Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary ...tumor lysates showed that Ron overexpression was associated with increases in β-catenin expression and tyrosine phosphorylation. β-Catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer and Fyn. However, the molecular and physiological roles of β-catenin and β-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and β-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of β-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both β-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of β-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced β-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/β-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the β-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or β-catenin. Finally, we show that genetic ablation of β-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis, following orthotopic transplantation into the mammary fat pad. Together, our data suggest that β-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.
Pancreatic cancer is an aggressive malignancy with a poor survival rate because it is difficult to diagnose the disease during its early stages. The currently available treatments, which include ...surgery, chemotherapy and radiation therapy, offer only limited survival benefit. Pharmacological interventions to inhibit Glycogen Synthase Kinase-3beta (GSK3β) activity is an important therapeutic strategy for the treatment of pancreatic cancer because GSK3β is one of the key factors involved in the onset, progression as well as in the acquisition of chemoresistance in pancreatic cancer. Here, we report the identification of MJ34 as a potent GSK3β inhibitor that significantly reduced growth and survival of human mutant KRas dependent pancreatic tumors. MJ34 mediated GSK3β inhibition was seen to induce apoptosis in a β-catenin dependent manner and downregulate NF-kB activity in MiaPaCa-2 cells thereby impeding cell survival and anti-apoptotic processes in these cells as well as in the xenograft model of pancreatic cancer. In vivo acute toxicity and in vitro cardiotoxicity studies indicate that MJ34 is well tolerated without any adverse effects. Taken together, we report the discovery of MJ34 as a potential drug candidate for the therapeutic treatment of mutant KRas-dependent human cancers through pharmacological inhibition of GSK3β.
Model showing impact of MJ34 on KRas dependent MiaPaCa-2 cancer cells under in vitro and in vivo conditions (A) MJ34 treatment induces apoptosis by inhibiting GSK3β and regulating its downstream targets like β-catenin, NF-kB and c- Myc as well as antiapoptotic mediators. (B) MJ34 induces c-Myc and β-catenin dependent tumor regression in MiaPaCa-2 xenograft tumor mice. Display omitted
This chapter focuses on Hepatocyte growth factor (HGF), and its receptor (MET) system. Interactions between HGF and MET can lead to a variety of responses including mitosis, motility, tubulogenesis, ...and cytotoxicity, because of a multifunctional docking site that is unique to the carboxy terminus of class IV tyrosine kinase receptors. Specific intra- and extracellular effectors also contribute to biologic outcome. Most HGF–MET interactions occur in paracrine endocrine manner. HGF must be cleaved to induce signaling, and there are multiple known molecules that are capable of performing this function, some of which (u-PA and u-PAR) that may contribute to the cell signaling process via complex formation. By generating mice with “floxed” HGF and/or MET genes, and then breeding them with mice that express the cre recombinase under organ-specific promoters, one can generate adult animals with regional ablation of the genes. There are six proteins that can facilitate the generation of mature HGE. The final area that will surely expand is the understanding of how a single ligand/receptor pair induces such a variety of responses. This last area of research will probably continue to explore signal transducers. However, it is also likely to expand heavily into the area of co-receptors.
Artificial extracorporeal support for hepatic failure has met with limited clinical success. In hepatocytes, nitric oxide (NO) functions as an antiapoptotic modulator in response to a variety of ...stresses. We hypothesized that NO administration would yield improved viability and hepatocellular restructuring in a four-compartment, hollow fiber-based bioreactor with integral oxygenation for dynamic three-dimensional perfusion of hepatic cells in bioartificial liver support systems.
Isolated adult rat liver cells were placed in culture medium alone (control) or medium supplemented with various concentrations of an NO donor (S-nitrosoglutathione GSNO) in the bioreactors. Media samples were obtained from the cell perfusion circuit to monitor cellular response. After 24 and 72 h, histology biopsies were taken to investigate spontaneous restructuring of the cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to quantify apoptotic nuclei.
Control bioreactors exhibited 47.9 +/- 2.9% (mean +/- standard error of the mean) apoptotic nuclei. In contrast, NO-treated bioreactors exhibited a biphasic response. Fewer apoptotic nuclei were seen in the 200 and 500 microM GSNO groups (14.4 +/- 0.4%). No effect was observed in the 10 microM GSNO group (47.3%), and increased TUNEL staining was observed in the 1000 microM GSNO group (82.6%). Media lactate dehydrogenase levels were lower in bioreactor groups treated with 200 or 500 microM GSNO (310 +/- 38 IU/L) compared with the control group (919 +/- 188 IU/L; p < 0.05). Protein synthesis was not affected, as measured by albumin levels in the media (115 +/- 19 microg/day/cell inoculum in GSNO-treated bioreactors at 24 h vs. 110 +/- 13 in controls; p = 0.851). Histologically, all of the bioreactor groups exhibited liver cell aggregates with some attached to the bioreactor capillaries. Increased numbers of cells in the aggregates and superior spontaneous restructuring of the cells were seen at 24 and 72 h in the bioreactor groups treated with either 200 or 500 microM GSNO compared with the control groups.
Addition of an NO donor reduces adult rat liver cell apoptosis during the initial 24 h after cell inoculation within a three-dimensional perfusion bioreactor system for liver support and promotes liver cell aggregation and spontaneous restructuring of the cells at 24 and 72 h. GSNO-treated bioreactors remain metabolically active and show significantly lower levels of cellular injury as compared with controls. Further studies will be required to evaluate the impact of NO treatment of liver support bioreactors for clinical studies.
The aim of our study was to isolate novel gene(s) involved in cell differentiation and embryonic liver development. Mouse cded/lior was identified from subtraction hybridization of embryonic liver ...cDNA libraries as well as an adult mouse liver genomic DNA library. The full open reading frame of cded/lior encodes a 131-amino acid protein with 71.88% overall similarity to the PH domain of rat PLC-gamma1. A gapped search with the C-terminal region of CDED/LIOR revealed a 36-41% similarity to several proteins related to signal transduction and cell replication, such as ORC1 and KSR. Northern blot analysis of adult mouse tissues shows a strong 2.6-kb transcript restricted to heart and skeletal muscle. RT-PCR utilizing cded/lior-specific primers demonstrates cded/lior mRNAs in heart, brain, and liver tissue throughout mid-embryonic mouse gestation. cded/lior maps to the distal end of mouse Chromosome (Chr) 2. Analysis of the genomic structure for cded/lior demonstrated a single exon gene that is not an alternatively spliced isoform of PLC-gamma1. Analysis of the cded/lior promoter region revealed a high GC-content, high ratio of CpG/GpC, multiple GC-boxes, the lack of a TATA box, CTF/NFI element, and two MyoD-MCK binding sites. These characteristics are also found in several genes important in the regulation of cell growth or DNA synthesis, such as transforming growth factor-beta1, c-Ha-ras, nerve growth factor, epidermal growth factor receptor, and DNA polymerase beta. These results suggest that cded/lior is a mesoderm/muscle-specific transcript that may be involved in the mesodermal inductive and regulatory interactions required for liver formation and embryonic development.
An essential feature of cell differentiation is the specificity of signal transduction events from extracellular cues, which are considered to be conferred by scaffold, anchoring and adaptor ...proteins. Our aim was to identify important scaffolding proteins required for liver development. Utilizing subtraction hybridization of embryonic liver cDNA libraries, here we report the full length cDNA sequence for mouse
itih-4 (Inter-
α-trypsin inhibitor H4).
Itih-4 encodes a 942 amino acid protein containing two EF-hand (helix–loop–helix) motifs with an unique short loop, with a potential calcium-binding function.
Itih-4 is expressed as a strong 3.1-kb transcript in liver, to a lesser extent in lung and heart tissue. RT–PCR demonstrates
itih-4 mRNAs abundantly in liver, less in heart and brain, during mid-embryonic gestation. These results suggest that
itih-4 is a potential regulator for extracellular matrix proteins and plays a role during early embryonic liver development.
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