neck (HNSCC) and of the lung (LSCC) share some important risk factors, but differ substantially in terms of prognosis and treatment. A pulmonary nodule developing in patients with surgically cured HNSCC may pose a diagnostic dilemma. Markers able to distinguish these two common malignancies would be of major clinical importance. In this work we compared the spectrum of antinuclear antibodies (ANA) from 22 patients with SCCL to that of 40 patients with HNSCC. Patient sera were used to probe immunoblots of nuclear extracts from all four major lung cancer cell types, normal lung fibroblasts, cells cultured from a HNSCC, and keratinocytes cultured from the field cancerization. The ability to classify retrospectively LSCC from HNSCC based on serum ANA reactivities was determined by recursive partitioning analyses. We found that while both malignancies share reactivities to a small group of nuclear antigens, other reactivities are directed against proteins uniquely or preferentially expressed in either SCCL or in SCCHN cells. Our work shows that autoimmunity is a prominent feature of squamous cell carcinoma and suggests that molecular characterization of nuclear antigens recognized by ANAs may lead to the discovery of markers valuable to distinguish LSCC from HNSCC.

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.