Henry M. Furneaux - US grants

The basis of this proposal is to investigate the molecular biology of antibody-associated paraneoplastic syndromes. Although rare, these neurological syndromes are of crucial importance since the patients harbor high-titer antibodies directed against human tumor antigens. Three antibody-associated paraneoplastic neurological syndromes which have been discovered and characterized in this laboratory will be further investigated at the molecular level. These syndromes include: the Yo syndrome associated with cerebellar dysfunction and antibodies directed against ovarian tumor antigens; the Hu syndrome associated with sensory neuronopathy and antibodies directed against small cell lung cancer antigens; the Ri syndrome associated with opsoclonus and antibodies directed against breast tumor antigens. The availability of these high-titer human antibodies provide a unique opportunity to: (a) Isolate human tumor antigens (b) Investigate tumor specific gene expression mechanisms. (c) Understand the molecular basis of the human anti-tumor immune response.

DESCRIPTION (Adapted from Investigator's Abstract): Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in diabetic retinopathy, cancer and coronary heart disease. The expression of VEGF is tightly controlled to the cellular oxygen tension. Previous studies have shown that VEGF mRNA is markedly stabilized under hypoxic conditions. The molecular mechanisms underlying the regulation of VEGF mRNA stability are not well understood. In the preliminary results the applicant showed that the stability of VEGF mRNA is controlled by a cis acting element in its 3'UTR. They have also identified a trans acting factor (HuR) that binds to this element and increases the expression of VEGF. In addition, they show that HuR itself is regulated by hypoxia. This proposal will focus on the role of HuR in hypoxic regulation of VEGF mRNA stability. The proposed studies may be important since they could lead to the generation of new drugs that will regulate VEGF expression at the post transcriptional level. The specific aims of the proposal are the following: (1) To determine the fine structure of the HuR/VEGF mRNA complex. To generate mutations in both the mRNA element and HuR and determine their effect on VEGF expression; (2) To design selective inhibitors of HuR/VEGF mRNA complexes; (3) To identify and clone HuR associate proteins that modify its activity; (4) To determine how hypoxia regulates HuR expression; and (5) To determine how HuR stabilizes VEGF mRNA.

[unreadable] DESCRIPTION (provided by applicant): Drug addiction is a complex neurological disorder likely to involve genetic components. For example, studies in mice have shown that elimination of the gene that encodes the receptor for cannabinoids blunts their "addictive response " to opiates and alcohol. Although many genes have been suspected to contribute to addictive behavior, unique human variants in these genes that unequivocally promote susceptibility to drug addiction have not been identified. So far, most attempts to identify such variants have focused on the protein coding segments of the suspect genes. However, it is quite possible that variants that alter the expression of such candidate genes may also render individuals susceptible to addictive disease. Recently, it has become clear that many human genes are regulated by a new epigenetic mechanism, which involves the specific annealing of a small RNA (microRNA) to a target element in the mRNA. In most cases, the annealing of the microRNA leads to a profound decrease in expression of the target mRNA. In our preliminary studies, we have identified a 50 nucleotide element in the 3'UTR of the human cannabinoid receptor mRNA that is likely to be the target of a microRNA. We have observed that insertion of this element into a luciferase reporter significantly reduces its expression. Importantly, a mutation in the target element that is predicted to compromise the binding of microRNAs abrogates the suppressive effect of the target element. Interestingly, this mutation has previously been identified as a rare polymorphism in African American populations. Thus, our overarching hypothesis is that the cannabinoid receptor is regulated by microRNAs and that variants in the cannabinoid receptor element may influence its expression and thus render the individual susceptible to drug addiction. In our first aim, we will confirm and extend our initial observations and identify the microRNA that mediates repression of the cannabinoid receptor. These studies will be aided by our experience in designing and using "Antagomir" reagents. Antagomirs are modified RNAs that can be introduced into cells and can potently down regulate the expression of specific microRNAs. In our second aim, we will examine whether any human variants in the cannabinoid receptor element correlate with susceptibility to addiction. To do this, we have established collaboration with Drs. Kranzler, Covault and Oncken who have access to DNA from control and affected patient populations. [unreadable] [unreadable] [unreadable]