MOLECULAR MEDICINE CONJ 514
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Date Created: 09/09/15
CANCER RESEARCH 62 564575650 October 15 2002 Advances in Brief Activated Mammalian Target of Rapamycin Pathway in the Pathogenesis of Tuberous Sclerosis Complex Renal Tumors1 Heidi L Kenerson Lauri D Aicher Lawrence D True and Raymond S Yeung2 Departments ofSurgery H L K L D A R s Y and Pathology 11 D 7 Umversrty of Washington Seattle Washington 98195 Ab stract Disruption of the TSC or TSC2 gene leads to the development of tumors in multiple organs most commonly affecting the kidney brain lung and heart Recent genetic and biochemical studies have identified a role for the tuberous sclerosis gene products in phosphoinositide 37kinase signaling On growth factor stimulation tuberin the TSC2 protein is phosphorylated by Akt thereby releasing its inhibitory effects on p7 0S6K Here we demonstrate that primary tumors from tuberous sclerosis come plex TSC patients and the Eker rat model of TSC expressed elevated levels of phosphorylated mammalian target of rapamycin mTOR and its effectors p70S6K S6 ribosomal protein 4EBP1 and eIF4G In the Eker rat shorteterm inhibition of mTOR by rapamycin was associated with a significant tumor response including induction of apoptosis and reduction in cell proliferation Surprisingly these changes were not accompanied by significant alteration in cyclin D1 and p27 levels Our data provide in viva evidence that the mTOR pathway is aberrantly activated in TSC renal patho ogy and that treatment with rapamycin appears effective in the preclinical setting Introduction TSC3 is an autosomal dominant syndrome associated with the multiorgan development of benign and occasional rrl ignant tumors most commonly affecting the central nervous system kidney and skin 1 Lesions such as cortical tubers subependymal giant cell astrocy toma cardiac rhabdomyomas and renal AML o en exhibit abnormal patterns of differentiation along with deregulated cell growth and proliferation The biochemical bases of these pathological alterations are not well understood but genetic studies in Drosophila indicate that the two genes implicated in tuberous sclerosis T SCI and T SC2 participate in the control of cell size via the insulinp7086K pathway reviewed in Ref 2 Epistasis experiments demonstrate that decl and decZ act upstream of dS K and downstream of dAkt Recent biochemical analyses con rmed that tuberin the T SC2 gene product is a substrate of Akt and can modulate P13Kdependent activation of p7086K 3 4 Phosphorylation of tuberin by Akt reduces the stability of tuberin and thereby releases its inhibitory function on p7086K signaling It has also been shown that diseasecausing T SC2 mutations can produce a reduced state of tuberin phosphorylation that causes it to interact less stably with the T SCI product hamartin 5 Beyond this T SCI and T SC2 have been implicated in other molecular path ways including regulation of lowmolecular weight GTPases Rapl Received 8102 accepted 9302 The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U S C Section 1734 solely to indicate this fact This work was supported by NIH Grant CA77882 and the Tuberous Sclerosis Alliance 2To whom requests for reprints should be addressed at Department of Surgery University of Washington 1959 NE Paci c Street Box 356410 Seattle WA 98195 Phone 206 6166405 Fax 206 6166406 Email ryeungu washington edu 3 breviations used are TSC tuberous sclerosis complex AML angiomyoli poma PIgK phosphoinositide 3kinase mTOR mammalian target of rapamycin lnc 39 stry PCNA proliferating cell nuclear antigen RCC renal cell carci noma LOH oss of heterozygosity TUNEL terminal deoxynucleotidyl transferas TdT39mediated nick end labeling RabS Rho p27 stability and steroiddependent transcription re viewed in Ref 6 As a large molecular complex TSClTSC2 could potentially mediate multiple pathways related to cell growth prolif eration differentiation and migration all of wllich are relevant to TSC biology In this study we examined the relevance of the P13KTSC286K pathway in the pathogenesis of TSC renal manifestations p7086K is a key effector of the PI3K pathway whose activity is regulated by sequential phosphorylation by multiple upstream kinases 7 Phos phorylation of the critical residue T111229 in the activation loop of p70 86K is mediated by PDKl and is most ef cient after prephospho rylation at Thr389 by mTOR and at the COOHterminal autoinllibitory domain by various kinases 7 On activation p70 86K phosphorylates 6 ribosomal protein to regulate translation of 539TOP mRNA and ribosome biogenesis Binding of mRNA with the 408 ribosomal subunit is so under the control of the elF4F complex consisting of e1F4E elF4A and e1F4G Stimulation of protein synthesis by amino acids releases elF4E from its inllibitory partner 4EBP1 on phospho rylation by mTOR 8 The latter cooperates with the P13K pathway to coordinate cellular responses to growth factors nutrients and energy sources Conserved through evolution TOR has been shown to con trol cell size by regulating protein synthesis mediated through down stream targets p7086K and 4EBP1 The importance of the mTOR pathway in human pathology is re ected in the overexpression of p7086K in a subset of breast cancers and its correlation with a poor prognosis 9 Moreover recent clinical studies have reported antiturnor response to rapamycin and its ester derivatives Rapamycin is a microbial product that binds the intracellular receptor F KBP12 to speci cally inllibit mTOR activity 10 We propose that the loss of tuberin function leads to activation of the mTOR pathway in TSCrelated renal tumors and that inhibition of mTOR signaling brings about reversal of the tumor phenotype In primary renal tumors derived from TSC patients and the Eker rat model of TSC multiple mTOR effectors and mTOR itself were found to be highly phosphorylated Treatment with rapamycin in the Eker rat elicited a signi cant biochemical and histological tumor response in keeping with the hypothesis that mTOR is a relevant target for therapeutic intervention in TSC patients Materials and Methods Antibodies and Chemicals The antibody for PHASI 4EBP1 was pur chased from Zymed San Francisco CA Kipl p27 was from Transduction Laboratories Los Angeles CA cyclin D1 was from Rockland Gilbertsville PA tuberin C20 was from Santa Cruz Biotechnology Santa Cruz CA actin was from Sigma St Louis MO and PCNA was from DAKO Carpinteria CA Antigelsolin antibody was a gi of David Kwiatkowski Brigham and Women s Hospital Boston MA All other antibodies were purchased from Cell Signaling Beverly MA Rapamycin was purchased from Calbiochem La Jolla CA An InSitu Cell Death Detection Kit peroxidase with a 33 diarninobenzidine substrate was obtained from Roche Diagnostics Indi anapolis 1N Secondary antibodies and electrocherniluminescence reagents were purchased from Arnersharn Pharrnacia Biotech Piscataway NJ The 5645 RAPAMYCIN IN39HIBITS TSC TUMORS IN VIVO Elite ABC kits 3339diaminobenzidine and Hematoxylin QS were purchased Vector Laboratories Burlingarne CA Eosin was obtained from Richard Allen Scientific Kalamazoo MI Ani Eker rat strain harboring a germline TSC2 mutation was as described previously 11 Fischer male carriers were identified by genotyping and housed and fed ad libitum until the age of 12 months at which point raparnycin was injected ip once daily for 3 consecutive days The control animal was given the same volume and concentration of DMSOvehicle and the treated rats were given raparnycin at three dose levels 016 04 and 1 mgkg Animals were sacrificed 24 h after the last injection and tissues were procured for IHC and Western blot analysis All work related to animals was in accordance with the protocol approved by the Animal Care Committee University of Washington Seattle IHC Kidney samples were fixed in formalin and paraffin embedded Fivepm sections were deparaffinized rehydrated and washed with PBS A er antigen retrieval in 10 mM sodium citrate pH 60 and quenching of endogenous peroxidase activity with 1 H202 samples were blocked with 5 normal goat serum before incubation with primary antibodies overnight at 4 C Negative controlsWere treated with 5 normal goat serum without the primary antibodies Signals were processed according to the supplied protocol Elite ABC Kit Slides were counterstained with Hernatoxylin QS dehydrated and mounted using Permount Fischer Scientific Santa Clara CA For the cell proliferation index PCNA r tumor cells were counted from 10 random non overlapping highpower fields within the tumors and the results were ex pressed as a percentage ofthe total number of tumor cells counted in the same dds Western Blotting Tissues were homogenized in icecold radioimmuno precipitation assay buffer 1 NP40 1 sodium deoxycholate 01 SDS 15 M aCl 10 mM Tris pH 72 0025 M Bglycophosphate pH 72 2 mM EDTA and 50 mM sodium uoride with protease and kinase inhibitors 005 mM 42aminoethylbenzenesulfonyl uoride 10 pugml aprotinin 10 pugml pepstatin 1 mM orthovanadate 10 pugml leupeptin 1 mM microc stin LR The protein concentration was measured using the BCA Protein Assay Pierce Rockford IL Equal amounts of protein were separated by SDSPAGE transferred to ImmobilonP membranes Millipore Bedford MA and blotted with antibodies according to the manufacturer s recommendations as de scribed previously 5 Results Expression of Phospho p7OSGK in Human TSC Renal Pathol ogy Patients with TSC are predisposed to the development of two forms of renal tumors RCC and AML 1 To determine the status of the p7OS K pathway in these tumors IHC was performed using antibodies that speci cally recognize phosphorylated p70 86K at Thr389 a modi cation that is necessary for activation of the protein and phosphorylated mTOR at Serum a site that has been associated with activity 7 12 Three cases of RCC derived from TSC patients were examined all demonstrating elevated levels of phosphomTOR and phosphop7OS K expression in the tumor cells compared with adjacent uninvolved kidney tissue Fig 1A The staining pattern of p7OS K was cytoplasmic whereas the mTOR signal could be seen in the cytoplasm and nucleus consistent with previous local ization studies l3 14 We did not nd elevated levels of phos phop7OS K in a few sporadic RCCs examined data not shown sporadic clearcell RCCs are knownto involve pathways other than TSCl and TSC2 15 The second and more common form of renal pathology in TSC patients is AML These tumors contain variable proportions of three histological components adipocytes smooth muscle cells and vas cular structures In ve of six AMLs from TSC patients we found robust expression of phosphop70 86K compared with adjacent tissues Fig 13 Speci cally staining was uniformly identi ed in the smooth muscle and lipomatous components whereas the vascular structures showed a heterogeneous pattern Many endothelial lined structures were surrounded by phosphop7OS Kpositive smooth musclelike cells that were contiguous with the myolipomatous com ponents of the tumor Fig 13 left However vessels with discrete walls were found to stain minimally for phosphop7OS K Fig 13 right The latter structures may represent preexis 39ng or reactive vessels A spora 39c AML was also found to aberrantly express phos phop70 86K data not shown This is consistent with the identi ca tion of LOH at the TSC2 locus in sporadic AMLs and suggests a common pathogenic mechanism of deregulated p7OS K activity in both the sporadic and familial forms of AML 16 Within the normal kidney discrete expression of phosphop7OS K was detected in the distal tubules and some collecting ducts the proximal tubules and glomeruli lacked immunoreactivity Fig 1C Approximately 5 of the cells in the normal kidney expressed de tectable levels of phosphop7OS K Because the latter has been im plicated in cell size control by regulating 539TOP translation it was unexpected to nd that phosphop70 S K positive distal tubular cells were consistently smaller than those in adjacent proximal tubules Collectively IHC analyses of human TSC renal pathology showed overexpression of activated mTOR and p7OS K To further investi gate the functional role of this pathway in turnorigenesis we exam ined spontaneous renal tumors derived from the Eker rat strain a wellcharacterized animal model of TSC 11 Expression of mTOR Effectors in Primary RCC of the Eker Rat The Eker rat carries a germline mutation of the T SC2 gene as a result of a retrotransposition of an endogenous IAP element As such the Eker T SC2 allele behaves as a null mutation Spontaneous renal cortical epithelial tumors in these animals have been shown to possess biallelic inactivation of T SC2 through LOH or nonsense or missense mutations reviewed in 17 In addition to p7OS K mTOR also regulates 4EBP1 in controlling capdependent translation 8 Using antibodies for 4EBP1 and phosphoS a substrate for p7OS K we analyzed the expression of these proteins in a panel of ve primary tumor lysates by Western blotting Compared with normal kidney all tumors showed marked phosphorylation of ribosomal protein 86 and 4EBP1 Fig 2A These tumors also have elevated levels of cyclin D1 accompanied by minimal expression of p27 and phosphoAkt Of the ve tumors three tumors 2 4 and 5 had lost expression of tuberin and the remaining two were expected to possess aberrant forms of the protein as a result of missense muta 39ons suggest that effectors downstream of mTOR were activated in the Eker renal lesions consistent with the IHC ndings in human TSC tumors To examine the distribution of these proteins in the Eker rat kidney immunostaining was performed using phosphospeci c antibodies in paraf nembedded tissues Intense uniform staining for phosphoS was found in tumors of all sizes compared with adjacent renal paren Fig 23 Lesions in their earliest form with only a few cells were also decorated with phosphoS immunoreactivity Fig targets of mTOR including p7OS K 4EBP1 and eIF4G were also expressed in their phosphorylated forms although their patterns showed greater degree of intratumoral heterogeneity Fig 2 C715 Conversely pathways such as that of mitogenactivated protein kinase did not appear affected as shown by the absence of phosphoErk expression in the tumors based on IHC Fig 2G and Western blotting data not shown The staining pattern of phosphoS in the nonturnor portion of the Eker rat kidney was speci c for cells within the distal tubules similar to that found in wildtype kidneys in rats data not w r own Inhibition of the mTOR Pathway in RCC Recent studies have shown that elevated levels of phosphop7OS K and phosphoS in cells lacking TSCl or TSC2 can be inhibited by raparnycin in vitro 18 19 Similarly we have observed that rat embryo broblasts in 5646 RAPAMYCIN iNHiEiTS TSC TUMORS m VIVD A Ekel39 Rat Kidney Tissue Normal Tumor Rapnmyun mgkg 0 016 04 D 015 04 phasphms Suns236 TnLIlSG Rihnrnmnl Pmlein AEBl39l Cyclin D1 B Rapamycin mgkg Total 6 Ribosomal Protein phosphoS Ser 235236 TUNEL Fig 3 Firmsquot l him r A rapamycln uf rap zmy n stained fur tutal so phusphurs Samm HM and apuptusrs using the TUNEL lot N nurmal kidney using rapamycin to prevent rejection in renal transplant patients The alone A recent study showed that p7056K signals cell survive by selective antitumoral effects on the Eker renal tumors seen a er a inactivating BAD through phosphorylation of Ser 5 21 Hence short exposure to rapamycin suggest mechanisms in addition o g acute downregulation of p7056K in tumor cells is expected to pro 39 39 39 svnth 39 39 39 39 39 e ap amycin has been shown to be a eltl mot optosis Furthermore rap in the tumors was unexpected on the basis of inhibition oftranslation potent cell cycle inhibitor by downregulating cyclin D 22 Given 49 RAPAMYCIN IN39HIBITS Tsc TUMORS IN VIVO Table 1 POMl Staining m Eer renal 174mm N0 of cells counted Rapamycin Positive Negative positivequot Control 258 238 53 0 15 mgkg 97 307 24 0 4 mgkg 88 581 13 1 0 mgkg 59 8 a X1 53 P lt 0 0001 There is a highly signi cant trend between the proportion of PCNA cells and rapamycin dose that the halflife of the cyclin D family of proteins is short 2 h the relatively stable levels of cyclin D and p27 in the treated renal tumors 0 no support 39s mechanism as the cause of reduced cell prolifer ation in this in vivo model Alternatively rapamycin may induce tumor response through an antiangiogenesis pathway Both hypoxia induced vascular proliferation and insulindependent stimulation of HIFl have been shown to be dependent on mTOR 23 24 In addition in muline models rapamycin inhibited vascular endothelial growth factor response angiogenesis andturnor growth 25 Collec tively our ndings implicate the mTOR pathway as a biologically relevant target in TSCrelated tumors Shortterm pharmacological manipulation of mTOR activity can bring about signi cant antitu moral effects by promoting cell death and reducing cell proliferation by a cyclin Dindependent pathway As a sensor of nutrients growth factors and ATP mTOR serves a critical role in regulating the translational macllinery and in doing so affects cellular responses to growth proliferation and differentiation all of which are abnormally manifested in TSC lesions To date little is known about the upstream regulators of mTOR Protein kinase B also known as Akt has been shown to phosphorylate mTOR Se12448 in vitro but its biological relevance remains unclear because disrup tion of this site does not affect mTOR signaling and deletion of this region enhances its kinase activity 26 However insulininduced phosphorylation of 4EBP1 was shown to be Aktmediated and de pendent on mTOR activity 27 Furthermore a functional link be tween Ser24 8 39 and muscle 1 A A was noted in vivo 12 In this study sustained phosphorylation of mTOR Serum and its downstream targets in TSC pathology support a role of TSC2 in regulating mTOR downstream of Akt Consistent with this model expression of tuberin was shown in a recent study to suppress mTOR activation of p7OS K and to modulate the level of Ser244g mTOR expression 28 However current data do not distinguish between a model where tuberin acts upstream of mTOR versus one that converges on a common downstream target In summary activated mTOR signaling in TSC renal pathology provides evidence that this pathway among others is relevant to their pathogenesis At least some of the classic TSC cellular phenotype e g abnormal cell size can now be explained y this mec 39sm Encouragingly induction of an in viva response to shortterm rapa mycin treatment in spontaneous renal tumors of the rat model lend support to its use in the clinical setting Acknowledgments We thank Jean Campbell and members ofDr Yeung s laboratory for critical reading and suggestions We are grate ll to David Ewalt Lisa an Laura Firm for providing TSCrelated pathology This Work is dedicated to Dr a Alfred Knudson Jr on his 80th blrthd 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and Kaelin w G Jr The von HippelLindau tumor suppressor gene xp Cell Res 254 1177125 2001 15 SmolarekT AWessnerL LMcConnackF xM letJ CMenonA Gand H that lymphangiomyomatosis is caused by TSC2 mutations chromosome 15p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis Am J Hum Genet52 81078111998 17 Kajino K and Hino o TSC1 and TSC2 gene mutations in human kidney tumors Contrib Nephrol 128 45750 1999 18 Kwiatkowski D I zhang H Bandura J L Heiberger K M Glogauer M elHashemite N and 0nda H A mouse model of TSC1 reveals sexdependent lethality from liver hemangiomas and upregulation ofp70S5 kinase activity in Tsc1 null cells Hum M Genet 11 5255342002 19 GoncharovaE AGoncharovD AEszterhasAHunterD SGlassbergM K Yeung R S Walker C L Noonan D Kwiatkowski D ou M 39 AJrand1ltrymskayav P Tuberinregulatesp70 S5 kinase activation andribosomal protein S5 phosphorylation a role for the TSC2 tumor suppressor gene 39 monary ymphangioleiomyomatosis LAM J Biol 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