Effects of PI3K/Akt/mTOR and MAPK pathway inhibition on cell growth, proliferation, apoptosis and cell viability.

<p>RT112 and T24 cells were treated with RAD001 (5 nM), NVP-BEZ235 (100 nM for T24, 500 nM for RT112) and U0126 (25 nM) alone or the indicated combination and a DMSO control (ctr). <b>A:</b> For cell counts, cells were stained with trypan blue and numbers of living cells were determined. <b>B:</b> Cell cycle analysis after 24 h treatment with chemical compounds by BrdU incorporation in combination with 7-AAD staining. <b>C:</b> Measurement of caspase 3/7 activity as a parameter for apoptosis and <b>D:</b> XTT-test for detection of cell viability performed 24 h after treatment. Values shown are the mean ± standard deviation from 3 independent experiments. Student t-test was performed for statistic analysis (*: p<0,05).</p

Combined activities of S6K1 and 4E-BP1 regulate cell growth.

<p><b>A:</b> Two days after transfection with siRNAs against 4E-BP-1, protein expression in RT112 and T24 cells was detected in immunoblots. β-actin was used as a loading control. <b>B:</b> Growth of living cells, silenced for S6K1 or 4E-BP1 expression or cells silenced for 4E-BP1 expression and incubated with everolimus (RAD001) were monitored over time. Mean values ± standard deviations are shown; statistical comparisons were performed using the student t-test (*: p<0,05).</p

Activation status of the PI3K signaling pathway and cellular profiling of RAD001 and NVP-BEZ235.

<p><b>A:</b> For protein analysis, cells were lysed in RIPA buffer applied to SDS-PAGE and transferred to PVDF membrane. Phosphorylation and expression status of proteins were analyzed in immunoblots. <b>B, C:</b> Cell lines were treated for 1 h with the indicated concentrations of RAD001 and NVP-BEZ235. The control (0) contained same concentrations of DMSO as in samples with chemical compounds. One representative result from 3 independent experiments is shown. <b>D:</b> The inhibitory concentration of 50% (IC50) was determined for target proteins of PI3K and mTORC1. Chemiluminescence signals were quantified using the ChemiDoc imaging system (BioRad Laboratories) and normalized to protein expression level. The average values from three or more independent experiments are shown.</p

Crosstalk between the PI3K and MAPK signaling pathways.

<p><b>A:</b> RT112 and T24 cells were treated with RAD001 or NVP-BEZ235 for 1 h or 24 h. Expression and phosphorylation status of ERK1/2 was analyzed in immunoblots on whole cell lysates. <b>B:</b> Two days after transfection with siRNA oligonucleotides against S6K1 or control siRNA (ctr siRNA), cells were lysed and phosphorylation and expression status of Raf and ERK1/2 was analyzed in immunoblots. <b>C:</b> Cells were treated for 24 h with RAD001, NVP-BEZ235 and U0126 alone or in combination and effects on expression and phosphorylation level of Akt, S6K1 and ERK1/2 was analyzed in immunoblots.</p

Long term treatment of cells with RAD001 and NVP-BEZ235 - S6K1 mediated activation of Akt.

<p><b>A, B:</b> Effect of RAD001 or NVP-BEZ235 treatment over 24 hours on phosphorylation level of Akt, S6K1, 4E-BP1 and GSK3-β in RT112 and T24 cells. Whole cell lysates were applied to SDS-PAGE and blotted on PVDF membranes followed by immunoblots to detect expression and phosphorylation level of depicted proteins. The control (0) contained same concentration of DMSO as in samples with chemical compounds. <b>C:</b> RT112 cells were transfected with two independent S6K1 specific siRNA oligonucleotides and one random siRNA oligonucleotide as control (ctr siRNA). Three days after transfection, expression and phosphorylation level of S6K1, Akt and GSK3-β were analyzed in immunoblots.</p

Schematic representation of the PI3K/Akt/mTOR and MAPK signaling pathways in urothelial carcinoma.

<p><b>A:</b> In the current model, activation of PI3K results in activation of Akt via mTORC2 and PDK1, a process that can be reversed by PTEN. Subsequently, Akt regulates amongst other proteins TSC1/2 that together with RHEB control mTORC1 and its downstream targets S6K1 and 4E-BP1. Both molecules are involved in the regulation of protein translation and cell proliferation. Crosstalk between the PI3K and the MAPK pathway has been demonstrated from PI3K/IRS to RAS and from ERK1/2 to TSC1/2 and PI3K. <b>B:</b> According to the present results, in urothelial carcinoma mTORC1 regulates activity of S6K1/S6RP whereas 4E-BP1 activation status is regulated via PI3K. Both, 4E-BP1 and S6K1 are involved in regulating cell proliferation. Inactivation of PI3K and also S6K1 results in phosphorylation of RAS and ERK1/2. The inhibition of MEK1/2 induces Akt but neither S6K1 nor 4E-BP1 phosphorylation. Potential target molecules and stratification marker are shaped elliptical.</p

Parallel PI3K, AKT and mTOR inhibition is required to control feedback loops that limit tumor therapy

<div><p>Targeting the PI3K pathway has achieved limited success in cancer therapy. One reason for the disappointing activity of drugs that interfere with molecules that are important player in this pathway is the induction of multiple feedback loops that have been only partially understood. To understand these limitations and develop improved treatment strategies, we comprehensively characterized molecular mechanisms of PI3K pathway signaling in bladder cancer cell lines upon using small molecule inhibitors and RNAi technologies against all key molecules and protein complexes within the pathway and analyzed functional and molecular consequences. When targeting either mTORC1, mTOR, AKT or PI3K, only S6K1 phosphorylation was affected in most cell lines examined. Dephosphorylation of 4E-BP1 required combined inhibition of PI3K and mTORC1, independent from AKT, and resulted in a robust reduction in cell viability. Long-term inhibition of PI3K however resulted in a PDK1-dependent, PIP3 and mTORC2 independent rephosphorylation of AKT. AKT rephosphorylation could also be induced by mTOR or PDK1 inhibition. Combining PI3K/mTOR inhibitors with AKT or PDK1 inhibitors suppressed this rephosphorylation, induced apoptosis, decreased colony formation, cell viability and growth of tumor xenografts. Our findings reveal novel molecular mechanisms that explain the requirement for simultaneous targeting of PI3K, AKT and mTORC1 to achieve effective tumor growth inhibition.</p></div

Multiple targeting of the PI3K signaling pathway inhibits 3-dimensional tumor growth.

<p>RT112 cells were grown on the CAM as xenografts and were treated with 1000 nM MK-2206, 2000 nM PIK-90, 5 nM RAD001, 200 nM NVP-BEZ235, their indicated combinations or control (ctrl). (A) Tumor weight shown as percentage of control from 7–21 tumors per condition in two independent experiments. Horizontal line indicates median, upper whisker indicates the difference between maximum and first quartile, and lower whisker indicates the difference between minimum and third quartile. * indicates p < 0.05. (B) Ki-67 positive cells were quantified from three fields from at least three tumors treated with the indicated inhibitors and expressed as a percentage. * indicates p < 0.05 (unpaired Student’s T-test). (C) Representative images of tumor sections treated with respective inhibitors and stained with Ki-67 antibody by immunohistochemistry. Scale bar indicates 20 um.</p

Inhibition of PI3K results in a positive feedback loop on AKT.

<p>RT112 cells were treated with 200 nM NVP-BEZ235 for the indicated duration or with control (ctrl). (A) Cells that were treated for 24 and 48 hours were additionally retreated with the same concentration for 1 hour (indicated by +, no retreatment indicated by -) and immunoblotting was performed with the respective antibodies (B) NVP-BEZ235 or control treatment was combined with 500 nM of GSK2334470 for the indicated duration or with control (ctrl). Immunoblotting was performed with the indicated antibodies. Results are representative of at least three independent experiments. (C) T24 cells were treated with control (ctrl) or 100 nM of NVP-BEZ235 for 1 or 24 hours. PIP3 and PI(4,5)P2 were extracted and the quantity was determined by ELISA. Results represent the mean +/- standard deviation of triplicate wells and indicate the PIP3/PI(4,5)P2 relative to control expressed as percentage. Results are representative of two independent experiments. * indicates p < 0.05 (unpaired Student’s T-test). (D-F) Cells were treated with control (ctrl), 500 nM GSK2334470, 500 nM PIK-90, 25 nM INK128 or 10 nM NVP-BEZ235 for the indicated duration and immunoblotting was performed on lysates with the indicated antibodies. Results are representative of at least three independent experiments.</p

Effect of PI3K and PI3K/mTOR inhibitors on bladder cancer cell lines.

<p>(A-C) Cells were treated with indicated concentrations of PIK-90, BKM-120 or INK128 for 1 hour and immunoblotting was performed on lysates with the denoted antibodies. (D) Cell lysates and immunoblotting were performed with inhibitor or control treated cells for 1 hour after transduction with control (ctrl) or mTOR shRNA for 72 hours. (E) Cells were treated with respective inhibitors using indicated concentrations for 72 hours and cell viability assay was performed. Results indicate the mean +/- standard error of relative cell fluorescence in arbitrary units expressed as a percentage of control. All results are representative of at least three independent experiments.</p