Data from Cytoplasmic Sequestration of p27 via AKT Phosphorylation in Renal Cell Carcinoma
Jean Hee KimEric JonaschAngela AlexanderJohn D. ShortShengli CaiSijin WenDimitra TsavachidouPheroze TamboliBogdan CzerniakKim‐Anh DoKevin J. WuLaura A. MarlowChristopher G. WoodJohn A. CoplandCheryl Lyn Walker
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<div>Abstract<p><b>Purpose:</b> p27 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.</p><p><b>Experimental Design:</b> RCC-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.</p><p><b>Results:</b> p27 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.</p><p><b>Conclusions:</b> In 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.</p></div>Keywords:
LY294002
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B7-H4 plays an important role in tumor immune evasion. In previous studies we have found that B7-H4 can translocate to the nucleus, and the exposure to PI3K inhibitor Ly294002 affects B7-H4 subcellular distribution. In this study we report the role of PI3K/Akt pathway in the B7-H4 subcellular distribution and the effect of PI3K/Akt inhibitors on B7-H4-mediated immunoresistance. The involvement of PI3K/Akt pathway in B7-H4 subcellular distribution was evident in experiments with wortmannin, while MDM2 inhibitor nutlin-3 and the mTOR inhibitor rapamycin were used to dissect the signaling downstream of Akt. Wortmannin and rapamycin demonstrated similar effects on B7-H4 subcellular distribution. Exposure to any of these inhibitors decreased levels of membrane B7-H4 while at the same time inducing its nuclear accumulation, while exposure to nutlin-3 had no effect on B7-H4 subcellular distribution. In the T cell proliferation assay, both wortmannin and rapamycin effectively inhibited B7-H4 WT/293 cells-mediated T cell proliferation while exerting no effect on Mock/293 cells. PI3K/Akt/mTOR plays a role in B7-H4 subcellular distribution, while MDM2 does not take part in it. Moreover, we show that wortmannin and rapamycin inhibit B7-H4-mediated tumor immunoresistance through regulating B7-H4 subcellular distribution. Taken together, these results suggest that PI3K/Akt/mTOR inhibitors might be used for adjuvant therapy aimed at inhibition of immune evasion.
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Phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), a downstream kinase, are both required for proliferation of splenic B cells. However, the functions of PI3K and mTOR in response to different stimuli and among B cell subsets have not been fully elucidated. We used flow cytometry and magnetic cell sorting to examine the requirement for PI3K and mTOR in responses of splenic B cell subsets to BCR and LPS stimulation. BCR-mediated phosphorylation of Akt and Erk is sensitive to the PI3K catalytic inhibitor wortmannin in both marginal zone (MZ) and follicular (FO) cells. BCR-mediated mTOR activation in both subsets is inhibited by wortmannin, though less strongly in MZ cells. In contrast, LPS-induced mTOR signaling is strikingly resistant to wortmannin in both subsets. Similarly, functional responses to LPS are partially wortmannin resistant yet sensitive to mTOR inhibition by rapamycin. We also observed mitogen-independent mTOR activity that is regulated by nutrient availability, and is significantly elevated in MZ cells relative to FO cells. These data define both similarities and differences in PI3K/mTOR signaling mechanisms in MZ and FO cells, and suggest that mTOR signaling can occur in the absence of PI3K activation to promote B cell responses to LPS.
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Voltage-dependent K+ (Kv) channels negatively regulate Ca2+ entry into pancreatic beta-cells by repolarizing glucose-stimulated action potentials. A role for phosphatidylinositol 3-kinase (PI3K) modulation of Kv channel function was investigated using the PI3K inhibitors wortmannin and LY294002, and LY303511, a negative control compound with respect to PI3K activity. In MIN6 insulinoma cells, wortmannin (100 nM) had no effect on whole-cell outward K+ currents, but LY294002 and LY303511 reversibly blocked currents in a dose-dependent manner (IC50=9.0+/-0.7 microM and 64.6+/-9.1 microM, respectively). Western blotting confirmed the specific inhibitory effects of LY294002 and wortmannin on insulin-stimulated PI3K activity. Kv currents in rat beta-cells at near physiological temperatures were inhibited 92% by 25 microM LY294002. Kv2.1 and Kv1.4 are highly expressed in beta-cells, and in Kv2.1-transfected tsA201 cells, 50 microM LY294002 and 100 microM LY303511 reversibly inhibited currents by 99% and 41%, respectively. In Kv1.4-transfected tsA201 cells, 50 microM LY294002 reduced the inactivation time constant from 73 to 18 ms. The insulinotropic properties of LY294002 and its effects in other excitable cells may be caused by inhibition of Kv currents rather than PI3K antagonism. Furthermore, LY294002 may represent a novel structure from which future Kv channel blockers may be developed.
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Angelica Sinensis (AS), a folk medicine, has long been used in ergogenic aids for athletes, but there is little scientific evidence supporting its effects. We investigated whether AS induces hypertrophy in myotubes through the phosphatidylinositol 3-kinase (PI3K)/Akt (also termed PKB)/mammalian target of the rapamycin (mTOR) pathway. An in vitro experiment investigating the induction of hypertrophy in myotubes was conducted. To investigate whether AS promoted the hypertrophy of myotubes, an established in vitro model of myotube hypertrophy with and without AS was used and examined using microscopic images. The role of the PI3K/Akt/mTOR signaling pathway in AS-induced myotube hypertrophy was evaluated. Two inhibitors, wortmannin (an inhibitor of PI3K) and rapamycin (an inhibitor of mTOR), were used. The results revealed that the myotube diameters in the AS-treated group were significantly larger than those in the untreated control group (P < 0.05). Wortmannin and rapamycin inhibited AS-induced hypertrophy. Furthermore, AS increased Akt and mTOR phosphorylation through the PI3K pathway and induced myotube hypertrophy. The results confirmed that AS induces hypertrophy in myotubes through the PI3K/Akt/mTOR pathway.
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Objective:To investigate whether phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) signal transduction pathway could be activated rapidly by trypsin in neutrophils and whether LY294002 and wortmannin,PI3K inhibitors,could inhibit such effect of trypsin. Methods:The levels of phosphorylated PKB (p-PKB) and total PKB were examined by Western blotting in neutrophils after stimulation with 40 nmol/L of trypsin. The inhibitory effects of LY294002 and wortmannin on the activation of PKB induced by trypsin were observed. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) protein and mRNA in the culture and neutrophils were determined by enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction,respectively. Results:The level of p-PKB in neutrophils and the levels of TNF-α and IL-1β protein in the culture after stimulation with trypsin were significantly higher than those in normal controls (P 0.01). Wortmannin and LY294002 could completely inhibit the activation of p-PKB induced by trypsin in neutrophils. With the inhibition of p-PKB,the levels of TNF-α and IL-1β protein and mRNA decreased. Conclusion:Trypsin could activate PI3K/PKB signal transduction pathway in neutrophils,and this action of trypsin is PI3K-dependent and could be inhibited or blocked by wortmannin and LY294002.
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