sirolimus and Urinary-Bladder-Neoplasms

Cancer is a complex disease characterized by a multitude of molecular and genetic abnormalities affecting cell proliferation and differentiation, apoptosis, and mobility (invasion). Each of these alterations represents a potential target for the development of targeted therapy. These new therapies inhibit cell growth and are said to be "cytostatic" in contrast with conventional "cytotoxic" chemotherapy. As a result of a better understanding of the molecular biology of bladder cancers, various signalling pathways involved in both carcinogenesis and tumour progression have been defined, and some of the key molecules in these pathways have been isolated and can be used as prognostic markers and as potential therapeutic targets. Locally advanced, and/or metastatic bladder cancer, is characterized by mutations of the p53 and retinoblastoma (Rb) genes, regulators of the cell cycle, which interact with the Ras-mitogen activated protein kinase (MPAK) transduction pathway. Overexpression of tyrosine kinase receptors, including EGFR, VEFGR and HER2/neu, is correlated with tumour progression and activation of the phosphatidyl-inositol-3 kinase (PI-3K) pathway is involved in tumour invasion and inhibition of apoptosis. Due to their molecular heterogeneity, optimal targeted therapy of bladder cancers will require the combined use of several molecules. Modulation of signalling pathways by these new molecules can restore chemosensitivity to cytotoxic drugs, which can then be associated with targeted therapy.

Topics: Angiogenesis Inhibitors; Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Disease Progression; Erlotinib Hydrochloride; Gefitinib; Genetic Therapy; Humans; Immunosuppressive Agents; Mutation; Piperidines; Protein Kinase Inhibitors; Pyridines; Quinazolines; Randomized Controlled Trials as Topic; Signal Transduction; Sirolimus; Targeted Gene Repair; Trastuzumab; Urinary Bladder Neoplasms

Cisplatin-based neoadjuvant chemotherapy (NAC) followed by cystectomy is the standard for muscle-invasive bladder cancer (MIBC), however, NAC confers only a small survival benefit and new strategies are needed to increase its efficacy. Pre-clinical data suggest that in response to DNA damage the tumor microenvironment (TME) adopts a paracrine secretory phenotype dependent on mTOR signaling which may provide an escape mechanism for tumor resistance, thus offering an opportunity to increase NAC effectiveness with mTOR blockade.. We conducted a phase I/II clinical trial to assess the safety and efficacy of gemcitabine-cisplatin-rapamycin combination. Grapefruit juice was administered to enhance rapamycin pharmacokinetics by inhibiting intestinal enzymatic degradation. Phase I was a dose determination/safety study followed by a single arm Phase II study of NAC prior to radical cystectomy evaluating pathologic response with a 26% pCR rate target.. In phase I, 6 patients enrolled, and the phase 2 dose of 35 mg rapamycin established. Fifteen patients enrolled in phase II; 13 were evaluable. Rapamycin was tolerated without serious adverse events. At the preplanned analysis, the complete response rate (23%) did not meet the prespecified level for continuing and the study was stopped due to futility. With immunohistochemistry, successful suppression of the mTOR signaling pathway in the tumor was achieved while limited mTOR activity was seen in the TME.. Adding rapamycin to gemcitabine-cisplatin therapy for patients with MIBC was well tolerated but failed to improve therapeutic efficacy despite evidence of mTOR blockade in tumor cells. Further efforts to understand the role of the tumor microenvironment in chemotherapy resistance is needed.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Cystectomy; Deoxycytidine; Gemcitabine; Humans; Muscles; Neoadjuvant Therapy; Neoplasm Invasiveness; Sirolimus; TOR Serine-Threonine Kinases; Tumor Microenvironment; Urinary Bladder Neoplasms

Although intravesical BCG is the standard treatment of high-grade non-muscle invasive bladder cancer (NMIBC), response rates remain unsatisfactory. In preclinical models, rapamycin enhances BCG vaccine efficacy against tuberculosis and the killing capacity of γδ T cells, which are critical for BCG's antitumor effects. Here, we monitored immunity, safety, and tolerability of rapamycin combined with BCG in patients with NMIBC.. A randomized double-blind trial of oral rapamycin (0.5 or 2.0 mg daily) versus placebo for 1 month was conducted in patients with NMIBC concurrently receiving 3 weekly BCG instillations (NCT02753309). The primary outcome was induction of BCG-specific γδ T cells, measured as a percentage change from baseline. Post-BCG urinary cytokines and immune cells were examined as surrogates for local immune response in the bladder. Secondary outcomes measured were adverse events (AEs) and tolerability using validated patient-reported questionnaires.. Thirty-one patients were randomized (11 placebo, 8 rapamycin 2.0 mg, and 12 rapamycin 0.5 mg). AEs were similar across groups and most were grade 1-2. One (12.5%) patient randomized to 2.0 mg rapamycin was taken off treatment due to stomatitis. No significant differences in urinary symptoms, bowel function, or bother were observed between groups. The median (IQR) percentage change in BCG-specific γδ T cells from baseline per group was as follows: -26% (-51% to 24%) for placebo, 9.6% (-59% to 117%) for rapamycin 0.5 mg (versus placebo, p=0.18), and 78.8% (-31% to 115%) for rapamycin 2.0 mg (versus placebo, p=0.03). BCG-induced cytokines showed a progressive increase in IL-8 (p=0.02) and TNF-α (p=0.04) over time for patients on rapamycin 2.0 mg, whereas patients receiving placebo had no significant change in urinary cytokines. Compared with placebo, patients receiving 2.0 mg rapamycin had increased urinary γδ T cells at the first week of BCG (p=0.02).. Four weeks of 0.5 and 2.0 mg oral rapamycin daily is safe and tolerable in combination with BCG for patients with NMIBC. Rapamycin enhances BCG-specific γδ T cell immunity and boosts urinary cytokines during BCG treatment. Further study is needed to determine long-term rapamycin safety, tolerability and effects on BCG efficacy.

Topics: Adjuvants, Immunologic; Administration, Intravesical; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; BCG Vaccine; Cytokines; Double-Blind Method; Female; Humans; Intraepithelial Lymphocytes; Male; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Staging; Phenotype; Sirolimus; Time Factors; Treatment Outcome; Tumor Microenvironment; Urinary Bladder Neoplasms; Urine

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (

Topics: Aged; Animals; B7-H1 Antigen; Cell Proliferation; Cystectomy; Disease Models, Animal; Female; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Phosphorylation; Postoperative Complications; Programmed Cell Death 1 Receptor; Ribosomal Protein S6; Sirolimus; T-Lymphocytes; Urinary Bladder; Urinary Bladder Neoplasms

Bladder cancer is the 7th cause of death from cancer in men and 10th in women. Metastatic patients have a poor prognosis with a median overall survival of 14 months. Until recently, vinflunine was the only second-line chemotherapy available for patients who relapse. Deregulation of the PI3K/AKT/mTOR pathway was observed in more than 40% of bladder tumors and suggested the use of mTOR as a target for the treatment of urothelial cancers.. This trial assessed the efficacy of temsirolimus in a homogenous cohort of patients with recurrent or metastatic bladder cancer following first-line chemotherapy. Efficacy was measured in terms of non-progression at two months according to the RECIST v1.1 criteria. Based on a two-stage optimal Simon's design, 15 non-progressions out of 51 evaluable patients were required to claim efficacy. Patients were treated at a weekly dose of 25 mg IV until progression, unacceptable toxicities or withdrawal.. Among the 54 patients enrolled in the study between November 2009 and July 2014, 45 were assessable for the primary efficacy endpoint. A total of 22 (48.9%) non-progressions were observed at 2 months with 3 partial responses and 19 stable diseases. Remarkably, 4 patients were treated for more than 30 weeks. Fifty patients experienced at least a related grade1/2 (94%) and twenty-eight patients (52.8%) a related grade 3/4 adverse event. Eleven patients had to stop treatment for toxicity. This led to recruitment being halted by an independent data monitoring committee with regard to the risk-benefit balance and the fact that the primary objective was already met.. While the positivity of this trial indicates a potential benefit of temsirolimus for a subset of bladder cancer patients who are refractory to first line platinum-based chemotherapy, the risk of adverse events associated with the use of this mTOR inhibitor would need to be considered when such an option is envisaged in this frail population of patients. It also remains to identify patients who will benefit the most from this targeted therapy.. ClinicalTrials.gov Identifier: NCT01827943 (trial registration date: October 29, 2012); Retrospectively registered.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Positron Emission Tomography Computed Tomography; Protein Kinase Inhibitors; Retreatment; Sirolimus; Treatment Outcome; Urinary Bladder Neoplasms

What's known on the subject? and what does the study add?: No recent advances have been made in the treatment of patients with advanced bladder cancer and, to date, targeted therapies have not resulted in an improvement in outcome. The mammalian target of rapamycin pathway has been shown to be up-regulated in bladder cancer and represents a rational target for therapeutic intervention. In the present phase II study of everolimus, one near-complete response, one partial response and several minor responses suggest that everolimus possesses biological activity in a subset of patients with bladder cancer. To maximize benefit from targeted agents such as everolimus, the preselection of patients based on molecular phenotype is required.. To assess the efficacy and tolerability of everolimus in advanced urothelial carcimoma (UC).. The present study comprised a single-arm, non-randomized study in which all patients received everolimus 10 mg orally once daily continuously (one cycle = 4 weeks). In total, 45 patients with metastatic UC progressing after one to four cytotoxic agents were enrolled between February 2009 and November 2010 at the Memorial Sloan-Kettering Cancer Center. The primary endpoints were 2-month progression-free survival (PFS) and the safety of everolimus, with the secondary endpoint being the response rate. A Simon minimax two-stage design tested the null hypothesis that the true two month PFS rate was ≤ 50%, as opposed to the alternative hypothesis of ≥ 70%.. The most common grade 3/4 toxicities were fatigue, infection, anaemia, lymphopaenia, hyperglycaemia and hypophosphataemia. There were two partial responses in nodal metastases, with one patient achieving a 94% decrease in target lesions and remaining on drug at 26 months. An additional 12 patients exhibited minor tumour regression. There were 23 of 45 (51%) patients who were progression-free at 2 months with a median (95% CI) PFS of 2.6 (1.8-3.5) months and a median (95% CI) overall survival of 8.3 (5.5-12.1) months. No clear association was observed between mammalian target of rapamycin pathway marker expression and 2-month PFS.. Although everolimus did not meet its primary endpoint, one partial response, one near-complete response and twelve minor regressions were observed. Everolimus possesses meaningful anti-tumour activity in a subset of patients with advanced UC. Studies aiming to define the genetic basis of everolimus activity in individual responders are ongoing.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Transitional Cell; Clinical Trials, Phase II as Topic; Everolimus; Female; Humans; Male; Middle Aged; Sirolimus; Urinary Bladder Neoplasms

This phase II trial assessed temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), as second-line therapy in patients with metastatic transitional carcinoma of the urothelium (TCCU) after failure of platinum containing therapy. From June/2009 to June/2011, we enrolled 15 patients in this trial. Primary endpoint was overall survival, as secondary endpoints we defined time to disease progression, safety and QoL along treatment. Patients with progressive TCCU after prior platinum-based chemotherapy received weekly 25 mg of temsirolimus for 8 weeks. Evaluation for response was accomplished every 8 weeks according to the RECIST criteria, QoL assessment was done every 4 weeks using the QLQ-C30 questionnaire, adverse events (AEs) were recorded and graded using NCI-CTC criteria. Fifteen patients were enrolled in this study, of whom 14 (93%) were available for activity, safety and QoL assessment. We treated 10 (71%) male and 4 female (29%) patients. Median age was 64,7 years (45-76). Patients received on average 13 (3-15) infusions of temsirolimus. As per protocol, no sufficient benefit on overall survival was observed, we early stopped the study after 14 patients. Median time to progression was 2.5 months (77 days), median overall survival was 3.5 months (107 days). Four patients with stable disease were observed. QoL assessment along treatment revealed a reduction of EORTC-QLQ-C30, Global Health Status subscale, from initial 7.86 to 5.00. Temsirolimus was well tolerated. As Grade 3-4 adverse events, we observed fatigue (n=2), leukopenia (n=2) and thrombopenia (n=2). All other adverse events were graded 1-2 in nature. Temsirolimus seems to have poor activity in patients with progressive metastasized TCCU after failure of platinum containing first-line therapy.

Topics: Aged; Antineoplastic Agents; Carcinoma, Transitional Cell; Female; Humans; Male; Middle Aged; Protein Kinase Inhibitors; Quality of Life; Sirolimus; Urinary Bladder Neoplasms

This phase II study assessed the safety and efficacy of everolimus, an oral mammalian target of rapamycin inhibitor in advanced transitional carcinoma cell (TCC) after failure of platinum-based therapy.. Thirty-seven patients with advanced TCC received everolimus 10 mg/day until progressive disease (PD) or unacceptable toxicity. The primary end point was the disease control rate (DCR), defined as either stable disease (SD), partial response (PR), or complete response at 8 weeks. Angiogenesis-related proteins were detected in plasma and changes during everolimus treatment were analyzed. PTEN expression and PIK3CA mutations were correlated to disease control.. Two confirmed PR and eight SD were observed, resulting in a DCR of 27% at 8 weeks. Everolimus was well tolerated. Compared with patients with noncontrolled disease, we observed in patients with controlled disease a significant higher baseline level of angiopoietin-1 and a significant early plasma decrease in angiopoietin-1, endoglin, and platelet-derived growth factor-AB. PTEN loss was observed only in patients with PD.. Everolimus showed clinical activity in advanced TCC. The profile of the plasma angiogenesis-related proteins suggested a role of the everolimus antiangiogenic properties in disease control. PTEN loss might be associated with everolimus resistance.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Transitional Cell; Everolimus; Female; Humans; Male; Middle Aged; Neoplasm Metastasis; Sirolimus; Urinary Bladder Neoplasms

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Everolimus; Humans; Paclitaxel; Sirolimus; Treatment Outcome; Urinary Bladder Neoplasms

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Everolimus; Humans; Paclitaxel; Sirolimus; Treatment Outcome; Urinary Bladder Neoplasms

Bladder cells are constantly exposed to multiple xenobiotics and bioactive metabolites. In addition to this challenging chemical environment, they are also exposed to shear stress originating from urine and interstitial fluids. Hence, physiological function of bladder cells relies on a high biochemical and biomechanical adaptive competence, which, in turn, is largely supported via autophagy-related mechanisms. As a negative side of this plasticity, bladder cancer cells are known to adapt readily to chemotherapeutic programs. At the molecular level, autophagy was described to support resistance against pharmacological treatments and to contribute to the maintenance of cell structure and metabolic competence. In this study, we enhanced autophagy with rapamycin (1-100 nM) and assessed its effects on the motility of bladder cells, as well as the capability to respond to shear stress. We observed that rapamycin reduced cell migration and the mechanical-induced translocation potential of Krüppel-like transcription factor 2 (KLF2). These effects were accompanied by a rearrangement of cytoskeletal elements and mitochondrial loss. In parallel, intracellular acetylation levels were decreased. Mechanistically, inhibition of the NAD + -dependent deacetylase sirtuin-1 (SIRT1) with nicotinamide (NAM; 0.1-5 mM) restored acetylation levels hampered by rapamycin and cell motility. Taken together, we described the effects of rapamycin on cytoskeletal elements crucial for mechanotransduction and the dependency of these changes on the mitochondrial turnover caused by autophagy activation. Additionally, we could show that targeted metabolic intervention could revert the outcome of autophagy activation, reinforcing the idea that bladder cells can easily adapt to multiple xenobiotics and circumvent in this way the effects of single chemicals.

Topics: Acetylation; Autophagy; Humans; Mechanotransduction, Cellular; Sirolimus; Sirtuin 1; Urinary Bladder; Urinary Bladder Neoplasms; Xenobiotics

To understand the effect of Nitazoxanide (NTZ), Rapamycin, Thalidomide, alone and in combination with BCG on bladder cancer (BC) histopathology and programmed death-ligand 1 (PD-L1) and anti-cytotoxic T lymphocyte antigen 4 (CTLA4) expression.. Female Fisher-344 rats underwent intravesical N-methyl-N-nitrosourea (MNU) followed by weekly intravesical treatment with saline (controls, n = 10), BCG (n = 10), NTZ (n = 8), BCG plus NTZ (n = 8), Rapamycin (n = 10) BCG plus Rapamycin (n = 10), Thalidomide (n = 10), and BCG plus Thalidomide (n = 10), and euthanized after 8 weeks and their bladders were investigated for BC and PD-L1 and CTLA4 expression.. Rapamicyn alone and in combination with BCG had the lowest number of bladder neoplasias in the histopathology exam (1/10). Neoplastic lesions were found in 4/10 BCG recipients, 5/10 Thalidomide recipients, 4/10 Thalidomide plus BCG recipients, 5/8 NTZ and 3/8 NTZ plus BCG recipients. Adding NTZ to BCG increased the expression of PD-L1 and adding Rapamycin or Thalidomide decreased PD-L1 and CTLA4 expression compared to BCG alone. Rapamycin alone significantly increased CTLA4 and slightly increased PD-L1 expression but its combination with BCG significantly decreased both markers. Thalidomide had a similar effect; however, it was only slightly different from the control and BCG alone groups.. Intravesical BCG combination treatment seems to effectively prevent BC development in an immunecompetent clinically relevant animal model, introducing Thalidomide, Nitazoxanide, and specially Rapamycin as candidates in the intravesical immunotherapy advancement. Our study contributes in understanding the mechanism of cancer immunotherapy.

Topics: Adjuvants, Immunologic; Administration, Intravesical; Animals; B7-H1 Antigen; BCG Vaccine; CTLA-4 Antigen; Female; Rats; Sirolimus; Thalidomide; Urinary Bladder Neoplasms

In this study, a novel class of thieno [2,3-d] pyrimidine derivatives containing resorcinol and morpholine fragments as Hsp90/mTOR dual inhibitors was designed, synthesized, and evaluated. In vitro anti-tumor assay results: the obtained compounds demonstrated effectiveness in suppressing the enzymatic activities of the Hsp90 and mTOR and inhibiting the proliferation of J82, T24, and SW780 cancer cell lines. Among these dual inhibitors, the most potent compound 17o, confirmed remarkable inhibitory activities on Hsp90, mTOR, and SW780 cell. Furthermore, the molecular dynamics simulation and a panel of mechanism studies revealed that inhibitor 17o suppressed the proliferation of SW780 cells through the over-activation of the PI3K/AKT/mTOR pathway regulated by mTOR inhibition and apoptosis regulated by the mitochondrial pathway. In subcutaneous J82 xenograft models, the compound 17o also presented considerable in vivo anti-tumor activity. Therefore, our investigations highlight that a new-found dual Hsp90/mTOR inhibitor by rational drug design strategies could be a promising lead compound for targeted bladder cancer therapy and deserves further studies.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; HSP90 Heat-Shock Proteins; Humans; Morpholines; MTOR Inhibitors; Oxygen Isotopes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Resorcinols; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Combined cisplatin-gemcitabine treatment causes rapid resistance development in patients with advanced urothelial carcinoma. The present study investigated the potential of the natural isothiocyanates (ITCs) allyl-isothiocyanate (AITC), butyl-isothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC) to suppress growth and proliferation of gemcitabine- and cisplatin-resistant bladder cancer cells lines. Sensitive and gemcitabine- and cisplatin-resistant RT112, T24, and TCCSUP cells were treated with the ITCs, and tumor cell growth, proliferation, and clone formation were evaluated. Apoptosis induction and cell cycle progression were investigated as well. The molecular mode of action was investigated by evaluating cell cycle-regulating proteins (cyclin-dependent kinases (CDKs) and cyclins A and B) and the mechanistic target of the rapamycin (mTOR)-AKT signaling pathway. The ITCs significantly inhibited growth, proliferation and clone formation of all tumor cell lines (sensitive and resistant). Cells were arrested in the G2/M phase, independent of the type of resistance. Alterations of both the CDK-cyclin axis and the Akt-mTOR signaling pathway were observed in AITC-treated T24 cells with minor effects on apoptosis induction. In contrast, AITC de-activated Akt-mTOR signaling and induced apoptosis in RT112 cells, with only minor effects on CDK expression. It is concluded that AITC, BITC, and PEITC exert tumor-suppressive properties on cisplatin- and gemcitabine-resistant bladder cancer cells, whereby the molecular action may differ among the cell lines. The integration of these ITCs into the gemcitabine-/cisplatin-based treatment regimen might optimize bladder cancer therapy.

Topics: Apoptosis; Carcinoma, Transitional Cell; Cell Line, Tumor; Cisplatin; Cyclin-Dependent Kinases; Cyclins; Deoxycytidine; Gemcitabine; Humans; Isothiocyanates; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Tumor cell-intrinsic programmed death-ligand 1 (PD-L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell-intrinsic PD-L1 signals in mouse MB49 and human RT4, UM-UC3, and UM-UC-14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α-PD-L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell-intrinsic PD-L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune-independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell-intrinsic PD-L1 signals also promoted basal and stress-induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell-intrinsic PD-L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis-platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell-intrinsic PD-L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof-of-concept, we showed that the autophagy inhibitor chloroquine improved cis-platinum treatment efficacy in vivo, with greater efficacy in PD-L1 null versus PD-L1-replete BC.

Topics: Animals; Antibiotics, Antineoplastic; Autophagy; B7-H1 Antigen; Cell Line, Tumor; Cell Proliferation; Chloroquine; Cisplatin; Deoxycytidine; Drug Resistance, Neoplasm; Female; Gemcitabine; Gene Expression; Humans; Mechanistic Target of Rapamycin Complex 1; Melanoma; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Ovarian Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus; Urinary Bladder Neoplasms

Transcriptional addiction plays a pivotal role in maintaining the hallmarks of cancer cells. Thus, targeting super-enhancers (SEs), which modulate the transcriptional activity of oncogenes, has become an attractive strategy for cancer therapy. As yet, however, the molecular mechanisms of this process in bladder cancer (BC) remain to be elucidated. Here, we aimed to provide detailed information regarding the SE landscape in BC and to investigate new potential pharmaceutical targets for BC therapy.. We employed THZ1 as a potent and specific CDK7 inhibitor. In vitro and in vivo studies were carried out to investigate the anticancer and apoptosis-inducing effects of THZ1 on BC cells. Whole-transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to investigate the mechanism and function of SE-linked oncogenic transcription in BC cells.. We found that THZ1 serves as an effective and potent inhibitor with suppressive activity against BC cells. An integrative analysis of THZ1-sensitive and SE-associated oncogenes yielded potential new pharmaceutical targets, including DDIT4, B4GALT5, PSRC1 and MED22. Combination treatment with THZ1 and the DDIT4 inhibitor rapamycin effectively suppressed BC cell growth. In addition, we found that THZ1 and rapamycin sensitized BC cells to conventional chemotherapy.. Our data indicate that exploring BC gene regulatory mechanisms associated with SEs through integrating RNA-seq and ChIP-seq data improves our understanding of BC biology and provides a basis for innovative therapies.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Drug Synergism; Enhancer Elements, Genetic; Female; Gene Expression Regulation, Neoplastic; Humans; Mice, Nude; Oncogenes; Phenylenediamines; Pyrimidines; RNA-Seq; Sirolimus; Transcription Factors; Tumor Burden; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Despite enormous scientific advancements in cancer treatment, there is a need for research to combat cancer, particularly bladder cancer. Drugs once proved to be effective in treating bladder cancer have shown reduced efficacy; hence, the cancer recurrence rate is increasing. To overcome this situation, several strategies have been considered, including the development of novel active drugs or modification of existing therapeutic regimens by combining two or more existing drugs. In recent years, atypical protein kinase Cs (PKCs), phospholipid‑dependent serine/threonine kinases, have been considered as a central regulator of various cancer‑associated signaling pathways, and they control cell cycle progression, tumorigenesis and metastasis. Additionally, the biologically crucial mTOR signaling pathway is altered in numerous types of cancer, including bladder cancer. Furthermore, despite independent activation, atypical PKC signaling can be triggered by mTOR. The present study examined whether the concurrent inhibition of atypical PKCs and mTOR using a combination of novel atypical PKC inhibitors (ICA‑I, an inhibitor of PKC‑ι; or ζ‑Stat, an inhibitor of PKC‑ζ) and rapamycin blocks bladder cancer progression. In the present study, healthy bladder MC‑SV‑HUCT2 and bladder cancer TCCSUP cells were tested and subjected to a WST1 assay, western blot analysis, immunoprecipitation, a scratch wound healing assay, flow cytometry and immunofluorescence analyses. The results revealed that the combination therapy induced a reduction in human bladder cancer cell viability compared with control and individual atypical PKC inhibitor and rapamycin treatment. Additionally, the concurrent inhibition of atypical PKCs and mTOR retards the migration of bladder cancer cells. These findings indicated that the administration of atypical PKC inhibitors together with rapamycin could be a useful therapeutic option in treating bladder cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Isoenzymes; Molecular Structure; Nucleosides; Protein Kinase C; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3'UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Renal Cell; Cell Movement; Cell Proliferation; Embryo, Mammalian; Female; Fibroblasts; Gene Expression Regulation; Humans; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; MicroRNAs; Receptors, Retinoic Acid; Sirolimus; Tuberous Sclerosis Complex 2 Protein; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

To develop an intravesical instillation system for the treatment of bladder cancer, rapamycin (Rap) was encapsulated into liposomes and then homogeneously dispersed throughout a poloxamer 407 (P407)-based hydrogel.. Rap-loaded conventional liposomes (R-CL) and folate-modified liposomes (R-FL) were prepared using a film hydration method and pre-loading technique, and characterized by particle size, drug entrapment efficiency, and drug loading. The cellular uptake behavior in folate receptor-expressing bladder cancer cells was observed by flow cytometry and confocal laser scanning microscopy using a fluorescent probe. In vitro cytotoxic effects were evaluated using MTT assay, colony forming assay, and Western blot. For in vivo intravesical instillation, Rap-loaded liposomes were dispersed in P407-gel, generating R-CL/P407 and R-FL/P407. Gel-forming capacities and drug release were evaluated. Using the MBT2/Luc orthotopic bladder cancer mouse model, in vivo antitumor efficacy was evaluated according to regions of interest (ROI) measurement.. Intravesical instillation of R-FL/P407 might represent a good candidate for bladder cancer treatment, owing to its enhanced retention and FR-targeting.

Topics: Administration, Intravesical; Animals; Antineoplastic Agents; Autophagy; Cell Line, Tumor; Cell Proliferation; Colloids; Disease Models, Animal; Drug Liberation; Female; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Hydrogels; Liposomes; Mice; Particle Size; Sirolimus; Temperature; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has significant potential for application in the treatment of urothelial carcinoma (URCa) of the bladder. Previous studies have shown that regulation of the AMP-activated serine/threonine protein kinase (AMPK)-mTOR signaling pathway enhances apoptosis by inducing autophagy or mitophagy in bladder cancer. Alteration of liver kinase B1 (LKB1)-AMPK signaling leads to mitochondrial dysfunction and the accumulation of autophagy-related proteins as a result of mitophagy, resulting in enhanced cell sensitivity to drug treatments. Therefore, we hypothesized that LKB1 deficiency in URCa cells could lead to increased sensitivity to rapamycin by inducing mitochondrial defect-mediated mitophagy. To test this, we established stable LKBI-knockdown URCa cells and analyzed the effects of rapamycin on their growth. Rapamycin enhanced growth inhibition and apoptosis in stable LKB1-knockdown URCa cells and in a xenograft mouse model. In spite of the stable downregulation of LKB1 expression, rapamycin induced AMPK activation in URCa cells, causing loss of the mitochondrial membrane potential, ATP depletion, and ROS accumulation, indicating an alteration of mitochondrial biogenesis. Our findings suggest that the absence of LKB1 can be targeted to induce dysregulated mitochondrial biogenesis by rapamycin treatment in the design of novel therapeutic strategies for bladder cancer.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Humans; Mice; Mice, Nude; Mitophagy; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Topics: Humans; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Sirolimus; Urinary Bladder Neoplasms

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Gene Deletion; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Neoplasm Proteins; Sirolimus; Urinary Bladder Neoplasms

Microarray analysis was used to investigate the lack of identified mammalian target of rapamycin (mTOR) pathway downstream genes to overcome cross-talk at non-muscle invasive high-grade (HG)-urothelial carcinoma (UC) of the bladder, gene expression patterns, gene ontology, and gene clustering by triple (p70S6K, S6K, and eIF4E) small interfering RNAs (siRNAs) or rapamycin in 5637 and T24 cell lines. We selected mTOR pathway downstream genes that were suppressed by siRNAs more than 2-fold, or were up-regulated or down-regulated by rapamycin more than 2-fold. We validated mTOR downstream genes with immunohistochemistry using a tissue microarray (TMA) of 125 non-muscle invasive HG-UC patients and knockout study to evaluate the synergistic effect with rapamycin. The microarray analysis selected mTOR pathway downstream genes consisting of 4 rapamycin up-regulated genes (FABP4, H19, ANXA10, and UPK3A) and 4 rapamycin down-regulated genes (FOXD3, ATP7A, plexin D1, and ADAMTS5). In the TMA, FABP4, and ATP7A were more expressed at T1 and FOXD3 was at Ta. ANXA10 and ADAMTS5 were more expressed in tumors ≤ 3 cm in diameter. In a multivariate Cox regression model, ANXA10 was a significant predictor of recurrence and ATP7A was a significant predictor of progression in non-muscle invasive HG-UC of the bladder. In an ATP7A knock-out model, rapamycin treatment synergistically inhibited cell viability, wound healing, and invasion ability compared to rapamycin only. Activity of the ANXA10 and ATP7A mTOR pathway downstream genes might predict recurrence and progression in non-muscle invasive HG-UC of the bladder. ATP7A knockout overcomes rapamycin cross-talk.

Topics: Aged; Cell Line, Tumor; Cell Movement; Cell Proliferation; Copper-Transporting ATPases; Disease Progression; Down-Regulation; Eukaryotic Initiation Factor-4E; Female; Humans; Male; Neoplasm Grading; Neoplasm Recurrence, Local; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation; Urinary Bladder Neoplasms

Temsirolimus is a mammalian target of rapamycin (mTOR) inhibitor that exhibits antitumor activity in renal cell carcinoma and mantle cell lymphoma. The metabolism of temsirolimus and its active metabolite sirolimus mainly depends on cytochrome P450 3A4/5 (CYP3A4/A5) and the ABCB1 transporter. Differently from sirolimus, no pharmacogenetic study on temsirolimus has been conducted. Therefore, the aim of this pilot study was to identify genetic determinants of the inter-individual variability in temsirolimus pharmacokinetics and toxicity.. Pharmacokinetic profiles were obtained for 16 patients with bladder cancer after intravenous infusion of 25 mg temsirolimus. Non-compartmental analysis was performed to calculate the pharmacokinetic parameters of temsirolimus and sirolimus, its main metabolite. The presence of single nucleotide polymorphisms (SNPs) in CYP3A5, ABCB1 and in their transcriptional regulator NR1I2 (PXR) was assessed by genotyping. Non-parametric statistical tests were used to assess associations between candidate SNPs and temsirolimus pharmacokinetics and toxicity.. The ratio between sirolimus AUC and temsirolimus AUC was 1.6-fold higher in patients who experienced serious toxic events (p = 0.034). The frequency of adverse events was significantly higher in patients homozygous for the NR1I2-rs6785049 A allele (OR = 0.065, p = 0.04) or NR1I2-rs3814055 C allele (OR = 0.032, p = 0.006). These NR1I2 SNPs were also predictive of temsirolimus half-life and global exposure to temsirolimus and sirolimus. Finally, the effect of the ABCB1-rs1128503, ABCB1-rs2032582 and CYP3A5*3 SNPs on sirolimus pharmacokinetics was confirmed.. Our findings suggest that SNPs of NR1I2 and its target genes CYP3A5 and ABCB1 are genetic determinants of temsirolimus pharmacokinetics and toxicity in patients with bladder cancer.

Topics: Aged; Antineoplastic Agents; Cytochrome P-450 CYP3A; Female; Genotype; Humans; Male; Middle Aged; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Sirolimus; Urinary Bladder Neoplasms

Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin-induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line; Cell Movement; Cell Proliferation; Embryo, Mammalian; Enzyme Activation; Fibroblasts; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Sirolimus; Stilbenes; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Urothelial tumors, accompanied by mutations of the tumor suppressor protein TP53 and dysregulation of mTOR signaling, are frequently associated with aggressive growth and invasiveness. We investigated whether targeting these two pathways would inhibit urothelial tumor growth and progression. Six-week-old transgenic UPII-SV40T male mice (n = 15/group) were fed control diet (AIN-76A) or experimental diets containing mTOR inhibitor (rapamycin, 8 or 16 ppm), p53 stabilizing agent [CP31398 (CP), 150 ppm], or a combination. Mice were euthanized at 40 weeks of age. Urinary bladders were collected and evaluated to determine tumor weight and histopathology. Each agent alone, and in combination, significantly inhibited tumor growth. Treatment with rapamycin alone decreased tumor weight up to 67% (P < 0.0001). Similarly, CP showed approximately 77% (P < 0.0001) suppression of tumor weight. The combination of low-dose rapamycin and CP led to approximately 83% (P < 0.0001) inhibition of tumor weight. There was no significant difference in tumor weights between rapamycin and CP treatments (P > 0.05). However, there was a significant difference between 8 ppm rapamycin and the combination treatment. Tumor invasion was also significantly inhibited in 53% (P < 0.005) and 66% (P < 0.0005) mice after 8 ppm and 16 ppm rapamycin, respectively. However, tumor invasion was suppressed in 73% (P < 0.0001) mice when CP was combined with 8 ppm rapamycin. These results suggest that targeting two or more pathways achieve better treatment efficacy than a single-agent high-dose strategy that could increase the risk of side effects. A combination of CP and rapamycin may be a promising method of inhibiting muscle-invasive urothelial transitional cell carcinoma.

Topics: Animals; Biomarkers, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Inflammation; Magnetic Resonance Imaging; Male; Mice; Mice, Transgenic; Muscles; Neoplasm Invasiveness; Neovascularization, Pathologic; Polyamines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Urinary Bladder; Urinary Bladder Neoplasms

We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 µM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.

Topics: Animals; Cell Line; Cell Proliferation; Cell Survival; Disease Progression; Eukaryotic Initiation Factor-4E; Female; Mice; Mice, Nude; Mucous Membrane; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

To analyze the cytotoxic action of temsirolimus using 3 established human bladder cancer cell lines and to assess whether temsirolimus potentiates the anticancer activity of gemcitabine and cisplatin.. Temsirolimus (500, 1,000, 2,000, and 4,000 nM), in isolation, and combined with gemcitabine (100 nM) and cisplatin (2.5 µg/ml), was given to 5637, T24, and HT1376 bladder cancer cell lines. Cell proliferation, autophagy, early apoptosis, and cell cycle distribution were analyzed after a 72-hour period. The expression of mammalian target of rapamycin baseline, Akt, and their phosphorylated forms, before and after treatment with temsirolimus, was evaluated by immunoblotting.. Temsirolimus slightly decreased the bladder cancer cell proliferation in all 3 cell lines. No significant differences in the expression of mammalian target of rapamycin, Akt, and their phosphorylated forms because of temsirolimus exposure were found in the 3 cell lines. As part of a combined regime along with gemcitabine, and especially with cisplatin, there was a more pronounced antiproliferative effect. This pattern of response was similar to the other parameters analyzed (increased autophagy and apoptosis). Also, in the combined regime, an enhanced cell cycle arrest in the G0/G1 phase was observed. The non-muscle invasive 5637 bladder cancer cell line was most sensitive to both combinations.. Temsirolimus makes a moderate contribution in terms of cell proliferation, apoptosis, and autophagy. However, it does potentiate the activity of gemcitabine and particularly cisplatin. Therefore, cisplatin- or gemcitabine-based chemotherapy regimen used in combination with temsirolimus to treat bladder cancer represents a novel and valuable treatment option, which should be tested for future studies in urinary bladder xenograft models.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Deoxycytidine; Dose-Response Relationship, Drug; Drug Synergism; Gemcitabine; Humans; Immunoblotting; Microscopy, Fluorescence; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

We investigated how dual inhibition of the molecular mechanism of mammalian target of rapamycin (mTOR) downstream of S6K1 and the eukaryotic initiation factor 4E (eIF4E) can lead to suppression of proliferation and progression of urothelial carcinoma.. We characterized the molecular mechanism of the mTOR pathway in the T24 and 5637 urothelial carcinoma cell lines by interfering with different molecular components using rapamycin and short interfering (siRNA) technology (S6K1 or elF4E) and analyzed the effects on molecular activation status, cell growth, proliferation, and invasion.. A high concentration of rapamycin (10 μM) blocked both S6K1 and elF4E phosphorylation and inhibited cell proliferation in T24 and 5637 cells. The inhibition of both S6K1 and elF4E phosphorylation by rapamycin reduced cell viability and proliferation more than transfection of siRNA against S6K1 or elF4E in 5637 and T24 cells. Cells silenced for S6K1 or elF4E expression exhibited significantly reduced cell migration and invasion compared with those of the control but inhibition of both S6K1 and elF4E phosphorylation by rapamycin reduced cell migration and invasion more than siRNA transfection against S6K1 or elF4E in 5637 and T24 cells.. These findings suggest that both the mTOR pathway downstream of eukaryotic initiation factor 4E and S6K1 can be successfully inhibited, therefore, the recurrence of bladder cancer can be prevented by high-dose rapamycin only, suggesting that 4E-BP1 might be still under mTORC1.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Eukaryotic Initiation Factor-4E; Humans; Neoplasm Invasiveness; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.

Topics: Adenine; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Deoxycytidine; DNA Fragmentation; Drug Resistance, Neoplasm; Everolimus; Gemcitabine; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Microtubule-Associated Proteins; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Securin; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

We previously reported both in vivo and in vitro effects of rapamycin on urothelial carcinoma. Clinically, the use of rapamycin could not completely prevent the recurrence of urothelial carcinoma. Therefore, we designed this study to compare the difference of efficacy between early and late use of rapamycin in a rat model of urothelial carcinoma.. The rat model of urothelial carcinoma was induced by adding 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to the drinking water for up to 20 weeks in male Fisher-344 rats. Rapamycin was fed orally from the 1st day, 5th week, 9th week, 13th week, and 17th week. The antitumor effects of different periods of rapamycin treatment were assessed grossly and microscopically.. Papillary tumors of urinary bladder were successfully induced in the BBN group. Simultaneous use of rapamycin and BBN from the 1st day of treatment significantly reduced the tumor growth in urinary bladder: 80% of the rats had no tumor and 20% had low-grade tumors. Adding rapamycin from the 5th week was associated with more tumor growth: 20% of the rats had no tumors, 20% had low-grade tumors, and 60% had high-grade tumors. Moreover, in the groups with rapamycin treatment from the 9th week, 13th week, and 17th week, all rats developed high-grade papillary tumors in urinary bladder, as did the control group that received no rapamycin.. The study results suggest that the anticancer effect of rapamycin on urothelial carcinoma is stage dependent. Early use of rapamycin provides better anticancer effect, whereas late use of rapamycin fails to inhibit the cancer growth.

Topics: Animals; Antineoplastic Agents; Butylhydroxybutylnitrosamine; Carcinoma, Papillary; Drug Administration Schedule; Male; Neoplasm Grading; Neoplasms, Experimental; Rats, Inbred F344; Sirolimus; Time Factors; Tumor Burden; Urinary Bladder Neoplasms; Urothelium

Cisplatin (CDDP)-based chemotherapy is a commonly treatment for advanced urothelial carcinoma. However, episodes of cisplatin resistance have been referenced. Recently it has been reported that everolimus (RAD001) could have an important role to play in bladder-cancer treatment and that mTOR inhibitors may restore chemosensitivity in resistant tumours. The aim of this study was to assess RAD001 in vitro ability to enhance CDDP cytotoxicity in three human bladder-cancer cell lines. Over the course of 72h, the cells were exposed to different concentrations of CDDP and RAD001, isolated or combined. Treatment with CDDP statistically (P<0.05) decreased cell proliferation in cell lines in a dose-dependent manner. The anti-proliferative activity of CDDP used in combination with RAD001 was statistically significant (P<0.05) in the cell lines at all concentrations tested. RAD001 had a therapeutic effect when used in combination with CDDP and could therefore be a useful anti-cancer drug combination for patients with bladder cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Dose-Response Relationship, Drug; Drug Synergism; Everolimus; Humans; In Situ Nick-End Labeling; Sirolimus; Urinary Bladder Neoplasms; Urothelium

Topics: Acid Anhydride Hydrolases; Aged; Antineoplastic Agents; Biomarkers, Pharmacological; Biomedical Research; Clinical Trials, Phase I as Topic; DNA Repair Enzymes; DNA-Binding Proteins; Drug Resistance, Neoplasm; Everolimus; Female; Humans; National Cancer Institute (U.S.); Remission Induction; Sirolimus; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; United States; Urinary Bladder Neoplasms; Uterine Neoplasms

Topics: Antineoplastic Agents; Carcinoma, Transitional Cell; Clinical Trials, Phase II as Topic; Everolimus; Humans; Sirolimus; Urinary Bladder Neoplasms

Topics: Antineoplastic Agents; Drug Resistance, Neoplasm; Humans; Sirolimus; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment.. Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors.. Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo.. Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.

Topics: Androstadienes; Animals; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Line, Tumor; Clinical Trials, Phase II as Topic; Disease-Free Survival; Drug Interactions; Enzyme Activation; Everolimus; Female; Humans; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Wortmannin; Xenograft Model Antitumor Assays

Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, can elicit anti-tumor effects in various malignancies. Here, we sought to clarify the role of autophagy in celecoxib-induced cytotoxicity in human urothelial carcinoma (UC) cells. The results shows celecoxib induced cellular stress response such as endoplasmic reticulum (ER) stress, phosopho-SAPK/JNK, and phosopho-c-Jun as well as autophagosome formation in UC cells. Inhibition of autophagy by 3-methyladenine (3-MA), bafilomycin A1 or ATG7 knockdown potentiated celecoxib-induced apoptosis. Up-regulation of autophagy by rapamycin or GFP-LC3B-transfection alleviated celecoxib-induced cytotoxicity in UC cells. Taken together, the inhibition of autophagy enhances therapeutic efficacy of celecoxib in UC cells, suggesting a novel therapeutic strategy against UC.

Topics: Adenine; Antineoplastic Agents; Autophagy; Autophagy-Related Protein 7; Carcinoma, Transitional Cell; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Macrolides; MAP Kinase Kinase 4; Phosphoproteins; Pyrazoles; RNA, Small Interfering; Signal Transduction; Sirolimus; Sulfonamides; Ubiquitin-Activating Enzymes; Urinary Bladder Neoplasms

To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines.. ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms.. The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P = 0.001), was only observed in the 5637 cell line.. RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Topics: Animals; Antineoplastic Agents; Apoptosis; Butylhydroxybutylnitrosamine; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Everolimus; Flow Cytometry; G1 Phase; Humans; Immunoblotting; Immunohistochemistry; Male; Mice; Mice, Inbred ICR; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small-molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon-based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT-4, HCV29, 97-1. Interestingly, only RT-4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97-1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP-AUY922, and we then found synergistic inhibitory effects of NVP-AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97-1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1-mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer.

Topics: Afatinib; Antibiotics, Antineoplastic; Cell Line, Tumor; ErbB Receptors; HSP90 Heat-Shock Proteins; Humans; Isoxazoles; Lapatinib; Loss of Heterozygosity; Naphthyridines; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Quinazolines; Resorcinols; Signal Transduction; Sirolimus; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Cells, Cultured; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

To compared the effects of three immunosuppressive agents, i.e. sirolimus (SRL), cyclosporine A (CsA) and mycophenolate mofetil (MMF), with different mechanisms of action on the in vitro growth of various tumor cell lines of human transitional cell carcinoma of bladder cell lines EJ and T24 and in vivo growth of cell line of EJ in nude mice model.. The effects of SRL, CsA and MMF on the proliferation of transitional cell carcinoma of bladder cell lines were examined with the method of methyl thiazolyl tetrazolium (MTT). The effects of these immunosuppressants on tumor growth and metastasis were explored in a nude mice model with human transitional cell carcinoma of bladder cell line EJ. Forty-two nude mice were divided into 7 groups to receive normal saline (control), SRL, CsA, MMF, SRL + CsA, SRL + MMF and CsA + MMF respectively (n = 6 each).. The in vitro cell proliferation was inhibited by SRL and MMF versus the control groups. But no obvious inhibition of proliferation was observed at < 1000 ng/ml in the CsA-treated group. In the in vivo nude mice mode, the tumor volume of SRL, CsA group were lower than that in control group ((441 ± 231), (463 ± 110) vs (1032 ± 382) mm(3), both P < 0.05). In the in vivo nude mice mode of EJ treated by SRL, CsA, SRL + CsA, SRL + MMF and CsA + MMF, tumor volume at Day 23 was the lowest in the SRL + CsA group ((191 ± 92) vs (1032 ± 382) mm(3), P < 0.05). There was an inhibition of 75.26% in SRL + CsA group versus the control groups.. SRL and MMF demonstrate dose-dependent antiproliferative effects in human transitional cell carcinoma of bladder cell both in vitro and in vivo. CsA can inhibit the growth of human transitional cell carcinoma of bladder cell lines EJ cells in vivo.

Topics: Animals; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cyclosporine; Humans; Mice; Mice, Nude; Mycophenolic Acid; Sirolimus; Urinary Bladder Neoplasms

The purpose of this study was to determine whether everolimus, a rapamycin derivative, might significantly enhance the cytotoxicity of gemcitabine, an antitumor drug, in two human bladder-cancer cell lines. Human bladder-cancer T24 and 5637 cells were incubated with gemcitabine and everolimus in a range of concentrations either alone or in combination for 72 h. Flow cytometry, comet assay, MTT method and optical microscopy were used to assess cell proliferation, cell cycle, DNA damage, and morphological alterations. Gemcitabine exerted an inhibitory effect on T24 and 5637 cell proliferation, in a concentration-dependent manner. Everolimus significantly reduced proliferation of 5637 bladder cancer cells (IC₃₀) at 1 μM), whereas T24 demonstrated marked resistance to everolimus treatment. A significant antiproliferative effect was obtained combining gemcitabine (100 nM) with everolimus (0.05-2 μM) with an arrest of cell cycle at S phase. Furthermore, an increase in frequency of DNA damage, apoptotic bodies, and apoptotic cells was observed when T24 and 5637 cancer cells were treated simultaneously with both drugs. Data show that in vitro combination produced a more potent antiproliferative effect when compared with single drugs.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Comet Assay; Deoxycytidine; DNA Damage; Drug Resistance, Neoplasm; Drug Synergism; Everolimus; Gemcitabine; Humans; Mutagens; Neoplasm Invasiveness; Osmolar Concentration; S Phase; Sirolimus; Urinary Bladder Neoplasms

Mutations that activate the PI3K/AKT/mTOR pathway are relatively common in urothelial (bladder) cancers, but how these pathway mutations affect AKT dependency is not known. We characterized the relationship between AKT pathway mutational status and sensitivity to the effects of the selective AKT kinase inhibitor AZ7328 using a panel of 12 well-characterized human bladder cancer cell lines.. Sequenome DNA sequencing was performed to identify mutations in a panel of 12 urothelial cancer cell lines. Drug-induced proliferative inhibition and apoptosis were quantified using MTT assays and propidium iodide staining with FACS analyses. Protein activation via phosphorylation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting.. AZ7328 inhibited proliferation and AKT substrate phosphorylation in a concentration-dependent manner but had minimal effects on apoptosis. Proliferative inhibition correlated loosely with the presence of activating PIK3CA mutations and was strengthened in combination with the mTOR inhibitor rapamycin. AZ7328 induced autophagy in some of the lines, and in the cells exposed to a combination of AZ7328 and chemical autophagy inhibitors apoptosis was induced.. The cytostatic effects of AZ7328 correlate with PIK3CA mutations and are greatly enhanced by dual pathway inhibition using an mTOR inhibitor. Furthermore, AZ7328 can interact with autophagy inhibitors to induce apoptosis in some cell lines. Overall, our results support the further evaluation of combinations of PI3K/AKT/mTOR pathway and autophagy inhibitors in pre-clinical in vivo models and ultimately in patients with PIK3CA mutant bladder cancers.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Humans; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

Topics: Antineoplastic Agents; Clinical Trials, Phase II as Topic; Codon, Nonsense; Disease-Free Survival; Drug Resistance, Neoplasm; Everolimus; Genome-Wide Association Study; Genome, Human; Humans; Mechanistic Target of Rapamycin Complex 1; Molecular Targeted Therapy; Multiprotein Complexes; Neoplasm Metastasis; Neurofibromin 2; Proteins; Sequence Deletion; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

To investigate the anticarcinogenic effects of sirolimus 2 mg/kg/day on a rat model of urinary bladder carcinogenesis induced with N-butyl-N(4-hydroxybutyl)nitrosamine (BBN).. Thirty-six male Wistar rats were divided into four groups: 1, a control group (eight), given tap water only; 2, a sirolimus control group (eight), given 2 mg/kg/day; 3, a carcinogen (BBN) group (12) exposed to 0.05% BBN; 4, a treatment group (sirolimus/BBN; eight) given 2 mg/kg/day + 0.05% BBN. In the tumour-induction phase, from week 1 to week 8, rats from groups 3 and 4 received BBN ad libitum in drinking water. In the treatment phase, from week 8 to week 20, rats from groups 2 and 4 received sirolimus 2 mg/kg/day by an oesophageal cannula. At week 20 the rats were killed humanely, and the number and size of tumours recorded. The bladders were collected for histological, immunohistochemical and gene expression evaluation. Blood was collected for the determination of several serum proliferative and inflammatory markers. Lipid peroxidation, through serum malondialdehyde (MDA) content, and total antioxidant status (TAS) were also evaluated.. Sirolimus caused a marked inhibition of bladder tumour growth. When compared with group 3, group 4 had a reduced proportion of rats with tumour (three of eight vs eight of 12), and significantly fewer tumours per rat, with a mean (sd) of 1.00 (0.0) vs 1.88 (0.35), and tumour volume per tumour, of 0.30 (0.11) vs 66.1 (48.9) mm³, with less aggressive histological changes, i.e. a marked reduction in hyperplasia (four of eight vs 12/12), high-grade dysplasia (four of eight vs 11/12) and urothelial tumour. Rats in group 4 had no infiltrative bladder cancers and had a lower incidence of high-grade tumours than rats in group 3. The rats in group 4 had decreased serum levels of transforming growth factor-β1, higher levels of tumour necrosis factor-α, and higher levels of serum TAS and a better serum MDA/TAS ratio, a marker of more favourable redox status. Furthermore, the down-regulation of bladder caspase 3 gene expression and the increased Ki67 immunostaining in group 3 were significantly attenuated in group 4.. Sirolimus given as an oral agent, 2 mg/kg/day, significantly inhibited rat bladder carcinogenesis. Sirolimus reduced the number and volume of tumours and induced a less aggressive histological behaviour. This might be due to antiproliferative and antioxidant properties, as well as to the restoration of apoptotic pathways.

Topics: Animals; Antibiotics, Antineoplastic; Butylhydroxybutylnitrosamine; Carcinogens; Caspase 3; Down-Regulation; Ki-67 Antigen; Male; Rats; Rats, Wistar; Sirolimus; Urinary Bladder; Urinary Bladder Neoplasms

To investigate the rationale for using targeted therapies against hypoxia-inducible factor (HIF) and mammalian target of rapamycin (mTOR) pathways in urothelial carcinoma of the bladder, by studying the immunohistochemical expression of molecules of these pathways in urothelial carcinoma, as recent pre-clinical studies and clinical trials have shown the potential utility of such targeted therapies.. Immunohistochemical stains were performed on a tissue microarray prepared from 92 cases of ≥ pT2 urothelial (transitional cell) carcinoma of bladder, using antibodies against HIF-1α and VEGF-R2, and phospho-S6 and phospho-4E BP1, molecules of HIF and activated mTOR pathways, respectively. Immunoreactivity was graded from 0 to 3+ (0, 0-5%; 1+, 6-25%; 2+, 26-50%; 3+, > 50% tumour cells positive).. In all, 58, 34, 35 and 17% of the tumours showed grade 2-3+ expression of phospho-4E BP1, phospho-S6, HIF-1α and VEGF-R2, respectively. Moderate correlation for immunoreactivity was observed between molecules within the same pathway [(phospho-4E BP1 with phospho-S6 (rho = 0.411), and HIF-1α with VEGF-R2 (rho = 0.265)], but not between molecules across pathways.. Urothelial carcinomas of the bladder express molecules of the HIF and mTOR pathways, providing a rationale for clinical trials evaluating agents targeting these pathways. Correlation between molecules within the same pathway, and not across pathways, suggests that investigating the usefulness of a specific targeted agent might benefit from pre-treatment evaluation of pathway marker expression.

Topics: Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Hypoxia; Cell Line, Tumor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

To investigate whether mammalian target of rapamycin (mTOR) inhibition by rapamycin is therapeutically efficacious in combination with cisplatin for bladder cancer.. Using a panel of human urothelial carcinoma cell lines, we determined the effect of rapamycin on cell viability, cell-cycle progression, signalling and apoptosis. The effect of mTOR inhibition on chemosensitivity was investigated by treating cells with rapamycin, alone, or with cisplatin. The effect of rapamycin or cisplatin treatment was assessed in xenograft mice inoculated with urothelial carcinoma cells. Expression of p-mTOR in human bladder cancer specimens was assessed using a tissue microarray.. Treatment with rapamycin significantly decreased cell viability in UMUC3 (P = 0.004) and 253J (P < 0.001) cells. It induced arrest in the G(0) -G(1) phase and decreased activation of p-mTOR and its downstream effector, p-S6K, in both cell lines. Treatment with rapamycin increased the ability of cisplatin to inhibit cell viability in UMUC3 (P = 0.002) and 253J (P = 0.03) cells. No evidence for apoptosis induction was noted after treatment with rapamycin alone. Mouse xenografts of UMUC3 cells revealed that rapamycin significantly prolonged survival and enhanced the therapeutic efficacy of cisplatin. In patient urothelial carcinoma specimens, p-mTOR expression was increased in cancer vs non-tumour bladder tissue in 65/203 (32.0%) tumours.. mTOR blockade inhibits urothelial carcinoma cell proliferation and enhances the effectiveness of cisplatin. Suppression of the mTOR pathway has the potential to be a therapeutic target in bladder cancer for selected patients.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Transitional Cell; Cell Cycle; Cell Proliferation; Cell Survival; Cisplatin; Humans; Male; Mice; Middle Aged; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

Bladder cancer and head and neck squamous cell carcinoma (HNSCC) are frequent but lack efficient therapies especially in advanced disease. Almost no studies on mTOR function and inhibition in these tumor entities have been reported. We examined the gene and protein expression levels of mTOR and its activated form (pmTOR) in three human bladder carcinoma cell lines (RT-4, T24, EJ28) and three HNSCC cell lines (PCI-1, PCI-13, BHY). Furthermore, the consequences of mTOR inhibition by mTOR-specific siRNAs and the mTOR inhibitor temsirolimus were analysed in vitro using immunohistochemical Ki-67 staining, mTOR and pmTOR western blot analysis, MTT assay, as well as cell cycle analysis with flow cytometry. Especially pmTOR protein expression levels showed marked differences between cell lines. siRNA transfection was associated with dose-dependent target protein reduction but not proliferation inhibition or apoptosis. On the contrary, temsirolimus significantly reduced cell viability and induced apoptosis and cell cycle arrest. According to these data, bladder cancer and HNSCC are promising tumor entities for mTOR inhibition with temsirolimus.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Protein Kinase Inhibitors; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We investigated the effect of the mTOR inhibitor RAD001 (everolimus) on human bladder cancer (BC) cells in vitro and in vivo.. 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 [(3)H]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 the mTOR pathway were analyzed by Western blotting. To test the effects of RAD001 in vivo, UM-UC-3, UM-UC-6, and UM-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.. 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 G(1)-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.. 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.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Transitional Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Everolimus; Female; Humans; Mice; Mice, Nude; Protein Biosynthesis; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

The purpose of this study was to determine the efficacy of a combination of gemcitabine and sirolimus in a mouse model of invasive bladder cancer.. Gemcitabine (50 mg/kg) and sirolimus (1.5 mg/kg) were administered to animals previously exposed to N-butyl-N-4(hydroxybutyl)nitrosamine in drinking water. Tumour development was determined by histopathological evaluation.. Both drugs were well tolerated by animals. The incidence of lesions in mice treated with gemcitabine was lower in comparison to those not treated, however this result was not statistically significant. The incidence of invasive bladder cancer in animals treated with sirolimus was statistically lower (20%) than in animals not treated (54%) (p=0.008). The results indicate that this drug combination has no statistical significance on the development of pre-neoplastic urothelial lesions and had only a minor impact on invasive bladder cancer incidence in mice.. The combination of gemcitabine and sirolimus had only a marginal impact on invasive bladder cancer in a mouse model.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Butylhydroxybutylnitrosamine; Deoxycytidine; Gemcitabine; Male; Mice; Mice, Inbred ICR; Neoplasm Invasiveness; Sirolimus; Survival Rate; Treatment Outcome; Urinary Bladder Neoplasms

Relapse after initial first-line chemotherapy shows a poor prognosis in metastatic urothelial cancer. Currently, several chemotherapeutic agents and targeted drugs are under evaluation for platin-resistant advanced urothelial carcinoma. Vinflunine has been approved for second-line treatment in this indication. We present a patient with initial T4 advanced and subsequently metastasized bladder cancer, who has shown prolonged survival of 44 months after radical cystectomy. During her clinical course, the patient received two different platinum-containing therapies, temsirolimus within a phase II protocol and subsequent vinflunine chemotherapy. Treatment duration was 15 weeks with temsirolimus and 9 weeks with vinflunine, respectively, with a stable disease period of 3.8 months under temsirolimus therapy. This case is an example of how patients can derive a survival benefit from adequate sequencing of surgery and medical treatment including the newest therapies, even in advanced disease.

Topics: Antineoplastic Agents; Carcinoma, Transitional Cell; Disease Progression; Female; Humans; Middle Aged; Sirolimus; Time Factors; Treatment Outcome; Urinary Bladder; Urinary Bladder Neoplasms; Vinblastine

Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Phosphoproteins; Proto-Oncogene Proteins c-akt; Quinolines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

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.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Transplantation, Heterologous; Urinary Bladder Neoplasms

To evaluate the in vitro effects of rapamycin in human transitional cell carcinoma and to clarify the possible rapamycin-hypoxia interactions.. Monolayer cultures of RT112 (G1), RT4 (G1-2), T24 (G3), and SUP (G4) cells were incubated in medium with or without rapamycin (10 microM, 100 microM) in different gaseous conditions (1% oxygen plus 5% carbon dioxide plus 94% nitrogen or 95% oxygen plus 5% carbon dioxide or 21% oxygen plus 78% nitrogen). Cell vitality and proliferation were determined using the microculture tetrazolium assay. Apoptotic cells were quantified by flow cytometry. Enzyme-linked immunosorbent assay was used to visualize inhibition of the mammalian target of rapamycin (mTOR) by rapamycin and synthesis of vascular endothelial growth factor.. Rapamycin delayed proliferation of cancer cell lines but did not induce apoptosis. No direct hypoxia interactions of rapamycin were found. Enzyme-linked immunosorbent assay gave evidence of mTOR inhibition in all cell lines and a significant decrease of hypoxia-induced vascular endothelial growth factor synthesis.. Because proliferation in bladder cancer cells was delayed, but no apoptosis was induced, cell cycle arrest caused by rapamycin was feasible. Rapamycin decreased hypoxia-induced synthesis of vascular endothelial growth factor. These findings should be substantiated in an animal model to verify the antiangiogenetic effects of rapamycin.

Topics: Angiogenesis Inducing Agents; Antibiotics, Antineoplastic; Carcinoma, Transitional Cell; Cell Hypoxia; Cell Proliferation; Humans; Sirolimus; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells.. T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis.. Sirolimus inhibits the growth of bladder carcinoma cells and decreases their viability. Significant correlations were found between cell proliferation and sirolimus concentration (r = 0.830; p < 0.01) as well as between cell viability and sirolimus concentration (r = -0.896; p < 0.01).. Sirolimus has an anti-proliferation effect on the T24 bladder carcinoma cell line. The information from our results is useful for a better understanding sirolimus's anti-proliferative activity in the T24 bladder cancer cell line.

Topics: Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Humans; Sirolimus; Urinary Bladder Neoplasms

Malignant tumor is the most common complication occurred in transplant recipients. It is widely recognized that immunosuppressive treatments increase the risk of cancer in transplant recipients. The efficacy and safety of rapamycin (RPM) in combination with low-dose calcineurin inhibitor (CNI) in treating 15 renal allograft recipients which developed urothelial carcinoma were observed.. Immunosuppressive regimen in all recipients was altered with rapamycin to replace mycophenolate mofetil (MMF) or azathioprine (Aza). The initial loading dosage was 2 mg/d, and the next dosage was 1 mg/d. The dosage of rapamycin was carefully adjusted according to the blood drug level and concentration of the drug was maintained at 4 - 6 microg/L. In all the 15 patients, the calcineurin inhibitor was reduced down to one third of the original dosage after the rapamycin blood concentration became stable. Surgical treatment and intravesical instillation chemotherapy were carried out in all patients. Recurrence of the tumor was monitored throughout the study. Post-transplant renal function and side effects were also closely monitored.. Among the 15 patients, 9 had no tumor recurrence in 2 years, 2 had tumor recurrences twice, and 4 had once. There was no acute rejection observed during RPM treatment. Post-transplant renal function in 11 patients was improved, with a decreased creatinine level. Hyperlipoidemia and thrombocytopenia were the most frequent adverse events which responded well to corresponding treatments.. Among the renal allograft recipients with urothelial carcinoma, combination of rapamycin and low dose calcineurin inhibitor treatment is effective and safe.

Topics: Adult; Azathioprine; Female; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; Sirolimus; Urinary Bladder Neoplasms; Urothelium

Sirolimus was originally used as an immunosuppressant drug but recent reports have indicated that it may have other potential biological effects as an anticancer drug. The chemopreventive efficacy of sirolimus was evaluated in an experimental model of invasive urinary bladder cancer.. ICR mice received N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water for a period of twelve weeks. Sirolimus was administered 5 days a week. Animals were sacrificed either one or four weeks after their final treatment. Ki-67 was immunohistochemically analysed in paraffin-embedded tissue.. No evidence of host toxicity was found. The incidence of BBN-induced invasive urothelial carcinoma was significantly reduced in mice treated with sirolimus. Preneoplastic and neoplastic lesions exhibited a significant decrease in cellular proliferation.. Histopathological and immunohistochemical studies showed that sirolimus reduced tumour incidence and proliferation. Sirolimus should be considered for further in vitro and in vivo studies in order to provide evidence of effectiveness.

Topics: Animals; Antibiotics, Antineoplastic; Butylhydroxybutylnitrosamine; Carcinoma in Situ; Carcinoma, Papillary; Hyperplasia; Immunoenzyme Techniques; Male; Mice; Mice, Inbred ICR; Sirolimus; Urinary Bladder Neoplasms

Topics: Antineoplastic Agents; Cell Line, Tumor; Dose-Response Relationship, Drug; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Models, Biological; Neoplasm Metastasis; Phosphorylation; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Early-stage bladder cancer occurs as two distinct forms: namely, low-grade superficial disease and high-grade carcinoma in situ (CIS), which is the major precursor of muscle-invasive bladder cancer. Although the low-grade form is readily treatable, few, if any, effective treatments are currently available for preventing progression of nonmuscle-invasive CIS to invasive bladder cancer. Based on our previous findings that the mammalian target of Rapamycin (mTOR) signaling pathway is activated in muscle-invasive bladder cancer, but not superficial disease, we reasoned that suppression of this pathway might block cancer progression. To test this idea, we performed in vivo preclinical studies using a genetically engineered mouse model that we now show recapitulates progression from nonmuscle-invasive CIS to muscle-invasive bladder tumors. We find that delivery of Rapamycin, an mTOR inhibitor, subsequent to the occurrence of CIS effectively prevents progression to invasive bladder cancer. Furthermore, we show that intravesical delivery of Rapamycin directly into the bladder lumen is highly effective for suppressing bladder tumorigenesis. Thus, our findings show the potential therapeutic benefit of inhibiting mTOR signaling for treatment of patients at high risk of developing invasive bladder cancer. More broadly, our findings support a more widespread use of intravesical delivery of therapeutic agents for treatment of high-risk bladder cancer patients, and provide a mouse model for effective preclinical testing of potential novel agents.

Topics: Administration, Intravesical; Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Disease Progression; Female; Integrases; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Knockout; Protein Serine-Threonine Kinases; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

This perspective on Seager et al. (beginning on p. 1008) considers an important advance in the effort to control bladder cancer. Frontline therapy for superficial transitional cell carcinoma of the bladder involves instillation of the crude immunomodulatory bacterial extract Bacillus Calmette-Guérin directly into the organ. Seager et al. now show that local administration of a chemical inhibitor of mammalian target of rapamycin strongly suppressed growth in a novel preclinical mouse model that develops carcinoma in situ, a particularly problematic form of transitional cell carcinoma of the bladder. The results not only support the clinical evaluation of mammalian target of rapamycin inhibitors in this setting, they open the door for the evaluation of additional molecular local therapies as well.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Transitional Cell; Humans; Intracellular Signaling Peptides and Proteins; Mice; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

We examined whether mTOR inhibition by RAD001 (Everolimus) could be therapeutically efficacious in the treatment of bladder cancer. RAD001 markedly inhibited proliferation of nine human urothelial carcinoma cell lines in dose- and sensitivity-dependent manners in vitro. FACS analysis showed that treatment with RAD001 for 48 h induced a cell cycle arrest in the G(0)/G(1) phase in all cell lines, without eliciting apoptosis. Additionally, RAD001 significantly inhibited the phosphorylation of S6 downstream of mTOR and VEGF production in all cell lines. We also found tumor weights from nude mice bearing human KU-7 subcutaneous xenografts treated with RAD001 were significantly reduced as compared to placebo-treated mice. This tumor growth inhibition was associated with significant decrease in cell proliferation rate and angiogenesis without changes in cell death. In conclusion inhibition of mTOR signaling in bladder cancer models demonstrated remarkable antitumor activity both in vitro and in vivo. This is the first study showing that RAD001 could be exploited as a potential therapeutic strategy in bladder cancer.

Topics: Animals; Apoptosis; Carcinoma; Cell Cycle; Cell Separation; Dose-Response Relationship, Drug; Everolimus; Female; Flow Cytometry; Humans; Intracellular Signaling Peptides and Proteins; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

Topics: Calcineurin Inhibitors; Cell Proliferation; Cyclosporine; Humans; Immunosuppressive Agents; Kidney Transplantation; Postoperative Care; Signal Transduction; Sirolimus; Urinary Bladder Neoplasms

Sirolimus (SRL), an immunosuppressant shown to possess anti-proliferative properties, was hypothesized to mitigate the occurrence of posttransplantation malignancy. We examined its effect on posttransplantation urothelial carcinoma (UC).. This retrospective case analysis included renal allograft recipients with UC treated with SRL in a single institute.. Among 90 renal recipients treated with SRL, 6 had previous/new-onset UC in the native kidney/ureter or bladder: at a mean period of 28 months (range, 7-49) of administering SRL for these recipients, UC occurred/recurred in 4 of the 6 patients. Individual cases are presented in detail.. SRL does not absolutely abolish the occurrence/recurrence of UC among renal transplant recipients. Its potency as an anti-cancerous immunosuppressant for transplant recipients with UC deserves to be further defined in larger studies, probably by controlling SRL blood levels at lower or much higher ranges than used herein.

Topics: Adult; Antibiotics, Antineoplastic; Female; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Recurrence; Retrospective Studies; Sirolimus; Transplantation, Homologous; Treatment Failure; Treatment Outcome; Ureteral Neoplasms; Urinary Bladder Neoplasms; Urologic Neoplasms