effects of mtor inhibitor everolimus (rad001) on bladder … · cancer therapy: preclinical effects...

Cancer Therapy: Preclinical

Effects of mTOR Inhibitor Everolimus (RAD001) on Bladder Cancer Cells

Edmund Chiong1, I-Ling Lee2, Ali Dadbin2, Anita L. Sabichi3, Loleta Harris2, Diana Urbauer4,David J. McConkey2, Rian J. Dickstein2, Tiewei Cheng2, and H. Barton Grossman2

AbstractPurpose: We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder

cancer (BC) cells in vitro and in vivo.

Experimental Design: The effect of RAD001 on the growth of UM-UC-3, UM-UC-6, UM-UC-9, and

UM-UC-14 BC cells were assessed by crystal violet and [3H]thymidine incorporation assays. Flow

cytometric cell-cycle analyses were done to measure the apoptotic cell fraction. Protein synthesis was

measured using tritium-labeled leucine incorporation assays. The effects of RAD001 on themTOR pathway

were analyzed byWestern blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, andUM-UC-

9 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with

RAD001 or placebo. Tumors were harvested for immunohistochemical analysis.

Results: In vitro, RAD001 transiently inhibited BC cell growth in a dose-dependent manner. This effect

was augmented by re-treatment of cells after 3 days. UM-UC-14 cells were the most sensitive to RAD001,

whereas UM-UC-9 cells were the least sensitive. After re-treatment with RAD001, only sensitive cell lines

showed G1-phase arrest, with no evidence of apoptosis. RAD001 significantly inhibited the growth of

tumors that were subcutaneously implanted in mice. Inhibition of protein synthesis through the S6K and

4EBP1 pathways seems to be the main mechanism for the RAD001-induced growth inhibition. However,

inhibition of angiogenesis was the predominant mechanism of the effect of RAD001 on UM-UC-9 cells.

Conclusions: The mTOR inhibitor RAD001 inhibits growth of BC cells in vitro. RAD001 is effective in

treating BC tumors in an in vivo nude mouse model despite the heterogeneity of in vitro responses. Clin

Cancer Res; 17(9); 2863–73. �2011 AACR.

Introduction

Bladder cancer is the fourth most common noncuta-neous malignancy and the eighth leading cause of cancerdeath among men in the United States, with an estimated13,750 deaths annually (1). Approximately 30% to 40% ofpatients with high-risk non–muscle-invasive bladder can-cer will have progression to more advanced disease within5 years, and up to 34% of these patients will ultimately dieof bladder cancer (2, 3). Radical cystectomy, which is themainstay of treatment of muscle-invasive bladder cancer,has a failure rate of 30% to 45%, largely because ofrecurrences in the form of distant metastases (4). Overall,

only 20% to 40% of patients with advanced bladder cancerhave a 5-year survival rate despite aggressive multimodaltherapy (5, 6). To improve survival rates, there is an urgentneed not only to improve current chemotherapeutic regi-mens but also to develop novel chemotherapeutic strate-gies to prevent or delay disease progression.

In recent years, mTOR, a key downstream protein kinaseof the phosphoinositide 3-kinase (PI3K)/AKT signalingpathway, has been recognized to play a central role incontrolling cancer cell growth (7). AKT andmTOR functionas "master switch" proteins in cancer cells to modulatemetabolism, the cell cycle, and apoptosis (8). Inhibition ofmTOR results in a wide variety of effects on normal andmalignant cells, including induction of apoptosis andinhibition of cell-cycle progression, cell growth, angiogen-esis, endothelial cell proliferation, and protein translation(7, 9). mTOR inhibitors such as RAD001 (everolimus;Novartis) and AP23573 (Ariad Pharmaceuticals) have beenshown to exhibit potent preclinical activities against a widevariety of cancers, including rhabdosarcoma, neuroblas-toma, glioblastoma, small cell lung cancer, renal cancer,osteosarcoma, pancreatic cancer, leukemias, B-cell lym-phoma, and breast and colon cancer (9). RAD001 hasbeen clinically approved for the treatment of renal cancer.

Given the breadth of its downstream effects on cancercells, the PI3K/AKT/mTOR signaling pathway may

Authors' Affiliations: 1Department of Urology, National University HealthSystem, Singapore; Departments of 2Urology and 3Clinical Cancer Pre-vention, and 4Division of Quantitative Sciences, The University of TexasM.D. Anderson Cancer Center, Houston, Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: H. Barton Grossman, Department of Urology,Unit 1373, The University of Texas M. D. Anderson Cancer Center, 1515Holcombe Boulevard Houston, TX 77030. Phone: 713-792-3250; Fax:713-794-4824. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-09-3202

�2011 American Association for Cancer Research.

ClinicalCancer

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represent a promising molecular target for bladder cancer.In support of this hypothesis, Wu and colleagues recentlyshowed that 55% of human bladder tumors analyzed hadincreased expression of phosphorylated AKT (p-AKT) andthat inhibition of the PI3K pathway could drasticallyreduce the invasive capacity of bladder cancer cell lines(10). PTENmutations are also reported to be present in upto 23% of patients with bladder transitional cell carcinoma(10). In addition, we reported that inhibition of the AKTpathway by forced expression of PTEN reduced the growthof bladder cancer cells and that the PTEN/AKT pathwaycould be an important therapeutic target for bladder cancer(11). AKT, a serine-threonine kinase that is dependent onPI3K signaling for activation, is known to be involved incell proliferation and a variety of antiapoptotic pathways(12). Inactivation of the tumor suppressor gene PTEN,which is mapped to chromosome 10q23, has also beensuggested to be involved in bladder cancer progression(13–16). Recently, a synergistic relationship between dele-tion of p53 and PTEN, which deregulates mTOR signalingand was shown to promote tumorigenesis in a murinemodel of bladder cancer (17), and rapamycin hasbeen shown to inhibit the proliferation of bladdercancer cells in vitro (18). Thus, we hypothesized thatRAD001, by inhibiting the mTOR pathway, may inhibitthe growth of bladder cancer cells. A recent preclinicalstudy had shown that mTOR inhibition by RAD001 has

antiproliferative activity in bladder cancer, supporting thishypothesis (19).

Here, we report the effects of RAD001 on bladder cancercell growth both in vitro in several bladder cancer cell lineswith different genetic backgrounds and in vivo in a nudemouse xenograft model.

Materials and Methods

Cell cultureThe transitional cell carcinoma cell lines UM-UC-3, UM-

UC-6, UM-UC-9, and UM-UC-14 (Table 1) were obtainedat source (H.B.G.) from cryopreserved cells frozen over aspan of more than 25 years. UM-UC-3 was also obtainedfrom the American Type Culture Collection (ATCC). Thecell lines were authenticated within 6 months of conduct-ing the experiments (20). These cell lines were maintainedin Eagle’s minimum essential medium (Mediatech Inc.)supplemented with 2 mmol/L L-glutamine, 10% heat-inac-tivated FBS, 100 U/mL penicillin, and 100 mg/mL strepto-mycin (20). All cultures were free of bacterial, fungal, andmycoplasmal contamination.

Reagents and drug preparationRAD001 and placebo were obtained from Novartis

Pharma AG. For in vitro experiments, RAD001was preparedin dimethyl sulfoxide (DMSO). For animal studies,RAD001 was prepared at 2% (w/w; 20 mg/g) in a micro-emulsion vehicle, which was diluted in 5% glucose indouble-distilled water just before administration by oralgavage.

In vitro cell growthCell growth was measured using a crystal violet assay as

previously described (21). Bladder cancer cells were platedinto 6-well plates at a density of 1.25 � 104 cells per well.After 24 hours, the cells were treated with 1 of 6 concen-trations of RAD001 (0.1, 0.5, 1, 10, 20, or 100 nmol/L).After 4 and 6 days of exposure to either RAD001 or thecontrol (DMSO), the medium was removed and the cellswere fixed with 1% glutaraldehyde for 15 minutes andstained with 0.5% crystal violet. The dye was eluted, trans-ferred to an ELISA 96-well plate, and the optical density wasread on a microplate autoreader (Bio-Tek Instruments) at540 nm. The optical density values of the RAD001-treated

Table 1. The characteristics of p53, PTEN, and p-AKT expression in bladder cancer cell lines aresummarized (11, 25)

Cell lines TP53 PTEN p-AKT

UM-UC-3 Mutated Mutated Increased expressionUM-UC-6 Wild type Wild type Decreased expressionUM-UC-9 Mutated Decreased expression Increased expressionUM-UC-14 Mutated Wild type Decreased expression

Translational Relevance

mTOR, a key downstream protein kinase in thephosphoinositide 3-kinase (PI3K)/AKT signaling path-way, plays a central role in promoting cancer cellgrowth. mTOR inhibitors have been shown to exhibitpotent preclinical activities against a wide variety ofsolid malignancies and are indicated for clinical usein some cancers. Only recently has it been reported thatthe PI3K/AKT pathway could be important in bladdercancer. Here, we show that themTOR inhibitor RAD001(everolimus) inhibits the growth of bladder cancer cellsin vitro and in vivo in a subcutaneous mouse model. Invivo activity was seen despite in vitro heterogeneity. Thiswork suggests that mTOR may be a new therapeutictarget for bladder cancer.

Chiong et al.

Clin Cancer Res; 17(9) May 1, 2011 Clinical Cancer Research2864




cells were normalized to the values obtained for theDMSO-treated control cells to determine the percentageof surviving cells. For the redosing experiments, the cellswere re-treated 3 days after the initial treatment with theIC50 dose of RAD001. Each assay was done in duplicate,and the experiments were repeated twice.

In vitro cell proliferationBladder cancer cells were plated into 96-well plates at a

density of 8 � 103 cells per well. After 24 hours, the cellswere treated with 1 of 5 concentrations of RAD001 (0.1,0.5, 1, 10, or 100 nmol/L). After 48 hours of exposure toRAD001 or the control or DMSO, the medium wasremoved and replaced with fresh cell culture mediumcontaining 1% FBS and 10 mCi/mL [3H]thymidine (MPBiomedicals). The cells were pulsed with [3H]thymidinefor 2 hours, and the media were subsequently removed.Cells were then analyzed by the addition of 0.1 mol/LKOH and harvested onto fiberglass filters. The incorpo-rated tritium was quantified in a scintillation counter(1450 MICROBETA Trilux liquid scintillation and lumi-nescence counter; PerkinElmer life sciences). Each assaywas carried out in duplicate, and the experiments wererepeated once.

Flow cytometryCells were grown in 6-well plates and after reaching 70%

confluence were exposed to various concentrations ofRAD001 for 24, 48, and 72 hours. Cells were harvestedby trypsinization and pelleted by centrifugation. The pelletswere then resuspended in PBS containing 50 mg/mL pro-pidium iodide (PI), 0.1% Triton X-100, and 0.1% sodiumcitrate. DNA staining with PI was measured by fluores-cence-activated cell-sorting analysis, using the FL-3 channel(FACSCalibur flow cytometer; Becton Dickinson), to deter-mine the cell-cycle distribution. Cells displaying a hypo-diploid DNA content, which is indicative of DNAfragmentation, were scored as apoptotic. PI exclusionwas carried out in a similar fashion 24 and 48 hours afterRAD001 exposure, without the addition of 0.1% Triton X-100. Annexin V–fluorescein isothiocyante (FITC) and PIflow cytometry were carried out following the man-ufacturer’s instructions (TACS Annexin V–FITC ApoptosisDetection Kit from Trevigen).

Protein synthesisThe bladder cancer cells were plated into 6-well plates

at a density of 1 � 105 cells per well. After 24 hours, thecells were treated with various concentrations ofRAD001. After 2 and 12 hours of exposure to eitherRAD001 or control (DMSO), the medium was removed,2 mCi/mL of L-[4,5-3H]leucine (GE Healthcare) and leu-cine-free media (Dulbecco’s modified Eagle’s mediumwithout L-glutamine and leucine; MP Biomedical) sup-plemented with 20 ml/L of 200 mmol/L L-glutamine and110 mg/L of sodium pyruvate were added, and the cellswere incubated at 37�C in a humidified chamber with5% CO2, for 2 hours. The radiolabeled protein–uptake

assay was done as previously described (22). The radio-activity of the solution was measured as disintegrationsper minute (DPM), using a liquid scintillation counter(Beckman LS 6500; Global Medical Instrumentation,Inc.). All experiments were carried out in triplicate andrepeated once.

Western blot analysisCells were harvested at 70% to 80% confluence, and

cell lysates were obtained as previously described (11).Protein extracts (30–50 mg per lane) were electrophor-etically separated on a 4% to 20% Tris-glycine gel (Invi-trogen) and transferred onto nitrocellulose membranes(11). The nitrocellulose membranes were incubated with1:200 to 1:1,000 dilutions of primary antibodies, fol-lowed by a horseradish peroxidase–linked secondaryantibody (anti-rabbit 1:3,000 or anti-mouse 1:5,000).The antibody-probed proteins were detected using anenhanced chemiluminescence kit (Amersham Bios-ciences) according to the manufacturer’s instructions.Anti-phosphorylated p70S6K (p-p70S6K; Thr 389),p70S6K, anti-phosphorylated mTOR (p-mTOR; Ser2448), mTOR (Ser 2448), and anti-phosphorylated4EBP1 (p-4EBP1; Ser 65) antibodies were purchasedfrom Cell Signaling. The secondary antibodies were pur-chased from Santa Cruz Biotechnology, and the anti-actin antibody was purchased from EMD Biosciences. Acell line from another organ system was used as a positivecontrol.

Animal studiesAll animal experiments were conducted according to

institutional guidelines established for the Animal CoreFacility at The University of Texas M. D. Anderson CancerCenter. UM-UC-3, UM-UC-6, andUM-UC-9 cells (1� 106)were injected subcutaneously into the right flank of 4- to 6-week-old female nude mice. Tumor growth was measuredwith calipers twice per week. The tumor volume wascalculated according to the following equation: volume(mm3) ¼ length � width2 � 0.5236. When the tumorsreached approximately 5 mm in diameter (1 week afterinjection), the animals received RAD001 by oral gavage at adose of 5 mg/kg in 100 mL 5% glucose in water or a placebo(Novartis Pharma AG) twice weekly for 1 month or untilthe tumor burden (approximately 1.5 cm in size) requiredeuthanasia (anesthetized with CO2 and euthanized bycervical dislocation). Harvested tumor specimens weredivided into 2 groups and either (i) fixed in 10% bufferedformalin and paraffin-embedded or (ii) embedded in OCT(optimum cutting temperature) and frozen in liquid nitro-gen. All experiments were repeated once.

Immunohistochemical analysisParaffin-embedded tissue sections were incubated in

citrate buffer, pH 6.0, at 100�C for 20 minutes for antigenretrieval. The Lab Vision Autostainer (Lab Vision Corp.;Thermo Fisher Scientific) was used for staining the sampleswith diaminobenzidene. Anti-p-p70S6K (Thr 389; Epi-

mTOR Inhibition (RAD001) on Bladder Cancer Cells

www.aacrjournals.org Clin Cancer Res; 17(9) May 1, 2011 2865




tomics; species reactivity: human and mouse), anti-CD31(Lab Vision Corp.; Thermo Fisher Scientific; species reac-tivity: human), anti-VEGF (Lab Vision Corp.; ThermoFisher Scientific; species reactivity: human), anti-p-AKT(Ser 473; Cell Signaling; species reactivity: human, mouse,rat, hamster, monkey, Drosophila melanogaster, bovine, andzebrafish), and anti-p-mTOR (Ser 2448; species reactivity:human, possibly mouse and rat) antibodies (Cell Signal-ing) were used for immunohistochemical analyses. Nega-tive control experiments were conducted in the absence ofthe primary antibody. Tumor tissue sections from all 3implanted cell lines were concurrently incubated with eachtype of antibody and stained with the autostainer. Addi-tional tumor tissue sections from each cell line were usedfor immunohistochemical staining as positive controls forthe other 2 cell lines (Supplementary Fig. S1). The tissuespecimens were evaluated by light microscopy, and theimmunostaining was scored by 2 independent observerswho were blinded to samples (i.e., drug-treated or placebo)that they were evaluating. Only cytoplasmic staining wasconsidered for scoring. The staining intensity was assigneda score for the most dominantly stained area, and thisstained area should occupy at least 20% of the specimen.The intensity of staining was scored on a scale of 0 to 3þ,with 0 for no staining, 1þ for weak staining intensity, 2þfor moderate staining intensity, and 3þ for the mostintense staining (examples of staining intensities are shownin Supplementary Fig. S2). The microvessel density (MVD)was measured by microscopy at 100� magnification bycounting the number of blood vessels (stained with anti-CD31) in each of 5 fields per tumor sample to obtain anaverage measurement (23). The fields with the highestdensity of blood vessels on the slide were selected foranalysis. The CD31 expression data were analyzed withrepeated-measures ANOVA. Statistical significance wasdetermined to be P < 0.05.

Results

Growth inhibitory effects of RAD001 in vitroThe bladder cancer cell lines UM-UC-3, UM-UC-6, UM-

UC-9, and UM-UC-14 were treated with increasing doses ofRAD001 (0.1–100 nmol/L) and incubated for up to 6 days.RAD001 inhibited the growth of all bladder cancer cellstested in a dose-dependent manner, with a partial loss ofresponse seen 4 days after initial treatment (Fig. 1A). TheUM-UC-14 cell line was themost sensitive to RAD001, withan IC50 of 0.1 nmol/L; the UM-UC-3 and UM-UC-6 celllines had moderate sensitivities, with IC50s of 0.5 nmol/L;and the UM-UC-9 cell line was the least sensitive, with anIC50 of 10 nmol/L. Re-treatment of cells with RAD001 (at adose corresponding to each cell line IC50) 3 days after theinitial therapy augmented the growth inhibition in all 4bladder cancer cell lines (Fig. 1B). The timing of redosing atday 3 was based on the observation that there was loss ofresponse to RAD001 in all 4 cell lines from day 4 ofincubation (Fig. 1A). The transient effect of a single doseof RAD001 is likely related to its half-life of 60 hours (24).

[3H]Thymidine incorporation assays showed that there wasa dose-dependent decrease in the number of bladder cancercells proliferating when treated with RAD001 (Fig. 1C).This was consistent with the growth inhibitory effects ofRAD001 on bladder cancer cells seen in the crystal violetassays (Fig. 1A).

Cell cycle and apoptosisAll 4 cell lines were treated once with increasing doses of

RAD001 (0.1, 0.5, 10, and 100 nmol/L), and cell-cycleanalysis was done at various time points (24, 48, and 72hours). We saw no specific growth-phase arrest with asingle treatment of RAD001 in any of the 4 cell lines tested(Fig 2A). When we re-treated UM-UC-6 and UM-UC-9 celllines with RAD001, 3 days after the initial dose, only theUM-UC-6 cells were arrested in the G0/G1 phase (Fig 2A).We did not see any evidence of apoptosis in any of the 4 celllines tested by Annexin V assays, which were carried out 12and 24 hours after a single treatment of cells with RAD001,even at doses as high as 100 nmol/L (data not shown). Wecorroborated these findings with PI exclusion assays 24 and48 hours after RAD001 treatment, which showed thatRAD001 induced nonapoptotic cell death at the IC50 dosesin all of the bladder cancer cell lines except UM-UC-9(Fig. 2B, data not shown for cell lines UM-UC-3 andUM-UC-14).

Protein synthesisUsing a radioactively labeled amino acid (leucine), we

showed that RAD001 inhibited protein synthesis in all 4bladder cancer cell lines (Supplementary Fig. S3, data notshown for cell lines UM-UC-3 and UM-UC-14).

Downstream effects of mTOR blockade by RAD001To investigate the effects of RAD001 treatment on

mTOR signaling, we used Western blotting to analyzethe levels of the phosphorylated forms of mTOR, p70S6K,4EBP1, and nonphosphorylated forms of mTOR andp70S6K after a single 24-hour exposure of UM-UC-3,UM-UC-6, UM-UC-9, and UM-UC-14 cells to variousdoses of RAD001. In all 4 cell lines, the levels of thephosphorylated forms of mTOR, p70S6K, and 4EBP1were reduced with increasing doses of RAD001, but theeffects of RAD001 on the nonphosphorylated form ofmTOR protein levels in each cell line were different, withonly UM-UC-3 and UM-UC-6 having a slight decrease inmTOR levels with increasing RAD001 dose (Fig. 3). Nodownstream effect of RAD001 was seen in the nonpho-sphorylated form of p70S6K in all 4 cell lines (Fig 3).Overall, the effects of RAD001 on the levels of theseproteins in the mTOR signaling pathway did not correlatewith the relative sensitivity of each cell line to the growthinhibitory effects of RAD001.

Effects of RAD001 in vivoUM-UC-3, UM-UC-6, and UM-UC-9 cells were

implanted subcutaneously into nude mice, and whenthe xenograft tumors reached 5 mm in diameter, the mice

Chiong et al.





were treated with RAD001 by oral gavage twice per week for1 month. RAD001 significantly inhibited the growth oftumors derived from all 3 cell lines (Fig. 4). Immunohis-tochemical analysis of tumor tissue samples showedreduced expression of p-p70S6K in the RAD001-treatedUM-UC-3- and UM-UC-6–derived tumors compared withcontrol-treated tumors, whereas the expression of p-p70S6K in RAD001-treated UM-UC-9–derived tumors

was unchanged (Table 2 and Fig. 5). There was no differ-ence in the expression level of p-mTOR between theRAD001-treated and control-treated tumors. For the UM-UC-3- and UM-UC-9–derived tumors, p-AKT expressionwas reduced in the RAD001-treated compared with con-trol-treated tumors, but the p-AKT levels were unchangedbetween the RAD001-treated and control-treated UM-UC-6–derived tumors.

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Figure 1. The inhibitory effects of RAD001 on bladder cancer cell growth and proliferation determined by crystal violet assays (A and B) and [3H]thymidineincorporation assays (C), respectively. UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 cells were treated with RAD001 and incubated for 6 days(A), with RAD001 initially at day 0 and then re-treated at day 3 with a dose equivalent to the IC50 value for each cell line and incubated for 5 days (B),and with RAD001 and incubated for 48 hours (C).






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Figure 2. Flow cytometric analysis of bladder cancer cell lines. A, cell-cycle analysis was done at 48 hours for control- and RAD001-treated (0.5 nmol/L)UM-UC-6 cells, control- and RAD001-treated (10 nmol/L) UM-UC-9 cells, UM-UC-6 cells treated with RAD001 (0.5 nmol/L) every 3 days, andUM-UC-9 cells treated with RAD001 (10 nmol/L) every 3 days. Bar charts comparing the differences in the percentage of G1-phase cells betweencontrols and RAD001-treated cells are shown. B, PI exclusion assays were conducted 24 hours after UM-UC-6 cells and UM-UC-9 cells were treatedwith RAD001. The flow cytometric profiles with the percentage of cells undergoing nonapoptotic cell death (G) are shown.

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The vascular density in the tumors was measured bycounting the number of blood vessels that expressed CD31.RAD001 treatment did not affect the vascular density ofUM-UC-6–derived tumors (P¼ 0.5503; Table 2 and Fig. 5).However, RAD001 treatment modestly reduced the vascu-lar density of the UM-UC-3–derived tumors (P ¼ 0.0597)and significantly reduced the vascular density of the UM-UC-9–derived tumors (P ¼ 0.036; Table 2 and Fig. 5).Expression levels of VEGF were similar in the RAD001- andcontrol-treated tumors derived from all 3 cell lines(Table 2), suggesting that the changes in vascular densitywere not due to changes in the VEGF pathway.

Discussion

In this study, we show that the inhibition of the mTORpathway effectively reduces the growth of bladder cancercells despite the variations in genetic backgrounds of thebladder cancer cell lines. This presents further evidence thatthe PI3K/AKT/mTOR signaling pathway is important inbladder cancer and that mTOR may be used as a moleculartarget for bladder cancer therapy.Like wild-type bladder cancer cells, the cell lines we

tested have different alterations in the PTEN/AKT and p53pathways. The UM-UC-3 cells have a PTEN deletion,constitutively active p-AKT, and mutated TP53; UM-UC-6 cells have wild-type PTEN and TP53; UM-UC-9cells have reduced PTEN expression and active p-AKT;and UM-UC-14 cells have wild-type PTEN and no p-AKTexpression (Table 1; refs. 11, 25). The effect of mTORinhibition on AKT activity has been reported to be vari-able, depending on the cell type, and inhibition of mTORhas led to increased activation, no change, or reducedphosphorylation of AKT after prolonged treatment (26,27). We show that RAD001 reduces the expression of AKTin the UM-UC-3- and UM-UC-9–derived tumors, bothcell lines constitutionally express p-AKT (Tables 1 and 2).This reduction in AKT expression was not seen in theRAD001-treated UM-UC-6–derived tumors, a cell line

that has a normal PTEN/AKT pathway (Tables 1 and2). Although it has been reported that in other tumors,PTEN and AKT status may correlate with the sensitivity ofcell lines to mTOR inhibitors, we did not find thisrelationship to be present in our panel of bladder cancercells despite having chosen cell lines with known variablePTEN/AKT status for our study (19, 28). mTOR comprises2 multiprotein complexes: mTOR complexes 1 and 2(mTORC1 and mTORC2; ref. 26). It is known thatmTORC1 is rapamycin sensitive and its activation resultsin phosphorylation of 4EBP1 and S6K1, whereasmTORC2 may be regulated by rapamycin to cause eitherAKT activation or inactivation (26). The variable effect ofRAD001 on p-AKT levels in different bladder cancer celltypes could be due in part to the differential effects ofmTOR inhibition on mTORC1 and mTORC2 in differentcell lines. The complexity of the multiple pathwaysinvolved may be one of the reasons for not seeing a directrelationship between the PTEN/AKT status and effects ofmTOR inhibition.

Our in vitro results show that RAD001 inhibits thegrowth of bladder cancer cells at concentrations that arephysiologically safe in humans (29). However, theresponse of bladder cancer cells to RAD001 in vitro washeterogeneous, with UM-UC-14 being the most sensitivecell line and UM-UC-9 being the least sensitive. The IC50

value of UM-UC-9 was approximately 20 times higher thanthat in the UM-UC-3 and UM-UC-6 cell lines, and 100times higher than in the UM-UC-14 cell line, respectively.The loss of the inhibitory effect of a single dose of RAD0014 days after treatment was overcome by re-treatment of cellson the third day following the first dose. This transienteffect of RAD001 provided the basis for the dosing schedulein our in vivo murine experiments. Because mTOR inhibi-tion was not seen to correlate with expression of upstreamtargets (PTEN and AKT), it is therefore likely that thevariability in sensitivity and the transient nature ofresponse of different cell types to single doses ofRAD001 was mainly due to the differential effects of mTOR

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Figure 3. Western blot analysis of UM-UC-3, UM-UC-6, UM-UC-9, and UM-UC-14 cells 24 hours after treatment with RAD001.






inhibition on downstream effectors. It is known that thedownstream effectors of mTOR are 4EBP1 and S6 kinase 1(S6K1), both of which are regulators of protein translation(7, 9). Activation of mTOR leads to phosphorylation of4EBP1, which prevents it from binding to eukaryotic initia-tion factor 4E (eIF4E), which, in turn, is then free to bind tomRNA transcripts and other translation initiation complexproteins and activate cap-dependent translation (7, 26, 30).S6K1, a key regulator of cell growth, phosphorylates ribo-somal protein S6 and enhances the translation of 50-term-

inal oligopyrimidine tract mRNAs in some models (26). Ithas also been reported to play a role in glucose homeostasisby phosphorylating insulin receptor substrate 1 and glyco-gen synthase kinase 3 and by regulating other factors suchas eukaryotic initiation factor 4B, programmed cell death 4,eukaryotic elongation factor-2 kinase, and S6K1 Aly/REF-like target (7, 26). Mansure and colleagues recently sug-gested that the reduction of phosphorylation levels of S6activation correlated with the sensitivity of bladder cancercells to RAD001 and that S6 inhibition may reflect thesensitivity of these cells to mTOR inhibition (19). In ourstudy, RAD001 reduced the expression of p-p70S6K and p-4EBP1 in vitro in all cell lines tested. However, we did notobserve a correlation between S6 phosphorylation levelsand sensitivity to mTOR inhibition by RAD001. Onereason for the difference may be due to the fact that thepanel of bladder cancer cell lines tested was different inboth studies. The differential sensitivity of bladder cancercells to mTOR inhibition may be due to a variety of otherfactors, such as variability of mTORC1 and mTORC2response or activation/inhibition of other pathways inresponse to mTOR inhibition. It has been shown thatmTORC1 and S6Kmay be activated independent ofmTOR,possibly through the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway (31, 32). It was also shown thatRAD001 can activate MAP kinase (MAPK), which ismediated by S6K/PI3K/Ras signaling, and that this feed-back could be a cause of the poor anticancer activity ofmTORC1 inhibitors (33). These complex relationshipsbetween mTOR and other pathways may account fordifferences in sensitivity of various cell lines to RAD001and the inability to reliably correlate expression of effectorssuch as S6K and response to mTOR inhibition. Suchrelationships deserve further investigation.

Contrary to other reports (7, 19), we observed that asingle treatment of RAD001 did not cause a specific growth-phase arrest in any of the cell lines we tested. Re-treatmentwith RAD001 induced G0/G1 arrest in UM-UC-3 and UM-UC-6 but not in UM-UC-9 cells, which are resistant toRAD001 in vitro. Because MAPK is known to regulatetranscription of genes that are important for cell cycle,effects of the MAPK feedback loop with mTOR may con-tribute to the differential effects on cell-cycle arrest seen invarious cell lines. In our study, we show that RAD001induced nonapoptotic cell death in all the cell lines tested.As autophagic activation by mTOR inhibition is known tooccur, this may be another possible mechanism of bladdercancer cell growth inhibition by RAD001 (34). Furtherevaluation of the relationship between mTOR inhibitionand autophagy is currently underway.

Our in vivo data confirm that mTOR inhibition byRAD001 is effective in inhibiting the growth of UM-UC-3, UM-UC-6, and UM-UC-9 bladder cancer cells subcuta-neously implanted into nude mice. Although the bladdercancer cell lines we tested in vitro responded to RAD001treatment with different sensitivities, this difference wasnot seen in vivo. We hypothesize that the differences in

UM-UC-3 implanted mice



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Figure 4. In vivo growth inhibitory effect of prolonged treatment ofsubcutaneous tumor–bearing nudemice. The graphs show tumor size withtime after RAD001 or placebo treatment in mice with UM-UC-3-, UM-UC-6-, and UM-UC-9–derived tumors, respectively.

Chiong et al.





sensitivity to RAD001 between the cell lines in vitro and invivo reflect the results of mTOR inhibition of alternatepathways such as the angiogenesis pathway, which areonly functional in vivo. Consistent with the in vitro findings,p-p70S6K expression in RAD001-treated tumors wasreduced in tumors derived from UM-UC-3 and UM-UC-

6 cell lines. However, contrary to our in vitro findingswherein expression was measured after a single RAD001dose, UM-UC-9–derived tumors exhibited similar levels ofp-p70S6K in RAD001- and control-treated tumors but hada significantly reduced mean vascular density (Table 2).Interestingly, VEGF expression was not different between

Table 2. Summary of the scoring results of immunohistochemical analysis for p-mTOR, p-p70S6K, p-AKT,CD31, and VEGF expression in UM-UC-3, UM-UC-6, and UM-UC-9 tumors from mice treated withRAD001

Cell lines Immunohistochemical marker

p-mTOR p-p70S6K p-AKT VEGF CD31

Staining intensity score Mean MVD (range) P

UM-UC-3Control treated 0 2þ 2þ 1þ 21.8 (18.6–25.0) 0.0597RAD001 treated 0/1þ 1þ 1þ 1þ 17.4 (14.1–20.7)

UM-UC-6Control treated 0 2þ 2þ 3þ 16.0 (14.0–18.0) 0.5503RAD001 treated 0 1þ 2þ 3þ 16.7 (14.7–18.7)

UM-UC9Control treated 1þ/2þ 1þ 3þ 2þ 31.1 (25.0–37.2) 0.0365RAD001 treated 1þ 1þ 2þ 2þ 20.9 (14.8–27.0)

NOTE: MVD was quantified at 100� magnification.

p-mTOR(400×)

UM-UC-3

Control RAD001 Control RAD001 Control RAD001

UM-UC-6 UM-UC-9

p-p70S6K(400×)

p-AKT(400×)

CD31(100×)

Figure 5. Immunohistochemical staining of representative tumor tissue sections harvested after prolonged treatment with either a placebo or RAD001.p-mTOR, p-p70S6K, and p-AKT are shown at 400� magnification, and CD31 is shown at 100� magnification.






the RAD001- and control-treated tumors in any of the 3 celllines tested (Table 2). On the basis of these findings, wehypothesize that inhibition of protein synthesis throughthe S6K and 4EBP1 pathways, rather than inhibition ofangiogenesis, seems to be the more important mechanismfor bladder cancer cell growth inhibition in UM-UC-3 andUM-UC-6 cells (Table 2 and Fig. 5). However, in the UM-UC-9 cells, inhibition of angiogenesis, possibly through anon–VEGF-related pathway, seems to play a dominantrole.

Although the cell lines we tested had different geneticbackgrounds, RAD001 was active against all cell linestested both in vitro and in vivo, but the sensitivities ofthe different cell lines to RAD001 in vitro did notcorrelate with the in vivo results. Thus, our results suggestthat the growth inhibitory action of RAD001 on bladdercancer cell lines is independent of genomic PTEN altera-tions in the AKT pathway or the AKT phosphorylationstatus.

Limitations of our study include the small number ofcell lines used, as responses of other cell lines to RAD001may yield additional insights in the differential responseto mTOR inhibition. Furthermore, there are potentiallymultiple mechanisms for differences in sensitivity tomTOR inhibition that have yet to be tested. Despite theselimitations, this study is one of very few preclinicalstudies that describe the use of mTOR inhibition inbladder cancer. p-mTOR expression was recently shownto be increased in 32% of human bladder cancers com-pared with noncancerous bladder tissue, and activation ofp-S6K was also seen in 25% of invasive human bladdercancers, which displayed altered p53 and PTEN expres-sion (17, 35). These findings, together with current lim-ited but growing preclinical evidence of the efficacy ofmTOR inhibition in bladder cancer cells, suggest mTOR

inhibition may have a future role in the treatment ofselected human bladder urothelial cancers.

Conclusions

The mTOR inhibitor RAD001 inhibits protein synth-esis and growth of bladder cancer cells in vitro at dosesranging from 0.1 to 100 nmol/L. Despite the hetero-geneity of responses seen in vitro, RAD001 is effective invivo, in part, because of changes in angiogenesis. Inconclusion, RAD001 affects tumor growth through dif-ferent mechanisms, depending on the genotype of thebladder cancer cells. This work suggests that mTOR ispotentially a new therapeutic target for bladder cancer.Future studies are required to determine its role inbladder cancer therapy.

Disclosure of Potential Conflicts of Interest

H.B. Grossman is on Novartis Advisory Board 2007. All other authorshave no conflict of interest.

Acknowledgments

We thank Kate Juliet Newberry for helping to edit the manuscript.

Grant Support

This study was supported by W.A. "Tex" and Deborah Moncrief, Jr.,Distinguished Chair in Urology, University of Texas M. D. Anderson CancerCenter. This study was also supported in part by the National Institutes ofHealth through University of Texas M. D. Anderson Cancer Center SupportGrant CA016672.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 7, 2009; revised January 19, 2011; accepted February9, 2011; published OnlineFirst March 17, 2011.

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2011;17:2863-2873. Published OnlineFirst March 17, 2011.Clin Cancer Res Edmund Chiong, I-Ling Lee, Ali Dadbin, et al. Cancer CellsEffects of mTOR Inhibitor Everolimus (RAD001) on Bladder

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