Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis

Abstract Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5−/− MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC

Autophagy : Moving Benchside Promises to Patient Bedsides

International audienceSurvival rates of patients with metastatic or recurrent cancers have remained virtually unchanged during the past 30 years. This fact makes the need for new therapeutic options even more urgent. An attractive option would be to target autophagy, an essential quality control process that degrades toxic aggregates, damaged organelles, and signaling proteins, and acts as a tumor suppressor pathway of tumor initiation. Conversely, other fascinating observations suggest that autophagy supports cancer progression, relapse, metastasis, dormancy and resistance to therapy. This review provides an overview of the contradictory roles that autophagy plays in cancer initiation and progression and discusses the promises and challenges of current strategies that target autophagy for cancer therapy

TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells

Rapamycin-induced miR-21 promotes mitochondrial homeostasis and adaptation in mTORC1 activated cells

mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro. miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells’ clonogenic and anchorage independent growth were reduced by ∼50% (p<0.01) and ∼75% (p<0.0001), respectively, and combined rapamycin treatment decreased soft agar growth by ∼90% (p<0.0001). miR-21 inhibition also increased sensitivity to apoptosis. Through a network biology-driven integration of RNAseq data, we discovered that miR-21 promotes mitochondrial adaptation and homeostasis in Tsc2-deficient cells. miR-21 inhibition reduced mitochondrial polarization and function in Tsc2-deficient cells, with and without co-treatment with rapamycin. Importantly, miR-21 inhibition limited Tsc2-deficient tumor growth in vivo, reducing tumor size by approximately 3-fold (p<0.0001). When combined with rapamcyin, miR-21 inhibition showed even more striking efficacy, both during treatment and after treatment cessation, with a 4-fold increase in median survival following rapamycin cessation (p=0.0008). We conclude that miR-21 promotes mTORC1-driven tumorigenesis via a mechanism that involves the mitochondria, and that miR-21 is a potential therapeutic target for TSC-associated hamartomas and other mTORC1-driven tumors, with the potential for synergistic efficacy when combined with rapalogs