buparlisib and dactolisib

The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signalling axis and androgen receptor (AR) pathways exhibit reciprocal feedback regulation in phosphatase and tensin homologue (PTEN)-deficient metastatic castration-resistant prostate cancer (CRPC) in preclinical models. This phase Ib study evaluated the pan-PI3K inhibitor buparlisib (BKM120) and the dual pan-PI3K/ mammalian target of rapamycin (mTOR) inhibitor dactolisib (BEZ235) in combination with abiraterone acetate (AA) in patients with CRPC.. Patients with CRPC who had progressed on AA therapy received escalating doses of either buparlisib or dactolisib, along with fixed doses of AA (1000 mg once daily (qd)) and prednisone (5 mg twice daily (bid)). The primary objective was to define the maximum tolerated dose (MTD) and/or the recommended dose for expansion (RDE) of either buparlisib or dactolisib in combination with AA. Secondary objectives included safety, antitumour activity (Prostate Cancer Working Group 2 (PCWG2) criteria; 30% of prostate-specific antigen (PSA) decline at ≥week 12) and pharmacokinetic (PK) profile.. Based on the assessment of available pharmacokinetics, safety, and efficacy data, no further study is planned for either buparlisib or dactolisib in combination with AA in CRPC.

Topics: Abiraterone Acetate; Aged; Aged, 80 and over; Aminopyridines; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Asthenia; Chills; Diarrhea; Fever; Humans; Hyperglycemia; Imidazoles; Kallikreins; Male; Maximum Tolerated Dose; Middle Aged; Morpholines; Prostate-Specific Antigen; Prostatic Neoplasms, Castration-Resistant; Quinolines; Stomatitis; Vomiting

Topics: Aminopyridines; Animals; Apoptosis; Cell Line; Cytokines; Hippocampus; Imidazoles; Immunosuppressive Agents; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Morpholines; Neuroinflammatory Diseases; Oxidative Stress; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Fibroblast growth factor receptor (FGFR)3 and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase, catalytic subunit alpha (PIK3CA) mutations are found in various types of cancer, such as urinary bladder cancer, human papillomavirus‑positive tonsillar and base of the tongue squamous cell carcinoma, breast cancer and some childhood sarcomas. Several drugs can target these genes, some of which have been used for the treatment of urinary bladder cancer. Much less is known about childhood cancer. For this reason, the present study investigated the presence of such mutations in neuroblastomas (NBs) and tested NB cell lines for sensitivity to FGFR and phosphoinositide 3‑kinase (PI3K) inhibitors. In total, 29 NBs were examined for the presence of the three most common FGFR3 and PIK3CA mutations using a competitive allele‑specific TaqMan PCR (CAST‑PCR). Furthermore, the SK‑N‑AS, SK‑N‑BE(2)‑C, SK‑N‑DZ, SK‑N‑FI and SK‑N‑SH NB cell lines (where SK‑N‑DZ had a deletion of PIK3C2G, none had FGFR mutations according to the Cancer Program's Dependency Map, but some were chemoresistant), were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BKM120) inhibitors by viability, cytotoxicity, apoptosis and proliferation assays. CAST‑PCR detected one FGFR3 mutation in 1/29 NBs. Following treatment with FGFR and PI3K inhibitors, a decrease in viability and proliferation was observed in the majority, but not all, the cell lines. Following combination treatment with both drugs, the sensitivity of all cell lines was increased. On the whole, the findings of this study demonstrate that FGFR3 and PIK3CA mutations are uncommon in patients with NB. However, certain NB cell lines are rather sensitive to both FGFR and PI3K inhibitors alone, and even more so when the different drugs are used in combination.

Topics: Aminopyridines; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Cell Line, Tumor; Cell Proliferation; Cell Survival; Child; Child, Preschool; Class I Phosphatidylinositol 3-Kinases; DNA Mutational Analysis; Drug Resistance, Neoplasm; Female; Humans; Imidazoles; Infant; Infant, Newborn; Male; Morpholines; Mutation; Neuroblastoma; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Pyrazoles; Quinolines; Receptor, Fibroblast Growth Factor, Type 3

Vascular-targeted photodynamic therapy (PDT) selectively disrupts vascular function by inducing oxidative damages to the vasculature, particularly endothelial cells. Although effective tumor eradication and excellent safety profile are well demonstrated in both preclinical and clinical studies, incomplete vascular shutdown and angiogenesis are known to cause tumor recurrence after vascular-targeted PDT. We have explored therapeutic enhancement of vascular-targeted PDT with PI3K signaling pathway inhibitors because the activation of PI3K pathway was involved in promoting endothelial cell survival and proliferation after PDT. Here, three clinically relevant small-molecule inhibitors (BYL719, BKM120, and BEZ235) of the PI3K pathway were evaluated in combination with verteporfin-PDT. Although all three inhibitors were able to synergistically enhance PDT response in endothelial cells, PDT combined with dual PI3K/mTOR inhibitor BEZ235 exhibited the strongest synergism, followed in order by combinations with pan-PI3K inhibitor BKM120 and p110α isoform-selective inhibitor BYL719. Combination treatments of PDT and BEZ235 exhibited a cooperative inhibition of antiapoptotic Bcl-2 family protein Mcl-1 and induced more cell apoptosis than each treatment alone. In addition to increasing treatment lethality, BEZ235 combined with PDT effectively inhibited PI3K pathway activation and consequent endothelial cell proliferation after PDT alone, leading to a sustained growth inhibition. In the PC-3 prostate tumor model, combination treatments improved treatment outcomes by turning a temporary tumor regrowth delay induced by PDT alone to a more long-lasting treatment response. Our study strongly supports the combination of vascular-targeted PDT and PI3K pathway inhibitors, particularly mTOR inhibitors, for therapeutic enhancement.

Topics: Aminopyridines; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Endothelial Cells; Humans; Imidazoles; Mice; Morpholines; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Recurrence, Local; Neoplasms; Neovascularization, Pathologic; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Photochemotherapy; Porphyrins; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Thiazoles; Verteporfin; Xenograft Model Antitumor Assays

The PI3K/AKT/mTOR pathway plays a crucial role in the development of leiomyosarcomas (LMSs). In this study, we tested the efficacy of dual PI3K/mTOR (BEZ235), PI3K (BKM120) and mTOR (everolimus) inhibitors in three human LMS cell lines. In vitro and in vivo studies using LMS cell lines showed that BEZ235 has a significantly higher anti-tumor effect than either BKM120 or everolimus, resulting in a greater reduction in tumor growth and more pronounced inhibitory effects on mitotic activity and PI3K/AKT/mTOR signaling. Strikingly, BEZ235 but neither BKM120 nor everolimus markedly enhanced the ERK pathway. This effect was reproduced by the combination of BKM120 and everolimus, suggesting the involvement of mTORC2 via a PI3K-independent mechanism. Silencing of RICTOR in LMS cells confirmed the role of mTORC2 in the regulation of ERK activity. Combined treatment with BEZ235 and GSK1120212, a potent MEK inhibitor, resulted in synergistic growth inhibition and apoptosis induction in vitro and in vivo. These findings document for the first time that dual PI3K/mTOR inhibition in leiomyosarcomas suppress a negative feedback loop mediated by mTORC2, leading to enhanced ERK pathway activity. Thus, combining a dual PI3K/mTOR inhibitor with MEK inhibitors may be a relevant approach to increase anti-tumor activity and prevent drug resistance in patients with LMS.

Topics: Aminopyridines; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Everolimus; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Leiomyosarcoma; MAP Kinase Kinase Kinases; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Quinolines; Rapamycin-Insensitive Companion of mTOR Protein; RNA Interference; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Leiomyosarcoma (LMS) is a common type of soft tissue sarcoma that responds poorly to standard chemotherapy. Thus the goal of this study was to identify novel selective therapies that may be effective in leiomyosarcoma by screening cell lines with a small molecule library comprised of 480 kinase inhibitors to functionally determine which signalling pathways may be critical for LMS growth.. LMS cell lines were screened with the OICR kinase library and a cell viability assay was used to identify potentially effective compounds. The top 10 % of hits underwent secondary validation to determine their EC50 and immunoblots were performed to confirm selective drug action. The efficacy of combination drug therapy with doxorubicin (Dox) in vitro was analyzed using the Calcusyn program after treatment with one of three dosing schedules: concurrent treatment, initial treatment with a selective compound followed by Dox, or initial treatment with Dox followed by the selective compound. Single and combination drug therapy were then validated in vivo using LMS xenografts.. Compounds that targeted PI3K/AKT/mTOR pathways (52 %) were most effective. EC50s were determined to validate these initial hits, and of the 11 confirmed hits, 10 targeted PI3K and/or mTOR pathways with EC50 values <1 μM. We therefore examined if BEZ235 and BKM120, two selective compounds in these pathways, would inhibit leiomyosarcoma growth in vitro. Immunoblots confirmed on-target effects of these compounds in the PI3K and/or mTOR pathways. We next investigated if there was synergy with these agents and first line chemotherapy doxorubicin (Dox), which would allow for earlier introduction into patient care. Only combined treatment of BEZ235 and Dox was synergistic in vitro. To validate these findings in pre-clinical models, leiomyosarcoma xenografts were treated with single agent and combination therapy. BEZ235 treated xenografts (n = 8) demonstrated a decrease in tumor volume of 42 % whereas combining BEZ235 with Dox (n = 8) decreased tumor volume 68 % compared to vehicle alone.. In summary, this study supports further investigation into the use of PI3K and mTOR inhibitors alone and in combination with standard treatment in leiomyosarcoma patients.

Topics: Aminopyridines; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Evaluation, Preclinical; Drug Synergism; Female; Humans; Imidazoles; Leiomyosarcoma; Mice, Inbred NOD; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Reproducibility of Results; TOR Serine-Threonine Kinases

Aggressive pituitary tumors are rare but difficult to manage, as there is no effective chemotherapy to restrict their growth and cause their shrinkage. Within these tumors, growth-promoting cascades, like the PI3K/mTOR pathway, appear to be activated. We tested the efficacy of two inhibitors of this pathway, NVP-BKM120 (Buparlisib; pan-PI3K) and NVP-BEZ235 (dual PI3K/mTOR), both in vitro on immortalized pituitary tumor cells (GH3) and on primary cell cultures of human pituitary tumors and in vivo on a rat model of prolactin (PRL) tumors (SMtTW3). In vitro, NVP-BEZ235 had a potent apoptotic and cytostatic effect that was characterized by decreased cyclin D/E and Cdk4/2 protein levels and subsequent accumulation of cells in G1 In vivo, the effect was transient, with a decrease in mitotic index and increase in apoptosis; long-term treatment had no significant inhibitory effect on tumor growth. In contrast, while NVP-BKM120 had little effect in vitro, it dramatically limited tumor growth in vivo Increased Akt phosphorylation observed only in the NVP-BEZ235-treated tumors may explain the differential response to the two inhibitors. Primary cell cultures of human PRL pituitary tumors responded to NVP-BEZ235 with reduced cell viability and decreased hormone secretion, whereas NVP-BKM120 had little effect. Altogether, these results show a potential for PI3K inhibitors in the management of aggressive pituitary tumors. Mol Cancer Ther; 15(6); 1261-70. ©2016 AACR.

Topics: Aminopyridines; Animals; Cell Cycle Proteins; Cell Proliferation; Cell Survival; Humans; Imidazoles; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Pituitary Neoplasms; Prolactin; Proto-Oncogene Proteins c-akt; Quinolines; Rats; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The PI3K/mTOR pathway is commonly deregulated in cancer. mTOR inhibitors are registered for the treatment of several solid tumors and novel inhibitors are explored clinically. Notably, this pathway also plays an important role in immunoregulation. While mTOR inhibitors block cell cycle progression of conventional T cells (Tconv), they also result in the expansion of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Tregs), and this likely limits their clinical antitumor efficacy. Here, we compared the effects of dual mTOR/PI3K inhibition (using BEZ235) to single PI3K (using BKM120) or mTOR inhibition (using rapamycin and everolimus) on Treg expansion and functionality. Whereas rapamycin, everolimus and BEZ235 effected a relative expansion benefit for Tregs and increased their overall suppressive activity, BKM120 allowed for similar expansion rates of Tregs and Tconv without altering their overall suppressive activity. Therefore, PI3K inhibition alone might offer antitumor efficacy without the detrimental selective expansion of Tregs associated with mTOR inhibition.

Topics: Aminopyridines; Antineoplastic Agents; Blotting, Western; Cell Proliferation; Cells, Cultured; Cytokines; Everolimus; Flow Cytometry; Humans; Imidazoles; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

To explore the molecular mechanisms of resistance to phosphatidyl inositol 3-kinase (PI3K) inhibitors in triple-negative breast cancer (TNBC) cells.. HCC70 cells (TNBC) were transfected with siFZD7, siWANT5B or siGSK3 using lipofectamine 2000 transfection reagent. The expression levels of key proteins of WNT/β-catenin and PI3K/AKT/mTOR pathways were determined by Western blot analysis. After HCC70, MCF-7 (ER-positive) and SK-BR3 (HER2-positive) cells were treated with PI3K/AKT/mTOR inhibitors, the inhibition rates of cell proliferation were measured by MTT assay, and half maximal inhibitory concentrations (IC50) were calculated. The altered activities of WNT/β-catenin and PI3K/AKT/mTOR proteins were detected by Western blot and luciferase report gene assay, respectively. The nuclear translocation of β-catenin protein was examined by immunofluorescence assay. Xenograft nude mouse model was used to evaluate the tumorigenicity of breast cancer cells treated with BKM120 in vivo. The expression levels of p-LRP6, p-4EBP1 and β-catenin proteins in the tumor tissues were determined by immunohistochemical staining.. The expression levels of FZD7, WANT5B and GSK3 proteins were significantly reduced in the HCC70 cells transfected with the target siRNAs. Meanwhile, the activity of WNT/β-catenin was enhanced and PI3K/AKT/mTOR pathway was inhibited. PI3K/AKT/mTOR inhibitors suppressed MCF-7 and SK-BR3 cell proliferation. The IC50 of GDC-094, BKM120, XL147, perifosine, everolimus, and BEZ235 in MCF-7 cells were 0.46 mmol/L, 1.44 mmol/L, 4.34 mmol/L, 11.35 μmol/L, 53.71 μmol/L and 12.87 μmol/L respectively, and 0.63 mmol/L, 0.58 mmol/L, 3.74 mmol/L, 13.22 μmol/L, 60.00 μmol/L and 11.38 μmol/L in the SK-BR3 cells, respectively. The results of luciferase report gene assay showed that the luciferase activities in HCC70, MCF-7 and SK-BR3 cells treated with BKM120 were 1.75±0.05, 1.13±0.02 and 0.43±0.01, respectively. The luciferase activities in HCC70 and SK-BR3 cells were significantly different from that of the control cells (1.00±0.02, P<0.05). The immunohistochemical analysis showed that BKM120 inhibited mTOR activity, and the enhanced WNT/β-catenin activity reversed the phenotype of inhibitory mTOR induced by BKM120. BKM120 suppressed the tumorigenic ability of MCF-7 and SK-BR3 cells in vivo, but had no effect on cultured HCC70 cells. The immunohistochemical analysis showed nuclear translocation of β-catenin protein and increased expression level of p-LRP-6 protein in transplanted tumor tissues from HCC70 cells treated with BKM120, increased the level of p-LRP-6 protein, and no changes of p-4EBP1 protein expression. However, no nuclear translocation of β-catenin protein and no decrease of p-LRP6 and p-4EBP1 protein levels in the transplanted tumor tissue of MCF-7 cells after treatment with BKM120.. The triple-negative breast cancer HCC70 cells have drugs-resistance to PI3K inhibitors. The WNT/β-catenin signaling pathway may regulate the PI3K/AKT/mTOR pathway, therefore, inducing the drug-resistance of TNBC cells to PI3K inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Aminopyridines; Animals; beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Imidazoles; Mice; Morpholines; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Phosphoproteins; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms

Graft-versus-host disease (GvHD) remains the major obstacle to successful allogeneic hematopoietic stem cell transplantation, despite of the immunosuppressive regimens administered to control T cell alloreactivity. PI3K/AKT/mTOR pathway is crucial in T cell activation and function and, therefore, represents an attractive therapeutic target to prevent GvHD development. Recently, numerous PI3K inhibitors have been developed for cancer therapy. However, few studies have explored their immunosuppressive effect.. The effects of a selective PI3K inhibitor (BKM120) and a dual PI3K/mTOR inhibitor (BEZ235) on human T cell proliferation, expression of activation-related molecules, and phosphorylation of PI3K/AKT/mTOR pathway proteins were analyzed. Besides, the ability of BEZ235 to prevent GvHD development in mice was evaluated.. Simultaneous inhibition of PI3K and mTOR was efficient at lower concentrations than PI3K specific targeting. Importantly, BEZ235 prevented naïve T cell activation and induced tolerance of alloreactive T cells, while maintaining an adequate response against cytomegalovirus, more efficiently than BKM120. Finally, BEZ235 treatment significantly improved the survival and decreased the GvHD development in mice.. These results support the use of PI3K inhibitors to control T cell responses and show the potential utility of the dual PI3K/mTOR inhibitor BEZ235 in GvHD prophylaxis.

Topics: Aminopyridines; Animals; Cell Proliferation; Graft vs Host Disease; Imidazoles; Lymphocyte Activation; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; T-Lymphocytes; TOR Serine-Threonine Kinases

A prominent role in the pathogenesis of squamous cell carcinoma of the lung (SQCLC) has been attributed to the aberrant activation of the PI3K signaling pathway, due to amplification or mutations of the p110α subunit of class I phosphatidylinositol 3-kinase (PIK3CA) gene. The aim of our study was to determine whether different genetic alterations of PIK3CA affect the biologic properties of SQCLC and to evaluate the response to specific targeting agents in vitro and in vivo. The effects of NVP-BEZ235, NVP-BKM120, and NVP-BYL719 on two-dimensional/three-dimensional (2D/3D) cellular growth, epithelial-to-mesenchymal transition, and invasiveness were evaluated in E545K or H1047R PIK3CA-mutated SQCLC cells and in newly generated clones carrying PIK3CA alterations, as well as in a xenograft model. PIK3CA mutated/amplified cells showed increased growth rate and enhanced migration and invasiveness, associated with an increased activity of RhoA family proteins and the acquisition of a mesenchymal phenotype. PI3K inhibitors reverted this aggressive phenotype by reducing metalloproteinase production, RhoA activity, and the expression of mesenchymal markers, with the specific PI3K inhibitors NVP-BKM120 and NVP-BYL719 being more effective than the dual PI3K/mTOR inhibitor NVP-BEZ235. A xenograft model of SQCLC confirmed that PIK3CA mutation promotes the acquisition of a mesenchymal phenotype in vivo and proved the efficacy of its specific targeting drug NVP-BYL719 in reducing the growth and the expression of mesenchymal markers in xenotransplanted tumors. These data indicate that PIK3CA mutation/amplification may represent a good predictive feature for the clinical application of specific PI3K inhibitors in SQCLC patients.

Topics: Aminopyridines; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Disease Models, Animal; Epithelial-Mesenchymal Transition; Humans; Imidazoles; Lung Neoplasms; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Thiazoles; Xenograft Model Antitumor Assays

Treatment for recurrent and aggressive meningiomas remains an unmet medical need in neuro-oncology, and chemotherapy exhibits limited clinical activity, if any. Merlin expression, encoded by the NF2 gene, is lost in a majority of meningiomas, and merlin is a negative regulator of mTORC1. The sst2 somatostatin receptor, targeted by octreotide, is highly expressed in meningiomas. To investigate new therapeutic strategies, we evaluated the activity of everolimus (mTOR inhibitor), BKM-120 and BEZ-235 (new Pi3K/Akt/mTOR inhibitors), octreotide and a combined treatment (octreotide plus everolimus), on cell proliferation, signaling pathways, and cell cycle proteins, respectively. The in vitro study was conducted on human meningioma primary cells extracted from fresh tumors, allowing the assessment of somatostatin analogs at the concentration levels used in patients. The results were correlated to WHO grades. Further, everolimus decreased cell viability of human meningiomas, but concomitantly, induced Akt activation, reducing the antiproliferative effect of the drug. The new Pi3K inhibitors were not more active than everolimus alone, limiting their clinical relevance. In contrast, a clear cooperative inhibitory effect of octreotide and everolimus was observed on cell proliferation in all tested meningiomas, including WHO grades II-III. Octreotide not only reversed everolimus-induced Akt phosphorylation but also displayed additive and complementary effects with everolimus on downstream proteins involved in translation (4EB-P1), and controlling cell cycle (p27Kip1 and cyclin D1). We have demonstrated a co-operative action between everolimus and octreotide on cell proliferation in human meningiomas, including aggressive ones, establishing the basis for a clinical trial.

Topics: Adult; Aged; Aged, 80 and over; Aminopyridines; Antineoplastic Agents; Cell Cycle; Cell Survival; Drug Therapy, Combination; Everolimus; Female; Humans; Imidazoles; Male; Meningeal Neoplasms; Meningioma; Middle Aged; Morpholines; Neurofibromin 2; Octreotide; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Receptors, Somatostatin; Signal Transduction; TOR Serine-Threonine Kinases

Non-Hodgkin lymphomas encompass a heterogeneous group of cancers, with 85-90% arising from B lymphocytes and the remainder deriving from T lymphocytes or NK lymphocytes. These tumors are molecularly and clinically heterogeneous, showing dramatically different responses and outcomes with standard therapies. Deregulated PI3K signaling is linked to oncogenesis and disease progression in hematologic malignancies and in a variety of solid tumors and apparently enhances resistance to antineoplastic therapy, resulting in a poor prognosis. Here, we have evaluated and compared the effects of the pan-PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitor BEZ235 on mantle, follicular, and T-cell lymphomas. Our results suggest that BKM120 and BEZ235 can effectively inhibit lymphoma cell proliferation by causing cell cycle arrest and can lead to cell death by inducing apoptosis and autophagy mediated by ROS accumulation. Despite great advances in lymphoma therapy after the introduction of monoclonal antibodies, many patients still die from disease progression. Therefore, novel treatment approaches are needed. BKM120 and BEZ235 alone and in combination are very effective against lymphoma cells in vitro. If further studies confirm their effectiveness in animal models, they may be promising candidates for development as new drugs.

Topics: Aminopyridines; Antineoplastic Agents; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Imidazoles; Lymphoma, Non-Hodgkin; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Prognosis; Quinolines; Reactive Oxygen Species; Signal Transduction

The precise involvement of the PI3K/mTOR and RAS/MEK pathways in carcinoid tumors is not well defined. Therefore, the purpose of our study was to evaluate the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion and to determine the effects of combined treatment on carcinoid tumor inhibition.. The human neuroendocrine cell lines BON (pancreatic carcinoid), NCI-H727 (lung carcinoid), and QGP-1 (somatostatinoma) were treated with either the pan-PI3K inhibitor, BKM120, or the dual PI3K-mTOR inhibitor, BEZ235, alone or in combination with the MEK inhibitor, PD0325901; proliferation, apoptosis, and protein expression were assessed. Peptide secretion was evaluated in BON and QGP-1 cells. The antiproliferative effect of BEZ235, alone or combined with PD0325901, was then tested in vivo.. Both BKM120 and BEZ235 decreased proliferation and increased apoptosis; combination with PD0325901 significantly enhanced the antineoplastic effects of either treatment alone. In contrast, neurotensin peptide secretion was markedly stimulated with BKM120 treatment, but not BEZ235. The combination of BEZ235 + PD0325901 significantly inhibited the growth of BON xenografts without systemic toxicity.. Both BKM120 and BEZ235 effectively inhibited neuroendocrine tumor (NET) cell proliferation and stimulated apoptosis. However, inhibition of the PI3K pathway alone with BKM120 significantly stimulated neurotensin peptide secretion; this did not occur with the dual inhibition of both PI3K and mTOR using BEZ235 suggesting that this would be a more effective treatment regimen for NETs. Moreover, the combination of BEZ235 and the MEK inhibitor PD0325901 was a safe and more effective therapy in vivo compared with single agents alone.

Topics: Aminopyridines; Animals; Apoptosis; Benzamides; Cell Line, Tumor; Cell Proliferation; Diphenylamine; Drug Synergism; Humans; Imidazoles; Male; Mice; Mitogen-Activated Protein Kinases; Morpholines; Neuroendocrine Tumors; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Quinolines; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Inhibitors of the phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathway are currently in clinical trials. In addition to antiproliferative and proapoptotic effects, these agents also diminish tumor hypoxia. Since hypoxia is a major cause of resistance to radiotherapy, we sought to understand how it is regulated by PI3K/mTOR inhibition.. Whole cell, mitochondrial, coupled and uncoupled oxygen consumption were measured in cancer cells after inhibition of PI3K (Class I) and mTOR by pharmacological means or by RNAi. Mitochondrial composition was assessed by immunoblotting. Hypoxia was measured in spheroids, in tumor xenografts and predicted with mathematical modeling.. Inhibition of PI3K and mTOR reduced oxygen consumption by cancer cell lines is predominantly due to reduction of mitochondrial respiration coupled to ATP production. Hypoxia in tumor spheroids was reduced, but returned after removal of the drug. Murine tumors had increased oxygenation even in the absence of average perfusion changes or tumor necrosis.. Targeting the PI3K/mTOR pathway substantially reduces mitochondrial oxygen consumption thereby reducing tumor hypoxia. These alterations in tumor hypoxia should be considered in the design of clinical trials using PI3K/mTOR inhibitors, particularly in conjunction with radiotherapy.

Topics: Aminopyridines; Animals; Cell Hypoxia; Cell Line, Tumor; HCT116 Cells; Humans; Imidazoles; Mice; Morpholines; Neoplasms; Oxygen Consumption; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Spheroids, Cellular; TOR Serine-Threonine Kinases

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in cancer, novel PI3K inhibitors such as ZSTK474, GDC-0941, NVP-BEZ235 and BKM-120 have been developed for cancer therapy. A high frequency of hotspot mutations known as E542K, E545K and H1047R in the PIK3CA gene, which encodes the catalytic subunit of PI3Kα, has been found in various types of human cancers. The hotspot PIK3CA mutations also lead to resistance to therapeutics targeting epidermal growth factor receptor (EGFR), further suggesting that inhibition of hotspot mutant PIK3CA be required for a PI3K inhibitor as anticancer drug candidate.. To investigate the activity of the novel PI3K inhibitors on the hotspot mutant PIK3CA, we determined the inhibition against the respective recombinant mutant PI3Kαs by biochemical assay. We further examined the activity at cellular background by determining the effect on phosphorylation of Akt (Ser473), and that on the growth of cancer cells. In addition, apoptosis and autophagy in cells with or without hotspot PIK3CA mutation induced by the four inhibitors were investigated.. Our results indicated that each inhibitor exhibit comparable activity on the hotspot mutant PI3Kα to that on the wild type, which was further demonstrated by the cell-based assays. No clear correlation was shown between the PIK3CA genetic status and the sensitivity for apoptosis or autophagy induction. Interestingly, among the 4 PI3K inhibitors, BKM-120 is the weakest in PI3K inhibitory potency, but induces most potent apoptosis, suggesting that BKM-120 might have a unique mode of action.. Our result shows that the PI3K inhibitors exhibit potent activity on both hotspot mutant and wild type PI3Kα, suggesting they might be used to treat patients with or without PIK3CA mutation when approved.

Topics: Aminopyridines; Antineoplastic Agents; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Humans; Imidazoles; Indazoles; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinolines; Sulfonamides; Triazines

Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.

Topics: Aminopyridines; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL12; Everolimus; Human Umbilical Vein Endothelial Cells; Humans; Imidazoles; Interleukin-4; Interleukin-6; Lymphoma, Mantle-Cell; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome; Tumor Microenvironment

To evaluate first-generation rapamycin analogs (everolimus, temsirolimus, and rapamycin) and second-generation drugs inhibiting mTOR kinase (AZD-8055), PI3K (BKM-120) or both (BEZ-235 and GDC-0980) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cells characterized for acquired resistance to sorafenib or sunitinib.. Anti-proliferative (MTT assay) and cell signaling (Western blot) effects of rapamycin analogs (1-20 μM) and second-generation drugs (0.03-20.0 μM) were assessed in human HCC SK-HEP1, RCC 786-0, and sorafenib- (SK-Sora) or sunitinib-resistant (786-Suni) cells.. In SK-HEP1 cells displaying high PTEN and Bcl2 expression, rapamycin analogs had poor anti-proliferative effects. However, SK-Sora cells were more sensitive to rapamycin analogs (≥1 μM) than SK-HEP1 cells. In 786-0 cells, lacking PTEN and Bcl2 expression, ≥1 μM rapamycin analogs blocked mTORC1 signaling, transiently activated Akt, and inhibited cell proliferation. Protracted sunitinib exposure in 786-Suni cells yielded an increase in p27 expression and a decreased sensitivity to rapamycin analogs, although mTORC1 function could be inhibited with rapamycin analogs. Second-generation drugs induced more potent growth inhibition than rapamycin analogs at concentrations >0.03 μM in parental cells, SK-Sora, and 786-Suni cells. Growth inhibitory concentrations of these new drugs also blocked mTORC1 downstream targets.. Rapamycin analogs inhibited mTORC1 downstream targets and yielded anti-proliferative effects in HCC and RCC cells. Second-generation drugs also appeared to be potent inhibitors of mTORC1 signaling; however, they appeared to be far more potent in inhibiting cellular proliferation in parental HCC and RCC cells and in cells developing resistance to sorafenib or sunitinib.

Topics: Aminopyridines; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Everolimus; Humans; Imidazoles; Indoles; Kidney Neoplasms; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Niacinamide; Phenylurea Compounds; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Pyrroles; Quinolines; Signal Transduction; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide with only few therapeutic options for patients with advanced disease. There is growing evidence indicating that activation of the PI3K/Akt/mTOR pathway plays an important role in HCC and therefore represents a promising target for novel therapeutic approaches. The aim of this study was to evaluate and compare the antitumour activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in vitro and in vivo.. The antitumour effects of RAD001, BEZ235 and BKM120 were analysed in seven hepatoma cell lines as mono and combination therapy with Doxorubicin, Cisplatin, Irinotecan or 5-Flourouracil in vitro and in xenografts. Cell-cycle progression, apoptosis, and autophagy were analysed. Furthermore, effects on mitochondrial respiration and glycolysis were assessed.. Treatment with RAD001, BEZ235 and BKM120 markedly reduced tumour cell viability. Combination of PI3K inhibitors with chemotherapy was most effective. RAD001, BEZ235 and BKM120 reduced tumour growth mainly by inhibiting cell-cycle progression rather than by inducing apoptosis. Interestingly, the antitumour effects were strongly associated with a reduction of mitochondrial respiration. BKM120, which exhibited the strongest antiproliferative effect, most strongly impaired oxidative phosphorylation compared with the other drugs.. In this study, BKM120 showed the strongest antitumour activity. Our findings suggest impairment of mitochondrial function as a relevant mechanism of BKM120. Moreover, combination of PI3K and mTOR inhibitors with cytotoxic agents could be promising option for non-cirrhotic HCC patients.

Topics: Aminopyridines; Analysis of Variance; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Camptothecin; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Cell Respiration; Cisplatin; Doxorubicin; Everolimus; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Irinotecan; Liver Neoplasms; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

The PI3K signaling pathway regulates diverse cellular processes, including proliferation, survival, and metabolism, and is aberrantly activated in human cancer. As such, numerous compounds targeting the PI3K pathway are currently being clinically evaluated for the treatment of cancer, and several have shown some early indications of efficacy in breast cancer. However, resistance against these agents, both de novo and acquired, may ultimately limit the efficacy of these compounds. Here, we have taken a systematic functional approach to uncovering potential mechanisms of resistance to PI3K inhibitors and have identified several genes whose expression promotes survival under conditions of PI3K/mammalian target of rapamycin (PI3K/mTOR) blockade, including the ribosomal S6 kinases RPS6KA2 (RSK3) and RPS6KA6 (RSK4). We demonstrate that overexpression of RSK3 or RSK4 supports proliferation upon PI3K inhibition both in vitro and in vivo, in part through the attenuation of the apoptotic response and upregulation of protein translation. Notably, the addition of MEK- or RSK-specific inhibitors can overcome these resistance phenotypes, both in breast cancer cell lines and patient-derived xenograft models with elevated levels of RSK activity. These observations provide a strong rationale for the combined use of RSK and PI3K pathway inhibitors to elicit favorable responses in breast cancer patients with activated RSK.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Expression; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; MCF-7 Cells; Mice; Mice, Nude; Molecular Targeted Therapy; Morpholines; Open Reading Frames; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Transcriptome; Tumor Burden; Xenograft Model Antitumor Assays

Everolimus, an mTOR inhibitor, showed great clinical efficacy in combination with tamoxifen, letrozole, or exemestane for the treatment of estrogen receptor-positive (ER+) breast cancer. However, its antitumor activity was shown to be compromised by a compensatory process involving AKT activation. Here, it was determined whether combining an additional PI3K inhibitor can reverse this phenomenon and improve treatment efficacy. In breast cancer cells (MCF-7 and BT474), everolimus inhibited the mTOR downstream activity by limiting phosphorylation of p70S6K and 4EBP1, which resulted in p-Ser473-AKT activation. However, addition of a LY294002, a PI3K inhibitor, to tamoxifen and everolimus treatment improved the antitumor effect compared with tamoxifen alone or the other two agents in combination. Moreover, LY294002 suppressed the activity of the PI3K/AKT/mTOR axis and mitigated the p-Ser473-AKT activation feedback loop in both cell lines. Critically, this combination scheme also significantly inhibited the expression of HIF-1a, an angiogenesis marker, under hypoxic conditions and reduced blood vessel sprout formation in vitro. Finally, it was shown that the three-agent cocktail had the greatest efficacy in inhibiting MCF-7 xenograft tumor growth and angiogenesis. Taken together, these results suggest that inhibition of PI3K and mTOR may further improve therapy in ER(+) breast cancer cells.. Combinatorial inhibition of the PI3K/AKT/mTOR signaling axis may enhance endocrine-based therapy in breast cancer.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Everolimus; Female; Humans; Imidazoles; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.. We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.. Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.. Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.

Topics: Aminopyridines; Antineoplastic Agents; Drug Synergism; Everolimus; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lymphocytes; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Morpholines; Multiprotein Complexes; Naphthyridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Prostate cancer is an ideal target for chemoprevention. To date, chemoprevention clinical trials with 5α-reductase inhibitors have yielded encouraging yet ultimately confounding results. Using a preclinical mouse model of high-grade prostatic intraepithelial neoplasia (HG-PIN) induced by PTEN loss, we observed unprecedented deteriorating effects of androgen deprivation, in which surgical castration or MDV3100 treatment accelerated disease progression of the otherwise stable HG-PIN to invasive castration-resistant prostate cancer (CRPC). As an alternative, targeting the phosphoinositide 3-kinase (PI3K) signaling pathway via either genetic ablation of genes encoding PI3K components or pharmacologic inhibition of the PI3K pathway reversed the PTEN loss-induced HG-PIN phenotype. Finally, concurrent inhibition of the PI3K and mitogen-activated protein kinase (MAPK) pathways was effective in blocking the growth of PTEN-null CRPC. Together, these data have revealed the potential adverse effects of antiandrogen chemoprevention in certain genetic contexts (such as PTEN loss) while showing the promise of targeted therapy in the clinical management of this complex and prevalent disease.. Chemoprevention with antiandrogen therapies is attractive for prostate cancer, given its prevalence and established hormonally mediated pathogenesis. However, because PTEN loss has been found in 9% to 45% of HG-PIN in the clinic, the current findings suggest that patients with PTEN-deficient prostate tumors might be better treated with PI3K-targeted therapies.

Topics: Aminopyridines; Androgen Receptor Antagonists; Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Castration; Imidazoles; Male; MAP Kinase Kinase Kinases; Mice; Mice, Transgenic; Morpholines; Nitriles; Phenylthiohydantoin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Quinolines; Testosterone

The phosphoinositide 3-kinase (PI3K) pathway is known to play an active role in many malignancies. The role of PI3K inhibition in the treatment of lymphomas has not been fully delineated. We sought to identify a role for therapeutic PI3K inhibition across a range of B-cell lymphomas.. We selected three small molecule inhibitors to test in a panel of 60 cell lines that comprised diverse lymphoma types. We tested the selective PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitors BEZ235 and BGT226 in these cell lines. We applied gene expression profiling to better understand the molecular mechanisms associated with responsiveness to these drugs.. We found that higher expression of the PAK1 gene was significantly associated with resistance to all three PI3K inhibitors. Through RNA-interference-mediated knockdown of the PAK1 gene, we showed a dramatic increase in the sensitivity to PI3K inhibition. We further tested a small-molecule inhibitor of PAK1 and found significant synergy between PI3K and PAK1 inhibition.. Thus, we show that PI3K inhibition is broadly effective in lymphomas and PAK1 is a key modulator of resistance to PI3K inhibition.

Topics: Aminopyridines; Biomarkers, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Gene Expression Profiling; Humans; Imidazoles; Lymphoma; Morpholines; Oligonucleotide Array Sequence Analysis; p21-Activated Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Small Molecule Libraries; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

Topics: Aminopyridines; Animals; Cell Proliferation; Cell Transformation, Neoplastic; Cerebellar Neoplasms; Cerebellum; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Genes, p53; Imidazoles; Medulloblastoma; Mice; Morpholines; Neural Stem Cells; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Quinolines; TOR Serine-Threonine Kinases

New targeted agents like antibodies or small molecules against tyrosine and lipid kinases clearly expand the standard therapy options in oncology. However, tumour resistance is still a challenge, often induced by mutations in growth-related signalling cascades. Twenty and ten percentage of all patients with colorectal and gastric cancers, respectively, carry phosphatidyl-3-kinase (PI3K) mutations and do not respond to receptor-blocking therapies. Recently, selective kinase inhibitors have been generated, which block the PI3K signalling pathway in tumour cells. So far, their therapeutic role for the treatment of mutated versus wild-type human gastrointestinal cancers has not been clarified in detail.. To define the inhibitory and pro-apoptotic effects of the two PI3K inhibitors BEZ235 and BKM120 in three human colon cancer (HT-29, HCT-116 and DLD-1) and three gastric cancer (NCI-n87, AGS and MKN-45), cell lines with different PIK3CA gene mutation status were used. Firstly, viability, apoptosis and caspase assays were performed during incubation with either the inhibitors alone or combined with different cytotoxic agents. Secondly, the molecular consequences for the cell cycle and signalling pathways were analysed by defining the protein levels by FACS and Western blot analysis.. Both the PI3K inhibitors BEZ235 and BKM120 induced a clear concentration-dependent reduction in cell viability and an increase in apoptotic cell death, with the mutated cells being more sensitive to treatment. However, single-agent BEZ235 caused a G1 arrest in tumour cells, whilst BKM120 induced a G2 shift in a half of the gastrointestinal cancer cell lines. There was a clear downregulation in the protein levels of the PI3K-AKT pathway at the concentrations of 100 nM for both agents and for BEZ235 the additional inhibition of the mTOR pathway. Furthermore, BEZ235 caused synergistic induction of apoptosis when combined with irinotecan in colon cancer cell lines. Human gastric cancer cells were less sensitive to both BEZ235 and BKM120.. BEZ235 and BKM120 induced pro-apoptotic effects in all cell lines and especially with an increased response in the PI3KCA mutated cells. Our data support the clinical development of these PI3K inhibitors for patients with wild-type or mutated colon cancers.

Topics: Aminopyridines; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Caspase 3; Cell Cycle Checkpoints; Cell Survival; Dose-Response Relationship, Drug; Gastrointestinal Neoplasms; HCT116 Cells; HT29 Cells; Humans; Imidazoles; Irinotecan; Morpholines; Mutation; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

[18F] Fluorodeoxyglucose Positron Emission Tomography ([18F]FDG-PET) is widely used to monitor response to therapy in the clinic and has, more recently, been proposed as an early marker of long term response. This relies on the assumption that a change in glucose consumption parallels a reduction in viability and long term growth potential. However, cells may utilise substrates other than glucose and as many therapeutics interfere with glucose metabolism directly, it is entirely plausible that a positive [18F]FDG-PET response may be unrelated to long term growth. Furthermore, changes in metabolism and proliferation may take place on different temporal scales, thus restricting the time window in which [18F]FDG-PET is predictive. The PI3K oncogenic signalling pathway is a master regulator of multiple cellular processes including glucose metabolism, proliferation and cell survival. Inhibition of PI3K has been shown to reduce [18F]FDG uptake in several tumour types but the relative influence of this pathway on glucose metabolism and proliferation is not fully established.. We proposed to (i) assess the suitability of [18F]FDG as a tracer for measuring response to PI3K inhibition and (ii) determine the optimum imaging schedule, in vitro. We used multicellular tumour spheroids, an excellent 3D in vitro model of avascular tumours, to investigate the effects of the PI3K inhibitors, NVP-BKM120 and NVP-BEZ235, on [18F]FDG uptake and its relation to 3D growth.. Spheroids were prepared from two cell lines with a constitutively active PI3K/Akt pathway, EMT6 (highly proliferative mouse mammary) and FaDu (moderately proliferate human nasopharyngeal). Treatment consisted of a 24h exposure to either inhibitor, and growth was monitored over the following 7 days. To mimic potential imaging regimens with [18F]FDG-PET, average [18F]FDG uptake per viable cell was measured (a) directly following the 24h exposure, (b) following an additional 24h recovery period, or (c) following a 48 h exposure.. Growth was restricted significantly (p<0.0001) in a dose-dependent fashion in spheroids from both cell lines treated with either inhibitor. In the highly proliferative cell line EMT6, [18F]FDG uptake was significantly reduced at all concentrations of inhibitor. For the moderately proliferative cell line FaDu, [18F]FDG was affected in a dose-dependent fashion, but to lesser degree. To assess the predictivity of [18F]FDG uptake for long term growth restriction, Pearson correlation coefficients were calculated for each imaging regimen. These indicated that the optimal imaging schedules differed between cell lines.. This study suggests that [18F]FDG may be a suitable marker of response to PI3K inhibition in the cell lines that we have studied. Our data support the hypothesis that imaging schedules should be optimised on a tumour type-specific basis.

Topics: Aminopyridines; Animals; Biological Transport; Cell Line, Tumor; Cell Proliferation; Feasibility Studies; Fluorodeoxyglucose F18; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Positron-Emission Tomography; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Spheroids, Cellular; Time Factors; Treatment Outcome

The secretin receptor (SR), a G protein-coupled receptor, mediates the effects of the gastrointestinal hormone secretin on digestion and water homeostasis. Recently, high SR expression has been observed in pancreatic ductal adenocarcinomas, cholangiocellular carcinomas, gastrinomas, and bronchopulmonary carcinoid tumors. Receptor overexpression associates with enhanced secretin-mediated signaling, but whether this molecule plays an independent role in tumorigenesis is currently unknown. We recently discovered that pheochromocytomas developing in rats affected by the MENX (multiple endocrine neoplasia-like) syndrome express at very high-level Sctr, encoding SR. We here report that SR are also highly abundant on the membranes of rat adrenal and extraadrenal pheochromocytoma, starting from early stages of tumor development, and are functional. PC12 cells, the best characterized in vitro pheochromocytoma model, also express Sctr at high level. Thus, we used them as model to study the role of SR in neoplastic transformation. Small interfering RNA-mediated knockdown of Sctr decreases PC12 cells proliferation and increases p27 levels. The proproliferative effect of SR in PC12 cells is mediated, in part, by the phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (AKT) pathway. Transfection of Sctr in Y1 adrenocortical carcinoma cells, expressing low endogenous levels of Sctr, stimulates cell proliferation also, in part, via the PI3K/AKT signaling cascade. Because of the link between SR and PI3K/AKT signaling, tumor cells expressing high levels of the receptor (MENX-associated primary pheochromocytoma and NCI-H727 human bronchopulmonary carcinoid cells) respond well and in a SR-dependent manner to PI3K inhibitors, such as NVP-BEZ235. The association between SR levels and response to PI3K inhibition might open new avenues for the treatment of tumors overexpressing this receptor.

Topics: Adrenal Gland Neoplasms; Adrenal Glands; Adult; Aged; Aminopyridines; Animals; Apoptosis; Cell Proliferation; Cell Survival; Female; Gastrointestinal Agents; Gene Knockdown Techniques; Humans; Imidazoles; Male; Morpholines; Neurites; PC12 Cells; Pheochromocytoma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Rats; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; RNA Interference; Secretin; Signal Transduction; Transcription, Genetic