Anti-MAGEA1 monoclonal antibody Made to order

Melanoma-associated antigen A1 (MAGEA1) and BORIS (also known as CTCFL) are members of the cancer testis antigen (CTA) family. Their functions and expression-regulation mechanisms are not fully understood. In this study, we reveal new functions and regulatory mechanisms of MAGEA1 and BORIS in breast cancer cells, which we investigated in parental and genetically manipulated breast cancer cells via gene overexpression or siRNA-mediated downregulation. We identified the interaction between MAGEA1 and CTCF, which is required for the binding of MAGEA1 to the BORIS promoter and is critical for the recruitment of DNMT3a. A protein complex containing MAGEA1, CTCF and DNMT3a was formed before or after conjunction with the BORIS promoter. The binding of this complex to the BORIS promoter accounts for the hypermethylation and repression of BORIS expression, which results in cell death in the breast cancer cell lines tested. Multiple approaches were employed, including co-immunoprecipitation, glutathione S-transferase pull-down assay, co-localization and cell death analyses using annexin V-FITC/propidium iodide double-staining and caspase 3 activation assays, chromatin immunoprecipitation and bisulfite sequencing PCR assays for methylation. Our results have implications for the development of strategies in CTA-based immune therapeutics.

Melanoma-associated antigen A1 (MAGEA1) is member of the MAGE gene family that is expressed in male germ line cells and placenta under normal physiological conditions. Although MAGEA1's expression levels have been evaluated as one of the cancer testis (CT) antigens for immunotherapy in melanoma and several other cancers, its functional role and signaling mechanisms are largely unknown. In this study, we examined the functional involvement and signaling mechanisms of MAGEA1 in breast and ovarian cancer cells. Inhibitory effects of MAGEA1 on cell proliferation and migration were observed using both gain-of-(MAGEA1 overexpression) and loss-of-(siRNA interference) function approaches in breast (MCF-7 and MDA-MB-231) and ovarian (SKOV3 and SKOV3ip) cancer cell lines. We revealed a novel interaction between MAGEA1 and the intracellular segment of NOTCH1 receptor (NICD1). MAGEA1 reduced NICD1's stability by promoting the ubiquitin modification of NICD1. MAGEA1 also interacted with FBXW7, a subunit of E3 ubiquitin protein ligase complex SCF, and the latter was functionally involved in NICD1 ubiquitination and degradation. In addition, siRNA interference of FBXW7 reversed the inhibitory effect of MAGEA1 on migration and proliferation of MCF-7 and MDA-MB-231 cells. These newly discovered MAGEA1-NICD1 and MAGEA1-FBXW7 interactions have potential clinical implications in breast and ovarian cancer treatment.