Regulation and function of tuberous sclerosis complex-2 tumor suppressor in renal cell carcinoma

Tuberous sclerosis complex-2 (TSC2) is an important tumor suppressor gene for multiple tumors associated with human TSC disease and rodent renal cell carcinoma (RCC) development. Germline mutation of Tsc2 in the Eker rats predisposes to spontaneous RCC, while von Hippel-Lindau (VHL) tumor suppressor gene is the major target for human RCC development. Using Eker rat animal model, I investigated the etiology of RCC development and the mechanism of TSC2 regulation by Akt phophorylation. In the first study, I characterized that VHL gene product pVhl has a unique distribution pattern in the rat nephron compared with human and mouse nephrons. I found that rat pVhl has two isoforms arising from different translation start sites. In contrast to human pVHL nuclear/cytoplasmic subcellular localization, both rat pVhl isoforms were expressed mainly in the membrane and cytosolic fraction. The difference of pVhl distribution/localization between human and rat may explain the species-specific function of Vhl in RCC development. In the second study, I provide evidence suggesting that human and rat RCC converge at the regulation of Hypoxia-inducible Factor alpha (HIFalpha). Eker rat RCC-derived cells exhibited high basal levels of HIF activity, similar to human RCC cell lines with VHL alterations. HIF2alpha was stabilized in Tsc2 null rat cells as well as in Eker rat RCC. High activity of HIF2alpha resulted in HIFalpha mediated upregulation of Vascular Endothelial Growth Factor (VEGF) expression and further angiogenic phenotype of rat RCC. These data identified dysregulation of HIF2alpha as a convergent pathway for both human and rat RCC development. Finally, I present data demonstrating that TSC2 gene product tuberin is a phosphorylated protein which contains multiple Akt phosphorylation sites overlapping with 14-3-3 binding sites. Phosphorylation of tuberin correlated with Akt phosphorylation. In addition, tuberin associated with 14-3-3 via Akt mediated phosphorylation. Interaction between tuberin and 14-3-3 could be abrogated by a tuberin polypeptide with the Akt site phosphorylated. These data suggest that Akt phosphorylation mediates tuberin/14-3-3 association and inhibits tuberin function. Taken together, these studies place TSC2/tuberin in the Akt mediated signaling pathway and elucidate the tumorigenesis mechanism for RCC development associated with loss of TSC2 function.