Phenotypic rescue via mTOR inhibition in neuron-specific Pten knockout mice reveals AKT and mTORC1-site specific changes
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PROVIDER: PRJNA1220130 | ENA |
REPOSITORIES: ENA
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Project description:Phosphatase and Tensin Homolog (PTEN) is a dual-specific protein and lipid phosphatase that regulates AKT and downstream signaling of the mechanistic target of rapamycin (mTOR). PTEN functions as a tumor suppressor gene whose mutations result in PTEN Hamartoma Tumor Syndrome (PHTS) characterized by increased cancer risk and neurodevelopmental comorbidity. Here, we generated a novel neuron-specific Pten knock-out mouse model (Syn-Cre/Pten HOM) to test the ability of pharmacologic mTOR inhibition to rescue Pten mutation-associated disease phenotypes in vivo and in vitro. We found that treatment with the mTOR inhibitor, everolimus, increased the survival of Syn-Cre/Pten HOM mice while some neurologic phenotypes persisted. Transcriptomic analyses revealed that in contrast to mice harboring a neuron-specific deletion of the Tuberous Sclerosis Complex 2 gene (Syn-Cre/Tsc2 KO), genes that are under AKT regulation were significantly increased in the Syn-Cre/Pten HOM mice. In addition, genes associated with synapse, extracellular matrix, and myelination were broadly increased in Syn-Cre/Pten HOM mouse neocortex. These findings were confirmed by immunostaining of cortical sections in vivo, which revealed excessive immunoreactivity of myelin basic protein and perineuronal nets (PNN), the specialized extracellular matrix surrounding fast-spiking parvalbumin (PV) interneurons. We also detected increased expression of Synapsin I/PSD95 positive synapses and network hyperactivity phenotypes in Syn-Cre/Pten HOM mice neurons compared to wild-type (WT) neurons in vitro. Strikingly, everolimus treatment rescued the number of synapses and network hyperactivity in the Syn-Cre/Pten HOM mice cortical neuron cultures. Taken together, our results revealed in vivo and in vitro molecular and neuronal network mechanisms underlying neurological phenotypes of PHTS. Notably, pharmacologic mTOR inhibition by everolimus led to successful downstream signaling rescue, including mTOR complex 1 (mTORC1) site-specific suppression of S6 phosphorylation, correlating with phenotypic rescue found in our novel neuron-specific Syn-Cre/Pten HOM mice.
2025-02-14 | GSE288965 | GEO
Project description:The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes so there is great interest in understanding mTOR regulatory networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin only inhibits a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. To examine the complete spectrum of mTOR responsive cellular processes, we compared the transcriptional profiles of mammalian cells treated with rapamycin versus the ATP-competitive inhibitor PP242. Our analysis provides a genome-wide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin. Gene expression in mouse NIH3T3 cells was measured after 18 hour treatment with DMSO (control), 50 nM rapamycin, or 2 uM PP242. Four independent experiments were performed for each condition.
2011-03-15 | E-GEOD-27784 | biostudies-arrayexpress
Project description:The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes so there is great interest in understanding mTOR regulatory networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin only inhibits a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. To examine the complete spectrum of mTOR responsive cellular processes, we compared the transcriptional profiles of mammalian cells treated with rapamycin versus the ATP-competitive inhibitor PP242. Our analysis provides a genome-wide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.
2011-03-15 | GSE27784 | GEO
Project description:To comprehensively analyze the effects of mTORC1 inhibition on GSK3, we employed the use of a PI3K/mTOR-specific phospho-antibody microarray that analyzed the site-specific phosphorylation of over 130 kinases within the PI3K/mTOR pathway. The phosphorylation levels of different kinases in monocytes were measured when stimulated with LPS in the presence or absence of a kind of mTORC1 inhibitor, rapamycin More than 130 highly specific and characterized phospho-antibodies for the human mTOR signaling pathway were immobilized and replicated six times on glass slides. The same non-phosphorylated target antibodies were included to allow the determination of the relative level of phosphorylatioin
2010-11-25 | E-GEOD-25520 | biostudies-arrayexpress
Project description:IL-7 r and Stat5 signaling drives early B lymphopoiesis, but it remains poorly understood how Stat5-dependent and independent pathways contribute to this process. We report the discrete effects of PI3K and mTOR signaling on Stat5 signaling and B cell development. PI3K was not engaged by IL-7 in pro-B cells but was actively suppressed by PTEN to ensure proper IL-7 r expression, Stat5 signaling and pro-B cell survival. Further, IL-7-mediated mTORC1 activation, which was uncoupled from PI3K signaling, orchestrated a unique program in early B cell development in a Stat5-independent but Myc-dependent manner. mTORC1 was also required for immunoglobulin heavy chain rearrangement and Myc-driven lymphomagenesis. Finally, mTORC2 was not essential for early B cell development but contributed to peripheral B cell maturation. Altogether, genetic dissection of PI3K, mTORC1, and mTORC2 reveals the distinct effects of these seemingly related pathways on B cell development and the intricate interplay between PI3K, mTOR and IL-7 r-Stat5 signaling.
2018-02-14 | GSE81844 | GEO
Project description:The mechanistic target of rapamycin (mTOR) pathway integrates diverse environmental inputs, including immune signals and metabolic cues, to direct T cell fate decisions1. Activation of mTOR, comprised of mTORC1 and mTORC2 complexes, delivers an obligatory signal for proper activation and differentiation of effector CD4+ T cells2,3, whereas in the regulatory T cell (Treg) compartment, the Akt-mTOR axis is widely acknowledged as a crucial negative regulator of Treg de novo differentiation4-8 and population expansion9. However, whether mTOR signaling affects the homeostasis and function of Tregs remains largely unexplored. Here we show that mTORC1 signaling is a pivotal positive determinant of Treg function. Tregs have elevated steady-state mTORC1 activity compared to naïve T cells. Signals via T cell receptor (TCR) and IL-2 provide major inputs for mTORC1 activation, which in turn programs suppressive function of Tregs. Disruption of mTORC1 through Treg-specific deletion of the essential component Raptor leads to a profound loss of Treg suppressive activity in vivo and development of a fatal early-onset inflammatory disorder. Mechanistically, Raptor/mTORC1 signaling in Tregs promotes cholesterol/lipid metabolism, with the mevalonate pathway particularly important for coordinating Treg proliferation and upregulation of suppressive molecules CTLA-4 and ICOS to establish Treg functional competency. In contrast, mTORC1 does not directly impact the expression of Foxp3 or anti- and pro-inflammatory cytokines in Tregs, suggesting a non-conventional mechanism for Treg functional regulation. Lastly, we provide evidence that mTORC1 maintains Treg function partly through inhibiting the mTORC2 pathway. Our results demonstrate that mTORC1 acts as a fundamental ‘rheostat’ in Tregs to link immunological signals from TCR and IL-2 to lipogenic pathways and functional fitness, and highlight a central role of metabolic programming of Treg suppressive activity in immune homeostasis and tolerance. We used microarrays to explore the gene expression profiles differentially expressed in regulatory T-cells from wild-type and CD4(cre) x Raptor(fl/fl) mice
2013-06-24 | E-GEOD-46693 | biostudies-arrayexpress
Project description:To comprehensively analyze the effects of mTORC1 inhibition on GSK3, we employed the use of a PI3K/mTOR-specific phospho-antibody microarray that analyzed the site-specific phosphorylation of over 130 kinases within the PI3K/mTOR pathway. The phosphorylation levels of different kinases in monocytes were measured when stimulated with LPS in the presence or absence of a kind of mTORC1 inhibitor, rapamycin
2010-11-25 | GSE25520 | GEO
Project description:Activation of the PI3K-mTOR pathway is central to breast cancer pathogenesis including resistance to many targeted therapies. The mTOR kinase forms two distinct complexes, mTORC1 and mTORC2, and understanding which is required for the survival of malignant cells has been limited by tools to selectively and completely impair either subcomplex. To address this, we used RMC-6272, a bi-steric molecule with a rapamycin-like moiety linked to an mTOR active-site inhibitor that displays >25-fold selectivity for mTORC1 over mTORC2 substrates. Complete suppression of mTORC1 by RMC-6272 causes apoptosis in ER+/HER2- breast cancer cell lines, particularly in those that harbor mutations in PIK3CA or PTEN, due to inhibition of the rapamycin resistant, mTORC1 substrate 4EBP1 and reduction of the pro-survival protein MCL1. RMC-6272 reduced translation of ribosomal mRNAs, MYC target genes, and components of the CDK4/6 pathway, suggesting enhanced impairment of oncogenic pathways compared to the partial mTORC1 inhibitor everolimus. RMC-6272 maintained efficacy in hormone therapy-resistant acquired cell lines and patient derived xenografts (PDX), showed increased efficacy in CDK4/6 inhibitor treated acquired resistant cell lines versus their parental counterparts, and was efficacious in a PDX from a patient experiencing resistance to CDK4/6 inhibition. Bi-steric mTORC1-selective inhibition may be effective in overcoming multiple forms of therapy-resistance in ER+ breast cancers.
2024-06-01 | GSE230627 | GEO
Project description:The RNA biding protein, LARP1, has been proposed to function downstream of mTORC1 to positively regulate the translation of 5M-bM-^@M-^YTOP mRNAs such as ribosome protein (RP) mRNAs. However, its regulatory roles in mTORC1-mediated translation remain unclear. PAR-CLIP of LARP1 revealed its direct and dynamic interactions with RP mRNAs through pyrimidine-enriched sequences in the 5M-bM-^@M-^YUTR of RP mRNAs when mTOR activity is inhibited. Importantly, this LARP1 is a direct substrate of mTORC1 and S6K1/Akt, and phosphorylated LARP1 scaffolds mTORC1 on translation-competent mRNAs to facilitate 4EBP1 and S6K1 phosphorylation. Ablation of LARP1 causes multiple defects in the processes of translation including abnormal eIF4G1 interaction with RP mRNAs and inefficient RP mRNA elongation thereby reducing ribosome biogenesis and cell proliferation. These observations illustrate that LARP1 functions both an effector and a regulator for local mTORC1 activity, and acts as a molecular switch for ribosome biogenesis by sensing growth factor/nutrient signaling. LARP1-bound RNA regions were sequenced from HEK293T cells under growing or mTOR-inactive conditions. In parallel, mRNA abundance was quantified, in biological duplicate, from HEK293T cells under the same conditions.
2014-07-31 | E-GEOD-59599 | biostudies-arrayexpress
Project description:Gene expression of hepatocyt-specific knockout of Pten and of Pten and Tgfbr2 in mice as a model for human cholangiocarcinoma was determined Affymetrix Mouse 1.0ST chips were used to measure gene expression Gene expression of the following mouse livers were characterized A. WT (n=3). B. Pten-/- (n=4). C. Pten-/- Tgfbr2-/- (n=4).
2016-02-26 | E-GEOD-66717 | biostudies-arrayexpress