Post on 15-Aug-2015
transcript
mTOR Transcriptional Regulation by Nrf2 by
Gabriel Bendavit
Principal Investigator
Dr. Gerald Batist
Submitted
April 2015
Department of Experimental Medicine McGill University Montreal, Quebec Canada
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of
MASTER OF SCIENCE
© Gabriel Bendavit 2015
ABSTRACT___________________________________________
Nuclear Erythroid 2-related factor (Nrf2) is a master transcription factor, and thereby is a
major regulator of cytoprotective responses to oxidative and electrophilic stress. This is
accomplished by recognition and binding to antioxidant response elements (ARE) in the
promoter of target genes, which triggers activation of genes encoding proteins that range
from drug metabolizing enzymes II family to drug efflux pumps. Numerous studies have
shown direct and indirect interactions between Nrf2 and different signaling pathways
including components of the Pi3K/AKT/mTOR signaling pathway.
The potential for a role for Nrf2 in cancer metabolism directed our study towards its
impact on mTOR, the metabolic maestro of this pathway. We observed that modulation
of Nrf2 levels in lung cancer cell lines regulates mTOR protein levels. In order to verify
if this regulation is present at the transcriptional level, we performed both RT-qPCR
analysis and a luciferase assay to functionally analyze the promoter region of this gene
for the presence of functional ARE motifs. We found that transcription of the Mtor
protein was directly modulated by Nrf2 levels in the non small cell lung cancer cell line
A549, as well as in the non-transformed human cell line HEK293. Mutation of the ARE
sequence in the promoter of the mTOR gene, decreased the effect of Nrf2 on an ARE-
luciferase construct’s activity by more than 50%. The physical binding of Nrf2 with the
ARE sequence in mTOR promoter was further confirmed in vitro via DNA pull-down
and EMSA and in vivo via in a ChIP assay. Additional studies show intimate interactions
between other components of the PI3K pathway and Nrf2.
RÉSUMÉ_____________________________________________
Nuclear Erythroid 2-related factor (Nrf2) est un facteur de transcription qui joue un rôle
primordial dans la défense cellulaire contre les stress oxydatif et électrophile. Il régule la
transcription en se fixant sur les éléments de réponse antioxidative (ARE) impliqués dans
la résistance et le métabolisme des médicaments. En outre, plusieurs études montrent des
intercactions directes ou indirectes de Nrf2 avec la voie de signalisation
Pi3K/AKT/mTOR
En se basant sur le rôle de Nrf2 dans le métabolisme du cancer et son interaction avec la
voie de signalisation mTOR, nous avons formulé l'hypothèse selon laquelle Nrf2
régulerait les niveaux de mTOR. Tout D'abord, nous avons observé que la modulation
des niveaux de Nrf2 dans les cellules du cancer du poumon régule mTOR au niveau
protéique. Ensuite, l'utilisation de la PCR quantitative à temps réel et l'essai de
transactivation sur un vecteur rapporteur luciférase contenant le promoteur de mTOR
nous a permis de montrer que Nrf2 régule mTOR au niveau transcriptionnel dans les
cellules HEK293 et A549.
D'autre part, l'introduction des mutations au sein de la séquence de l'ARE du promoteur
de mTOR réduit l'activité luciférase par plus de 50%. Ceci confirme que malgré sa
séquence différente de la séquence consensus, cet ARE est requis pour la liaison et la
régulation de l'expression de mTOR.
l'interaction physique de Nrf2 avec l'ARE du promoteur de mTOR a été confirmé in vitro
par DNA pull down et par retard sur gel (EMSA) et in vivo par immunoprécipitation de la
chromatine. En conclusion, nos résultats suggèrent que le rôle de Nrf2 dans la sensibilité
aux traitements cytotoxiques pourrait découler de sa capacité à réguler l'expression de
mTOR.
TABLE OF CONTENTS_________________________________
Abstract........................................................................................................................... 2
Table of Contents......................................................................................................... 4
1. Introduction......................................................................................................... 7
1.1 Nrf2 and the cap ‘n’ collar (Cnc) family........................................................... 7
1.1.1 Discovering Nrf2………………………................................................... 7
1.1.2 Nrf2 molecular structure ……................................................................... 8
1.2.3 Nrf2 regulation………............................................................................... 9
1.2 Cytoprotective apparatus of cellular detoxification ...................................... 11
1.3 Antioxidant Response Element (ARE)………………………………………12
1.3.1 Discovering the ARE ………………………....…..………....................12
1.4 Nrf2 clinical relevance..………………………………………...................... 13
1.4.1 Nrf2 and carcinogenesis ………………................................................. 14
1.5 Nrf2 cross talk with various pathways involved in cancer………...……….. 15
1.6 The PI3K/Akt/mTOR pathway……………………………………………... 16
1.6.1 Nrf2 interactions with the PI3K pathway….…….……...……..…..….. 18
1.6.2 Clinical relevance of the interaction between Nrf2 and the Pi3K/AKT pathway…………………………………………………………………………. 18
1.7 Nrf2 enhance the PI3K pathway in systems with high metabolic state.……. 19 1.8 mTOR………………………………………………….………………….... 20
1.8.1 mTORC1………………………………………...………..…………... 20
1.8.2 mTORC2……………………………………………..……………….. 21 1.9 Role of Nrf2 on mTOR expression ……..………...…...…………………... 22
2. Hypothesis.......................................................................................................... 22
3. Materials and Methods.................................................................................. 23
3.1 Cell Lines and Tissue Culture/ Transient Transfection…….......................... 23
3.2 Western blot …………………………………………..…..………..………. 24
3.3 Quantitative RT-PCR…………………………………………………………........ 25
3.4 Bioinformatic Analysis………………….…………………………………. 25
3.5 Molecular Cloning and Vector Construction………….……………………. 25
3.6 Nrf2 modulation …………………………………………………..…........... 26
3.7 Luciferase assay constructs……………………………………………...….. 26
3.8 Luciferase Assay…………………………………………….……………… 27
3.9 Electrophoretic Mobility Shift Assay (EMSA)……………………............... 28
3.10 DNA Pull-Down Assay ………….………………………………………... 28
3.11 Chromatin immunoprecipitation ………………………………………..… 29
4. Results................................................................................................................. 30
4.1 Nrf2 modulates mtor expression in A549 cells……..………….….………... 31
4.1.1 mTOR expression when Nrf2 is up-regulated………..……………… 31
4.1.2 mTOR expression when Nrf2 is down-regulated…………………….. 33
4.2 Functional ARE present on mTOR promoter activates its transcription in Nrf2 inducible condition……………………………………………………………… 34
4.3 Nrf2 binds to mTOR promoter region at basal conditions in vitro…………. 36
4.3.1 Nrf2 binding to mTOR promoter region decreases in Nrf2 silencing conditions……………………………………………………………………….. 39
4.3.2 Nrf2 binds to mTOR promoter in vivo at inducible conditions…........ 40
4.4 Expression analyses of the other elements of PI3K pathway due to Nrf2 modulation ……………………………………………………………………... 41
4.4.1 TSC2, S6K and AKT expression when Nrf2 is up-regulated….…… 42
4.4.1.1 TSC2 is a potential indirect Nrf2 transcriptional target at inducible conditions on H460 cells ……………………………………………….. 42
4.4.1.2 At Nrf2 inducible conditions AKT is a possible indirect Nrf2 transcriptional target on H460 cells and posttranslational target on A549 cells……………………………………………………………………... 43
4.4.2 TSC2, S6K and AKT expression when silencing Nrf2…...……..…… 49
4.4.2.1 TSC2, S6K and AKT may be affected post translationally, when Nrf2 is silenced ………………………………………………………… 49
5. Discussion & Conclusions................................................................................. 53
6. Future Directions................................................................................................. 63
7. Acknowledges........................................................................................................ 66
8. References.............................................................................................................. 66
9. Appendix................................................................................................................ 77
1. INTRODUCTION_____________________________________________________
1.1 Nrf2 and the cap ‘n’ collar (Cnc) family
Nrf2 is a basic leucine zipper (bZIP) transcription factor from the cap ‘n’ collar (Cnc)
family. The Cnc domain has 43 conserved amino acids located N-terminal to the DNA
binding domain. Prior to interaction with their target genes, the Cnc family of
transcription factors binds to Maf-recognition elements (MAREs), also known as the
erythroid transcription factor NF-E2 binding sequence(1). Maf (musculo-aponeurotic
fibrosarcoma oncogene) are a family of proteins that lack transcriptional activation
domains. In the nucleus CNC factors function via heterodimerizing with small Maf
proteins, which provide high affinity, sequence-specific DNA-binding activity of the
CNC factors to the MARE element(2).
The Cnc protein family is composed of SKN-1 (Skinhead family member 1) in
Caenorhabditis elegans and Cnc in Drosophila. In vertebrates this family is represented
by, p45 NFE2 subunit(3) and the NFE2-related factors, known as “Nrf” proteins,
Nrf1(NFE2L1/LCRF1/TCF11)(4), Nrf2(NFE2L2) (Itoh et al., 1995)(5), and Nrf3
(NFE2L3)(6). Bach1 and Bach2 (7) are other members of this family, witch however have
no transactivation capacity and instead function as transcriptional repressors. Bach1 is a
truncated isoform of Nrf1, while Bach2 is a caspase-cleaved form of Nrf2. The p45 NFE2
acts during development and is present only in hematopoietic progenitor cells. Besides
their role in early development, the Nrf proteins have a broad and sometimes overlapping
function as stress-activated transcription factors.
1.1.1 Discovering Nrf2
Nrf2 was first isolated and characterized in 1994 by Moi et al,(8) who identified closely
regulated proteins of erythroid-derived 2 (NF-E2). NF-E2, a member of the family of
bZIP transcription factors is a dimeric protein involved in the regulation of the β- globin
gene expression in hematopoietic cells. Nrf2 was named for its ability to bind to the
nuclear factor, NF-E2/ activating protein 1 (AP-1) repeat in the promoter of the β -globin
gene. Tandem Binding of Nrf2 to NF-E2/ AP1 was achieved via expression cloning of
the consensus sequence (5'GCACAGCAATGCTGAGTCATGATGAGTCATGCTG-3')
in K562 erythroid cell line. This repeat sequence is a oligonucleotide containing double-
strand concatemers of the tandem NF-E2/ AP1 repeat of the β- globin locus control
region, DNase I-hypersensitive site 2 (HS2).
1.2.2 Nrf2 molecular structure
The Nrf2 protein, has a molecular weight ranging from 95 to 110 kDa(9), and is
composed of 605 amino acids with 6 functional domains called Neh1-6 (Nrf2-ECH
<chicken Nrf2> homologous domain). The Neh1 holds the CNC homology region and a
basic-leucine zipper domain. It is responsible for heterodimerisation between Nrf2 and
small Maf proteins .The C terminal Neh3 motif is also responsible for Nrf2
transactivation activity (10) The Neh4 and Neh5 are conserved acidic domains that interact
with CBP [CREB cyclic AMP- response element binding protein (CREB) binding
protein], and are responsible for Nrf2 transcription activation strengths(11). Neh6 is a
serine-rich conserved region and serves as a target for a GSK 3 mediated phosphorylation
and consequently proteasomal degradation via ubiquitination(12).
Neh2 is a composite domain that is structurally divisible into two subregions. The
carboxy-terminal of Neh2 (amino acid residues 33–73) is hydrophilic and with no present
functional importance, while the amino-terminal region of Neh2 has 32 amino acids,
which are rich in hydrophobic residues, and shows conservation with Nrf1 and the C.
elegans Skn-1. It is an important functional domain, working as a negative regulator of
Nrf2, proved via domain deletion by Itoh et al(13). They also identified Kelch-like ECH-
associated protein1 (Keap1) responsible for post translational control of Nrf2.
Keap1 is an actin-binding cytoplasmic protein with four main domains, a intervening
region (IVR), double glycine repeat (DGR), C-terminal region (CTR) and broad
complex–tramtrack–bric-a-brac (BTB) domain. The DGR domain, also called Kelch
domain owing to its homology with Drosophila Kelch protein, is important for the
interaction with Nrf2 and for binding to actin. The BTB domain, present in Keap1 C-
terminus, is required for Nrf2 cytoplasmic sequestration and is involved in dimer
formation(14). The IVR domain, which is cysteine-rich protein with 27 cysteine residues,
is important for its reactivity to electrophilic and oxidative stimuli. In the presence of
oxidative stress 10 of these cysteines are activated by positively charged amino acids(15),
which leads to conformational changes in Keap1.
1.2.3 Nrf2 regulation
Keap1 is an important interacting protein of Nrf2 and they form a “hinge and latch”
structure with one another as shown by X-ray crystallography(16). The “hinge” structure is
formed due to a high-affinity interaction of ETGE motif, a stretch of four amino acids
present in the Neh2 domain of Nrf2, with keap1 kelch domain. While the “latch”
structure is generated via low-affinity interaction of DLG motif of nrf2-neh2 domain with
other keap1 monomers(17).
Under basal conditions, the redox–sensitive protein, Keap1 binds Nrf2 to form a
Keap1/Nrf2 complex, and anchors it in the cytoplasm. This cytoplasmic localization was
proved by confocal laser microscopic immunohistochemical analysis, where Keap1 was
shown to be tethered to the actin cytoskeleton(18). As others broad complex–tramtrack–
bric-a-brac (BTB)-containing proteins, Keap1 is an adaptor protein for the Cullin 3
ubiquitin E3 ligase (Cul3) which is a scaffold protein in the E3 ligase complex and forms
a catalytic core complex together with roc1/rbx1/Hrt1.The cognate E2 enzyme is then
recruited by Roc 1. This way, Nrf2 is specifically targeted (Lawah Zellers) for
degradation by the ubiquitin-proteasome pathway by 26 S proteasome(14).
In situations of oxidative stress, Keap1 undergoes conformational changes, which result
in the breakdown of the Nrf2-Keap1 complex. This occurs due to the difference in
affinity of “hinge” and “latch,” interactions, which have a difference of 2 orders of
magnitude, caused by the variance in the number of electrostatic interactions between
each domain and Keap1. This difference in affinity, weakens the interaction of the DLG
motif leading to the Nrf2-Keap1 complex disruption(17,19). This culminates in the release
of Nrf2 and its translocation to the nucleus, where it accumulates and activates the
cytoprotective program. Prior binding to its target genes, Nrf2 forms a heterodimer with
members of the small Maf family. This hetero-dimerization happen in the Nrf2 Neh1
domain. The complex Nrf2/small Maf then binds antioxidant response elements (AREs)
localized in the promoter region of its target genes(20).
Apart from the Keap1 mechanism of post-translational regulation of Nrf2, it is known
that some kinases, such as p38 kinase (21)and PTEN, can inhibit Nrf2. Kensuke Sakamoto
et al(22) showed via chromatin immunoprecipitation of Jurkat human leukemia, baring a
PTEN mutation, that the PI3K inhibitor LY294002 blocks CBP and Nrf2 recruitment to
ARE while it releases Bach1 to ARE. Glycogen synthetase kinase 3 (GSK-3ß) is also a
Kinase that can inhibit Nrf2.(12,23,24)
The serine/threonine GSK-3ß protein regulates glycolytic metabolism and directs the
ubiquitination and proteasomal of a variety of transcription factors(24). GSK-3ß is
involved in metabolic processes such as glycogen metabolism, Wnt signaling and
sensitization to oxidative-stress-mediated apoptosis. GSK-3ß is negatively regulated by
the Ser/Thr kinase Akt(25). AKT phosphorylates GSK-3ß’s Ser-9 in its pseudosubstrate
domain which inactivates GSK-3ß and consequently inhibits apoptosis. In order to
understand the mechanistic connection between the phase II genes’ cyto protection
against oxidative stress and the PI3K survival pathway, Salazar et al (23) focused on
control of nuclear Nrf2 accumulation. They suggested that Nrf2 was negatively regulated
via GSK-3ß phosphorylation in the nucleus post-translation. This study found that Nrf2
contains a consensus sequence for GSK-3ß phosphorylation (S/T)XXX(S/T) which was
confirmed by both immunocytochemistry and subcellular fractionation analyses. In a
following study by Rada P et al(24), it was demonstrated in mouse, that GSK-3ß acts as an
adapter protein for Nrf2 by phosphorylating a group of Ser residues in its Neh6 domain
and consequently targeting it to the SCF/ ß -TrCP SCF protein.
There is thus evidence for interaction between elements of the PI3Kinase pathway and
Nrf2 transcription factor. To date that data demonstrates regulation of Nrf2 by proteins
such as p38 Kinase, PTEN and GSK-3ß
1.2 Cytoprotective apparatus of cellular detoxification
In normal physiological conditions, nuclear factor NRF2 is essential for cell homeostsis
against endogenous and exogenous redox stress. This master cytoprotective transcription
factor is responsible for the activation of phase II detoxifying enzymes, antioxidants,
phase III drug efflux pumps and transporters(26).
The cytoprotective apparatus of cellular detoxification has been stratified into 3
categories phase I, II and III drug metaboling enzymes (DMEs). The phase I and II
enzyme systems are localized in the endoplasmic reticulum (ER) while the phaseIII is
present in the cytoplasmic membrane.(27) Phase I is composed of cytochrome
P450s(CYPs) gene superfamily. These large hydrophobic organic molecules are
responsible for oxidation and reduction by introducing polar functional groups into
nonpolar molecules. This group of enzymes are regulated by, ligand activated, Aryl
hydrocarbon receptor (AHR) transcription factor. DNA sequences called xenobiotic
response elements (XREs) are present in the promoter region of Phase I DMEs and are
essential for the regulation of these classes of enzymes. XREs are the target regions for
AHR binding, which activate transcription, after chaperoning with a nuclear transporter
called ARNT. There are growing evidences that Nrf2 regulates AHR, thus also phase I
DMEs.(27,28)
The phase II DMEs are Nrf2-dependent gene battery that includes enzymes acting on
cellular redox status and cell protection against oxidative damage, cytotoxicity,
mutagenicity and carcinogenicity. Phase II DMEs works synergistically with phase III
DMEs transporters in various metabolic reactions. Together, their functions involves
disposition of xenobiotics, and endogenous substances (26). Some of the phase II DMEs
are glutathione S-transferases (GSTs), sulfotransferases (SULTs) UDP-glucuronosyl
transferases (UGTs) FAD containing flavoprotein NAD(P)H:Quinone
Oxidoreductase(NQO1), Heme oxygenase (HO-1). These are involved in catalyzing
conjugation reactions through covalent linkage of xenobiotics or phase reaction products,
to groups that are more functionally polar (glucuronate, sulfate, amino acids and
glutathione) which occurs via nucleophilic trapping. In this context, GSTs assign
glutathione, a cellular nucleophile, to electrophilic xenobiotics (9). Similar mechanism is
also seen in SULTs and methyltransferases(29,30). The other category of enzymes present
in the DME phase II is represented by UGTs. These conjugate adenosine-containing
cofactors with nucleophilic xenobiotics. Superoxide dismutases, glutathione peroxidase,
and catalase such as the NQO1 function in a similar manner. The detoxification
mechanism of NQO1 involves catalyzing quinone to hydroquinones via two electron
reduction, bypassing the formation of highly reactive semiquinone(30). Phase II DMEs are
also represented by thiol-containing molecules, such as, glutathione and thioredoxin and
HO-1. HO-1 is an essential enzyme in heme catabolism and is responsible for cleaving
heme to form biliverdin, which is ultimately converted to bilirubin. (27)
The third category is composed of membrane efflux transporters such as the multidrug
resistance associated proteins (MRPs 1,2,3 and 4).). The MRPs are adenosine
triphosphate-dependent drug transporters. They are responsible for the excretion of
endogenous substances, such as bilirubin and xenobiotis, together their conjugated
metabolites products from the DME phase II enzymes.
1.3 Antioxidant Response Element (ARE).
The phase II and III DMEs reach their highest level of expression primarily through
activation of a specific enhancer in their respective promoter region. These enhancers are
cis-acting regulatory elements, called antioxidant response element (ARE). Present in
phase II and III enzymes, ARE regulate the expression of genes involved in the cellular
redox status and are present as a single or multiple copies(27).
1.3.1 Discovering the AREs
The ARE pathway was originally observed by Talalay et al(31), when analyzing the
different ways by which some xenobiotics regulate Phase I and Phase II drug-
metabolizing enzymes. This was the first evidence of a Phase II enzyme induction.
Further studies were done(32) in order to identify trans-acting proteins that interact with
these cis-acting regulatory elements. These were classified and characterized by
Rushmore and Pickett(33) after identification of oxidative responsive elements and basal
promoter elements in the rat GST Ya subunit (Gsta2) gene. This novel Cis-acting element
in the 5'-flanking region element, when used in a reporter construct was shown to induce
the activity of the phenolic antioxidant tert-Butylhydroquinone(tBHQ), hence the name
antioxidant response element. The ARE core sequence (cARE), 5′-TGACnnnGC-3′ was
determined via deletion and mutational analysis. Jaiswal el al (34) established the role of
Nrf2 as a transcription factor for genes containing ARE in their promoter region, hence
regulating expression of genes affecting xenobiotic metabolism. The NQO1 induction by
Nrf2 and Nrf1 was shown via supershift assay after transient transfection of these
transcription factors into human hepatoblastoma HepG2 cells. More experiments(35)
involving a broad spectrum of Nrf2 inducers demonstrated the activation of various phase
II DMEs by Nrf2. Sternberg et al 2006 (36) used high-performance liquid chromatography
(HPLC) to show that retinal pigment epithelium (RPE) cells, when treated with zinc,
increased the levels of glutathione synthesis through Nrf2. The cARE motif was further
confirmed as a binding site for Nrf2 via numerous ChIP-seq methodologies followed by
global transcriptional profiling, which demonstrated the variety of Nrf2 proteins
interactions. In recent literature, Biswal et al 2010(37) performed a global Nrf2 ChIP-seq
analysis of mouse embryonic fibroblasts (MEF) with either constitutive nuclear
accumulation (Keap1-/-) or depletion (Nrf2-/-) of Nrf2. Integrating ChIP-Seq and
microarray analyses, they identified 645 basal and 654 inducible direct targets of Nrf2,
with 244 genes overlapping a microarray datasets used to identify Nrf2 direct
transcriptional targets. Also, Chorley et al 2012(38) performed another ChIP-seq analysis
of NRF2-regulated genes utilizing the same cARE motif. Utilizing lymphoid cells with
Nrf2 induced by isothiocyanate, sulforaphane (SFN) they were able to identify 242 high
confidence genomic regions to which Nrf2 binds.
1.4 Nrf2 clinical relevance
There is abundant evidence of Nrf2 involvement in direct protein interactions and
pathway cross talk. This complex regulatory system generated by Nrf2 interactions is
reflected in the clinic by the vast variety of pathologies in which it is involved. In mice
Nrf2 was shown to play a role in carcinogenesis, chronic obstructive pulmonary disease,
obesogenesis, and neurodegeneration(39). Although, Nrf2 knock out was shown to be
nonessential for the normal development in mice(40), the Nrf2/ARE interaction is vital in
humans for normal cell homeostasis promoting cellular antioxidant defenses and
increased capacity to detoxify drugs. Previous studies with Nrf2 _ / _ mouse models(41)
have shown a high sensitivity of mice to chemical and physical insults. As previously
mentioned, these insults have a strong correlation with the incidence of cancer via
oxidative and electrophilic stressors, or drugs that induce the production of free radicals.
It was also shown that Nrf2-deficient mice seemed to be more sensitive to
carcinogenesis,(42,43) and are at an enhanced risk of metastasis(44),(45).Consequently, Nrf2
was considered to work only as tumor suppressor and so the benefits of Nrf2 signaling in
cancer chemoprevention were largely explored (46).
However, this increase in cellular protection, via high Nrf2 levels, leads to unwanted side
effects in some cancer types(47), as constitutive activation or augmented signaling of the
Nrf2 pathway may promote tumorigenesis and be involved in resistance to chemo- and
radiotherapeutic treatments, showing that the transcription factor could have a proto-
oncogenic role(48).
1.4.1 Nrf2 and carcinogenesis
The role of Nrf2 in cancer promotion was first found in an hepatocellular carcinoma
model by Ikeda et al in 2004(49). In this study both levels of Nrf2 and GSTP1, a neoplastic
marker, were elevated. It was also found that Nrf2 was regulating GSTP1 through an
ARE, present in the promoter region of the gene. Additional studies have proven Nrf2
relation to tumorigenesis, chemoresistance, increased cell survival, metastasis, and cell
growth (47)-(49,50)-(51).
While much focus remains on enhancing Nrf2 as a cancer chemoprevention strategy
against genotoxic agents(52), (53) or inflammation(54), participation of Nrf2 in the process of
carcinogenesis is also strongly demonstrated in many papers in the literature. Nrf2
together with its downstream genes, is elevated in many cancers cell lines and human
cancer tissues, resulting in chemoresistance(50) and a poor prognosis in patients (55,56, 59,60)
thus providing the cancer cells an advantage for survival and growth.
One of the principal reasons for the constitutively high levels of active Nrf2 in cancer is
due to loss-of-function mutations in Keap1.(16,55) which causes its inactivation or reduced
expression. This results in increased Nrf2 stability and its translocation to the nucleus and
consequently transcriptional activation of its target genes. Constitutive stabilization of
NRF2, due to Keap1 mutations, was found in various human cancers, with increased Nrf2
activity in lung (~40%), head and neck (~20%), gallbladder (~30%), liver, and breast
cancers(56). There are also some cell lines in which gain-of-function mutations in the Nrf2
gene is observed, (56-58)like in advance Esophageal squamous cancer (ESC) with
occurrence of (18/82, 22%)(50)
In both, in-vivo and in clinical specimens of non-small cell lung cancer (NSCLC)(55),
loss-of-function Keap1 mutations resulted in constitutively high levels of active Nrf2 and
subsequent resistance to chemotherapeutic drugs (taxanes, platinums) and radiotherapy.
Keap1 mutations are reported in up to 60% of papillary lung adenocarcinoma, as well as
in other cancers including ovarian, gall bladder and others(59).
The inverse of the abovementioned is also the case. A low level of Nrf2 within the cancer
cells is responsible for chemo sensitisation. Batist et al 2009(51) found very low Nrf2
levels in breast cancer cell lines and in the majority of a 200-sample tissue microarray,
which is consistent with the high response rates of breast cancer to many cytotoxic
therapies.
1.5. Nrf2 cross talk with various pathways involved in cancer
As mentioned before Nrf2 can block cell damage induced by oxidative and electrophilic
drugs and also reduce their accumulation in the cell via MDR protein. However, Nrf2
chemoresistance can also occur, due to its interaction with other pathways present in
cancer, which are related to metastasis, increase in cell survival and cell growth.
Some Nrf2 target genes, such as HO-1, were shown to be related to cellular metastatic
potential. HO-1 is overexpressed in various solid tumors(60) and is related with
angiogenesis and acceleration of prostate cancer progression(54). The HO-1 protein is also
related with increased cell survival via apoptosis inhibition in chronic myelogenous
leukemia (CML). Nrf2, also, regulates proteins from the Bcl-2 family through
transcriptional control of the antiapoptotic proteins Bcl-2 and Bcl-XL. Additionally, Nrf2
was shown to increase cell survival via inhibition of p53-dependent apoptosis(61). In
response to stress stimuli, the tumor suppressor p53, control the expression of the
cycling-dependent kinase inhibitor p21 via cell cycle G1 arrest(62). Nrf2 is stabilized by
p21 via direct interaction of the DLG and the ETGE Nrf2’s motifs with the KRR motif in
p21, which displaces the Nrf2-Keap1 interaction(63). In a ROS-dependent mechanism,
p53 induces apoptosis via a two-phase Nrf2 response. Under conditions where ROS
levels are low, in a phase called induction, p53 is also low and it enhances the protein
level of Nrf2 transcriptionally via the target gene p21. The other side of this biphasic
regulation is called the repression phase, and it is present when ROS, and consequently
p53 levels, are high. In this phase p53 binds to a sequence near the ARE which repress
Nrf2 transcription by displacing it from the ARE(61,64). P53 was also showed to negatively
regulate TSC2, PTEN, consequently inhibiting the IGF-1-AKT-mTOR axis. (65) This
suggest at least an indirect relationship between Nrf2 biding to its cognate sequence
(ARE) and elements of the PI3K pathway, including mTOR.
1.6 The PI3K/Akt/mTOR pathway
The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway is important for cell
survival and is involved in metabolism, apoptosis, cell growth, differentiation, calcium
signaling, and insulin signaling(66). In addition to those cited above, a variety of recent
studies suggest that this pathway interacts with Nrf2(67,68). PI3K/AKT pathway has a role
in tumor development and has shown potential in tumor treatment, through the PI3K
pathway inhibitor Wortmannin(66) (69). Multiple molecules that target this pathway are
currently in clinical development.
PI3Ks are part of a lipid Kinase family with main distinctive feature is its capability to
phosphorylate inositol ring 3’-OH group in inositol phospholipids. The mechanism of
action of this signaling pathway starts with PI3K activation. One mode of activation is
through binding of an extracellular growth factor to the RPTK (Receptor Protein
Tyrosine Kinase). Binding of this receptor by growth factors lead to dimerization of
RPTK monomers along with heterologous auto phosphorylation of this receptor
monomers, the IRS-1 (insulin receptor substrate I) then binds to a phosphorylated IGF
receptor. This complex function as binding and activation site for PI3K. Another mode of
activation is via direct binding to a phosphorylated receptor Tyrosine Kinase. This
pathway can also be activated by binding of PI3K to a small membrane bound, active
GTP-Ras(66).
The next step of this pathway involves activation of the second messenger
phosphatidylinositol-3,4,5-trisphosphate (PIP3) and AKT (a serine/treonine kinase
protein also known as protein kinase B). Migration of PI3K to the inner membrane and
binding to PIP2 (Phosphatidylinositol 4,5-bisphosphate) leads to phosphorylation of PIP2
to PIP3 which then activates AKT. The Pi3K pathway is negatively regulated by the
presence of phosphatases capable of dephosphorylating PIP3 back to PIP2. Inhibition of
this pathway can be achieved via chromosome 10 (PTEN) barring a homologue deletion
of phosphatase and tensin. Decrease in PTEN expression indirectly stimulates PI3K
activity and is largely seen in cancer(66).
There are at least four main downstream effects of AKT activation. The first one is the
inhibition of apoptosis via binding with BAX (BCL2-associated X protein) which in-turn
stops BAX from creating holes in the mitochondria inner membrane, responsible for
generating apoptosis by the Caspase cascade. The second effect is the phosphorylation of
Forkhead box O (FoxO) which serves as a substrate for the enzyme ubiquitin ligase,
resulting in its degradation in the proteasome. In the absence of this process FoxO
inhibits cell proliferation. The third effect is the inhibition of Glycogen synthase kinase-
3ß (GSK-3ß). The fourth effect is its role in translation by a multi step protein cascade.
This cascade begins with the activation of Rheb by AKT, which activates the protein
kinase mechanistic target of rapamycin (mTOR; formerly known as mammalian TOR)(70).
Another mechanism of mTOR activation via AKT is by phosphorylation o the mTOR
inhibitor PRAS40 (proline-rich Akt/PKB substrate 40 kDa)(71).
1.6.1 NRF2 interactions with the PI3K pathway
As noted, several studies showed evidence for interactions between the PI3K pathway
and NRF2 using different techniques and models. In the previously mentioned global
mapping of Nrf2 biding sites(37), TSC2 was shown to be a basal target for Nrf2; since the
cells were not “stimulated” in any way with respect to Nrf2 function or nuclear
accumulation, this type of study is mute on Nrf2’s potential role in the transcription of
these proteins in conditions of redox stress.
In an in silico analyses of Nrf2 interactome and regulome, that includes 289 protein–
protein, 7469 TF–DNA and 85 miRNA interactions, shown in a manually curated
network of Nrf2, it was observed that AKT functions as an indirect activator of Nrf2 (67).
Biological evidence of this interaction was also observed in previous studies where
human dopaminergic neuroblastoma SH-SY5Y cells(72) showed PI3K involvement in the
Nrf2 regulation of antioxidative proteins HO-1, Trx, and PrxI, According to the paper,
after treating the cells with hemin, a dose dependent nuclear translocation of Nrf2 was
observed together with PI3K phosphorylation. Also, PI3K inhibitors, wortmannin and
LY294002, lead to inhibition of Nrf2 nuclear translocation. In another study(68), Nrf2 up
regulation via the PI3K and the Extracellular Regulated Kinase (Erk) pathways was
observed after cell treatment with eckol, which is a phlorotannin component of brown
algae such as Ecklonia cava (Laminariaceae), and is known to upregulate ERK and AKT
individually. In this paper it was also shown that treatments with any of the drugs (
U0126, an Erk kinase inhibitor, or LY294002) or short interfering RNAs (Erk1 siRNA,
and Akt siRNA) suppressed Nrf2 activity, which was observed by decrease of HO-1
levels.
1.6.2 Clinical relevance of the interactions between Nrf2 and the Pi3K/AKT
pathway
Interaction between the PI3K/AKT pathway and Nrf2 might well be clinically relevant,
as the pharmacological inhibition of this pathway suppresses the nuclear translocation of
Nrf2 in cancer cells (73,74). This was also shown by Ling Wang et al,(75) who working on
age-related macular degeneration (AMD) caused by accumulated oxidative injury, found
that cultured human retinal pigment epithelium (RPE) cells treated with PI3K inhibitors
were able to decrease Nrf2 levels. Additionally, a study by Papaiahgari et al 2006(76)
showed that PI3K/Akt signaling regulates Nrf2 activation by hyperoxia. Lung injury due
oxygen supplementation (hyperoxia) is currently used in the treatment of pulmonary
diseases such as respiratory distress syndrome (ARDS) and emphysema. PI3K inhibition
blocked hyperoxia-stimulated Akt and ERK1/2 kinase activation, which activate Nrf2
transcriptional activity. Nrf2 regulation by AKT was later shown to occur via inactivation
of GSK-3b(12).
1.7 Nrf2 enhances the PI3K pathway in systems with high metabolic state
There is growing evidence that Nrf2 also enhance the PI3K pathway in systems with a
high metabolic state (74-77). A hyperproliferative phenotype is a fundamental feature of
tumor growth, and this depends on the metabolic reorganization of elements involved in
bioenergetics, macromolecular synthesis, and cell division(77). Besides Nrf2’s role in
cancer cell resistance to cytotoxic agents, it also cross-talks with other pathways
responsible for modulating metabolism and cell growth, including PI3K/AKT/mTOR and
MAP/ERK pathways. In this context, Nrf2 was observed to mediate NSCLC cell
proliferation via activation of the epidermal growth factor receptor EGFR/MEK1-2/ERK
axis. In the NSCLC H292 cell line, which expresses both wild-type EGFR and Keap1,
EGFR ligand was shown to increase Nrf2 levels in a dose-dependent manner via the
MAP/ERK pathway(78). Also, when EGFR is constitutively active, due to gain of function
mutations, Nrf2 is permanently active(78).
Nrf2 was shown to reinforce the metabolic reprogramming triggered by proliferative
signals. Mitsuishi, Y et al(79) has shown that in the presence of active PI3K-Akt signaling,
combined with high Nrf2 levels in the cell, higher than the ones required for the
transcription of antioxidant target genes, Nrf2 redirects glucose and glutamine into
anabolic pathways. Direct Nrf2 transcriptional targets are associated with de novo
nucleotide synthesis via the pentose phosphate pathway (PPP). AKT activation via Nrf2
was observed in another study of liver repair in mice NRF2 KO mice(80) . As expected,
Mitsuishi, Y et al(79) also found AKT to be phosphorylated in a Nrf2 dependent manner,
thus activating the AKT/mTORC1/Sterol Regulatory Element-Binding Proteins (SREBP)
axis. SREBP is a transcription factor known to induce the PPP genes when mTORC1 is
activated (81).
1.8 mTOR
mTor (also known as RAFT1 or FRAP) is a vital cell metabolic regulatory component of
the PI3K pathway, indirectly activated by AKT via Rheb. mTOR plays a central role in
various signaling pathways, is responsible for the intra and extra cellular detection of
nutrients levels, and functions as a metabolic regulator of cellular anabolic and catabolic
processes coupling growth signals to nutrient availability via ribosome biogenesis and
autophagy(82-84)
The mTOR protein has a molecular weight, of 289 kDa and contains 2549 amino acids
with several conserved structural domains. The N terminus possesses 20 tandem
Huntington, EF3, A subunit of PP2A, TOR1 (HEAT) repeats, forming two α helices of
40 amino acids with hydrophobic and hydrophilic residues. These HEAT repeats are
responsible for protein-protein interactions. The kinase domain of mTOR is located in the
C-terminal. The FKBP12-rapamycin-binding (FRB) domain is located upstream of its
catalytic domain and is, responsible for the formation of the rapamycin inhibitory
complex. Near FRB domain a large FRAP, ATM, TRAP (FAT) domain is present. This
FAT domain is essential for mTOR activity because of its interaction with another FAT
domain, present in the end of the C terminal domain, called FATC. The interaction
between those two domains produces a configuration that exposes the catalytic domain.
Between the FATC and the catalytic domain there is a putative negative regulatory
domain (NRD)(82).
1.8.1 mTORC1
mTOR is part of two functionally and structurally distinct complexes, namely,
rapamycin-sensitive mTOR complex 1 (mTORC1) and rapamycin-insensitive mTOR
complex 2 (mTORC2). mTORC1 is related to regulation of translation, autophagy, cell
growth, lipid biosynthesis, mitochondria biogenesis, and ribosome biogenesis. The
downstream effects of mTORC1 are initiated by its interaction with the accessory protein
regulatory-associated protein of mTOR (Raptor). This interaction mediates the
association of this complex to a conserved short sequence called the TOS motif of S6K
and the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E–BP1 and 2). Once
bound, the raptor–mTORC1 complex phosphorylates S6K, and 4E–BP, which are
markers for mTORC1 activity. S6K is phosphorylated on its Thr389 site, and functions to
enhance the translation of 5′-terminal oligopolypyrimidine (5′-TOP) mRNA’s via
activation of 40S ribosomal subunit. These activated mRNA’s encode anabolic elements
such as, ribosomal proteins, elongation factors and insulin growth factor 2(83,84).
In its non-phosphorylated form 4E-BP binds to eIF4E at the 5 ́-cap of mRNAs, inhibiting
the interaction of eIF4E with eIF-4G protein, consequently arresting initiation of
translation. The 4E-BP/ eIF4E complex is released after 4E-BP phosphorylation by the
raptor–mTOR complex. Therefore enhancing cap-dependent protein translation via eIF4E
activation, resulting in a global boost of cellular protein synthesis and ribosome
biogenesis. Anabolic processes generated by mTORC1 also involve stimulation of
glucose uptake, glycolysis and NADPH production. One of the mechanisms that generate
these effects is the increase in translation of hypoxia-inducible factor 1α (HIF1α),
resulting in higher levels of glucose transporters and glycolytic enzymes(83,84).
1.8.2 mTORC2
The second mTOR complex, mTORC2, interacts with rapamycin-insensitive companion
of mTOR (RICTOR) which is a hydrophobic motif kinase for Akt/PKB activation. Akt is
a vital element of the insulin/PI3K signaling pathway and regulates the influx of nutrients
that activate the raptor–mTOR pathway. The role of mTORC2 in cancer is well
documented (79,80). This complex is hyper activated in cancers via inactivation of the
tumor suppressor PTEN. mTORC2 is known to control cell survival and proliferation by
enhancing the p53-regulator mdm2 and transcription factors from the FOXO family(83,84).
There are a myriad of known mTOR regulators such as growth factors, amino acids,
glucose, energy status, stress (e.g. osmotic stress, DNA damage) and, the tumor
suppressors TSC1 (hamartin) and TSC2 (tuberin). The TSC1/2 complex indirectly
inhibits raptor–mTOR by working as a GTPase-activating protein (GAP) for rheb, a
GTP-binding protein from the ras-family that activates raptor–mTOR by direct biding(69).
mTOR complex 1 activity is also regulated by Rheb via RagD. This member of the
small G-protein family binds directly to the mTOR complex, recruiting it to the
endosomal fraction where mTOR is activated(85).
Using the UCSC genome browser we identified an extended list of ubiquitous
transcription factors acting on mTOR including SP-1, C-MYC and C-FOS. From this list,
the activating factor (ATF-5) was mentioned in the literature. ATF-5 is a member of the
cAMP response element binding (CREB)/ATF subfamily of basic leucine zipper
transcription factors(86). It was shown that the oncoprotein BCR-ABL suppresses
authophagy by up regulating ATF-5 via PI3K/AKT/FOXO4 signaling(87). ATF-5 then
activates mTOR by a direct binding to its promoter, which is in a region between 1560-
2227 bp upstream of the transcription start site, as demonstrated via ChIP assay(87).
Interestingly, a member of the same group of transcription factors, ATF3, is known to
inhibit Nrf2 via direct ATF3-Nrf2 protein-protein interactions(88). Nrf2 belongs to the
same family of transcription factors as ATF and has already been shown to indirectly
interact with mTOR via TSC2 and AKT.
1.9 Role of Nrf2 on mTOR expression
Due to the multi-level interaction of Nrf2 with the PI3K pathway we were interested to
know if Nrf2 could directly act on different components of this pathway. Recent studies
of Nrf2 participation on translation and in cancer anabolism focused our attention to the
metabolic regulator of this pathway, mTOR.
2. HYPOTHESIS________________________________________________________
From literature it is observed that Nrf2 interacts with different components of the PI3K
pathway and regulate specific processes. Recently, Nrf2 has been shown to be involved in
the regulation of metabolic processes in the cell(78) - (80) and hence, we hypothesized that
Nrf2 might also be interacting directly with mTOR, which has not previously been
shown. If demonstrated, this would be one of the possible pathways in which Nrf2
directly regulates the metabolic processes of the cell, positioning it as a link between cell
metabolism and cytoprotection.
To examine this hypothesis, mTOR expression was analyzed using western blot and RT-
PCR in conditions where Nrf2 levels are modulated. Our experiments were focused in
three different cell lines, selected according to the mutations present in them. We used
the non-tranformed Human Embryonic Kidney (HEK293) cells, as well as two human
non-small-cell lung cancer (NSCLC) cell lines. A549 cells have a Kras mutation in
addition to mutations in keap1. Another NSCLC cell line H460, contains a loss of
function mutation on keap1(55) and gain of function mutation on PIK3CA (E545K) and
Kras(89). To further study the Nrf2/mTOR interaction we performed mutation analysis in
dual luciferase assay, as well as DNA pulldown, electroctrophoretic mobility shift assay
(EMSA) and ChIP assay. Additionally, we analyzed the expression of the other elements
of the PI3K pathway (TSC2, S6K and AKT), under Nrf2 silencing and inducing
conditions, via western blot, RT PCR and luciferase assay.
3. MATERIALS & METHODS____________________________________________
3.1 Cell Lines and Tissue Culture/ Transient Transfection
The cell lines A549, HEK293 and H460 (Sigma) were cultured in RPMI (Sigma) media,
supplemented with 10% fetal bovine serum (Sigma), 5% antibiotic/antimycotic (Life
Technologies) and grown in 5% CO2 at 37°C. The cell lines were storage at -80oC in
cryogenic vials containing 106 cell in 1 ml solution of 90% FBS plus 10% DMSO.
Twenty-four hours prior to transfection, 9 X 104 cells were plated in 6 well dish plates
and were transfected when they were approximately 60% confluent. The cells were
incubated with fresh media 1 hour before transfection. The transfections, except for the
ones utilized on ChIP assay, were carried out using Lipofectamine LTX Reagent PLUS™
(Life Technologies) as per manufacturer protocol, utilizing Opti-MEM with a 1:5 ratio of
plasmid to LTX and Plus reagent. The transfection mix was vortexed thoroughly and
incubated for 30 min before addition to cells. Cells were incubated 24 hours before
collection. The internal control used for Luciferase assay, pRL Vector, was co transfected
with the modulatory reagents (pCDNA_Nrf2 or siNrf2 with their respective controls
pCDNA 4.0 or scrambled RNA) and the construct containing the sequence of interest, in
1:1 ratio. 24 hours after transfection the cells were harvested and split for Western blot,
qPCR and luceferace applications.
3.2 Western blot
Protein expression analysis of the cells A549, H460 and HEK293 were performed by
Western blot. Cells were disrupted with lysis buffer (20mM Tris pH 7.5, 420mM NaCl,
2mM MgCl2, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.5% Triton, 1x P8340 (Sigma),
1mM PMSF, 1mM DTT, 2mM NaF, 10mM BGP) for 30 min on ice followed by a 20
min spin at 13000rpm to pellet debris. The supernatant was then removed and quantified
using the Bradford reagent. The OD595 of each sample was then measured using a
spectrophotometer and compared to a standard curve prepared with bovine serum
albumin. An equal concentration of sample was then separated using standard Sodium
Dodecyl Sulfate-Polyacrilamide Gel Electrophoresis (SDS-PAGE) techniques. 40 µg of
cell protein/lysate per each sample was loaded and run through a 10% SDS-PAGE gel
before transferring electrophoretically at 400mA for 2 hours onto a BioRad
nitrocellulose membrane. For the incubation with antibodies, the membrane was first
blocked with 10% fat-free milk solution in 1x Tris Buffered Saline and 0.1% Tween
(TBS-T) for 1 hour at room temperature and probed overnight at 4oC with the antibodies
listed below at the dilutions provided by the manufactor. The day after, membranes were
washed three times in TBS-T and were then incubated with secondary anti-mouse or anti-
rabbit horseradish-peroxidase for 1hour at room temperature. This was followed by three
additional washes with TBS-T.
The results were documented on x-ray film with ECL detection and autophotography to
capture the differences in protein levels in the cells between samples. The antibodies used
as probes for Western were as follows; Nrf2 (abcam) all the others antibodies, beta-Actin,
TSC2, AKT, S6K and Nqo1 were purchased from Cell signaling.
3.3 Quantitative RT-PCR
Total RNA was isolated from, HEK293, A549 and H460 using EZ-10 DNAaway RNA
Mini-Preps Kit (Bio Basic Canada INC.) according to the manufacturer's protocol.
cDNAs were synthesized from total RNA (1 µg) of each sample using , SuperScript® II
Reverse Transcriptase (Invitrogen™)), diluted 4 times with water. The cDNA was used
as the template for quantitative PCR detection using the GoTaq® qPCR Master Mix
(Promega). The real-time PCR conditions were optimized as 95 °C for 7 min and 40
cycles of 95°C for 10 s, 61°C for 5 s, and 72°C for 20 s followed by melting curve cycle.
The amplification reactions were carried out with the AB Applied Biosystems 7500 Fast
Real-Time PCR System. The primers for amplifying human genes (Nrf2, mTOR,Nqo1,
HMOX1, TSC2, AKT, S6K and Gapdh)appendix(Table 1). The comparative ΔΔCt method was
used for relative quantification of the amount of mRNA in each sample normalized to
GAPDH transcript levels. Fold induction is expressed as the ratio of induction from
treated cells versus untreated. Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparison of
treated and untreated cells (*, p < 0.05).
3.4 Bioinformatic Analysis
We screened for the presence of the core ARE sequence (TGAxxxxGC) up to 5kb
upstream of the transcription start site of the target genes. This ARE motif analysis was
performed using BlAST, SCOPE and InSilicase algorithms.
3.5 Molecular Cloning and Vector Construction
Primers were designed using the Primer3 software (http://fokker.wi.mit.edu)2,
synthesized by Integrated DNA Technologies, Montreal, QC. PCR was done according to
the Phusion® High-Fidelity DNA Polymerase protocol (Thermo). Sanger DNA
sequencing at the Innovation Centre, located at McGill University, confirmed the
presence of the desired promoters. The restriction enzymes used on molecular cloning
were purchased from Invitrogen™
3.6 Nrf2 modulation
Inducible Nrf2 construct – The inducible construct PC_Nrf2 appendix (figure 1A) containing
1925bp of the Nrf2 coding sequence was obtained by amplifying the coding sequence of
Nrf2 from A549 RNA (cDNA). Restriction sites for BamHI and XbaI were included in
the primers used for Nrf2 amplification, and enabled the insertion of Nrf2 cDNA into the
pCDNA 4.0 plasmid (Life Technologies). The resulting construct, PC_Nrf2, was
sequenced to validate the plasmid identity. Nrf2 induction was generated via transient
transfection of inducible PC_Nrf2 plasmid. pcDNA 4.0 was used as a negative control for
the cells transfected with inducible Nrf2.
siNrf2 – Nrf2 silencing was generated via transient transfection of Small interfering RNA
targeting Nrf2 (siNrf2) NFE2L2HSS181505 (Invitrogen). Scramble RNA (Invitrogen)
was used as a negative control.
3.7 Luciferase assay constructs
pRL – The internal control used was pRL Vector, which is wildtype Renilla luciferase
(Rluc) control reporter vectors that is used for the purpose of normalizing the luciferase
values.
PCR cloning was used to amplify the target regions and clone into PGL3 basic vector. In
short, the constructs were digested with the restriction enzymes Kpn1 and Xho1 with the
exception of Mtor, which was digested by SacI and MluI.
For site directed mutagenesis the TGA portion of the ARE’s analyzed were deleted using
the Quickchange II XL Stie-directed mutagenesis Kit. The primers sequence for Nqo1,
mTOR, TSC2, and S6K mutations are listed at appendix (Table 1).
Molecular Cloning of Nqo1 Promoter – The ARE site at 550bp upstream of start of
transcription is shown to be active on Nqo1(90). This region was cloned on the
PGL3_basic vector used as positive control (Nqo1_PGL3) Appendix (figure 1B). Nqo1_Pgl3
with the deleted TGA sequence (Nqo1_Pgl3 mut) was used as negative control.
Molecular Cloning of mTOR Promoter –The screened mTOR promoter region contained
eight ARE binding sites. I studied the closest ARE site present at 723bp upstream of the
TSS. The promoter region of mTOR, 1231 bp upstream from TSS, was cloned into the
Pgl3 basic vector (mTOR_Pgl3) Appendix (figure 1C) and used in subsequent functional
analyses. For site-directed deletion analyses the mTOR_Pgl3 mut was created.
Molecular Cloning of TSC2 Promoter –The screened TSC2 promoter region contained 6
ARE binding sites. I studied the closest ARE site present at 756bp upstream of the TSS.
The promoter region of TSC2, 1079 bp upstream from TSS, was cloned into the pgl3
basic vector (TSC2 _Pgl3) Appendix (figure 1D) and used in subsequent functional analyses.
For site-directed deletion analyses the TSC2_Pgl3 mut was created.
Molecular Cloning of S6K Promoter –The screened S6K promoter region contained 12
ARE binding sites. I studied the firsts 5 closest ARE sites, present at 255bp, 285bp,
324bp, 432bp and 2543bp upstream of the TSS. The promoter region of S6K, 2660bp
upstream from TSS, was cloned into the pgl3 basic vector (S6K_Pgl3) Appendix (figure 1E) and
used in subsequent functional analyses. For site-directed deletion analyses the two
closest ARE’s to TSS were mutated at the TGA (S6K_Pgl3 mut).
Molecular Cloning of AKT Promoter –The screened AKT promoter region contained 3
ARE binding sites. I studied those Are’s were present at 1191 bp, 1403 bp and 1681 bp
upstream of the TSS. The promoter region of AKT, 2200 bp upstream from TSS, was
cloned into the pgl3 basic vector (AKT _Pgl3) Appendix (figure 1F) and used in subsequent
functional analyses.
3.8 Luciferase Assay
Cells were lysed with Passive Lysis Buffer, and kept at -80ºC overnight. Luciferase
activities were analyzed in 20-µl cell extracts with the dual luciferase assay kit
(Promega).
Firefly and Renilla luciferase activities were then determined in triplicates for each
sample on the EnSpire multimode plate reader (PerKinElmer). The luciferase activities
reported were expressed as a ratio of the pGL3 reporter activity to that of the control
plasmid pRL. -Fold induction (Relative Luciferase activity) is expressed as the ratio of
induction from treated cells(PC_Nrf2 and siNrf2) versus untreated (pcDNA 4.0 and
Scramble RNA) respectively. Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparing treated
and non-treated cells (*, p < 0.05).
3.9 Electrophoretic Mobility Shift Assay (EMSA)
A549 cells (4 x106), were plated in four 175cm2 flasks with RPMI for 24 hours. The cells,
were transfected with Nrf2 siRNA or scrambled SiRNA and were harvested 24 hours
later. Nuclear extracts of A549 cells were prepared using 1M tris ph 7.5, 100mM Mgcl2,
3M Kcl, 500mM EDTA, 1M sucrose, 100% Glycerol, 1MDTT, 1M orthvanadate, 0.5M
BGlyc-phos, 100mM PMSF and 100x protease cocktail. The annealed primers for Nqo1
wild type, Nqo1 mutant, mTOR wild type, mTOR mutant 1, mTOR mutant 2, and mTOR
mutant 3 composed the probes used for the experiment appendix (table1). The primers were
annealed by heating at 95°C for 10 minutes followed by overnight incubation at 4 °C.
The probes were then labeled with the radioactive isotope g-[32P]ATP at 30°C for 30
minutes following 10 minutes incubation at 65C. For DNA-protein binding reactions, 10
µg of nuclear extract was incubated at room temperature for 30 min with 20 mM HEPES-
KOH (pH 7.9), 60 mM KCL, 1 mM MgCL2, 1 mM EDTA, 1 µg poly(dI-dC)
dithiothreitol, 10% glycerol, 0.2 mM ZnSo4 and 10,000 cpm g-[32P]ATP-labeled probe.
Protein-DNA complexes were resolved through a 4% polyacrylamide gel. The gel was
then dried and subjected to autoradiography with an intensifying screen at -80°C.
3.10 DNA Pull-Down Assay
Tissue culture, transient transfection and the nuclear extraction were performed for both
the DNA pull down as it was for the EMSA assay. This assay was performed via a
modified protocol described by Benoit Grondin et all 2006(91). The biotinylated primers
Nqo1 wild type, NQO1 mutant, Mtor wild type, Mtor mutant appendix (table1) were generated
at IDT (Integrated DNA Technologies). Annealing reaction of the primers was performed
as described for EMSA experiment. For DNA-protein binding reactions, 200 µg of
nuclear protein extraction was incubated over night at – 10C on a shaker with 10 µg of
biotinylated probes on 1 ml of biding/washing buffer (20 mM Tris [pH 8.0], 10%
glycerol, 6.25 mM MgCl2, 5 mM dithiothreitol, 0.1 mM EDTA, 0.01% NP-40) in a final
concentration of 200 mM NaCl. After 1 hour incubation with 50 µl of the magnetic beads
(Dynabeads® MyOne™ Streptavidin C1), immobilized templates were washed three
times with 0.5 ml of binding buffer, dried and resuspended on SDS and loading dye. The
samples were than boiled and resolved on a 10% SDS-PAGE gel for immunoblot
analysis with Nrf2 antibody (abcam).
3.11 Chromatin immunoprecipitation
This experiment was carried with as a modified protocol previously described by Donner
et al 2007(92), 2010(93). Briefly, A549 cells were grown until 80% confluence in 15 cm
plates and were transfected with 15µg of PC_Nrf2 using GenJet Plus transfection reagent
(SignaGen Laboratories). Before harvesting, the cells were cross-linked with 1%
formaldehyde for 10 mins at room temperature on a rocker. The cross-linking reaction
was quenched using 125mM glycine and washed twice with ice-cold phosphate-buffered
saline. The cells were harvested by scraping in RIPA buffer (150mM NaCl;1% v/v
Nonidet P-40;0.5% w/v deoxycholate; 0.1% w/v SDS;50mM Tris pH 8.0;5mM EDTA)
supplemented with protease inhibitor cocktail(Fisher), phosphatase inhibitors and PMSF.
These cells were sonicated on ice with 15 pulses of 15 seconds(20% amplitude) with
30second intervals to obtain an average chromatin length of 500 to 1,000 bps using a
Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pa.) and centrifuged. The supernatant,
containing the chromatin, was collected and quantified alongside BSA standards and
equalized to a final concentration of 1mg/ml. The chromatin (1mg/ml) was pre-cleared
using 25μl of IgG magnetic beads (Dynabeads Invitrogen), previously washed with
RIPA, for 2 hrs at 4°C on a rocker. 10μl of pre-cleared chromatin was reserved as input
sample. The rest was immunoprecipitated with 25μl IgG magnetic beads, blocked with
salmon sperm DNA(0.3mg/ml) and BSA(1mg/ml), and with either anti-Nrf2 antibody
(Santa Cruz, Santa Cruz, Calif.), anti-RNA pol II antibody (Active Motif), or no antibody
overnight at 4°C with rotation. The next day, the beads were washed with RIPA and wash
buffer (100mM TrisHCl pH 8.8;500mM LiCl;1% v/v Nonidet P-40; 1% w/v deoxycholic
acid) and were resuspended in 100μl of 1X TE buffer. To elute the imunocomplexes,
200μl of elution buffer (70mM Tris HCl pH8.0;1mM EDTA;1.5% w/v SDS) was added
and the samples were incubated for 10min at 65°C with occasional vortexing. To reverse
cross-linked chromatin, 200mM NaCl is added to the eluted complexes and input samples
and incubated at 65°C for 6hrs. All the samples were then treated with 20 mg/ml
proteinase K (Fisher) and extracted with phenol-chloroform-isoamyl alcohol (25:24:1).
DNA was precipitated with ethanol and 3M sodium acetate and re-suspended in 100μl of
water. 2μl of purified DNA was used for qPCR appendix (table1).
4. RESULTS__________________________________________
Evidence from the literature shows that Nrf2 interacts with PI3K pathway at different
locations and regulates various functions of the cell(23, (37), (67)-80). The aim of this study
was to determine if Nrf2 transcriptionally controls the expression of the mTOR gene and
to illustrate whether this regulation is through direct or indirect binding of Nrf2 to the
mTOR promoter. To achieve these goals, western blot and qPCR analysis in conditions of
induced and silenced Nrf2 protein levels were performed. This was followed by
luciferase assays to confirm the presence of functionally active AREs in the mTOR
promoter. Lastly, we performed DNA pull down, EMSA and ChIP assays to confirm
direct binding of Nrf2 to elements in the mTOR promoter. The possibility of an Nrf2
impact on the other elements of the PI3K pathway (TSC2, S6K and AKT), was also
analyzed via western blot, qPCR and luciferase assay.
4.1 Nrf2 modulates mTOR expression in A549 cells
4.1.1 mTOR expression when Nrf2 is up-regulated
Expression analysis of mTOR was performed in A549, H460 and HEK293 cell lines.
Induction of Nrf2 was carried out by transiently transfecting Nrf2 cDNA (PC_Nrf2) appendix (figure 1A) for 24h. pcDNA 4.0 was used as a negative control for the cells .
The transiently transfected cell lines (figure 1) have significant increase in Nrf2 mRNA and
protein level, however the basal levels differ amongst the three cell lines. A549 cells have
the lowest basal Nrf2 protein levels such that the effect of transfection was most dramatic
in these cells. In A549 cells, mTOR expression was significantly increased, by
approximately five folds at both transcriptional and protein levels. In HEK 293 cells, an
increase in mTOR transcription was observed while protein levels showed no change. In
H460 cell lines there was 1.6 fold increase in mTOR protein, although thre was no
observable increase in transcriptional activity.
Figure 1. mTOR (Nrf2 inducible) expression analysis- A. mTOR protein levels were not increased in HEK293 cells. B and C. mTOR protein levels were increased five fold in A549 cells and 1.6 folds increased on H460 cells, respectively. D and E. mTOR transcription was increased two folds in HEK 293 cells and four folds in A549 cells. F. No increase in mTOR transcription was observed in H460 cells. G. The relative Luciferase activity of mTOR-WT in HEK293 cells was 20 folds increased and five folds increased in mTOR- mut. H. The A549 cells presented three folds increase of mTOR-WT relative luciferase activity with no change in mTOR-mut. I. The H460 cell lines did not present a significant change of relative Luciferase activity in both mTOR-WT and mTOR-mut. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of induction from treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible(mTOR)
Western blot
mTOR
β-Actin
mTOR
mTORβ-Actin
1 : 0.96
1 : 1.6
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 1.73
Nqo1 1 : 1.3
Control Pc_Nrf2
1 : 5.3
Nrf2 1 : 1.63
Control Pc_Nrf2
Nqo1 1 : 60
Nrf2 1: 1.5Control Pc_Nrf2
Nqo1 1 : 1.6
qPCR
D)
E)
F)
Control Nrf2 Nqo1 mTOR 0.00.51.01.52.02.5
mR
NA
expr
essi
onle
vels
*
Control Nrf2 Nqo1 mTOR 0.00.51.01.52.0
2345678
mR
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expr
essi
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Control Nrf2 Nqo1 mTOR 0.00.51.01.52.0
2345678
mR
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exp
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leve
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Luciferase
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut0.00.51.01.52.0
510152025
Rel
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cife
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Ac
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Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut0.0
0.5
1.0
1.5
2.02345678
Rel
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Ac
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Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut0.0
0.5
1.0
1.5
2.02345678
Rel
ativ
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Ac
tivity
G)*
*
**
H)*
**
*
*
**
*
**
*
*
I)
4.1.2 mTOR expression when Nrf2 is down-regulated
Figure 2. mTOR (Nrf2 silencing) expression analysis. A, B and C. mTOR protein levels were significantly transiently decreased in the three cell lines. D and E. mTOR transcription was decreased proximately 1.5 folds on HEK293 cells and 2 folds in A549 cells. F. No change was observed on mTOR transcription in H460 cell lines. G, H and I. No change in the luciferase activity was observed for Mtor-WT and mtor mut in all the three cell lines. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of silencing from treated cells (siNrf2) versus Control (Scramble RNA). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing(mTOR)
Western blot
mTOR
β-Actin
mTOR
mTORβ-Actin
1 : 0.03
1 : 0.65
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 0.03
Nqo1 1 : 0.07
Control Si_Nrf2
1 : 0.51
Nrf2 1 : 0.02
Control Si_Nrf2
Nqo1 1 : 0.53
Nrf2 1 : 0.77Control Si_Nrf2
Nqo1 1 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
Control Nrf2 Nqo1 mTOR 0.0
0.5
1.0
1.5
mR
NA
exp
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Control Nrf2 Nqo1 mTOR 0.0
0.5
1.0
1.5
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Control Nrf2 Nqo1 mTOR 0.0
0.5
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Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut0.0
0.5
1.0
1.5
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Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut0.0
0.5
1.0
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Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut0.0
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1.0
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Rel
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*
*
*
**
**
*
* *
*
*
In conditions where Nrf2 is silenced (figure 2), all the three cell lines presented a significant
decrease of Nrf2 at both transcriptional and protein levels, with the most significant
effects seen at protein levels in HEK 293 and A549 cells. Silencing Nrf2 transcription
resulted in a two-fold decrease in mTOR, both its transcription and protein levels. In
HEK293 cells, a small decrease in mTOR transcription was observed.
4.2 Functional ARE present on mTOR promoter activates its transcription in Nrf2
inducible conditions.
Luciferase assay was performed in order to verify if the regulation of mTOR gene
expression was due to the presence of a functional ARE binding site in the mTOR
promoter region. Biswal et al(37), performed ChIP-Seq experiment to explore the network
of Nrf2 regulated genes and in this work they used the consensus core ARE sequence
TGANNNNGC. Here the mTOR promoter region was screened for ARE sites that had
the same motif sequence. Biswal et al, also screened 5225 background sequences relative
to the closest gene transcription starting site (TSS) in order to identify ARE sites. They
identified the highest peaks at AREs closest to the genes’ TSS. Similarly, in another Nrf2
ChIP-seq study performed by Chorley BN et al (38), based on 39 currently known
functional human AREs, NRF2-binding sites were found to be cis-acting elements more
commonly located at an average distance of ~1800 bp from the gene TSS. For these
reasons, in this study, from the eight ARE’s found within 5000bp of mTOR promoter
region, the “TGACCAGGC” ARE, located closest to mTOR TSS (723 bp upstream from
TSS), was cloned into an expression vector. The PRL-mTOR vector contained 1231 bp
of the mTOR promoter was then used on Luciferase assay (mTOR WT) appendix (figure1C).
As shown by Biswal et al(37) via alignment of 20 known ARE binding sites and MEME
motif discovery algorithm on their Nrf2 ChIP-Seq dataset, the “TGA” portion of the ARE
is the most recurrent portion of the sequence. For this reason, in this study, site-directed
deletion was performed in the mTOR WT construct where the “TGA” of the ARE biding
site was deleted (mTOR Mut). Both mTOR WT and mTOR Mut constructs were
analyzed by luciferase activity assay at inducible and silencing conditions. Promoter of
the Nqo1 gene, a known target of Nrf2, was used as a positive control for this assay
b
(Nqo1 WT) appendix (figure 1B).
When transfected with the inducible PC_Nrf2 construct Nqo1 was substantially increased
at the protein and transcription level on all the cell lines (figure 1). In Nrf2 inducible
conditions, A549 cell line showed a 60 fold increase in the Nqo1 protein and a three fold
increase in the transcription of Nqo1 gene, compared to basal conditions. Whereas, in
Nrf2 silencing conditions (figure 2), Nqo1 expression was reduced in all the three cell lines.
Both transcription and protein levels of the control were decreased two fold in A549
cells.
The negative control consisted of the same Nqo1 promoter region with a mutated ARE
(Nqo1 Mut). At the basal level (Graph 1), the luciferase assay showed that the negative
control, when compared with Nqo1 WT activity, decreased five fold in A549 cells and
two fold in both of HEK293 and H460 cells. In this same condition, the activity of the
mTOR Mut was two folds lower than the mTOR WT in A549 and HEK293 cells while
no change was recorded on H460 cells.
Analysis of Nrf2 modulation was performed by comparing the fold change of the
luciferase activity of the Pgl3 constructs at basal Nrf2 levels (control) with cells
transfected with the same construct and Pc_Nrf2(figure 1) or Si_Nrf2 (figure 2). Induction or
silencing of Nrf2 was validated with Nqo1 WT activity following Nrf2 up and down
patterns of expression in the three cell lines, with three folds increase and 7 folds
decrease on A549 cells. The negative control was not affected by Nrf2 variations in the
cells. The one exception was HEK293 cells in Nrf2 inducible condition, where there was
a four folds increase. Nevertheless, Nqo1 Mut activity was 6 fold lower than Nqo1 WT
in these conditions in HEK293 cells, so the Nrf2 is playing a regulatory role through its
interaction with ARE. When transfecting the cells with the inducible construct (figure 1) it
was observed that the luciferase activity of the mTOR wild type (mTOR WT) construct
was increase 20 folds in HEK293 and four folds on A549 cells, but there is no change on
H460 cells. mTOR Mut activity remained unchanged during Nrf2 up regulation in A549
but not in HEK293 cells. In silencing conditions (figure 2) no change in activity for the wild
type and mutant mTOR constructs were observed in any of the cell lines. From the cell
lines analyzed, A549 cells presented a clearer correlation between Nrf2 levels and mTOR
expression. For this reason, additional analyses of the Nrf2/mTOR interaction were
performed in this cell line.
Graph1. Nqo1 and mTOR (Nrf2 basal levels) Luciferase activity. A. Nqo1 Mut presented a 2 fold decrease in HEK293 and H460 cells and 4 fold decrease in A549 cells. B. mTOR Mut presented 2 fold decrease in HEK293 and A549 cell and no change on HEK293 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity was represented as the fold change of the ratio from cells transfected with mutant constructs (Nqo1-Mut and mTOR-Mut) versus cells transfected with wild type constructs (Nqo1-WT and mTOR-WT). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of mutant and wild type constructs expression (*, p < 0.05).
4.3 Nrf2 binds to mTOR promoter region at basal conditions in vitro
Nrf2 binding to the mTOR promoter was demonstrated in vitro using DNA pull-down
and EMSA experiments. In the DNA pull down assay the mTOR promoter region was
used as a probe to selectively obtain a protein-DNA complex from an A549 nuclear
extract. The high affinity tag, biotin, was present in both extremities of the probe and the
complex purification was performed with streptavidin magnetic beads. The proteins were
eluted from DNA and detected via western blot (figure 3). Assessment of the biding capacity
of the ARE sequence present in this promoter region was performed via a mTOR probe
with a scrambled ARE site appendix (table 1). Nqo1 promoter region was used as a positive
control, and scrambled ARE site was used as a negative control
Nqo1-W
T
Nqo1-M
ut
Nqo1-W
T
Nqo1-M
ut
Nqo1-W
T
Nqo1-M
ut0.0
0.5
1.0
1.5
2.0HEK A549 H460
**
*
Rel
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tivity
A)
mTOR-WT
mTOR-Mut
mTOR-WT
mTOR-Mut
mTOR-WT
mTOR-Mut
0.0
0.5
1.0
1.5
2.0HEK A549 H460
* *
Rel
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B)
Figure 3. Western blot from DNA pull-down samples using Nrf2 antibody - Blot analysis of (Input) nuclear extract from A549 cells, (No Probes) negative control comprising of reaction mix alone incubated with magnetic beads and probed samples. The probed samples consisted of (Nqo1 wt) Nqo1 promoter region containing functional ARE which was used as a positive control, scramble ARE from Nqo1 promoter region which was used as a negative control (Nqo1mutant), mTOR promoter region containing ARE (mTOR WT) and scramble ARE from mTOR promoter region which was used as a negative control (mTor mutant). It was observed an 2 folds decrease of Nrf2 protein pulled down with mTOR mutant probe when compered with the amount of protein pulled down with mTOR WT probe, as it was for the controls, Nqo1 mutant and Nqo1 WT.
On western blot analysis, our results suggest that Nrf2 binds to an element(s) in mTOR
promoter region. The fact that the amount of Nrf2 protein pulled down with the WT
mTOR probe was 2 folds higher than the amount pulled down with mutant mTOR probe
and with negative control (no probe), adds our speculation that the ARE is the biding site
of the Nrf2.
EMSA was carried out in order to further verify the Nrf2 biding is at the mTOR’s ARE
located 1231 bp upstream from the TSS (mTOR wild type). For this experiment a
mutation was done by removing the entire ARE sequence TGACCAGGC and adding 5
bp in both 5’ and 3’ prime extremities (mTOR Mut) (figure 4). The mTOR wt, mTOR
mutants as well as the positive(Nqo1 wild type) and the negative control (Nqo1 mutant)
were end labeled with [32P] ATP and incubated with nuclear extract isolated from A549
cells.
It was observed that the predicted Nrf2 site was present in the sample incubated with
mTOR wild type and not in mTOR mutant. It was also observed that an additional biding
was present in the mutated mTOR probe at an adjacent site (figure 5).
Figure 4. mTOR probes used on EMSA assay. mTOR WT sequence containing the “ TGACCAGGC” ARE and mTOR Mut with deleted ARE sequence and 5 bp extension at 5’and 3’ ends. The primers were annealed, with it respective reverse complementary sequence, end labeled with [32P] ATP and used on EMSA experiments.
5’-TTCACCATGTTGACCAGGCTGGTCTCGAC-3’
5’-GGGAATTTCACCATGT********* TGGTCTCGACTCCTC-3’
Figure 5. ARE dependent biding of nuclear components to mTOR-WT- EMSA was performed using labeled promoter fragment of Nqo1-WT (positive control), Nqo1-Mut (negative control), mTOR –WT (mTOR promoter region containg ARE site) and mTOR –Mut (mTOR promoter region containg deleted ARE site plus addiction of 5bp on 5’ and 3’ ends) incubated with nuclear extracts (10 µg per lane ) from A549 cells. Top red arrow indicate shift of predicted Nrf2 biding site and bottom black arrows indicate new and unknown biding appeared on cells incubated with labeled mTOR –Mut probes. Predicted Nrf2 biding site (red arrow) was presented on samples incubated with mTOR-WT and Nqo1-WT and not on samples incubated with Nqo1-Mut and mTOR –Mut
4.3.1 Nrf2 binding to mTOR promoter region decreases in Nrf2 silencing conditions
EMSA assay was also performed in A549 cells in which Nrf2 was silencing (figure 6). After
incubation nuclear extract of the Nrf2 down regulated A549 cell with radioactive labeled
mTOR WT probe a significant decrease in bound protein was observed. Intensity of the
blots present on samples incubated with mTOR WT probes suggests that in basal
conditions the Nrf2-mTOR biding is weak.
Figure 6. Biding of nuclear components to mTOR-WT at Nrf2 silencing conditions. A. EMSA was done on nuclear extract (NE) of transiently transfect A549 cell with SiNrf2 or scrambled RNA (control). SiNrf2 A549cells NE and Scramble A549cells NE were incubated with labeled promoter fragment of Nqo1-WT (positive control), Nqo1-mut (negative control ). The films containing shift of predicted Nrf2 biding site (red arrow) were developed after over nigh or four days gel incubation at -80oC. Once incubated over nigh the SiNrf2 A549cells NE samples that contained Nqo1-WT probes presented decreased blot intensity when compared with Scramble A549 cells NE. After four days incubation, the SiNrf2 A549cells NE samples that contained mTOR-WT probes presented decreased blot
4.3.2 Nrf2 binds to mTOR promoter in vivo at inducible conditions
In order to clarify the in vitro results of the Nrf2/mTOR binding, this interaction was
analyzed in vivo. One of the factors that can influence the assays performed in vitro
assays is the lack of the natural DNA conformational topology on those assays(94). In
order for genomic regulation and recombination to occur, these processes require DNA
bending, twisting, and looping as well as wrapping around histone octamers in order to
occur. Thus, in vitro assays, such as the ones performed in this study, may not give the
precise representation of the actual intracellular processes. Also, in addition to DNA
structure, molecular crowding caused by the presence of particles on the cytoplasmic
microenviroment may influence local and distal interactions(95). Biochemical reactions in
vivo occur at crowding conditions with high concentrations of biomacromolecules. While
the majority of the biochemical reactions in vitro are performed in solutions containing
low concentrations of biomacromolecules.
ChIP assay followed by qPCR amplification enables the capture of protein–DNA
interactions in vivo and is considered a definitive confirmatory method when analyzing
Nrf2 transcriptional targets(96). This assay was used in the past to identify important Nrf2
targets such as antiapoptotic protein Bcl-2, catalytic subunit of glutamylcysteine ligase
(GCLC) and Aldose reductase (AR)(97-99) among others. Nrf2/mTOR biding in vivo
Chromatin ImmunoPrecipitation (ChIP) coupled to detection by quantitative real-time
PCR was performed on A549 cells (Graph 2). The samples were immunoprecipitated with
either anti-Nrf2 antibody, anti-RNA pol II antibody or no antibody. The experiment
compared the fold enrichment, with respect to no antibody control, of crosslinked
protein-DNA complexes in two Nrf2 conditions, basal and inducible. At the basal levels,
the ChIP performed using anti-Nrf2 antibody, showed a 2.5 fold enrichment compered to
no antibody control, of the mTOR promoter, which denotes a weak binding at basal
levels. Whereas in Nrf2 inducible conditions, the enrichment of the same mTOR
promoter was seen to increase to 13 folds. Anti-RNA polII antibody was used as a
positive control antibody to confirm successfulness of the ChIP assay. Nqo1 promoter
region was used as a positive control for the anti-nrf2 antibody, while GAPDH served as
a positive control for anti RNA Pol II antibody and as a negative control for anti Nrf2
antibody.
Graph2. ChIP assay. Crosslinked protein-DNA complexes were immunoprecipitated using either anti-RNA polymerase II antibody (Pol II, positive control), anti-Nrf2 antibody or no antibody in A549 cells transfected with inducible or basal (empty vector) constructs (Nrf2 cDNA containing plasmid). Enrichment was measured as fold increase of antibody vs the no antibody control by q-PCR.
4.4 Expression analyses of the other elements of PI3K pathway due to Nrf2
modulation
Expression of other components of the PI3K pathway elements including TSC2, S6K and
AKT as well as luciferase assay on promoters of these genes, in which ARE core
sequence were identified, were also analyzed in conditions of Nrf2 modulation. The Nrf2
inducible and silencing conditions as well as the control were the same as the ones
ChIP Assay A549 cells
Nrf2-ba
sal
Nrf2-in
ducib
le
Pol II-b
asal
Pol II-i
nduc
ible
0102030405060708090
100110120130140150200400600800
100012001400
MtorNqo1Gapdh
Antibody used for ChIP
Fold
Enr
ichm
ent t
o N
o ab
performed for mTOR. The presence of Nrf2 affected the expression of the targeted
proteins in a very heterogenous fashion across the three cell lines. Also, for some of the
above-mentioned genes, protein expression and transcriptional activity did not followed
the same pattern in all the three cell lines.
Luciferase assay was performed on the promoter regions containing the ARE sites. As
was the case for mTOR, 5000bps upstream from the TSS of each of the respective genes
were screened for the presence of AREs. TSC2 promoter region contained 6 ARE
binding sites. Luciferase assay was performed on the closest ARE present at 756bp
upstream of the TSS (TSC2 WT)(figure1Dappendix). S6K promoter region contained 12
ARE binding sites. The firsts 5 closest AREs, present at 255bp, 285bp, 324bp, 432bp and
2543bp upstream of the TSS were used in this assay (S6K WT) (figure1Eappendix). AKT
promoter region contained 3 ARE’s present at 1191 bp, 1403 bp and 1681 bp upstream of
the TSS witch were cloned and also used for this assay (AKT WT) (figure1Fappendix). For
site-directed deletion analyses the TGA site of the TSC2 ARE was mutated (TSC2 Mut),
and on S6K the two closest ARE’s to TSS were mutated as well ( S6K Mut )(table
1appendix). The activity of the abovementioned AREs showed great variation amongst the
three cell lines and in many cases did not followed the same pattern of the transcription
levels observed via qPCR.
4.4.1 TSC2, S6K and AKT expression when Nrf2 is up-regulated
4.4.1.1 TSC2 is a potential indirect Nrf2 transcriptional target at inducible
conditions on H460 cells
When upregulating Nrf2 (figure 7), TSC2 protein expression was induced only in H460 cells
while transcription was increase in all the three cell lines. The ARE present on TSC2
promoter region (graph 3) showed, in basal conditions, a small decrease in activity for TSC2
mut (A549 and H460). When Nrf2 is induced (figure 7), this ARE driven construct had
increased activity for when the ARE was WT (TSC2 WT) in A549 cells and HEK cells
and also in TSC2 mut in A549 cells. This could indicate that TSC2 is potentially an
indirect Nrf2 transcriptional target of increased Nrf2 as opposed to at basal conditions. In
H460 cells where TSC2 protein levels and transcription were increased. Although TSC2
transcription levels where increased by 10 fold in A549 cells no change was observed at
the protein level, perhaps suggesting a post-translational level of regulation of TSC2 in
these cells.
As observed for TSC2, when Nrf2 is increased (figure 8), S6K transcription is increased in
A549 and H460 cells. However, although, at basal Nrf2 levels (graph 4), luciferase activity
of S6K-mut was decreased 2 fold in the two cell lines, at Nrf2 inducible conditions, both
luciferase activity of S6K-WT and S6K-mut were increased in the A549 cells. This
implies that, while the ARE present on S6K promoter region is important for
transcription at Nrf2 basal levels, it is probable not induced by increased Nrf2 levels.
4.4.1.2 At Nrf2 inducible conditions AKT is a possible indirect Nrf2 transcriptional
target on H460 cells and posttranslational target on A549 cells
In H460 cell lines, at Nrf2 inducible conditions (figure 9), AKT transcription was increased
2 fold and proteins levels by over 5 fold. Since, no change was observed on the AKT
luciferase activity in this cell line, the results suggest that Nrf2 regulates this gene
indirectly, probably at the protein level. The increase of AKT luciferase activity on
HEK293 and A549 cells were also deceptive, since no significant changes were observed
at the transcription and protein levels in HEK293 cells and at the transcription level in
A549 cells. At protein level however, AKT was proximately 2 folds decreased in A549
cells. Hence, high Nrf2 levels affect some post-translational regulation of AKT protein
expression in A549 cells.
Figure 7. TSC2 (Nrf2 inducible) expression analysis. A and B. No significant change on Tsc2 protein levels were observed in HEK293 cells and A549 cells. C. Tsc2 protein levels were 6.78 fold increased in H460 cells. D, E and F. Tsc2 transcription was increased proximately two fold in HEK293 cells, 10 fold on A549 cells and two fold in H460 cells. G. The relative Luciferase activity of TSC2-WT in HEK293 cells was 1.5 fold increased with no change in activity on TSC2- mut. H. The A549 cell lines shown proximately two fold increase of TSC2-WT relative Luciferase activity with 1.5 folds increase on TSC2-mut. I. No significant change was observed in H460 cells for the relative Luciferase activities of TSC2-WT and TSC2-mut. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of induction from treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible(TSC2)
Western blot
TSC2
β-Actin
TSC2
TSC2β-Actin
1 : 1.16
1 : 6.78
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 1.73
Nqo1 1 : 1.3
Control Pc_Nrf2
1 : 0.91
Nrf2 1 : 1.63
Control Pc_Nrf2
Nqo1 1 : 60
Nrf2 1: 1.5Control Pc_Nrf2
Nqo1 1 : 1.6
qPCR
D)
E)
F)
Luciferase
G)
*
*
*
H)
*
I)
Control Nrf2 Nqo1 TSC20.00.51.01.52.02.53.03.54.0
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Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut0.00.51.01.52.02.53.03.54.0
6
9
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Graph 3. TSC2 (Nrf2 basal) Luciferase activity. TSC2 Mut presented a small decrease on A549 and H460 cells and no change on HEK cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity was represented as the fold change of the ratio from cells transfected with mutant construct (TSC2-Mut) versus cells transfected with wild type constructs (TSC2-WT). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of mutant and wild type constructs expression (*, p < 0.05).
TSC2-WT
TSC2-Mut
TSC2-WT
TSC2-Mut
TSC2-WT
TSC2-Mut
0.0
0.5
1.0
1.5
2.0HEK A549 H460
* *
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Figure 8. S6K (Nrf2 inducible) expression analysis. A, B and C. No significant change was observed on S6K protein levels on the three cell lines D, E and F. S6K transcription did not changed in HEK293 cells and it was 2 fold increased in A549 and H460 cells. G. The relative Luciferase activity of S6K –WT and S6K-mut were 1.5 fold increased in HEK293 cells H.The relative Luciferase activity of S6K –WT and S6K-mut were 2 fold increased in A549 cells I. No change was observed on the relative Luciferase activity of S6K –WT and S6K-mut in H460 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of induction from treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible(S6K)
Western blot
S6Kβ-Actin
S6K
S6Kβ-Actin
1 : 0.95
1 : 0.85
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 1.73
Nqo1 1 : 1.3
Control Pc_Nrf2
1 : 1.32
Nrf2 1 : 1.63
Control Pc_Nrf2
Nqo1 1 : 60
Nrf2 1: 1.5Control Pc_Nrf2
Nqo1 1 : 1.6
qPCR
D)
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Luciferase
G)*
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Graph 4. S6K (Nrf2 basal ) Luciferase activity. S6K Mut presented 2 fold decrease in A549 and H460 cells and no change in HEK293 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity was represented as the fold change of the ratio from cells transfected with mutant construct (S6K-Mut) versus cells transfected with wild type constructs (S6K-WT). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of mutant and wild type constructs expression (*, p < 0.05).
S6K-W
T
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ut0.0
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Figure 9. AKT (Nrf2 inducible) expression analysis. A, B and C. AKT protein levels were 5.15 fold increased in H460 cells, proximately 2 fold decreased in A549 cells and no significant change was observed in HEK293 cells. D, E and F. No significant change in AKT transcription was observed in HEK293 and A549 cells and it was two folds increased in H460 cell lines. G. Luciferase activity of AKT-WT was 1.5 fold increased in HEK293 cells, proximately 2 fold increased in A549 cells and no change was observed in H460 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of induction from treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible(AKT)
Western blot
AKTβ-Actin
AKT
AKTβ-Actin
1 : 0.85
1 : 5.15
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 1.73
Nqo1 1 : 1.3
Control Pc_Nrf2
1 : 0.56
Nrf2 1 : 1.63
Control Pc_Nrf2
Nqo1 1 : 60
Nrf2 1: 1.5Control Pc_Nrf2
Nqo1 1 : 1.6
qPCR
D)
E)
F)
Luciferase
G)
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4.4.2 TSC2, S6K and AKT expression when silencing Nrf2
4.4.2.1 TSC2, S6K and AKT may be affected post translationally, when Nrf2 is
silenced.
Decreasing Nrf2 has no significant effect on the observed on TSC2 (figure 10) and S6K (figure 11) transcription and luciferase activity. However, Tsc2 protein levels where
decreased 2.64 folds in HEK293 cells and S6K was 5.84 folds increased on A549 cells.
This suggests that at low cellular Nrf2 levels, TSC2 (HEK293 cells) and S6K (A549
cells) protein levels are in some way affected.
When silencing Nrf2 in A549 cells (figure 12), luciferase activity of AKT WT decreased
four folds alongside with two folds decrease in AKT transcription. These findings imply
that AKT could be a direct Nrf2 transcriptional target. However, the small increase in
AKT protein levels suggests that those changes in transcription and luciferase activity
may not be biological relevant. In both H460 and HEK293 cell, the changes in AKT
transcription was also probably misleading since they did not followed the same pattern
observed in the AKT Western blot. However, because AKT protein levels were
proximately two fold decreased in HEK293 cells, we believe that AKT may be a potential
Nrf2 post-translational target.
Figure 10. TSC2 (Nrf2 silencing) expression analysis. A, B and C. TSC2 protein levels were 2.64 fold decreased in HEK293 cells with no significant change observed in A549 and H460 cells. D-I.In all three cell line, no significant change was observed on TSC2 transcription and Luciferase activity of TSC2-WT and TSC2-mut. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of silencing from treated cells (siNrf2) versus Control (Scramble RNA). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing(TSC2)
Western blot
TSC2
β-Actin
TSC2
TSC2β-Actin
1 : 0.34
1 : 0.78
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 0.03
Nqo1 1 : 0.07
Control Si_Nrf2
1 : 1.01
Nrf2 1 : 0.02
Control Si_Nrf2
Nqo1 1 : 0.53
Nrf2 1 : 0.77Control Si_Nrf2
Nqo1 1 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
*
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Figure 11. S6K (Nrf2 silencing) expression analysis. A, B and C. S6K protein levels were 5.84 fold increased in A549 and no significant change was observed in HEK293 and H460 cells D, E and F. no significant change was detected in S6K transcription on the three cell lines G, H and I. Relative Luciferase activity of both wild type and mutant S6K constructs were 1.7 fold increased in HEK293 and H460 cells, no significant change was observed on A549 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of silencing from treated cells (siNrf2) versus Control (Scramble RNA). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing(S6K)
Western blot
S6K
β-Actin
S6K
S6Kβ-Actin
1 : 1.09
1 : 1.35
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 0.03
Nqo1 1 : 0.07
Control Si_Nrf2
1 : 5.84
Nrf2 1 : 0.02
Control Si_Nrf2
Nqo1 1 : 0.53
Nrf2 1 : 0.77Control Si_Nrf2
Nqo1 1 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
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Figure 12. AKT (Nrf2 silencing) expression analysis. A, B and C. AKT protein levels were proximately two fold decreased in HEK293 cells and no significant change was observed in A549 and H460 cells. D, E and F. AKT transcription was proximately 1.5 fold increased in HEK293 cells, 2 fold decreased in A549 cells and proximately 2 fold decreased in H460 cells. G, H and I. The Relative Luciferase activity for AKT-WT was four fold decreased on A549 cells and no significant change was observed in HEK293 and H460 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented as the fold change of the ratio of silencing from treated cells (siNrf2) versus Control (Scramble RNA). Values represent the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing(AKT)
Western blot
AKTβ-Actin
AKT
AKTβ-Actin
1 : 0.54
1 : 1.26
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf2 1 : 0.03
Nqo1 1 : 0.07
Control Si_Nrf2
1 : 1.38
Nrf2 1 : 0.02
Control Si_Nrf2
Nqo1 1 : 0.53
Nrf2 1 : 0.77Control Si_Nrf2
Nqo1 1 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
**
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Nrf2 has been shown to have impact of sensitivity to some cancer cytotoxic drugs, by
virtue of its regulation of a broad cyto-protective gene battery that includes cellular
defense against a variety of chemotoxins reactive oxygen species. A variety of studies
have shown that Nrf2 interacts with a wide variety of cellular proteins in different
pathways, among which is the PI3K pathway. The PI3K pathway is involved in various
mechanisms important for tumor development and growth, such as cell survival,
differentiation, and metabolism. Nrf2 is expressed predominantly in metabolic organs(40)
and there is evidence that Nrf2 enhances the PI3K pathway in systems with a high
metabolic state(79). mTOR is a crucial metabolic regulatory component of the PI3K
pathway, controlling cellular anabolic and catabolic processes coupling growth signals to
nutrient availability, via ribosome biogenesis and autophagy. The biological processes
determined by proteins in this cell-signaling pathway are commonly deregulated in
human cancers. Due to the importance of mTOR in cancer, and the previously described
interactions between Nrf2, PI3K pathway proteins including mTOR, the present studies
aimed to clarify a precise role of Nrf2 in the regulation of the PI3K pathway, including
mTOR.
The most important finding of this study is that modulation of Nrf2 levels regulates the
levels of mTOR at the protein level; further analysis confirmed that Nrf2 regulates the
transcription of mTOR on A549 cells. While the Luciferase assay and DNA pull down
results suggest that this regulation is a direct interaction of Nrf2 with elements in the
mTOR promoter, EMSA and ChIP assay did not allow us to definitely confirm that, as
the binding of Nrf2 to mTOR promoter in all the conditions studied is weak. There is still
much to explore about this interaction since it mechanism of action was not yet
established. An interesting aspect of the Nrf2/mTOR interaction, as well as the Nrf2
interaction with the other elements of the PI3K pathway, is that they may be cell line
specific, as differences were observed between the 3 cell lines used in our study. In A549
cells, mTOR transcription and protein translation correlated with Nrf2 levels. In HEK
cells, while Nrf2 upregulation did not lead to an increase in mTOR pretein levels, despite
the fact that an increase in mTOR transcription was observed. However, Nrf2 silencing
gave a similar expression profile as in A549 cells, which is two-fold decrease of mTOR
transcription along with decrease of its protein translation. In this condition, it was
observed a two-fold decrease of mTOR protein in A549 cells and total silencing of
mTOR in HEK293 cells. In H460 cell lines, which have an activating mutation PI3K and
therefore an activated pathway, as well as higher (than in A549) basal Nrf2 levels, we
observed no change at either mRNA or at Mtor protein levels when Nrf2 expression was
modulated.
These expression and promoter region analysis have shown that A549 cells presented a
more distinct correlation between Nrf2 levels and mTOR expression compared to the
other analyzed cell lines. Therefore, A549 cells were utilized in the subsequent studies of
determining the nature of the regulation of mTOR by Nrf2, using DNA pull down,
EMSA and ChIP assay.
The different results in the three cell lines reflects important heterogeneity amongst them.
HEK293 is a non-transformed Human Embryonic Kidney cell line. The absence of
mutations known to alter Nrf2 expression, can probably explain the low Nrf2
level and the different results when compared with the NSCLC cell lines. In
a study performed by Zhu L et al (100) using BEAS-2B cell line, which is a normal human
bronchial epithelium, Nrf2 up-regulation did not increase mTOR protein levels. On this
basis, it was affirmed that mTOR is not a target of Nrf2 activation. These results,
combined with our observations in HEK cells, suggest that Nrf2 does not cause a direct
increase in mTOR expression in non-cancerous cell lines.
Both A549 and H460 are cancer cell lines derived from NSCLC. The most common
NSCLC histologies include; epidermoid or squamous cell carcinoma, adenocarcinoma
and large cell carcinoma. The cell lines A549 and H460 are derived from
adenocarcinoma and large cell carcinoma respectively. Although diagnosis, staging,
prognosis, and treatment are similar for these different types of NSCLC, distinct set of
mutations, present, even within the same NSCLC histologic group, provide specific
molecular profile for each cell line(101). Both A549 and H460 cell lines have K-RAS and
Keap1 mutations. The K-RAS mutations are present at codon V12 on A549 cells and at
the codon V61 on H460 cells and Keap1 mutations are D236H and G333C in H460 and
A549, respectively (Singh et al 2006). K-RAS is known to generate an oncogene-directed
increased expression of Nrf2(102), while Keap1 when mutated liberates Nrf2 from
proteasomal ubiquitination(7,55). Western blot analysis showed higher Nrf2 protein in the
nucleus of NSCLC cell lines A549 and H460 than in the cytoplasm (55). The combination
of these mutations on K-RAS and Keap1 generates high constitutive levels of active
Nrf2, providing an ideal condition to study this transcription factor. Although, both H460
and A549 cells share similar mutations, they are distinct one from another, at least in part
due to the presence of a PIK3CA gain of function mutation in H460 cells(89). This may
cause a differential regulation of the elements of the PI3K pathway in H460 cells as
suggested by expression analysis assays of mTOR in this cell line as compared to A549.
In a study by ZU-QUAN ZOU(103) these NSCLC cell lines were expose to GDC-0941, a
dual inhibitor of class I PI3K and mTOR. It was found that due activating PIK3CA
mutations, H460 cells were more sensitive to GDC-0941 compared to A549 cells, likely
reflecting cellular “ addiction” to the PI3K pathways, which is driving cell growth. Their
study showed that the molecular profile present in H460 cells generates a distinct
phenotype, when compared to A549 cells, with respect to mTOR regulation. A
constitutively highly expressed PI3K pathway could possibly bypass regulatory
mechanisms of its elements, such as the proposed Nrf2/ mTOR interaction, making this
cell line more Nrf2- independent.
The results found here in A549 cells, in western blot and qPCR studies imply that mTOR
is either a direct or an indirect target of Nrf2. To our knowledge, there is no documented
direct Nrf2 targeting of mTOR transcription. The literature only describes indirect
interactions where the intermediate proteins act post-translationally on mTOR levels.
Shibata T et al (104) described an indirect interaction between Nrf2 mutant and mTOR via
RagD. Utilized gene set enrichment analysis (GSEA) they determined that a mutant Nrf2
induced pathways associated with mTOR signaling; Peng Rapamycin DN, Peng Leucine
DN and Peng Glutamine DN. Additionally they found that mutant Nrf2 indirectly
upregulates the mTOR activator RagD and further that decreased Nrf2 reduced RagD
expression. This could indicate that Nrf2 directly regulates RagD expression, however,
the bioinformatic algorithms we use to identify potential ARE sequences found no such
motifs present in the promoter region of RagD. Thus, the link between Nrf2 and Rag D
remains unknown. Sasaki H et al (56) also found a correlation between mutant Nrf2 and
RagD gene expression in a study that involved 90 cases of surgically-treated lung
squamous cell cancer patients. Among all the patients 14 cases were positive for a Nrf2
gene promoter polymorphism, which generated a gain of function mutation. In this group
of patients RagD expression was 3 times higher, indicating at least a strong correlation
between Nrf2 and RagD.
The somatic mutations present on mutant Nrf2 also occur in its coding region and it
modifies amino acids in the DLG or ETGE motifs, causing abnormal cellular
accumulation of Nrf2, likely because the mutant does not bind efficiently do Keap1, the
chaperone for proteosomal degradation(105). The mutations present in Nrf2 in those
studies may explain why mTOR was not found on GSEA, as structural changes at the
protein level could have affected the detection of mTOR enrichment. Little is known
about the many effects that this mutation cold have on Nrf2 binding and, as it was
observed in our EMSA and ChIP results, the Nrf2/mTOR promoter binding is probably
weak. Perhaps, for this reason, the genome wide profile in the study Shibata T et al (104)
did not observed a direct interaction between Nrf2 and mTOR . Also, as already
described by Shibata et al 2008(105), the Nrf2 present in A549 cells is not mutated. The
Nrf2 mutation described in the literature occurs more frequently in lung cancers that do
not have an EGFR and Kras mutations(105). While, A549 cells express a wild type EGFR
and a mutated Kras gene (106), this NSCLC is an adeneocarcinomic alveolar basal sub-
type, whereas the Nrf2 gene somatic mutation was show to be more prevalent in lung
squamous cell carcinomas(107).
It is possible that an unknown element, which is influenced by Nrf2 levels in the cell is
responsible for the observed mTOR transcription regulation. In order to establish whether
mTOR is a direct Nrf2 transcriptional target, bioinformatics analyses were performed on
this gene promoter region looking for ARE biding sites. The ARE site TGACTCAGC is
believed to be the canonical ARE binding site, although some variations of that canonical
binding site have also shown to be functional. In our study, site directed deletion of the
“TGA” portion of the ARE showed that this ARE is essential for transcription as the
luciferase activity, as the mutated ARE showed a two fold decrease on A549 cells. When
Nrf2 is decreased using SiRNA, no change was observed on mTOR luciferase activity for
this cell line. However, when Nrf2 expression is increased, luciferase activity is increased
three fold in A549 cells transfected with mTOR-WT, whereas there was no change in
cells transfected with mTOR-mut. These data suggest that in A549 cells, while Nrf2 does
not affect basal mTOR expression, when Nrf2 is increased above basal levels it has a
direct effect on mTOR expression.
The nature of the Nrf2/mTOR promoter binding in A549 cell line, however, was unclear
as it contradicted the western blot and qPCR results, which demonstrated that Nrf2 down
regulation results in decreased mTOR protein levels. Various factors could have
contributed to this; one limitation of the luciferase assay is that only a small portion of the
mTOR promoter region was analyzed, excluding further upstream regions of the
promoter that could contain binding sites for interacting proteins and other regulatory
elements that could play a role on mTOR expression. Additionally, not yet identified
Nrf2 targets might bind to the mTOR promoter region and alter its transcription.
Therefore, for more clarification additional analyses on A549 cells .
Through DNA pull-down and EMSA studies, we found that Nrf2 binds to the mTOR
promoter region at the ARE present at 723 bp upstream from TSS under basal Nrf2
conditions. In the DNA pull down assay, the mTOR probe containing a scrambled ARE
site presented two fold decrease of the amount of mTOR protein bound when compared
with the wild type mTOR probe. In EMSA, the addition of the wild type mTOR probe
generated a blot with similar characteristic to the one presented in Nqo1. After removing
the ARE site from the mTOR promoter sequence and adding five bp in the extremities of
the probe, mutant mTOR the predicted Nrf2 binding site disappeared. These results
confirmed that the transcriptional activity observed on A549 cells at Nrf2 basal
conditions in the luciferase assay was truly due to Nrf2 binding. However, the
Nrf2/mTOR binding observed in the EMSA assay suggests that it is a weak binding as it
was only observed after four days of film exposure.
Additionally, It was shown via EMSA that Nrf2 binding to the mTOR promoter region
decreases in Nrf2 silencing conditions. Since no change was observed in the luciferase
assay when Nrf2 was silenced, the decrease in binding observed via EMSA assay, most
likely, does not affect mTOR transcription. Hence, possibly, the decrease in the mTOR
protein and transcriptional levels, observed via immunoblot and qPCR assay at Nrf2
silencing conditions, could be due to an unknown mTOR regulatory element that is
affected by Nrf2 levels in the cell.
An interesting finding, that is outside the scope of this study, is the putative presence of
NF-ΚΒ in mTOR promoter region. In the EMSA assay we observed that protein was
bound to another biding site present on the mTOR mutant probe. This protein or complex
was not present in the wild type mTOR probe, but was seen in the mutant. There is strong
evidence that this protein could be NF-ΚΒ. This transcription factor binds to the
consensus DNA sequence 5’-GGGRNYYYC-‘3 (in which R is a purine, Y is a
pyrimidine and N is any nucleotide) known as the ΚΒ site(108) .The 5’ end of the mutated
probe after the five bp extension is 5’- GGGAATTTC-‘3, which fits the specification of
the ΚΒ biding site. Also, when analyzing the mTOR promoter region using the UCSC
genome browser, NF-ΚΒ is present in the same position of the portion of the promoter
region of this study. The potential for influence of this transcription factor on mTOR
regulation by Nrf2 has never been studied.
Finally, in A549 cells, ChIP analysis confirmed the presence of a weak Nrf2/mTOR
biding under basal Nrf2 conditions along with establishing mTOR as an inducible target
of Nrf2, as mTOR enrichment was seen to increase 13 folds after Nrf2 up regulating in.
The basal cellular level of Nrf2 can be seen as an indication of low cellular stress, due to
redox or chemical toxicity (13,14). The Nrf2 levels are increased in response to any of these
stresses(13,14) Our data thus suggest that cell stressors can result in enhanced mTOR
transcription, which is of interest since mTOR activity can itself generate ROS(82,109).
For the first time it is shown that, on a specific type of NSCLC, Nrf2 directly interacts
with a putative ARE binding site present in the mTOR promoter region. It was
demonstrated that this biding resulted in the induction of mTOR expression at the
transcriptional level. Additionally, it was confirmed via ChIP assay that this binding
occurs at Nrf2 inducible conditions. The next step in order to fully understand this
interaction would be to identify the biological conditions that lead to mTOR
transcriptional activation via Nrf2. There are descriptions in the literature of regulatory
mechanisms between Nrf2 and mTOR(110). Although not yet fully understood, these
interactions were shown to generate a positive loop that regulates autophagy(110). On one
side Nrf2 inactivates mTOR activity through an entirely different, and yet undefined
mechanism that determines mTOR phosphorylation(110) and in the other side this decrease
in mTOR activity indirectly increases Nrf2 expression(111).
Nrf2 is known to participate in autophagy and modulation of hepatic regenerative
response to liver mass loss(112). It is persistently active whenever autophagy is deficient in
the cell, which was shown to be due to Nrf2 up-regulation of Bcl-2(97) and p62(100).
The p62 protein, also called polyubiquitin-binding protein p62/SQSTM1(sequestosome
1), is encoded by the SQSTM1 gene and is responsible for protein aggregation and for
facilitating the passage of those cellular components into autophagosomes for lysosomal
degradation(113) mTOR is also related with autophagy and control of liver architecture.
When activated, this protein decreases lysosomal degradation of intracellular
components. In order to regulate the number and size of maternal hepatocytes, mTOR
needs to be activated in a stage-dependent phosphorylation pattern. When exploring the
role of Nrf2 in the regulation of maternal hepatic adaptation to pregnancy, Yuhong Zou et
al(110) stated that Nrf2 is a negative regulator of mTOR signaling. Their study showed that
the progressive decrease in Nrf2 activity was correlated with a gradual increase in mTOR
phosphorylation in the maternal liver during the second half of pregnancy. Also, Nrf2
deficient mice caused hyperphosphorylation of mTOR in the non-pregnant state. The
mechanism by which Nrf2 inhibit mTOR phosphorylation was not approached in this
study.
The p62 protein along with being transcriptionally up regulated by Nrf2, also increases
Nrf2 stabilization through Keap1 docking, which blocks the Nrf2 binding, thus
generating a positive feedback loop(114). Lerner et al (111) presented mTOR as part of the
p62 interaction with Nrf2 when they showed that reduction in mTOR activity leads to
increased turnover of p62/SQSTM1. Therefore, Nrf2 decreases mTOR activity which in-
turn increases p62/SQSTM1turnover, resulting in increase of Nrf2 activity.
The putative regulatory loop between Nrf2 and mTOR indicates that the two proteins can
interact in a complex biological system and in various ways. These interactions however,
are indirect and occurs at the posttranslational level. In our study we showed that Nrf2
can bind directly to the mTOR promoter region and that it can activate its expression at
the transcriptional level. It is known that depending on the cell type and stress conditions
ROS can serve as a signal for autophagy through various pathways(115). Based on the
literature(77, 116, 117) , we believe that the mTOR transcriptional regulation via Nrf2 is also
dictated by ROS. Both Nrf2 and mTOR activities are altered by ROS levels in the cell.
While regulatory loop of ROS in Nrf2 is well known; an increase in ROS activates Nrf2
which in turn transcriptionally upregulats anti-oxidants genes thereby decreasing ROS.
However, mTOR regulation by ROS is an intricate processes. Chen et al (116) have shown,
in PC12 cells and primary murine neurons, that apoptosis of neural cells via oxidative
stress could be due to the H2O2 inhibition of the mTOR-mediated phosphorylation of
S6K1 and 4E-BP1. This inhibition of mTOR activity occurs indirectly, through the
activation of AMPK and inhibition of AKT and PDK1 phosphorylation. Another
condition where mTOR is inactivated by ROS is via activation of LKB1/AMPK/TSC2 in
Ataxia-telangiectasia mutated (ATM) cells. Mutation of the ATM protein kinase in
ataxia- telangiectasia (AT) is known to generate a high ROS environment in the cell. In
this condition the LKB1 tumor suppressor activate AMPK which activate TSC2 and
therefore inhibiting mTORC1(109) While these are examples of ROS decreasing mTOR
activity, there is much evidence for the opposite effect, and we believe that our findings
regarding Nrf2 here are relevant (118,119).
The role of mTOR as a regulator of metabolism its attributed to its capacity to integrate
signals from the cell environment, such as nutrients and oxygen availability, with protein
translation(77). Arsham et al (118) demonstrated that oxygen is a modulator of the mTOR
pathway signaling with oxygen activating mTOR while hypoxia inhibiting it. Other
studies have shown that O2 byproducts can also activate mTOR, and ROS can even work
as a messenger in nutrient-sensing pathway as it was illustrated by the activation of the
mTOR pathway via Leucine generated ROS(120). In the early stage of incubation with this
amino acid mTOR phosphorylation was independent of the PI3K pathway and at a later
stage mTOR was activated via a ROS mediated IR/IGF-IR phosphorylation which
activate the PI3K/Akt/mTOR and ERK signaling pathways.
The ROS mediated mTOR activation was shown by some groups to be via AKT while
other groups stated that it could also occur as an AKT-independent process. Radisavljevic
et al (121) showed mTOR activation by ROS is a processes that occurs downstream from
the PI3K/Akt signaling pathway, in the study on H2O2 exposed mice type II pneumocytes.
In this study they found that ROS-mediated mitosis was due to H2O2 phosphorylation of
AKT at Ser473, which activated mTOR. Huang C et al (119) demonstrated an AKT-
independent model of ROS mediated mTOR activation in mouse epidermal JB6 Cl41
cells. In their study, the PI3K/TOR /S6K pathway was activated after UV- generated
H2O2 exposure. They found, after combining H2O2 treatment with Rapamycin, that
phosphorylation of p70S6K at Thr389 and Thr421/Ser424 was an AKT independent and
mTOR-dependent processes. The molecular mechanism responsible for mTOR activation
was not revealed(119). The mechanism of AKT-independent of mTOR activation was
shown later by Sanchez Canedo C. et al (122) They showed that PDK1 is required for
PRAS40 phosphorylation in a Leucine mediated activation of the cardiac mTOR/p70S6K
pathway. Our study has shown that mTOR can be activated at the transcriptional level via
Nrf2. In theory, this PI3K independent activation of mTOR might also be mediated by
ROS.
The central role of ROS regulation by both mTOR and Nrf2, their importance in cancer
metabolic reprogramming together with the identification of mTOR as an Nrf2 inducible
transcriptional target, suggest that a ROS mediated feedback loop is possible between
these two proteins. On the one hand, mTORC1 is important for stimulation of
transcription of genes involved in mitochondrial biogenesis, consequently increasing the
levels of ROS in the cell as a byproduct of the respiratory chain(117). On the other hand,
oxidative stress generated by ROS would lead to breakdown of the Nrf2-Keap1 complex
and subsequently transcription of anti oxidants genes and mTOR by Nrf2 in the nucleus.
The ROS inhibition via Nrf2 activation and the mTOR inhibition by ROS could
potentially work as self-regulatory mechanisms of this feedback loop. This novel
interaction between mTOR and Nrf2 could help us better understand the ROS regulatory
system in cancer.
The other elements of the Pi3K pathway were analyzed only in a preliminary fashion,
using western blot, qpcr and luciferase assays, in order to determine if Nrf2 also
interacted with the different elements of the pathway, apart from mTOR. The results
showed varying degrees of regulation of Nrf2 on the other elements of the pathway.
However, further assays need to be performed in order to accurately evaluate the role of
Nrf2 on the regulation of TSC2, AKT and S6K activities and/or expression.
It has been shown previously by Malhotra, D. et al (37) that TSC2 is a possible target of
Nrf2 at basal levels. Our data suggest that in Nrf2-inducible conditions TSC2 is a indirect
Nrf2 transcriptional target. However, when silencing Nrf2, only TSC2 protein levels
where decreased. Therefore, Nrf2 may act on TSC2 expression at the transcriptional level
at inducible conditions and at the protein level when downregulated.
The discordance with Malhotra, D. et al results could be due the cell type specificity of
these effects that we found, since the ChIP-Seq study utilized MEFs cells and our
experiments were done on human cancer cell lines. Furthermore, interacting proteins,
which could be directly or indirectly influenced by Nrf2 levels in the cell, may regulate
TSC2 expression. MAPK is involved in activation of Nrf2 by various pathways and is
also responsible for TSC2 phosphorylation. Perhaps, this indirect interaction observed
between Nrf2 and TSC2 is mediated by MAPK.
To our knowledge, there is no description in the literature showing that Nrf2 directly
interacts with AKT. There are however, examples where Nrf2 enhances, via AKT
interactions, the metabolic reprogramming triggered by proliferative signals. This was
observed in the Nrf2 mediated AKT phosphorylation during PPP(79) and in the redirection
of glucose and glutamine into anabolic pathways in cells with high Nrf2 levels and active
PI3K-AKT signaling. In the present study AKT was shown to be a possible indirect Nrf2
transcriptional target in Nrf2 inducible conditions. This interaction however may be also
cell line dependent as it is the case for mTOR. The higher metabolic state of H460 cells
lines when compared to A549 cell, is primarily due to the PI3K gain of function
mutation, and may explain the presence of a possible interaction between Nrf2 and AKT
in H460 cells. When silencing Nrf2, AKT was two folds decreased at the protein level on
HEK293 cells. In a previous study(80) on ROS mediated insulin/IGF1 resistance, NRF2
KO mice were shown to decrease AKT activity but not at the proteins level. Therefore,
this interaction is yet to be clarified.
Also, to our knowledge, there is no previous mention elsewhere of a direct Nrf2
interaction with S6K. This well-known substrate of mTORC1 is not affected at the
transcription or protein level by Nrf2. Therefore the data obtained in our study are the
result of an unknown interaction. While no significant change was observed on S6K
levels after Nrf2 upregulation, Nrf2 silencing induced a 5.84 folds increased in A549
cells, suggesting that Nrf2 may have a direct or indirect inhibitory effect on S6K at the
protein level. It would be interesting to know in the future how Nrf2 inhibit S6K and if
this inhibition plays a role in the proposed ROS mediated feedback loop between Nrf2
and mTOR.
In order to establish if the Nrf2 transcriptional activation of mTOR is mediated by ROS,
lactate and ROS levels will be measure in a time course experiment together with
proteins activity. This study may utilize AKT inhibitors so as to observe if the PI3K
pathway participates in the proposed feedback loop.
In the future, others transcription factors that may interact with Nrf2 at the mTOR
promoter region will be studied in order to better understand this regulatory system.
It was observed in preliminary ChIP results, but not yet confirmed, (data not shown) that
Nrf2 may interact in the mTOR’s ARE binding site with JundD, a member of the AP-1
family.
Like Nrf2, the AP-1 family of transcription factors also belong to the bZip family and
they are comprised by the Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and
Fra-2) families. Among the functions from which these transcription factors are involved
are proliferation, differentiation, apoptosis and development(123). The core sequence of
those transcription factors biding site is 5’-TGA(G/C)TCA-3’ and it is called TPA
response element (TRE) (123). Similary, the Nrf2 partner small Maf ( Maf G/F/K) as well
as the other members of the Maf family (c-Maf, MafB, MafA, and Nrl) also bind to the
TRE(124).
The TRE closely resembles the Nrf2 transcription factor binding site ARE. The main
difference between ARE and TRE is the presence of the GC box at the end of the ARE,
which is essential for the ARE-driven gene expression. Jaiswal et al(34) showed that the
human NQO1 ARE contained TRE and TRE like elements and, AP-1 family members,
Jun and Fos, bind to the human Nqo1 ARE. They also demonstrated that overexpression
of combinations of nuclear proteins Jun and c-Fos or Jun and Fra1 downregulated hARE-
mediated gene expression, however, Jun proteins alone did not exert any significant
effect on the hARE. Similar effects of AP-1 proteins were also seen on γ-
Glutamylcysteine synthetase (γ-GCS) by Jaiswal et al. (125) They showed that Nrf2
heterodimerized with c-Jun to significantly upregulate. The binding of Nrf2 and AP-1
proteins to ARE is believed to be competitive. For example, the γ-GCS ARE-mediated
expression is differentially regulated by AP-1(Jun+Fos; negative) and Nrf2(positive
effect) and the dynamic is thought to be determined by the amount of each nuclear
protein present.
It is presumed from preliminary ChIP results that JunD acts as a co-factor for Nrf2 when
binding to mTOR. However, further ChIP analyses are necessary in order to confirm this
interaction. Also, a EMSA super shift experiment will be performed with A549 cells line
in Nrf2 basal, silencing and inducible conditions. In this experiment the mTOR wt and
mTOR mutant probes will be utilized together with JunD. This experiment will further
confirm the presence of Nrf2 on mTOR biding site and also demonstrate the interaction
of this members of the AP1 family with Nrf2 at the mTOR promoter region.
The Nrf2/mTOR interaction explored in this study may also have future application in the
clinic. With various studies focusing on inhibitiors of mTOR activity no studies have
been done on its inhibition at the transcription level. Recent attempts to develop mTOR
inhibitors largely focus on rapamycin analogs. Sadly, clinical trials suggest that
rapamycin is effective in few cancers only (B cell lymphoma, endometrial cancer, and
renal cell carcinoma)(126).This restricted therapeutic effect is due the presence of a feed
back loop, as activation of mTORC1 signaling strongly represses PI3K- AKT signaling
upstream in the PI3K pathway (reviewed in Manning, 2004). This inhibition is done via
mTORC1 dependent S6K activation, which inhibits the insulin receptor substrate 1(IRS-
1) ultimately blocking the PI3K/AKT pathway. This class of drugs not only activates
proliferative effectors, such as AKT, but also incompletely inhibits mTORC1. Therefore,
rapamycin analogs could potentially be responsible for hyperactivation of rapamycin-
resistant mTORC1. Also, mTORC2 works as a PI3K by its direct activation of AKT by
phosphorylation in the hydrophobic Ser473(127). The mTORC2 activity is blocked only at
high toxic levels of Rapamacin(128).
Dual inhibition of the PI3K pathway or other signaling pathways and mTOR could be an
effective strategy (Fan and Weiss, 2006; Wan et al., 2007). Among the drugs that explore
this approach are gefitinib (Iressa, an EGFR inhibitor), imatinib mesylate (Gleevec, a
BCR-ABL inhibitor), tamoxifen (estrogen receptor modulator), cisplatin (DNA damaging
agent), and paclitaxel (microtubule stabilizer) (reviewed in Faivre et al., 2006; Granville
et al., 2006). This line of treatment seems effective in cancer cell lines and tumors with
hyperactivated PI3K pathway. However, presence of K-Ras hyperactivation in Ras-
driven tumorogeneses made necessary the additional blocking of downstream RAS
mediators. This combination of signaling disturbance could lead to toxicity in normal
cells(129).
The abovementioned reflects that the mechanism of action responsible for mTOR
activation is still not fully understood. Possibly, Nrf2 as an mTOR transcriptional
activator could be explored in the future as a new mechanism for the development of a
combined therapy for cancer treatment, with mTOR inhibitors and the blockage of Nrf2
activity.
Lastly, a newly discovered Nrf2 function will be explored alongside with mTOR. Since it
targets mTOR, Nrf2 may also therefore play a role in protein translation (not published
data). In order to better understand the role of Nrf2 on translation and the effects of the
mTOR induced transcription via Nrf2 transactivation, a Polisome fraction assay will be
performed at Nrf2 basal, induction and silencing conditions. Simultaneously, mTOR
inhibitors will also be used so as to evaluate if the translation is due mTOR induction.
7. Acknowledges________________________________________________________
I would like to acknowledge Dr. Batist for giving me the opportunity to work with him in this exhilarating field of research and providing me with his knowledge throughout the project. I am obliged to Dr. Tahar Aboulkassim for his patience and guidance and my lab colleagues Dr. Liu Qiang and Sujay Shah for their assistance. Lastly, I would like to express thanks to the members of Dr. Witcher lab, Dr. Maud Marques, Dr. Khalid Hilmi and Dr. Tiejun Zhao and also the members of Dr. Alaoui-Jamali lab Dr. Sabrina Wurzba and Amine Saad for their support.
Figure 1. Nrf2 inducible construst plus constructs used for Luciferase assay. A. The inducible construct PC_Nrf2 contained 1925bp of Nrf2 coding sequence (green line) cloned on the expression vector pcDNA 4.0. B –F. All the constructs used on Luciferase assay comprised of the promoter region of the gene of interest (green line) cloned on pGL3-basic Luciferase reporter vectors, C terminally to the Luciferace reporter sequence (purple line). B. PGL3-Nqo1 contained 550 bp of Nqo1 promoter region C. PGL3-mTOR contained 1231bp of mTOR promoter region D. PGL3-TSC2 contained 1079 bp of TSC2 promoter region E. PGL3-S6K contained 2660bp of S6K promoter region F. PGL3-AKT contained 2200 bp of AKT promoter region
Table 1. Primers used for qPCR, Luciferse constructs, DNA pulldown, EMSA and ChIP assay
5’-TGAAGGTCGGAGTCAACGGA-3’ 5’-GAGGGATCTCGCTCCTGGAAG-3’5’-GAGAGCCCAGTCTTCATT-3’ 5’-TGCTCAATGTCCTGTTGCAT-3’5’-GCTGGTTGAGCGAGTGTTC-3’ 5’- CTGCCTTCTTACTCCGGAAGG-3’5’-GACTTCGCCCATAAGAGGCA-3’ 5’-TAGCTGTGGAATCTGACGGC-3’5’-GTCTCTTCCGAGATTTTCGACG-3’ 5’-ATCCCCAACATTGTTAAGCGT-3’5’-GGACGCTGGAGAAGTTCAAG-3’ 5’-CGGATTTTTGGTTCAAAGGA-3’5’-TCTATGGCGCTGAGATTGTG-3’ 5’-CTTAATGTGCCCGTCCTTGT-3’
5'-GGTGGGTACCCAAAACCAATTA-'3 5'-GGTG CTCGAG G CCGTTTGAGGTGGACAGCCTA-3'5'-CCTTCCAAATCCGCAGCAGTGACTCAGCA5'-TTCTGCTGAGTCACTGCTGCGGATTTGGAAGG-3'5'-GGTG ACGCGTGTGCCAGGCCCTAGA-3'5'- GTCGAGACCAGCCTGGACATGGTGAAAT5'-GGTGGGTACCGGGAAAAAGGCGCAAGGT5'-CATGCGCCCCGCGTGATGCAAGG-3'5'-GGTG GGTACC GCCAGCAGGGTCCTCTTT GGGCCCGGACCCGCCGC-3' 5'-CCGAACGCTCCAGCCAATGCGCATGCTC-5'-ATATGGTACGCATGCCTGTCCACCGAACG5'-GGTGGGTACCGCCAGCAGGGTCCTCTTTT
5’ -/Biosq/GCAGTCACAGTGACTCAGCAGAA 5’ -/Biosq/TTCTGCTGAGTCACTGTGACTGC- 3’5’ -/Biosq/GCAGTCACAGACTCTCAATAGAAT5’ -/Biosq/ATTCTATTGAGAGTCTGTGACTGC-3’5’- /Biosq/GTCGAGACCAGCCTGGTCAACAT 5’- /Biosq/ATGTTGACCAGGCTGGTCTCGAC-3’5’- Biosq/GTCGAGACCAGCCTGGTAAAT GG 5’ /Biosq/CCATTTACCAGGCTGGTCTCGAC-3’
5'-GCAGTCACAGTGACTCAGCAGAATCTG A5’-CTCAGATTCTGCTGAGTCACTGTGACTGC-3'5’-GCA GTC ACAGACTCTCAATAGAAT-3’ 5’-CTCAGATTCTATTGAGAGTCTGTGACTGC-3’5'-GTCGAGACCAGCCTGGTCAACATGGTGA 5’-TTCACCATGTTGACCAGGCTGGTCTCGAC-3’5’-CAGGAGTCGAGACCAACATGGTGAAATT5’-GGGAATTTCACCATGTTGGTCTCGACTCC-3’
CHIP5'-CGGTGGCTCACGCCCATAATT-'3 5'-CAGCCTCCCGAGTATC-'35-AAGTGTGTTGTATGGGCCCC-'3 5'-GTGGAAGTCGTCCCAAGAGA''3