Inactivating mutations of the neurofibromatosis 2 (NF2) gene, NF2, result predominantly in benign neurological tumors, schwannomas and meningiomas, in humans; however, mutations in murine Nf2 lead to a broad spectrum of cancerous tumors. The tumor-suppressive function of the NF2 protein, merlin, a membranecytoskeleton linker, remains unclear. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a novel mediator of merlin's tumor suppressor activity. Merlin-deficient human meningioma cells and merlin knockdown arachnoidal cells, the nonneoplastic cell counterparts of meningiomas, exhibit rapamycin-sensitive constitutive mTORC1 activation and increased growth. NF2 patient tumors and Nf2-deficient mouse embryonic fibroblasts demonstrate elevated mTORC1 signaling. Conversely, the exogenous expression of wild-type merlin isoforms, but not a patient-derived L64P mutant, suppresses mTORC1 signaling. Merlin does not regulate mTORC1 via the established mechanism of phosphoinositide 3-kinase-Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase-mediated TSC2 inactivation and may instead regulate TSC/mTOR signaling in a novel fashion. In conclusion, the deregulation of mTORC1 activation underlies the aberrant growth and proliferation of NF2-associated tumors and may restrain the growth of these lesions through negative feedback mechanisms, suggesting that rapamycin in combination with phosphoinositide 3-kinase inhibitors may be therapeutic for NF2.
The tumor suppressor tuberin, encoded by the Tuberous Sclerosis Complex (TSC) gene TSC2, negatively regulates the mammalian target of rapamycin (mTOR) pathway, which plays a key role in the control of cell growth and proliferation. In addition to naturally occurring mutations, several kinases including Akt, RSK1, and ERK are known to phosphorylate and inactivate tuberin. We demonstrate a novel mechanism of tuberin inactivation through ubiquitination by Pam, a putative RING finger-containing E3 ubiquitin (Ub) ligase in mammalian cells. We show that Pam associates with E2 ubiquitin-conjugating enzymes, and tuberin can be ubiquitinated by Pam through its RING finger domain. Tuberin ubiquitination is independent of its phosphorylation by Akt, RSK1, and ERK kinases. Pam is also self-ubiquitinated through its RING finger domain. Moreover, the TSC1 protein hamartin, which forms a heterodimer with tuberin, protects tuberin from ubiquitination by Pam. However, TSC1 fails to protect a disease-associated missense mutant of TSC2 from ubiquitination by Pam. Furthermore, Pam knockdown by RNA interference (RNAi) in rat primary neurons elevates the level of tuberin, and subsequently inhibits the mTOR pathway. Our results provide novel evidence that Pam can function as an E3 Ub ligase toward tuberin and regulate mTOR signaling, suggesting that Pam can in turn regulate cell growth and proliferation as well as neuronal function through the TSC/mTOR pathway in mammalian cells.
Purpose: Chordomas are rare, malignant bone neoplasms in which the pathogenic mechanisms remain unknown. Interestingly, tuberous sclerosis complex (TSC) is the only syndrome in which the incidence of chordomas has been described. We previously reported the pathogenic role of the TSC genes in TSC-associated chordomas. In this study, we investigated whether aberrant TSC/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is associated with sporadic chordomas. Experimental Design: We assessed the status of mTORC1signaling in primary tumors/cell lines of sacral chordomas and further examined upstream of mTORC1 signaling, including the PTEN (phosphatase and tensin homologue deleted on chromosome ten) tumor suppressor. We also tested the efficacy of the mTOR inhibitor rapamycin on signaling and growth of chordoma cell lines. Results: Sporadic sacral chordoma tumors and cell lines examined commonly displayed hyperactivated Akt and mTORC1 signaling. Strikingly, expression of PTEN, a negative regulator of mTORC1signaling, was not detected or significantly reduced in chordoma-derived cell lines and primary tumors. Furthermore, rapamycin inhibited mTORC1 activation and suppressed proliferation of chordoma-derived cell line. Conclusions: Our results suggest that loss of PTEN as well as other genetic alterations that result in constitutive activation of Akt/mTORC1signaling may contribute to the development of sporadic chordomas. More importantly, a combination of Akt and mTORC1 inhibition may provide clinical benefits to chordoma patients.Chordomas are uncommon, slow-growing, malignant neoplasms that are thought to originate from notochordal remnants along the axial skeleton in the sacrococcygeal/sacral, sphenooccipital/clivus, and spinal regions (1). Chordomas are usually sporadic, but a few families have been reported with multiple family members affected by chordoma (2 -6), suggesting that inherited variations may predispose to these tumors. Intriguingly, several investigations identified chordomas in tuberous sclerosis complex (TSC) patients (7 -11). We previously reported two cases of TSC with concomitant sacrococcygeal chordomas and identified somatic inactivation of the TSC genes, thus providing the first evidence of a pathogenic role of the TSC genes in sacrococcygeal chordomas (12).TSC is a tumor suppressor syndrome characterized by abnormal tissue growths, known as hamartomas, in many organs (13). TSC is caused by inactivating mutations in tumor suppressor genes TSC1 or TSC2, which encode hamartin (TSC1) and tuberin (TSC2), respectively. Of note, somatic mutations in these genes are also found in sporadic lymphangioleiomyomatosis (14,15). TSC proteins form a heterodimeric TSC1-TSC2 complex and function to inhibit mTORC1 signaling, which critically regulates cell growth and proliferation (16,17). Activation of the phosphoinositide-3-kinase (PI3K)/ Akt or Ras/mitogen-activated protein kinase (MAPK) pathway by growth factors leads to TSC2 phosphorylation and inhibition of the TSC protein comple...
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