Rab GTP-binding proteins are members of the Ras supergene family. Rab proteins are generally involved in protein transport/trafficking and determine the specificity of membrane transport steps within cell. We have identified a novel member of the Rab protein family, which we have named Nu-Rab, for its unique nuclear localization. Nu-Rab mRNA is highly expressed in most of cancer cell lines examined, and it is overexpressed in more than 60% of human primary breast cancers when compared to their matching normal tissues. The GFP- and Myc-tagged Nu-Rab mainly localizes to nucleus with some distribution noted in the cytoplasmic subcellular compartment. Immnunochemistry using Nu-Rab antibodies generated in our laboratory also detects that the endogenous Nu-Rab predominantly localizes to nucleus. Nu-Rab expression is down-regulated by apoptosis inducing agents such as ultraviolet radiation (UV), thapsigargin and sulindac sulfide. The PI3-kinase inhibitor LY294002, PI3-K phosphotase PTEN and dominant-negative mutant-Akt also down-regulate Nu-Rab mRNA expression, suggesting that Nu-Rab is a potential downstream target of the PI3-K/Akt signaling pathway. We are now proposing to further investigate the role of Nu-Rab in cell growth and/or survival and to elucidate the molecular basis of its overexpression in human breast cancer.
In specific aim 1, we will analyze a larger pool of fresh-frozen and paraffin-embedded tissue specimens to evaluate the expression of Nu-Rab at the mRNA and protein levels.
In specific aim 2, we will initiate functional studies to explore the effect of wild type-and GTP-binding mutants of Nu-Rab protein in oncogenic transformation. We will also explore whether Nu-Rab overexpression or deficiency effects apoptosis induced by genotoxic and non-genotoxic agents. Studies in specific aim 3 will be initiated to identify potential Nu-Rab effectors/regulator proteins in order to explore whether Nu-Rab plays a role in nuclear-cytoplasmic protein trafficking and study potential signaling events modulated by Nu-Rab. These are exploratory/developmental studies that, upon completion, will generate sufficient new data and reagents that will form the basis of future in-depth studies investigating the molecular mechanisms (s) of action of this novel GTP-binding protein in breast cancer development and/or progression. ? ? ?
