HGF/c-met/Stat3 signaling during skin tumor cell invasion: indications for a positive feedback loop

<p>Abstract</p> <p>Background</p> <p>Stat3 is a cytokine- and growth factor-inducible transcription factor that regulates cell motility, migration, and invasion under normal and pathological situations, making it a promising target for cancer therapeutics. The hepatocyte growth factor (HGF)/c-met receptor tyrosine kinase signaling pathway is responsible for stimulation of cell motility and invasion, and Stat3 is responsible for at least part of the c-met signal.</p> <p>Methods</p> <p>We have stably transfected a human squamous cell carcinoma (SCC) cell line (SRB12-p9) to force the expression of a dominant negative form of Stat3 (S3DN), which we have previously shown to suppress Stat3 activity. The <it>in vitro </it>and <it>in vivo </it>malignant behavior of the S3DN cells was compared to parental and vector transfected controls.</p> <p>Results</p> <p>Suppression of Stat3 activity impaired the ability of the S3DN cells to scatter upon stimulation with HGF (c-met ligand), enhanced their adhesion, and diminished their capacity to invade <it>in vitro </it>and <it>in vivo</it>. Surprisingly, S3DN cells also showed suppressed HGF-induced activation of c-met, and had nearly undetectable basal c-met activity, as revealed by a phospho-specific c-met antibody. In addition, we showed that there is a strong membrane specific localization of phospho-Stat3 in the wild type (WT) and vector transfected control (NEO4) SRB12-p9 cells, which is lost in the S3DN cells. Finally, co-immunoprecipitation experiments revealed that S3DN interfered with Stat3/c-met interaction.</p> <p>Conclusion</p> <p>These studies are the first confirm that interference with the HGF/c-met/Stat3 signaling pathway can block tumor cell invasion in an <it>in vivo </it>model. We also provide novel evidence for a possible positive feedback loop whereby Stat3 can activate c-met, and we correlate membrane localization of phospho-Stat3 with invasion <it>in vivo</it>.</p

Identification of the B-Raf/Mek/Erk MAP kinase pathway as a target for all-trans retinoic acid during skin cancer promotion

<p>Abstract</p> <p>Background</p> <p>Retinoids have been studied extensively for their potential as therapeutic and chemopreventive agents for a variety of cancers, including nonmelanoma skin cancer (NMSC). Despite their use for many years, the mechanism of action of retinoids in the prevention of NMSC is still unclear. In this study we have attempted to understand the chemopreventive mechanism of all-<it>trans </it>retinoic acid (ATRA), a primary biologically active retinoid, in order to more efficiently utilize retinoids in the clinic.</p> <p>Results</p> <p>We have used the 2-stage dimethylbenzanthracene (DMBA)/12-<it>O</it>-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model to investigate the chemopreventive effects of ATRA. We have compared the gene expression profiles of control skin to skin subjected to the 2-stage protocol, with or without ATRA, using Affymetrix 430 2.0 DNA microarrays. Approximately 49% of the genes showing altered expression with TPA treatment are conversely affected when ATRA is co-administered. The activity of these genes, which we refer to as 'counter-regulated', may contribute to chemoprevention by ATRA. The counter-regulated genes have been clustered into functional categories and bioinformatic analysis has identified the B-Raf/Mek/Erk branch of the MAP kinase pathway as one containing several genes whose upregulation by TPA is blocked by ATRA. We also show that ATRA blocks signaling through this pathway, as revealed by immunohistochemistry and Western blotting. Finally, we found that blocking the B-Raf/Mek/Erk pathway with a pharmacological inhibitor, Sorafenib (BAY43-9006), induces squamous differentiation of existing skin SCCs formed in the 2-stage model.</p> <p>Conclusion</p> <p>These results indicate that ATRA targets the B-Raf/Mek/Erk signaling pathway in the 2-stage mouse skin carcinogenesis model and this activity coincides with its chemopreventive action. This demonstrates the potential for targeting the B-Raf/Mek/Erk pathway for chemoprevention and therapy of skin SCC in humans. In addition our DNA microarray results provide the first expression signature for the chemopreventive effect of ATRA in a mouse skin cancer model. This is a potential source for novel targets for ATRA and other chemopreventive and therapeutic agents that can eventually be tested in the clinic.</p

Alzheimer\u27s Therapeutics Targeting Amyloid Beta 1-42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors is Displaced by Drug Candidates That Improve Cognitive Deficits

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer\u27s disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer\u27s disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer\u27s therapeutics

Effects of ATRA combined with citrus and ginger-derived compounds in human SCC xenografts

<p>Abstract</p> <p>Background</p> <p>NF-κB is a survival signaling transcription factor complex involved in the malignant phenotype of many cancers, including squamous cell carcinomas (SCC). The citrus coumarin, auraptene (AUR), and the ethno-medicinal ginger (Alpinia galanga) phenylpropanoid, 1'-acetoxychavicol acetate (ACA), were previously shown to suppress 12-<it>O</it>-tetradecanoylphorbol-13-acetate (TPA) induced mouse skin tumor promotion. The goal of the present study was to determine whether AUR and ACA are effective either alone or in combination with all-<it>trans </it>retinoic acid (ATRA) for suppressing SCC tumor growth.</p> <p>Methods</p> <p>We first determined the effects of orally administered ACA (100 mg/kg bw) and AUR (200 mg/kg bw) on lipopolysaccharide (LPS)-induced NF-κB activation in NF-κB-RE-luc (Oslo) luciferase reporter mice. Dietary administration of AUR and ACA ± ATRA was next evaluated in a xenograft mouse model. Female SCID/bg mice were fed diets containing the experimental compounds, injected with 1 × 10⁶SRB12-p9 cells s.c., palpated and weighed twice a week for 28 days following injection.</p> <p>Results</p> <p>Both ACA and AUR suppressed LPS-induced NF-κB activation in the report mice. In the xenograft model, AUR (1000 ppm) and ACA (500 ppm) modestly suppressed tumor volume. However, in combination with ATRA at 5, 10, and 30 ppm, ACA 500 ppm significantly inhibited tumor volume by 56%, 62%, and 98%, respectively. The effect of ATRA alone was 37%, 33%, and 93% inhibition, respectively. AUR 1000 ppm and ATRA 10 ppm were not very effective when administered alone, but when combined, strongly suppressed tumor volume by 84%.</p> <p>Conclusions</p> <p>Citrus AUR may synergize the tumor suppressive effects of ATRA, while ACA may prolong the inhibitory effects of ATRA. Further studies will be necessary to determine whether these combinations may be useful in the control of human SCC.</p

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics

Characterization of synthetic human Abeta 1–42 oligomers by non-denaturing Western blot, MALDI-TOF.

<p><b>A</b>, Freshly prepared solutions of synthetic human Abeta 1–42 (lane 1) or 1–40 (lane 3) peptide loaded onto non-denaturing western gels immediately after reconstitution contain large amounts of monomer (arrow; fainter lower molecular weight band represents peptide degradation product) and little higher molecular weight material. In contrast, the same solution of Abeta 1–42 peptide that is allowed to oligomerize for 24 hours (lane 2) contains much larger amounts of higher molecular weight material >50 kDa, and less monomeric protein. The full length of gel lanes are shown from loading well to dye front. Note that oligomers run differently on non-denaturing gels than globular molecular weight protein size standards <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111898#pone.0111898-Tseng1" target="_blank">[49]</a>. <b>B</b>. The presence of significant amounts of monomer in oligomer preparations is also confirmed by MALDI-TOF analysis of the same Abeta 1–42 oligomer preparation that shows both a 4.5 kDa monomer peak and multiple lower abundance peaks corresponding to oligomers of various sizes. MALDI-TOF (detection range 3–100 kDa) of vehicle (media without Abeta) is shown below for comparison (<b>C</b>).</p