Neural Crest Origin of TSC Tumors

Tuberous sclerosis (TS) remains an incurable disease partly because the time when these tumors begin to form and the source of cells that form these tumors are not known. We are also interested in understanding why TS tumors occur in multiple organs. We recently discovered that tumors in TS are derived from a neural crest stem cell, and this type of cell can generate various tumors, which could explain the reason why TS lesions are comprised of cells of multiple phenotypes. Neural crest cells, during early development, also form multiple organs of the body, which might explain why TS patients exhibit lesions in multiple organs. Furthermore, HMGA2, a gene identified by our group to be important in TS and lymphangioleiomyomatosis (LAM), has recently been shown to be important for stem cell renewal and tumor formation. We will therefore undertake studies to determine when tumors begin to occur in TS mice and track the growth of these tumors over an extended 6-month period. Further experiments will explore the importance of HMGA2 in this process.

We will detect the presence of tumors in mice by mating the TS mice with reporter mice such that the tumor fluoresces red when neural crest cells are present. This mating will yield daughter TS mice that show tumors containing red fluorescing cells. This cellular epifluorescence will allow us to detect tumors non-invasively and follow their growth over time as the mouse ages. We will sacrifice the mice at monthly intervals over the 6-month tumor growth measurement period, remove the tumors, and separate the tumor cells that fluoresce red to identify neural crest cells occurring in these tumors. We will then sequence RNA extracted from the tumor neural crest cells to determine changes in the patterns of genes involved in the formation of tumors and differentiation of neural crest cells.

In the second series of studies, we will assess the function of HMGA2 in the development of tumors in TS using human induced pluripotent stem cells (iPSCs) derived from skin fibroblasts of diseased TS patients. Induced pluripotent stem cells will be further differentiated into neural crest cells in vitro, and CRISPR/Cas9-targeted mutations will generate cells heterozygote at the TSC locus. HMGA2 will also be expressed in these cells, which hitherto do not express HMGA2 in their adult form. Sequencing of RNA isolated from single cells expressing HMGA2 and heterozygote at the TSC2 loci will then be performed to define the signaling interactions between HMGA2 and TSC2. These studies sufficiently fall within the purview of the following Tuberous Sclerosis Complex Research Program Focus Areas: (a) Understanding phenotypic heterogeneity in TS complex and (b) gaining a deeper knowledge of TS signaling pathways and the cellular consequences of TS complex deficiency. As described above, the stem cell nature of the TS tumor cell explains the phenotypic heterogeneity of the tumors. The studies in the proposal will not only characterize the nature of the neural crest stem cells causing TS disease but will allow us to understand the signaling pathways promoting growth of the neural crest stem cells and how to regulate the growth of these cells. If successful, we anticipate identifying novel targets in which to block the growth of TS tumors to ultimately deliver to the clinic. By the end of this proposal, we will have identified novel therapeutic targets to stymy growth of neural crest cells causing tumors in TS.