STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) andThe nonreceptor tyrosine kinase breast tumor kinase (Brk) 2 was originally isolated from a human breast carcinoma (1). Brk is also known as PTK6, having been identified as a highly expressed protein-tyrosine kinase in human melanocytes (2). In addition, a cDNA for the mouse orthologue, Ptk6 (previously termed Sik), which has 80% amino acid identity to Brk/ PTK6, was cloned from mouse intestinal crypt cells (3). Brk contains an Src homology (SH) 3 domain, an SH2 domain, and a tyrosine kinase catalytic domain, but it lacks an N-terminal myristoylation site for membrane targeting (1). Brk is expressed in many malignancies, such as metastatic melanomas and colon and prostate tumors (4 -6). Brk expression is also detected in a large proportion of human mammary gland tumors, whereas it is not expressed in the normal mammary gland (1, 7). It is noteworthy that small interfering RNA-mediated down-regulation of Brk expression in breast cancer cells results in their decreased growth capacity (8). Furthermore, it has been demonstrated that overexpression of Brk sensitizes human mammary epithelial cells to EGF and/or heregulin stimuli and increases anchorage-independent growth (9, 10). Down-regulation of Brk can also influence EGF-and heregulin-induced cell proliferation, suggesting a contribution of Brk to signaling induced by members of the EGF receptor family (11). However, the molecular mechanism by which Brk participates in tumorigenesis remains poorly characterized.STAT3 and STAT5, which play crucial roles in cell proliferation and differentiation, are believed to be activated by Brk (12,13). Another Brk substrate is STAP-2 (signal transducing adaptor protein-2), whose tyrosine residues are phosphorylated by Brk (14, 15). STAP-2, which we isolated as a c-Fmsinteracting protein, contains an N-terminal pleckstrin homology (PH) domain and a region distantly related to the SH2 domain (16). In its C-terminal region, a proline-rich region and a STAT3-binding YXXQ motif are also present (16). Our previous experiments have suggested that STAP-2 interacts with and influences several signaling molecules, including STAT3 and STAT5 (16,17). The dysregulated activation of STAT3 is a possible mechanism of tumorigenesis in breast cancers; therefore, it would be very informative to analyze the interactions among Brk, STAP-2, and STAT3.In the present study, we found that STAP-2 acts as an endogenous positive regulator of breast cancer cell growth. Manipulation of STAP-2 expression also indicated that STAP-2 is essential for Brk-mediated STAT3 activation. In addition, we investigate the domains responsible for the effects of STAP-2. EXPERIMENTAL PROCEDURESReagents and Antibodies-Expression vectors, STAP-2, STAT3, and their mutants were described previously (15)(16)(17)(18)

Krüppel-associated box-associated protein 1 (KAP1) is thought to act mainly as a scaffold for protein complexes, which together silence transcription by triggering the formation of heterochromatin. Using small interfering RNA-mediated KAP1 knockdown, we found that endogenous KAP1 negatively regulated TNF-α–induced IL-6 production in HeLa cells. KAP1 is likely to modulate the binding of NF-κB to the IL-6 promoter because KAP1 knockdown enhanced TNF-α–induced NF-κB-luciferase activity, but not IκBα degradation. Of importance, we found negative regulatory effects of KAP1 on the serine phosphorylation of STAT3, the acetylation of NF-κB/p65 by p300, and the nuclear localization of NF-κB/p65. In addition, KAP1 associated with NF-κB/p65 and inhibited the binding between NF-κB/p65 and p300. Thus, KAP1 is likely to negatively control the acetylation of NF-κB/p65, which is critical for its nuclear retention. Taken together, KAP1 modulated the acetylation of NF-κB/p65 by interfering with the interactions among STAT3, p300, and NF-κB/p65, resulting in reduced IL-6 production after TNF-α stimulation. Our findings that KAP1 directly interacts with transcriptional factors are new, and will inform further research to elucidate KAP1 function.

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With recent advances in cancer vaccination therapy targeting tumor-associated antigens (TAAs), dendritic cells (DCs) are considered to play a central role as a cell-based drug delivery system in the bioactive immune environment. Ex vivo generation of monocyte-derived DCs has been conventionally applied in adherent manufacturing systems with separate loading of TAAs before clinical use. We developed DCs pre-pulsed with Wilms’ tumor (WT1) peptides in low-adhesion culture maturation (WT1-DCs). Quality tests (viability, phenotype, and functions) of WT1-DCs were performed for process validation, and findings were compared with those for conventional DCs (cDCs). In comparative analyses, WT1-DCs showed an increase in viability and recovery of the DC/monocyte ratio, displaying lower levels of IL-10 (an immune suppressive cytokine) and a similar antigen-presenting ability in an in vitro cytotoxic T lymphocytes (CTLs) assay with cytomegalovirus, despite lower levels of CD80 and PD-L2. A clinical study revealed that WT1-specific CTLs (WT1-CTLs) were detected upon using the WT1-DCs vaccine in patients with cancer. A DC vaccine containing TAAs produced under an optimized manufacturing protocol is a potentially promising cell-based drug delivery system to induce acquired immunity.

Given the recent advancements of immune checkpoint inhibitors, there is considerable interest in cancer immunotherapy provided through dendritic cell (DC)-based vaccination. Although many studies have been conducted to determine the potency of DC vaccines against cancer, the clinical outcomes are not yet optimal, and further improvement is necessary. In this study, we evaluated the potential ability of human platelet lysate (HPL) to produce interferon-α-induced DCs (IFN-DCs). In the presence of HPL, IFN-DCs (HPL-IFN-DCs) displayed high viability, yield, and purity. Furthermore, HPL-IFN-DCs displayed increased CD14, CD56, and CCR7 expressions compared with IFN-DCs produced without HPL; HPL-IFN-DCs induced an extremely higher number of antigen-specific cytotoxic T lymphocytes (CTLs) than IFN-DCs, which was evaluated with a human leukocyte antigen (HLA)-restricted melanoma antigen recognized by T cells 1 (MART-1) peptide. Additionally, the endocytic and proteolytic activities of HPL-IFN-DCs were increased. Cytokine production of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α was also elevated in HPL-IFN-DCs, which may account for the enhanced CTL, endocytic, and proteolytic activities. Our findings suggest that ex-vivo-generated HPL-IFN-DCs are a novel monocyte-derived type of DC with high endocytic and proteolytic activities, thus highlighting a unique strategy for DC-based immunotherapies.