IGF-I-induced FoxO3a nuclear-cytoplasmic pulsing dynamics in mammary epithelial cell lines following treatment with increasing doses of an AKT inhibitor. Dataset 1 of 3: single-cell reporter measurements.
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ABSTRACT:
SUBMITTER: Somponnat Sampattavanich, Bernhard Steiert
Project description:Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with a highly variable prognosis. Activation of the PI3K/AKT pathway in neuroblastoma is correlated with poor patient prognosis, but the precise downstream effectors mediating this effect have not been determined. Here, we identify the forkhead transcription factor FOXO3a as a key target of the PI3K/AKT pathway in neuroblastoma. FOXO3a expression was elevated in low stage neuroblastoma tumors and normal embryonal neuroblasts, but reduced in late stage neuroblastoma. Inactivation of FOXO3a by AKT was essential for neuroblastoma cell survival. Treatment of neuroblastoma cells with the dual PI3K/mTOR inhibitor PI-103 activated FOXO3a and triggered apoptosis. This effect was rescued by FOXO3a silencing. Conversely, apoptosis induced by PI-103 or the AKT inhibitor MK-2206 was potentiated by FOXO3a overexpression. Further, levels of total or phosphorylated FOXO3a correlated closely with apoptotic sensitivity to MK-2206. In clinical specimens, there was an inverse relationship between gene expression signatures regulated by PI3K signaling and FOXO3a transcriptional activity. Moreover, high PI3K activity and low FOXO3a activity were each associated with an extremely poor prognosis. Our work indicates that expression of FOXO3a and its targets offer useful prognostic markers as well as biomarkers for PI3K/AKT inhibitor efficacy in neuroblastoma. Affymetrix U133 Plus 2.0 profiling of SY5Y-TetR-FOXO3A cells treated with doxycycline and/or the PI3K/mTOR inhibitor PI-103. Each condition profiled in triplicate.
Project description:Pulmonary arterial hypertension (PAH) is a group of severe, progressive, and debilitating diseases with limited therapeutic options. This study aimed to explore novel therapeutic targets in PAH through bioinformatics and experiments. Weighted gene co-expression network analysis (WGCNA) was applied to detect gene modules related to PAH, based on the GSE15197, GSE113439, and GSE117261. GSE53408 was applied as validation set. Subsequently, the validated most differentially regulated hub gene was selected for further ex vivo and in vitro assays. PARM1, TSHZ2, and CCDC80 were analyzed as potential intervention targets for PAH. Consistently with the bioinformatic results, our ex vivo and in vitro data indicated that PARM1 expression increased significantly in the lung tissue and/or pulmonary artery of the MCT-induced PAH rats and hypoxia-induced PAH mice in comparison with the respective controls. Besides, a similar expression pattern of PARM1 was found in the hypoxia- and PDGF--treated isolated rat primary pulmonary arterial smooth muscle cells (PASMCs). In addition, hypoxia/PDGF--induced PARM1 protein expression could promote the elevation of phosphorylation of AKT, phosphorylation of FOXO3A and PCNA, and finally the proliferation of PASMCs in vitro, whereas PARM1 siRNA treatment inhibited it. Mechanistically, PARM1 promoted PAH via AKT/FOXO3A/PCNA signaling pathway-induced PASMC proliferation.
Project description:BACKGROUND:FOXO3a has been widely regarded as a tumor suppressor. It also plays a paradoxical role in regulating the cancer stem cells (CSCs), responsible for tumor-initiation, chemo-resistance, and recurrence in various solid tumors, including oral squamous cell carcinoma (OSCC). This study aims to uncover the role of FOXO3a and its importance for a non-canonical pathway of TGFβ in regulating the OSCC stemness. METHODS:We identified FOXO3a expression in OSCC tissues and cell lines using immunohistochemistry and western blot. The correlation between FOXO3a and stemness was evaluated. Stable cell lines with differential expression of FOXO3a were constructed using lentiviruses. The effects of FOXO3a on stem-cell like properties in OSCC was further evaluated in vitro and in vivo. We also explored the effect of TGFβ on FOXO3a with respect to its expression and function. FINDINGS:Our findings suggest that FOXO3a was widely expressed and negatively correlated with the stemness in OSCC. This regulation can be abolished by TGFβ through phosphorylation, nuclear exclusion, and degradation in the non-Smad pathway. We also observed that non-Smad AKT-FOXO3a axis is essential to regulate stemness of CSCs by TGFβ. INTERPRETATION:TGFβ induces stemness through non-canonical AKT-FOXO3a axis in OSCC. Our study provides a foundation to understand the mechanism of CSCs and a possible therapeutic target to eliminate CSCs.
Project description:Purposep27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy.Experimental designRCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated.Resultsp27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not.ConclusionsIn RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.