[CANCER RESEARCH 62, 5645–5650, 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 of Surgery [H. L. K., L. D. A., R. S. Y.] and Pathology [L. D. T.], University of Washington, Seattle, Washington 98195
`
`Abstract
`
`Disruption of the TSC1 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 3-kinase
`signaling. On growth factor stimulation, tuberin, the TSC2 protein, is
`phosphorylated by Akt, thereby releasing its inhibitory effects on p70S6K.
`Here we demonstrate that primary tumors from tuberous sclerosis com-
`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, 4E-BP1, and eIF4G. In the Eker
`rat, short-term 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 vivo
`evidence that the mTOR pathway is aberrantly activated in TSC renal
`pathology 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 malignant 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 often 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, TSC1 and TSC2,
`participate in the control of cell size via the insulin/p70S6K pathway
`(reviewed in Ref. 2). Epistasis experiments demonstrate that dTsc1
`and dTsc2 act upstream of dS6K and downstream of dAkt. Recent
`biochemical analyses confirmed that tuberin, the TSC2 gene product,
`is a substrate of Akt and can modulate PI3K-dependent activation of
`p70S6K (3, 4). Phosphorylation of tuberin by Akt reduces the stability
`of tuberin and thereby releases its inhibitory function on p70S6K
`signaling. It has also been shown that disease-causing TSC2 mutations
`can produce a reduced state of tuberin phosphorylation that causes it
`to interact less stably with the TSC1 product, hamartin (5). Beyond
`this, TSC1 and TSC2 have been implicated in other molecular path-
`ways, including regulation of low-molecular weight GTPases (Rap1,
`
`Rab5, Rho), p27 stability, and steroid-dependent transcription (re-
`viewed in Ref. 6). As a large molecular complex, TSC1-TSC2 could
`potentially mediate multiple pathways related to cell growth, prolif-
`eration, differentiation, and migration, all of which are relevant to
`TSC biology.
`In this study, we examined the relevance of the PI3K/TSC2/S6K
`pathway in the pathogenesis of TSC renal manifestations. p70S6K 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, Thr229, in the activation loop of
`p70S6K is mediated by PDK1 and is most efficient after prephospho-
`rylation at Thr389 by mTOR and at the COOH-terminal autoinhibitory
`domain by various kinases (7). On activation, p70S6K phosphorylates
`S6 ribosomal protein to regulate translation of 5⬘-TOP mRNA and
`ribosome biogenesis. Binding of mRNA with the 40S ribosomal
`subunit is also under the control of the eIF4F complex, consisting of
`eIF4E, eIF4A, and eIF4G. Stimulation of protein synthesis by amino
`acids releases eIF4E from its inhibitory partner, 4E-BP1, on phospho-
`rylation by mTOR (8). The latter cooperates with the PI3K 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, p70S6K and 4E-BP1.
`The importance of the mTOR pathway in human pathology is
`reflected in the overexpression of p70S6K in a subset of breast
`cancers and its correlation with a poor prognosis (9). Moreover, recent
`clinical studies have reported antitumor response to rapamycin and its
`ester derivatives. Rapamycin is a microbial product that binds the
`intracellular receptor FKBP12 to specifically inhibit mTOR activity
`(10). We propose that the loss of tuberin function leads to activation
`of the mTOR pathway in TSC-related 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 significant 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
`
`Received 8/1/02; accepted 9/3/02.
`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.
`1 This work was supported by NIH Grant CA77882 and the Tuberous Sclerosis
`Alliance.
`2 To whom requests for reprints should be addressed, at Department of Surgery,
`University of Washington, 1959 NE Pacific Street, Box 356410, Seattle, WA 98195.
`Phone: (206) 616-6405; Fax: (206) 616-6406; E-mail: ryeung@u.washington.edu.
`3 The abbreviations used are: TSC, tuberous sclerosis complex; AML, angiomyoli-
`poma; PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; IHC,
`immunohistochemistry; PCNA, proliferating cell nuclear antigen; RCC, renal cell carci-
`noma; LOH,
`loss of heterozygosity; TUNEL,
`terminal deoxynucleotidyl
`transferase
`(TdT)-mediated nick end labeling.
`
`Antibodies and Chemicals. The antibody for PHAS-I (4E-BP1) was pur-
`chased from Zymed (San Francisco, CA), Kip1 (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 gift 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 In-Situ Cell Death Detection Kit (peroxidase) with a
`3,3⬘-diaminobenzidine substrate was obtained from Roche Diagnostics (Indi-
`anapolis, IN). Secondary antibodies and electrochemiluminescence reagents
`were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The
`5645
`
`
`
`Downloaded from on June 4, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`Breckenridge Exhibit 1108
`Kenerson - Page 001
`
`

`

`RAPAMYCIN INHIBITS TSC TUMORS IN VIVO
`
`Elite ABC kits, 3,3⬘-diaminobenzidine, and Hematoxylin QS were purchased
`from Vector Laboratories (Burlingame, CA). Eosin was obtained from Richard
`Allen Scientific (Kalamazoo, MI).
`Animals. The Eker rat strain harboring a germ-line 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
`rapamycin was injected i.p. once daily for 3 consecutive days. The control
`animal was given the same volume and concentration of DMSO-vehicle, and
`the treated rats were given rapamycin at three dose levels (0.16, 0.4, and 1
`mg/kg). 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.
`Five-␮m sections were deparaffinized, rehydrated, and washed with PBS.
`After antigen retrieval in 10 mM sodium citrate (pH 6.0) and quenching of
`endogenous peroxidase activity with 1% H2O2, samples were blocked with 5%
`normal goat serum before incubation with primary antibodies overnight at 4°C.
`Negative controls were 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 Hematoxylin QS, dehydrated, and
`mounted using Permount (Fischer Scientific, Santa Clara, CA). For the cell
`proliferation index, PCNA⫹ tumor cells were counted from 10 random, non-
`overlapping high-power fields within the tumors, and the results were ex-
`pressed as a percentage of the total number of tumor cells counted in the same
`fields.
`Western Blotting. Tissues were homogenized in ice-cold radioimmuno-
`precipitation assay buffer [1% NP40, 1% sodium deoxycholate, 0.1% SDS,
`0.15 M NaCl, 10 mM Tris (pH 7.2), 0.025 M ␤-glycophosphate (pH 7.2), 2 mM
`EDTA, and 50 mM sodium fluoride] with protease and kinase inhibitors [0.05
`mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 ␮g/ml aprotinin, 10 ␮g/ml
`pepstatin, 1 mM orthovanadate, 10 ␮g/ml leupeptin, 1 mM microcystin LR].
`The protein concentration was measured using the BCA Protein Assay (Pierce,
`Rockford, IL). Equal amounts of protein were separated by SDS-PAGE,
`transferred to Immobilon-P membranes (Millipore, Bedford, MA), and blotted
`with antibodies according to the manufacturer’s recommendations, as de-
`scribed previously (5).
`
`ponents of the tumor (Fig. 1B, left). However, vessels with discrete
`walls were found to stain minimally for phospho-p70S6K (Fig. 1B,
`right). The latter structures may represent preexisting or reactive
`vessels. A sporadic AML was also found to aberrantly express phos-
`pho-p70S6K (data not shown). This is consistent with the identifica-
`tion of LOH at the TSC2 locus in sporadic AMLs and suggests a
`common pathogenic mechanism of deregulated p70S6K activity in
`both the sporadic and familial forms of AML (16).
`Within the normal kidney, discrete expression of phospho-p70S6K
`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 phospho-p70S6K. Because the latter has been im-
`plicated in cell size control by regulating 5⬘-TOP translation, it was
`unexpected to find that phospho-p70S6K-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 p70S6K. To further investi-
`gate the functional role of this pathway in tumorigenesis, we exam-
`ined spontaneous renal tumors derived from the Eker rat strain, a
`well-characterized 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 TSC2 gene as a
`result of a retrotransposition of an endogenous IAP element. As such,
`the Eker TSC2 allele behaves as a null mutation. Spontaneous renal
`cortical epithelial tumors in these animals have been shown to possess
`biallelic inactivation of TSC2 through LOH or nonsense or missense
`mutations (reviewed in 17). In addition to p70S6K, mTOR also
`regulates 4E-BP1 in controlling cap-dependent translation (8). Using
`antibodies for 4E-BP1 and phospho-S6, a substrate for p70S6K, we
`analyzed the expression of these proteins in a panel of five primary
`tumor lysates by Western blotting. Compared with normal kidney, all
`tumors showed marked phosphorylation of ribosomal protein S6 and
`4E-BP1 (Fig. 2A). These tumors also have elevated levels of cyclin
`D1 accompanied by minimal expression of p27 and phospho-Akt. Of
`the five tumors, three (tumors 2, 4, and 5) had lost expression of
`Expression of Phospho-p70S6K in Human TSC Renal Pathol-
`tuberin, and the remaining two were expected to possess aberrant
`ogy. Patients with TSC are predisposed to the development of two
`forms of the protein as a result of missense mutations. Our results
`suggest that effectors downstream of mTOR were activated in the
`forms of renal tumors: RCC and AML (1). To determine the status of
`Eker renal lesions, consistent with the IHC findings in human TSC
`the p70S6K pathway in these tumors, IHC was performed using
`tumors.
`antibodies that specifically recognize phosphorylated p70S6K at
`Thr389, a modification that is necessary for activation of the protein,
`To examine the distribution of these proteins in the Eker rat kidney,
`and phosphorylated mTOR at Ser2448, a site that has been associated
`immunostaining was performed using phospho-specific antibodies in
`paraffin-embedded tissues. Intense, uniform staining for phospho-S6
`with activity (7, 12). Three cases of RCC derived from TSC patients
`was found in tumors of all sizes compared with adjacent renal paren-
`were examined, all demonstrating elevated levels of phospho-mTOR
`chyma (Fig. 2B). Lesions in their earliest form with only a few cells
`and phospho-p70S6K expression in the tumor cells compared with
`were also decorated with phospho-S6 immunoreactivity (Fig. 2F).
`adjacent uninvolved kidney tissue (Fig. 1A). The staining pattern
`This suggests that the mTOR pathway is activated in the primary
`of p70S6K was cytoplasmic, whereas the mTOR signal could be
`stages of renal tumorigenesis in the Eker rat. Other downstream
`seen in the cytoplasm and nucleus, consistent with previous local-
`targets of mTOR, including p70S6K, 4E-BP1, and eIF4G, were also
`ization studies (13, 14). We did not find elevated levels of phos-
`expressed in their phosphorylated forms, although their patterns
`pho-p70S6K in a few sporadic RCCs examined (data not shown);
`showed greater degree of intratumoral heterogeneity (Fig. 2, C–E).
`sporadic clear-cell RCCs are known to involve pathways other than
`Conversely, pathways such as that of mitogen-activated protein kinase
`TSC1 and TSC2 (15).
`did not appear affected, as shown by the absence of phospho-Erk
`The second and more common form of renal pathology in TSC
`expression in the tumors based on IHC (Fig. 2G) and Western blotting
`patients is AML. These tumors contain variable proportions of three
`(data not shown). The staining pattern of phospho-S6 in the nontumor
`histological components: adipocytes, smooth muscle cells, and vas-
`portion of the Eker rat kidney was specific for cells within the distal
`cular structures. In five of six AMLs from TSC patients, we found
`tubules, similar to that found in wild-type kidneys in rats (data not
`robust expression of phospho-p70S6K compared with adjacent tissues
`shown).
`(Fig. 1B). Specifically, staining was uniformly identified in the
`Inhibition of the mTOR Pathway in RCC. Recent studies have
`smooth muscle and lipomatous components, whereas the vascular
`shown that elevated levels of phospho-p70S6K and phospho-S6 in
`structures showed a heterogeneous pattern. Many endothelial lined
`cells lacking TSC1 or TSC2 can be inhibited by rapamycin in vitro
`structures were surrounded by phospho-p70S6K-positive smooth
`(18, 19). Similarly, we have observed that rat embryo fibroblasts in
`muscle-like cells that were contiguous with the myolipomatous com-
`5646
`
`Results
`
`
`
`Downloaded from on June 4, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`Breckenridge Exhibit 1108
`Kenerson - Page 002
`
`

`

`RAPAMYCIN INHIBITS TSC TUMORS IN VIVO
`
`Fig. 1. Expression of mTOR and p70S6K in
`human TSC tumors and normal kidney. A, papillary
`RCC from a TSC patient stained with phospho-
`specific antibodies for p70S6K (Thr389) and mTOR
`(Ser2448). Note negatively stained stromal cells (S)
`and adjacent kidney tissue (N) compared with
`brown-stained tumor cells (magnification, ⫻400
`for left panel, ⫻200 for right panel). B, TSC-
`related AMLs stained with phospho-p70S6K anti-
`body. Left panel shows uniform staining in all three
`cellular components, including the central vessel
`(V; magnification, ⫻200). Right panel shows
`negative-staining vessels (arrows; magnification,
`⫻400). A, adipocyte; SM, smooth muscle. C, nor-
`mal human kidney stained with phospho-p70S6K
`antibody. Arrow indicates prominently stained dis-
`tal tubule (magnification, ⫻200). G, glomerulus.
`
`change in the overall expression of S6 in the renal tumors. To rule out
`the absence of tuberin have constitutive activation of mTOR effectors,
`nonspecific response to rapamycin, serial sections were stained with
`including 4E-BP1, eIF4G, S6K, and S6, as seen in the Eker rat renal
`gelsolin, an actin-binding protein that is expressed in intercalated cells
`tumors; all of these effectors can be down-regulated by rapamycin and
`of the distal tubules and has been shown previously to be a marker for
`LY294002 but not by Wortmannin or PD98059 (data not shown).
`TSC-related pathology (20). The level of gelsolin immunoreactivity
`To investigate the dependence of in vivo tumor growth on the
`did not change significantly after rapamycin treatment (data not
`mTOR pathway, we treated animals with rapamycin and monitored
`shown). In the normal kidney adjacent to the tumors, phospho-S6
`tumor responses. After three daily i.p. doses of rapamycin at three
`immunostaining of the distal tubules was also decreased with rapa-
`dose levels, none of the animals appeared ill or behaved abnormally.
`mycin administration.
`On the fourth day, the Eker rats were sacrificed and the kidney tumors
`To determine tumor response to rapamycin, we examined the
`were analyzed for phosphorylation of the mTOR effectors by use
`histology, cell proliferation, and apoptosis in the renal tumors. Vehicle
`of phospho-specific antibodies. At
`the highest
`rapamycin dose
`control-treated lesions were indistinguishable from those that were
`(1 mg/kg), we did not find tumors large enough for tissue homoge-
`untreated (data not shown). However, rapamycin-treated tumors
`nization. On Western blotting, the level of phospho-S6 in the tumors
`showed significantly more condensed, fragmented, and pyknotic nu-
`treated with vehicle only was highly elevated compared with the
`clei compared with vehicle-treated lesions (Fig. 3B, H&E). TUNEL
`adjacent kidney tissue (Fig. 3A). Treatment with rapamycin dramati-
`staining confirmed the greater extent of apoptosis in the treated
`cally reduced phospho-S6 expression in the tumors even at the lowest
`tumors compared with control (Fig. 3B). With increasing doses of
`dose (0.16 mg/kg). Phosphorylation of 4E-BP1 was partially sup-
`rapamycin, the degree of apoptosis decreased as tumor necrosis in-
`pressed by rapamycin as shown by the increasing intensity of the
`creased. Lymphoid infiltration did not change significantly with ra-
`faster mobility band with higher doses. Surprisingly, the levels of
`pamycin dose, but did correlate in a direct manner with tumor size.
`cyclin D1 and p27 expression, when corrected for protein loading, did
`The percentage of PCNA⫹ nuclei was used as an index of cell
`not change significantly with rapamycin treatment (Fig. 3A). These
`findings correlated well with IHC. Fig. 3B shows specific reduction of
`proliferation (Table 1). Rapamycin-treated tumors were associated
`with a smaller proportion of PCNA⫹ cells. There was a highly
`phospho-S6 reactivity with rapamycin treatment without significant
`5647
`
`
`
`Downloaded from on June 4, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`Breckenridge Exhibit 1108
`Kenerson - Page 003
`
`

`

`RAPAMYCIN INHIBITS TSC TUMORS IN VIVO
`
`Fig. 2. mTOR effectors in the Eker renal tumors. A, Western blot analysis of primary tumor lysates showing expression of the indicated proteins. Actin was used as loading control.
`B–G, immunohistochemical staining of renal tumors with phospho-specific antibodies: B, S6 (Ser235/236; magnification, ⫻100); C, p70S6K (Thr389; magnification, ⫻100); D, 4E-BP1
`(Thr70; magnification, ⫻100); E, eIF4G (Ser1108; magnification, ⫻100); F, S6 (Ser235/236) staining of an early lesion (magnification, ⫻400); G, Erk (Thr202/Tyr204), T, tumor tissue
`(magnification, ⫻200).
`
`significant inverse correlation between the rapamycin dose and the
`percentage of PCNA⫹ cells.
`Overall, rapamycin, even at the lowest dose administered, produced
`profound inhibition of p70S6K activity, which correlated with in-
`creased tumor cell death and necrosis. Importantly, rapamycin did not
`cause any significant histological alteration to the nontumor portions
`of the kidneys. If these observed effects are sustained, rapamycin is
`expected to be an effective therapy for the renal manifestations
`of TSC.
`
`phenotype. Clear-cell and papillary RCCs are known to involve path-
`ways independent of TSC1 or TSC2 (15). Interestingly, sporadic
`AMLs exhibit evidence of increased phospho-S6 expression consist-
`ent with earlier reports of TSC1/2 LOH in these lesions (16). Immuno-
`staining with the phospho-specific antibodies used in this study may
`aid in the classification of sporadic AMLs and RCCs with respect
`to their underlying pathogenesis. In the setting of TSC, abnormal
`“tumor” cells can be recognized by their expression of phosphorylated
`p70S6K, S6, or 4E-BP1. As potential surrogate markers of TSC
`pathology, further studies are needed to address their specificity.
`The kidney tumors in the Eker rat have an immunophenotype
`similar to the human lesions, thus further validating the use of the
`In this study, we showed that tumors associated with TSC gene
`Eker rat as a model of human TSC. Importantly, the fact that rapa-
`mutations are accompanied by activation of the mTOR pathway,
`mycin treatment can down-regulate mTOR effectors and induce tumor
`including p70S6K, 4E-BP1, and eIF4G. In humans, RCC is an infre-
`response in vivo points to the biological dependence of renal lesions
`quent component of TSC, whereas AML is a common manifestation.
`on the effects of the activated mTOR pathway. Of note, adjacent
`Both types of tumors were shown to express phosphorylated mTOR/
`normal kidneys showed minimal cellular toxicity from rapamycin
`p70S6K and their substrates. These alterations appear specific to TSC
`because sporadic RCCs do not share the same immunohistological
`treatment. This is in agreement with the vast clinical experience in
`5648
`
`Discussion
`
`
`
`Downloaded from on June 4, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`Breckenridge Exhibit 1108
`Kenerson - Page 004
`
`

`

`RAPAMYCIN INHIBITS TSC TUMORS IN VIVO
`
`Fig. 3. Effects of rapamycin on Eker renal tumors. A, Western blot analysis of primary tumor lysates showing biochemical responses to i.p. rapamycin. The tumor samples were
`compared with adjacent normal kidney tissue for each dose level. Actin served as loading control. B, representative sections of renal tumors from animals treated with various doses
`of rapamycin stained for total S6, phospho-S6 (Ser235/236), H&E, and apoptosis using the TUNEL kit. N, normal kidney.
`
`alone. A recent study showed that p70S6K signals cell survive by
`using rapamycin to prevent rejection in renal transplant patients. The
`inactivating BAD through phosphorylation of Ser136 (21). Hence,
`“selective” antitumoral effects on the Eker renal tumors seen after a
`short exposure to rapamycin suggest mechanisms in addition to reg-
`acute down-regulation of p70S6K in tumor cells is expected to pro-
`ulation of protein synthesis. The rapid induction of apoptosis/necrosis
`mote apoptosis. Furthermore, rapamycin has been shown to be a
`in the tumors was unexpected on the basis of inhibition of translation
`potent cell cycle inhibitor by down-regulating cyclin D (22). Given
`5649
`
`
`
`Downloaded from on June 4, 2017. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org
`
`
`
`Breckenridge Exhibit 1108
`Kenerson - Page 005
`
`

`

`RAPAMYCIN INHIBITS TSC TUMORS IN VIVO
`
`Table 1 PCNA staining in Eker renal tumors
`
`No. of cells counted
`
`Rapamycin
`
`Positive
`
`Negative
`
`% positivea
`
`53
`238
`268
`Control
`24
`307
`97
`0.16 mg/kg
`13
`581
`88
`0.4 mg/kg
`8
`683
`59
`1.0 mg/kg
`a ␹2 ⫽ 353; P ⬍ 0.0001. There is a highly significant trend between the proportion of
`PCNA⫹ cells and rapamycin dose.
`
`that the half-life 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
`do not support this 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 insulin-dependent stimulation of
`HIF-1 have been shown to be dependent on mTOR (23, 24). In
`addition, in murine models, rapamycin inhibited vascular endothelial
`growth factor response, angiogenesis, and tumor growth (25). Collec-
`tively, our findings implicate the mTOR pathway as a biologically
`relevant target in TSC-related tumors. Short-term pharmacological
`manipulation of mTOR activity can bring about significant antitu-
`moral effects by promoting cell death and reducing cell proliferation
`by a cyclin D-independent pathway.
`As a sensor of nutrients, growth factors, and ATP, mTOR serves a
`critical role in regulating the translational machinery 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 Ser2448
`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, insulin-induced
`phosphorylation of 4E-BP1 was shown to be Akt-mediated and de-
`pendent on mTOR activity (27). Furthermore, a functional link be-
`tween Ser2448 phosphorylation and muscle hypertrophy/atrophy was
`noted in vivo (12). In this study, sustained phosphorylation of mTOR
`(Ser2448) 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 p70S6K and to modulate the level of Ser2448
`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 by this mechanism.
`Encouragingly, induction of an in vivo response to short-term 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 of Dr. Yeung’s laboratory for critical
`reading and suggestions. We are grateful to David Ewalt, Lisa Henske, and
`Laura Finn for providing TSC-related pathology. This work is dedicated to Dr.
`Alfred Knudson, Jr. on his 80th birthday.
`
`References
`
`1. Gomez, M. R. Tuberous Sclerosis, 3rd Ed., pp. 10 –23. New York: Oxford University
`Press, 1999.
`2. Montagne, J., Radimerski, T., and Thomas, G. Insulin signaling: lessons from the
`Drosophila tuberous sclerosis complex, a tumor suppressor. Sci. STKE, 105: PE36,
`2001.
`
`3. Manning, B., Tee, A., Logsdon, M., Blenis, J., and Cantley, L. Identification of the
`tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the
`phosphoinositide 3-kinase/Akt pathway. Mol. Cell, 10: 151–162, 2002.
`4. Dan, H., Sun, M., Yang, L., Feldman, R., Sui, X., Yeung, R., Halley, D., Nicosia, S.,
`Pledger, W., and Cheng, J. PI3K/AKT pathway regulates TSC tumor suppressor
`complex by phosphorylation of tuberin. J. Biol. Chem., 277: 35364 –35370, 2002.
`5. Aicher, L. D., Campbell, J. S., and Yeung, R. S. Tuberin phosphorylation regulates its
`interaction with hamartin. Two proteins involved in tuberous sclerosis. J. Biol. Chem.,
`276: 21017–21021, 2001.
`6. Hengstschlager, M., Rodman, D. M., Miloloza, A., Hengstschlager-Ottnad, E.,
`Rosner, M., and Kubista, M. Tuberous sclerosis gene products in proliferation control.
`Mutat. Res., 488: 233–239, 2001.
`7. Templeton, D. J. Protein kinases: getting NEKed for S6K activation. Curr. Biol., 11:
`R596 –R599, 2001.
`8. Rohde, J., Heitman, J., and Cardenas, M. E. The TOR kinases link nutrient sensing to
`cell growth. J. Biol. Chem., 276: 9583–9586, 2001.
`9. Barlund, M., Forozan, F., Kononen, J., Bubendorf, L., Chen, Y., Bittner, M. L.,
`Torhorst, J., Haas, P., Bucher, C., Sauter, G., Kallioniemi, O. P., and Kallioniemi, A.
`Detecting activation of ribosomal protein S6 kinase by complementary DNA and
`tissue microarray analysis. J. Natl. Cancer Inst. (Bethesda), 92: 1252–1259, 2000.
`10. Heitman, J., Movva, N. R., and Hall, M. N. Targets for cell cycle arrest by the
`immunosuppressant rapamycin in yeast. Science (Wash. DC), 253: 905–909, 1991.
`11. Yeung, R. S., Xiao, G. H., Jin, F., Lee, W. C., Testa, J. R., and Knudson, A. G.
`Predisposition to renal carcinoma in the Eker rat is determined by germ-line mutation
`of the tuberous sclerosis 2 (TSC2) gene. Proc. Natl. Acad. Sci. USA, 91: 11413–
`11416, 1994.
`12. Reynolds, T. H., Bodine, S. C., and Lawrence, J. C., Jr. Control of Ser2448 phos-
`phorylation in the mammalian target of rapamycin by insulin and skeletal muscle
`load. J. Biol. Chem., 277: 17657–17662, 2002.
`13. Johanson, S. O., Naccache, P. A., and Crouch, M. F. A p85 subunit-independent
`p110␣ PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates
`thrombin-stimulated stress fiber formation in Swiss 3T3 cells. Exp. Cell Res., 248:
`223–233, 1999.
`14. Zhang, X., Shu, L., Hosoi, H., Murti, K. G., and Houghton, P. J. Predominant nuclear
`localization of mammalian target of rapamycin in normal and malignant cells in
`culture. J. Biol. Chem., 277: 28127–28134, 2002.
`15. Kondo, K., and Kaelin, W. G., Jr. The von Hippel-Lindau tumor suppressor gene.
`Exp. Cell Res., 264: 117–125, 2001.
`16. Smolarek, T. A., Wessner, L. L., McCormack, F. X., Mylet, J. C., Menon, A. G., and
`Henske, E. P. Evidence that lymphangiomyomatosis is caused by TSC2 mutations:
`chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes
`from women with lymphangiomyomatosis. Am. J. Hum. Genet., 62: 810 – 815, 1998.
`17. Kajino, K., and Hino, O. TSC1 and TSC2 gene mutations in human kidney tumors.
`Contrib. Nephrol., 128: 45–50, 1999.
`18. Kwiatkowski, D. J., Zhang, H., Bandura, J. L., Heiberger, K. M., Glogauer, M.,
`el-Hashemite, N., and Onda, H. A mouse model of TSC1 reveals sex-dependent
`lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1
`null cells. Hum. Mol. Genet., 11: 525–534, 2002.
`19. Goncharova, E. A., Goncharov, D. A., Eszterhas, A., Hunter, D. S., Glassberg, M. K.,
`Yeung, R. S., Walker, C. L., Noonan, D., Kwiatkowski, D. J., Chou, M. M.,
`Panettieri, R. A., Jr., and Krymskaya, V. P. Tuberin regulates p70 S6 kinase activation
`and ribosomal protein S6 phosphorylation: a role for the TSC2 tumor suppressor gene
`in pulmonary lymphangioleiomyomatosis (LAM). J. Biol. Chem., 277: 30958 –
`30967, 2002.
`20. Onda, H., Lueck, A., Marks, P. W., Warren, H. B., and Kwiatkowski, D. J.
`Tsc2(⫹/⫺) mice develop tumors in multiple sites that express gelsolin and are
`influenced by genetic background. J. Clin. Investig., 104: 687– 695, 1999.
`21. Harada, H., Andersen, J. S., Mann, M., Terada, N., and Korsmeyer, S. J. p70S6 kinase
`signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD.
`Proc. Natl. Acad. Sci. USA, 98: 9666 –9670, 2001.
`22. Muise-Helmericks, R. C., Grimes, H. L., Bellacosa, A., Malstrom, S. E., Tsichlis,
`P. N., and Rosen, N. Cyclin D expression is controlled post-transcriptionally via a
`phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem., 273: 29864 –
`29872, 1998.
`23. Humar, R., Kiefer, F. N., Berns, H., Resink, T. J., and Battegay, E. J. Hypoxia
`enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-
`dependent signaling. FASEB J., 16: 771–780, 2002.
`24. Treins, C., Giorgetti-Peraldi, S., Murdaca, J., Semenza, G. L., and Van Obberghen,