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• W2101967561 abstract "Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth. Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth. Bladder cancer occurs in multiple forms, the most common of which is urothelial carcinoma (UCC), which represents >90% of all bladder cancers.1Eble JN SG Epstein JI Sesterhenn IA Pathology and genetics of tumours of the urinary system and male genital organs.in: Kleiheus P SL World Health Organization Classification of Tumours. IARC Press, Lyon, France2004Google Scholar Approximately 30 to 50% of patients with invasive bladder cancer into the muscular wall of the bladder will develop metastatic disease and die within 2 years of diagnosis.2Stoter G Splinter TA Child JA Fossa SD Denis L van Oosterom AT de Pauw M Sylvester R Combination chemotherapy with cisplatin and methotrexate in advanced transitional cell cancer of the bladder.J Urol. 1987; 137: 663-667Abstract Full Text PDF PubMed Scopus (80) Google Scholar In addition, virtually all patients diagnosed with distant UCC metastases will succumb to disease.3Wu XR Urothelial tumorigenesis: a tale of divergent pathways.Nat Rev Cancer. 2005; 5: 713-725Crossref PubMed Scopus (561) Google Scholar Currently, the standard treatment modality for muscle-invasive bladder cancer is radical cystectomy; systemic chemotherapy is generally reserved for patients with metastatic disease, although these treatment regimens provide only a limited long-term benefit with only rare reports of complete remission.4Saxman SB Propert KJ Einhorn LH Crawford ED Tannock I Raghavan D Loehrer Sr, PJ Trump D Long-term follow-up of a phase III intergroup study of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a cooperative group study.J Clin Oncol. 1997; 15: 2564-2569Crossref PubMed Scopus (479) Google Scholar, 5Sternberg CN Donat SM Bellmunt J Millikan RE Stadler W De Mulder P Sherif A von der Maase H Tsukamoto T Soloway MS Chemotherapy for bladder cancer: treatment guidelines for neoadjuvant chemotherapy, bladder preservation, adjuvant chemotherapy, and metastatic cancer.Urology. 2007; 69: 62-79Abstract Full Text Full Text PDF PubMed Google Scholar In light of these clinical outcomes, identification of new therapeutic targets is needed to define potential additional treatment avenues for these patients. Activation of the mammalian target of rapamycin (mTOR) signaling pathway occurs in many cancers and has recently been shown to correlate with more aggressive disease behavior,6Gildea JJ Herlevsen M Harding MA Gulding KM Moskaluk CA Frierson HF Theodorescu D PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity.Oncogene. 2004; 23: 6788-6797Crossref PubMed Scopus (73) Google Scholar, 7Atkins MB Hidalgo M Stadler WM Logan TF Dutcher JP Hudes GR Park Y Liou SH Marshall B Boni JP Dukart G Sherman ML Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma.J Clin Oncol. 2004; 22: 909-918Crossref PubMed Scopus (891) Google Scholar, 8Chan S Scheulen ME Johnston S Mross K Cardoso F Dittrich C Eiermann W Hess D Morant R Semiglazov V Borner M Salzberg M Ostapenko V Illiger HJ Behringer D Bardy-Bouxin N Boni J Kong S Cincotta M Moore L Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer.J Clin Oncol. 2005; 23: 5314-5322Crossref PubMed Scopus (438) Google Scholar, 9Drakos E Rassidakis GZ Medeiros LJ Mammalian target of rapamycin (mTOR) pathway signalling in lymphomas.Expert Rev Mol Med. 2008; 10: e4Crossref PubMed Scopus (30) Google Scholar although it has not been examined in great detail in UCC. Activation of mTOR occurs via a multistep process that includes upstream phosphoinositide-3 kinase (PI3K) and AKT activation, leading to phosphorylation and inactivation of the tuberous sclerosis complex 1 and 2 (TSC1/TSC2) heterodimer.10Hay N The Akt-mTOR tango and its relevance to cancer.Cancer Cell. 2005; 8: 179-183Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 11Sabatini DM mTOR and cancer: insights into a complex relationship.Nat Rev Cancer. 2006; 6: 729-734Crossref PubMed Scopus (1130) Google Scholar Inactivation of this heterodimer results in release of Rheb inhibition and subsequent mTOR activation by means of Rheb GTPase activity. Once activated, mTOR can induce increased mRNA translation or regulate the actin cytoskeleton via differential association Rictor and Raptor proteins.10Hay N The Akt-mTOR tango and its relevance to cancer.Cancer Cell. 2005; 8: 179-183Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 11Sabatini DM mTOR and cancer: insights into a complex relationship.Nat Rev Cancer. 2006; 6: 729-734Crossref PubMed Scopus (1130) Google Scholar Ultimately, mTOR activity regulates the effects of a number of downstream molecules important in cellular growth, including p70 S6 kinase-1 (S6K) and elongation-initiation factor 4E binding protein-1 (4E-BP1). Selective inhibition of the mTOR pathway can be achieved using rapamycin or rapamycin analogs temsirolimus (CCI-779, Wyeth Pharmaceuticals) and everolimus (RAD001, Novartis), which are currently in use in numerous clinical trials for solid tumors, with promising results in patients with advanced renal cell carcinoma.12Hanna SC Heathcote SA Kim WY mTOR pathway in renal cell carcinoma.Expert Rev Anticancer Ther. 2008; 8: 283-292Crossref PubMed Scopus (44) Google Scholar, 13Hudes G Carducci M Tomczak P Dutcher J Figlin R Kapoor A Staroslawska E Sosman J McDermott D Bodrogi I Kovacevic Z Lesovoy V Schmidt-Wolf IG Barbarash O Gokmen E O'Toole T Lustgarten S Moore L Motzer RJ Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma.N Engl J Med. 2007; 356: 2271-2281Crossref PubMed Scopus (3279) Google Scholar To further investigate the potential role of mTOR signaling and inhibition in UCC of the bladder, we used human cancer specimens, xenograft models, and in vitro analysis to determine the effects of mTOR on cellular proliferation, apoptosis, tumor growth, and clinical outcomes in this cancer population. Permission for this study was obtained from The Cleveland Clinic Institutional Review Board. Specimens included archived paraffin blocks from patients who underwent radical cystectomy or cystoprostatectomy for muscle-invasive UCC (pathological stage pT2 or greater) between the years 1998 to 2007. All specimens were received in the surgical pathology suite on ice within 10 minutes postcystectomy, and tissue was immediately placed into 10% buffered formalin for routine processing. Primary bladder tumors that were either nonmetastatic (n = 52) or metastatic to regional lymph nodes (n = 69) were used for analysis. Paired lymph node metastases from the latter group were available in 59 cases for analysis. Patient demographics, clinicopathologic features, and outcomes are presented in Table 1. In addition, noninvasive low- (n = 20) and high-grade (n = 20) papillary UCCs identified on biopsy were used for comparison for P-mTOR and P-S6 expression.Table 1Patient Demographics and Clinical OutcomesTotal primary carcinomasNonmetastatic subsetMetastatic subsetPatient number, n1215269Patient age, y Mean676866 Median687165 Range33–8933–8943–84Male:female ratio98:2345:753:16Pathologic stage, pT pT13 (2%)2 (4%)1 (2%) pT229 (24%)16 (31%)13 (19%) pT384 (69%)34 (65%)50 (72%) pT45 (4%)0 (0%)5 (7%)Positive margin32 (27%)12 (23%)20 (29%)Clinical follow-up available118 (98%)50 (96%)68 (99%) Mean follow-up, mo283324 Median follow-up, mo222717Subsequent metastases38 (32%)15 (30%)23 (33%)Dead of disease74 (63%)25 (50%)49 (71%) Open table in a new tab Tissue microarrays were prepared from both nonmetastatic and metastatic UCCs. Each specimen was represented by four 1.0-mm cores on tissue microarray to obtain adequate representation of different regions of neoplastic cells to assess for intratumoral heterogeneity and which has been demonstrated to adequately represent tissue variation within UCC.14Nocito A Bubendorf L Tinner EM Süess K Wagner U Forster T Kononen J Fijan A Bruderer J Schmid U Ackermann D Maurer R Alund G Knönagel H Rist M Anabitarte M Hering F Hardmeier T Schoenenberger AJ Flury R Jäger P Fehr JL Schraml P Moch H Mihatsch MJ Gasser T Sauter G Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade.J Pathol. 2001; 94: 349-357Crossref Scopus (271) Google Scholar Slides were deparaffinized in fresh xylenes and rehydrated through sequential-graded ethanol steps. For P-S6, antigen retrieval was performed by citrate buffer incubation [18 mmol/L sodium citrate (pH 6.0)] using a household vegetable steamer for 60 minutes. Slides were incubated for 5 minutes with 3% hydrogen peroxide, washed in TBS/T [20 mmol/L Tris, 140 mmol/L NaCl, 0.1% Tween 20 (pH 7.6)], and incubated in appropriate antibody dilution for P-S6 (Thr389, 1:50, Cell Signaling Technologies) overnight at 4°C. P-S6 immunostaining was validated by a secondary analysis with Ser240/244 antibody (1:50, Cell Signaling Technologies) with no significant differences in pathway activation identified. Normal saline was substituted for the primary antibody in control sections. The avidin-biotin-peroxidase complex method from DAKO (Glostrup, Denmark) was used, and slides were subsequently counterstained with hematoxylin. P-mTOR immunohistochemistry was performed using an automated stainer (Ventana Medical Systems, Tucson, AZ) with monoclonal antibody against P-mTOR (clone 49F9; Cell Signaling Technologies). Assessment of immunohistochemical labeling of the tissue microarrays was performed by one of the authors (D.E.H.). Immunostaining was scored semiquantitatively including both intensity of stain (0, no staining; 1, weak staining; 2, moderate staining; 3, intense staining) and percentage of immunoreactive cells within the lesion. Comparison of immunolabeling between populations was performed using the Fisher exact test. Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL) and Statistica (Statsoft Inc., Tulsa, OK). All tests were two-tailed, analyzed at a significance level of P ≤ 0.05, with a statistical trend defined as P ≤ 0.1. The Mann–Whitney test was used to compare the probability distributions from two independent groups, one-way analysis of variance was used to compare means between independent groups. Pearson correlation was used to determine the linear dependence between two variables. Regression (Logistic, Ordinal) was used for predicting probability for data, and survival analysis was performed using the Cox Proportional Hazards model. The end point in survival analysis was disease-related death, measured in time as a function of months from surgery until death. The groups mTOR and S6 intensity were both dichotomized along their median (0–1) into 0, and (2–3) into 2. UCC cell lines T24, RT4, UMUC3, and J82 were purchased from the American Type Culture Collection cell line bank (ATCC, Manassas, VA). Cells were grown on 60-mm Falcon dishes (Becton Dickinson Labware, Franklin Lakes, NJ) in RPMI-1640 (GIBCO, Invitrogen Corp., Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO) and, for labeling analysis, subsequently plated onto LAB-TEK II 4-well chambered slides (Fisher Scientific, Pittsburgh, PA). Cells were maintained at 50% confluence. Twenty-four hours before rapamycin addition (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), the cell culture medium was replaced with RPMI/0.5% fetal bovine serum. After serum starvation, graded doses of rapamycin (1 picomolar to 1 micromolar concentration for 48 hours) were applied to cells. For proliferation assays, cells were subsequently labeled for 6 hours using the BrdU Labeling Kit I (Roche Diagnostics, Indianapolis, IN) and stained per protocol. Experiments were performed in triplicate, and cell counts per treatment condition included, on average, 1291 (J82), 1015 (T24), 1738 (RT4), and 588 (UMUC3) cells. For apoptosis analysis, cells were analyzed using the in Situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN) following the same treatment protocol described for proliferation assays. Cell migration assays were performed using the Boyden Chamber motility assay (Costar transwell permeable support, 8 μmol/L) using 60,000 cells per well. Cells were treated with DMSO or varying concentrations of rapamycin in DMSO for 36 hours, followed by fixation with absolute methanol for 10 minutes and visualization by crystal violent stain (Mallinckrodt Baker, Phillipsburg, NJ). Comparison between treatment populations was performed by two-tailed Student t test analysis. For immunohistochemical analysis, cells were trypsinized, resuspended in RPMI-1640, and spun to collect the cell pellet, which was fixed with 10% cold neutral buffered saline overnight at 4°C. The following day, cells were centrifuged, washed twice in PBS, and incubated with plasma and thrombin to form a cell clot, which was embedded in paraffin using a standard tissue-processing cassette and processor, and 5 μmol/L sections were obtained on charged slides (Cardinal Health, Dublin, OH). Immunohistochemical analysis was performed as described for paraffin-embedded tissue specimens. Scrambled control and mTOR siRNA were obtained from Cell Signaling Technologies and cells were transfected with OPTI-MEMI (Gibco). Confluent cells were extracted into lysis buffer consisting of 25 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 1% Triton-X-100, 1 EDTA, 0.1% SDS, protease inhibitor cocktail (Sigma, St. Louis, MO), and phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), centrifuged at 10,000g for 30 minutes at 4°C, and the supernatant containing soluble proteins was collected. Protein concentration was measured using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Twenty-five μg of total protein per sample was fractionated on a standard SDS-polyacrylamide gel containing 10% acrylamide/0.8% bis-acrylamide and transferred via semidry transfer cell (Trans-blot SD, Bio-Rad) to nitrocellulose membranes (Millipore, Bedford, MA) in 48 mmol/L Tris, 39 mmol/L glycine, 0.0375% SDS, and 20% methanol. Prestained molecular weight standards were run concurrently with protein samples (Lonza, Rockland, ME). Blots were blocked with 1% bovine serum albumin diluted in 10 mmol/L Tris-HCl (pH 7.5) and 150 mmol/L NaCl containing 0.05% Tween 20 (TBS/T; phosphorylated p70S6kinase-1 antibody) or in 5% Carnation instant milk in TBS/T (for remaining antibodies) for 1 hour at room temperature. Antibodies included P-mTOR (1:1000), total mTOR (1:1000), P-p70S6kinase-1 (P-S6K; 1:1000), total S6K (1:1000), P-S6 (1:1000), total S6 (1:1000), P-AKT (Ser473; 1:500), P-AKT (Thr308; 1:500); total AKT (1:1000), Rictor (1:1000), Raptor (1:1000), TSC1 (1:1000), TSC2 (1:1000), and PARP (1:2000), all from Cell Signaling Technologies. Antibodies for PTEN (clone 6H2.1) were obtained from Cascade Bioscience (Winchester, MA) and used at a dilution of 1:500. Blots were incubated with antibody overnight at 4°C in blocking solution and rinsed, followed by incubation for 2 hours at room temperature with alkaline phosphatase conjugated anti-rabbit IgG antibody (1:30,000; Amersham Corp.) and visualized using the Enhanced Chemiluminescence Kit (Amersham Corp.). Proteins were visualized using chemifluorescence scanning Storm 860 Molecular Imager (GMI, Ramsey, MN). After primary antibody incubation, blots were stripped in 62.5 mmol/L Tris, pH 6.8, containing 100 mmol/L [β]-mercaptoethanol and 2% SDS for 30 minutes at 50°C. Western blots were reprobed using a 1:1000 dilution of either total mTOR or total S6K (Cell Signaling Technologies) and reprobed with a 1:1000 dilution of rabbit polyclonal anti-actin antibody (Sigma Chemical Company, St. Louis, MO) for 1 hour. Genomic DNA was extracted from T24 and UMUC3 cells and the PTEN gene was analyzed via standard PCR, using GC-clamped intronic primers that encompass each of the 9 PTEN exons and flanking intronic sequences, followed by denaturing gradient gel electrophoresis analysis,15Mutter GL Lin MC Fitzgerald JT Kum JB Baak JP Lees JA Weng LP Eng C Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.J Natl Cancer Inst. 2000; 92: 924-930Crossref PubMed Google Scholar which has proven more than 99% sensitive and specific for intragenic “small” mutations. Identified denaturing gradient gel electrophoresis variants were next subjected to DNA sequencing16Liaw D Marsh DJ Li J Dahia PL Wang SI Zheng Z Bose S Call KM Tsou HC Peacocke M Eng C Parsons R Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.Nat Genet. 1997; 16: 64-67Crossref PubMed Scopus (1692) Google Scholar to determine specific sequence variations within targeted regions. Xenograft studies were performed as a collaborative effort with University of Virginia under institutional IACUC approval. Subcutaneous inoculation of 6-week-old nude mice was performed using 106 T24 cells in 50% matrigel (BD Biosciences, San Jose, CA) for both control (n = 9) and rapamycin (n = 8) groups. Mice were examined and weighed weekly until the establishment of visible tumors. Once established, mice were given twice-weekly intraperitoneal injections of either 2.5 mg/kg rapamycin in DMSO or vehicle alone.17Birle DC Hedley DW Signaling interactions of rapamycin combined with erlotinib in cervical carcinoma xenografts.Mol Cancer Ther. 2006; 5: 2494-2502Crossref PubMed Scopus (30) Google Scholar Subcutaneous tumors were measured twice weekly for 4 weeks using calipers, and tumor volume was calculated in mm3, with statistical significance calculated via the Wilcoxon Signed Ranks Test Tumors were embedded in formalin, sectioned, and subsequently immunostained with Ki-67 (predilute; Ventana Medical Systems, Inc., Tucson, AZ) using the Ventana Benchmark XT. Statistical analysis of proliferation indices between populations was performed using the Student t test. To examine the potential role of the mTOR pathway in UCC, we first examined the frequency of mTOR activation in a consecutive cohort of 121 muscle-invasive UCCs using well-characterized antibodies targeting phosphorylated mTOR (P-mTOR, Ser2448), a marker of mTOR activation,18Altomare DA Wang HQ Skele KL De Rienzo A Klein-Szanto AJ Godwin AK Testa JR AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth.Oncogene. 2004; 23: 5853-5857Crossref PubMed Scopus (349) Google Scholar, 19Chiang GG Abraham RT Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase.J Biol Chem. 2005; 280: 25485-25490Crossref PubMed Scopus (432) Google Scholar and phosphorylated S6 (P-S6), a downstream target of mTOR. Specimens included 52 cases of primary UCCs without metastases (“non-metastatic UCCs”) and 69 cases of primary UCCs (“metastatic UCCs”) with concurrent regional lymph node metastases (Table 1). Of these latter cases, 59 paired lymph node metastases were available for analysis. No significant differences in patient age, gender, pathological stage, margin status, or clinical follow-up were present between populations. Semiquantitative analysis of tumor cells expressing either P-mTOR or P-S6 was performed and was tallied as a continuous score from 0 to 100% and as a range of immunoreactivity (0 to 3+ intensity; Figure 1, A–I). Phosphorylation of mTOR was identified in 90/121 (74%) primary UCCs, with the majority of tumors demonstrating 2 to 3+ immunoreactivity (82/121; 68%; Figure 2A). P-mTOR expression was present in at least 25% of cells in approximately half of all P-mTOR-expressing UCCs (19/52 non-metastatic and 26/69 metastatic UCCs; Figure 2B), with the remainder demonstrating more focal P-mTOR expression. No significant difference was apparent either between nonmetastatic or metastatic primary UCC or between primary UCCs and lymph node metastases when comparing either intensity of expression (P = 0.56 and P = 0.85, respectively) or percentage of positive cells (P = 1.0 and P = 0.29, respectively). Although phosphorylation of mTOR has been used extensively as a biomarker to indicate activation of the mTOR pathway in cancer cells, we additionally examined the phosphorylation of ribosomal S6 protein, a downstream target of mTOR. In contrast to P-mTOR levels, P-S6 demonstrated a slight reduction in both intensity of expression and percentage of positive cells, with the greatest difference identified in the primary UCC populations. Overall, expression of P-S6 was identified in 66/121 (55%) of all primary UCCs examined. P-S6 intensity of 2 to 3+ was reduced to 52% (27/52) of nonmetastatic and 35% of metastatic primary UCCs (Figure 2C). Similarly, fewer cases demonstrated at least 25% tumor cell immunoreactivity in the primary UCC component, which was present in only 27% (14/52) of nonmetastatic and 22% (15/69) of metastatic lesions (Figure 2D). Robust expression of P-S6, however, remained constant in lymph node metastases, with a significant increase in P-S6 status, defined as both intensity of staining and percentage of positive cells, in the lymph node metastases versus the paired primary carcinoma (P < 0.001 and P = 0.01, respectively). Concordance between P-mTOR and P-S6 status, defined as identical intensity and %positive cells within 20%, was present in 31/52 non-metastatic (60%) and 49/69 (71%) of metastatic primary carcinomas, with the majority of discordant cases representing a reduction in P-S6 levels relative to P-mTOR, as demonstrated in Figure 2. We next evaluated a set of noninvasive lesions, including 20 low-grade papillary UCCs and 20 high-grade papillary UCCs. In low-grade lesions, P-mTOR intensity of 2 to 3 was present in 13/20 (65%) lesions and was present in approximately 50% of cells on average (range, 5 to 80%). Similar to that identified in muscle-invasive UCCs, P-S6 expression was more limited, present in 36% of cells with an average intensity of 2. Of note, P-S6 expression in noninvasive low-grade papillary UCC appeared to be restricted in many cases to the upper half of the urothelium (Figure 3A). Similar results were identified for noninvasive high-grade papillary UCC (P-mTOR mean %cells 31%, range 5 to 80%, mean intensity 2; P-S6 mean %cells 38%, range 10 to 100%, mean intensity 2). In contrast to low-grade lesions, high-grade papillary UCC often demonstrated full-thickness expression of P-S6 (Figure 3B). As phosphorylation of mTOR and S6 appeared to be increased in lymph node metastases, an indicator of aggressive disease, we next examined the relationship between P-mTOR and P-S6 status and clinicopathologic parameters, including positive margin status, pathological stage (depth of invasion), development of subsequent metastases, and disease-specific survival. P-mTOR intensity, defined and dichotomized as 0 to 1+ (no to weak staining) or 2 to 3+ (moderate to strong staining), was evaluated against pathological parameters using the Mann–Whitney test. The probability distributions were found to differ for depth of invasion (P < 0.001), and ordinal regression analysis was performed. Increased P-mTOR intensity was found to be significantly associated across all categorical levels (0 to 2)) of invasion depth relative to the highest level of 3 (P < 0.01, P = 0.05, P = 0.02, respectively), with a stronger P-mTOR intensity yielding a more aggressive invasion. P-mTOR intensity also demonstrated a significant inverse relationship with length of survival using one-way analysis of variance analysis (P = 0.04), which is graphically represented (Figure 4A). In addition, increased percentage of P-mTOR–positive cells was also independently associated with increased depth of invasion on a continuous regression (P < 0.01) and showed a trend toward negative correlation with length of disease-specific survival using Cox Proportional Hazards model (P = 0.08, Figure 4B). No significant association between either P-mTOR intensity or percentage of P-mTOR positive cells was identified for margin status at cystectomy or the presence of subsequent metastatic disease. A similar analysis was performed for P-S6 in primary UCCs of the bladder. P-S6 intensity was found to be positively associated with increased depth of invasion across most ordinal levels relative to 3 (P < 0.01, P < 0.01, P = 0.77, respectively) No significant association was identified between percentage of P-S6–positive cells and increased depth of invasion (P = 0.47). As mTOR activity appeared to correlate with pathological stage, we assessed the effects of mTOR activity on UCC migration in vitro. Using the Boyden Chamber assay, we plated T24 and UMUC3 cells into control (DMSO-solvent containing) or rapamycin-containing (10 nmol/L, 1 μmol/L) media for 36 hours. Both T24 and UMUC3 cells treated with rapamycin, a general mTOR inhibitor, demonstrated a reduction in cells that were successfully able to migrate through the insert. Under control conditions, 285 cells (±35) T24 cells and 935 UMUC3 cells (±40) migrated through the insert. Cell migration was reduced in T24 and UMUC3 cells, respectively, to 39% and 70% with 10 nmol/L rapamycin and 35% and 45% with 1 μmol/L rapamycin. These findings suggest that mTOR activity may play a role in potentially mediating cell migration in UCC cells. Because of the frequent nature of mTOR pathway activation in UCC, we next sought to determine the role of mTOR inhibition in mediating cell proliferation in vitro. We used the immortalized UCC cell lines T24, RT4, J82, and UMUC3, which demonstrated baseline phosphorylation of mTOR and S6 by Western blot analysis. Cells were serum-starved for 24 hours and incubated with 1 pM to 1 μmol/L rapamycin for 48 hours. Control cells were incubated under parallel conditions with the solvent DMSO. Rapamycin treatment resulted in a dose-dependent decrease in proliferation within the nanomolar range of drug20Shor B Zhang WG Toral-Barza L Lucas J Abraham RT Gibbons JJ Yu K A new pharmacologic action of CCI-779 involves FKBP12-independent inhibition of mTOR kinase activity and profound repression of global protein synthesis.Cancer Res. 2008; 68: 2934-2943Crossref PubMed Scopus (114) Google Scholar(Figure 5A). Control cells demonstrated a baseline proliferative index of 41.9% (J82), 58.4% (T24), 13.1% (RT4), and 26.2% (UMUC3) of the total cell population. Addition of 1 nmol/L rapamycin reduced the proliferation index by 57% (J82), 94% (T24), 45% (RT4), and 29% (UMUC3) of control, which was statistic" @default.
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• W2101967561 date "2010-06-01" @default.
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• W2101967561 title "Mammalian Target of Rapamycin (mTOR) Regulates Cellular Proliferation and Tumor Growth in Urothelial Carcinoma" @default.
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