Despite the central role of endothelial cells (ECs) in cancer biology, relatively little is known about the molecular perturbations that occur in tumor endothelium and their relationship to disease. Tumor ECs are particularly difficult to study because their behavior is influenced by characteristics of the tumor microvascular environment that cannot be recapitulated ex vivo, including EC functional interactions with adjacent stromal and tumor cells and their dependence on blood flow and extracellular matrix for stable differentiation. This project seeks to develop a robust technology platform enabling analysis of tumor endothelium cell gene expression within native tissues. The technology is based on the ability to co-immunoprecipitate RNA binding proteins together with their associated mRNA species from cell and tissue extracts. The primary implementation of the technology will rely on engineering a transgenic mouse expressing an epitope-tagged poly(A) binding protein under control of an endothelium-specific promoter. Anti-epitope tag antibodies can then be used to recover endothelium mRNAs from both normal and tumor transgenic tissue extracts. Recovered mRNAs can be detected and quantitated in high throughput fashion using microarray technologies. In addition, the mRNAs will be used as input for construction of SAGE and EST libraries that will serve to validate the microarray data as well as to guide the construction of EC-targeted custom microarrays. Human homologues will be identified for any genes that are differentially expressed in murine tumor endothelium, and the homologues will be validated for specificity of expression in human tumor endothelium. As a secondary goal, a parallel effort will be conducted to identify naturally occurring human endothelium-specific ribonucleoprotein (RNP) epitope tags so that the technology can be practiced directly on human tissues. RNP-tag technologies will be used to study murine and human EC gene expression in a variety of tumors and normal tissues across a range of anatomic sites. Detected alterations in tumor EC gene expression that may affect disease progression or that can serve as diagnostic markers or therapeutic targets will be validated in ECs from different tumor and tissue types. This project will generate reagents, information, and technologies that are likely to lead to improved detection and treatment of cancer.
| Mansfield, Kyle D; Keene, Jack D (2009) The ribonome: a dominant force in co-ordinating gene expression. Biol Cell 101:169-81 |
| Tiruchinapalli, Dhanrajan M; Caron, Marc G; Keene, Jack D (2008) Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain. J Neurochem 107:1529-43 |
