Tuberous sclerosis complex (TSC) is a commonly inherited disorder manifested by benign tumors most typically in brain, kidney, heart, lung, skin, and eye. Generally, loss or malfunction of one of two tumor suppressor proteins, TSC1 or TSC2, causes the growth of these tumors. This suggests that their normal physiological function is to moderate or stop cell growth. Among the variety of components necessary for cell growth, protein synthesis plays an essential role. Recent studies have shown that a complex of TSC1 and TSC2 negatively regulates this process through the inactivation of a cellular signaling pathway known as mTOR/S6K. However, the molecular mechanism by which they achieve this inactivation was, until recent months, unknown. We and others have now found that TSC2 inactivates a protein known as Rheb. Rheb belongs to a protein family that functions as molecular switches to regulate a vast array of cellular processes that include cell growth. Furthermore, unlike the inhibitory TSC1/2 complex, Rheb activates the mTOR/S6K pathway. Overall, these findings suggest that Rheb is the physiological target that is inhibited by the TSC1/2 complex. However, the significance of these findings in the development of TSC remains to be defined. It is also still unclear how they relate to the clinical manifestations of the disease that seem to depend on steroid hormone regulation. For example, rhabdomyomas of the heart are largest at birth and have a second peak of incidence during puberty, while lymphangioleiomyomatosis (LAM) of the lung is almost exclusively found in women and exacerbated by pregnancy. The involvement of Rheb in LAM may explain this phenomenon because it is an inducible protein. This means that certain cellular stimuli can promote the expression of more Rheb protein. Because estrogen promotes protein expression, it is very possible that estrogen-dependent upregulation of Rheb accelerates cell growth, providing an explanation for age- and sex-dependent manifestations of the disease.

Our objective is to establish the relevance of Rheb in the development of TSC. To accomplish this goal, we will use a rat model TSC cell line that lacks TSC2, and importantly, cells and tissues obtained from TSC patients. Three different approaches will allow us to conclusively establish the involvement of Rheb: we will (1) silence the expression of Rheb by introducing into the cells specific molecules known as small interference RNA; (2) inhibit the normal function of Rheb by the introduction of proteins known as dominant negatives; and (3) re-introduce a normal TSC2 protein to inactivate Rheb. If Rheb is responsible for the aberrant growth of these cells, we expect to observe cell growth inhibition. To well understand the relevance of these findings in disease, we will extend these studies to human TSC cells obtained from LAM lesions normally found in the lung of female patients (LAM cells). To address the estrogen-dependent upregulation of Rheb during some clinical manifestations of the disease, we will use the TSC model cell line and LAM cells. We will study the upregulation of Rheb expression in response to estrogen stimulation and its correlation with increased cell growth. Again, the inclusion of experiments on LAM cells will give strong disease relevance to the observations. To strengthen these conclusions, we will next study overexpression of Rheb in human LAM and renal angiomyolipoma specimens from young female TSC patients. Overall, the studies described will indicate the importance of blocking Rheb function in the treatment of TSC. Because Rheb needs to attach to cellular membranes to function, its activity can be blocked by drugs known as farnesyl transferase inhibitors (FTIs) that prevent this attachment. Thus, we will test the efficacy of FTIs to block the growth of TSC cells. These experiments will establish the potential use of FTIs in the clinical treatment of TSC.

Characterization of the signaling events responsible for tumor formation is essential for understanding and treating TSC. The identification of proteins involved in the initiation and propagation of growth signals will provide targets for the rationale design of effective drugs/therapies to reverse the clinical manifestations of the disease. The identification of Rheb as the initiator of aberrant cell growth in TSC patients is an example of the importance of these studies. In fact, our recent findings suggest that FTIs, which inhibit Rheb function, might provide an effective treatment for TSC. Finally, understanding the implications of increased Rheb expression during the progression of TSC will help in the design of future strategies to manage the disease and identify new molecular targets to combat it.