Abstract
TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1−/− MEFs have decreased migration compared to littermate-derived Tsc1+/+ MEFs. Migration of Tsc1−/− MEFs with re-expressed TSC1 was comparable to Tsc1+/+ MEF migration. In contrast, Tsc2−/− MEFs showed an increased migration compared to Tsc2+/+ MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1−/− MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2−/− MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1−/− or Tsc2−/− MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s) by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2−/− MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2-null cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.
Highlights
Mutations of tumor suppressor genes tuberous sclerosis complex 1 (TSC1) and TSC2 are linked to the pathobiology of hamartoma syndrome Tuberous Sclerosis (TSC) and pulmonary lymphangioleiomyomatosis (LAM) [1,2,3,4,5]
In addition to abnormal proliferation, smooth muscle-like cells from LAM lungs have increased motility and invasiveness [7], and LAM nodule recurrence was reported after single-lung transplantation in patients without renal angiomyolipoma [8], suggesting a metastatic nature of cells with mutational inactivation of TSC1/TSC2
We show that TSC2 deficiency promotes cell migration and invasiveness while loss of TSC1 attenuates cell migration and reduces cellular invasive potential
Summary
Mutations of tumor suppressor genes tuberous sclerosis complex 1 (TSC1) and TSC2 are linked to the pathobiology of hamartoma syndrome Tuberous Sclerosis (TSC) and pulmonary lymphangioleiomyomatosis (LAM) [1,2,3,4,5]. Pulmonary LAM, a rare disease that can be sporadic or associated with TSC, is characterized by the neoplastic growth of smooth-muscle like lesions in the lungs, destruction of the lung parenchyma, loss of pulmonary function, and is associated with increased occurrence of renal angiomyolipomas [6]. In addition to abnormal proliferation, smooth muscle-like cells from LAM lungs have increased motility and invasiveness [7], and LAM nodule recurrence was reported after single-lung transplantation in patients without renal angiomyolipoma [8], suggesting a metastatic nature of cells with mutational inactivation of TSC1/TSC2. The major breakthrough in understanding the functions of TSC1 and TSC2 came with identifying that TSC2 binds TSC1 via its N-terminal domain and forms the TSC1/TSC2 tumor suppressor complex that acts as a negative regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a key regulator of cell growth, proliferation, metabolism, and autophagy [9,10,11]. TSC2 GAP inhibits Rheb, leading to Rheb-dependent inhibition of mTORC1, its downstream effectors S6 kinase 1 (S6K1)-ribosomal protein S6, and suppression of cell growth and proliferation [12]
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