sirolimus and Glioblastoma

Activation of PI3K/Akt/mTOR (mechanistic target of rapamycin) signaling cascade has been shown in tumorigenesis of numerous malignancies including glioblastoma (GB). This signaling cascade is frequently upregulated due to loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is responsive to growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases like Akt and SGK. mTORC2 plays a crucial role in maintenance of normal and cancer cells through its association with ribosomes, and is involved in cellular metabolic regulation. Both complexes control each other as Akt regulates PRAS40 phosphorylation, which disinhibits mTORC1 activity, while S6K regulates Sin1 to modulate mTORC2 activity. Another significant component of mTORC2 is Sin1, which is crucial for mTORC2 complex formation and function. Allosteric inhibitors of mTOR, rapamycin and rapalogs, have essentially been ineffective in clinical trials of patients with GB due to their incomplete inhibition of mTORC1 or unexpected activation of mTOR via the loss of negative feedback loops. Novel ATP binding inhibitors of mTORC1 and mTORC2 suppress mTORC1 activity completely by total dephosphorylation of its downstream substrate pS6K

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Movement; Cell Proliferation; Clinical Trials as Topic; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Indoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Purines; Signal Transduction; Sirolimus

Temozolomide (TMZ), an alkylating agent, is widely used for treating primary and recurrent high-grade gliomas. However, the efficacy of TMZ is often limited by the development of resistance. Recently, studies have found that TMZ treatment could induce autophagy, which contributes to therapy resistance in glioma. To enhance the benefit of TMZ in the treatment of glioblastomas, effective combination strategies are needed to sensitize glioblastoma cells to TMZ. In this regard, as autophagy could promote cell survival or autophagic cell death, modulating autophagy using a pharmacological inhibitor, such as chloroquine, or an inducer, such as rapamycin, has received considerably more attention. To understand the effectiveness of regulating autophagy in glioblastoma treatment, this review summarizes reports on glioblastoma treatments with TMZ and autophagic modulators from in vitro and in vivo studies, as well as clinical trials. Additionally, we discuss the possibility of using autophagy regulatory compounds that can sensitive TMZ treatment as a chemotherapy for glioma treatment.

Topics: Antineoplastic Combined Chemotherapy Protocols; Autophagy; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Chloroquine; Clinical Trials as Topic; Dacarbazine; Drug Resistance, Neoplasm; Drug Synergism; Glioblastoma; Humans; Sirolimus; Temozolomide

Atypical serine-threonine kinase, mTOR (mechanistic target of Rapamycin; originally coined "mammalian TOR"), exists in two distinct multi-protein complexes termed mTOR complex 1 (mTORC1) and 2 (mTORC2), that senses and integrates a variety of environmental signals to control organism growth and homeostasis via non-overlapping signaling pathways. mTOR belongs to the phosphoinositide 3-kinase (PI3-K)-related kinase family, and an aberrant activation of mTORC1 is a potential contributing factor in uncontrolled cell growth, proliferation, and survival of tumor cells via specific effects on cap-dependent translation initiation, as well as in a more sustained manner via advancing ribosome biogenesis. It is thereby shown to be deregulated in numerous pathological conditions including cancer, obesity, type 2 diabetes, and neurodegeneration. Notably, mTOR itself, or through its substrates, regulates stem cell differentiation and maintenance of plueropotency. mTORC2 has been linked to cytoskeletal reorganization and cell survival through Akt, and is crucial to many divergent physiological functions, which may include stem cell regulation.

Topics: Cell Differentiation; Glioblastoma; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Neoplasms; Proto-Oncogene Proteins c-akt; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases

Kinase inhibitors have emerged as effective cancer therapeutics in a variety of human cancers. Glioblastoma (GBM), the most common malignant brain tumor in adults, represents a compelling disease for kinase inhibitor therapy because the majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. Attempts to target the Ras-Phosphatidylinositol 3-kinase (PI3K)-mammalian Target of Rapamycin (mTOR) axis in GBM with first generation receptor tyrosine kinase (RTK) inhibitors and rapalogs have been disappointing. However, there is reason for renewed optimism given the now very detailed knowledge of the cancer genome in GBM and a wealth of novel compounds entering the clinic, including next generation RTK inhibitors, class I PI3K inhibitors, mTOR kinase inhibitors (TORKinibs), and dual PI3(K)/mTOR inhibitors. This chapter reviews common genetic alterations in growth factor signaling pathways in GBM, their validation as therapeutic targets in this disease, and strategies for future clinical development of kinase inhibitors for high grade glioma.

Topics: Antineoplastic Agents; Clinical Trials as Topic; Drug Resistance, Neoplasm; ErbB Receptors; Glioblastoma; Humans; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; ras Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Clinical Trials as Topic; Combined Modality Therapy; Drug Delivery Systems; Drug Evaluation, Preclinical; ErbB Receptors; Glioblastoma; Humans; Neoplasm Proteins; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) plays a critical role in the regulation of cell growth, proliferation and survival. Components of the mTOR pathway are activated in a variety of tumors, including glioblastoma multiforme (GBM), and we have found that one surprising consequence of mTOR pathway activation is resistance of GBMs to the proapoptotic effects of agents such as APO2L/TRAIL. mTOR inhibition has become feasible following the development of rapamycin and comparable analogs with improved pharmacological properties, including CCI-779, RAD001 and AP23573. Numerous studies have also demonstrated promising proapoptotic activity, with relatively mild side effects, using rapamycin analogs in vitro and in vivo in conjunction with APO2L/TRAIL. These studies suggest that mTOR inhibitors can be combined with APO2L/TRAIL as a potential tumor-selective therapy.

Topics: Antineoplastic Agents; Glioblastoma; Humans; Protein Kinase Inhibitors; Protein Kinases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Sirolimus; TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases

Temsirolimus (CCI779), an intravenous analog of rapamycin, presents immunosuppressive properties and also antiproliferative activity. Its principal target is the mTOR serine/threonin kinase which controls the initiation of the transcription of many ARNm implicated in carcinogenesis. Breast cancers, glioblastoma and renal cell carcinoma were particularly studied with response rates from 10 to 20 %. In haematology, mantle-cell lymphoma is of particular interest because of constitutional activation of cyclin D1 (response rate of 40 %). As a whole these data define temsirolimus as a promising new drug. Current and further developments are based on its association with chemotherapy in a concomitant or sequential way.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclin D1; Glioblastoma; Humans; Kidney Neoplasms; Lymphoma, Mantle-Cell; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Glioblastoma, the most highly aggressive and lethal form of brain cancer, has been a particular challenge to treat in terms of improving a patient's quality of life and outcome. Each of the current treatment options is limited due to factors intrinsic to the tumor's biology and the special microenvironment of its location within the brain. Surgical resection is limited by the non-circumscribed borders that can be detected. Radiation therapy has to contend with neurotoxicity to adjacent normal tissues. Chemotherapy is constrained by the blood-brain barrier, which is a very real constraint of systemic therapy -- producing minimal benefit with substantial toxicity in order to administer therapeutic dosages. In part, such hurdles explain the reasons why survival has changed little over many decades of research in this field. The newest generation of treatments includes more effective cytotoxic agents, so-called targeted compounds, and biologics/immunotherapeutics. This article summarizes the preclinical proof-of-concept research and human studies involving some of the agents creating the most positive buzz in the medical community. The advantages and limitations of each are described.

Topics: Antineoplastic Agents; Antineoplastic Agents, Alkylating; Benzamides; Biocompatible Materials; Brain Neoplasms; Cancer Vaccines; Clinical Trials as Topic; Combined Modality Therapy; Dacarbazine; Decanoic Acids; ErbB Receptors; Erlotinib Hydrochloride; Farnesyltranstransferase; Gefitinib; Glioblastoma; Humans; Imatinib Mesylate; Immunotoxins; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Piperazines; Polyesters; Protein Kinase Inhibitors; Proteins; Pyrimidines; Quinazolines; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases; Transcription Factors

Malignant glioma (MG) is the most deadly primary brain cancer. Signaling though the PI3K/AKT/mTOR axis is activated in most MGs and therefore a potential therapeutic target. The mTOR inhibitor temsirolimus and the AKT inhibitor perifosine are each well-tolerated as single agents but with limited activity reclinical data demonstrate synergistic anti-tumor effects from combined treatment. Therefore, we initiated a phase I trial of combined therapy in recurrent MGs to determine safety and a recommended phase II dose.. Adults with recurrent MG, Karnofsky Performance Status ≥ 60 were enrolled, with no limit on the number of prior therapies. Temsirolimus dose was escalated using standard 3 + 3 design from 15 mg to 170 mg administered once weekly. Perifosine was fixed as a 600 mg load on day 1 followed by 100 mg nightly (single agent MTD) until dose level 7 when the load increased to 900 mg.. We treated 35 patients with with glioblastoma (17) or other MGs (18; including nine anaplastic astrocytoma, nine anaplastic oligodendroglioma, one anaplastic oligoastrocytoma, and two low grade astrocytomas with radiographic transformation to MG). We observed five dose-limiting toxicities (DLTs): one at dose level 3 (50mg temsirolimus), then two at dose level 7 expansion (170 mg temsirolimus), and then two more at dose level 6 expansion (170 mg temsirolimus). DLTs included thrombocytopenia (n = 3), intracerebral hemorrhage (n = 1) and lung infection (n = 1).. Combining the mTOR inhibitor temsirolimus dosed at 115 mg weekly and the AKT inhibitor perifosine dosed at 100 mg daily (following 600 mg load) is tolerable in heavily pretreated adults with recurrent MGs.

Topics: Adult; Aged; Antineoplastic Agents; Brain Neoplasms; Drug Therapy, Combination; Female; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Phosphorylcholine; Prospective Studies; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Young Adult

Mitogen-activated protein kinase (MAPK) activation and mammalian target of rapamycin (mTOR)-dependent signaling are hallmarks of glioblastoma. In the current study, the authors conducted a phase 1/2 study of sorafenib (an inhibitor of Raf kinase and vascular endothelial growth factor receptor 2 [VEGFR-2]) and the mTOR inhibitor temsirolimus in patients with recurrent glioblastoma.. Patients with recurrent glioblastoma who developed disease progression after surgery or radiotherapy plus temozolomide and with ≤2 prior chemotherapy regimens were eligible. The phase 1 endpoint was the maximum tolerated dose (MTD), using a cohorts-of-3 design. The 2-stage phase 2 study included separate arms for VEGF inhibitor (VEGFi)-naive patients and patients who progressed after prior VEGFi.. The MTD was sorafenib at a dose of 200 mg twice daily and temsirolimus at a dose of 20 mg weekly. In the first 41 evaluable patients who were treated at the phase 2 dose, there were 7 who were free of disease progression at 6 months (progression-free survival at 6 months [PFS6]) in the VEGFi-naive group (17.1%); this finding met the prestudy threshold of success. In the prior VEGFi group, only 4 of the first 41 evaluable patients treated at the phase 2 dose achieved PFS6 (9.8%), and this did not meet the prestudy threshold for success. The median PFS for the 2 groups was 2.6 months and 1.9 months, respectively. The median overall survival for the 2 groups was 6.3 months and 3.9 months, respectively. At least 1 adverse event of grade ≥3 was observed in 75.5% of the VEGFi-naive patients and in 73.9% of the prior VEGFi patients.. The limited activity of sorafenib and temsirolimus at the dose and schedule used in the current study was observed with considerable toxicity of grade ≥3. Significant dose reductions that were required in this treatment combination compared with tolerated single-agent doses may have contributed to the lack of efficacy. Cancer 2018;124:1455-63. © 2018 American Cancer Society.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Female; Follow-Up Studies; Glioblastoma; Humans; Male; Maximum Tolerated Dose; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Sirolimus; Sorafenib; Survival Rate

EORTC 26082 assessed the activity of temsirolimus in patients with newly diagnosed glioblastoma harboring an unmethylated O6 methylguanine-DNA-methyltransferase (MGMT) promoter.. Patients (n = 257) fulfilling eligibility criteria underwent central MGMT testing. Patients with MGMT unmethylated glioblastoma (n = 111) were randomized 1:1 between standard chemo-radiotherapy with temozolomide or radiotherapy plus weekly temsirolimus (25 mg). Primary endpoint was overall survival at 12 months (OS12). A positive signal was considered >38 patients alive at 12 months in the per protocol population. A noncomparative reference arm of 54 patients evaluated the assumptions on OS12 in a standard-treated cohort of patients. Prespecified post hoc analyses of markers reflecting target activation were performed.. Both therapies were administered per protocol with a median of 13 cycles of maintenance temsirolimus. Median age was 55 and 58 years in the temsirolimus and standard arms, the WHO performance status 0 or 1 for most patients (95.5%). In the per protocol population, 38 of 54 patients treated with temsirolimus reached OS12. The actuarial 1-year survival was 72.2% [95% confidence interval (CI), 58.2-82.2] in the temozolomide arm and 69.6% (95% CI, 55.8-79.9) in the temsirolimus arm [hazard ratio (HR) 1.16; 95% CI, 0.77-1.76; P = 0.47]. In multivariable prognostic analyses of clinical and molecular factors, phosphorylation of mTORSer2448 in tumor tissue (HR 0.13; 95% CI, 0.04-0.47; P = 0.002), detected in 37.6%, was associated with benefit from temsirolimus.. Temsirolimus was not superior to temozolomide in patients with an unmethylated MGMT promoter. Phosphorylation of mTORSer2448 in the pretreatment tumor tissue may define a subgroup benefitting from mTOR inhibition. Clin Cancer Res; 22(19); 4797-806. ©2016 AACR.

Topics: Adult; Aged; Brain Neoplasms; Chemoradiotherapy; Dacarbazine; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; Female; Glioblastoma; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Promoter Regions, Genetic; Proportional Hazards Models; Sirolimus; Temozolomide; Tumor Suppressor Proteins; Young Adult

Targeting specific molecular alterations in glioblastoma (GBM) might more effectively kill tumor cells and increase survival. Vandetanib inhibits epidermal growth factor receptor and vascular endothelial growth factor receptor 2. Sirolimus inhibits mammalian target of rapamycin (mTOR), a member the phosphoinositide 3-Kinase signaling pathway. We sought to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of vandetanib combined with sirolimus. Twenty-two patients (14 men; 8 women) with recurrent GBM enrolled. Median age and KPS were 52.5 years and 90 %, respectively. Patients were naive to anti-VEGF and anti-EGF therapy and mTOR inhibitors, and not on CYP3A4-inducing drugs. Vandetanib and sirolimus were orally administered on a continuous daily dosing schedule in escalating dose cohorts. Ten patients enrolled in the dose escalation phase. Twelve more enrolled at the MTD to explore progression-free survival at 6 months (PFS6) in a single arm, single stage phase II-type design. In total, 19 patients received at least one dose at the MTD, and 15 completed at least 1 cycle at MTD. MTD was 200 mg vandetanib plus 2 mg sirolimus. The DLT was elevated AST/SGOT. The most common toxicities were lymphopenia, fatigue, rash, and hypophosphatemia. For 19 patients who received at least one dose at the MTD, including seven from the phase I group, two had a partial response [10.5 %; 95 % CI (1, 33 %)] and PFS6 was 15.8 % [95 % CI (3.9, 34.9 %)]. Vandetanib and sirolimus can be safely co-administered on a continuous, daily dosing schedule.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cohort Studies; Disease-Free Survival; Female; Glioblastoma; Humans; Male; Maximum Tolerated Dose; Middle Aged; Neoplasm Recurrence, Local; Piperidines; Quinazolines; Sirolimus

Bevacizumab combined with chemotherapy has recently shown promising efficacy in recurrent high-grade glioma. Phosphatase and tensin homolog (PTEN) mutation in glioblastoma multiforme (GBM) patients causes abnormally high activity of the pathways of Phosphatidylinositide 3-kinases (PI3K), Protein Kinase B (AKT), and the mammalian target of rapamycin (mTOR) and is associated with unfavorable prognosis. Temsirolimus, an mTOR inhibitor, has been well-tolerated in monotherapy, but with limited effects. The combination of temsirolimus and antibodies to vascular endothelial factor (VEGF) has not yet been investigated, but with the hypothesis that temsirolimus might provide complimentary therapeutic benefit in combination with bevacizumab, we included patients with progressive GBM after bevacizumab in an open phase II study.. Adult patients with GBM recurrence after standard temozolomide chemoradiotherapy and bevacizumab-containing second-line therapy, received temsirolimus (25 mg i.v.) on days 1 and 8 and bevacizumab (10 mg/kg) on day 8, every two weeks. Assessments were performed every eight weeks. Blood samples for biomarkers were collected weekly for the first eight weeks and at progression. The primary end-point was median progression-free survival (PFS) and secondary end-points were radiographic response, overall survival (OS), and safety of the bevacizumab-temsirolimus combination.. Thirteen patients were included, whereof three went off-study during the first four weeks and were replaced. The trial was terminated at 13 patients, according to the planned two-stage design, because 0/10 patients obtained partial remission (PR). Two out of 10 patients obtained radiological stable disease (SD). The median PFS survival was eight weeks, and OS was 15 weeks. One patient had an serious adverse event (SAE) with a hypersensitive reaction to temsirolimus; overall, side-effects were mild, and the most common grade III side-effect was hypercholesterolaemia (4/10). Other grade III side-effects included hypertriglyceridaemia (1/10), thrombocytopenia (1/10), infection (1/10), hypertension (1/10), and hyperglycemia (1/10).. Temsirolimus can be safely administered in combination with bevacizumab. This study failed to detect activity of such a combination in patients with progressive GBM beyond bevacizumab therapy.

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Brain Neoplasms; Female; Follow-Up Studies; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Sirolimus; Survival Rate; Young Adult

To determine the safety of the mammalian target of rapamycin inhibitor everolimus (RAD001) administered daily with concurrent radiation and temozolomide in newly diagnosed glioblastoma patients.. Everolimus was administered daily with concurrent radiation (60 Gy in 30 fractions) and temozolomide (75 mg/m(2) per day). Everolimus was escalated from 2.5 mg/d (dose level 1) to 5 mg/d (dose level 2) to 10 mg/d (dose level 3). Adjuvant temozolomide was delivered at 150 to 200 mg/m(2) on days 1 to 5, every 28 days, for up to 12 cycles, with concurrent everolimus at the previously established daily dose of 10 mg/d. Dose escalation continued if a dose level produced dose-limiting toxicities (DLTs) in fewer than 3 of the first 6 evaluable patients.. Between October 28, 2010, and July 2, 2012, the Radiation Therapy Oncology Group 0913 protocol initially registered a total of 35 patients, with 25 patients successfully meeting enrollment criteria receiving the drug and evaluable for toxicity. Everolimus was successfully escalated to the predetermined maximum tolerated dose of 10 mg/d. Two of the first 6 eligible patients had a DLT at each dose level. DLTs included gait disturbance, febrile neutropenia, rash, fatigue, thrombocytopenia, hypoxia, ear pain, headache, and mucositis. Other common toxicities were grade 1 or 2 hypercholesterolemia and hypertriglyceridemia. At the time of analysis, there was 1 death reported, which was attributed to tumor progression.. Daily oral everolimus (10 mg) combined with both concurrent radiation and temozolomide followed by adjuvant temozolomide is well tolerated, with an acceptable toxicity profile. A randomized phase 2 clinical trial with mandatory correlative biomarker analysis is currently under way, designed to both determine the efficacy of this regimen and identify molecular determinants of response.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Chemotherapy, Adjuvant; Dacarbazine; Dose Fractionation, Radiation; Everolimus; Female; Glioblastoma; Humans; Male; Maximum Tolerated Dose; Middle Aged; Sirolimus; Temozolomide

Treating glioblastoma through the simultaneous inhibition of multiple transduction pathways may prove more effective than single-pathway inhibition. We evaluated the safety, biologic activity, and pharmacokinetic profile of oral AEE788, a selective inhibitor of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), plus oral RAD001, a mammalian target of rapamycin inhibitor, in glioblastoma patients.. This phase IB/II, open-label, multicenter, 2-arm, dose-escalation study enrolled adult glioblastoma patients at first or second recurrence/relapse. Primary objective was to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of AEE788 combined with RAD001. Secondary objectives included determining the safety/tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of the combination.. Sixteen patients were enrolled (AEE788 200 mg/day + RAD001 5 mg/day, 2 patients; AEE788 150 mg/day + RAD001 5 mg every other day [qod], 14); all patients discontinued the study most commonly because of disease progression. Four patients experienced DLT (AEE788 200 mg/day + RAD001 5 mg/day, 1 patient; AEE788 150 mg/day + RAD001 5 mg qod, 3). Both patients receiving AEE788 (200 mg/day) plus RAD001 (5 mg/day) experienced clinically significant thrombocytopenia requiring a dose reduction/interruption. AEE788 appeared to inhibit the metabolism of RAD001. The study was terminated prematurely before an MTD was determined because of safety findings in other studies evaluating AEE788 monotherapy.. The coadministration of AEE788 and RAD001 in glioblastoma patients caused a clinically significant thrombocytopenia and a higher-than-expected RAD001 area under the curve concentration when dosed at 200 and 5 mg/day, respectively. After a dose reduction to AEE788 (150 mg/day) and RAD001 (5 mg qod), the combination appeared to be better tolerated.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Dose-Response Relationship, Drug; Drug Interactions; Everolimus; Female; Glioblastoma; Humans; Male; Maximum Tolerated Dose; Middle Aged; Purines; Sirolimus; Thrombocytopenia; Treatment Outcome

This phase I trial was designed to determine the recommended phase II dose(s) of everolimus (RAD001) with temozolomide (TMZ) in patients with glioblastoma (GBM). Patients receiving enzyme-inducing antiepileptic drugs (EIAEDs) and those not receiving EIAEDs (NEIAEDs) were studied separately.. Enrollment was restricted to patients with proven GBM, either newly diagnosed or at first progression. Temozolomide was administered at a starting dose of 150 mg/m(2)/day for 5 days every 28 days, and everolimus was administered continuously at a starting dose of 2.5 mg orally on a daily schedule starting on day 2 of cycle 1 in 28-day cycles.. Thirteen patients receiving EIAEDs and 19 not receiving EIAEDs were enrolled and received 83 and 116 cycles respectively. Everolimus 10 mg daily plus TMZ 150 mg/m(2)/day for 5 days was declared the recommended phase II dose for the NEIAEDs cohort. In the EIAEDs group, doses were well tolerated without DLTs, and pharmacokinetic parameters indicated decreased everolimus exposure. Temozolomide pharmacokinetic parameters were unaffected by EIAEDs or everolimus. In the subset of 28 patients with measurable disease, 3 had partial responses (all NEIAEDs) and 16 had stable disease.. A dosage of 10 mg everolimus daily with TMZ 150 mg/m(2)/day for five consecutive days every 28 days in patients is the recommended dose for this regimen. Everolimus clearance is increased by EIAEDs, and patients receiving EIAEDs should be switched to NEIAEDs before starting this regimen.

Topics: Adult; Aged; Anticonvulsants; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Dacarbazine; Drug Combinations; Everolimus; Female; Glioblastoma; Humans; Immunosuppressive Agents; Male; Middle Aged; PTEN Phosphohydrolase; Sirolimus; Temozolomide; Young Adult

To evaluate the efficacy of adding bevacizumab, a vascular endothelial growth factor (VEGF) inhibitor, and everolimus, a mammalian target of rapamycin (mTOR inhibitor), to standard radiation therapy/temozolomide in the first-line treatment of patients with glioblastoma.. Following surgical resection or biopsy, patients with newly diagnosed glioblastoma received standard radiation therapy/temozolomide plus bevacizumab 10 mg/kg intravenously (IV) every 2 weeks. Four weeks after the completion of radiation therapy, patients began oral everolimus 10 mg daily, and continued bevacizumab every 2 weeks; therapy continued until tumor progression or unacceptable toxicity.. Sixty-eight patients were treated, 82% of whom had previously undergone partial or complete surgical resection. Sixty-four patients completed combined modality therapy, and 57 patients began maintenance therapy with bevacizumab/everolimus. Thirty-one of 51 patients (61%) with measurable tumor had objective responses. After a median follow-up of 17 months, the median progression-free survival (PFS) was 11.3 months (95% confidence interval [CI], 9.3-13.1 months); median overall survival was 13.9 months. Toxicity was consistent with the known toxicity profile of bevacizumab; grade 3/4 toxicities during maintenance therapy related to everolimus included fatigue (27%), pneumonitis (7%), and stomatitis (5%).. The use of bevacizumab and everolimus as part of first-line combined modality therapy for glioblastoma was feasible and efficacious. The PFS compared favorably to previous reports with standard radiation therapy/temozolomide therapy, and is similar to results achieved in other phase II trials in which bevacizumab was added to fist-line treatment. Ongoing randomized phase III trials will clarify the role of bevacizumab in this setting.

Topics: Adult; Aged; Aged, 80 and over; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Alkylating; Bevacizumab; Brain Neoplasms; Combined Modality Therapy; Dacarbazine; Disease-Free Survival; Everolimus; Female; Glioblastoma; Humans; Immunosuppressive Agents; Male; Middle Aged; Sirolimus; Temozolomide; Young Adult

The activity of single-agent targeted molecular therapies in glioblastoma has been limited to date. The North American Brain Tumor Consortium examined the safety, pharmacokinetics, and efficacy of combination therapy with sorafenib, a small molecule inhibitor of Raf, vascular endothelial growth factor receptor 2, and platelet-derived growth factor receptor-β, and temsirolimus (CCI-779), an inhibitor of mammalian target of rapamycin. This was a phase I/II study. The phase I component used a standard 3 × 3 dose escalation scheme to determine the safety and tolerability of this combination therapy. The phase II component used a 2-stage design; the primary endpoint was 6-month progression-free survival (PFS6) rate. Thirteen patients enrolled in the phase I component. The maximum tolerated dosage (MTD) for combination therapy was sorafenib 800 mg daily and temsirolimus 25 mg once weekly. At the MTD, grade 3 thrombocytopenia was the dose-limiting toxicity. Eighteen patients were treated in the phase II component. At interim analysis, the study was terminated and did not proceed to the second stage. No patients remained progression free at 6 months. Median PFS was 8 weeks. The toxicity of this combination therapy resulted in a maximum tolerated dose of temsirolimus that was only one-tenth of the single-agent dose. Minimal activity in recurrent glioblastoma multiforme was seen at the MTD of the 2 combined agents.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Disease-Free Survival; Female; Glioblastoma; Humans; Male; Maximum Tolerated Dose; Middle Aged; Neoplasm Recurrence, Local; Niacinamide; Phenylurea Compounds; Sirolimus; Sorafenib; Young Adult

The mammalian target of rapamycin (mTOR) functions within the PI3K/Akt signaling pathway as a critical modulator of cell survival. On the basis of promising preclinical data, the safety and tolerability of therapy with the mTOR inhibitor RAD001 in combination with radiation (RT) and temozolomide (TMZ) was evaluated in this Phase I study.. All patients received weekly oral RAD001 in combination with standard chemoradiotherapy, followed by RAD001 in combination with standard adjuvant temozolomide. RAD001 was dose escalated in cohorts of 6 patients. Dose-limiting toxicities were defined during RAD001 combination therapy with TMZ/RT.. Eighteen patients were enrolled, with a median follow-up of 8.4 months. Combined therapy was well tolerated at all dose levels, with 1 patient on each dose level experiencing a dose-limiting toxicity: Grade 3 fatigue, Grade 4 hematologic toxicity, and Grade 4 liver dysfunction. Throughout therapy, there were no Grade 5 events, 3 patients experienced Grade 4 toxicities, and 6 patients had Grade 3 toxicities attributable to treatment. On the basis of these results, the recommended Phase II dosage currently being tested is RAD001 70 mg/week in combination with standard chemoradiotherapy. Fluorodeoxyglucose (FDG) positron emission tomography scans also were obtained at baseline and after the second RAD001 dose before the initiation of TMZ/RT; the change in FDG uptake between scans was calculated for each patient. Fourteen patients had stable metabolic disease, and 4 patients had a partial metabolic response.. RAD001 in combination with RT/TMZ and adjuvant TMZ was reasonably well tolerated. Changes in tumor metabolism can be detected by FDG positron emission tomography in a subset of patients within days of initiating RAD001 therapy.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Combined Modality Therapy; Dacarbazine; Everolimus; Female; Fluorodeoxyglucose F18; Follow-Up Studies; Glioblastoma; Humans; Male; Middle Aged; Positron-Emission Tomography; Radiopharmaceuticals; Sirolimus; Temozolomide

We evaluated the anti-tumor activity and safety of erlotinib, a receptor tyrosine kinase inhibitor of the epidermal growth factor receptor, plus sirolimus, an inhibitor of the mammalian target of rapamycin, among patients with recurrent glioblastoma (GBM) in a phase 2, open-label, single-arm trial. Thirty-two patients received daily erlotinib and sirolimus. The doses of erlotinib and sirolimus were 150 mg and 5 mg for patients not on concurrent CYP3A-inducing anti-epileptics (EIAEDS), and 450 mg and 10 mg for patients on EIAEDS. Evaluations were performed every two months. The primary endpoint was 6-month progression-free survival and secondary endpoints included safety and overall survival. Archival tumor samples were assessed for EGFR, EGFRvIII, PTEN, pAKT and pS6. Enrolled patients were heavily pre-treated including 53% who had received three or more prior chemotherapy agents and 28% who had received prior bevacizumab therapy. The most common grade > or = 2 adverse events were rash (59%), mucositis (34%) and diarrhea (31%). Grade 3 or higher events were rare. Best radiographic response included stable disease in 15 patients (47%); no patients achieved either a CR or PR. The estimated 6-month progression-free survival was 3.1% for all patients. Progression-free survival was better for patients not on EIAEDs (P = 0.03). Tumor markers failed to show an association with PFS except for increased pAKT expression which achieved borderline significance (P = 0.045). Although neither rash nor diarrhea had an association with outcome, hyperlipidemia was associated with longer PFS (P = 0.029). Erlotinib plus sirolimus was well tolerated but had negligible activity among unselected recurrent GBM patients. (ClinicalTrials.gov number: NCT0062243).

Topics: Adult; Aged; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Central Nervous System Neoplasms; Disease-Free Survival; Dose-Response Relationship, Drug; Erlotinib Hydrochloride; Female; Glioblastoma; Humans; Magnetic Resonance Imaging; Male; Middle Aged; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Quinazolines; Sirolimus; Treatment Outcome

The mammalian target of rapamycin (mTOR) functions within the phosphoinositide 3-kinase/Akt signaling pathway as a critical modulator of cell survival.. The mTOR inhibitor temsirolimus (CCI-779) was combined with chemoradiotherapy in glioblastoma multiforme (GBM) patients in a dose-escalation phase I trial. The first 12 patients were treated with CCI-779 combined with radiation/temozolomide and adjuvant temozolomide. A second cohort of 13 patients was treated with concurrent CCI-779/radiation/temozolomide followed by adjuvant temozolomide monotherapy.. Concomitant and adjuvant CCI-779 was associated with a high rate (3 of 12 patients) of grade 4/5 infections. By limiting CCI-779 treatment to the radiation/temozolomide phase and using antibiotic prophylaxis, the rate of infections was reduced, although 2 of 13 patients developed exacerbation of pre-existing fungal or viral infections. Dose-limiting toxicities were observed in 2 of 13 patients with this modified schedule. Weekly CCI-779 (50 mg/week) combined with radiation/temozolomide is the recommended phase II dose and schedule. The immune profile of patients in the second cohort was assessed before, during, and after CCI-779 therapy. There was robust suppression of helper and cytotoxic T cells, B cells, natural killer, cells and elevation of regulatory T cells during CCI-779/radiation/temozolomide therapy with recovery to baseline levels during adjuvant temozolomide of cytotoxic T cells, natural killer cells, and regulatory T cells.. The increased infection rate observed with CCI-779 combined with chemoradiotherapy in GBM was reduced with antibiotic prophylaxis and by limiting the duration of CCI-779 therapy. The combined suppressive effects of CCI-779 and temozolomide therapy on discrete immune compartments likely contributed to the increased infectious risks observed.

Topics: Aged; Antineoplastic Agents; Brain Neoplasms; Combined Modality Therapy; Dose-Response Relationship, Drug; Female; Glioblastoma; Humans; Male; Maximum Tolerated Dose; Middle Aged; Protein Kinase Inhibitors; Risk Factors; Sirolimus; Treatment Outcome

Twenty-two patients with recurrent glioblastoma (GBM) were prospectively treated with everolimus and gefitinib, designed to test the combined inhibition of mammalian target of rapamycin (mTOR) and epidermal growth factor receptor (EGFR) as part of a larger clinical trial. The primary endpoint was radiographic response rate. Secondary endpoints included progression-free survival and correlation of molecular profiles with treatment response. 36% of patients had stable disease and 14% a partial response; however, responses were not durable and only one patient was progression-free at six months. Radiographic changes were not well characterized by conventional response criteria, and implied differential effects of therapy within the tumor and/or antiangiogenic effects. EGFR and PTEN status did not clearly predict response to treatment.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Disease-Free Survival; ErbB Receptors; Everolimus; Female; Gefitinib; Glioblastoma; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Recurrence, Local; Pilot Projects; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Quinazolines; Sirolimus

There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.. Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).. Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.. http://www.ClinicalTrials.gov (#NCT00047073).

Topics: Adult; Aged; Antineoplastic Agents; Brain Neoplasms; Cell Division; Combined Modality Therapy; Disease Progression; Feedback, Physiological; Female; Glioblastoma; Humans; Male; Middle Aged; Neoadjuvant Therapy; Neoplasm Proteins; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6; Salvage Therapy; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Temsirolimus (CCI-779) is a small-molecule inhibitor of the mammalian target of rapamycin (mTOR) and represents a rational therapeutic target against glioblastoma multiforme (GBM).. Recurrent GBM patients with < or = 1 chemotherapy regimen for progressive disease were eligible. Temsirolimus was administered in a 250-mg intravenous dose weekly.. Sixty-five patients were treated. The incidence of grade 3 or higher nonhematologic toxicity was 51%, and consisted mostly of hypercholesterolemia (11%), hypertriglyceridemia (8%), and hyperglycemia (8%). Grade 3 hematologic toxicity was observed in 11% of patients. Temsirolimus peak concentration (Cmax), and sirolimus Cmax and area under the concentration-time curve were decreased in patients receiving p450 enzyme-inducing anticonvulsants (EIACs) by 73%, 47%, and 50%, respectively, but were still within the therapeutic range of preclinical models. Twenty patients (36%) had evidence of improvement in neuroimaging, consisting of decrease in T2 signal abnormality +/- decrease in T1 gadolinium enhancement, on stable or reduced steroid doses. Progression-free survival at 6 months was 7.8% and median overall survival was 4.4 months. Median time to progression (TTP) for all patients was 2.3 months and was significantly longer for responders (5.4 months) versus nonresponders (1.9 months). Development of grade 2 or higher hyperlipidemia in the first two treatment cycles was associated with a higher percentage of radiographic response (71% v 31%; P = .04). Significant correlation was observed between radiographic improvement and high levels of phosphorylated p70s6 kinase in baseline tumor samples (P = .04).. Temsirolimus is well tolerated in recurrent GBM patients. Despite the effect of EIACs on temsirolimus metabolism, therapeutic levels were achieved. Radiographic improvement was observed in 36% of temsirolimus-treated patients, and was associated with significantly longer TTP. High levels of phosphorylated p70s6 kinase in baseline tumor samples appear to predict a patient population more likely to derive benefit from treatment. These findings should be validated in other studies of mTOR inhibitors.

Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Brain Neoplasms; Disease-Free Survival; Female; Genes, erbB-1; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Phosphoric Monoester Hydrolases; Prognosis; Protein Kinase Inhibitors; Protein Kinases; PTEN Phosphohydrolase; Salvage Therapy; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Tumor Suppressor Proteins

Loss of PTEN, which is common in glioblastoma multiforme (GBM), results in activation of the mammalian target of rapapmycin (mTOR), thereby increasing mRNA translation of a number of key proteins required for cell-cycle progression. CCI-779 is an inhibitor of mTOR. The primary objectives of this study were to determine the efficacy of CCI-779 in patients with recurrent GBM and to further assess the toxicity of the drug.. CCI-779 was administered weekly at a dose of 250 mg intravenously for patients on enzyme-inducing anti-epileptic drugs (EIAEDs). Patients not on EIAEDs were initially treated at 250 mg; however, the dose was reduced to 170 mg because of intolerable side effects. Treatment was continued until unacceptable toxicity, tumor progression, or patient withdrawal. The primary endpoint was 6-month progression-free survival.. Forty-three patients were enrolled; 29 were not on EIAEDs. The expected toxicity profile of increased lipids, lymphopenia, and stomatitis was seen. There were no grade IV hematological toxicities and no toxic deaths. One patient was progression free at 6 months. Of the patients assessable for response, there were 2 partial responses and 20 with stabilization of disease. The median time to progression was 9 weeks.. CCI-779 was well tolerated at this dose schedule; however, there was no evidence of efficacy in patients with recurrent GBM. Despite initial disease stabilization in approximately 50% of patients, the durability of response was short. Because of the low toxicity profile, CCI-779 may merit exploration in combination with other modalities.

Topics: Adult; Aged; Anemia; Anticonvulsants; Antineoplastic Agents; Brain Neoplasms; Disease-Free Survival; Drug Interactions; Female; Glioblastoma; Humans; Hypercholesterolemia; Infusions, Intravenous; Lymphopenia; Male; Middle Aged; Neoplasm Recurrence, Local; Sirolimus

Topics: Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Phosphatidylinositol 3-Kinases; Signal Transduction; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases

Initially introduced in therapy as immunosuppressants, the selective inhibitors of mTORC1 have been approved for the treatment of solid tumors. Novel non-selective inhibitors of mTOR are currently under preclinical and clinical developments in oncology, attempting to overcome some limitations associated with selective inhibitors, such as the development of tumor resistance. Looking at the possible clinical exploitation in the treatment of glioblastoma multiforme, in this study we used the human glioblastoma cell lines U87MG, T98G and microglia (CHME-5) to compare the effects of a non-selective mTOR inhibitor, sapanisertib, with those of rapamycin in a large array of experimental paradigms, including (i) the expression of factors involved in the mTOR signaling cascade, (ii) cell viability and mortality, (iii) cell migration and autophagy, and (iv) the profile of activation in tumor-associated microglia. We could distinguish between effects of the two compounds that were overlapping or similar, although with differences in potency and or/time-course, and effects that were diverging or even opposite. Among the latter, especially relevant is the difference in the profile of microglia activation, with rapamycin being an overall inhibitor of microglia activation, whereas sapanisertib was found to induce an M2-profile, which is usually associated with poor clinical outcomes.

Topics: Cell Line, Tumor; Cell Proliferation; Glioblastoma; Glioma; Humans; Mechanistic Target of Rapamycin Complex 1; Microglia; Sirolimus; TOR Serine-Threonine Kinases

Topics: Autophagy; Cell Death; Chloroquine; Glioblastoma; Glioma; Humans; Lysosomes; Monoterpenes; Rolipram; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases

On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates

Topics: Brain; Drug Therapy, Combination; Glioblastoma; Humans; Ligands; MTOR Inhibitors; Sirolimus; Tacrolimus Binding Protein 1A; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Mechanistic target of rapamycin (mTOR)-signaling is one key driver of glioblastoma (GBM), facilitating tumor growth by promoting the shift to an anti-inflammatory, pro-cancerogenic microenvironment. Even though mTOR inhibitors such as rapamycin (RAPA) have been shown to interfere with GBM disease progression, frequently chaperoned toxic drug side effects urge the need for developing alternative or supportive treatment strategies. Importantly, previous work document that taste-immune associative learning with RAPA may be utilized to induce learned pharmacological placebo responses in the immune system. Against this background, the current study aimed at investigating the potential efficacy of a taste-immune associative learning protocol with RAPA in a syngeneic GBM rat model. Following repeated pairings of a novel gustatory stimulus with injections of RAPA, learned immune-pharmacological effects could be retrieved in GBM-bearing animals when re-exposed to the gustatory stimulus together with administering 10 % amount of the initial drug dose (0.5 mg/kg). These inhibitory effects on tumor growth were accompanied by an up-regulation of central and peripheral pro-inflammatory markers, suggesting that taste-immune associative learning with RAPA promoted the development of a pro-inflammatory anti-tumor microenvironment that attenuated GBM tumor growth to an almost identical outcome as obtained after 100 % (5 mg/kg) RAPA treatment. Together, our results confirm the applicability of taste-immune associative learning with RAPA in animal disease models where mTOR overactivation is one key driver. This proof-of-concept study may also be taken as a role model for implementing learning protocols as alternative or supportive treatment strategy in clinical settings, allowing the reduction of required drug doses and side effects without losing treatment efficacy.

Topics: Animals; Disease Progression; Glioblastoma; Rats; Sirolimus; Taste; TOR Serine-Threonine Kinases; Tumor Microenvironment

Psychiatric symptoms as seen in affective and anxiety disorders frequently appear during glioblastoma (GBM) treatment and disease progression, additionally deteriorate patient's daily life routine. These central comorbidities are difficult to recognize and the causes for these effects are unknown. Since overactivation of mechanistic target of rapamycin (mTOR)- signaling is one key driver in GBM growth, the present study aimed at examining in rats with experimentally induced GBM, neurobehavioral consequences during disease progression and therapy. Male Fisher 344 rats were implanted with syngeneic RG2 tumor cells in the right striatum and treated with the mTOR inhibitor rapamycin (3 mg/kg; once daily, for eight days) before behavioral performance, brain protein expression, and blood samples were analyzed. We could show that treatment with rapamycin diminished GBM tumor growth, confirming mTOR-signaling as one key driver for tumor growth. Importantly, in GBM animals' anxiety-like behavior was observed but only after treatment with rapamycin. These behavioral alterations were moreover accompanied by aberrant glucocorticoid receptor, phosphorylated p70 ribosomal S6 kinase alpha (p-p70s6k), and brain derived neurotrophic factor protein expression in the hippocampus and amygdala in the non-tumor-infiltrated hemisphere of the brain. Despite the beneficial effects on GBM tumor growth, our findings indicate that therapy with rapamycin impaired neurobehavioral functioning. This experimental approach has a high translational value. For one, it emphasizes aberrant mTOR functioning as a central feature mechanistically linking complex brain diseases and behavioral disturbances. For another, it highlights the importance of elaborating the cause of unwanted central effects of immunosuppressive and antiproliferative drugs used in transplantation medicine, immunotherapy, and oncology.

Topics: Animals; Antibiotics, Antineoplastic; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Glioblastoma; Male; Maze Learning; Rats; Rats, Inbred F344; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Tumor Burden

Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy (

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Glioblastoma; Humans; Mitochondria; Mitochondrial Dynamics; Mitophagy; Sirolimus; TOR Serine-Threonine Kinases

Tumor angiogenesis is critical for the growth and progression of cancer. As such, angiostasis is a treatment modality for cancer with potential utility for multiple types of cancer and fewer side effects. However, clinical success of angiostatic monotherapies has been moderate, at best, causing angiostatic treatments to lose their early luster. Previous studies demonstrated compensatory mechanisms that drive tumor vascularization despite the use of angiostatic monotherapies, as well as the potential for combination angiostatic therapies to overcome these compensatory mechanisms. We screened clinically approved angiostatics to identify specific combinations that confer potent inhibition of tumor-induced angiogenesis. We used a novel modification of the ex ovo chick chorioallantoic membrane (CAM) model that combined confocal and automated analyses to quantify tumor angiogenesis induced by glioblastoma tumor onplants. This model is advantageous due to its low cost and moderate throughput capabilities, while maintaining complex in vivo cellular interactions that are difficult to replicate in vitro. After screening multiple combinations, we determined that glioblastoma-induced angiogenesis was significantly reduced using a combination of bevacizumab (Avastin®) and temsirolimus (Torisel®) at doses below those where neither monotherapy demonstrated activity. These preliminary results were verified extensively, with this combination therapy effective even at concentrations further reduced 10-fold with a CI value of 2.42E-5, demonstrating high levels of synergy. Thus, combining bevacizumab and temsirolimus has great potential to increase the efficacy of angiostatic therapy and lower required dosing for improved clinical success and reduced side effects in glioblastoma patients.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Chickens; Chorioallantoic Membrane; Drug Screening Assays, Antitumor; Drug Synergism; Glioblastoma; Humans; Neovascularization, Pathologic; Rats; Sirolimus; Tumor Cells, Cultured

The failure of immune checkpoint inhibitor (ICPi) on glioblastoma (GBM) treatment underscores the need for improving therapeutic strategy. We aimed to change tumor associated macrophage (TAM) from M2 type (anti-inflammatory) to M1 (pro-inflammatory) type to increase the therapeutic response of ICPi. We proposed that combined rapamycin (R) and hydroxychloroquine (Q) preferentially induce M2 cells death, as fatty acid oxidation was their major source of energy.. Macrophage polarization was characterized on mice and human macrophage cell lines by specific cytokines stimulation with or without RQ treatment under single culture or co-culture with GBM cell lines. Tumor sizes were evaluated on subcutaneous and intracranial GL261 mice models with or without RQ, anti-PD1 mAb treatment. Tumor volumes assessed by MRI scan and proportions of tumor infiltrating immune cells analyzed by flow cytometry were compared.. In vitro RQ treatment decreased the macrophages polarization of M2, increased the phagocytic ability, and increased the lipid droplets accumulation. RQ treatment decreased the expression levels of CD47 and SIRPα on tumor cells and macrophage cells in co-culture experiments. The combination of RQ and anti-PD1 treatment was synergistic in action. Enhanced the intra-tumoral M1/M2 ratio, the CD8/CD4 ratio in the intracranial GL261 tumor model after RQ treatment were evident.. We provide a rationale for manipulating the macrophage phenotype and increased the therapeutic effect of ICPi. To re-educate and re-empower the TAM/microglia opens an interesting avenue for GBM treatment.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Polarity; Cells, Cultured; Female; Glioblastoma; Humans; Hydroxychloroquine; Macrophages; Mice; Mice, Inbred C57BL; Programmed Cell Death 1 Receptor; Sirolimus

Glioblastoma (GBM) is the most common type of brain cancer and has the highest mortality. Dysregulated expression of wild‑type isocitrate dehydrogenase 1 (IDH1) has been demonstrated to promote the progression of primary GBM without accumulating D‑2‑hydroxyglutarate, which differs from IDH1 mutation‑related mechanisms of tumorigenesis. Previous studies have revealed several roles of wild‑type IDH1 in primary GBM, involving proliferation and apoptosis. However, the function of IDH1 in cell migration has not been investigated. In the current study, the results of bioinformatics analysis revealed that IDH1 expression was significantly upregulated in patients with primary GBM. Wound healing and Transwell assays demonstrated that IDH1 overexpression promoted cell migration in primary GBM cells and that IDH1 knockdown hindered this process. Furthermore, α‑ketoglutarate (α‑KG), which is the main product of IDH1‑catalyzed reactions, was significantly decreased by IDH1 knockdown and upregulated by IDH1 overexpression. α‑KG treatment significantly increased the migration of primary GBM cells. Additionally, RNA sequence analysis of patients with primary GBM reported significant alterations in the expression of phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway‑regulated genes, including Myc, Snail family transcriptional repressor 2 and Twist‑related protein 1, which are primarily cell migration regulatory factors. Western blotting revealed that the overexpression or knockdown of IDH1 promoted or inhibited the PI3K/AKT/mTOR pathway, respectively. α‑KG treatment of primary GBM cells also promoted the PI3K/AKT/mTOR pathway. Furthermore, IDH1‑overexpressing and α‑KG‑treated U87 cells were incubated with rapamycin, an mTOR‑specific inhibitor, and the results revealed that rapamycin treatment reversed the increased cell migration caused by IDH1 overexpression and α‑KG treatment. The results indicated that IDH1 regulated the migration of primary GBM cells by altering α‑KG levels and that the function of the IDH1/α‑KG axis may rely on PI3K/AKT/mTOR pathway regulation.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Humans; Isocitrate Dehydrogenase; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Sequence Analysis, RNA; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

PI3K/Akt/mTOR signaling pathway activity is highly elevated in glioblastoma (GBM). Although rapamycin is known to inhibit this pathway, GBM patients are resistant to rapamycin monotherapy. This may be related to mutations of tumor suppressor phosphatase and tensin homolog (PTEN). Here, we show that higher expression of E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) in GBM is correlated with poor prognosis. Smurf1 promotes cell growth and colony formation by accelerating cell cycle and aberrant signaling pathways. In addition, we show that Smurf1 ubiquitylates and degrades PTEN. We further demonstrate that the oncogenic role of Smurf1 is dependent on PTEN. Upregulated Smurf1 impairs PTEN activity, leading to consistent activation of PI3K/Akt/mTOR signaling pathway; and depletion of Smurf1 dramatically inhibits cell proliferation and tumor growth. Moreover, loss of Smurf1 abolishes the aberrant regulation of PTEN, causing negative feedback on PI3K/Akt/mTOR signaling pathway, and thus leading to rescue of tumor sensitivity to rapamycin in an orthotopic GBM model. Taken together, we show that Smurf1 promotes tumor progression via PTEN, and combined treatment of Smurf1 knockdown with mammalian target of rapamycin (mTOR) inhibition reduces tumor progression. These results identify a unique role of Smurf1 in mTOR inhibitor resistance and provide a strong rationale for combined therapy targeting GBM.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioblastoma; Heterografts; Humans; Immunohistochemistry; Mice; Mutation; Oncogenes; Prognosis; PTEN Phosphohydrolase; Sirolimus; Synthetic Lethal Mutations; Ubiquitin-Protein Ligases; Ubiquitination

Glioblastoma exhibits high mortality rates due to challenges with drug delivery to the brain and into solid tumors. This two-pronged barrier necessitates high doses of systemic therapies, resulting in significant off-target toxicities. Recently, dendrimer-nanomedicines (without ligands) have shown promise for targeting specific cells in brain tumors from systemic circulation, for improved efficacy and amelioration of systemic toxicities. A dendrimer-rapamycin conjugate (D-Rapa) is presented here that specifically targets tumor-associated macrophages (TAMs) in glioblastoma from systemic administration. D-Rapa improves suppression of pro-tumor expression in activated TAMs and antiproliferative properties of rapamycin in glioma cells

Topics: Brain Neoplasms; Dendrimers; Drug Delivery Systems; Glioblastoma; Humans; Sirolimus; Tumor Microenvironment; Tumor-Associated Macrophages

Mammalian target of rapamycin (mTOR) is upregulated in a high percentage of glioblastomas. While a well-known mTOR inhibitor, rapamycin, has been shown to reduce glioblastoma survival, the role of mitochondria in achieving this therapeutic effect is less well known. Here, we examined mitochondrial dysfunction mechanisms that occur with the suppression of mTOR signaling. We found that, along with increased apoptosis, and a reduction in transformative potential, rapamycin treatment significantly affected mitochondrial health. Specifically, increased production of reactive oxygen species (ROS), depolarization of the mitochondrial membrane potential (MMP), and altered mitochondrial dynamics were observed. Furthermore, we verified the therapeutic potential of rapamycin-induced mitochondrial dysfunction through co-treatment with temzolomide (TMZ), the current standard of care for glioblastoma. Together these results demonstrate that the mitochondria remain a promising target for therapeutic intervention against human glioblastoma and that TMZ and rapamycin have a synergistic effect in suppressing glioblastoma viability, enhancing ROS production, and depolarizing MMP.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Drug Synergism; Glioblastoma; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Dynamics; Reactive Oxygen Species; Signal Transduction; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases

BACKGROUND Glioblastoma, the most common and malignant glial tumor, often has poor prognosis. Tivantinib has shown its potential in treating c-Met-high carcinoma. No studies have explored whether tivantinib inhibits the development of glioblastoma. MATERIAL AND METHODS The correlation between c-Met expression and clinicopathological characteristics of glioblastoma was investigated. U251 and T98MG glioblastoma cells treated with tivantinib, PI3K inhibitor (LY294002), PI3K activator (740 Y-P), and/or mammalian target of rapamycin (mTOR) inhibitor were subjected to MTT assay or colony formation assay to evaluate cell proliferation. The expression of mTOR signaling and caspase-3 in tivantinib-treated glioblastoma cells was differentially measured by western blotting. RESULTS In a group of Chinese patients, expression of c-Met was elevated with the size of glioblastoma, but not with the other clinicopathological characteristics, including gender, age, grade, IDH status, 1p/19q status, and Ki67 status. High dose of tivantinib (1 μmol/L) obviously repressed the proliferation and colony formation of U251 and T98MG glioblastoma cells, but low dose (0.1 μmol/L) of tivantinib failed to retard cell proliferation. Tivantinib blocked PI3K/Akt/mTOR signaling but did not change the expression of cleaved caspase-3. PI3K activator 740 Y-P (20 μmol/L) significantly rescued tivantinib-induced decrease of cell proliferation. Tivantinib (1 μmol/L) in combination with PI3K inhibitor LY294002 (0.5 μmol/L) and mTOR inhibitor rapamycin (0.1 nmol/L) largely inhibited the proliferation of glioblastoma cells. CONCLUSIONS c-MET inhibitor tivantinib blocks PIKE/Akt/mTOR signaling and hampers the proliferation of glioblastoma cells, which endows the drug a therapeutic effect.

Topics: Adult; Aged; Cell Line, Tumor; Cell Proliferation; China; Chromones; Female; Gene Expression; Glioblastoma; Humans; Male; Middle Aged; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pyrrolidinones; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin is an enzyme that regulates cell metabolism and proliferation. It is up-regulated in aggressive tumors, such as glioblastoma, leading to increased glucose uptake and consumption. It has been suggested that glucose CEST signals reflect the delivery and tumor uptake of glucose. The inhibitor rapamycin (sirolimus) has been applied as a glucose deprivation treatment; thus, glucose CEST MRI could potentially be useful for monitoring the tumor responses to inhibitor treatment.. A human U87-EGFRvIII xenograft model in mice was studied. The mice were treated with a mammalian target of Rapamycin inhibitor, rapamycin. The effect of the treatment was evaluated in vivo with dynamic glucose CEST MRI.. Rapamycin treatment led to significant increases (P < 0.001) in dynamic glucose-enhanced signal in both the tumor and contralateral brain as compared to the no-treatment group, namely a maximum enhancement of 3.7% ± 2.3% (tumor, treatment) versus 1.9% ± 0.4% (tumor, no-treatment), 1.7% ± 1.1% (contralateral, treatment), and 1.0% ± 0.4% (contralateral, no treatment). Dynamic glucose-enhanced contrast remained consistently higher in treatment versus no-treatment groups for the duration of the experiment (17 min). This was confirmed with area-under-curve analysis.. Increased glucose CEST signal was found after mammalian target of Rapamycin inhibition treatment, indicating potential for dynamic glucose-enhanced MRI to study tumor response to glucose deprivation treatment.

Topics: Animals; Antibiotics, Antineoplastic; Brain; Brain Chemistry; Brain Neoplasms; Cell Line, Tumor; Female; Glioblastoma; Humans; Magnetic Resonance Imaging; Mice; Mice, SCID; Sirolimus; Xenograft Model Antitumor Assays

Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine's anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects.

Topics: Brain Neoplasms; Carnosine; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy.. To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures.. An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization.. These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Autophagy is a vital process that involves degradation of long-lived proteins and dysfunctional organelles and contributes to cellular metabolism. Glioma-initiating cells (GICs) have the ability to self-renew, differentiate into heterogeneous types of tumor cells, and sustain tumorigenicity; thus, GICs lead to tumor recurrence. Accumulating evidence indicates that autophagy can induce stem cell differentiation and increase the lethality of temozolomide against GICs. However, the mechanism underlying the regulation of GIC self-renewal by autophagy remains uncharacterized. In the present study, autophagy induced by AZD8055 and rapamycin treatment suppressed GIC self-renewal in vitro. We found that autophagy inhibited Notch1 pathway activation. Moreover, autophagy activated Notch1 degradation, which is associated with maintenance of the self-renewal ability of GICs. Furthermore, autophagy abolished the tumorigenicity of CD133 + U87-MG neurosphere cells in an intracranial model. These findings suggest that autophagy regulating GICs self-renewal and tumorigenicity is probably bound up with Notch1 degradation. The results of this study could aid in the design of autophagy-based clinical trials for glioma treatments, which may be of great value.

Topics: Animals; Antineoplastic Agents; Autophagy; Brain Neoplasms; Calcium-Binding Proteins; Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Membrane Proteins; Mice; Mice, Nude; Morpholines; Neoplastic Stem Cells; Proteolysis; Receptor, Notch1; Signal Transduction; Sirolimus; Sodium-Potassium-Exchanging ATPase; Spheroids, Cellular; Survival Analysis; Xenograft Model Antitumor Assays

The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors-rapamycin, temsirolimus, torin 1, and PP242-suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as "stemness-inhibiting drugs" and a promising therapeutic approach to target glioma stem cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Fibronectins; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Naphthyridines; Neoplasm Recurrence, Local; Neural Stem Cells; Oncostatin M; Phosphatidylinositol 3-Kinases; Signal Transduction; Sirolimus; STAT3 Transcription Factor; Tetradecanoylphorbol Acetate; TOR Serine-Threonine Kinases; Vimentin

Glioblastoma cells feature mammalian target of rapamycin (mTOR) up-regulation which relates to a variety of effects such as: lower survival, higher infiltration, high stemness and radio- and chemo-resistance. Recently, it was demonstrated that mTOR may produce a gene shift leading to altered protein expression. Therefore, in the present study we administered different doses of the mTOR inhibitor rapamycin to explore whether the transcription of specific genes are modified. By using a variety of methods we demonstrate that rapamycin stimulates gene transcription related to neuronal differentiation while inhibiting stemness related genes such as nestin. In these experimental conditions, cell phenotype shifts towards a pyramidal neuron-like shape owing long branches. Rapamycin suppressed cell migration when exposed to fetal bovine serum (FBS) while increasing the cell adhesion protein phospho-FAK (pFAK). The present study improves our awareness of basic mechanisms which relate mTOR activity to the biology of glioblastoma cells. These findings apply to a variety of effects which can be induced by mTOR regulation in the brain. In fact, the ability to promote neuronal differentiation might be viewed as a novel therapeutic pathway to approach neuronal regeneration.

Topics: Antigens, Nuclear; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Endopeptidases; Gelatinases; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioblastoma; Humans; Membrane Proteins; Nerve Tissue Proteins; Nestin; Serine Endopeptidases; Signal Transduction; Sirolimus; Tubulin

Ketamine is a potent anti-depressive agent. Nitric oxide plays an essential role in neuronal transmission and cerebral blood flow and has been implicated in the pathophysiology of major depressive disorder as well as cardiovascular functioning. We investigated the effect of ketamine on eNOS expression in human A172 astroglial cells. Ketamine (50-500μM) increased eNOS expression at 4-24h in a concentration-dependent manner. This effect was mediated by NMDA receptor, Akt inhibition and ERK1/2 activation and was synergistically augmented by rapamycin. The combined effect on the vascular, immune and neuronal systems may be relevant to the rapid antidepressive effect of ketamine.

Topics: Astrocytes; Cell Line, Transformed; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glioblastoma; Humans; Immunosuppressive Agents; Ketamine; MAP Kinase Signaling System; N-Methylaspartate; Nitric Oxide Synthase Type III; RNA, Messenger; Sirolimus; Time Factors; Up-Regulation; Valine

Although signaling from phosphatidylinositol 3-kinase (PI3K) and AKT to mechanistic target of rapamycin (mTOR) is prominently dysregulated in high-grade glial brain tumors, blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Allosteric mTOR inhibitors, such as rapamycin, incompletely block mTORC1 compared with mTOR kinase inhibitors (TORKi). Here, we compared RapaLink-1, a TORKi linked to rapamycin, with earlier-generation mTOR inhibitors. Compared with rapamycin and Rapalink-1, TORKi showed poor durability. RapaLink-1 associated with FKBP12, an abundant mTOR-interacting protein, enabling accumulation of RapaLink-1. RapaLink-1 showed better efficacy than rapamycin or TORKi, potently blocking cancer-derived, activating mutants of mTOR. Our study re-establishes mTOR as a central target in glioma and traces the failure of existing drugs to incomplete/nondurable inhibition of mTORC1.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Female; Glioblastoma; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Multiprotein Complexes; Protein Kinase Inhibitors; Sirolimus; Tacrolimus Binding Protein 1A; TOR Serine-Threonine Kinases

Glioblastoma is the deadliest brain tumor in humans. High systemic toxicity of conventional chemotherapies prompted the search for natural compounds for controlling glioblastoma. The natural flavonoids luteolin (LUT) and silibinin (SIL) have anti-tumor activities. LUT inhibits autophagy, cell proliferation, metastasis, and angiogenesis and induces apoptosis; while SIL activates caspase-8 cascades to induce apoptosis. However, synergistic anti-tumor effects of LUT and SIL in glioblastoma remain unknown. Overexpression of tumor suppressor microRNA (miR) could enhance the anti-tumor effects of LUT and SIL. Here, we showed that 20 µM LUT and 50 µM SIL worked synergistically for inhibiting growth of two different human glioblastoma U87MG (wild-type p53) and T98G (mutant p53) cell lines and natural combination therapy was more effective than conventional chemotherapy (10 µM BCNU or 100 µM TMZ). Combination of LUT and SIL caused inhibition of growth of glioblastoma cells due to induction of significant amounts of apoptosis and complete inhibition of invasion and migration. Further, combination of LUT and SIL inhibited rapamycin (RAPA)-induced autophagy, a survival mechanism, with suppression of PKCα and promotion of apoptosis through down regulation of iNOS and significant increase in expression of the tumor suppressor miR-7-1-3p in glioblastoma cells. Our in vivo studies confirmed that overexpression of miR-7-1-3p augmented anti-tumor activities of LUT and SIL in RAPA pre-treated both U87MG and T98G tumors. In conclusion, our results clearly demonstrated that overexpression of miR-7-1-3p augmented the anti-tumor activities of LUT and SIL to inhibit autophagy and induce apoptosis for controlling growth of different human glioblastomas in vivo.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Brain Neoplasms; Carmustine; Caspase 8; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dacarbazine; Female; Glioblastoma; Humans; Luteolin; Mice; Mice, Nude; MicroRNAs; Signal Transduction; Silybin; Silymarin; Sirolimus; Temozolomide; Xenograft Model Antitumor Assays

The PI3K-AKT-mTOR signaling axis is central to the transformed phenotype of glioblastoma (GBM) cells, due to frequent loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10). The mechanistic target of rapamycin (mTOR) kinase is present in two cellular multi-protein complexes, mTORC1 and mTORC2, which have distinct subunit composition, substrates and mechanisms of action. Targeting the mTOR protein is a promising strategy for GBM therapy. However, neither of these complexes is fully inhibited by the allosteric inhibitor of mTOR, rapamycin or its analogs. Herein, we provide evidence that the combined inhibition of mTORC1/2, using the ATP-competitive binding inhibitor PP242, would effectively suppress GBM growth and dissemination as compared to an allosteric binding inhibitor of mTOR. GBM cells treated with PP242 demonstrated significantly decreased activation of mTORC1 and mTORC2, as shown by reduced phosphorylation of their substrate levels, p70 S6K(Thr389) and AKT(Ser473), respectively, in a dose-dependent manner. Furthermore, insulin induced activation of these kinases was abrogated by pretreatment with PP242 as compared with rapamycin. Unlike rapamycin, PP242 modestly activates extracellular regulated kinase (ERK1/2), as shown by expression of pERK(Thr202/Tyr204). Cell proliferation and S-phase entry of GBM cells was significantly suppressed by PP242, which was more pronounced compared to rapamycin treatment. Lastly, PP242 significantly suppressed the migration of GBM cells, which was associated with a change in cellular behavior rather than cytoskeleton loss. In conclusion, these results underscore the potential therapeutic use of the PP242, a novel ATP-competitive binding inhibitor of mTORC1/2 kinase, in suppression of GBM growth and dissemination.

Topics: Adenosine Triphosphate; Binding, Competitive; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chemotaxis; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; Indoles; Insulin; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Phenotype; Phosphorylation; Protein Binding; Purines; S Phase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and the RAF/mitogen-activated and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are frequently deregulated in cancer. Temsirolimus (TEM) and its primary active metabolite rapamycin allosterically block mTOR complex 1 substrate recruitment. The context-/experimental setup-dependent opposite effects of rapamycin on the multiple centrosome formation, aneuploidy, DNA damage/repair, proliferation, and invasion were reported. Similarly, the context-dependent either tumor-promoting or suppressing effects of RAF-MEK-ERK pathway and its inhibitors were demonstrated. Drug treatment-mediated stress may promote chromosomal instability (CIN), accelerating changes in the genomic landscape and phenotype diversity. Here, we characterized the genomic and phenotypic changes of U251 and T98G glioblastoma cell lines long-term treated with TEM or U0126, an inhibitor of MEK1/2. TEM significantly increased clonal and non-clonal chromosome aberrations. Both TEM and U0126 affected copy number alterations (CNAs) pattern. A proliferation rate of U251TEM and U251U0126 cells was lower and higher, respectively, than control cells. Colony formation efficiency of U251TEM significantly decreased, whereas U251U0126 did not change. U251TEM and U251U0126 cells decreased migration. In contrast, T98GTEM and T98GU0126 cells did not change proliferation, colony formation efficiency, and migration. Changes in the sensitivity of inhibitor-treated cells to the reduction of the glucose concentration were observed. Our results suggest that CIN and adaptive reprogramming of signal transduction pathways may be responsible for the cell type-dependent phenotype changes of long-term TEM- or U0126-treated tumor cells.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosomal Instability; Glioblastoma; Glucose; Humans; MAP Kinase Signaling System; Nitriles; Phenotype; Protein Kinase Inhibitors; Sirolimus

SOX2 and SOX9 are commonly overexpressed in glioblastoma, and regulate the activity of glioma stem cells (GSCs). Their specific and overlapping roles in GSCs and glioma treatment remain unclear.. SOX2 and SOX9 levels were examined in human biopsies. Gain and loss of function determined the impact of altering SOX2 and SOX9 on cell proliferation, senescence, stem cell activity, tumorigenesis and chemoresistance.. SOX2 and SOX9 expression correlates positively in glioma cells and glioblastoma biopsies. High levels of SOX2 bypass cellular senescence and promote resistance to temozolomide. Mechanistic investigations revealed that SOX2 acts upstream of SOX9. mTOR genetic and pharmacologic (rapamycin) inhibition decreased SOX2 and SOX9 expression, and reversed chemoresistance.. Our findings reveal SOX2-SOX9 as an oncogenic axis that regulates stem cell properties and chemoresistance. We identify that rapamycin abrogate SOX protein expression and provide evidence that a combination of rapamycin and temozolomide inhibits tumor growth in cells with high SOX2/SOX9.

Topics: Adult; Animals; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Sirolimus; SOX9 Transcription Factor; SOXB1 Transcription Factors; Temozolomide; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Glioblastoma (GBM) is the most aggressive type of brain tumors in adults with survival period <1.5 years of patients. The role of mTOR pathway is documented in invasion and migration, the features associated with aggressive phenotype in human GBM. However, most of the preclinical and clinical studies with mTOR inhibitors are focused on antiproliferative and cytotoxic activity in GBM. In this study, we demonstrate that mTOR inhibitors-rapamycin (RAP), temisirolimus (TEM), torin-1 (TOR) and PP242 suppress invasion and migration induced by Tumor Necrosis Factor-α (TNFα) and tumor promoter, Phorbol 12-myristate 13-acetate (PMA) and also reduce the expression of the TNFα and IL1β suggesting their potential to regulate factors in microenvironment that support tumor progression. The mTOR inhibitors significantly decreased MMP-2 and MMP-9 mRNA, protein and activity that was enhanced by TNFα and PMA. The effect was mediated through reduction of Protein kinase C alpha (PKC-α) activity and downregulation of NFκB. TNFα- induced transcripts of NFκB targets -VEGF, pentraxin-3, cathepsin-B and paxillin, crucial in invasion were restored to basal level by these inhibitors. With limited therapeutic interventions currently available for GBM, our findings are significant and suggest that mTOR inhibitors may be explored as anti-invasive drugs for GBM treatment.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Glioblastoma; Humans; Indoles; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthyridines; Neoplasm Invasiveness; NF-kappa B; Phenylacetates; Protein Kinase C-alpha; Purines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Microenvironment; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

There are still unmet medical needs for the treatment of glioblastoma (GBM), the most frequent and aggressive brain tumor worldwide. EGFRvIII, overexpressed in approximately 30% of GBM, has been regarded as a potential therapeutic target. In this study, we demonstrated that CH12, an anti-EGFRvIII monoclonal antibody, could significantly suppress the growth of EGFRvIII+ GBM in vivo; however, PTEN deficiency in GBM reduced the efficacy of CH12 by attenuating its effect on PI3K/AKT/mTOR pathway. To overcome this problem, CH12 was combined with the mTOR inhibitor rapamycin, leading to a synergistic inhibitory effect on EGFRvIII+PTEN- GBM in vivo. Mechanistically, the synergistic antitumor effect was achieved via attenuating EGFR and PI3K/AKT/mTOR pathway more effectively and reversing the STAT5 activation caused by rapamycin treatment. Moreover, the combination therapy suppressed angiogenesis and induced cancer cell apoptosis more efficiently. Together, these results indicated that CH12 and rapamycin could synergistically suppress the growth of EGFRvIII+PTEN- GBM, which might have a potential clinical application in the future.

Topics: Animals; Anti-Bacterial Agents; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Line, Tumor; Drug Synergism; ErbB Receptors; Glioblastoma; Humans; Mice; PTEN Phosphohydrolase; Sirolimus; Xenograft Model Antitumor Assays

Rapamycin, a mammalian target of rapamycin inhibitor and anti-proliferative agent, is used to treat glioma and other malignancies, but its effectiveness is limited by the fact that it cannot be delivered in a targeted manner to the site of the tumor. To address this issue, we fabricated a mesh via electrospinning using two biodegradable materials, poly(lactic acid) (PLA) and polyethylene oxide (PEO) as a carrier for rapamycin delivery to the tumor. Nanofiber diameter decreased with increasing PLA concentration in the mixed solution. Scanning electron microscopy analysis revealed the smooth and uniform surface morphology of hybrid fibers. Fourier transform infrared spectroscopy analysis demonstrated that rapamycin was encapsulated in the polymer solution; encapsulation efficiency was high and stable over the range of drug concentrations from 0.5-2wt%. A correlation was observed between sustained release of the drug in vitro and cytotoxicity in cultured glioma cells. These results indicate that the PEO/poly(d,l-lactic acid) nanofiber mesh can be used as a targeted delivery system for rapamycin that can limit side effects and prevent locoregional recurrence following surgical resection of glioma.

Topics: Cell Line, Tumor; Drug Delivery Systems; Drug Liberation; Glioblastoma; Humans; Lactates; Nanofibers; Polyethylene Glycols; Sirolimus; Spectroscopy, Fourier Transform Infrared; Treatment Outcome; X-Ray Diffraction

Glioblastoma is a highly aggressive, common brain tumor with poor prognosis. Therefore, this study examines a new therapeutic approach targeting oncogenic and survival pathways combined with common chemotherapeutics. The RIST (rapamycin, irinotecan, sunitinib, temozolomide) and the variant aRIST (alternative to rapamycin, GDC-0941) therapy delineate growth inhibiting effects in established glioblastoma cell lines and primary cultured patient material. These combinations significantly decreased cell numbers and viability compared to inhibitors and chemotherapeutics alone with aRIST being superior to RIST. Notably, RIST/aRIST appeared to be apoptogenic evoked by reduction of anti-apoptotic protein levels of XIAP and BCL-2, with concomitant up-regulation of pro-apoptotic protein levels of p53 and BAX. The treatment success of RIST therapy was confirmed in an orthotopic mouse model. This combination treatment revealed significantly prolonged survival time and drastically reduced the tumor burden by acting anti-proliferative and pro-apoptotic. Surprisingly, in vivo, aRIST only marginally extended survival time with tumor volumes comparable to controls. We found that aRIST down-regulates the microvessel density suggesting an insufficient distribution of chemotherapy. Further, alterations in different molecular modes of action in vivo than in vitro suggest, that in vivo RIST therapy may mimic the superior aRIST protocol's pro-apoptotic inhibition of pAKT in vitro. Of note, all substances were administered in therapeutically relevant low doses with no adverse side effects observed. We also provide evidence of the potential benefits of the RIST therapy in a clinical setting. Our data indicates RIST therapy as a novel treatment strategy for glioblastoma achieving significant anti-tumorigenic activity avoiding high-dose chemotherapy.

Topics: Adolescent; Animals; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Camptothecin; Cell Line, Tumor; Child; Dacarbazine; Female; Glioblastoma; Humans; Indoles; Irinotecan; Male; Membrane Potential, Mitochondrial; Mice, Inbred NOD; Molecular Targeted Therapy; Pyrroles; Sirolimus; Sunitinib; Temozolomide; Xenograft Model Antitumor Assays

The present in vitro study aimed to assess the effects of combining the mTOR inhibitor RAD001 and temozolomide (TMZ) together with irradiation by either low-linear energy transfer (LET) radiation (γ-rays) or high-LET radiation (fast neutrons) on the growth and cell survival of the human glioblastoma cell line U-87. We observed a strong decrease in cell proliferation along with a concomitant increase in cell death as a function of the radiation dose. As expected, high-LET radiation was more effective and induced more sustained damage to DNA than low-LET radiation. While RAD001 in association with TMZ induced autophagic cell death, additional combination with either type of radiation did not further increase its occurrence. On the contrary, apoptosis remained at a low level in all experimental groups.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Cell Proliferation; Cell Survival; Dacarbazine; DNA Damage; Dose-Response Relationship, Radiation; Everolimus; Gamma Rays; Glioblastoma; Histones; Humans; Linear Energy Transfer; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells.

Topics: Allosteric Regulation; Antibiotics, Antineoplastic; Catalytic Domain; Cell Line, Tumor; Cell Movement; Cell Survival; Everolimus; Glioblastoma; Humans; Immunosuppressive Agents; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mitosis; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Glioblastoma is the most fatal type of brain tumor, requiring a better chemotherapy regime to prolong the survival of the patient. The toxicity associated with a high dose of a single drug can be overcome by a combination of different anticancer drugs. Methoxy poly(ethylene glycol)-poly (ε-caprolactone) block copolymeric micelles loaded with two different anticancer drugs such as curcumin and rapamycin were used to assess their therapeutic efficacy in glioblastoma cell lines. In addition, the combination of curcumin and rapamycin was also encapsulated in micelles for better anticancer activity. The in-vitro cellular uptake studies and cytotoxicity studies showed that the drugs encapsulated in the micelles showed ∼3.3 times more uptake than a mixture of native drugs as well as a single drug. The enhanced mitochondrial membrane potential depolarization by drug-loaded micelles leads to higher cell death compared with native drugs in T98G glioblastoma cell culture experiments. The drug combination downregulates P13/AKT and serine/threonine kinase proteins, and leads to programmed cell death. Thus, curcumin and rapamycin-loaded micelles can be used effectively for the treatment of glioblastoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Curcumin; Cyclooxygenase 2; Drug Delivery Systems; Drug Synergism; Glioblastoma; Humans; Membrane Potential, Mitochondrial; Micelles; NF-kappa B; Polyesters; Polyethylene Glycols; Sirolimus

Glioblastoma multiforme (GBM) is the most malignant histological type of glioma. It exhibits an extremely aggressive action including invasion of large zones of brain parenchyma. Even after the application of surgery, radio and chemotherapy, the effect and survival for patients with GBM continue to be very poor. The PI3K/AKT/mTOR is a key pathway in the regulation of the proliferation of cancer cells. This is the reason to consider the mTOR inhibitors such as rapamycin analogs as an encouraging therapy for malignant glioma, but current investigations suggest that single inhibition of mTOR may be insufficient. For this reason, there is a need for the use of more than one agent rationally combined.. In this study, we have evaluated the therapeutic potential of the combination of two different drugs: intraperitoneal rapamycin and convection enhanced delivery of nanoliposomes containing the topoisomerase I inhibitor CPT-11. The effect was analyzed by flow cytometry, cell growth, immunocytochemistry and immunohistochemistry, and rodent orthotopic xenograft survival analysis.. The combination presented remarkable efficacy in a survival study. We present an increase in survival of 6-fold in xenotransplanted animals without rise in toxicity.. In summary, we propose a very powerful new combination therapy for glioma.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Camptothecin; Cell Line, Tumor; Drug Monitoring; Glioblastoma; Humans; Infusions, Parenteral; Irinotecan; Liposomes; Rats; Sirolimus; Topoisomerase I Inhibitors; TOR Serine-Threonine Kinases; Treatment Outcome; Xenograft Model Antitumor Assays

The treatment of glioblastoma multiforme (GBM) is an unmet clinical need. The 5-year survival rate of patients with GBM is less than 3%. Temozolomide (TMZ) remains the standard first-line treatment regimen for gliomas despite the fact that more than 90% of recurrent gliomas do not respond to TMZ after repeated exposure. We have also independently shown that many of the Asian-derived glioma cell lines and primary cells derived from Singaporean high-grade glioma patients are indeed resistant to TMZ. This issue highlights the need to develop new effective anti-cancer treatment strategies. In a recent study, wild-type epidermal growth factor receptor (wtEGFR) has been shown to phosphorylate a truncated EGFR (known as EGFRvIII), leading to the phosphorylation of STAT proteins and progression in gliomagenesis. Despite the fact that combination of EGFR targeting drugs and rapamycin has been used before, the effect of mono-treatment of Nimotuzumab, rapamycin and combination therapy in human glioma expressing different types of EGFR is not well-studied. Herein, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an mTOR inhibitor (rapamycin) in Caucasian patient-derived human glioma cell lines, Asian patient-derived human glioma cell lines, primary glioma cells derived from the Mayo GBM xenografts, and primary short-term glioma culture derived from high-grade glioma patients.. The combination effect of Nimotuzumab and rapamycin was examined in a series of primary human glioma cell lines and glioma cell lines. The cell viability was compared to TMZ treatment alone. Endogenous expressions of EGFR in various GBM cells were determined by western blotting.. The results showed that combination of Nimotuzumab with rapamycin significantly enhanced the therapeutic efficacy of human glioma cells compared to single treatment. More importantly, many of the Asian patient-derived glioma cell lines and primary cells derived from Singaporean high-grade gliomas, which showed resistance to TMZ, were susceptible to the combined treatments.. In conclusion, our results strongly suggest that combination usage of Nimotuzumab and rapamycin exert higher cytotoxic activities than TMZ. Our data suggest that this combination may provide an alternative treatment for TMZ-resistant gliomas regardless of the EGFR status.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dacarbazine; Drug Resistance, Neoplasm; ErbB Receptors; Glioblastoma; Humans; Mutation; Sirolimus; Temozolomide

The cancer stem cell (CSC) hypothesis suggests a hierarchical organization of cells within the tumor, in which only a subpopulation of stem-like cells is responsible for the rise and progression of the tumor. Glioblastomas (GBM), a lethal brain tumor, may contain a variable proportion of active CSCs. On the other hand, the phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) pathway is highly active in up to 70 % of GBM. The kinase mTOR is a key component of the PI3K pathway that mediates the regulation of growth and cell survival signaling. However, clinical trials with rapamycin, an effective inhibitor of mTOR, have not been up to the created expectations and a plausible explanation is missing. In this work, we analyze the effect of rapamycin on the GBM-CSC population.. The efficacy of rapamycin in vitro was tested on two primary cell lines derived from human GBM surgical resections that fulfill the criteria to be considered as CSCs. We confirmed the inhibition state of the PI3K/Akt/mTOR pathway analyzing the mTOR direct target ribosomal protein S6. We assayed the growth rate, CD133 expression and ability of forming colonies in soft agar of the CSCs under different doses of rapamycin. The efficacy of rapamycin in vivo was assayed in a CSCs-based orthotopic xenograft.. We report the efficacy of rapamycin by reducing CSCs proliferation and tumorigenic potential in vitro. Despite these encouraging results, the efficacy in vivo was very poor. This finding confirms the limited use of rapamycin as a monotherapy for glioblastomas.

Topics: Antibiotics, Antineoplastic; Glioblastoma; Humans; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ-resistant cells. In order to elucidate the involvement of the autophagy process as pro-death or pro-survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR-silenced T98G cells, suggesting a pro-death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin-mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR-autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863-1873, 2014. © 2014 Wiley Periodicals, Inc.

Topics: Autophagy; Autophagy-Related Protein 7; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Clone Cells; Dacarbazine; Drug Resistance, Neoplasm; ErbB Receptors; Gene Silencing; Glioblastoma; Humans; Radiation Tolerance; Radiation, Ionizing; RNA, Small Interfering; Sirolimus; Temozolomide; Transfection; Ubiquitin-Activating Enzymes

The success of immunotherapeutic approaches targeting glioblastoma multiforme (GBM) demands a robust antiglioma T-cell cytotoxic and memory response. Recent evidence suggests that rapamycin regulates T-cell differentiation. Herein, we tested whether administration of rapamycin could enhance the efficacy of immunotherapy utilizing Fms-like tyrosine kinase 3 ligand (Ad-Flt3L) and thymidine kinase/ganciclovir (Ad-TK/GCV). Using the refractory rat RG2 glioma model, we demonstrate that administration of rapamycin with Ad-Flt3L + Ad-TK/GCV immunotherapy enhanced the cytotoxic activity of antitumor CD8(+) T cells. Rats treated with rapamycin + Ad-Flt3L + Ad-TK/GCV exhibited massive reduction in the tumor volume and extended survival. Rapamycin administration also prolonged the survival of Ad-Flt3L + Ad-TK/GCV-treated GL26 tumor-bearing mice, associated with an increase in the frequency of tumor-specific and IFNγ(+) CD8(+) T cells. More importantly, rapamycin administration, even for a short interval, elicited a potent long-lasting central memory CD8(+) T-cell response. The enhanced memory response translated to an increased frequency of tumor-specific CD8(+) T cells within the tumor and IFNγ release, providing the mice with long-term survival advantage in response to tumor rechallenge. Our data, therefore, point to rapamycin as an attractive adjuvant to be used in combination with immunotherapy in a phase I clinical trial for GBM.

Topics: Adenoviridae; Animals; Antigens; Antigens, Surface; Cell Line, Tumor; Cytokines; Dendritic Cells; Disease Models, Animal; Ganciclovir; Genetic Therapy; Genetic Vectors; Glioblastoma; Glioma; Immunologic Memory; Immunophenotyping; Immunotherapy; Membrane Proteins; Mice; Rats; Signal Transduction; Sirolimus; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Thymidine Kinase; TOR Serine-Threonine Kinases

Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor-initiating cells (BTICs).. We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts.. The cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing "advanced" BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo.. Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Brain Neoplasms; Cell Proliferation; Combined Modality Therapy; Female; Flow Cytometry; Fluorescent Antibody Technique; Glioblastoma; Green Fluorescent Proteins; Humans; Immunoenzyme Techniques; Luciferases; Mice; Mice, SCID; Myxoma virus; Neoplastic Stem Cells; Oncolytic Virotherapy; Poxviridae Infections; Sirolimus; Tumor Cells, Cultured; Tumor Virus Infections; Virus Replication; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Brain Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Glioblastoma; Humans; Leukemia, Promyelocytic, Acute; Oxides; Quinazolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

We have studied the consequences of the combination of the mammalian target of rapamycin (mTOR) inhibitor RAD001 and temozolomide on the growth and cell death of the glioblastoma cell line U-87 in vitro. A progressive decrease of cell proliferation was recorded with increasing concentrations of temozolomide, which was markedly reinforced and prolonged by the addition of RAD001. While this combination treatment resulted in only a low level of apoptosis, it led to a pronounced enhancement of autophagic cell death. When combined with γ-ray irradiation, a significant reinforcement of the overall cytotoxicity was obtained, suggesting the efficacy of such a multipronged approach for the treatment of glioblastoma. RAD001 strongly contributes to the reinforcement of temozolomide-induced autophagy, which appears to represent a major form of cell death in glioblastoma. The association of such combined chemotherapies with radiotherapy could be useful for the management of these hard-to-treat malignancies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Blotting, Western; Brain Neoplasms; Cell Proliferation; Cesium Radioisotopes; Dacarbazine; Drug Synergism; Everolimus; Flow Cytometry; Gamma Rays; Glioblastoma; Humans; Sirolimus; Temozolomide; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, portends a poor prognosis despite current treatment modalities. Recurrence of tumor growth is attributed to the presence of treatment-resistant cancer stem cells (CSCs). The targeting of these CSCs is therefore essential in the treatment of this disease. Mechanistic target of rapamycin (mTOR) forms two multiprotein complexes, mTORC1 and mTORC2, which regulate proliferation and migration, respectively. Aberrant function of mTOR has been shown to be present in GBM CSCs. All-trans retinoic acid (ATRA), a derivative of retinol, causes differentiation of CSCs as well as normal neural progenitor cells. The purpose of this investigation was to delineate the role of mTOR in CSC maintenance, and to establish the mechanism of targeting GBM CSCs using differentiating agents along with inhibitors of the mTOR pathways. The results demonstrated that ATRA caused differentiation of CSCs, as demonstrated by the loss of the stem cell marker Nestin. These observations were confirmed by western blotting, which demonstrated a time-dependent decrease in Nestin expression following ATRA treatment. This effect occurred despite combination with mTOR (rapamycin), PI3K (LY294002) and MEK1/2 (U0126) inhibitors. Expression of activated extracellular signal-regulated kinase 1/2 (pERK1/2) was enhanced following treatment with ATRA, independent of mTOR pathway inhibitors. Proliferation of CSCs, determined by neurosphere diameter, was decreased following treatment with ATRA alone and in combination with rapamycin. The motility of GBM cells was mitigated by treatment with ATRA, rapamycin and LY29002 alone. However, combination treatment augmented the inhibitory effect on migration suggesting synergism. These findings indicate that ATRA-induced differentiation is mediated via the ERK1/2 pathway, and underscores the significance of including differentiating agents along with inhibitors of mTOR pathways in the treatment of GBM.

Topics: Antibiotics, Antineoplastic; Brain Neoplasms; Butadienes; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromones; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glioblastoma; Humans; MAP Kinase Kinase 1; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Morpholines; Multiprotein Complexes; Neoplastic Stem Cells; Nestin; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tretinoin

An essential mode of acquired resistance to radiotherapy (RT) appears to be promotion of tumor cell motility and invasiveness in various cancer types, including glioblastoma, a process resembling 'evasive resistance'. Hence, a logical advancement of RT would be to identify suitable complementary treatment strategies, ideally targeting cell motility. Here we report that the combination of focal RT and mammalian target of rapamycin (mTOR) inhibition using clinically relevant concentrations of temsirolimus (CCI-779) prolongs survival in a syngeneic mouse glioma model through additive cytostatic effects. In vitro, the mTOR inhibitor CCI-779 exerted marked anti-invasive effects, irrespective of the phosphatase and tensin homolog deleted on chromosome 10 status and counteracted the proinvasive effect of sublethal irradiation. Mechanistically, we identified regulator of G-protein signaling 4 (RGS4) as a novel target of mTOR inhibition and a key driver of glioblastoma invasiveness, sensitive to the anti-invasive properties of CCI-779. Notably, suppression of RGS4-dependent glioma cell invasion was signaled through both mTOR complexes, mTORC1 and mTORC2, in a concentration-dependent manner, indicating that high doses of CCI-779 may overcome tumor-cell resistance associated with the sole inhibition of mTORC1. We conclude that combined RT and mTOR inhibition is a promising therapeutic option that warrants further clinical investigation in upfront glioblastoma therapy.

Topics: Adult; Aged; Animals; Astrocytoma; Cell Line, Tumor; Cell Movement; Female; Glioblastoma; Humans; Male; Mice; Mice, Inbred Strains; Middle Aged; Neoplasm Invasiveness; Protein Kinase Inhibitors; RGS Proteins; Sirolimus; TOR Serine-Threonine Kinases

The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild-type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3-kinase (PI3-K)/AKT pathway-focused gene profiles were examined by real-time polymerase chain reaction-based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen-activated protein kinase kinase inhibitor U0126. The dual PI3-K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal-regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.

Topics: Butadienes; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Furans; Gene Expression; Glioblastoma; Humans; Nitriles; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyridines; Pyrimidines; Quinazolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The molecular target of rapamycin (mTOR) is up-regulated in glioblastoma (GBM) and this is associated with the rate of cell growth, stem cell proliferation and disease relapse. Rapamycin is a powerful mTOR inhibitor and strong autophagy inducer. Previous studies analyzed the effects of rapamycin in GBM cell lines. However, to our knowledge, no experiment was carried out to evaluate the effects of rapamycin neither in primary cells derived from GBM patients nor in vivo in brain GBM xenograft. These data are critical to get a deeper insight into the effects of such adjuvant therapy in GBM patients. In the present study, various doses of rapamycin were tested in primary cell cultures from GBM patients. These effects were compared with that obtained by the same doses of rapamycin in GBM cell lines (U87Mg). The effects of rapamycin were also evaluated in vivo, in brain tumors developed from mouse xenografts. Rapamycin, starting at the dose of 10nm inhibited cell growth both in U87Mg cell line and primary cell cultures derived from various GBM patients. When administered in vivo to brain xenografts in nude mice rapamycin almost doubled the survival time of mice and inhibited by more than 95% of tumor volume.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Flow Cytometry; Glioblastoma; Humans; Mice; Mice, Nude; Microscopy, Electron, Transmission; Sirolimus; Xenograft Model Antitumor Assays

Development of novel patient stratification tools for cancer is a challenge that require advanced molecular screening and a detailed understanding of tumour signalling networks. Here, we apply phospho-specific flow cytometry for signal profiling of primary glioblastoma tumours after preservation of single-cell phosphorylation status as a strategy for evaluation of tumour signalling potential and assessment of rapamycin-mediated mTOR inhibition. The method has already enhanced insight into cancers and disorders of the immune system, and our study demonstrate a great potential to improve the understanding of aberrant signalling in glioblastoma and other solid tumours.

Topics: Antibiotics, Antineoplastic; Base Sequence; Brain Neoplasms; Cell Proliferation; Epidermal Growth Factor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Library; Glioblastoma; Humans; Phosphorylation; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Cediranib (AZD2171) is a potent small molecule inhibitor of vascular endothelial growth factor (VEGF) receptors. Cediranib has demonstrated single agent activity in several adult cancers and is being studied in combination with standard cytotoxic agents in multiple disease settings.. Cediranib was tested in vivo against six childhood tumor xenograft models (four sarcomas, one glioblastoma, one neuroblastoma) alone or combined with cyclophosphamide (two models), vincristine (three models) or cisplatin (one model), each administered at its maximum tolerated dose, or rapamycin (six models).. The combination of cediranib with standard cytotoxic agents was superior to the cytotoxic agent used alone for a single xenograft (one of the three xenografts evaluated for the vincristine-cediranib combination). The cediranib-cyclophosphamide combination was inferior to single agent cyclophosphamide in time to event for both models studied and was significantly inferior for one of the models. Cediranib combined with rapamycin was superior to each of the agents used alone in two of the six models and was determined to be additive or supra-additive with rapamycin in four models, although the effects were not large.. Cediranib combined with cytotoxic chemotherapy agents demonstrated little or no benefit (and in one case was significantly inferior) compared to chemotherapy alone for the six pediatric cancer xenografts studied. By contrast, the combination of cediranib with rapamycin was additive or supra-additive in four of the six models in terms of prolongation of time to event, though tumor regressions were not observed for this combination.

Topics: Adolescent; Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Female; Glioblastoma; Humans; Infant; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Neuroblastoma; Quinazolines; Receptors, Vascular Endothelial Growth Factor; Sarcoma; Sirolimus; Transplantation, Heterologous; Xenograft Model Antitumor Assays

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the po

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Carbon Radioisotopes; Disease Models, Animal; Everolimus; Glioblastoma; Humans; Magnetic Resonance Spectroscopy; Male; Neuroimaging; Rats; Rats, Nude; Sirolimus; Xenograft Model Antitumor Assays

Amplification of the gene encoding the epidermal growth factor receptor (EGFR) occurs commonly in glioblastoma (GBM), leading to activation of downstream kinases, including phosphatidylinositol 3'-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). A serine-threonine kinase, mTOR controls cell growth by regulating mRNA translation, metabolism, and autophagy; acting as both a downstream effector and upstream regulator of PI3K. These signaling functions are distributed between at least two distinct complexes, mTORC1 and mTORC2 with respect to pathway specificity. We have investigated mTOR signaling in glioma cells with the allosteric mTORC1 inhibitor rapamycin, the mTORC1/2 inhibitor Ku-0063794, a dual PI3K/mTORC1/2 kinase inhibitor PI-103, and siRNA against raptor, rictor, or mTOR, and evaluated the value of mTOR inhibitors for the treatment of glioblastoma.

Topics: Adaptor Proteins, Signal Transducing; Carrier Proteins; Cell Line, Tumor; ErbB Receptors; Furans; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proteins; Pyridines; Pyrimidines; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

Topics: Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Everolimus; Glioblastoma; Humans; Lactic Acid; Magnetic Resonance Spectroscopy; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylcholine; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment.

Topics: Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Dacarbazine; Glioblastoma; Humans; Membrane Proteins; Microtubule-Associated Proteins; Radiation-Sensitizing Agents; RNA Interference; RNA, Small Interfering; Sirolimus; Temozolomide

These findings emphasize that the mTOR pathway may contribute to maintenance of quiescence of CSCs, and provide a basis for manipulating CSCs in the treatment of GBM. Future research should focus on further defining the PI3K/Akt/mTOR molecular network in the regulation of stem cell quiescence and provide rationale for targeting the cancer-initiating cells of GBM.

Topics: Animals; Antibiotics, Antineoplastic; Butadienes; Cell Line; Enzyme Inhibitors; Glioblastoma; Humans; MAP Kinase Signaling System; Neoplastic Stem Cells; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Platelet derived growth factor receptor (PDGFR) activity is deregulated in human GBM due to amplification and rearrangement of the PDGFR-alpha gene locus or overexpression of the PDGF ligand, resulting in the activation of downstream kinases such as phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Aberrant PDGFR signaling is observed in approximately 25-30% of human GBMs, which are frequently molecularly classified as the proneural subclass. It would be valuable to understand how PDGFR driven GBMs respond to Akt and mTOR inhibition.. Using genetically engineered PTEN-intact and PTEN-deficient PDGF-driven mouse models of GBM that closely mimic the histology and genetics of the human PDGF subgroup, we investigated the effect of inhibiting Akt and mTOR alone or in combination in vitro and in vivo. We used perifosine and CCI-779 to inhibit Akt and mTOR, respectively. Here, we show in vitro data demonstrating that the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null primary glioma cell cultures is obtained when using both inhibitors in combination. We next investigated if the effects we observed in culture could be duplicated in vivo by treating mice with gliomas for 5 days. The in vivo treatments with the combination of CCI-779 and perifosine resulted in decreased Akt and mTOR signaling, which correlated to decreased proliferation and increased cell death independent of PTEN status, as monitored by immunoblot analysis, histology and MRI.. These findings underline the importance of simultaneously targeting Akt and mTOR to achieve significant down-regulation of the PI3K pathway and support the rationale for testing the perifosine and CCI-779 combination in the human PDGF-subgroup of GBM.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Drug Synergism; Glioblastoma; Mice; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Sirolimus

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Flow Cytometry; G1 Phase; Glioblastoma; Histones; Humans; Inhibitor of Apoptosis Proteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Survivin; Time Factors; TOR Serine-Threonine Kinases

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by increased proliferation and resistance to chemotherapy and radiotherapy. Recently, the identification of tumor-initiating cells with stem-like properties in diverse human cancers including GBM represents an important conceptual advance in cancer biology with therapeutic implications. However, the factors determining the differential development and radiosensitization of glioma-initiating cells (GICs) remain poorly defined. Here, we report that rapamycin induced differentiation of GICs and increased their sensitivity to radiation by activating autophagy. Transient in vitro exposure to rapamycin and radiation abolished the capacity of transplanted GICs to establish intracerebral GBMs. Most importantly, in vivo combination of rapamycin and radiation effectively blocked the tumor growth and associated mortality that occurs in mice after intracerebral grafting of human GICs. We demonstrate that rapamycin activated their autophagy and triggers the differentiation cascade in GICs isolated from human GBMs. This was followed by a reduction in proliferation, cell viability, clonogenic ability and increased expression of neural differentiation markers after radiation. Our results suggest that autophagy plays an essential role in the regulation of self-renewal, differentiation, tumorigenic potential and radiosensitization of GICs, suggesting autophagy could be a promising therapeutic target in a subset of GBMs. We propose that autophagy defect in GICs contributes to radioresistance of GICs by desensitizing GICs to normal differentiation cues. Activating autophagy may abrogate the resistance of GICs to radiation and could lead to the development of novel therapeutic approaches for the treatment of GBMs.

Topics: Animals; Antibiotics, Antineoplastic; Autophagy; Blotting, Western; Brain Neoplasms; Cell Differentiation; Cell Proliferation; Glioblastoma; Humans; Mice; Neoplastic Stem Cells; Radiation Tolerance; Sirolimus; Tumor Cells, Cultured; Tumor Stem Cell Assay; X-Rays

Glioblastoma multiforme (GBM) is the most common aggressive brain cancer with a median survival of approximately 1 year. In a search for novel molecular targets that could be therapeutically developed, our kinome-focused microarray analysis identified the MAP (mitogen-activated protein) kinase-interacting kinase 1 (MNK1) as an attractive theranostic candidate. MNK1 overexpression was confirmed in both primary GBMs and glioma cell lines. Inhibition of MNK1 activity in GBM cells by the small molecule CGP57380 suppressed eIF4E phosphorylation, proliferation, and colony formation whereas concomitant treatment with CGP57380 and the mTOR inhibitor rapamycin accentuated growth inhibition and cell-cycle arrest. siRNA-mediated knockdown of MNK1 expression reduced proliferation of cells incubated with rapamycin. Conversely, overexpression of full-length MNK1 reduced rapamycin-induced growth inhibition. Analysis of polysomal profiles revealed inhibition of translation in CGP57380 and rapamycin-treated cells. Microarray analysis of total and polysomal RNA from MNK1-depleted GBM cells identified mRNAs involved in regulation of TGF-β pathway. Translation of SMAD2 mRNA as well as TGF-β-induced cell motility and vimentin expression was regulated by MNK1 signaling. Tissue microarray analysis revealed a positive correlation between the immunohistochemical staining of MNK1 and SMAD2. Taken together, our findings offer insights into how MNK1 pathways control translation of cancer-related mRNAs including SMAD2, a key component of the TGF-β signaling pathway. Furthermore, they suggest MNK1-controlled translational pathways in targeted strategies to more effectively treat GBM.

Topics: Adolescent; Adult; Aged; Aniline Compounds; Antibiotics, Antineoplastic; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Synergism; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Protein Serine-Threonine Kinases; Purines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sirolimus; Smad2 Protein; Transforming Growth Factor beta

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.

Topics: Amino Acid Substitution; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Drug Resistance, Neoplasm; Glioblastoma; Heterogeneous Nuclear Ribonucleoprotein A1; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Mutation, Missense; Phosphorylation; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.

Topics: Blotting, Western; Brain; Endothelial Cells; Flow Cytometry; Furans; Glioblastoma; Humans; Microscopy, Fluorescence; Pyridines; Pyrimidines; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases; Transfection

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol 3-kinase (PI3K) pathway are in clinical trials. However, it remains challenging to assess responses by traditional imaging methods. Here, we show for the first time the efficacy of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K inhibition by monitoring hyperpolarized [1-(13)C]lactate levels produced from hyperpolarized [1-(13)C]pyruvate through lactate dehydrogenase (LDH) activity. In GS-2 glioblastoma cells, PI3K inhibition by LY294002 or everolimus caused hyperpolarized lactate to drop to 42 +/- 12% and to 76 +/- 5%, respectively. In MDA-MB-231 breast cancer cells, hyperpolarized lactate dropped to 71 +/- 15% after treatment with LY294002. These reductions were correlated with reductions in LDH activity to 48 +/- 4%, 63 +/- 4%, and 69 +/- 12%, respectively, and were associated with a drop in levels of LDHA mRNA and LDHA and hypoxia-inducible factor-1alpha proteins. Supporting these findings, tumor growth inhibition achieved by everolimus in murine GS-2 xenografts was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio detected by in vivo MRS imaging, whereas an increase in this ratio occurred with tumor growth in control animals. Taken together, our findings illustrate the application of hyperpolarized (13)C MRS of pyruvate to monitor alterations in LDHA activity and expression caused by PI3K pathway inhibition, showing the potential of this method for noninvasive imaging of drug target modulation.

Topics: Animals; Carbon Isotopes; Chromones; Drug Delivery Systems; Enzyme Inhibitors; Everolimus; Glioblastoma; Humans; Lactic Acid; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Monitoring, Physiologic; Morpholines; Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Sirolimus; Treatment Outcome; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Glioblastoma Multiforme (GBM) is a malignant primary brain tumor associated with poor survival rate. PI3K/Akt pathway is highly upregulated in gliomas due to deletion or mutation of PTEN and its activation is associated with tumor grade. mTOR is downstream from PI3K/Akt pathway and it initiates translation through its action on S6K and 4E-BP1. mTOR is an important therapeutic target in many cancers, including glioblastomas. Rapamycin and its analogues are known to inhibit mTOR pathway; however, they also show simultaneous upregulation of Akt and eIF4E survival pathways on inhibition of mTOR, rendering cells more resistant to rapamycin treatment. In this study we investigated the effect of combination treatment of rapamycin with isoflavones such as genistein and biochanin A on mTOR pathway and activation of Akt and eIF4E in human glioblastoma (U87) cells. Our results show that combination treatment of rapamycin with isoflavones, especially biochanin A at 50 muM, decreased the phosphorylation of Akt and eIF4E proteins and rendered U87 cells more sensitive to rapamycin treatment when compared to cells treated with rapamycin alone. These results suggest the importance of combining chemopreventive with chemotherapeutic agents in order to increase the efficacy of chemotherapeutic drugs.

Topics: Anticarcinogenic Agents; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Eukaryotic Initiation Factor-4E; Genistein; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; Isoflavones; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

A number of important studies were presented at the CNS tumors section of the 2010 American Society of Oncology (ASCO) Annual Meeting. There was particular interest in a Phase II study showing that the mTOR inhibitor everolimus had significant activity in tuberous patients with subependymal giant cell astrocytomas. Two Phase III studies reported on the relative benefits of radiotherapy and chemotherapy in elderly patients with glioblastomas. Other studies focused on promising new agents such as XL184 and ANG1005.

Topics: Aged; Astrocytoma; Central Nervous System Neoplasms; Clinical Trials as Topic; Everolimus; Glioblastoma; Humans; Immunosuppressive Agents; Sirolimus; Tuberous Sclerosis

Glioblastoma, the most intractable cerebral tumor, is highly lethal. Recent studies suggest that cancer stem-like cells (CSLCs) have the capacity to repopulate tumors and mediate radio- and chemoresistance, implying that future therapies may need to turn from the elimination of rapidly dividing, but differentiated, tumor cells to specifically targeting the minority of tumor cells that repopulate the tumor. However, the mechanism by which glioblastoma CSLCs maintain their immature stem-like state or, alternatively, become committed to differentiation is poorly understood. Here, we show that the inactivation of mammalian target of rapamycin (mTor) by the mTor inhibitor rapamycin or knockdown of mTor reduced sphere formation and the expression of neural stem cell (NSC)/progenitor markers in CSLCs of the A172 glioblastoma cell line. Interestingly, combination treatment with rapamycin and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, not only reduced the expression of NSC/progenitor markers more efficiently than single-agent treatment, but also increased the expression of βIII-tubulin, a neuronal differentiation marker. Consistent with these results, a dual PI3K/mTor inhibitor, NVP-BEZ235, elicited a prodifferentiation effect on A172 CSLCs. Moreover, A172 CSLCs, which were induced to undergo differentiation by pretreatment with NVP-BEZ235, exhibited a significant decrease in their tumorigenicity when transplanted either subcutaneously or intracranially. Importantly, similar results were obtained when patient-derived glioblastoma CSLCs were used. These findings suggest that the PI3K/mTor signaling pathway is critical for the maintenance of glioblastoma CSLC properties, and targeting both mTor and PI3K of CSLCs may be an effective therapeutic strategy in glioblastoma.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromones; Drug Therapy, Combination; Enzyme Inhibitors; Glioblastoma; Humans; Imidazoles; Immunosuppressive Agents; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathways are aberrantly activated in many tumors, including highly proliferative glioblastomas, but how they are wired with the cell cycle remains imperfectly understood. Inhibitors of MEK/ERK and mTOR pathways are tested as anticancer agents. They are generally considered to induce a G(1) cell cycle arrest through down-regulation of D-type cyclins and up-regulation of p27(kip1). Here, we examined the effect of targeting mTOR by rapamycin and/or MEK by PD184352 in human glioblastoma cell lines. In combination, these drugs cooperatively and potently inhibited the G(1)-S transition and retinoblastoma protein phosphorylation. Their cooperation could not be explained by their partial and differential inhibitory effects on cyclin D1 or D3 but instead by their synergistic inhibition of the activating T172 phosphorylation of cyclin-dependent kinase (CDK) 4. This appeared independent of p27 and unrelated to weak modulations of the CDK-activating kinase activity. The T172 phosphorylation of CDK4 thus appears as a crucial node integrating the activity of both MEK/ERK and mTOR pathways. Combined inhibition of both pathways should be considered as a promising strategy for treatment of tumors harboring a deregulated CDK4 activity.

Topics: Adaptor Proteins, Signal Transducing; Benzamides; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 4; DNA Replication; Drug Combinations; Drug Evaluation, Preclinical; Glioblastoma; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Proteins; Regulatory-Associated Protein of mTOR; Retinoblastoma Protein; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tumor Cells, Cultured

The EGFR/PI3K/Akt/mTOR signaling pathway is activated in many cancers including glioblastoma, yet mTOR inhibitors have largely failed to show efficacy in the clinic. Rapamycin promotes feedback activation of Akt in some patients, potentially underlying clinical resistance and raising the need for alternative approaches to block mTOR signaling. AMPK is a metabolic checkpoint that integrates growth factor signaling with cellular metabolism, in part by negatively regulating mTOR. We used pharmacological and genetic approaches to determine whether AMPK activation could block glioblastoma growth and cellular metabolism, and we examined the contribution of EGFR signaling in determining response in vitro and in vivo. The AMPK-agonist AICAR, and activated AMPK adenovirus, inhibited mTOR signaling and blocked the growth of glioblastoma cells expressing the activated EGFR mutant, EGFRvIII. Across a spectrum of EGFR-activated cancer cell lines, AICAR was more effective than rapamycin at blocking tumor cell proliferation, despite less efficient inhibition of mTORC1 signaling. Unexpectedly, addition of the metabolic products of cholesterol and fatty acid synthesis rescued the growth inhibitory effect of AICAR, whereas inhibition of these lipogenic enzymes mimicked AMPK activation, thus demonstrating that AMPK blocked tumor cell proliferation primarily through inhibition of cholesterol and fatty acid synthesis. Most importantly, AICAR treatment in mice significantly inhibited the growth and glycolysis (as measured by (18)fluoro-2-deoxyglucose microPET) of glioblastoma xenografts engineered to express EGFRvIII, but not their parental counterparts. These results suggest a mechanism by which AICAR inhibits the proliferation of EGFRvIII expressing glioblastomas and point toward a potential therapeutic strategy for targeting EGFR-activated cancers.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Glioblastoma; Humans; Lipogenesis; Mice; Protein Kinases; PTEN Phosphohydrolase; Ribonucleotides; Ribosomal Protein S6 Kinases; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The AKT/mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in glioblastoma multiforme (GBM) oncogenesis due to activation of AKT. We studied two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), through which mTOR controls cell survival, growth and motility. Inhibition of mTOR by rapamycin (RAPA) resulted in time-dependent suppression of S6 ribosomal protein (pS6KSer235/236; mTORC1 substrate) and caused transient suppression of pAKTSer473 (mTORC2 substrate) at 1 to 3 h followed by a consistent increase from 6 to 24 h. Inhibition of mTOR or phosphoinositide 3-kinase (PI3K) suppressed platelet-derived growth factor (PDGF)- or fibronectin (FN)-induced activation of p70S6KThr389. Combined inhibition of mTOR and PI3K abolished PDGF- or FN-induced activation of STAT3Ser727. Expression of pAKT was suppressed by siRNA silencing of mTORC2 co-protein rictor, but not by mTORC1 co-protein raptor. GBM cell proliferation and motility paralleled the activation of mTORC2. Combined inhibition of mTOR and PI3K had an additive effect on suppressing cell growth and motility. PDGF-induced nuclear localization of mTOR was blocked by pre-treatment with RAPA. The results demonstrated that an activation of mTORC2 occurs when mTORC1 is inhibited by RAPA. Therefore, simultaneous suppression of mTORC1 and mTORC2 may provide novel therapy for GBM.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromones; Enzyme Activation; Fibronectins; Glioblastoma; Humans; Morpholines; Multiprotein Complexes; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Serine; Signal Transduction; Sirolimus; STAT3 Transcription Factor; Threonine; Time Factors; TOR Serine-Threonine Kinases

Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIP(S), and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIP(S) protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIP(S) polyubiquitination. FLIP(S) half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIP(S) E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIP(S) or with the FLIP(S)-containing death inducing signal complex. Small interfering RNA-mediated suppression of AIP4 levels in PTEN WT cells decreased FLIP(S) ubiquitination, prolonged FLIP(S) half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIP(S) steady-state levels, and suppressed FLIP(S) ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIP(S) ubiquitination, FLIP(S) stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability.

Topics: Animals; Apoptosis; Blotting, Western; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Drug Resistance, Neoplasm; Glioblastoma; Half-Life; Humans; Immunoprecipitation; Mice; Mice, Knockout; Protein Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Repressor Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Xenograft Model Antitumor Assays

Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element-binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1, possibly explaining rapamycin's poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl coenzyme A reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated prosurvival metabolic pathway and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.

Topics: Antineoplastic Agents; Brain Neoplasms; ErbB Receptors; Fatty Acids; Gene Knockdown Techniques; Glioblastoma; Humans; Hydrolysis; Lapatinib; Lipogenesis; Phosphatidylinositol 3-Kinases; Protein Transport; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Sirolimus; Sterol Regulatory Element Binding Protein 1

Inhibition of the protein kinase mammalian target of rapamycin (mTOR) is being evaluated for treatment of a variety of malignancies. However, the effects of mTOR inhibitors are cytostatic and standard size criteria do not reliably identify responding tumors. The aim of this study was to evaluate whether response to mTOR inhibition could be assessed by positron emission tomography (PET) imaging of tumor metabolism.. Glucose, thymidine, and amino acid utilization of human glioma cell lines with varying degrees of sensitivity to mTOR inhibition were assessed by measuring in vitro uptake of [18F]fluorodeoxyglucose ([18F]FDG), [18F]fluorothymidine ([18F]FLT), and [3H]l-tyrosine before and after treatment with the mTOR inhibitor rapamycin. The tumor metabolic activity in vivo was monitored by small-animal PET of tumor-bearing mice. The mechanisms underlying changes in metabolic activity were analyzed by measuring expression and functional activity of enzymes and transporters involved in the uptake of the studied imaging probes.. In sensitive cell lines, rapamycin decreased [18F]FDG and [18F]FLT uptake by up to 65% within 24 hours after the start of therapy. This was associated with inhibition of hexokinase and thymidine kinase 1. In contrast, [3H]l-tyrosine uptake was unaffected by rapamycin. The effects of rapamycin on glucose and thymidine metabolism could be imaged noninvasively by PET. In sensitive tumors, [18F]FDG and [18F]FLT uptake decreased within 48 hours by 56 +/- 6% and 52 +/- 8%, respectively, whereas there was no change in rapamycin-resistant tumors.. These encouraging preclinical data warrant clinical trials evaluating [18F]FDG and [18F]FLT-PET for monitoring treatment with mTOR inhibitors in patients.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Fluorine Radioisotopes; Fluorodeoxyglucose F18; Glioblastoma; Glucose; Humans; Mice; Positron-Emission Tomography; Protein Kinases; Sirolimus; Thymidine; Thymidine Kinase; TOR Serine-Threonine Kinases; Tyrosine

Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus).. To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN.. Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas 1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy.. These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Proliferation; ErbB Receptors; Everolimus; Glioblastoma; Humans; Mice; Mice, Nude; Neovascularization, Pathologic; Prognosis; Protein Kinases; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterotopic; Xenograft Model Antitumor Assays

Oncolytic adenoviruses are a promising tool in cancer therapy. In this study, we characterized the role of autophagy in oncolytic adenovirus-induced therapeutic effects. OBP-405, an oncolytic adenovirus regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad, OBP-301) with a tropism modification (RGD) exhibited a strong antitumor effect on glioblastoma cells. When autophagy was inhibited pharmacologically, the cytotoxicity of OBP-405 was attenuated. In addition, autophagy-deficient Atg5(-/-) mouse embryonic fibroblasts (MEFs) were less sensitive than wild-type MEFs to OBP-405. These findings indicate that OBP-405-induced autophagy is a cell killing effect. Moreover, autophagy-inducing therapies (temozolomide and rapamycin) synergistically sensitized tumor cells to OBP-405 by stimulating the autophagic pathway without altering OBP-405 replication. Mice harboring intracranial tumors treated with OBP-405 and temozolomide survived significantly longer than those treated with temozolomide alone, and mice treated with OBP-405 and the rapamycin analog RAD001 survived significantly longer than those treated with RAD001 alone. The observation that autophagy inducers increase OBP-405 antitumor activity suggests a novel strategy for treating patients with glioblastoma.

Topics: Adenoviridae; Animals; Autophagy; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Genetic Therapy; Glioblastoma; Mice; Oncolytic Virotherapy; Oncolytic Viruses; Sirolimus; Temozolomide

Despite recent advances in cancer immunotherapy, cellular mechanisms controlling expression of tumor-associated antigens are poorly understood. Mutations in cancer cells, such as loss of PTEN, may increase expression of tumor-associated antigens. The authors investigated the relationship between PTEN status and the expression of a glioma-associated antigen, adenosine diphosphate-ribosylation factor 4-like (ARF4L) protein.. Human glioma cell lines with confirmed PTEN status were examined by Northern blot analysis and quantitative polymerase chain reaction. Western blot analysis was used to measure ARF4L protein levels across multiple cell lines.. The loss of PTEN was shown to lead to increased levels of ARF4L protein but no change in transcript levels. Cell lines with serial mutations, including activation of Ras and Akt pathways, also demonstrated increased levels of ARF4L protein, which decreased after treatment with rapamycin. The ARF4L transcript preferentially localized to the polysomal compartment after PTEN loss in glioma or activation of Akt in human astrocytes.. Expression of ARF4L is controlled by the activated Akt/mTOR pathway, which is a downstream effect of the loss of PTEN function. Mutations leading to oncogenesis may impact the regulation and expression of tumor specific antigens. Screening of mutation status in glioma may be helpful in selecting patients for immunotherapy trials in the future.

Topics: ADP-Ribosylation Factors; Antibiotics, Antineoplastic; Antigens, Neoplasm; Astrocytes; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Genes, ras; Glioblastoma; Humans; Mutation; Polymerase Chain Reaction; Polyribosomes; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; Transcription, Genetic

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.

Topics: Autophagy; Calcium Channel Blockers; Cell Line, Tumor; Drug Evaluation, Preclinical; Fluspirilene; Glioblastoma; Green Fluorescent Proteins; Humans; Intracellular Membranes; Loperamide; Microtubule-Associated Proteins; Mycotoxins; Peptides; Phagosomes; Phosphatidylinositol Phosphates; Pimozide; Protein Kinases; Recombinant Fusion Proteins; Sirolimus; Small Molecule Libraries; TOR Serine-Threonine Kinases; Trifluoperazine; Zinc Fingers

Previously, we have shown that PKC-eta (protein kinase C-eta) positively regulates glioblastoma proliferation and confers resistance to irradiation-induced apoptosis. In this study, we investigated the efficacy of rapamycin in inhibiting cell proliferation in two glioblastoma cell lines U-251MG (PKC-eta expressing) and U-1242MG (PKC-eta deficient) following PKC-eta activation. In U-251MG cells, rapamycin (10 nM) treatment was less effective as an antiproliferative agent when cells were concurrently stimulated with 10% serum and phorbol 12-myristate 13-acetate (PMA, 100 nM), a potent activator of PKC isozymes. Rapamycin-insensitive growth was owing to PKC-eta, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC-eta (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC-eta antisense oligonucleotides were sensitive to rapamycin. PKC-eta-expressing cells stimulated with PMA maintained p70S6K phosphorylation on Thr389 and phosphorylation of rpS6 (ser235/36), suggesting p70S6K kinase activity was still intact. Inhibition of p70S6K expression with small interfering RNA oligonucleotides inhibited cell proliferation greater than 50% in the presence of a combination of PMA and serum. Additionally, p70S6K co-precipitated with PKC-eta, suggesting a physical interaction between PKC-eta and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC-eta signaling.

Topics: Blotting, Western; Carcinogens; Cell Proliferation; Drug Synergism; Enzyme Inhibitors; Glioblastoma; Humans; Immunoprecipitation; Immunosuppressive Agents; Oligonucleotides, Antisense; Phosphorylation; Protein Kinase C; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Serum; Signal Transduction; Sirolimus; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Rapamycin, a highly specific mTOR inhibitor, has shown anti-proliferative and anti-angiogenic properties, as well as an enhancement in tumour growth delay when used in combination with radiation in mouse xenograft models. Our goal was to determine if rapamycin can also have a positive effect on the local tumour control achieved by radiotherapy.. Nude mice bearing U87 glioblastoma xenografts were treated with concomitant rapamycin and radiotherapy over a 5 day fractionation schedule. Animals received graded total doses ranging from 24 to 100 Gy. Experimental endpoints were tumour growth delay and local tumour control. In addition, histological evaluation of tumour sections was performed to examine changes occurring within the tumour microenvironment as a result of treatment. Analysis of proliferation, mTOR signalling, hypoxia, and vessel thrombosis was conducted.. As a single agent, rapamycin reduced the in vitro growth of U87 cells by 70% and caused a 4 day growth delay of tumour xenografts. In combination with radiation, no further increase in tumour growth delay was observed when compared to radiation alone. The tumour control dose 50% (TCD(50)) was 46.8 Gy (95% CI 41; 53 Gy) in tumours treated with radiation alone and was slightly but not significantly lower at 42.8 Gy (95% CI 36; 49 Gy) after simultaneous treatment with rapamycin. Histological evaluation revealed evidence of elevated hypoxia following rapamycin treatment that may be due to vessel thrombosis.. The influence of rapamycin on thrombosis and tumour hypoxia may be a confounding factor limiting its effectiveness in combination with radiotherapy.

Topics: Animals; Antibiotics, Antineoplastic; Cell Hypoxia; Cell Line, Tumor; Combined Modality Therapy; Dose Fractionation, Radiation; Glioblastoma; Mice; Mice, Nude; Neoplasm Transplantation; Radiotherapy Dosage; Signal Transduction; Sirolimus; Thrombosis; Transplantation, Heterologous

Regulated gene expression may be required for the clinical development of certain gene therapies. Several approaches have been developed that allow pharmacologic control of transgene expression, including the dimerizer-regulated transcriptional system in which rapamycin or its analogs function as transcriptional inducers. These compounds can also act as direct antitumor agents via inhibition of mammalian target of rapamycin (mTOR). We describe the development of an optimized recombinant adeno-associated virus (AAV) expression cassette that allows dimerizer-regulated gene expression from a single vector in vitro and in vivo. After demonstrating multiple cycles of rapamycin-dependent transgene induction following a single administration of an AAV vector in vivo, application of this regulated AAV gene expression system to the pharmacologic control of antiangiogenic therapy was evaluated in preclinical tumor models. Dimerizer-regulated vectors were constructed encoding a soluble inhibitor of the vascular endothelial growth factor (VEGF) pathway. In two subcutaneous models of glioblastoma, regulated expression of the VEGF inhibitor via recombinant AAV-mediated gene transfer, in combination with rapamycin, was shown to decrease tumor growth rate significantly. The dual properties of rapamycin--as a transcriptional inducer and mTOR inhibitor--are exploited in combination with an AAV-encoded antiangiogenic agent to provide a novel approach for the treatment of malignant diseases.

Topics: Angiostatins; Animals; Blotting, Western; Cell Line; Cell Line, Tumor; Dependovirus; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glioblastoma; Humans; Immunosuppressive Agents; Mice; Mice, Nude; Neoplasms; Prealbumin; Promoter Regions, Genetic; Receptors, Vascular Endothelial Growth Factor; Sirolimus; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The oncogenic epidermal growth factor receptor (EGFR) pathway triggers downstream phosphatidylinositol 3-kinase (PI3K)/RAS-mediated signaling cascades. In transgenic mice, glioblastoma cannot develop on single but only on simultaneous activation of the EGFR signaling mediators RAS and AKT. However, complete blockade of EGFR activation does not result in apoptosis in human glioblastoma cells, suggesting additional cross-talk between downstream pathways. Based on these observations, we investigated combination therapies using protein kinase inhibitors against EGFR, platelet-derived growth factor receptor, and mammalian target of rapamycin, assessing glioblastoma cell survival. Clinically relevant doses of AEE788, Gleevec (imatinib), and RAD001 (everolimus), alone or in combinations, did not induce glioblastoma cell apoptosis. In contrast, simultaneous inactivation of the EGFR downstream targets mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and PI3K by U0126 and wortmannin triggered rapid tumor cell death. Blocking EGFR with AEE788 in combination with sublethal concentrations of the microtubule stabilizer patupilone also induced apoptosis and reduced cell proliferation in glioblastoma cells, accompanied by reduced AKT and ERK activity. These data underline the critical role of the PI3K/AKT and the RAS/RAF/mitogen-activated protein/ERK kinase/ERK signaling cascades in the cell-intrinsic survival program of sensitive glioblastoma cell lines. We conclude that drug combinations, which down-regulate both ERK and protein kinase B/AKT activity, may prove effective in overcoming cell resistance in a subgroup of glioblastoma.

Topics: Apoptosis; Benzamides; Blotting, Western; Cell Cycle; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Therapy, Combination; Epothilones; ErbB Receptors; Everolimus; Glioblastoma; Humans; Imatinib Mesylate; Immunosuppressive Agents; MAP Kinase Kinase 1; Microtubules; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phorbol Esters; Phosphatidylinositol 3-Kinases; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purines; Pyrimidines; Signal Transduction; Sirolimus; Tumor Cells, Cultured

Malignant gliomas of which glioblastomas represent the ultimate grade of malignancy are characterized by dismal prognoses because malignant glioma cells present both important proliferation and neoangiogenesis processes and can actively migrate through the narrow extra-cellular spaces in the brain, often travelling relatively long distances, making them elusive targets for effective surgical management. Invasive malignant glioma cells show a decrease in their proliferation rates and a relative resistance to apoptosis (type I programmed cell death) but seem less resistant to autophagic cell death (type II programmed cell death). Autophagic cell death represents an alternative mechanism to overcome, at least partly, the dramatic resistance of glioblastomas to pro-apoptotic-related therapies. Another way to potentially overcome apoptosis resistance is to decrease the migration of malignant glioma cells in the brain, which then should restore a level of sensitivity to pro-apoptotic drugs. We conclude this work with an algorithm showing the optimal current treatment for glioblastoma with the potent future innovations.

Topics: Algorithms; Antibiotics, Antineoplastic; Antineoplastic Agents, Alkylating; Apoptosis; Autophagy; Brain Neoplasms; Cell Movement; Clinical Trials as Topic; Dacarbazine; Forecasting; Glioblastoma; Humans; Prognosis; Sirolimus; Temozolomide; Transcription Factors

The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN-deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblastoma patients, particularly those with PTEN-deficient tumors.

Topics: Cell Division; Cell Line, Tumor; Enzyme Activation; ErbB Receptors; Erlotinib Hydrochloride; Glioblastoma; Humans; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Protein Kinases; PTEN Phosphohydrolase; Quinazolines; Sirolimus; TOR Serine-Threonine Kinases; Transfection

The phosphoinositide 3-kinase (PI3-kinase) signaling pathway is frequently aberrantly activated in glioblastoma multiforme (GM) by mutation or loss of the 3' phospholipid phosphatase PTEN. PTEN abnormalities result in inappropriate signaling to downstream molecules including protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR). PI3-kinase activation increases resistance to radiation-induced cell death; conversely, PI3-kinase inhibition enhances the sensitivity of tumors to radiation. The effects of LY294002, a biochemical inhibitor of PI3-kinase, on the response to radiation were examined in the PTEN mutant glioma cell line U251 MG. Low doses of LY294002 sensitized U251 MG to clinically relevant doses of radiation. In contrast to LY294002, rapamycin, an inhibitor of mTOR, did not result in radiosensitization. We demonstrate that among multiple known targets of LY294002, PI3-kinase is the most likely molecule responsible for LY294002-induced radiosensitization. Furthermore, using a myristoylated PKB/Akt construct, we identified PKB/Akt as the downstream molecule that mediates the synergistic cytotoxicity between LY294002 and radiation. Thus PI3-kinase dysregulation may contribute to the notable radioresistance of GM tumors and inhibition of PKB/Akt offers an excellent target to enhance radiosensitivity.

Topics: Agammaglobulinaemia Tyrosine Kinase; Analysis of Variance; Antibiotics, Antineoplastic; Brain Neoplasms; Cell Line, Tumor; Chromones; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoric Monoester Hydrolases; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Radiation-Sensitizing Agents; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Combined activation of Ras and AKT leads to the formation of astrocytic glioblastoma multiforme (GBM) in mice. In human GBMs, AKT is not mutated but is activated in approximately 70% of these tumors, in association with loss of PTEN and/or activation of receptor tyrosine kinases. Mechanistic justification for the therapeutic blockade of targets downstream of AKT, such as mTOR, in these cancers requires demonstration that the oncogenic effect of PTEN loss is through elevated AKT activity. We demonstrate here that loss of Pten is similar to AKT activation in the context of glioma formation in mice. We further delineate the role of mTOR activity downstream of AKT in the maintenance of AKT+KRas-induced GBMs. Blockade of mTOR results in regional apoptosis in these tumors and conversion in the character of surviving tumor cells from astrocytoma to oligodendroglioma. These data suggest that mTOR activity is required for the survival of some cells within these GBMs, and mTOR appears required for the maintenance of astrocytic character in the surviving cells. Furthermore, our study provides the first example of conversion between two distinct tumor types usually thought of as belonging to specific lineages, and provides evidence for signal transduction-mediated transdifferentiation between glioma subtypes.

Topics: Animals; Apoptosis; Blotting, Western; Brain; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Cells, Cultured; Enzyme Activation; Exons; Glioblastoma; Green Fluorescent Proteins; Immunohistochemistry; In Situ Nick-End Labeling; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Genetic; Mutation; Neurons; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Plasmids; Polymerase Chain Reaction; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Elevated epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) signaling are known to contribute to the malignant properties of glioblastoma multiforme (GBM), which include uncontrolled cell proliferation and evasion of apoptosis. Small molecule inhibitors that target these protein kinases have been evaluated in multiple clinical trials for cancer patients, including those with GBM. Here we have examined the cellular and molecular effects of a combined kinase inhibition of mTOR (rapamycin) and EGFR (EKI-785) in U87 and U251 GBM cells. Simultaneous treatment with rapamycin and EKI-785 results in synergistic antiproliferative as well as proapoptotic effects. At a molecular level, rapamycin alone significantly decreases S6 phosphorylation, whereas EKI-785 alone promotes substantially reduced signal transducer and activator of transcription (STAT3) phosphorylation. Treatment with rapamycin alone also increases Akt phosphorylation on Ser-473, but this effect is blocked by a simultaneous administration of EKI-785. Individually, EKI-785 diminishes while rapamycin promotes the binding of the translation inhibitor eukaryotic initiation factor 4E binding protein (4EBP1) to the eukaryotic translation initiation factor 4E (eIF4E). In spite of these opposing effects, the highest level of 4EBP1-eIF4E binding occurs with the combination of the two inhibitors. These results indicate that the inhibition of EGFR and mTOR has distinct as well as common signaling consequences and provides a molecular rationale for the synergistic antitumor effects of EKI-785 and rapamycin administration.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Dose-Response Relationship, Drug; ErbB Receptors; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Immunoblotting; Immunoprecipitation; Models, Biological; Phosphoproteins; Phosphorylation; Protein Kinases; Quinazolines; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Thymidine; TOR Serine-Threonine Kinases

We previously demonstrated that protein kinase C-eta (PKC-eta) mediates a phorbol 12-myristate-13-acetate (PMA)-induced proliferative response in human glioblastoma (GBM) cells. In this report, we show that PMA-stimulated activation of PKC-eta in U-251 GBM cells resulted in activation of both Akt and the mammalian target of rapamycin (mTOR) signaling pathways and an increase in cell proliferation. Expression of a kinase dead PKC-eta (PKC-etaKR) construct reduced the basal and PMA-evoked proliferation of PKC-eta-expressing U-251 GBM cells, as well as abrogated the PMA-induced activation of Akt, mTOR, and the mTOR targets 4E-BP1 and STAT-3. Treatment of cells with the PI-3 kinase inhibitor LY294002 (10 muM) or the mTOR inhibitor rapamycin (10 nM) also reduced PMA-induced proliferation and cell-cycle progression. Expression of a constitutively active PKC-eta (PKC-etaDeltaNPS) construct in a GBM cell line with no endogenous PKC-eta (U-1242) also provided evidence that PKC-eta targets the Akt and mTOR signaling pathways. Moreover, activation of 4E-BP1 and STAT-3 in both PMA-treated U-251 and PKC-etaDeltaNPS-expressing U-1242 GBM cells was inhibited by rapamycin. However, activation of Akt, but not mTOR was inhibited by the PI-3 kinase inhibitor LY294002. This study identifies Akt and mTOR as downstream targets of PKC-eta that are involved in GBM cell proliferation.

Topics: Brain Neoplasms; Cell Cycle; Chromones; Enzyme Inhibitors; Glioblastoma; Humans; Morpholines; Protein Kinase C; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Tetradecanoylphorbol Acetate; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) modulates key signaling pathways that promote uncontrolled proliferation of glioblastoma multiforme (GBM). Because rapid tumor proliferation may contribute to the clinical radioresistance of GBM tumors, the combination of rapamycin, a selective mTOR inhibitor, and radiation was studied in vitro and in vivo in a GBM model. In monolayer cultures of U87 and SKMG-3 cells, rapamycin had no impact on radiation sensitivity. In contrast, rapamycin significantly enhanced the efficacy of fractionated radiation of established U87 xenografts in nude mice. Similar effects were seen in U87 spheroids treated with rapamycin and radiation, which suggests that the sensitizing effects of this drug are dependent on disruption of mTOR signaling pathways specifically within tumor cells. Inhibition of these signaling pathways can lead to inhibition of G(1)-specific cyclin-dependent kinase activities, and this could contribute to the sensitizing effects of rapamycin. Consistent with this idea, roscovitine, a specific cyclin-dependent kinase inhibitor, also enhanced the efficacy of fractionated radiation in U87 spheroids. These data demonstrate that inhibition of tumor proliferation does not diminish the efficacy of fractionated radiation and suggest that disruption of key signal transduction pathways may significantly enhance the effectiveness of radiation therapy in malignant gliomas.

Topics: Animals; Cell Division; Combined Modality Therapy; Female; Glioblastoma; Humans; Mice; Mice, Nude; Protein Kinase Inhibitors; Protein Kinases; Radiation Tolerance; Radiation-Sensitizing Agents; Signal Transduction; Sirolimus; Spheroids, Cellular; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line (<2-fold variation). The activity of p70(S6k), activated downstream of mTOR, was similar in four cell lines (range, 11.75-41. 8 pmol/2 x 10(6) cells/30 min), but activity was equally inhibited in cells that were highly resistant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intrinsically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 and Rh1 cells, the addition of serum rapidly induced c-MYC (protein) levels. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells, selected for high level acquired resistance to rapamycin, showed >/=10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Carrier Proteins; Cell Cycle Proteins; Child; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Female; Glioblastoma; Humans; Insulin-Like Growth Factor I; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Proto-Oncogene Proteins c-myc; Rhabdomyosarcoma; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured