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Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival. [ABSTRACT FROM AUTHOR]

Lack of molecular targets in estrogen receptor-negative (ER- negative) breast cancer is a major therapeutic hurdle. We studied NF-κB activation in human breast tumors and in carcinoma cell lines. Activated NF-κB was detected predominantly in ER-negative vs. ER-positive breast tumors and mostly in ER-negative and ErbB2-positive tumors (86%). These in vivo results demonstrate association of activated NF-κB with a subgroup of human breast tumors and are consistent with previously reported in vitro observations using similar classes of human breast cancer cell lines. Finding such an association suggested functional and biological significance. Immunofluorescence demonstrated increased nuclear p65, a component of the active NF-κB complex, in cytokeratin 19 (CK19)-positive epithelial cells of ER-negative/ErbB2-positive tumor samples. In contrast, nuclear NF-κB was detected mostly in stroma of ER-negative and ErbB2-negative tumors, suggesting a role of activated NF-κB in intercellular signaling between epithelial and stromal cells in this type of breast cancers. To elucidate roles of activated NF-κB, we used an ER-negative and ErbB2-positive human breast tumor cell line (SKBr3). The polypeptide heregulin β1 stimulated, and herceptin, the anti-ErbB2 antibody, inhibited, NE-κB activation in SKBr3 cells. The NF-κB essential modulator (NEMO)-binding domain (NBD) peptide, an established selective inhibitor of IκB-kinase (IKK), blocked heregulin-mediated activation of NF-κB and cell proliferation, and simultaneously induced apoptosis only in proliferating and not resting cells. These results substantiate the hypothesis that certain breast cancer cells rely on NF-κB for aberrant cell proliferation and simultaneously avoid apoptosis, thus implicating activated NF-κB as a therapeutic target for distinctive subclasses of ER-negative breast cancers. [ABSTRACT FROM AUTHOR]

Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC. [ABSTRACT FROM AUTHOR]