Tuberous sclerosis complex (TSC) is a genetic condition characterized by tumors in different organs: brain, eye, skin, kidney, and heart. The most symptomatic involves brain lesions, where affected individuals can develop seizures, epilepsy, developmental brain defects, mental retardation, and autism. It is estimated that TSC affects one in 6,000 live births in this country. This disease is caused by the loss of normal function of the TSC1 and TSC2 proteins because of mutations in the genes that encode these proteins. Apparently, TSC proteins regulate the motility of neurons during brain development. Loss or dysfunction of either the TSC1 or TSC2 protein leads to aberrant neuronal motility, which is critical during brain development where correct positioning of neurons is achieved by their directional movement from place of origin to the eventual place of residence within brain. Thus, TSC represents a major cause of developmental disorders and epilepsy in the pediatric population.

Individuals with TSC, particularly women, frequently develop another disease, lymphangioleiomyomatosis (LAM), which is also caused by the loss of function of the TSC2 protein. LAM is a progressive disease characterized by lung tumors consisting of abnormal smooth muscle-like LAM cells; this growth leads to the cystic destruction of the lungs and loss of pulmonary function. Clinical and genetic evidence suggests that LAM cells have the ability to spread by metastasis, suggesting that dysfunction of the TSC2 protein in LAM cells may lead to abnormal cell adhesion and motility.

The cellular and molecular mechanism that regulates neuronal motility in TSC and metastatic LAM cell growth in TSC patients with LAM remains poorly understood. Our preliminary data show that LAM cells derived from lung nodules of LAM patients have abnormally high motility, which is reversed by the reintroduction of TSC2, suggesting that TSC2 regulates the motility of LAM cells. Additionally, we found that the TSC2 protein regulates LAM cell adhesion, which is critical for the attachment of cells to the extracellular matrix and for motility. Importantly, our data show that phosphatidylinositol 3-kinase (PI3K), a regulator of TSC proteins, is necessary and sufficient for TSC2-deficient cell motility. Furthermore, we found that PI3K regulates the motility of human neuronal cells. Based on published studies and our preliminary data, we hypothesize that TSC proteins regulate cell adhesion and motility, and loss of either TSC1 or TSC2 function alters cell adhesion and induces aberrant motility, which promotes the pathological conditions associated with TSC and LAM. In this proposal, we will determine the role of the TSC1 and TSC2 proteins in regulating cell adhesion and motility. This study will fill a gap in the current understanding of cytoskeleton remodeling, such as cell adhesion and motility in LAM and TSC.

Elucidation of the cellular function of the TSC tumor suppressors is an emerging field in signal transduction. We have discovered that TSC2 is a negative regulator of S6 kinase, and that loss of TSC2 function promotes abnormal cell growth in LAM. We found that rapamycin, a specific inhibitor of S6 kinase, abrogates abnormal LAM cell growth. Based on our findings, the rapamycin clinical trial for the treatment of kidney and lung manifestations of TSC and LAM has been initiated. Our discovery that TSC2 is a negative regulator of S6 kinase has placed us in a unique position. We feel that this research proposal can advance not only the field of TSC research, but also help us to better understand and potentially treat epilepsy which affects almost 2.5 millions Americans.

We have an established record of investigation in signal transduction pathways regulating LAM, pulmonary arterial and airway smooth muscle cell growth and motility; these studies are a logical extension of our work and build on our expertise. Our studies will provide insight into the molecular mechanisms that may be important in the pathogenesis of TSC and LAM, and they will improve our understanding of the molecular signaling pathways regulating neuronal and LAM cell motility. Mechanistic insight into the role of TSC proteins in disease pathogenesis should advance the development of therapeutic strategies to treat TSC and LAM.