ABSTRACT
Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2-/- mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2-/- MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2-/- MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2-/- MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.
Subject(s)
Diacylglycerol Kinase/antagonists & inhibitors , Lung Neoplasms/drug therapy , Lymphangioleiomyomatosis/drug therapy , Pinocytosis/drug effects , Ritanserin/pharmacology , Tuberous Sclerosis Complex 2 Protein/deficiency , Tuberous Sclerosis/drug therapy , Angiolipoma/genetics , Animals , Autophagy/drug effects , Cell Proliferation , Chloroquine/pharmacology , Diacylglycerol Kinase/genetics , Diacylglycerol Kinase/metabolism , Down-Regulation , Drug Synergism , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression , Kidney Neoplasms/genetics , Lung Neoplasms/etiology , Lung Neoplasms/pathology , Lymphangioleiomyomatosis/etiology , Lymphangioleiomyomatosis/pathology , Lysosomes/drug effects , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Nude , Nutrients/metabolism , Phosphatidic Acids/metabolism , Phospholipids/metabolism , Pinocytosis/physiology , Tuberous Sclerosis/complicationsABSTRACT
The skin of aquatic vertebrates surrounds all the mechanical lineages of the body and must, therefore, play an important role in locomotion. A cross-woven collagenous dermal design has converged across several clades of vertebrates. Despite this intriguing pattern, the biomechanical role of skin in swimming fishes remains largely unknown. A direct force transmission role for fish skin has been proposed, a hypothesis that is supported by the arrangement of the connective tissues linking the skin to the axial musculature. To evaluate this direct force-transmission hypothesis, we undertook hundreds of uniaxial tensile tests on skin samples from coho salmon (Oncorhynchus kisutch), Florida pompano (Trachinotus carolinus), and red snapper (Lutjanus campechanus). To do this, we developed highly precise, low-cost, custom-built material testing units. To augment our data, we also assembled a data set of skin stiffness of four additional species of actinopterygians fishes from previously published studies. We found that stiffness varies significantly between species and that the skin of our study species was increasingly stiff along a rostrocaudal gradient. Placing our results in the context of the limited body of previous work, we found that species with lower skin stiffness exhibit shorter propulsive wavelengths and low thrust production at the caudal fin and species with higher skin stiffness possess longer propulsive wavelengths and high thrust production at the caudal fin. In addition, we found that mean collagen fiber angle was close to 50° and that fiber angle was lower in posterior samples than in anterior and midlateral samples. Taken as a whole, our mechanical and morphological results support the hypothesis that the skin functions as an important direct force-transmission device in actinopterygians whereby muscular force generated in anterior myotomes is transmitted to the posterior of the body through the increasingly stiff skin.
