Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

NF-κB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-κB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-κB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-κB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-κB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-κB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.

No abstract

Current treatment options for triple-negative breast cancers (TNBCs) is limited by the absence of well-defined biomarkers, excluding a targeted therapy. Notably, epidermal growth factor receptor (EGFR) is overexpressed in a great proportion of TNBCs and is a negative prognostic factor. In clinical trials, however, existing EGFR inhibitors showed disappointing outcome. Oligonucleotide aptamers are a valid alternative to antibodies for diagnostic and therapeutic uses. Here, we prove that, when applied to aggressive TNBC cell lines with unique stem-like plasticity, the anti-EGFR CL4 aptamer, but not erlotinib or cetuximab, prevents the vasculogenic mimicry (VM) capability of the cells and destroys previously formed channels in three-dimensional culture. Notably, we found that CL4 impairs the matrix-induced integrin αvβ3 interaction with EGFR and integrin αvβ3-dependent cell adhesion. Consistently, the aptamer strongly inhibits VM and tumor growth in a xenograft TNBC model. These data suggest that in TNBC cells, EGFR may cooperate with integrin αvβ3 to regulate integrin binding to extracellular ligands required for VM, and EGFR-targeting by CL4 aptamer may counteract this event. Overall, we demonstrate a novel mechanism of action for CL4 related with integrin αvβ3-EGFR interaction, that may help to develop new oligonucleotide-based strategy addressing unmet need for TNBCs therapy.

No abstract

No abstract

Glioblastoma Multiforme (GBM) is the most common and aggressive human brain tumor, associated with very poor survival despite surgery, radiotherapy and chemotherapy.The epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor β (PDGFRβ) are hallmarks in GBM with driving roles in tumor progression. In approximately half of the tumors with amplified EGFR, the EGFRvIII truncated extracellular mutant is detected. EGFRvIII does not bind ligands, is highly oncogenic and its expression confers resistance to EGFR tyrosine kinase inhibitors (TKIs). It has been demonstrated that EGFRvIII-dependent cancers may escape targeted therapy by developing dependence on PDGFRβ signaling, thus providing a strong rationale for combination therapy aimed at blocking both EGFRvIII and PDGFRβ signaling.We have recently generated two nuclease resistant RNA aptamers, CL4 and Gint4.T, as high affinity ligands and inhibitors of the human wild-type EGFR (EGFRwt) and PDGFRβ, respectively.Herein, by different approaches, we demonstrate that CL4 aptamer binds to the EGFRvIII mutant even though it lacks most of the extracellular domain. As a consequence of binding, the aptamer inhibits EGFRvIII autophosphorylation and downstream signaling pathways, thus affecting migration, invasion and proliferation of EGFRvIII-expressing GBM cell lines.Further, we show that targeting EGFRvIII by CL4, as well as by EGFR-TKIs, erlotinib and gefitinib, causes upregulation of PDGFRβ. Importantly, CL4 and gefitinib cooperate with the anti-PDGFRβ Gint4.T aptamer in inhibiting cell proliferation.The proposed aptamer-based strategy could have impact on targeted molecular cancer therapies and may result in progresses against GBMs.

Purpose: Premature or stress-induced senescence is a major cellular response to chemotherapy in solid tumors and contributes to successful treatment. However, senescent tumor cells are resistant to apoptosis and may also reenter the cell cycle.We set out to find a means to specifically induce senescent tumor cells to undergo cell death and not to reenter the cell cycle that may have general application in cancer therapy. Experimental Design: We investigated the mechanisms regulating cell survival in drug-induced senescent tumor cells. Using immunofluorescence and flow cytometry^based techniques, we established the status of the ataxia telangiectasia mutated (ATM) signaling pathway in these cells.We assayed the requirement of ATM signaling and p21 CIP1 expression for survival in premature senescent tumor cells using pharmacologic inhibitors and antisense oligonucleotides. Results: The ATM/ATR (ATM-and Rad3-related) signaling pathway was found to be constitutively active in drug-induced senescent tumor cells. We found that blocking ATM/ATR signaling with pharmacologic inhibitors, including the novel ATM inhibitors KU55933 and CGK733, induced senescent breast, lung, and colon carcinoma cells to undergo cell death. We show that the mechanism of action of this effect is directly via p21 CIP1