The ever-evolving story of Rapamycin and the mTOR pathway

By Caitlin Davies

Rapamycin is a potent anti-fungal metabolite produced by a bacterial strain called Streptomyces Hygroscopius. The metabolite exhibits a macrocylic lactone structure and was first identified in a soil sample collected from Easter Island, or as natively known, Rapa Nui in 1972 which is where the inspiration for the metabolite’s name originated (Wullschleger, Loewith and Hall, 2006). Interest in the compound piqued when it was found to inhibit mammalian cell proliferation and the race to find the target and mechanism of action began. Subsequently, the target of Rapamycin was first discovered within mammalian cells by Sabatini et al (Sabatini et al., 1994) and separately by Schreiber et al. (Brown et al., 1994) in 1994 and was assigned to the phosphoinositide-3’ kinase (P13K)-related kinase family. The target is known today as the mammalian target of rapamycin/mechanistic target of rapamycin or mTOR for short, but it was originally referred to as RAFT1 or FRAP, names given by Sabatini and Schreiber respectively (Sabatini, 2017). It wasn’t until Abraham et al identified the target a few months after that the name we use today came into existence (Sabers et al., 1995), the inspiration for the name being drawn from similar kinases called TOR1 and TOR2 which had been identified in yeast (Hall, Heitman and Movva, 1991), (Livi et al., 1991), (Sabatini, 2017). In 2009, the HUGO Gene Nomenclature Committee accepted mTOR as the official name for the target and the previous names become obsolete (Sabatini, 2017).

Once the target of Rapamycin was discovered, the research effort moved onto unveiling mTOR’s function and Rapamycin’s mechanism of action. During the search for mTOR’s function, two distinct complexes containing mTOR as a catalytic subunit were discovered, named mTORC1 (Hara et al., 2002) and mTORC2 (Dos D. Sarbassov et al., 2004). The components of mTORC1 were revealed to be mLST8 (mammalian lethal with sec-13 protein 8 or alternatively known as G-protein B-subunit-like protein GBL), PRAS40 (proline-rich Akt substrate 40 kDa) and Raptor (regulatory-associated protein of mTOR) which acts as the scaffold protein that holds the complex together (Sabatini and Laplante, 2012). mTORC2 on the other hand was found to be composed of mLST8, mSin1 (mammalian stress-activated protein kinase-interacting protein 1), Protor (protein observed with Rictor) and Rictor (rapamycin insensitive companion of mTOR) which acts as the scaffold protein instead (Sabatini and Laplante, 2012). In terms of functions, more is understood about mTORC1 than mTORC2. mTORC1 is associated with regulating cell growth and proliferation through effects on protein synthesis, lipid synthesis and lysosome biogenesis whereas mTORC2 is associated with cell survival and cytoskeletal organisation (Wullschleger, Loewith and Hall, 2006) . mTORC1 is regulated by a higher number of extracellular and intracellular inputs than mTORC2 which include responding to nutrient levels, growth factors, oxidative stress, oxygen levels and amino acids (Sabatini, and Laplante, 2012). mTORC2 on the other hand responds to growth factors but does not respond to nutrient levels (Sabatini and Laplante, 2012). It is clear that mTOR signalling is crucial to the survival of mammalian cells.

With regards to Rapamycin’s mechanism of action, it was first elucidated in yeast by Heitman et al who demonstrated Rapamycin forming a complex with a cytosolic protein called FK506 binding protein 12 (FKBP12) which binds to a domain present on mTORC1 called the FK506-binding FRB domain, ultimately leading to the inhibition of cellular proliferation and growth (Heitman, Movva and Hall, 1991). A phenomenon which was later additionally demonstrated in mammalian cells in 1996 (Choi et al., 1996). The identification of Rapamycin’s mechanism of action was important as this led to Rapamycin being approved for use within a medical setting as an immunosuppressant (Dumont and Su, 1995). Later on after the discovery of hyperactive mTORC1 signalling present in the majority of cancers, Rapamycin began to be used as a chemotherapeutic agent as its effects on mammalian cellular proliferation and growth were noted once more (Xie, Wang and Proud, 2016).

Unfortunately, the use of Rapamycin to treat cancer does have disadvantages. Rapamycin does not inhibit all of the functions of mTORC1 thought to be due to the Rapamycin-FKBP12 complex binding to a mTOR domain adjacent to its catalytic kinase domain and this is believed to be responsible for its only modest efficacy (Xie, Wang and Proud, 2016). Additionally, Rapamycin treatment only affects mTORC2 indirectly after prolonged treatment. This is because the formation of the mTOR-Rapamycin-FKBP12 complex leads to gradual depletion of free mTOR levels, leading to reduced association between mTOR and Rictor which subsequently prevents the assembly of mTORC2 (Sarbassov et al., 2006). Furthermore, many malignancies have shown resistance to Rapamycin’s anti-proliferative effects through a number of different mechanisms which are still being investigated, limiting its efficacy further (Gruppuso, Boylan and Sanders, 2011).

The downsides of Rapamycin as a chemotherapeutic agent spurred the development of the next generation of mTOR inhibitors referred to as Rapalogs. These possess superior pharmacological characteristics such as increased aqueous solubility to facilitate oral administration (Xie, Wang and Proud, 2016). However, even Rapalogs have faced limited clinical efficacy and the approach has now moved forward to using Rapmaycin/Rapalogs alongside other drugs in a combination therapy in an attempt to inhibit any compensatory pathways or targets activated by the inhibition of mTOR feedback loops. The aim being to improve efficacy and delay the onset of drug resistance (Li, Kim and Blenis, 2014).

However, the puzzle of the mTOR pathway continues to evolve. Scientists began to question whether the composition of the established mTOR complexes differed between the types of somatic tissues and in 2016, the first of three papers claiming to have identified a third mTOR complex with a different composition was published. Smithson et al claimed to have identified GIT1 as a new novel mTOR binding-partner within brain neural tissues (Smithson and Gutmann, 2016). The complex did not contain either Raptor, Rictor, mLST8, PRAS40 or mSIN1 suggesting that GIT1 did not belong to either of the two previously established complexes (Smithson and Gutmann, 2016). In 2018, the second paper was published by Harwood et al., who stated that they had also found a third mTOR complex but this complex contained ETV7 and mTOR and was found using myeloid and lymphoid cell lines instead (Harwood et al., 2018). They also believed this complex to be a contributor to Rapamycin/Rapalog resistance in many different types of tumour, as tumour cell lines that lose mTORC3 expression become rapamycin-sensitive (Harwood et al., 2018). In 2019, the third paper was published by Nguyen et al., claiming to have additionally identified a third mTOR complex within a few human cancer cell lines composed of mEAK-7 and DNA-PKcs, however, this paper failed to rule out that this finding may be an mTORC2 variant due to the lack of a Rictor control when performing immunoprecipitations (Nguyen, Haidar and Fox, 2019). On the other hand, no such paper has been published by Sabatini et al., who are considered to be the leading experts within the field of mTOR signalling. In a personal communication to the author, Sabatini stated he did not believe a third complex existed, contradicting the previous papers discussed. 

To conclude, the story of the mTOR pathway is still being unveiled. As research moves on to developing further mTOR inhibitors that are capable of inhibiting both established mTOR complexes in order to improve efficacy and to fully elucidating the function of the mTOR complexes, it is likely we will continue to see novel research published within this field for a long time to come yet.

References:

Brown, E. J. et al. (1994) ‘A mammalian protein targeted by G1-arresting rapamycin-receptor complex’, Nature, 369(6483), pp. 756–758. doi: 10.1038/369756a0.

Choi, J. et al. (1996) ‘Structure of the FKBP12-Rapamycin Complex Interacting with Binding Domain of Human FRAP’, Science, 273(5272), pp. 239–242. doi: 10.1126/science.273.5272.239.

Dos D. Sarbassov et al. (2004) ‘Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton’, Current Biology, 14(14), pp. 1296–1302. doi: 10.1016/j.cub.2004.06.054.

Francis J. Dumont and Qingxiang Su (1995) Mechanism of action of the immunosuppressant Rapamycin. doi: 10.1016/0024-3205(95)02233-3.

Gruppuso, P. A., Boylan, J. M. and Sanders, J. A. (2011) ‘The physiology and pathophysiology of rapamycin resistance’, Cell Cycle, 10(7), pp. 1050–1058. doi: 10.4161/cc.10.7.15230.

Hall, M., Heitman, J. and Movva, N. (1991) ‘Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast’, Science, 253(5022), pp. 905–909. doi: 10.1126/science.1715094.

Hara, K. et al. (2002) ‘Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action’, Cell, 110(2), pp. 177–189. doi: 10.1016/s0092-8674(02)00833-4.

Harwood, F. C. et al. (2018) ‘ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer’, Science Advances, 4(9), p. eaar3938. doi: 10.1126/sciadv.aar3938.

Heitman, J., Movva, N. R. and Hall, M. N. (1991) ‘Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast’, Science, 253(5022), pp. 905–909. doi: 10.1126/science.1715094.

Li, Jing, Kim, Sang Gyun and Blenis, John (2014) Rapamycin: One Drug, Many Effects | Elsevier Enhanced Reader. doi: 10.1016/j.cmet.2014.01.001.

Livi, G. P. et al. (1991) ‘Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.’, Molecular and Cellular Biology, 11(3), pp. 1718–1723. doi: 10.1128/MCB.11.3.1718.

Nguyen, Joe Truong, Haidar, Fatima Sarah and Fox, Lucienne (2019) mEAK-7 Forms an Alternative mTOR Complex with DNA-PKcs in Human Cancer | Elsevier Enhanced Reader. doi: 10.1016/j.isci.2019.06.029.

Sabatini, David M. et al. (1994) ‘RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs’, Cell, 78(1), pp. 35–43. doi: 10.1016/0092-8674(94)90570-3.

Sabatini, David M (2017) Twenty-five years of mTOR: Uncovering the link from nutrients to growth | PNAS. Available at: https://www.pnas.org/content/114/45/11818#ref-12 (Accessed: 17 August 2020).

Sabatini, David M and Laplante, Mathieu (2012) mTOR Signaling in Growth Control and Disease | Elsevier Enhanced Reader. doi: 10.1016/j.cell.2012.03.017.

Sabers, C. J. et al. (1995) ‘Isolation of a Protein Target of the FKBP12-Rapamycin Complex in Mammalian Cells’, Journal of Biological Chemistry, 270(2), pp. 815–822. doi: 10.1074/jbc.270.2.815.

Sarbassov, D. D. et al. (2006) ‘Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB’, Molecular Cell, 22(2), pp. 159–168. doi: 10.1016/j.molcel.2006.03.029.

Smithson, L. J. and Gutmann, D. H. (2016) ‘Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival’, Genes & Development, 30(12), pp. 1383–1388. doi: 10.1101/gad.279661.116.

Wullschleger, S., Loewith, R. and Hall, M. N. (2006) ‘TOR Signaling in Growth and Metabolism’, Cell, 124(3), pp. 471–484. doi: 10.1016/j.cell.2006.01.016.

Xie, J., Wang, X. and Proud, C. G. (2016) ‘mTOR inhibitors in cancer therapy’, F1000Research, 5. doi: 10.12688/f1000research.9207.1.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s