Scientific Report 2005

Immunology

Expression of a Novel Large Noncoding RNA in Neoplasia, Proliferation, and Differentiation

R. Lin, C. Liu, T.S. Edgington, S. Maeda,* M. Karin*

* University of California, San Diego, California

Recent genomic and transcriptomic studies have revealed the existence of a substantial number of large noncoding RNAs. Only a few of these RNAs have been characterized, and little is known of their functional roles. Using differential display analysis of CD31-enriched tumor microvascular endothelial cells from a syngeneic murine colon carcinoma, we discovered a novel large noncoding RNA of approximately 360 base pairs. The full-length gene is a 7000-base mRNA-like transcript but lacks extensive or conserved open reading frames.

Bioinformatics analysis suggested that the full-length gene is highly conserved among vertebrates, implying functional significance. More important, Northern blot analysis and in situ hybridization indicated that expression of the full-length transcript is highly upregulated in carcinogen-induced primary hepatomas and is a novel marker for hepatomas and for other neoplasms so far analyzed.

We designated this noncoding RNA hepatomin. We found that hepatomin transcription is induced in liver undergoing regeneration, skin undergoing wound healing, hair follicles during growth of hair, and cultured cells synchronized at the mitosis stage.

These results suggest that hepatomin plays roles in proliferation and differentiation. We conclude that hepatomin is a member of the new class of large noncoding RNAs and a new marker for hepatomas. This discovery can provide unique murine models for analysis of the role of this noncoding RNA in neoplasia, proliferation, and differentiation as an apparent regulator of the changes in gene expression associated with these processes.

Novel Redirected Molecular Expression, Architecture, and Properties in the Microvasculature of Tumors

A. El-Sheikh, G. Bhattacharjee, Z. Ruggeri,* T.S. Edgington, P. Borgstrom**

* Department of Molecular and Experimental Medicine, Scripps Research
** Sidney Kimmel Cancer Center, La Jolla, California

Neoplastic cells induce remarkable changes in their new angiogenic microvasculature as they seek nutrients, oxygen, and the freedom to invade and metastasize. The loss of normal vascular development and architecture results in a bizarre, functionally incompetent maze of capillary-like vessels lined by endothelium with strikingly divergent gene transcription and aberrant display of cell-surface proteins. Understanding the aberrations may broaden our knowledge of the molecular biology of these complex angiogenic and postangiogenic vascular mazes, and some of the molecules involved may provide novel targets for therapeutic intervention.We discovered an endothelial receptor complex that appears unique for the tumor microvascular environment. Vascular endothelial growth factor receptor 2 and its coreceptor neuropilin-1 can be found elsewhere within the vascular tree, but the association of an oligosaccharide (chondroitin C sulfate) to create a ternary complex is unique to the tumor microvasculature. This complex is a novel target for therapeutic strategies.

We developed a thrombogen specific for tumor vasculature that recognizes this ternary complex selectively on tumor neoangiogenic vessels. This soluble hybrid protein, HBDt-TFt, incorporates the modified exon 7–encoded heparin-binding domain of the gene for vascular endothelial growth factor at its N terminus with the extracellular domain of tissue factor at the C terminus. The hybrid can selectively dock on the surface of tumor endothelium at this trimolecular complex and initiate the coagulation protease cascade. Subsequently, the tumor microvasculature is thrombosed, resulting in local infarctive eradication of tumor. Using intravital microscopy, we optimized the dose and rate of infusion of HBDt-TFt and monitored microcirculation within the tumor as well as thrombus formation. In addition, using fluorescein-labeled platelets, we showed a central participation of platelets in tumor-specific microvascular thrombosis.

Regulation of Tissue Factor–Mediated Initiation of the Coagulation Cascade by Grp78

G. Bhattacharjee, J. Ahamed, B. Pedersen, A. El-Sheikh, N. Mackman, W. Ruf, C. Liu, T.S. Edgington

We used biopanning with phage-displayed peptidyl libraries to discover peptide probes that bind selectively to the surface of the endothelium in atherosclerotic plaques. EKO130, the peptide with the highest affinity among those tested, binds the 78-kD glucose-regulated protein (Grp78). This protein participates in many pathologic processes, including regulation of the coagulation cascade. To characterize how Grp78 regulates coagulation, we analyzed the effect of the protein on tissue factor–mediated procoagulant activity in mouse brain endothelial cells and in macrophage-like cells.

We found that Grp78 is present on the endothelium and on monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 increased procoagulant activity. We also found that Grp78 negatively regulates procoagulant activity by interacting directly with the extracellular domain of tissue factor on the cell surface.

These findings indicate that Grp78 negatively regulates the functional activity of tissue factor by binding directly to tissue factor and subsequently inhibiting it. Determining how Grp78 regulates the function of tissue factor may provide insight into the pathobiology of atherosclerosis and associated arterial thrombosis.