Inhibition of PI-3K restores nuclear p27(Kip1) expression in a mouse model of Kras-driven lung cancer.

Reduced expression of the CDK inhibitor p27(Kip1) (p27) in human lung cancer correlates with tumor aggressiveness and poor prognosis. However, the regulation of p27 expression and the role of p27 during lung cancer are poorly understood. Urethane-induced lung tumors in mice frequently harbor mutations in the Kras oncogene, and in this study, we use this model to address the regulation of p27 during tumorigenesis. The Ras effector Akt is known to regulate p27 mRNA abundance by phosphorylating and inactivating the FOXO transcription factors. Phosphorylated Akt and FOXO proteins were both increased in lung tumors, correlating with a reduction in p27 mRNA transcript. Akt also directly phosphorylates p27 and regulates its nuclear/cytoplasmic localization. Tumors showed a reduced nuclear/cytoplasmic ratio of p27 protein, together with an increase in phosphorylated Thr197 p27 in the cytoplasmic pool. Treatment of lung tumor-bearing mice with the phosphoinositol-3 kinase inhibitor LY294002 induced a rapid decrease in phosphorylated Akt and phosphorylated p27, concomitant with an increase in nuclear p27. Germline p27 deficiency accelerated both the growth and malignant progression of urethane-induced lung tumors, and did so in a cell autonomous manner, confirming a causal role of p27 in tumor suppression. These results show that p27 is a potent barrier to the growth and malignant progression of Kras-initiated lung tumors. Further, the reduction of nuclear p27 in tumors is mediated by oncogene signaling pathways, which can be reversed by pharmacological agents.Oncogene advance online publication, 3 August 2009; doi:10.1038/onc.2009.226

Compartment-Specific and Sequential Role of MyD88 and CARD9 in Chemokine Induction and Innate Defense during Respiratory Fungal Infection

Aspergillus fumigatus forms ubiquitous airborne conidia that humans inhale on a daily basis. Although respiratory fungal infection activates the adaptor proteins CARD9 and MyD88 via C-type lectin, Toll-like, and interleukin-1 family receptor signals, defining the temporal and spatial pattern of MyD88- and CARD9-coupled signals in immune activation and fungal clearance has been difficult to achieve. Herein, we demonstrate that MyD88 and CARD9 act in two discrete phases and in two cellular compartments to direct chemokine- and neutrophil-dependent host defense. The first phase depends on MyD88 signaling because genetic deletion of MyD88 leads to delayed induction of the neutrophil chemokines CXCL1 and CXCL5, delayed neutrophil lung trafficking, and fatal pulmonary damage at the onset of respiratory fungal infection. MyD88 expression in lung epithelial cells restores rapid chemokine induction and neutrophil recruitment via interleukin-1 receptor signaling. Exogenous CXCL1 administration reverses murine mortality in MyD88-deficient mice. The second phase depends predominately on CARD9 signaling because genetic deletion of CARD9 in radiosensitive hematopoietic cells interrupts CXCL1 and CXCL2 production and lung neutrophil recruitment beyond the initial MyD88-dependent phase. Using a CXCL2 reporter mouse, we show that lung-infiltrating neutrophils represent the major cellular source of CXCL2 during CARD9-dependent recruitment. Although neutrophil-intrinsic MyD88 and CARD9 function are dispensable for neutrophil conidial uptake and killing in the lung, global deletion of both adaptor proteins triggers rapidly progressive invasive disease when mice are challenged with an inoculum that is sub-lethal for single adapter protein knockout mice. Our findings demonstrate that distinct signal transduction pathways in the respiratory epithelium and hematopoietic compartment partially overlap to ensure optimal chemokine induction, neutrophil recruitment, and fungal clearance within the respiratory tract

Rapid Pharmacokinetic and Biodistribution Studies Using Cholorotoxin-Conjugated Iron Oxide Nanoparticles: A Novel Non-Radioactive Method

Recent advances in nanotechnology have led to the development of biocompatible nanoparticles for in vivo molecular imaging and targeted therapy. Many nanoparticles have undesirable tissue distribution or unacceptably low serum half-lives. Pharmacokinetic (PK) and biodistribution studies can help inform decisions determining particle size, coatings, or other features early in nanoparticle development. Unfortunately, these studies are rarely done in a timely fashion because many nanotechnology labs lack the resources and expertise to synthesize radioactive nanoparticles and evaluate them in mice.To address this problem, we developed an economical, radioactivity-free method for assessing serum half-life and tissue distribution of nanoparticles in mice. Iron oxide nanoparticles coated with chitosan and polyethylene glycol that utilize chlorotoxin as a targeting molecule have a serum half-life of 7-8 hours and the particles remain stable for extended periods of time in physiologic fluids and in vivo. Nanoparticles preferentially distribute to spleen and liver, presumably due to reticuloendothelial uptake. Other organs have very low levels of nanoparticles, which is ideal for imaging most cancers in the future. No acute toxicity was attributed to the nanoparticles.We report here a simple near-infrared fluorescence based methodology to assess PK properties of nanoparticles in order to integrate pharmacokinetic data into early nanoparticle design and synthesis. The nanoparticles tested demonstrate properties that are excellent for future clinical imaging strategies and potentially suitable for targeted therapy