第二代 mTOR 抑制剂的抗肿瘤研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)是 PI3K/Akt/mTOR 等多种信号通路的下游分子,在细胞增殖、分化、转移和存活中发挥 重要作用,已成为癌症治疗的一个重要靶标。传统的 mTOR 抑制剂主要是雷帕霉素及其衍生物,能特异性抑制 mTORC1,但在部分癌 症临床治疗中未达到预期疗效,且易产生耐药性。第二代 mTOR 抑制剂即双重或多重 mTOR 抑制剂能与 mTOR 的催化位点竞争 ATP, 高度选择性地抑制 mTORC1 和 mTORC2,比单靶点 mTOR 抑制剂具有更大的治疗优势。此外,某些天然来源产物也具有对 mTOR 的 抑制作用,且毒性、副作用更小。综述近几年有关 mTOR 及其抑制剂在抗肿瘤方面的研究进展。     

Sulfamethizole attenuates poloxamer 407-induced atherosclerotic neointima formation via inhibition of mTOR in C57BL/6 mice

Mammalian target of Rapamycin C1 (mTORC1) inhibition limits plaque progression in atherosclerosis. The present study evaluated the protective effect of sulfamethizole on poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition. Poloxamer 407 (P-407) (0.5 g/kg body weight) was administered intraperitoneally to male C57BL/6 mice every third day for 148 days to induce chronic hyperlipidemia. From Day 121 to 148, animals were additionally administered Sulfamethizole (5, 10, and 50 mg/kg, p.o.), Rapamycin (0.5 mg/kg, positive control), or vehicle (1 ml/kg). Plasma lipid levels were measured on Days 120 and 148. Upon sacrifice, histological studies were performed, and aortic tissue interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and mTOR levels were evaluated. A molecular docking study was carried out to mimic the interaction of sulfamethizole with mTOR protein. Chronic P-407 administration significantly (p < 0.001) elevated plasma lipid levels, compared with those of the normal control group. Chronic hyperlipidemia resulted in increased tunica intima thickness, collagen deposition, and IL-6, TNF-α, and mTOR levels. Treatment with Sulfamethizole attenuated these parameters significantly in a dose-dependent manner. Molecular docking studies showed a significant interaction of Sulfamethizole with mTOR. In conclusion, this study suggests that sulfamethizole significantly limits poloxamer 407-induced atherosclerotic neointima formation in C57BL/6 mice via mTOR inhibition.     

The inhibition of mammalian target of rapamycin (mTOR) in improving inflammatory response after traumatic brain injury

Traumatic brain injury (TBI) provokes primary and secondary damage on endothelium and brain parenchyma, leading neurons die rapidly by necrosis. The mammalian target of rapamycin signalling pathway (mTOR) manages numerous aspects of cellular growth, and it is up-regulated after moderate to severe traumatic brain injury (TBI). Currently, the significance of this increased signalling event for the recovery of brain function is unclear; therefore, we used two different selective inhibitors of mTOR activity to discover the functional role of mTOR inhibition in a mouse model of TBI performed by a controlled cortical impact injury (CCI). Treatment with KU0063794, a dual mTORC1 and mTORC2 inhibitor, and with rapamycin as well-known inhibitor of mTOR, was performed 1 and 4 hours subsequent to TBI. Results proved that mTOR inhibitors, especially KU0063794, significantly improved cognitive and motor recovery after TBI, reducing lesion volumes. Also, treatment with mTOR inhibitors ameliorated the neuroinflammation associated with TBI, showing a diminished neuronal death and astrogliosis after trauma. Our findings propose that the involvement of selective mTORC1/2 inhibitor may represent a therapeutic strategy to improve recovery after brain trauma.     

Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice

Inhibition of the mTOR (mechanistic Target Of Rapamycin) signaling pathway robustly extends the lifespan of model organisms including mice. The precise molecular mechanisms and physiological effects that underlie the beneficial effects of rapamycin are an exciting area of research. Surprisingly, while some data suggest that mTOR signaling normally increases with age in mice, the effect of age on mTOR signaling has never been comprehensively assessed. Here, we determine the age‐associated changes in mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) signaling in the liver, muscle, adipose, and heart of C57BL/6J.Nia mice, the lifespan of which can be extended by rapamycin treatment. We find that the effect of age on several different readouts of mTORC1 and mTORC2 activity varies by tissue and sex in C57BL/6J.Nia mice. Intriguingly, we observed increased mTORC1 activity in the liver and heart tissue of young female mice compared to male mice of the same age. Tissue and substrate‐specific results were observed in the livers of HET3 and DBA/2 mouse strains, and in liver, muscle and adipose tissue of F344 rats. Our results demonstrate that aging does not result in increased mTOR signaling in most tissues and suggest that rapamycin does not promote lifespan by reversing or blunting such an effect.     

The integral role of mTOR in lipid metabolism

Comment on: Soliman GA, et al. Lipids 2010; 45:1089-100.     

Conditional and domain‐specific inactivation of the Tsc2 gene in neural progenitor cells

Tuberous sclerosis complex (TSC) is a genetic disease characterized by multiorgan benign tumors as well as neurological manifestations. Epilepsy and autism are two of the more prevalent neurological complications and are usually severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse outcomes. Tuberin contains a highly conserved GTPase‐activating protein (GAP) domain that indirectly inhibits mammalian target of rapamycin complex 1 (mTORC1). mTORC1 dysregulation is currently thought to cause much of the pathogenesis in TSC but mTORC1‐independent mechanisms may also contribute. We generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with loxP sites. Mice homozygous for this knock‐in Tsc2 allele are viable and fertile with normal appearing growth and development. Exposure to Cre recombinase then creates an in‐frame deletion involving critical residues of the GAP domain. Homozygous conditional mutant mice generated using Emx1^Cre have increased cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA, though GAP‐deficient tuberin protein, appear unstable and rapidly degraded. This novel animal model will allow further study of tuberin function including the requirement of the GAP domain for protein stability. genesis 51:284–292. © 2013 Wiley Periodicals, Inc.     

The role of S6K1 in ER-positive breast cancer

The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases, which also includes Akt and many others. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1). S6K1 is sensitive to a wide range of signaling inputs, including growth factors, amino acids, energy levels and hypoxia. S6K1 relays these signals to regulate a growing list of substrates and interacting proteins in control of oncogenic processes, such as cell growth and proliferation, cell survival and apoptosis and cell migration and invasion. Several lines of evidence suggest an important role for S6K1 in estrogen receptor (ER)-positive breast cancer. S6K1 directly phosphorylates and activates ERα. Furthermore, S6K1 expression is estrogenically regulated. Therefore, hyperactivation of mTORC1/S6K1 signaling may be closely related to ER-positive status in breast cancer and may be utilized as a marker for prognosis and a therapeutic target.     

mTORC2: The other mTOR in autophagy regulation

The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best‐described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex‐specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.     

cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity

cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP]_i in mouse embryonic fibroblasts (MEFs) and human embryonic kidney 293 (HEK293) cells inhibits mTORC1 activation via a PKA-dependent mechanism. Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades (i.e. PKB, MAPK and AMPK) that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2^−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]_i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.     

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR^Ser2481 exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR^Ser2481 was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR^Ser2481 staining could be clearly observed among condensed chromosomes. Phospho-mTOR^Ser2481 then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR^Ser2481 displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR^Ser2481 largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR^Ser2481 was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR^Ser2481 to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR^Ser2448 with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR^Ser2481 to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR^Ser2481 to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.     

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

Energy- and nutrient-sensing proteins such as AMPK, mTOR and S6K1 are now recognized as novel regulators of mitotic completion in proliferating cells. We investigated the cellular distribution of the Ser2481 autophosphorylation of mTOR, which directly monitors mTORC-specific catalytic activity, during mammalian cell mitosis and cytokinesis. Automated immunofluorescence experiments in human carcinoma cell lines revealed that phospho-mTOR^Ser2481 exhibited profound spatial and temporal dynamics during cell division. Phospho-mTOR^Ser2481 was strikingly enriched in mitotic cells, and in prophase, bright phospho-mTOR^Ser2481 staining could be clearly observed among condensed chromosomes. Phospho-mTOR^Ser2481 then redistributes from diffuse cytosolic staining that partially colocalizes with the mitotic spindle during the early phases of mitosis to the furrow at the onset of cytokinesis. Like the bona fide chromosomal passenger proteins (CPPs) INCENP and Aurora B, phospho-mTOR^Ser2481 displayed noteworthy accumulation in the central spindle midzone and the midbody regions, which persisted during the furrowing process. Accordingly, double-staining experiments confirmed that phospho-mTOR^Ser2481 largely colocalized with CCPs in the midbodies. The CPP-like mitotic localization of phospho-mTOR^Ser2481 was fully prevented by the microtubule-depolymerizing drug nocodazole; mitotic traveling of phospho-mTOR^Ser2481 to the midbody during telophase and cytokinesis, where it appears to be integrated into the CPP-driven cytokinetic machinery, may therefore require dynamic microtubules. Although the Ser2448-phosphorylated form of mTOR was also found at high levels during M-phase in human cancer cells, we failed to observe a significant association of phospho-mTOR^Ser2448 with CCP-positive mitotic and cytokinetic structures. Our findings add phospho-mTOR^Ser2481 to the growing list of phospho-active forms of proteins belonging to the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus. Future studies should elucidate how the specific ability of phospho-mTOR^Ser2481 to spatially and temporally couple to the cleavage furrow and midbody region as a CPP-like protein can signal to or from adjacent signaling complexes and/or with the basic machinery of cell abscission.     

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.     

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.     

The role of S6K1 in ER-positive breast cancer

The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases, which also includes Akt and many others. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1). S6K1 is sensitive to a wide range of signaling inputs, including growth factors, amino acids, energy levels and hypoxia. S6K1 relays these signals to regulate a growing list of substrates and interacting proteins in control of oncogenic processes, such as cell growth and proliferation, cell survival and apoptosis and cell migration and invasion. Several lines of evidence suggest an important role for S6K1 in estrogen receptor (ER)-positive breast cancer. S6K1 directly phosphorylates and activates ERα. Furthermore, S6K1 expression is estrogenically regulated. Therefore, hyperactivation of mTORC1/S6K1 signaling may be closely related to ER-positive status in breast cancer and may be utilized as a marker for prognosis and a therapeutic target.     

MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis

Cadmium (Cd) may be accumulated in human body through long-term exposure to Cd-polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd-induced neurodegeneration, PC12 and SH-SY5Y cells were exposed to Cd. We observed that Cd-induced apoptosis in the cells in a time- and concentration-dependent manner. Cd rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c -Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd-induced apoptosis. Consistently, over-expression of dominant negative c- Jun or down-regulation of Erk1/2, but not p38 MAPK, partially prevented Cd-induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)-mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd-induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd-induced apoptosis. Down-regulation of mTOR by RNA interference also in part, rescued cells from Cd-induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd-induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd-induced neurodegeneration.     

4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspaselike domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.