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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.

Background 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. Methods 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 in vitro and in vivo malignant behavior of the S3DN cells was compared to parental and vector transfected controls. Results 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 in vitro and in vivo . 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. Conclusion These studies are the first confirm that interference with the HGF/c-met/Stat3 signaling pathway can block tumor cell invasion in an in vivo 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 in vivo .

Background 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- trans retinoic acid (ATRA), a primary biologically active retinoid, in order to more efficiently utilize retinoids in the clinic. Results We have used the 2-stage dimethylbenzanthracene (DMBA)/12- O -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. Conclusion 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.

The purpose of the current study was to determine whether a tropical ginger derived compound 1’-acetoxychavicol acetate (ACA), suppresses skin tumor promotion in K5.Stat3C mice. In a two-week study in which wild-type (WT) and K5.Stat3C mice were co-treated with either vehicle, ACA, galanga extract, or fluocinolone acetonide (FA) and tetradecanoyl phorbol acetate (TPA), only the galanga extract and FA suppressed TPA-induced skin hyperproliferation and wet weight. None of these agents were effective at suppressing p-Tyr 705 Stat3 expression. However, ACA and FA showed promising inhibitory effects against skin tumorigenesis in K5.Stat3C mice. ACA also suppressed phospho-p65 NF-κB activation, suggesting a potential mechanism for its action.

Background 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- O -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- trans retinoic acid (ATRA) for suppressing SCC tumor growth. Methods 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 × 10 6 SRB12-p9 cells s.c., palpated and weighed twice a week for 28 days following injection. Results 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%. Conclusions 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.

The primary goal of these studies is to gain a better understanding of the role of Stat3 signaling during skin cancer development. Stat3 has been shown to be important in the regulation of apoptosis, proliferation, motility, and angiogenesis, all hallmarks of malignancy. Our laboratory and others have shown that Stat3 activity is constitutive in several malignant cell types and is required for initiation, promotion and progression to a more malignant phenotype in skin SCC. We aim to address the following three problems: (1) How does all-trans retinoic acid (ATRA) administration affect Stat3 activity and tumor formation in 2-stage chemical carcinogenesis mouse models of skin cancer? We have previously used the well-established mouse skin 2-stage chemical carcinogenesis protocol to test the ability of ATRA to suppress skin tumor formation. Mice of the SENCAR strain develop benign papillomas which convert to malignant SCCs at a very low frequency after 30 weeks of TPA treatment. ATRA is a highly efficient suppressor of tumor formation in this model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/ERK pathway. Interestingly, ATRA treatment also suppresses Stat3 activity during early stages of tumor promotion, but is unable to do so beyond 7 weeks. Unlike SENCAR mice, TPA-induced tumor formation in K5.Stat3C mice, which express a constitutively active form of Stat3, called Stat3C, under the control of the skin basal cell-specific cytokeratin 5 promoter, is not inhibited by ATRA. However, ATRA is still able to reduce the activity of Stat3 in the skin of these mice. These studies are the first description of the effect of ATRA treatment on Stat3 activity in the 2-stage skin chemical carcinogenesis model. (2) What role does Stat3 activation play in the motility and invasion of SCC cells and how is this activity related to the HGF/cmet signaling axis? In order to investigate the role of Stat3 in the induction of cell motility and invasion in greater detail, we have employed a cell culture model in which we have expressed a dominant-negative form of Stat3 in a tumorigenic squamous cell carcinoma cell line, SRB 12-p9. We showed that SRB12-p9 cell clones expressing FLAG-tagged Stat3β-Y705F (S3DN cells) were significantly less invasive in both in vitro and in vivo assays, and this phenotype was accompanied by inhibition of HGF-induced motility. We also described a likely role for Stat3 at the cell membrane that contributes to the malignant phenotype of invading tumor cells. We determine that the HGF/cmet/Stat3 signaling cascade is critically involved in the motility and invasion of cancer cells, and importantly, disruption of this pathway by expression of S3DN diminishes cmet activity and inhibits cmet/Stat3 interaction. We speculate that Stat3 alone may not be sufficient for enhanced invasion, but a combination of high cmet and Stat3 activity, especially in an unregulated positive feedback loop, can drive cells toward invasive behavior. We successfully suppress this invasive potential with the expression of S3DN, which blocks cmet activity. Based on these results, we suggest that targeting the HGF/cmet/Stat3 pathway may be an especially effective target strategy for cancer therapy. (3) What effect does overexpression of S3DN have on the survival of SCC cells and which genes can we identify as key regulators of Stat3 mediated survival? In the present study we demonstrated that S3DN cells had reduced expression of survivin, an inhibitor of apoptosis that is often up-regulated during tumorigenesis and is associated with chemotherapy resistance and poor patient survival. S3DN cells progressively lost expression of survivin during a time course of serum starvation, correlating with cell death. In vivo mouse experiments indicate that tumors generated from S3DN cells also have reduced expression of survivin. DNA microarray analysis reveals that several genes involved in the regulation of apoptosis, including survivin, as well as numerous cell cycle regulators, are differentially expressed in control and S3DN cells under conditions of serum deprivation. Further bioinformatics analysis of this microarray data has organized the differentially expressed genes into functional gene networks. Using arrays designed to detect secreted chemokines, we were able to analyze which chemokines differ between cells undergoing apoptosis (S3DN cells) and those able to survive (SRB12-p9 parental cells) in serum deprivation conditions. The chemokines identified in this study are promising candidates for future experiments, since they may be critical Stat3 regulated survival genes. (Abstract shortened by UMI.)