Irene Khavin

mTORC2 is required for proliferation and survival of TSC2-null cells.

ABSTRACT Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia

Pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysi‐ological component of vascular remodeling in pulmonary arterial hypertension (PAH) for which cellular and molecular mechanisms are poorly understood. The goal of our study was to determine the role of mammalian target of rapamycin (mTOR) in PAVSM cell proliferation, a major pathological manifestation of vascular remodeling in PAH. Our data demonstrate that chronic hypoxia promoted mTOR(Ser‐2481) phosphorylation, an indicator of mTOR intrinsic catalytic activity, mTORC1‐specific S6 and mTORC2‐specific Akt (Ser‐473) phosphorylation, and proliferation of human and rat PAVSM cells that was inhibited by siRNA mTOR PAVSM cells derived from rats exposed to chronic hypoxia (VSM‐H cells) retained increased mTOR(Ser‐2481), S6, Akt (Ser‐473) phosphorylation, and DNA synthesis compared to cells from nor‐moxia‐exposed rats. Suppression of mTORC2 signaling with siRNA rictor, or inhibition of mTORC1 signaling with rapamycin and metformin, while having little effect on other complex activities, inhibited VSM‐H and chronic hypoxia‐induced human and rat PAVSM cell proliferation. Collectively, our data demonstrate that up‐regulation of mTOR activity and activation of both mTORC1 and mTORC2 are required for PAVSM cell proliferation induced by in vitro and in vivo chronic hypoxia and suggest that mTOR may serve as a potential therapeutic target to inhibit vascular remodeling in PAH.—Krymskaya, V. P., Snow, J., Cesarone, G., Khavin, I., Goncharov, D. A., Lim, P. N., Veasey, S. C, Ihida‐Stansbury, K., Jones, P. L., Goncharova, E. A. mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia. FASEB J. 25, 1922‐1933 (2011). www.fasebj.org

Prevention of Alveolar Destruction and Airspace Enlargement in a Mouse Model of Pulmonary Lymphangioleiomyomatosis (LAM)

In a mouse model of the human disease pulmonary lymphangioleiomyomatosis, treatment with rapamycin plus simvastatin prevented alveolar space enlargement and growth of TSC2-null lesions. On the LAM, in Search of Treatments Typically diagnosed in women of childbearing age or in patients with tuberous sclerosis (a genetic disease associated with nonmalignant tumors in the brain and other organs), pulmonary lymphangioleiomyomatosis (LAM) is a rare disease that results in proliferation of smooth muscle–like cells in the lung and destruction of the surrounding normal lung tissue, leading to progressive respiratory problems. LAM can also cause benign tumors in other organs such as the kidneys. Although antiestrogen medications have been used to treat this disorder, these drugs have major side effects and have to be used indefinitely because they do not cure the disease. Now, Goncharova and colleagues have developed a mouse model that recapitulates the key clinical features of LAM and shows promising results after treatment with a combination of medications. Even in patients who do not have tuberous sclerosis, LAM is associated with inactivating mutations in tuberous sclerosis complex (TSC) genes, which encode tumor suppressor proteins. The authors found that injection of kidney tumor cells derived from mice lacking one of these genes, TSC2, into nude mice produced symptoms that are similar to those seen in human LAM disease. These mice developed LAM-like lung lesions, which accumulated around blood vessels and airways, as well as inflammation and destruction of surrounding normal lung tissue. Using this mouse model, the authors demonstrated that simvastatin (a commonly used cholesterol-lowering drug) and rapamycin (an immunosuppressive medication) displayed an additive effect on LAM lesions, inhibiting their growth. In addition, the authors showed that simvastatin decreased the destruction of normal lung tissue, which rapamycin alone did not do. The rapamycin-simvastatin treatment combination did not cure LAM in the mice, and more research is needed to determine whether these promising findings will translate to human patients. However, the two drugs are already approved for use in human subjects for other indications. Thus, the current study brings this treatment regimen one step closer to the clinic—and to a more tolerable long-term therapy for LAM. Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic disease characterized by neoplastic growth of atypical smooth muscle–like LAM cells, destruction of lung parenchyma, obstruction of lymphatics, and formation of lung cysts, leading to spontaneous pneumothoraces (lung rupture and collapse) and progressive loss of pulmonary function. The disease is caused by mutational inactivation of the tumor suppressor gene tuberous sclerosis complex 1 (TSC1) or TSC2. By injecting TSC2-null cells into nude mice, we have developed a mouse model of LAM that is characterized by multiple random TSC2-null lung lesions, vascular endothelial growth factor–D expression, lymphangiogenesis, destruction of lung parenchyma, and decreased survival, similar to human LAM. The mice show enlargement of alveolar airspaces that is associated with progressive growth of TSC2-null lesions in the lung, up-regulation of proinflammatory cytokines and matrix metalloproteinases (MMPs) that degrade extracellular matrix, and destruction of elastic fibers. TSC2-null lesions and alveolar destruction were differentially inhibited by the macrolide antibiotic rapamycin (which inhibits TSC2-null lesion growth by a cytostatic mechanism) and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin (which inhibits growth of TSC2-null lesions by a predominantly proapoptotic mechanism). Treatment with simvastatin markedly inhibited MMP-2, MMP-3, and MMP-9 levels in lung and prevented alveolar destruction. The combination of rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction by inhibiting MMP-2, MMP-3, and MMP-9. Our findings demonstrate a mechanistic link between loss of TSC2 and alveolar destruction and suggest that treatment with rapamycin and simvastatin together could benefit patients with LAM by targeting cells with TSC2 dysfunction and preventing airspace enlargement.

Interferon β Augments Tuberous Sclerosis Complex 2 (TSC2)-Dependent Inhibition of TSC2-Null ELT3 and Human Lymphangioleiomyomatosis-Derived Cell Proliferation

Lymphangioleiomyomatosis (LAM), a rare pulmonary disorder, manifests as an abnormal neoplastic growth of smooth muscle-like cells within the lungs. Mutational inactivation of tumor suppressor tuberous sclerosis complex 2 (TSC2) in LAM constitutively activates the mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) signaling pathway and promotes neoplastic growth of LAM cells. In many cell types, type I interferon β (IFNβ) inhibits proliferation and induces apoptosis through signal transducers and activators of transcription (STAT)-dependent and STAT-independent signaling pathways, one of which is the mTOR/S6K1 signaling pathway. Our study shows that IFNβ is expressed in LAM tissues and LAM-derived cell cultures; however, IFNβ attenuates LAM-derived cell proliferation only at high concentrations, 100 and 1000 U/ml (IC50 value for IFNβ is 20 U/ml compared with 1 U/ml for normal human mesenchymal cells, human bronchus fibroblasts and human airway smooth muscle cells). Likewise, IFNβ only attenuates proliferation of smooth muscle TSC2-null ELT3 cells. Analysis of IFNβ signaling in LAM cells showed expression of IFNβ receptor α (IFNβRα) and IFNβRβ, activation and nuclear translocation of STAT1, and phosphorylation of STAT3 and p38 mitogen-activated protein kinase (MAPK), but IFNβ had little effect on S6K1 activity. However, the re-expression of TSC2 or inhibition of mTOR/S6K1 with rapamycin (sirolimus) augmented antiproliferative effects of IFNβ in LAM and TSC2-null ELT3 cells. Our study demonstrates that IFNβ-dependent activation of STATs and p38 MAPK is not sufficient to fully inhibit proliferation of cells with TSC2 dysfunction and that TSC2-dependent inhibition of mTOR/S6K1 cooperates with IFNβ in inhibiting human LAM and TSC2-null ELT3 cell proliferation.

Differential effects of formoterol on thrombin- and PDGF-induced proliferation of human pulmonary arterial vascular smooth muscle cells

BackgroundIncreased pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysiological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PH). The long-acting β2-adrenergic receptor (β2AR) agonist formoterol, a racemate comprised of (R,R)- and (S,S)-enantiomers, is commonly used as a vasodilator in chronic obstructive pulmonary disease (COPD). PH, a common complication of COPD, increases patients’ morbidity and reduces survival. Recent studies demonstrate that formoterol has anti-proliferative effects on airway smooth muscle cells and bronchial fibroblasts. The effects of formoterol and its enantiomers on PAVSM cell proliferation are not determined. The goals of this study were to examine effects of racemic formoterol and its enantiomers on PAVSM cell proliferation as it relates to COPD-associated PH.MethodsBasal, thrombin-, PDGF- and chronic hypoxia-induced proliferation of primary human PAVSM cells was examined by DNA synthesis analysis using BrdU incorporation assay. ERK1/2, mTORC1 and mTORC2 activation were determined by phosphorylation levels of ERK1/2, ribosomal protein S6 and S473-Akt using immunoblot analysis.ResultsWe found that (R,R) and racemic formoterol inhibited basal, thrombin- and chronic hypoxia-induced proliferation of human PAVSM cells while (S,S) formoterol had lesser inhibitory effect. The β2AR blocker propranolol abrogated the growth inhibitory effect of formoterol. (R,R), but not (S,S) formoterol attenuated basal, thrombin- and chronic hypoxia-induced ERK1/2 phosphorylation, but had little effect on Akt and S6 phosphorylation levels. Formoterol and its enantiomers did not significantly affect PDGF-induced DNA synthesis and PDGF-dependent ERK1/2, S473-Akt and S6 phosphorylation in human PAVSM cells.ConclusionsFormoterol inhibits basal, thrombin-, and chronic hypoxia-, but not PDGF-induced human PAVSM cell proliferation and ERK1/2, but has little effect on mTORC1 and mTORC2 signaling. Anti-proliferative effects of formoterol depend predominantly on its (R,R) enantiomer and require the binding with β2AR. These data suggest that (R,R) formoterol may be considered as potential adjuvant therapy to inhibit PAVSM cell proliferation in COPD-associated PH.

Interferon beta augments tuberous sclerosis complex 2 (TSC2)-dependent inhibition of TSC2-null ELT3 and human lymphangioleiomyomatosis-derived cell proliferation.

Lymphangioleiomyomatosis (LAM), a rare pulmonary disorder, manifests as an abnormal neoplastic growth of smooth muscle-like cells within the lungs. Mutational inactivation of tumor suppressor tuberous sclerosis complex 2 (TSC2) in LAM constitutively activates the mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) signaling pathway and promotes neoplastic growth of LAM cells. In many cell types, type I interferon β (IFNβ) inhibits proliferation and induces apoptosis through signal transducers and activators of transcription (STAT)-dependent and STAT-independent signaling pathways, one of which is the mTOR/S6K1 signaling pathway. Our study shows that IFNβ is expressed in LAM tissues and LAM-derived cell cultures; however, IFNβ attenuates LAM-derived cell proliferation only at high concentrations, 100 and 1000 U/ml (IC50 value for IFNβ is 20 U/ml compared with 1 U/ml for normal human mesenchymal cells, human bronchus fibroblasts and human airway smooth muscle cells). Likewise, IFNβ only attenuates proliferation of smooth muscle TSC2-null ELT3 cells. Analysis of IFNβ signaling in LAM cells showed expression of IFNβ receptor α (IFNβRα) and IFNβRβ, activation and nuclear translocation of STAT1, and phosphorylation of STAT3 and p38 mitogen-activated protein kinase (MAPK), but IFNβ had little effect on S6K1 activity. However, the re-expression of TSC2 or inhibition of mTOR/S6K1 with rapamycin (sirolimus) augmented antiproliferative effects of IFNβ in LAM and TSC2-null ELT3 cells. Our study demonstrates that IFNβ-dependent activation of STATs and p38 MAPK is not sufficient to fully inhibit proliferation of cells with TSC2 dysfunction and that TSC2-dependent inhibition of mTOR/S6K1 cooperates with IFNβ in inhibiting human LAM and TSC2-null ELT3 cell proliferation.

Interferon β augments TSC2-dependent inhibition of TSC2-null ELT3 and human LAM-derived cell proliferation*

Lymphangioleiomyomatosis (LAM), a rare pulmonary disorder, manifests as an abnormal neoplastic growth of smooth muscle-like cells within the lungs. Mutational inactivation of tumor suppressor tuberous sclerosis complex 2 (TSC2) in LAM constitutively activates the mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) signaling pathway and promotes neoplastic growth of LAM cells. In many cell types, type I interferon β (IFNβ) inhibits proliferation and induces apoptosis through signal transducers and activators of transcription (STAT)-dependent and STAT-independent signaling pathways, one of which is the mTOR/S6K1 signaling pathway. Our study shows that IFNβ is expressed in LAM tissues and LAM-derived cell cultures; however, IFNβ attenuates LAM-derived cell proliferation only at high concentrations, 100 and 1000 U/ml (IC50 value for IFNβ is 20 U/ml compared with 1 U/ml for normal human mesenchymal cells, human bronchus fibroblasts and human airway smooth muscle cells). Likewise, IFNβ only attenuates proliferation of smooth muscle TSC2-null ELT3 cells. Analysis of IFNβ signaling in LAM cells showed expression of IFNβ receptor α (IFNβRα) and IFNβRβ, activation and nuclear translocation of STAT1, and phosphorylation of STAT3 and p38 mitogen-activated protein kinase (MAPK), but IFNβ had little effect on S6K1 activity. However, the re-expression of TSC2 or inhibition of mTOR/S6K1 with rapamycin (sirolimus) augmented antiproliferative effects of IFNβ in LAM and TSC2-null ELT3 cells. Our study demonstrates that IFNβ-dependent activation of STATs and p38 MAPK is not sufficient to fully inhibit proliferation of cells with TSC2 dysfunction and that TSC2-dependent inhibition of mTOR/S6K1 cooperates with IFNβ in inhibiting human LAM and TSC2-null ELT3 cell proliferation.