Research Interests
The central theme of our laboratory involves identifying novel pathways in gastro-intestinal cancer, from discovery to mechanistic insight as well as translational roles. Earlier studies involved cloning elf, Praja, cded, itih-4 and generating mouse knockouts of these genes. Current studies in the Mishra lab involve the elf knockout mice generated by the lab, and dissection of the gastro-intestinal phenotype. These studies have demonstrated a crucial role for ELF in gastro-intestinal carcinogenesis, epithelial cell morphogenesis, as well as new interactions as an adaptor protein to TGF-beta signaling molecules, Smad3 and Smad4. Other studies have revealed an important role for PRAJA ubiquitination as an E3 for ELF, and have led to key studies demonstrating the role of RING finger proteins in ubiquitination. Similarly, studies with a liver specific protease inhibitor, ITIH-4 suggests a prominent role for this protein in IL-6 mediated hepatocyte proliferation. For instance, gastric cancer remains the second most common cancer worldwide. Recent findings implicating Smad4 as a major tumor suppressor gene in the gastrointestinal tract are confirmed by the gastric cancer phenotype in Smad4+/- mutant mice.
We have utilized this model to investigate gastric tumorigenesis and molecular pathways that accompany the inactivation of TGF-beta signaling. Our studies have involved TGF-beta stimulation of normal antral mucosal cells, resulting in endogenous binding of ELF to Smad4 as seen by co-immunoprecipitation studies combined with colocalization by confocal microscopy.
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ELF, ß-Spectrins and TGF-ß
Beta-spectrins play essential roles in cell-cell interactions and in the maintenance of cell polarity. Our aim was to identify beta-spectrin genes important for the establishment of hepatocyte polarity and differentiation. Using subtractive screening of cDNA libraries from early embryonic mouse livers (post-coital days 10, 11, and 12), we have isolated elf1 (embryonic liver fodrin 1), a differentially expressed beta-spectrin or fodrin. Elf1 encodes a 220-amino acid protein with an NH2 terminal actin-binding domain. In situ hybridization studies demonstrate elf1 expression initially in day 10 embryonic heart tissue, then in day 11-11.5 hepatic tissue. These studies suggest that elf1 may play a role in the emergence of hepatocyte polarity during liver development.
Disruption of the adaptor protein ELF, a beta-spectrin, leads to disruption of transforming growth factor–beta (TGF-beta) signaling by Smad proteins in mice. Elf-/- mice exhibit a phenotype similar to smad2+/- / smad3+/- mutant mice of midgestational death due to gastrointestinal, liver, neural, and heart defects. We show that TGF-beta triggers phosphorylation and association of ELF with Smad3 and Smad4, followed by nuclear translocation.
ELF deficiency results in mislocalization of Smad3 and Smad4 and loss of the TGF-beta–dependent transcriptional response, which could be rescued by overexpression of the COOH terminal region of ELF. This study reveals an unexpected molecular link between a major dynamic scaffolding protein and a key signaling pathway.
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Explants & Stem Cells
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. Northern blot analysis demonstrated three transcripts in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos. 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.
Smads serve as intracellular mediators of transforming growth factor beta (TGF-beta) signaling. After phosphorylation by activated type I TGF-beta receptors, Smad proteins translocate to the nucleus, where they serve as transcription factors and increase or decrease expression of TGF-beta target genes. Mice lacking one copy each of Smad2 and Smad3 suffered midgestation lethality due to liver hypoplasia and anemia, suggesting essential dosage requirements of TGF-beta signal components. Culture of mutant livers in vitro revealed the existence of a parallel developmental pathway mediated by hepatocyte growth factor (HGF), which could rescue the mutant phenotype independent of Smad activation. These pathways merge at the beta 1-integrin, the level of which was increased by HGF in the cultured mutant livers. HGF treatment reversed the defects in cell proliferation and hepatic architecture in the Smad2+/-; Smad3+/- livers.
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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 library. RT-PCR utilizing cded/lior-specific primers demonstrates cded/lior mRNAs in heart, brain, and liver tissue throughout mid-embryonic gestation. 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 astransforming growth factor-beta1, c-HA-ras, nerve growth factor, epidermal growth factor receptor, and DNA polymerase beta. These results suggest 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.
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Clinical: Esophogeal & Gastric Cancer
The 5-year survival in patients with gastric cancer is low (20%), even among those undergoing surgical
resection. Moreover, the use of adjuvant chemotherapeutic treatments has not been established and is restricted to controlled trials. Palliative treatment includes surgery, ethanol injection, and Nd:YAG laser photocoagulation. These modalities may provide temporary relief of gastric outlet obstruction caused by the cancer in patients who cannot undergo curative treatment. The identification of new palliative chemomodulatory forms of treatment for the disease may therefore permit alternative approaches to therapy that could improve the outcome. This is a report of a patient with advanced gastric carcinoma considered poor surgical candidate, in whom local tumor control was achieved after intratumoral chemotherapy with cisplatin/epinephrine injectable gel administered under endoscopy.
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Lack of a suitable model has hindered efforts to understand inflammation and granuloma formation in Crohn’s disease. To identify features of in vitro aggregates, which are similar to tissue granulomas of Crohn’s disease, the gross morphology and immunohistological appearance of the aggregates produced peripheral blood mononuclear cells from patients with Crohn’s disease were analyzed, and the size of in vitro aggregates was correlated with clinical activity of the disease. Blocking antibodies were used to evaluate the role of cell-adhesion molecules in the formation of in vitro aggregates. In active Crohn’s disease, in vitro aggregates show immunohistological features of hypersensitivity type granulomas.
Blocking antibodies against leukocyte function associated antigen LFA-2 (CD2), LFA-3 (CD58), and Mac-1 (CD11b/CD18) inhibit in vitro 
aggregate formation. In vitro aggregates model in vivo granulomas in size and organization. Cell adhesion molecules like CD2, CD58, and CD11b/CD18 may be involved in granuloma-formation of Crohn’s disease.
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Spectrins & Neural Development
Spectrins play a pivotal role in axonal transport, neurite extension, the organization of synaptic vesicles, as well as for protein sorting in the Golgi apparatus and cell membrane. In this study we demonstrate the genomic structure of elf-3, as well as the expression of ELF, a beta spectrin, in the developing mouse brain using a peptide specific antibody against its distinctive amino-terminal end. At E12 ± 14, anti-ELF localized to axonal sprouts in the developing neuroblasts of cortex and purkinje cell layer of the cerebellum, as well as in cell bodies in the 
diencephalon and metencephalon. Double labeling identified significant co-localization of anti-ELF, nestin and dystrophin in subventricular zone cells and in stellate-like cells of the developing forebrain. These studies define clearly the expression of ELF, a new isoform of beta-G-spectrin in the developing brain. Based on its expression pattern, ELF may have a role in neural stem cell development and is a marker of axonal sprouting in mid stages of embryonic development.
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PRAJA1 protein contains a C3HC4 zinc-binding motif found in many regulatory proteins as well as in several human proto-oncogenes such as PML, RFP and BRCA1. PRAJA1 is specifically inhibited during apoptotic death of ILI3 dependent 32Dc13 cells and therefore may act as an inhibitor of apoptosis in these cells. In this study we identify fetal neuronal precursors that express PRAJA1 at different embryonic developmental stages in order to test its use as a marker of neuronal differentiation. Cells of the marginal layer of the telencephalon were labeled in e14 embryonic brain; reactivity was present in the cytoplasm and its filamentous extensions. Scattered ovoid cells with large cytoplasm and small central nuclei in the prosencephalic mantle layer in e12 embryonic brain showed slight cytoplasmic reactivity. PRAJA1 is a marker for specific subsets of neuroblasts. Its zinc-binding and RING motifs as well as its restricted and specific expression and role in apoptosis suggest an important physiological function in development of the brain.