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Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor β (RARβ).

ABSTRACT: miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3'UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.

SUBMITTER: Liu HJ

PROVIDER: S-EPMC6895402 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor β (RARβ).

Liu Heng-Jia HJ Lam Hilaire C HC Baglini Christian V CV Nijmeh Julie J Cottrill Alischer A AA Chan Stephen Y SY Henske Elizabeth P EP

Oncogene 20190816 49

miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment ...[more]

PMID: 31420607

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Project description:BackgroundThe signaling pathways imposing hormonal control over adipocyte differentiation are poorly understood. While insulin and Akt signaling have been found previously to be essential for adipogenesis, the relative importance of their many downstream branches have not been defined. One direct substrate that is inhibited by Akt-mediated phosphorylation is the tuberous sclerosis complex 2 (TSC2) protein, which associates with TSC1 and acts as a critical negative regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Loss of function of the TSC1-TSC2 complex results in constitutive mTORC1 signaling and, through mTORC1-dependent feedback mechanisms and loss of mTORC2 activity, leads to a concomitant block of Akt signaling to its other downstream targets.Methodology/principal findingsWe find that, despite severe insulin resistance and the absence of Akt signaling, TSC2-deficient mouse embryo fibroblasts and 3T3-L1 pre-adipocytes display enhanced adipocyte differentiation that is dependent on the elevated mTORC1 activity in these cells. Activation of mTORC1 causes a robust increase in the mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master transcriptional regulator of adipocyte differentiation. In examining the requirements for different Akt-mediated phosphorylation sites on TSC2, we find that only TSC2 mutants lacking all five previously identified Akt sites fully block insulin-stimulated mTORC1 signaling in reconstituted Tsc2 null cells, and this mutant also inhibits adipogenesis. Finally, renal angiomyolipomas from patients with tuberous sclerosis complex contain both adipose and smooth muscle-like components with activated mTORC1 signaling and elevated PPARgamma expression.Conclusions/significanceThis study demonstrates that activation of mTORC1 signaling is a critical step in adipocyte differentiation and identifies TSC2 as a primary target of Akt driving this process. Therefore, the TSC1-TSC2 complex regulates the differentiation of mesenchymal cell lineages, at least in part, through its control of mTORC1 activity and PPARgamma expression.

Project description:RationaleThe mTORC1 (mechanistic target of rapamycin complex-1) controls metabolism and protein homeostasis and is activated following ischemia reperfusion (IR) injury and by ischemic preconditioning (IPC). However, studies vary as to whether this activation is beneficial or detrimental, and its influence on metabolism after IR is little reported. A limitation of prior investigations is their use of broad gain/loss of mTORC1 function, mostly applied before ischemic stress. This can be circumvented by regulating one serine (S1365) on TSC2 (tuberous sclerosis complex) to achieve bidirectional mTORC1 modulation but only with TCS2-regulated costimulation.ObjectiveWe tested the hypothesis that reduced TSC2 S1365 phosphorylation protects the myocardium against IR and is required for IPC by amplifying mTORC1 activity to favor glycolytic metabolism.Methods and resultsMice with either S1365A (TSC2SA; phospho-null) or S1365E (TSC2SE; phosphomimetic) knockin mutations were studied ex vivo and in vivo. In response to IR, hearts from TSC2SA mice had amplified mTORC1 activation and improved heart function compared with wild-type and TSC2SE hearts. The magnitude of protection matched IPC. IPC requited less S1365 phosphorylation, as TSC2SE hearts gained no benefit and failed to activate mTORC1 with IPC. IR metabolism was altered in TSC2SA, with increased mitochondrial oxygen consumption rate and glycolytic capacity (stressed/maximal extracellular acidification) after myocyte hypoxia-reperfusion. In whole heart, lactate increased and long-chain acylcarnitine levels declined during ischemia. The relative IR protection in TSC2SA was lost by lowering glucose in the perfusate by 36%. Adding fatty acid (palmitate) compensated for reduced glucose in wild type and TSC2SE but not TSC2SA which had the worst post-IR function under these conditions.ConclusionsTSC2-S1365 phosphorylation status regulates myocardial substrate utilization, and its decline activates mTORC1 biasing metabolism away from fatty acid oxidation to glycolysis to confer protection against IR. This pathway is also engaged and reduced TSC2 S1365 phosphorylation required for effective IPC. Graphic Abstract: A graphic abstract is available for this article.

Dataset Information

Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor β (RARβ).

Publications

Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor β (RARβ).

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