sirolimus and Carcinoma--Transitional-Cell

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

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

There is little evidence about whether mammalian target of rapamycin (mTOR) inhibitor could prevent post-kidney transplant (KT) urothelial carcinoma (UC) or not. The aim of this study is to analyze the role of mTOR inhibitor add-on in tacrolimus-based kidney transplant recipients.. The data were obtained from the Kaohsiung Chang Gung Memorial Hospital using the Chang Gung Research Database and retrospectively reviewed from January 2000 to December 2015. Patients then were categorized into 2 groups: group FK (more than 2-year tacrolimus [FK] prescription) and group FK + mTOR inhibitor (more than 2-year tacrolimus plus at least 6-month continued sirolimus prescription). The primary end point is post-KT UC development. The secondary end point is mTOR inhibitor add-on effect on renal function deterioration episode.. There were 140 patients with tacrolimus-based immunosuppressant (group FK) and 82 patients with tacrolimus-based and add-on mTOR inhibitor regimen (group FK + mTOR inhibitor). The follow-up duration, sex distribution, and combined mycophenolate mofetil rate are similar in both groups. Younger age, lower tacrolimus trough level, lower UC incidence, and longer KT-to-UC interval were observed. Short- to intermediate-term results revealed noninferior graft outcome by creatinine level or creatinine deterioration.. In our preliminary result, mTOR inhibitor add-on in patients with tacrolimus-based regimen revealed less post-KT UC occurrence. In addition, noninferior graft outcome was also observed. In Taiwan, a high UC prevalence area, mTOR inhibitor add-on strategy can be considered as a preventive strategy for UC after KT.

Topics: Adult; Carcinoma, Transitional Cell; Female; Graft Rejection; Graft Survival; Humans; Immunocompromised Host; Immunosuppressive Agents; Incidence; Kidney Transplantation; Male; Middle Aged; Postoperative Complications; Retrospective Studies; Sirolimus; Tacrolimus; Taiwan; TOR Serine-Threonine Kinases

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

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

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

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

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

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

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