The role of TGF-β signaling in cell-cell interactions in the pathogenesis of lymphangioleiomyomatosis

RESEARCH SUMMARY Pulmonary lymphangioleiomyomatosis (LAM) is a rare, female-predominant lung disease that is caused by inactivating mutations in the tuberous sclerosis complex (TSC1/2) tumor suppressor genes and consequent downstream hyperactivation of the mammalian target of rapamycin (mTOR) pathway. TSC1/2 mutations in LAM- initiating cells are central to disease pathogenesis, in collaboration with non-mutated native lung cells, including fibroblasts, endothelial, and alveolar epithelial cells. Rapamycin, the only FDA-approved treatment for LAM is cytostatic rather than cytotoxic and is associated with acquired resistance and adverse effects resulting its discontinuation. My proposal addresses a critical need to identify new therapeutic targets in LAM, and this will require the development of tissue-level in vitro LAM models that represent the cellular complexity of the disease. Lung cells within and surrounding clusters of LAM cells exhibit notable changes, particularly in fibroblasts, where markers of activation are significantly upregulated. Additionally, in alveolar epithelial cells the number of KRT8+ transitional-state cells is abnormally high. Using TSC2-/- renal angiomyolipoma cells (TSC2-/-AML) as a surrogate for LAM cells, my preliminary data demonstrates that secreted levels of several growth factors, including TGF- β1 and TGF-β2 are upregulated. These growth factors are known to promote fibroblast activation and influence alveolar differentiation, however they have yet to be evaluated in the context of LAM pathogenesis. The objective of my study is, therefore, to elucidate the role of TGF-β signaling in LAM pathogenesis. My central hypothesis is that TGF-β acts as a critical regulator of intercellular signaling, and its upregulation drives LAM progression by promoting fibroblast activation and alveolar simplification. To test this hypothesis, in Aim 1, I will determine the extent to which TSC2 inactivation and subsequent mTOR hyperactivation, using TSC2-/-AML cells, influence intra- and intercellular TGF-β signaling. I will use 2D and 3D co-culture models seeded with TSC2-/- or TSC2+ AML cells and non-diseased fibroblasts to determine how TSC2 inactivation affects TGF-β signaling activation and fibroblast phenotypes. Quantitative methods including live cell imaging, protein and RNA quantification will be used to measure changes in TGF-β signaling and fibroblast activation. Functional assays, such as wound healing assays and electrical cell impedance sensing (ECIS), will quantify the impact of TSC2-/- cells on fibroblast function. In Aim 2, I will develop a trans-well model of co-cultured TSC2-/- cells and primary AT2 cells and a LAM- on-chip model, to recapitulate interactions among TSC2-/- cells, fibroblasts, and AT2 cells; and use this model in conjunction with genetic and pharmacologic manipulation of mTOR and TGF-β signaling to both determine the extent to which TSC2-dependent TGF-β signaling influences differentiation of the alveolar epithelium and identify mechanisms by which LAM causes alveolar dysfunction. The proposed study is expected to provide mechanistic insights into the role of TGF-β signaling in LAM and its impact on various cell types within the disease microenvironment, identifying new therapeutic targets.