S6K1 and 4E-BP1 Are Independent Regulated and Control Cellular Growth in Bladder Cancer

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

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

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

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

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

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