Oncogene (2014) 33, 4352–4358 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

SHORT COMMUNICATION Pivotal role of augmented aB-crystallin in tumor development induced by deficient TSC1/2 complex

F Wang1, X Chen1,CLi1, Q Sun2, Y Chen3, Y Wang1, H Peng4, Z Liu1, R Chen1, K Liu5,HYan3,BHYe6, DJ Kwiatkowski7 and H Zhang1

Tuberous sclerosis complex 1 (TSC1) and TSC2 are suppressors of mechanistic target of rapamycin (mTOR). mTOR is the major component of two protein complexes: mTOR complex 1 (mTORC1) and mTORC2. Inactive mutation of either TSC1 or TSC2 unleashes mTOR signaling and consequently causes TSC, a benign tumor syndrome affecting multiple organs. We report here that expression of aB-crystallin was upregulated in Tsc1 À / À or Tsc2 À / À mouse embryonic fibroblasts, Eker rat uterine leiomyoma-derived Tsc2- deficient ELT3 cells, mutant Tsc2-associated mouse kidney tumors, and human lung lymphangioleiomyomatosis nodules. aB-crystallin was transcriptionally activated by mTOR complex 2 (mTORC2): nuclear factor-kappa B (NFkB) signaling cascade. The augmented aB-crystallin was critical for the migration, invasion and apoptotic resistance of Tsc2-defective cells. Disruption of aB-crystallin suppressed Tsc2-null cell proliferation and tumorigenesis. Therefore, enhanced aB-crystallin has an essential role in TSC1/2 complex deficiency-mediated tumorigenesis, and inhibition of aB-crystallin may complement the current therapy for TSC.

Oncogene (2014) 33, 4352–4358; doi:10.1038/onc.2013.401; published online 30 September 2013 Keywords: aB-crystallin; mTOR; NFkB; TSC; tumor

INTRODUCTION mutation of one of these two genes is the major cause for TSC, The receptor tyrosine kinase/phosphatidylinositol 3-kinase/AKT/ a benign tumor syndrome afflicting multiple organs.5,15–18 TSC is mechanistic target of rapamycin (RTK/PI3K/AKT/mTOR) signaling featured with cerebral cortical tubers, subependymal nodules, cascade, which has pivotal roles in cell metabolism, autophagy, renal angiomyolipomas, pulmonary lymphangiomyomatosis growth, proliferation and survival, is one of the most frequently (LAM), cardiac rhabdomyomas and facial angiofibroma.19 LAM is deregulated signaling pathways in cancers.1–4 The constitutive characterized by the abnormal proliferation of smooth muscle-like activation of this network is often caused by gain-of-function cells and cystic lung destruction. It is thought to be initiated by mutations of proto-oncogenes, such as epidermal growth factor histologically benign cells carrying TSC1 or TSC2 mutations that receptor (EGFR), PI3K and AKT, and by loss-of-function mutations of migrated from renal angiomyolipomas.20,21 tumor suppressors, such as phosphatase and tensin homolog In early 1990s, aB-crystallin had been found elevated in TSC (PTEN), tuberous sclerosis complex 1 (TSC1) and TSC2.5–8 As the lesions, including the brain, kidney, lung and heart.22,23 The major effector of this pathway, mTOR can integrate signals from putative role of enhanced aB-crystallin in inactive TSC1/2 growth factors, nutrients and energy supplies, and then regulate a complex-mediated tumorigenesis is unknown. Besides being a subset of gene expression and protein synthesis/modification in major component in eye lens, aB-crystallin is a ubiquitous small controlling cell growth, proliferation and survival.9 Being a serine/ heat-shock protein (sHSP) and can be induced by stress in threonine kinase, mTOR is the catalytic subunit of two distinct counteracting the deleterious aggregation of misfolded proteins complexes named mTOR complex 1 (mTORC1) and mTORC2.10 and thus helping cell survival.24 Another cytoprotective function mTORC1 consists of mTOR, mLST8, Raptor and PRAS4011,12 and of aB-crystallin is antiapoptotic effect. aB-crystallin overexpression controls cell growth and proliferation by modulating mRNA can protect cells from apoptosis induced by a large panel of translation through phosphorylation of eukaryotic translation stimuli, whereas aB-crystallin deficiency sensitizes cells to initiation factor 4E binding protein (4E-BP) and ribosomal apoptosis.25,26 Overexpression of aB-crystallin has also been protein S6 kinase (S6K).13 mTORC2 contains mTOR, mLST8, Rictor detected in several human cancers.27–29 and SIN1 and regulates actin cytoskeleton dynamics.14 In contrast In this study, we investigated the significance of augmented to the well-documented mTORC1, the function and mechanism of aB-crystallin in TSC1/2 deficiency-induced tumorigenesis. We mTORC2 are less characterized. found that TSC1 and TSC2 were negative regulators of aB-crys- TSC1 and TSC2 tumor suppressors form a protein complex in tallin expression, and the upregulation of aB-crystallin was suppression of mTOR. mTOR hyperactivation due to inactive mTORC2 dependent. We then identified NFkB as the transducer

1State Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences and School of Basic Medicine, Graduate School of Peking Union Medical College, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China; 2Department of Immunology, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin, China; 3Center for Coronary Heart Disease, Cardiovascular Institute and Fu Wai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China; 4Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL, USA; 5Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China; 6Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA and 7Division of Translational Medicine, Brigham and Women’s Hospital and Dana-Faber Cancer Institute, Harvard Medical School, Boston, MA, USA. Correspondence: Dr H Zhang, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences and School of Basic Medicine, Graduate School of Peking Union Medical College, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100005, China. E-mail: [email protected] Received 11 November 2012; revised 22 July 2013; accepted 23 August 2013; published online 30 September 2013 mTORC2 upregulation of aB-Crystallin in TSC F Wang et al 4353 of mTORC2 upregulation of aB-crystallin. Disruption of aB-crys- we hypothesized that loss of TSC1/TSC2 complex promoted tallin suppressed the cell proliferation and tumorigenesis caused aberrant cell proliferation through induction of aB-crystallin expres- by TSC1/2 deficiency. sion. We found that both aB-crystallin mRNA and protein were markedly elevated in mouse embryonic fibroblasts (MEFs) with mTOR hyperactivation caused by lack of either Tsc1 or Tsc2 gene RESULTS AND DISCUSSION (Figures 1a and b), indicating that the upregulation of aB-crystallin mTORC2 positively regulates aB-crystallin expression in TSC expression occurs at the transcriptional level. Mouse kidney tumors TSC is featured with tumor lesions in multiple organs. aB-crystallin with hyperactive mTOR due to loss of heterozygosity for the has been found to be highly expressed in TSC lesions. Therefore, wild-type Tsc2 allele in Tsc2 þ / À mice17 exhibited enhanced

Figure 1. mTORC2 positively regulates aB-crystallin expression in TSC. Total RNA and protein lysates were extracted from WT, Tsc1 À / À (a)or Tsc2 À / À (b) MEFs for qRT-PCR (left) and immunoblotting (right), respectively. Data are presented as mean±s.e.m. from three independent experiments. *Po0.05 compared with control (t-test). (c) Kidney tumors from three Tsc2 þ /– mice were immunoblotted.17 (d)WT,Tsc2 À / À MEFs (left) and ELT3 cells (right) were treated with 10 nM rapamycin (R, Sigma-Aldrich, St Louis, MO, USA) or 3 mM WYE-354 (W, Merck) for 24 h and then subjected to immunoblotting. The bottom bar graph represents aB-crystallin levels upon normalization for b-actin as fold change over control. Tsc2 À / À MEFs (left) and ELT3 cells (right) were transfected with siRNA of mTOR (e), rictor (f), raptor (g) or scramble siRNA using Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturer’s instructions and then subjected to immunoblotting. aB-crystallin levels are represented as in panel d. CAKRRRLpSpSLRA peptides were injected into rabbit to produce polyclonal antibody against phospho-S6 (Ser235/236). TSC2, b-actin and ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). aB-crystallin antibody was purchased from Stressgen-Enzo Life Sciences (Farmingdale, NY, USA). TSC1 antibody was purchased from Abcam (Cambridge, MA, USA). mTOR, raptor, rictor and phospho-AKT (Ser473) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). (d–g) All experiments were repeated at least two times with similar results.

& 2014 Macmillan Publishers Limited Oncogene (2014) 4352 – 4358 mTORC2 upregulation of aB-Crystallin in TSC F Wang et al 4354 aB-crystallin expression (Figure 1c). By analyzing transcript profiling Table 1. Human LAM with hyperactive mTOR/aB-crystallin signaling of human LAM nodules compared with normal pulmonary artery smooth muscle cells from the Gene Expression Omnibus database Gene Fold changea tb P-valuec (GEO accession number GSE12027, deposited by Y. Zhang and VEGFD 30.2 À 6.28 0.001 G. Pacheco-Rodriguez) (Supplementary Table 1), we found VEGFD o 30 aB-crystallin 6.7 À 6.71 o0.001 (a marker of mTOR activation in LAM) and aB-crystallin were significantly increased in LAM nodules (Table 1). Taken together, Transcript profiling of 14 human LAM nodules and 7 pulmonary artery these results demonstrate that TSC1/TSC2 complex negatively smooth muscle cell lines is from the Gene Expression Omnibus database. aFold change is presented as LAM versus pulmonary artery smooth muscle regulates aB-crystallin expression both in vitro and in vivo. b c mTOR exists in two multiprotein complexes, rapamycin- cells. t-statistic. Po0.01 and a 1.5-fold change were considered 10 significant. sensitive mTORC1 and rapamycin-insensitive mTORC2. Because loss of TSC1 or TSC2 induces both mTOR hyperactivation and

Figure 2. mTORC2 stimulates aB-crystallin expression through induction of NFkB. (a) Protein lysates were extracted from WT or Tsc2 À / À MEFs for immunoblotting. Tsc2 À / À MEFs (left) and ELT3 cells (right) were transfected with siRNA of rictor (b), p65 (c) or scramble siRNA in Lipofectamine 2000 and then subjected to immunoblotting. p65 and phospho-p65 (ser536) antibodies were purchased from Cell Signaling Technology. (a-c) All experiments were repeated at least two times with similar results. (d) Schematic representation of the putative wt and mutated NFkB-binding sites within the promoter region of mouse aB-crystallin gene. (e) HEK293T cells were transfected with paB-crystallin- Luc reporter plasmid encoding firefly luciferase and p65 expression vector (pDsRed-p65) or control vector (pDsRed-Express-N1, Clontech, Palo Alto, CA, USA) using Lipofectamine 2000. pRL-TK plasmid (Promega, Madison, WI, USA) encoding Renilla luciferase was cotransfected as internal control. Relative luciferase activity was evaluated 24 h after transfection, by a Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity: firefly/Renilla luciferase activity. (f) Tsc2 À / À MEFs were cotransfected with paB-crystallin-Luc or pmutNFkB-Luc reporter plasmid and pRL-TK plasmid in Lipofectamine 2000. Relative luciferase activity was evaluated 48 h after transfection. Data are presented as mean±s.d. from three independent experiments. **Po0.01; ***Po0.001 compared with control (t-test).

Oncogene (2014) 4352 – 4358 & 2014 Macmillan Publishers Limited mTORC2 upregulation of aB-Crystallin in TSC F Wang et al 4355 aB-crystallin overexpression, we speculated that mTOR was a all of these cells (Figure 1d). Then we cloned a 2193-bp fragment positive regulator of aB-crystallin expression. To elucidate which of mouse aB-crystallin gene to luciferase reporter plasmid mTOR complex is responsible for the upregulation of aB-crystallin, (Figure 2d) and found that the augmented aB-crystallin promoter we first evaluated the effect of rapamycin on aB-crystallin activity in Tsc2 À / À MEFs was significantly suppressed with expression. Rapamycin treatment led to marked reduction in S6 WYE-354 treatment, whereas rapamycin treatment did not alter phosphorylation (a marker of mTORC1 activity), but no reduction aB-crystallin promoter activity (Supplementary Figures 1a and b). in aB-crystallin expression in WT MEFs, Tsc2 À / À MEFs or rat Furthermore, we knocked down mTOR, rictor or raptor, respec- uterine leiomyoma-derived Tsc2-null ELT3 cells (Figure 1d).31 We tively, in Tsc2 À / À MEFs and ELT3 cells. Reduction in mTOR or also treated these cells with WYE-354, an ATP-competitive mTOR rictor significantly decreased aB-crystallin (Figures 1e and f), kinase inhibitor that could suppress both mTORC1 and mTORC2 whereas reduced raptor did not alter the level of aB-crystallin activities.32 WYE-354 markedly reduced aB-crystallin expression in (Figure 1g). Consistently, knockdown of rictor, but not raptor,

Figure 3. Augmented aB-crystallin is critical for migration, invasion and resistance to serum deprivation-induced apoptosis of Tsc2-defective cells. (a) Tsc2 À / À MEFs (left) and ELT3 cells (right) stably expressing the shRNA for aB-crystallin or scramble shRNA were serum-starved for 24 h and then subjected to immunoblotting. Cleaved caspase-3 (Asp175) antibody was purchased from Cell Signaling Technology. WT and Tsc2 À / À MEFs (b), Tsc2 À / À MEFs (c) or ELT3 cells (d) stably expressing the shRNA for aB-crystallin or scramble shRNA (shV) suspended in serum-free DMEM were added to the upper wells of transwell chamber (8.0 mM pore size, Costar, Tewksbury, MA, USA) with or without ECM gel (Sigma-Aldrich). DMEM with 20% FBS was used as chemoattractant in the lower well. Cells were incubated at 37 1C and 5% CO2 for 12–20 h. The cells invading or migrating through the membrane were stained with crystal violet. Representative images (left) were presented. Scale bars, 50 mM. Number of migrating or invading cells was counted in at least five fields (right). Data are presented as mean±s.e.m. **Po0.01; ***Po0.001 compared with control (t-test). (a-d) All experiments were repeated at least two times with similar results.

& 2014 Macmillan Publishers Limited Oncogene (2014) 4352 – 4358 mTORC2 upregulation of aB-Crystallin in TSC F Wang et al 4356 inhibited aB-crystallin promoter activity (Supplementary Figures 1c aB-crystallin gene expression. As aB-crystallin can suppress the and d). Taken together, mTORC2 activates aB-crystallin expression NFkB pathway in astrocytes following TNF stimulation,40 the in TSC1/2-deficient cells. It has been reported that mTORC2 has an negative feedback regulation of NFkBonaB-crystallin may essential role in TSC2-null cell growth and survival.33 As partially contribute to the benign nature of TSC-associated tumor. aB-crystallin is a downstream target of mTORC2, aB-crystallin may be targeted for the treatment of TSC and other hyperactive mTOR-mediated cancers. Essential role of aB-crystallin for Tsc2-null cells in resistant to serum deprivation-induced apoptosis It has been reported that TSC1/2 deficiency exerts resistance to mTORC2 stimulates aB-crystallin expression through induction of serum deprivation-induced apoptosis.41 As both aB-crystallin and NFkB loss of TSC1/2 inhibit apoptosis, we postulated that loss of TSC1/2 NFkB is a key transcriptional factor that regulates expression of a abrogated serum withdrawal-induced apoptosis at least partially large number of genes involved in apoptosis, proliferation, through induction of aB-crystallin. In comparison with Tsc2 / 34 À À tumorigenesis and metastasis. The cross talk between PI3K- MEFs, WT MEFs were more susceptible to serum deprivation AKT-mTOR and NFkB signaling cascade has been extensively (Supplementary Figure 3a), independent of p53 as both cells are 35,36 studied. NFkB activity may be potentiated by both mTORC1 deficient of p53.17 We knocked down aB-crystallin expression in 37–39 and mTORC2. NFkB signaling was enhanced in Tsc2 À / À Tsc2 À / À MEFs and ELT3 cells (p53 þ / þ ) and evaluated the MEF cells (Figure 2a). The augmented signaling was suppressed apoptosis induced by serum withdrawal in these cells. Although the with inhibition of either mTORC1 by raptor knockdown or basal level of apoptotic cells was higher in non-specific siRNA- rapamycin treatment or mTORC2 by rictor knockdown in both transfected cells than in non-transfected cells (Supplementary Tsc2 À / À MEFs and ELT3 cells (Supplementary Figure 2 and Figures 3b versus 3a), reduction in aB-crystallin expression Figure 2b). Therefore, similar to mTOR and mLST8, NFkB signaling sensitized Tsc2 À / À MEFs or ELT3 cells to apoptosis induced by is a component of both mTORC1 signaling cascade and mTORC2 serum withdrawal (Figure 3a and Supplementary Figure 3b). There- signaling network in the cells. fore, aB-crystallin contributes to resistance to serum deprivation- As aB-crystallin was upregulated at transcriptional level in induced apoptosis in Tsc2-deficient cells with or without p53. mTOR-activated cells, we tested the potential role of NFkBin As rapamycin or raptor knockdown failed to counteract the regulation of aB-crystallin expression. Knockdown of NFkB apoptotic resistance of Tsc2-null cells to serum limitation,41 this markedly reduced aB-crystallin expression (Figure 2c). To inves- newly dissected TSC1/2 regulation of apoptosis through mTORC2- tigate whether NFkB transactivates aB-crystallin expression, we NFkB-aB-crystallin signaling cascade may attribute to this phenotype identified two putative NFkB-binding sites within the promoter of Tsc2-null cells. Moreover, we identify aB-crystallin as a therapeutic region on mouse aB-crystallin gene (Figure 2d). Luciferase reporter target for induction of cell death in the treatment of TSC. assay in HEK293T cells showed that overexpression of p65 markedly enhanced the transcriptional activity of the aB-crystallin promoter (Figure 2e). Moreover, aB-crystallin promoter activity in aB-crystallin is essential for the migration and invasion of TSC1/2 Tsc2 À / À MEFs was markedly attenuated when either one of its complex-deficient cells putative NFkB-binding sites was mutated (Figure 2f). Collectively, TSC1 and TSC2 can inhibit cell migration and invasion in vitro.42,43 these results demonstrate that mTORC2-NFkB axis transactivates In our study, Tsc2 À / À MEFs showed an B30-fold increase in

Figure 4. Inhibition of aB-crystallin suppresses cell proliferation and tumorigenesis mediated by TSC2 deficiency. (a) NTC/T2-null cells are stably transduced with shRNA silencing aB-crystallin or scramble shRNA (shV) lentiviruses and then subjected to immunoblotting. (b) The proliferation of NTC/T2-null cells stably expressing shV or shaB-crystallin was examined using an MTT assay for up to 3 days. Data are presented as mean±s.d. from one experiment performed in four replicates. **Po0.01; ***Po0.001 compared with control (t-test). (c) Colony formation of NTC/T2-null cells stably expressing shV or shaB-crystallin.(a-c) All experiments were repeated at least two times with similar results. (d)NTC/ T2-null cells stably expressing shV or shaB-crystallin were injected s.c. into nude mice for analysis of tumor development (left) and survival of the tumor-bearing mice (right). Subcutaneous tumors were established as previously described.49 Immunodeficient nude mice (strain BALB/c, 6 weeks old) were obtained from the Vital River Laboratory Animal Technology (Beijing, China). Eight mice (four male and four female) were used in each cohort. All animals were maintained and used in accordance with the guidelines of the Animal Center of the Institute of Basic Medical Sciences, Peking Union Medical College & Chinese Academy of Medical Sciences, and was compliant with the regulation of Beijing Administration Office of Laboratory Animal on the care of experimental animals. (e) Tumors were collected from euthanized mice, and the formalin-fixed and paraffin-embedded tumor tissue sections were subjected to immunohistochemical staining. Representative images were presented. Scale bar, 50 mM.

Oncogene (2014) 4352 – 4358 & 2014 Macmillan Publishers Limited mTORC2 upregulation of aB-Crystallin in TSC F Wang et al 4357 transwell migration and an B5-fold increase at invading the ECM gel 3 Ma J, Sun Q, Mi R, Zhang H. Avian influenza A virus H5N1 causes autophagy- compared with WT MEFs (Figure 3b). As both aB-crystallin over- mediated cell death through suppression of mTOR signaling. J Genet Genomics expression and loss of TSC1/2 promote cell migration and invasion, 2011; 38: 533–537. we assumed that loss of TSC1/2 promoted cell migration and 4 Sun Q, Chen X, Ma J, Peng H, Wang F, Zha X et al. Mammalian target of rapamycin invasion at least in part through enhancement of aB-crystallin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic expression. To test this possibility, aB-crystallin was knocked down in glycolysis and tumor growth. Proc Natl Acad Sci USA 2011; 108: 4129–4134. 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