The expanding relevance of nuclear mTOR in carcinogenesis

Abstract

Deregulated mTOR signaling drives the growth of various human cancers, making mTOR a major target for development of cancer chemotherapeutics. The role of mTOR in carcinogenesis is thought to be largely a consequence of its activity in the cytoplasm, resulting in increased translation of pro-tumorigenic genes. However, emerging data locate mTOR in various subcellular compartments, including Golgi, mitochondria, endoplasmic reticulum and the nucleus, implying the presence of compartment-specific mTOR substrates and functions. Efforts to identify mTOR substrates in these compartments and the mechanisms by which mTOR recruits these substrates and affects downstream cellular processes will add to our understanding of the diversity of roles played by mTOR in carcinogenesis.

Mammalian target of rapamycin, mTOR, is a PI3K-related Ser/Thr kinase that regulates cell growth, proliferation and survival by integrating various external signals into intracellular machinery via two known mTOR-containing complexes, mTOR complex 1 (mTORC1) and mTORC2.^1,2 Aberrant mTOR signaling is often associated with human cancers³ and is implicated in a wide range of pathogenic conditions and processes, including metabolic diseases, epileptogenesis and aging.^1,4,5 Because of its primary role in translation, mTOR has been viewed as an exclusively cytoplasmic protein. However, this view is changing as a result of emerging data showing that mTOR resides in numerous cellular compartments, including the Golgi apparatus,^6,7 the endoplasmic reticulum,^7,8 the mitochondrial outer membrane,⁹ lysosomes¹⁰ and the nucleus.^11–14 Compartment-dependent substrate specificity and functions have been shown; for example, mTORC1 has been shown to directly interact with YY1, a zinc-finger GLI-Kruppel class of transcription factor that regulates the expression of mitochondrial genes.¹⁵ Inhibition of mTOR leads to defective YY1 activation and interaction with PGC-1α (peroxisome-proliferator-activated receptor coactivator), which then inhibits the transcriptional control of mitochondrial oxidative function.¹⁵ mTOR has also been shown to accumulate at a specific location in Golgi together with autophagic components during Rasinduced senescence, a phenomenon that was shown to be associated with interleukin-6/8 synthesis.⁶ The upstream signals and mechanisms leading to the spatial arrangement of mTOR, the functions of mTOR in these cellular compartments and whether mTOR forms complexes distinct from mTORC1 and mTORC2 are all yet to be fully understood. Clearly, identification of new compartment-specific mTOR substrates will expand our view of the functional diversity of mTOR signaling.

mTOR and its Nuclear Activities: A Link to Epigenetic Regulation of Cancer Cell Survival

The nuclear activities of mTOR have been associated with increases in its cytoplasmic functions in the control of protein translation.¹⁶ Shuttling of mTOR between cytosol and the nucleus has been shown to increase its kinase capacity in the cytoplasm.^13,17 mTOR binding to the promoters of rDNA (rDNA) and tRNA (tRNA) genes have been shown to increase protein biosynthetic capacity.¹⁶ mTOR was also shown to interact with TFIIIC, a transcription factor that binds to pol III promoters.¹⁸ Binding of mTOR to TFIIIC may allow tRNA and 5S rRNA expression, resulting in enhanced protein synthesis in response to nutrients and growth factors.¹⁸ In another example, sequestration of mTOR in the nucleus through physical interaction with promyelocytic leukemia protein (PML) has been shown to suppress the synthesis rate of hypoxia-inducible factor 1α (HIF-1α).¹⁹ However, accumulating data indicate additional role(s) of mTOR in the nucleus. A potential link between mTOR signaling and increased mutagenesis was shown in a yeast system in which TOR signaling is required for S-phase progression and yeast cell survival in response to genotoxic stress.²⁰ Levels of dNTPs have been shown to increase following DNA damage, and this increase, which is necessary for the function of error-prone translesion DNA polymerases,²⁰ dramatically promotes cell survival—at the cost, however, of an increased mutation rate.²¹ Thus, it is postulated that increased mutation rates accompanying TOR-dependent cell survival in response to DNA damaging agents may contribute to the acquisition of chemotherapeutic drug resistance.²⁰ Given the current recognition that there are significant levels of nuclear mTOR in cancer cells,¹² this has important implications in cancers where chemoresistance is a major impediment to successful treatment outcomes. The question is, does nuclear mTOR activity correlate with chemoresistant cancer cell survival? Using A431 human squamous cell carcinoma (SCC) cells, we recently demonstrated that DNA damage-induced nuclear mTOR signaling, via inhibitory phosphorylation of Sirtuin 1 (SIRT1), a NAD-dependent deacetylase, mediates chemoresistant cell survival.²² The direct phosphorylation-dependent inhibition of SIRT1 by mTOR increases expression of the anti-apoptotic Bcl-2 family member, Bfl-1/A1, via increased p65 NFκB acetylation, which renders cells resistant to DNA damage-induced apoptosis.²² SIRT1 forms complexes with mTORC1 in response to treatment with genotoxic agents (Fig. 1);²² furthermore, this complex is persistently sustained in chemoresistant variants of A431 cells (Fig. 2). The formation of mTORC1-SIRT1 complexes, mTORC1-mediated signaling and the resulting apoptosis resistance are blocked in the presence of rapamycin (Figs. 1 and 2). It is of interest that nuclear mTORC1 signaling is associated with a p53-independent premature senescence-like cell cycle arrest in A431 cells.²² mTOR activation was recently shown to favor senescence in p53-arrested cells by fostering the ongoing cell growth in the arrested cells, while inhibition of mTOR leads to quiescence.²³ However, in response to genotoxic stress, cells that are deficient in p53 and already have active cytoplasmic mTOR signaling, as in the case of A431 cells, may transition from an mTOR-regulated growth state to a survival state. We show, in A431 cells, that this transition requires the nuclear translocation of cytoplasmic mTOR.²² Given the nuclear activities of mTOR in the control of protein biosynthetic capacity,¹⁶ it remains to be elucidated whether nuclear mTORC1 signaling regulates, in part, senescence induction. Furthermore, p53-deficient human colon cancer cells were shown to have increased resistance and anchorage-independent growth upon mTOR activation, which involved the activation of a DNA damage response,²⁴ raising questions regarding the cellular consequences of genotoxic stress and mTOR activation—cytoplasmic vs. nuclear—and whether nuclear mTORC1 signaling requires a p53-independent environment. Moreover, SIRT1 deacetylates diverse proteins, including histones, transcription factors and cofactors. Whether persistent mTORC1-dependent repression of SIRT1 deacetylase leads to broad modifications in chromatin function through acetylation of histones as well as by promoting alterations in the methylation of histones and DNA are important issues to be addressed. It is noteworthy that mTOR has been shown to form a functional transcriptional complex with CBP/p300, a coactivator of HIF-1α, leading to increased transcriptional activity of HIF-1α.²⁵ mTOR has also been shown to bind to the promoters of RNA polymerase I- and III-transcribed genes in β-TC6 cells.²⁶ These data suggest that mTOR may have specific roles in gene regulation, and that proteins and substrates with which mTOR interacts may play functional roles in mediating epigenetic modifications.

Figure 1.

Inhibition of DNA damage-induced formation of mTOR-SIRT1 complex by rapamycin in A431 cells. Direct detection of nuclear mTOR-pS47 SIRT1 complexes in response to genotoxic agents (DOX, doxorubicin, 1 µM; CIS, cisplatin, 20 µM) and in the presence of rapamycin (RAP),assessed by in situ proximity ligation technology (Duolink in situ PLA kit, Olink Bioscience), which allows for detection of native complexes with minimal cellular disruption, using anti-mTOR and anti-pS47 SIRT 1 antibodies and species-specific secondary PLA probes attached with a unique short DNA strand, followed by DNA strand ligation and rolling circle amplification. Red, mTOR-pS47 SIRT 1 interaction; green, actin; blue, dapi. A signal is obtained only if oligonucleotides coupled to anti-mTOR and anti-pS47 SIRT 1 antibodies are sufficiently close to allow enzymatic ligation; detection then relies on amplification from the ligated template, followed by hybridization with a fluorescent probe.¹⁸

Figure 2.

Sustained mTOR-SIRT1 complexes in chemoresistant A431 variants. Chemoresistant A431 clones (DR6 and DR7) derived from A431 cells treated with 1 µM DOX for 48 h then cultured in the absence of DOX for >30 d have reduced SIRT 1 activity (A, yellow bars) and are resistant to cisplatin (A, blue bars), retained mTOR-SIRT1 interaction (B) and SIRT1 phosphorylation and Bfl-1/A1 expression (C).

Putative Nuclear Substrates Identified by TOS Motif

Perhaps the most important structural element in the cellular processes mediated by mTORC1 signaling is a highly conserved TOS (TOR signaling) motif present in mTOR substrates. mTORC1 consists of mTOR, raptor and mLst8/GβL. The TOS motif allows substrates to bind to mTOR through raptor.^27,28 The TOS motif is present in known mTOR substrates, such as S6K1, 4EBP1 and HIF-1α^25,27–29. In addition to SIRT1,²² STAT3 (signal transducer and activator of transcription 3) is also known to contain two putative TOS motifs, although the importance of these has not been confirmed experimentally.³⁰ Identification of new mTOR substrates in the nucleus will likely reveal additional role(s) of mTOR in the nucleus. A search of the protein sequence database for proteins containing known variations of TOS motifs (FDVEL, FDIEL, FEMDI, FEMDV, FDIDL, FDVDL, FEIDL) identified 39 candidates containing at least one TOS motif. These include proteins located in various subcellular structures including the cell membrane, endoplasmic reticulum and mitochondria as well as secreted proteins. Table 1 shows a list of candidates localized in the nucleus. A majority are proteins involved in transcription regulation and epigenetic modification; they include hEaf6, JARID1B (KDM5B/PLU-1/RBP2-H1) and ATF6α. hEaf6 is a component of HBO1 HAT complex, which is likely responsible for the majority of histone H4 acetylation, and plays key roles in gene regulation and DNA replication.³¹ JARID1B (KDM5B/PLU-1/RBP2-H1) is a member of the highly conserved family of jumonji/ARID1 (JARID1) histone 3 K4 (H3K4) demethylases and was recently shown to be essential for continuous melanoma growth.³² ATF6α is a transcription factor essential for the adaptation of dormant cells to chemotherapy and nutritional stress. It was recently shown to be critical for survival of quiescent squamous carcinoma cells via the ATF6α-Rheb-mTOR pathway.³³ Interestingly, three putative mTOR phosphorylation sites are present in ATF6α (Table 1), suggesting possible translational modification of ATF6α by mTORC1. The physical interaction between these proteins and mTORC1, specifically with raptor, needs further investigation. Furthermore, studies into the functional implication of these interactions will be important for our appreciation of the full engagement of mTORC1 in various cellular activities, such as chromatin modification, transcription regulation, metabolism, hypoxia and regulation of cell cycle progression, all of which contribute to the relevance of mTOR in carcinogenesis.

Table 1.

Nuclear proteins containing putative TOS motifs

Gene ID	Gene Name	TOS Motif Sequence	Putative mTOR phosphorylation sites (R/K)X(R/K)XX(S*T*)	Subcellular Location	Functional Description
10765	KDM5B	(aa1037) FDVEL	153T, 298S, 300S, 304T, 754S	Nucleus	Lysine (K)-specific demethylase B Histone demethylase that demethylates ‘Lys-4’ of histone H3; A transcriptional co-repressor for FOXG1B and PAX 9
23216	TBC1D1	(aa1037) FEMDI	595T, 640S	Nucleus	TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1, Diabetes; Putative GTPase-activating protein for Rab family protein(s); Putative role in the cell cycle and differentiation
27161	EIF2C2	(aa45) FEMDI	None	Nucleus/Cytoplasm	Eukaryotic translation initiation factor 2C
29945	ANAPC4	(aa744) FEMDI	None	Nucleus	Anaphase-promoting complex subunit 4
4089	SMAD4	(aa329) FEMDV	None	Nucleus/Cytoplasm	SMAD family member 4
64769	MEAF6	(AA135) FEIDL	None	Nucleus/Nucleolus	MYST/Esa1-associated factor 6, HAT Component of the HBO1 complex, which has a histone H4-specific acetyltransferase activity
57178	ZMIZ1	(aa69) FDLDL	849S	Nucleus speckle/Cytoplasm	Zinc finger, MIZ-type containing 1; Retinoic acid-induced protein 17, PIA S-like protein Zimp10
22926	ATF6	(aa62) FDLDL	373S, 501T, 565T	Nucleus/Endoplasmic reticulum	Cyclic AMP-dependent transcription factor AT F-6 alpha; Binds to the endoplasmic reticulum stress response elements
64344	HIF3A	(AA96) FVMVL	12T, 526S	Nucleus/Cytoplasm	Hypoxia-inducible factor 3alpha subunit; Suppresses hypoxia-inducible expression of HIF1A and EPAS1

aa, the position of the first amino acid of TOS motif.