Urologic Oncology: Seminars and Original Investigations
Volume 30, Issue 1 , Pages 69-77, January 2012

Rictor-dependent AKT activation and inhibition of urothelial carcinoma by rapamycin

  • Ming-Ju Wu, M.D., Ph.D.

      Affiliations

    • Division of Nephrology, Taichung Veterans General Hospital, Taichung, Taiwan
    • Chung-Shan Medical University, Taichung, Taiwan
    • Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan
    • Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan
    • Corresponding Author InformationCorresponding author. Tel.: +886-4-23592525, ext 3057; fax: +886-4-23594980
    • ,
  • Chi-Hao Chang, M.Sc.

      Affiliations

    • Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan
    • Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan
    • ,
  • Yung-Tsung Chiu, Ph.D.

      Affiliations

    • Department of Research, Taichung Veterans General Hospital, Taichung, Taiwan
    • ,
  • Mei-Chin Wen, M.D.

      Affiliations

    • Department of Pathology, Taichung Veterans General Hospital, Taichung, Taiwan
    • ,
  • Kuo-Hsiung Shu, M.D.

      Affiliations

    • Division of Nephrology, Taichung Veterans General Hospital, Taichung, Taiwan
    • Chung-Shan Medical University, Taichung, Taiwan
    • ,
  • Jian-Ri Li, M.D.

      Affiliations

    • Division of Urology, Taichung Veterans General Hospital, Taichung, Taiwan
    • ,
  • Kun-Yuan Chiu, M.D.

      Affiliations

    • Division of Urology, Taichung Veterans General Hospital, Taichung, Taiwan
    • ,
  • Yen-Ta Chen, M.C.

      Affiliations

    • Division of Urology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan

Received 11 September 2009; received in revised form 8 November 2009; accepted 10 November 2009. published online 08 March 2010.

Article Outline

Abstract 

Objective

We previously reported a very high cumulative incidence of urothelial carcinoma in Taiwanese kidney transplant recipients. Rapamycin, the inhibitor of mTOR Complex 1, provides alternative immunosuppressive therapy after kidney transplantation with less neoplastic potential. We examined the in vivo and in vitro effects of rapamycin on urothelial carcinoma.

Materials and methods

The rat model of urothelial carcinoma was induced by 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in Fischer F344 rats. The anti-tumor effect of rapamycin was assessed grossly, microscopically, and by Western blot analysis. The mechanism of rapamycin's attenuation of urothelial carcinoma was also evaluated by T24 cells.

Results

Rapamycin significantly reduced urinary bladder tumor growth in the rat model of 0.05% BBN-induced urothelial carcinoma (P < 0.001). The blood trough levels of rapamycin were correlated with the occurrence of urothelial carcinoma. In vitro, rapamycin also inhibited the cell proliferation, migration, and invasion, as well as the protein expression of vascular endothelial growth factor-A of T24 urothelial carcinoma cells, whereas rapamycin did not induce significant apoptosis in T24 cells. Rapamycin decreased the expression of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A. Rapamycin also activated AKT in T24 cells in the rat model of urothelial carcinoma. The rapamycin-associated activation of AKT was inhibited by rictor siRNA, but not raptor siRNA.

Conclusions

This study provides in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings also suggest rictor-dependent AKT activation as a consequence of mTORC1 inhibition.

Keywords:  AKT , Rapamycin , Rictor , Urothelial carcinoma , VEGF

 

Back to Article Outline

1. Introduction 

Post-kidney transplant malignancy is one of the most serious complications of general immunosuppressive drugs [1], [2]. Cancer has, therefore, become a major cause of death in kidney transplant recipients [3]. One approach to addressing this problem is to identify drugs that have potent immunosuppressive effects but low proneoplastic effects, or even some antineoplastic properties [4]. The present study presents evidence that rapamycin may fulfill these diverse needs.

Rapamycin is a bacterial macrolide with potent immunosuppressive activities. mTOR is an important regulator of cell growth and proliferation [5]. mTORC1 blocks the activation of signal transduction pathways and induces arrest of the cell cycle in the G1 phase. Through the blockade of interleukin-2-induced proliferation of T cells, mTOR inhibitors preserve renal allograft function, while producing excellent rejection-free and graft survival rates [6]. Beyond their immunosuppressive properties, the mTOR inhibitors have proven anticancer activities [2]. There is growing evidence that mTOR inhibitors, alone or in combination with other calcineurin inhibitors, may reduce the risk of cancer development after kidney transplantation [7], [8]. Temsirolimus, an ester analog of rapamycin, has been approved as an anticancer agent [9].

Skin tumors, followed by lymphoma, are the most common tumors arising after kidney transplantation in most Western countries [1], [2]. However, unlike in Western countries, there is a very high cumulative incidence of urothelial carcinoma in Chinese kidney transplant recipients [10], [11], [12], [13], [14]. Similar to previous reports, in our retrospective study we found that a rapamycin-based regimen was associated with a lower incidence of post-transplant malignancy, including urothelial carcinoma [15]. Potentially, mTOR inhibitors could be considered the drug of choice in groups at high risk of developing de novo malignancies after kidney transplantation. However, conversion of calcineurin inhibitors to rapamycin failed to rescue all post-kidney transplant malignancies, including urothelial carcinoma [16]. The negative mTORC1/S6K/IRS/PI3K feedback loop might be part of the answer. The current in vitro and in vivo study was designed to clarify the effect and mechanism of rapamycin on urothelial carcinoma.

Back to Article Outline

2. Materials and methods 

2.1. Animal model 

The rat model of bladder cancer was induced by adding 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to the drinking water for 20 weeks [17]. Forty male Fisher-344 rats were randomly divided into 4 groups: (1) control group, 10 rats treated only with vehicle (PBS) instead of rapamycin by gavage once daily; (2) rapamycin group, 10 rats treated with 2 mg/kg/d rapamycin (Wyeth-Ayerst, Taipei, Taiwan); (3) BBN-vehicle, 10 rats treated with both BBN in drinking water and vehicle instead of rapamycin; and (4) BBN-rapamycin 2 mg/kg/d, 10 rats treated with both BBN in drinking water and 2 mg/kg/d rapamycin. All rats were sacrificed under pentobarbital anesthesia after 20 weeks of treatment. Whole-blood samples were collected before harvesting. A high performance liquid chromatography (HPLC)-UV method was used to measure blood trough levels of rapamycin.

2.2. Morphometric analysis 

After fixing the bladder in 10% formalin, the lumen was inspected for grossly visible lesions, and the whole bladder was submitted for microscopic examination. The stage of urothelial carcinoma was recorded by observation under a dissecting microscope, and sections were examined histologically by 2 reviewers who were blinded to the treatment. All immunohistochemical studies were performed on paraffin-embedded sections [18]. The 4-μm-thick deparaffinized sections were incubated with the primary antibodies of VEGF-A (1:50) and phosphor-AKT (Ser473, 1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA). As a negative control, the primary antibody was replaced with normal rabbit IgG, without staining.

2.3. Cell culture and rapamycin treatment 

The human transitional cell carcinoma cell line T24 (G3, p53 mutant type) was obtained from American Type Culture Collection (Rockville, MD). The T24 cells were maintained in McCoy's 5A medium (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated fetal calf serum supplemented with 100 U/ml penicillin-G, 100 g/ml streptomycin, and 2 mM L-glutamine (CCS; HyClone, Logan, UT). To evaluate the inhibitory effect of rapamycin on T24 cell viability, migration, VEGF secretion, and protein activity, T24 cells were cultured in 60-mm dishes for 24 h with rapamycin (Sigma-Aldrich).

2.4. MTT assay for cell proliferation activity 

T24 cells (5 × 104) were plated into each well (500 μl/well) of 24-well culture plates for 24 hours and treated with rapamycin at concentrations of 10 ng/ml and 100 ng/ml, respectively, or vehicle alone for another 24 to 48 hours. MTT solution (50 μl at 5 mg/ml) was then added to each well, and the plates further incubated at 37°C for 4 hours in an incubator at 5% CO2. The medium was then aspirated to ease the formation of the formazan product, which was then solubilized with the addition of DMSO. An aliquot of 200 μl was measured and transferred into 96-well plates. The optical density was measured at OD570–OD630 with a microplate autoreader (Dynex Technologies, Chantilly, VA). Experiments were repeated in triplicate.

2.5. Wound scratch assay for cell migration activity 

T24 cells (2 × 104) were seeded into 6-well tissue culture dishes and cultured in medium containing 10% FBS to a confluent cell monolayer, then carefully wounded using sterile 200-μl pipette tips. Any cell debris was removed with PBS. The cells were then incubated in 100 ng/ml rapamycin or vehicle alone for 24 hours and photographed under a phase contrast microscope. Experiments were repeated in triplicate.

2.6. Transwell motility assay for cell migration activity 

T24 cells were plated at 105 cells/cm2 in the upper compartment of an 8-μm pore-size transwell migration chamber (Corning, Acton, MA) and cultured in medium containing rapamycin (10 and 100 ng/ml) or vehicle alone for 72 hours. The cells on the upper surface were then removed by wiping with a cotton swab, the filter gently removed from the chamber, and mounted on glass slides. The cells that had invaded the filter and attached to its lower surface were fixed, stained with Giemsa stain solution (Sigma), and counted in 10 randomly selected microscopic fields (at 200× magnification) per filter. Experiments were repeated in triplicate.

2.7. Cell cycle analysis 

Cell cycle analysis was performed with a flow cytometer (FACS Callibur; BD, San Jose, CA). T24 cells were cultured and treated with rapamycin or vehicle alone for 48 hours. The floating and adherent T24 cells were collected, fixed, and permeabilized with 70% ethanol at 4°C, then incubated with 30 μg/ml propidium iodide and 100 μg/ml RNase. Data acquisition was performed in the flow cytometer with the accompanying software (CellQuest, BD). Appropriate gating was used to select the easily distinguished single population. Ten thousand events per sample were counted, and determinations were made at least in triplicate to assure the distribution of each cell cycle.

2.8. Vascular endothelial growth factor-A ELISA assay 

T24 cells were cultured with McCoy's 5A medium containing 10% FBS and treated with vehicle alone or 10, 100, or 1,000 ng/mL rapamycin for 48 hours. T24 condition media were collected at different time points, and the Human-VEGF-A ELISA kit (BioSource, Camarillo, CA) was used to detect the concentration of VEGF-A in T24 condition medium. Experiments were repeated in triplicate.

2.9. Western blot analysis 

For Western blots, cells and tissue were prepared with primary antibodies, as previously reported [18]. Primary antibody was detected using horseradish peroxidase-linked goat anti-mouse or goat anti-rabbit IgG antibody at a 1:1,000 dilution (Santa Cruz) and visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL). Primary antibodies used included mTOR, phospho-mTOR (Ser2448), ERK, phospho-ERK (Thr202/Tyr204), regulator associated protein of mTOR (raptor), and AKT (Cell Signaling, Boston, MA), phospho-AKT (Ser473), p70S6K, phospho-p70S6K (Thr421/Ser424), cyclin D1, and VEGF-A (Santa Cruz).

2.10. siRNA inhibition 

One day before transfection, the T24 cells (1 × 105) were seeded in 6-well plates in cell culture medium without antibiotics. T24 cells were transfected by the 10 nmol/l regulator associated protein of mTOR (raptor) 10 nmol/l siRNA and rapamycin-insensitive companion of mTOR (rictor) siRNA (Santa Cruz) by the INTERFERin siRNA reagent according to the manufacturer's instructions (Polyplus-transfection, New York, NY). Then the T24 cells were treated with vehicle or 100 ng/ml rapamycin for 24 hours. The T24 cell lysates were collected by RIPA buffer (Upstate Biotechnology, Lake Placid, NY) containing protease inhibitor cocktail (Complete, Roche, Mannheim, Germany) and subjected to Western blot analyses. Similar experiments were repeated in triplicate.

2.11. Statistics 

All data are expressed as mean ± SEM. Statistical calculations were performed using the SPSS software for statistical analysis (SSPS, Inc, Chicago, IL). Statistical differences between 2 groups were analyzed using unpaired, two-tailed t-tests, and multiple groups were compared using one-way ANOVA with post hoc Bonferroni correction. Statistical significance was defined as a P < 0.05.

Back to Article Outline

3. Results 

3.1. Rapamycin exhibits potent anti-tumor properties against BBN-induced urothelial carcinoma 

In vivo, no tumor was noted in control rats and rats treated with rapamycin alone. Typical papillary bladder tumors were noted in all Fisher-344 rats treated with 0.05% BBN in drinking water for 20 weeks. All rats treated with 0.05% BBN in drinking water for 20 weeks had high-grade urothelial carcinoma in the urinary bladder. Compared with the BBN group, combined treatment with rapamycin and BBN had significantly reduced occurrences of urothelial carcinoma. As shown in Table 1, 60% of rats treated with BBN and rapamycin did not develop urothelial carcinoma, while another 30% had only low-grade, noninvasive tumors (stage pTa). Only 10% had high-grade, invasive papillary tumors (stage pT1) (P < 0.001). All pathologic findings were confirmed by 2 independent reviewers. At the start of the study, there was no difference in weights of the 4 groups of rats; their average weight was 174.5 ± 9.9 g. The final weight of the rats was 382 ± 17.3 g in the control group, 361.9 ± 12.7 g in the rapamycin group, 375.4 ± 18.5 g in the BBN group, and 354.1 ± 20 g in the BBN + rapamycin group. Combined treatment with rapamycin and BBN was associated with less weight gain; P values were 0.006 and 0.056 compared with the control group and the BBN group, respectively.

Table 1. The tumor growth induced by 0.05% BBN-induced uroepithelial carcinoma in Fisher-344 rats
GroupsControlRapamycin0.05% BBN0.05% BBN + rapamycin
High grade tumor (stage pT1)0%0%100%10%
Low grade tumor (stage pTa)0%0%0%30%
No tumor detected100%100%0%60%⁎

⁎ P < 0.05, compared with BBN group.

The blood trough level of rapamycin in the BBN + rapamycin group was 5.7 ± 1.4 ng/mL. Lower blood trough levels of rapamycin were associated with the occurrence of urothelial carcinoma (4.3 ± 0.5 vs. 6.7 ± 0.8 ng/ml, P < 0.05). Fig. 1 shows the representative gross morphology and histologic findings of the rat bladders with hematoxylin and eosin staining, VEGF-A staining, and phosphor-AKT (Ser473) staining. Rats treated with both rapamycin and BBN showed significantly decreased VEGF-A expression, but increased phosphor-AKT, compared with BBN-treated rats.

  • View full-size image.
  • Fig. 1. 

    Effects of rapamycin on 0.05% BBN-induced urothelial carcinoma. Representative photographs show the gross pictures of bladders (A–D), hematoxylin-eosin staining (E–H), and vascular endothelial growth factor-A staining (I–L), phosphor-AKT (Ser473) staining (M–P) from the control vehicle group (A, E, I), 2 mg/kg/day rapamycin group (B, F, J), BBN-vehicle group (C, G, K), and BBN-rapamycin 2 mg/kg/day group (D, H, L). Original magnification × 400. (Color version of figure is available online.)

Based on the anti-tumor effects of rapamycin in the rat model of urothelial carcinoma induced by BBN, we examined whether the effects of this potential therapeutic agent were reflected by a decrease in the phosphorylation of mTOR and p70S6K in vivo. As shown in Fig. 2, when lysates obtained from rats treated by BBN + rapamycin were examined by Western blotting, there was a clear reduction in the accumulation of phospho-mTOR and phoshpo-p70S6K without any consistent variation in the total levels of mTOR, p70S6K, and p27. Furthermore, the expressions of VEGF-A and cyclin D1 were markedly increased in the BBN-treated group. Again, the effect of rapamycin was documented by the almost complete disappearance of the band corresponding to VEGF-A and cyclin D1. Of interest, the phosphorylation of AKT and ERK was enhanced with rapamycin treatment (Fig. 2A). These results are consistent with the ability of the mTOR-p70S6K pathway to control the expression of VEGF-A, and they indicate that inhibition of mTORC1 by rapamycin may activate the PI3K-AKT pathway. Moreover, the expression of phospho-AKT was notably higher in rat bladder tumors after rapamycin treatment.

  • View full-size image.
  • Fig. 2. 

    Western blot analyses for the effects of rapamycin on the 0.05% BBN-induced urothelial carcinoma (A), the effects of rapamycin, raptor siRNA, and rictor siRNA on T24 cells (B, C). The expressions of phospho-mTOR and cyclin D1 are significantly reduced by 100 ng/mL rapamycin. Rapamycin also increased AKT phosphorylation (B). Raptor siRNA strongly increases AKT phosphorylation, even more than rapamycin treatment. Rictor siRNA, but not raptor siRNA, prevents rapamycin-induced AKT phosphorylation in T24 cells (C). The same blots were stripped and reprobed with actin to confirm equal loading.

3.2. Rapamycin inhibits T24 cell proliferation, migration, and invasion 

The in vitro anticancer effect of rapamycin was tested using a human invasive urothelial carcinoma T24 cell line. On the MTT assay, rapamycin treatment (10 and 100 ng/ml) for 24 to 48 hours significantly inhibited T24 cell viability in a dose- and time-dependent manner (Fig. 3A). All OD values are relative to the OD at 0 hour. The ratios of OD at 570 nm are shown at 24 and 48 hours incubation. Each value is the mean ± SEM of 3 experiments. Compared with vehicle control, T24 cell viability was reduced to 76.8% at 24 hours and 75.9% at 48 hours with 10 ng/ml rapamycin. With 100 ng/ml rapamycin, T24 cell viability was further reduced to 53.6% at 24 hours and 44.8% at 48 hours.

  • View full-size image.
  • Fig. 3. 

    Effect of rapamycin on T24 cell proliferation, migration, and invasion. MTT assay in the presence of vehicle and rapamycin at 10 ng/mL and 100 ng/mL for T24 cells (A). Representative photographs show the result of wound scratch assay that the scratch gap dimension with rapamycin treatment for 24 hours was significantly bigger than in control T24 cells (B). The T24 cells spreading along the wound edges were significantly delayed (44.9%) by rapamycin treatment (C). Representative photographs show the nuclei of T24 cells which invaded the filter and attached on the lower surface of the filter, stained with Giemsa stain after treatment of rapamycin 10, 100 ng/ml or vehicle alone for 72 h (D). Invasion capability after rapamycin treatments are expressed as the ratio of cell nuclei numbers of rapamycin to vehicle groups. Rapamycin significantly inhibited the invasion capability of T24 cells in a dose-dependent manner (E). Each value is the mean ± S.E.M of three experiments.

The ability of tumor cells to migrate is closely associated with their metastatic potential; hence, the wound scratch assay was used to assess this potential. The scratch gap dimension with rapamycin treatment for 24 hours was significantly bigger than in control T24 cells (Fig. 3B). This result implies that the migration capability of T24 cells spreading along the wound edges was significantly delayed (44.9%) by rapamycin treatment (Fig. 3C).

Because of the scratch-space limitation in the wound scratch assay, the transwell motility assay was used in parallel to investigate the metastatic potential of T24 cells at a further 72 hours post-rapamycin treatment. The invasion capability was also inhibited in a dose-dependent manner without the addition of any chemoattractant, as has often been cited elsewhere, to be required (Fig. 3D). Compared with vehicle control, the cell nuclei numbers were reduced by rapamycin to 63.2% (10 ng/ml) and 17.8% (100 ng/ml), respectively (Fig. 3E).

3.3. Effect of rapamycin on cell cycle arrest in T24 cells 

To evaluate the effect of rapamycin on cell cycle arrest in T24 cells, flow cytometric analysis was performed on T24 cells that had been treated by rapamycin. After cultivation for 48 hours, flow-cytometric analysis showed more phase G1 delay, as shown in Fig. 4A, in the 10 and 100 ng/ml rapamycin-treated groups. The percentages of cells delayed in phase G1 were increased by 12.1% and 12.9%, using doses of 10 and 100 ng/mL rapamycin, respectively.

  • View full-size image.
  • Fig. 4. 

    (A) The flow cytometric analysis of T24 cells in the presence of rapamycin 100 ng/mL or vehicle alone. Significant cell arrest occurs at phase G1 with rapamycin. The percentages arrested in phase G1 by rapamycin dosages of 10 and 100 ng/mL were reduced by 12.1% and 12.9%, respectively. (B, C) The expression of VEGF-A in human bladder cancer T24 cells treated with rapamycin or vehicle alone. The concentration of VEGF-A was determined by enzyme-linked immunosorbent assay. Each value is the mean ± S.E.M of three experiments. (B) Treatment with rapamycin 10, 100 and 1000 ng/mL for 24 h significantly inhibits the production of VEGF-A in T24 cells. (C) Rapamycin 100 ng/mL significantly inhibits the production of VEGF-A in T24 cells on a time dependent manner.

3.4. Rapamycin abolishes the expression of VEGF-A in T24 cells 

To determine whether rapamycin inhibits the in vitro expression of VEGF-A in T24 cells, the expression of VEGF-A in culture medium was measured using an ELISA kit after rapamycin treatment. VEGF-A expression in T24 cells increased gradually during the 48 hours culture period. Fig. 4B and C show that rapamycin treatment significantly abolished the VEGF-A increase in a dose- and time-dependent manner.

3.5. Inhibition of mTOR phosphorylation leads to rictor-dependent AKT phosphorylation in T24 cells 

The effect of rapamycin on the PI3K/AKT/mTOR/S6K pathway and the possible feedback loop after rapamycin treatment was explored in T24 cells. On Western blot analysis, Fig. 2B shows that the expressions of phospho-mTOR and cyclin D1 were significantly reduced by 100 ng/ml rapamycin, while the expressions of total mTOR, total AKT, total ERK, phosphor-ERK, and p27 were unaffected. In agreement with the in vivo findings, rapamycin treatment increased AKT activation as judged by its phosphorylation of serine 473. Next, the effect of the binding partners of mTOR, raptor, and rictor on rapamycin-induced phosphor-AKT expression was examined. Raptor siRNA treatment strongly increased the phosphorylation of AKT, even more than rapamycin treatment. Treatment with rictor siRNA, but not raptor siRNA, prevented rapamycin-induced AKT phosphorylation in T24 cells (Fig. 2C).

Back to Article Outline

4. Discussion 

In Taiwan, urothelial carcinoma is the most common type of cancer after kidney transplantation, with a prevalence of 4.1%, followed by hepatocellular carcinoma observed in 2.7% of the patients. The cumulative incidence of urothelial carcinoma was 3% after 3 years of graft survival, increasing to 7.2% at 6 years, and 17.5% at 10 years. The age at the time of kidney transplantation, female gender, compound analgesics use, Chinese herb use, and underground water intake contaminated by arsenic, but not cigarette smoking, had statistical significance as risk factors for developing urothelial carcinoma after kidney transplantation [10]. Kidney transplant recipients in mainland China also have a high incidence of urothelial carcinoma [13], [14]. Since early 2003, increasing numbers of kidney transplant recipients have received a rapamycin-based regimen in our hospital. In agreement with data analysis from United Network for Organ Sharing (UNOS) [8], we found that maintenance immunosuppression with rapamycin was associated with a reduced incidence of de novo malignancies, including urothelial carcinoma, in our ongoing, randomized, controlled trial and cumulative data analysis. One episode of post-kidney transplant malignancy per 130 patient-years was noted in patients on rapamycin, compared with 1 in 60.4 patient-years for patients without. The incidence of urothelial carcinoma in our kidney transplant recipients was also decreased.

Rapamycin has been proven to be effective in inhibiting some types of cancers [19], [20], [21], [22], [23]. Boffa and coworkers demonstrated that rapamycin, but not cyclosporine, inhibited tumor growth and prevented the formation of distant metastases in murine KLN-205 non-small cell lung cancer [19]. Luan and colleagues reported that rapamycin conditioning of renal cancer cells up-regulated E-cadherin expression and induced phenotypic transition from invasive spindle to noninvasive cuboidal cells. In vivo, rapamycin prevented tumor growth and metastatic progression of renal cell carcinoma. Although they did not show the details, they mentioned that rapamycin treatment alone, or with cyclosporine, prolonged the survival of mice inoculated with urothelial carcinoma T24 cells [20]. Pinto-Leite and colleagues reported that rapamycin had an anti-proliferation effect on the T24 bladder carcinoma cell line [21]. The present study provides both in vivo and in vitro evidence that rapamycin inhibits urothelial carcinoma. Urothelial carcinoma is not a common cancer post-kidney transplantation in most Western countries, but it is very significant in kidney transplant recipients in Taiwan.

Using a rat model of urothelial carcinoma induced by 0.05% BBN in drinking water for 20 weeks, it was first demonstrated that rapamycin exhibits potent anti-tumor properties against 0.05% BBN-induced urothelial carcinoma. The anti-tumor effect of rapamycin on urothelial carcinoma correlates with the blood trough level of rapamycin. The blood trough levels of rapamycin in the present study were very close to the clinical levels achieved in kidney transplant recipients. It was also observed that treatment with rapamycin was associated with less weight gain in rats with or without 0.05% BBN in drinking water. Although no significant malnutrition was observed after rapamycin treatment in the present study, the role of weight change in the rapamycin-related anti-tumor effect deserves further study.

The present in vivo experiments showed that rapamycin treatment also induced a similar decrease of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A in urothelial carcinoma, as in other cancers [22], [23]. The present in vitro studies showed that rapamycin inhibited cell proliferation, migration, and invasion in invasive urothelial carcinoma T24 cells. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells [24]. In the present study, it was demonstrated that the anticancer effect of rapamycin on T24 cells is independent of apoptosis or G1 phase arrest. Beyond the antiproliferative effect, anti-angiogenic activity is another crucial mechanism responsible for the rapamycin's efficacy against tumor progression. Rapamycin, as an alternative to cyclosporine, has been shown to inhibit primary and metastatic tumor growth by angiogenesis [22]. In the present study, rapamycin reduced the T24 cell expression of VEGF-A into culture medium on ELISA. Immunohistochemical staining and Western blot analysis showed the expression of VEGF-A in the rat bladder cancer model was significantly inhibited by rapamycin.

Moreover, activation of AKT by Ser473 phosphorylation was observed in both T24 cells and rat bladder tissues after rapamycin treatment. The mTOR pathway is a very complex system, and various negative feedback loops may exist in different cancers [25]. Although the phenomenon of AKT activation after rapamycin treatment has been observed in a number of cancers, the biological and therapeutic relevance of this AKT phosphorylation in urothelial carcinoma is not well known [26], [27], [28], [29], [30]. mTOR is associated with several proteins, most critically, regulator associated protein of mTOR (raptor) and rapamycin insensitive companion of mTOR (rictor) [2], [31]. Although several investigators have provided basic knowledge of the PI3K-AKT-mTORC1 pathway, it remains uncertain what input triggers mTORC2 [32]. The knockdown of rictor suppresses AKT phosphorylation in 3T3-L1 adipocytes, which offered us the rationale for the use of rictor siRNA [33]. In agreement with a previous report, rictor-dependent AKT activation was associated with rapamycin treatment in the urothelial carcinoma T24 cell line. A recent report from Breuleux and co-workers showed that the increase of AKT S473 phosphorylation after mTORC1 inhibition is rictor-dependent and does not predict tumor cell response to PI3K/mTOR inhibition [26]. However, they did not test the finding in urothelial adenocarcinoma cell lines. Fig. 5 provides a schematic outline of the mechanism by which rapamycin inhibits bladder cancer cell proliferation, VEGF-A expression, and feedbacks of rictor-dependent AKT activation.

  • View full-size image.
  • Fig. 5. 

    Schematic representation of rapamycin's antitumor effect and AKT activation in bladder cancer cells. Inhibition of mTORC1 by rapamycin attenuates the phosphorylation of p70S6K and the expression of cyclin D1, and inhibits T24 cell proliferation and VEGF-A expression. On the other hand, rapamycin treatment leads to rictor-dependent AKT activation. The bold arrow lines indicate the main effects of rapamycin in T24 cells.

Back to Article Outline

5. Conclusions 

Taken together, the data presented provide both in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings highlight the value of a rapamycin-based immunosuppressive regimen in kidney transplant recipients. Randomized prospective clinical trials are needed to ensure the preventive effect of rapamycin in the development of urothelial carcinoma in Taiwanese kidney transplant recipients.

Back to Article Outline

Acknowledgments 

The authors thank Miss Chia-Fang Hung for her excellent technical assistance on the animal experiments. This work was supported by grant number TCVGH-963604C and 973604C from Taichung Veterans General Hospital.

Back to Article Outline

References 

  1. Vajdic CM , McDonald SP , McCredie MRE , et al.  Cancer incidence before and after kidney transplantation . JAMA . 2006;296:2823–2831
  2. Geissler EK , Schlitt HJ , Thomas G . mTOR, cancer, and transplantation . Am J Transplant . 2008;8:2212–2218
  3. Webster AC , Wong G , Craig JC , et al.  Managing cancer risk and decision making after kidney transplantation . Am J Transplant . 2008;8:2185–2191
  4. Dantal J , Pohanka E . Malignancies in renal transplantation: An unmet medical need . Nephrol Dial Transplant . 2007;22(Suppl 1):i4–i10
  5. Tsang CK , Qi H , Liu LF , et al.  Targeting mammalian target of rapamycin for health and disease . Drug Discov Today . 2007;12:112–114
  6. MacDonald AS . A worldwide, phase III, randomized, controlled, safety and efficacy study of a sirolimus/cyclosporine regimen for prevention of acute rejection in recipients of primary mismatched renal allografts . Transplantation . 2001;71:271–280
  7. Campistol JM , Eris J , Oberbauer R , et al.  Sirolimus therapy after early cyclosporine withdrawal reduces the risk for cancer in adult renal transplantation . J Am Soc Nephro . 2006;17:581–589
  8. Kauffman HM , Cherikh WS , Cheng Y , et al.  Maintenance immunosuppression with target-of-rapamycin inhibitors is associated with a reduced incidence of de novo malignancies . Transplantation . 2005;80:883–889
  9. Hudes G , Carducci M , Tomczak P , et al.  Temsirolimus, interferon α, or both for advanced renal-cell carcinoma . N Engl J Med . 2007;356:2271–2281
  10. Wu MJ , Lian JD , Yang CR , et al.  High cumulative incidence of urinary tract transitional cell carcinoma after kidney transplantation in Taiwan . Am J Kidney Dis . 2004;43:1091–1097
  11. Hung YM , Chou KJ , Hung SY , et al.  De novo malignancies after kidney transplantation . Urology . 2007;69:1041–1044
  12. Chiang YJ , Wang HH , Liu KL , et al.  Hepatocellular carcinoma following renal transplantation: Experience in northern Taiwan . Transplant Proc . 2008;40:2397–2399
  13. Tian Y , Xiao J , Du LD , et al.  Clinical study of 44 cases of urological malignant tumors following kidney transplantation . Chin J Organ Transplant . 2007;28:417–419
  14. Zhou MS , Zhu YH , Wang LM , et al.  Malignant tumor of urinary system in renal allograft recipients in one center . Chin J Organ Transplant . 2007;28:624–626
  15. Wu MJ . The role of mTOR inhibitors in renal disease . Acta Nephrologica . 2008;22:209–210
  16. Lin J , Tian Y , Tang YW , et al.  Nine cases of conversion from calcineurin inhibitor to sirolimus in the treatment of urological neoplasm in kidney allograft recipients . Chin J Organ Transplant . 2007;28:205–207
  17. Hattori K , Iida K , Joraku A , et al.  Chemopreventive effects of cyclooxygenase-2 inhibitor and epidermal growth factor-receptor kinase inhibitor on rat urinary bladder carcinogenesis . BJU Int . 2006;97:640–643
  18. Wu MJ , Wen MC , Chiu YT , et al.  Rapamycin attenuates unilateral ureteral obstruction-induced renal fibrosis . Kidney Int . 2006;69:2029–2036
  19. Boffa DJ , Luan F , Thomas D , et al.  Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer . Clin Cancer Res . 2004;10:293–300
  20. Luan FL , Hojo M , Maluccio M , et al.  Rapamycin blocks tumor progression: Unlinking immunosuppression from antitumor efficacy . Transplantation . 2002;73:1565–1572
  21. Pinto-Leite R , Botelho P , Ribeiro E , et al.  Effect of sirolimus on urinary bladder cancer T24 cell line . J Exp Clin Cancer Res . 2009;28:3
  22. Guba M , Breitenbuch PV , Steinbauer M , et al.  Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: Involvement of vascular endothelial growth factor . Nature Med . 2002;8:128–135
  23. Amornphimoltham P , Patel V , Sodhi A , et al.  Mammalian target of rapamycin, a molecular target in squamous cell carcinomas of the head and neck . Cancer Res . 2005;65:9953–9961
  24. Decker T , Hipp S , Ringshausen I , et al.  Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin . Blood . 2003;101:278–285
  25. Di Paolo S , Teutonico A , Leogrande D , et al.  Chronic inhibition of mammalian target of rapamycin signaling down-regulated insulin receptor substrates 1 and 2 and AKT activation: A crossroad between cancer and diabetes? . J Am Soc Nephrol . 2006;17:2236–2244
  26. Breuleux M , Klopfenstein M , Stephan C , et al.  Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition . Mol Cancer Ther . 2009;8:742–753
  27. O'Reilly KE , Rojo F , She QB , et al.  mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates AKT . Cancer Res . 2006;66:1500–1508
  28. Sun SY , Rosenberg LM , Wang X , et al.  Activation of AKT and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition . Cancer Res . 2005;65:7052–7058
  29. Zhang H , Bajraszewski N , Wu E , et al.  PDGFRs are critical for PI3K/AKT activation and negatively regulated by mTOR . J Clin Invest . 2007;117:730–738
  30. Sarbassov DD , Ali SM , Sengupta S , et al.  Prolonged rapamycin treatment inhibits mTORC2 assembly and ATK/PKB . Mol Cell . 2006;22:159–168
  31. Abraham R , Gibbons JJ . The mammalian target of rapamycin signaling pathway: Twists and turns in the road to cancer therapy . Clin Cancer Res . 2007;13:3109–3114
  32. Wan X , Harkavy B , Shen N , et al.  Rapamycin induces feedback activation of AKT signaling through an IGF-1R-dependent mechanism . Oncogene . 2007;26:1932–1940
  33. Fan QW , Knight ZA , Goldenberg DD , et al.  A dual PI3 Kinase/mTOR inhibitor reveals emergent efficacy in glioma . Cancer Cell . 2006;9:341–349

PII: S1078-1439(09)00362-7

doi:10.1016/j.urolonc.2009.11.009

Urologic Oncology: Seminars and Original Investigations
Volume 30, Issue 1 , Pages 69-77, January 2012

Article Tools

* Image Usage & Resolution