Radiotherapy kills tumor-cells by inducing DNA double strand breaks (DSBs). However, the efficient repair of tumors frequently prevents successful treatment. Therefore, identifying new practical sensitizers is an essential step towards successful radiotherapy. In this study, we tested the new hypothesis: identifying the miRNAs to target DNA DSB repair genes could be a new way for sensitizing tumors to ionizing radiation. Here, we chose two genes: DNA-PKcs (an essential factor for non-homologous end-joining repair) and ATM (an important checkpoint regulator for promoting homologous recombination repair) as the targets to search their regulating miRNAs. By combining the database search and the bench work, we picked out miR-101. We identified that miR-101 could efficiently target DNA-PKcs and ATM via binding to the 3′- UTR of DNA-PKcs or ATM mRNA. Up-regulating miR-101 efficiently reduced the protein levels of DNA-PKcs and ATM in these tumor cells and most importantly, sensitized the tumor cells to radiation in vitro and in vivo. These data demonstrate for the first time that miRNAs could be used to target DNA repair genes and thus sensitize tumors to radiation. These results provide a new way for improving tumor radiotherapy.

Perturbations in the adipocytokine profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1), and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with the small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion, and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of the GSK3β-LKB1-Axin complex that leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of the Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation, and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors shows increased expression of Wnt1, pGSK3β, and vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized cross-talk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.

Significance: Respiratory syncytial virus (RSV) causes major disease in pediatric and elderly patients, urging the development of efficacious therapeutics. This study establishes a recombinant RSV reporter strain for drug discovery and identifies an entry inhibitor class targeting the viral fusion (F) protein. Biochemical, structural, and functional characterization of the inhibitor spotlights two microdomains governing the conformational stability of prefusion F. Mutations in these domains cause broad cross-resistance against the compound and RSV entry inhibitors in preclinical and clinical development, without mandatory loss of in vivo pathogenicity, challenging the possible clinical benefit of current RSV entry inhibitor classes. Anti-RSV campaigns should better target postentry steps or proactively circumvent resistance to entry inhibition. The resistant RSV reporter strain developed here establishes a strategy toward this goal.

Interestingly, some lymphoma cells, expressing high levels of transmembrane (tm)TNF-α, are resistant to secretory (s)TNF-α-induced necrosis but sensitive to tmTNF-α-mediated apoptosis. As tmTNF-α mediates “forward” as well as “reverse” signaling, we hypothesize that a balanced signaling between forward and reverse directions may play a critical role in determining the fate of cells bearing tmTNF-α. Using Raji cells as a model, we first added exogenous tmTNF-α on fixed, transfected NIH3T3 cells onto Raji cells to examine tmTNF-α forward signaling and its effects, showing that constitutive NF-κB activity and cellular inhibitor-of-apoptosis protein 1 transcription were down-regulated, paralleled with Raji cell death. As Raji cells express tmTNF-α, an inhibition of their tmTNF-α expression by antisense oligonucleotide caused down-regulation of NF-κB activity. Conversely, increasing tmTNF-α expression by suppressing expression of TNF-α-converting enzyme that cleaves tmTNF-α led to an enhanced activation of NF-κB, indicating that tmTNF-α, but not sTNF-α, contributes to constitutive NF-κB activation. We next transfected Raji cells with a mutant tmTNF-α lacking the intracellular domain to competitively suppress reverse signaling via tmTNF-α; as expected, constitutive NF-κB activity was decreased. In contrast, treating Raji cells with sTNFR2 to stimulate reverse signaling via tmTNF-α ehanced NF-κB activation. We conclude that tmTNF-α, when highly expressed on tumor cells and acting as a receptor, promotes NF-κB activation through reverse signaling, which is helpful to maintain tumor cell survival. On the contrary, tmTNF-α, when acting as a ligand, inhibits NF-κB activity through forward signaling, which is inclined to induce tumor cell death.