Depletion of protein kinase N3 (PKN3) impairs actin and adherens junctions dynamics and attenuates endothelial cell activation.

Several pathways are involved in the control of endothelial cell morphology, endothelial permeability and function in order to maintain vascular homeostasis. Here we report that protein kinase N3 (PKN3) appears to play a pivotal role in maintaining endothelial cell morphology, cell-cell junctions and motility. An RNAi-based cell biological approach in cultured human endothelial cells (HUVEC) revealed that knockdown of PKN3 expression gave rise to cells with divergent cell morphology, impaired locomotion, disturbed adherens junctions (AJ) integrity and irregular actin organization. Notably, knockdown of PKN3 cells led to improper stress fiber formation and marked adhesiveness of intercellular adherens junctions when cells became stimulated with the pro-inflammatory cytokine TNF-α. Moreover, TNF-α-induced ICAM-1 expression on the cell surface was reduced in cells with suppressed PKN3 expression. Finally, loss-of-function for PKN3 appeared to affect Pyk2 phosphorylation in endothelial cells. These observations suggest that PKN3 can be considered a novel protein implicated in remodeling the actin-adherens junction, possibly by linking ICAM-1-signaling with actin/AJ dynamics. We propose that loss of PKN3 function and concomitant aberrations in actin rearrangement may attenuate pro-inflammatory activation of endothelial cells.

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge.

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.

Atu027 prevents pulmonary metastasis in experimental and spontaneous mouse metastasis models.

PURPOSE: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node metastasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the pharmacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four different preclinical mouse models. EXPERIMENTAL DESIGN: Atu027 compared with vehicle or control small interfering RNA lipoplexes was tested in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous metastasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules (repeated low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. Primary tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistochemistry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to provide an insight into molecular changes on depletion of PKN3, in support of efficacy results. RESULTS: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formation of spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well as in mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an increase in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change endothelial function, resulting in reduced colonization and micrometastasis formation. CONCLUSION: Atu027 can be considered as a potent drug for preventing lung metastasis formation, which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy.

Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression.

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density.

Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking.

The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases.