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mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation  [PDF]
James Yu,Aylin Yaba,Corinna Kasiman,Travis Thomson,Joshua Johnson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0021415
Abstract: We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.
PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines
Jianwen Que, Qizhou Lian, Reida M El Oakley, Bing Lim, Sai-Kiang Lim
Journal of Molecular Signaling , 2007, DOI: 10.1186/1750-2187-2-9
Abstract: We investigated the signaling pathway that maintains proliferation of these cells in an undifferentiated state, and demonstrate that PI3 K/Akt/mTOR, but not Raf/MEK/Erk, signaling in these cells was active during proliferation and was downregulated during endothelial differentiation. Inhibition of PI3 K/Akt/mTOR signaling, but not Raf/MEK/Erk, reduced proliferation and induced expression of endothelial specific proteins. During differentiation or inhibition of PI3 K/Akt/mTOR signaling, cyclinD2 transcript abundance in ribosome-enriched RNA but not in total RNA was reduced with a corresponding reduction in protein level. In contrast, transcript abundance of endothelial-specific genes e.g. Kdr, Tek and Pdgfrα in ribosome-enriched RNA fraction was not reduced and their protein levels were increased. Together these observations suggested that translational control mediated by PI3K/Akt/mTOR signaling was critical in regulating proliferation and endothelial differentiation of lineage-restricted RoSH-like stem cell lines.This study highlights translation regulation as a critical regulatory mechanism during proliferation and differentiation in stem cells.Embryonic stem cells (ESCs) are pluripotent stem cells capable of differentiating into cells of all three germ layers, making ESCs an ideal source of cells for regenerative therapy for many diseases and tissue injuries [1,2]. However, this property of ESCs poses a unique challenge of having to generate therapeutically efficacious quantity of appropriate cell types without being contaminated by potentially deleterious cell types. Recently, we have generated lineage-restricted stem cell lines with endothelial potential termed RoSH and E-RoSH lines from mouse embryos and mouse ESCs (mESCs), respectively [3,4]. RoSH and E-RoSH lines are derived from CD9hi, SSEA-1- cells in embryo and ESC-derived embryoid body cultures. Despite their different tissue source of origin, both RoSH and E-RoSH cell lines are highly similar with an al
The mTOR Pathway Controls Cell Proliferation by Regulating the FoxO3a Transcription Factor via SGK1 Kinase  [PDF]
Shunsuke Mori, Shigeyuki Nada, Hironobu Kimura, Shoji Tajima, Yusuke Takahashi, Ayaka Kitamura, Chitose Oneyama, Masato Okada
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088891
Abstract: The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.
Hepatitis B Virus X Protein Upregulates mTOR Signaling through IKKβ to Increase Cell Proliferation and VEGF Production in Hepatocellular Carcinoma  [PDF]
Chia-Jui Yen, Yih-Jyh Lin, Chia-Sheng Yen, Hung-Wen Tsai, Ting-Fen Tsai, Kwang-Yu Chang, Wei-Chien Huang, Pin-Wen Lin, Chi-Wu Chiang, Ting-Tsung Chang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041931
Abstract: Hepatocellular carcinoma (HCC), a major cause of cancer-related death in Southeast Asia, is frequently associated with hepatitis B virus (HBV) infection. HBV X protein (HBx), encoded by a viral non-structural gene, is a multifunctional regulator in HBV-associated tumor development. We investigated novel signaling pathways underlying HBx-induced liver tumorigenesis and found that the signaling pathway involving IκB kinase β (IKKβ), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1), was upregulated when HBx was overexpressed in hepatoma cells. HBx-induced S6K1 activation was reversed by IKKβ inhibitor Bay 11-7082 or silencing IKKβ expression using siRNA. HBx upregulated cell proliferation and vascular endothelial growth factor (VEGF) production, and these HBx-upregulated phenotypes were abolished by treatment with IKKβ inhibitor Bay 11-7082 or mTOR inhibitor rapamycin. The association of HBx-modulated IKKβ/mTOR/S6K1 signaling with liver tumorigenesis was verified in a HBx transgenic mouse model in which pIKKβ, pS6K1, and VEGF expression was found to be higher in cancerous than non-cancerous liver tissues. Furthermore, we also found that pIKKβ levels were strongly correlated with pTSC1 and pS6K1 levels in HBV-associated hepatoma tissue specimens taken from 95 patients, and that higher pIKKβ, pTSC1, and pS6K1 levels were correlated with a poor prognosis in these patients. Taken together, our findings demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ, which is crucially linked to HBV-associated HCC development.
Influences of surface coatings and components of FePt nanoparticles on the suppression of glioma cell proliferation
Sun H, Chen X, Chen D, Dong M, Fu X, Li Q, Liu X, Wu Q, Qiu T, Wan T, Li S
International Journal of Nanomedicine , 2012, DOI: http://dx.doi.org/10.2147/IJN.S32678
Abstract: fluences of surface coatings and components of FePt nanoparticles on the suppression of glioma cell proliferation Original Research (2463) Total Article Views Authors: Sun H, Chen X, Chen D, Dong M, Fu X, Li Q, Liu X, Wu Q, Qiu T, Wan T, Li S Published Date July 2012 Volume 2012:7 Pages 3295 - 3307 DOI: http://dx.doi.org/10.2147/IJN.S32678 Received: 06 April 2012 Accepted: 14 May 2012 Published: 06 July 2012 Haiming Sun,1,* Xiaohui Chen,2,* Dan Chen,1 Mingyan Dong,1 Xinning Fu,1 Qian Li,1 Xi Liu,1 Qingzhi Wu,1 Tong Qiu,1 Tao Wan,1 Shipu Li1 1State Key Laboratory of Advanced Technology for Materials Synthesis and Processing and Biomedical Materials and Engineering Center, Wuhan University of Technology, Wuhan, China; 2Department of Prosthetics, School of Stomatology, Wuhan University, Wuhan, China *Both authors contributed equally to this work Abstract: Malignant gliomas are primary brain tumors with high rates of morbidity and mortality; they are the fourth most common cause of cancer death. Novel diagnostic and therapeutic techniques based on nanomaterials provide promising options in the treatment of malignant gliomas. In order to evaluate the potential of FePt nanoparticles (NPs) for malignant glioma therapy, FePt NPs with different surface coatings and components were tunably synthesized using oleic acid/oleylamine (OA/OA) and cysteines (Cys) as the capping agents, respectively. The samples were characterized using X-ray diffraction, transmission electron microscopy (TEM), X-ray photon spectroscopy, Fourier transform infrared spectroscopy, atomic absorption spectrum, and zeta potential. The influence of the surface coatings and components of the FePt NPs on the proliferation of glioma cells was assessed through MTT assay and TEM observation using three typical glioma cell lines (glioma U251 cells, astrocytoma U87 cells, and neuroglioma H4 cells) as in vitro models. The results showed that the proliferation of glioma cells was significantly suppressed by lipophilic FePt-OA/OA NPs in a time- and/or dose-dependent manner, while no or low cytotoxic effects were detected in the case of hydrophilic FePt-Cys NPs. The IC50 value of FePt-OA/OA NPs on the three glioma cell lines was approximately 5–10 μg mL-1 after 24 hours’ incubation. Although the cellular uptake of FePt NPs was confirmed regardless of the surface coatings and components of the FePt NPs, the suppression of FePt NPs on glioma cell proliferation was dominantly determined by their surface coatings rather than their components. Therefore, these results demonstrate that, through engineering of the surface coating, FePt NPs can potentially be developed as novel therapeutic agents for malignant gliomas.
Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors
Yi-Wen Hsieh, Xian-Jie Yang
Neural Development , 2009, DOI: 10.1186/1749-8104-4-32
Abstract: We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates.These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase re-entry, whereas rapid accumulation or reduction of Pax6 protein during the G2/M phase of the cell cycle may be required for specific neuronal fates. These findings thus provide novel insights on the dynamic regulation of Pax6 protein among neurogenic progenitors and the temporal frame of neuronal fate determination.The Pax6 gene encodes an evolutionarily conserved paired homeobox protein critically involved in eye development and retinogenesis [1-5]. In both Drosophila and vertebrates, ectopic expression of Pax6 induces ectopic eyes or
Response to mTOR inhibition: activity of eIF4E predicts sensitivity in cell lines and acquired changes in eIF4E regulation in breast cancer
Sampoorna Satheesha, Victoria J Cookson, Louise J Coleman, Nicola Ingram, Brijesh Madhok, Andrew M Hanby, Charlotte AB Suleman, Vicky S Sabine, E Jane Macaskill, John MS Bartlett, J Michael Dixon, Jim N McElwaine, Thomas A Hughes
Molecular Cancer , 2011, DOI: 10.1186/1476-4598-10-19
Abstract: We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition.Estimates of eIF4E activity predict sensitivity to mTOR inhibition in cell lines but breast tumours with high estimated eIF4E activity gain changes in eIF4E regulation in order to enhance resistance.Rapamycin is an immunosuppressant drug prescribed for prophylaxis of organ rejection following renal transplant [1]. Recently it, and derivatives such as everolimus, have been tested as cancer therapeutics with some success [2-5]. The drugs inhibit the serine/threonine-specific protein kinase mTOR (mammalian Target Of Rapamycin) by forming a complex with another protein, FKBP12 (FK 506-binding protein of 12 kDa), that then associates with mTOR.
The Role of AKT/mTOR Pathway in Stress Response to UV-Irradiation: Implication in Skin Carcinogenesis by Regulation of Apoptosis, Autophagy and Senescence  [PDF]
Elwira Strozyk,Dagmar Kulms
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140815260
Abstract: Induction of DNA damage by UVB and UVA radiation may generate mutations and genomic instability leading to carcinogenesis. Therefore, skin cells being repeatedly exposed to ultraviolet (UV) light have acquired multilayered protective mechanisms to avoid malignant transformation. Besides extensive DNA repair mechanisms, the damaged skin cells can be eliminated by induction of apoptosis, which is mediated through the action of tumor suppressor p53. In order to prevent the excessive loss of skin cells and to maintain the skin barrier function, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway. However, AKT/mTOR not only prevents cell death, but is also active in cell cycle transition and hyper-proliferation, thereby also counteracting p53. In turn, AKT/mTOR is tuned down by the negative regulators being controlled by the p53. This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance. Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence. Conclusively, AKT/mTOR via an extensive cross talk with p53 influences the UV response in the skin with no black and white scenario deciding over death or survival.
Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells  [cached]
Grabinski Nicole,Ewald Florian,Hofmann Bianca T,Staufer Katharina
Molecular Cancer , 2012, DOI: 10.1186/1476-4598-11-85
Abstract: Background Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients. Methods The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells. Results AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient. Conclusion Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.
Mimicking p14ARF Phosphorylation Influences Its Ability to Restrain Cell Proliferation  [PDF]
Maria Vivo, Michela Ranieri, Federica Sansone, Cristina Santoriello, Raffaele A. Calogero, Viola Calabrò, Alessandra Pollice, Girolama La Mantia
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0053631
Abstract: The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered loci in human cancer. It is generally accepted that ARF is involved in oncogenic checkpoint pathways by sensitizing incipient cancer cells to undergo growth arrest or apoptosis through both p53-dependent and independent pathways. While intensive studies have been focused on ARF activation at the transcriptional level, only recently mechanisms governing ARF turnover have been identified. Here, we show for the first time that p14ARF is a PKC target. Prediction analysis showed many potential phosphorylation sites in PKC consensus sequences within ARF protein, and, among them, the threonine at position 8 was the most conserved. Substitution of this threonine influences both ARF stability and localization. Furthermore, a phosphomimetic ARF mutation reduces the ability to arrest cell growth although the ability to bind MDM2 and stabilize p53 result unaffected. Thus we propose that phosphorylation of ARF in both immortalized and tumor cell lines could be a mechanism to escape ARF surveillance following proliferative and oncogenic stress.
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