For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.