ß-Catenin is required for Ron receptor-induced mammary tumorigenesis.

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 ß-catenin expression and tyrosine phosphorylation. ß-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 ß-catenin and ß-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and ß-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 ß-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both ß-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of ß-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 ß-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/ß-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the ß-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 ß-catenin. Finally, we show that genetic ablation of ß-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 ß-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.

Itih-4, a serine protease inhibitor regulated in interleukin-6-dependent liver formation: role in liver development and regeneration.

Inter-alpha-trypsin inhibitor-4 (Itih-4) is a liver-restricted member of the serine protease inhibitor family with diverse functions as an anti-apoptotic and matrix stabilizing molecule that are important throughout development. We investigate the functional role of Itih-4 in liver formation, regeneration (LR) and examine its role in calcium and hyaluronic acid binding. Itih-4 expression is prominent in early liver development at E9 and later at E16, being restricted to hepatoblasts, immature hepatocytes, and differentiated hepatocytes. We note a marked and differential increase in Itih-4 labeling in proliferating hepatocytes, compared with bile duct cells in liver explant cultures treated with interleukin-6 (IL-6). After partial hepatectomy, maximal Itih-4 expression occurs in a bimodal manner at 30 min and at 12 hr, with a predominant centrizonal distribution. There is no detectable binding of glutathione transferase-fusion Itih-4 protein to calcium and hyaluronic acid, indicating a possible requirement for posttranslational modifications for these functions. These results suggest that in LR, Itih-4 expression corresponds to that of immediate early genes and may contribute to the entry of normally quiescent hepatocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggests a prominent role for Itih-4 at key points in liver formation.

Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration.

The wnt/beta-catenin pathway is important during embryogenesis and carcinogenesis. beta-Catenin interaction with E-cadherin has been shown to be crucial in cell-cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt-1 and beta-catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in beta-catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of beta-catenin (decrease in serine phosphorylated beta-catenin) as seen by immunoprecipitation studies. We observed activation of beta-catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine-phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E-cadherin protein during liver regeneration are, in general, reverse to those seen in beta-catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine-phosphorylated beta-catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic beta-catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell-cell adhesion for structural reorganization during the process of liver regeneration.

Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants.

Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-beta leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.

Intratumoral therapy of cisplatin/epinephrine injectable gel for palliation in patients with obstructive esophageal cancer.

Obstructing esophageal cancer produces severe dysphagia with ensuing death within 90 days. Palliation is possible with modalities like stent placement, laser, and photodynamic therapy. However, these treatments have a high rate of complications, and the overall mortality is not altered. A new alternative treatment evaluated in this study is endoscopic intratumoral injection with cisplatin/epinephrine (CDDP/epi) gel. CDDP/epi gel injections were administered weekly for 3 to 8 weeks in nine patients, median age, 72 years; mean tumor volume (+/-SEM), 41.44 (+/-22.4) cm3. Eight patients had stage IV, and one had stage III esophageal carcinoma. The mean dysphagia score (+/-SEM) was 3.5 (+/-0.17). All patients were followed up until death. Dysphagia resolved in eight patients with reduction in mean dysphagia score (+/-SEM) from 3.5 (+/-0.17) to 0.75 (+/-0.28; p = 0.005). Tumor volume was reduced by 75% in one patient and by 50% in two patients. The median survival was 4 months. The longest follow-up has been 15 months (458 days). In this pilot study, intratumoral injection of CDDP/epi gel restored swallowing in eight of nine patients and was an effective and safe outpatient treatment in patients with obstructive esophageal cancer.

Elf3 encodes a novel 200-kD beta-spectrin: role in liver development.

beta-spectrins are crucial for the maintenance of cell shape, the establishment of cell polarity, and the formation of distinct membrane domains. Our strategy for identifying genes important for hepatocyte polarity has been to utilize subtractive hybridization of early embryonic mouse cDNA liver libraries. As a result, we have cloned three isoforms of a novel beta-spectrin elf (embryonic liver beta-fodrin), and here we report the analysis of elf3, the longest isoform (8172 nt). ELF3 comprises 2154 residues with an overall similarity of 89.0% and 95.3% to mouse beta-spectrin (betaSpIIsigma1) at the nucleotide and amino acid level, respectively. ELF3 is characterized by an actin-binding domain, a long repeat domain, and a short regulatory domain remarkable for the absence of a PH domain. Linkage analysis reveals that elf3 maps to mouse chromosome 11 between D11Bir6 and D11Xrf477, a different chromosomal locus from that of the other four spectrin genes. Northern blot analysis utilizing an elf3 3'-UTR probe demonstrates an abundant 9.0-kb transcript in brain, liver, and heart tissues. Western blot with a polyclonal antibody against ELF identifies a 200 kD protein in mouse liver, brain, kidney, and heart tissues. Immunohistochemical studies demonstrate ELF labeling of the basolateral or sinusoidal membranes surface as well as a granular cytoplasmic pattern in hepatocytes. Antisense studies utilizing cultured liver explants show a vital role of elf3 in hepatocyte differentiation and intrahepatic bile duct formation. The differential expression, tissue localization, and functional studies demonstrate the importance of elf3 in modulating interactions between various components of the cytoskeleton proteins controlling liver and bile duct development.

Praja1, a novel gene encoding a RING-H2 motif in mouse development.

As part of a cloning strategy to identify genes involved in early mouse liver development we have isolated Praja1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl) which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja1 is a 2.1 kb gene encoding a putative 396 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized Praja1 on chromosome X at 36 cM, which may be a candidate gene for mouse sla (sex linked sideroblastic anemia), near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, p.c.). In vitro transcription/translation yielded a product with an Mr of 59 kD. Immunohistochemical staining of in vitro liver explant cultures using a heterologous antibody against praja1 demonstrated cytoplasmic staining of cuboidal cells that have hepatocyte morphology and organization. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja1 product is possibly involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of Praja1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.