Diverse signaling mechanisms of mTOR complexes: mTORC1 and mTORC2 in forming a formidable relationship

Abstract

Activation of Mechanistic target of rapamycin (mTOR) signaling plays a crucial role in tumorigenesis of numerous malignancies including glioblastoma (GB). The Canonical PI3K/Akt/mTOR signaling cascade is commonly upregulated due to loss of the tumor suppressorm PTEN, a phosphatase that acts antagonistically to the kinase (PI3K) in conversion of PIP2 to PIP3. mTOR forms two multiprotein complexes, mTORC1 and mTORC2 which are composed of discrete protein binding partners to regulate cell growth, motility, and metabolism. These complexes are sensitive to distinct stimuli, as mTORC1 is sensitive to nutrients while mTORC2 is regulated via PI3K and growth factor signaling. The main function of mTORC1 is to regulate protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. On the other hand, mTORC2 is responsive to growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases like Akt and SGK and it also plays a crucial role in maintenance of normal and cancer cells through its association with ribosomes, and is involved in cellular metabolic regulation. mTORC1 and mTORC2 regulate each other, as shown by the fact that Akt regulates PRAS40 phosphorylation, which disinhibits mTORC1 activity, while S6K regulates Sin1 to modulate mTORC2 activity.

Allosteric inhibitors of mTOR, rapamycin and rapalogs, remained ineffective in clinical trials of Glioblastoma (GB) patients, in part due to their incomplete inhibition of mTORC1 as well as unexpected activation of mTOR via the loss of negative feedback loops. In recent years, novel ATP binding inhibitors of mTORC1 and mTORC2 suppress mTORC1 activity completely by total dephosphorylation of its downstream substrate pS6K^Ser235/236, while effectively suppressing mTORC2 activity, as demonstrated by complete dephosphorylation of pAKT^Ser473. Furthermore by these novel combined mTORC1/mTORC2 inhibitors reduced the proliferation and self-renewal of GB cancer stem cells. However, a search of more effective way to target mTOR has generated a third generation inhibitor of mTOR, “Rapalink”, that bivalently combines rapamycin with an ATP-binding inhibitor, which effectively abolishes the mTORC1 activity. All in all, the effectiveness of inhibitors of mTOR complexes can be judged by their ability to suppress both mTORC1/mTORC2 and their ability to impede both cell proliferation and migration along with aberrant metabolic pathways.

Introduction

Mechanistic target of Rapamycin (mTOR; AKA Mammalian target of Rapamycin) is a large protein product of 289kDA and a product of a single gene localized to the chromosome 1p36.2. The Rapamycin was first discovered in the soil bacterium Streptomyces hygroscopicus present in soil samples from the Easter Island (also locally termed Rapa Nui) in the 1970s, and found to have potent antifungal activity. The target of rapamycin (TOR) was identified in the budding yeast Saccharomyces Cerevisiae in 1991 (Heitman et al., 1991). mTOR, a serine-threonine protein kinase, belongs to phosphoinositide 3-kinase-related kinase (PI3K) family with homologs in all eukaryotes (Laplante and Sabatini, 2012; Russell et al., 2011). Structurally, the N-terminus of mTOR contains several huntingtin elongation factor 3 protein phosphatase 2A TOR1 (HEAT) repeats to mediate the majority of interactions with other proteins. The C- terminus contains FKB-binding domain and a kinase domain that places it in the phosphatidylinositol-3-kinase (PI3K) family. The PI3K/Akt/mTOR signaling axis regulates various physiological functions such as cell cycle progression, transcription, mRNA translation, differentiation, apoptosis, autophagy, motility, and metabolism (Guertin and Sabatini, 2009; Jacinto et al., 2004; Laplante and Sabatini, 2012; Saxton and Sabatini, 2017). Hyperactivation of mTOR activity results in an increase in cell growth, and can cause some cell types to enter the cell cycle (Laplante and Sabatini, 2012). Constitutive activation of mTOR via single point mutations has been shown in many cancers including adenocarcinoma and renal cell carcinoma (Sato et al., 2010).

Aberrant PI3K/Akt signaling has been reported in many human cancers, which is a major cause of hyperactivation of the mTOR pathway contributing to both cancer pathogenesis as well as therapy resistance. Loss-of-function due to mutations in tumor suppressors, such as phosphatase and tensin homolog (PTEN), tuberous sclerosis 1/2 (TSC1/2), neurofibromin 1/2 (NF1/2), or oncogenic mutations in KRAS, PIK3CA, or AKT are the most common causes of mTOR signaling hyperactivity. Moreover, constitutive activation of the PI3K/Akt/mTOR signaling network is seen in patients with acute myeloid leukemia (AML) (Martelli et al., 2007). Additionally, studies suggest that tumor suppressors including PTEN and p53 may regulate stem cell populations by controlling self-renewal of cancer stem cells (Korkaya and Wicha, 2007).

It has been demonstrated that mTOR signaling is aberrantly regulated in Glioblastoma (GB), leading to abnormalities in protein synthesis, metabolism and motility, thereby resulting in uncontrolled growth and dissemination (Jhanwar-Uniyal et al., 2011, 2015a, 2015b). GB is WHO defined Grade IV astrocytoma, is the most common and aggressive CNS malignancy. Despite current treatment modalities survival rate remains dismal. As such, GB tumors are mostly PTEN and/or p53 deregulated (Ohgaki and Kleihues, 2009). Mutations of PTEN are found in approximately 70–90% of GB. Accordingly, there is up-regulation of the PI3K/Akt pathway in GB (Hay and Sonenberg, 2004; Phillips et al., 2006). The role of Akt in formation of gliomagenesis has been demonstrated in animal models and along with its downstream target mTOR, controls distinct cellular functions (Guertin et al., 2009; Holland, 2001). Because of overactivation of mTOR pathways, Rapamycin and its analogue, have been considered for the treatment of many cancers (under the trade name Sirolimus), as an FDA-approved immunosuppressant and chemotherapeutic agent (see Fig. 1).