We are investigating the function of tumor suppressor genes and growth factor signaling pathways involved in the molecular pathogenesis of two different human genetic disorders, both of which are members of the phakomatoses characterized by formation of benign and malignant tumors. We have previously shown that Ras proteins play an important role in the tumors of patients with neurofibromatosis type 1 (NF1), which affects the peripheral nervous system and other organs. We are currently investigating other events that occur in the process of tumor development in NF1, using both human tumors and animal model systems. In addition, we have found that the small GTPase Rap1, which is closely related to Ras, interacts with the protein product (tuberin) encoded by one of the genes (TSC2) mutated in patients with tuberous sclerosis (TSC). TSC patients develop lesions in the brain, kidney, heart, and other organs, and some of these lesions progress to malignancy. One interesting aspect of TSC is the fact that this disease can develop as a rssult of the inactivation of either of two tumor suppressor genes, designated TSC1 and TSC2. Consistent with these proteins acting in the same pathway, it has been recently shown that the products of TSC1 (hamartin) and TSC2 (tuberin) physically associate in cells. Thus we have been exploring the role of this complex in the molecular pathology of TSC. NF1 Loss of expression of the NF1 product neurofibromin leads to activation of cellular Ras proteins, as neurofibromin functions as a negative regulatory, GTPase-activating protein (GAP) for Ras. This is particularly true in Schwann cells, which are critical to the formation of benign and malignant tumors in NF1 patients. Our recent NF1 studies have focused on the role of other genetic or epigenetic events that may drive tumorigenesis in NF1 patients. Specifically, we have found that the epidermal growth factor receptor (EGFR) is aberrantly expressed in cells of both benign and malignant NF1 tumors. This result, which we have observed in NF1 patient tumor cell lines, primary benign and malignant tumors, and animal models of NF1, is surprising in that Schwann cells do not normally express the EGFR. Instead, these cells express erbB2 and erbB3, and respond to neuregulin-stimulated growth. Nevertheless, cell lines derived from NF1 malignant peripheral nerve sheath tumors (MPNST), as well as Schwann cells from mice lacking a functional copy of the NF1 gene respond to EGF by activation of mitogenic signaling pathways and increased growth. In addition, we found that anti-EGFR agents inhibited the growth of cell lines derived from NF1 patient MPNSTs. These findings may represent a new avenue for therapeutic intervention in NF1 patients, and we are addressing this possibility through the use of additional animal model systems. Recently, we have found EGFR expression in tumors from compound heterozygous mice bearing lesions in p53 and NF1, and shown that cell lines from these tumors display EGF-dependent growth. TUBEROUS SCLEROSIS (TSC) We previously found that that tuberin acts as a negative regulator of Rap1 in vitro, and co-localizes with Rap1 in the cis/medial region of the Golgi apparatus. More recently, we have found that overexpression of TSC1 directly inhibits cell growth, and that this inhibition is associated with stabilization of tuberin. We found that the cellular tuberin that is bound to hamartin is prevented from being degraded by the ubiquitin-mediated proteolytic system. These results suggest that the stabilization of tuberin by hamartin is critical for its tumor suppressor function. We are currently investigating the structural requirements for the hamartin-tuberin complex, and we have found that specific TSC patient mutations can disrupt the association of these proteins, suggesting that their binding is critical for tumor suppression.
