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Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
Molecular
Cancer
Therapeutics
Cancer Biology and Signal Transduction
Aromatase Inhibitor–Mediated Downregulation of
INrf2 (Keap1) Leads to Increased Nrf2 and
Resistance in Breast Cancer
Raju Khatri1, Preeti Shah1, Rupa Guha1, Feyruz V. Rassool1, Alan E. Tomkinson2,
Angela Brodie1, and Anil K. Jaiswal1
Abstract
Aromatase inhibitors are effective drugs that reduce or eliminate hormone-sensitive breast cancer. However, despite their
efficacy, resistance to these drugs can occur in some patients. The
INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiationinduced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/
Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress,
Nrf2 dissociates from INrf2, is stabilized, translocates to the
nucleus, and coordinately induces a battery of cytoprotective gene
expression. Current studies investigated the role of Nrf2 in aromatase inhibitor resistance. RT-PCR and immunoblot assays
showed that aromatase inhibitor–resistant breast cancer LTLTCa
and AnaR cells express lower INrf2 and higher Nrf2 protein levels,
as compared with drug-sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/
degradation of Nrf2 in aromatase inhibitor–resistant cells. Higher
Nrf2-mediated levels of biotransformation enzymes, drug transporters, and antiapoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance.
shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells
reduced resistance and sensitized cells to aromatase inhibitor
exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed
reduced levels of aldehyde dehydrogenase, a marker of tumorinitiating cells and significantly decreased mammosphere formation, as compared with LTLTCa-Vector control cells. The results
together suggest that persistent aromatase inhibitor treatment
downregulated INrf2 leading to higher expression of Nrf2 and
Nrf2-regulated cytoprotective proteins that resulted in increased
aromatase inhibitor drug resistance. These findings provide a
rationale for the development of Nrf2 inhibitors to overcome
resistance and increase efficacy of aromatase inhibitors. Mol Cancer
Introduction
biosynthesis; the aromatization of androgens to estrogens (8, 9).
Breast cancer tissues have been shown to express aromatase and
produce higher levels of estrogens than noncancerous cells (7).
Estrogens stimulate breast cancer cell growth and proliferation.
Aromatase inhibitors became the choice of treatment for breast
cancer in postmenopausal women because they block the synthesis of estrogens required by cancer cells to grow (10). Currently,
there are three aromatase inhibitors approved by the FDA, letrozole, anastrozole, and exemestane (Supplementary Fig. S1). These
are approved for postmenopausal women with hormone receptor–positive breast cancer in both the adjuvant and metastatic
setting. Letrozole is more potent than other aromatase inhibitors
in reducing plasma estrogen levels (11). While aromatase inhibitors are a very effective treatment, their benefit is often limited
by the emergence of resistance that occurs in a significant number
of patients in the adjuvant setting and is inevitable in the metastatic setting.
The INrf2 (Keap1):Nrf2 complex acts as a cellular sensor of
xenobiotics, drugs, and radiation-induced ROS/electrophilic
stress (12). Nuclear factor Nrf2 controls the expression and
coordinated induction of a battery of genes encoding detoxifying
enzymes [quinone oxidoreductases (NQO1 and NQO2), glutathione S-transferases, heme oxygenase 1 (HO-1)], glutathione
and related proteins [glutathione, thioredoxins, g-glutamyl cysteinyl synthetase (g-GCS)], ubiquitination enzymes and proteasomes (12, 13), drug transporters (MRP; refs. 14, 15), and antiapoptotic proteins (16). Nrf2 is retained in the cytoplasm by an
inhibitor INrf2 or Keap1 (17, 18). INrf2 functions as an adapter
Drug resistance is the major obstacle to the successful treatment
of many cancers (1). The factors that contribute to the development of drug resistance include alterations in drug intake, efflux,
metabolism, and excretion. Deregulation of cell death by evasion
of apoptosis, necrosis, mitotic catastrophe, or senescence also
contributes to drug resistance (1–3). In addition, the differential
expression of membrane proteins such as solute carriers, channels, and ATP-binding cassette transporters have all been demonstrated to play important role in drug resistance (4, 5).
Breast cancer is the most common cancer among women (6).
Aromatase inhibitors are an effective first line of treatment for
ERa-positive breast cancer that constitutes three-fourth of all
types of breast cancers (7). Aromatase (cytochrome P450
CYP19A1) catalyzes the rate-limiting and final step of estrogen
1
Department of Pharmacology and Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, Maryland.
2
Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Anil K. Jaiswal, University of Maryland School of
Medicine, 655 West Baltimore Street, Baltimore, MD 21201. Phone: 410-7062285; Fax: 410-706-5692; E-mail: [email protected].
doi: 10.1158/1535-7163.MCT-14-0672
2015 American Association for Cancer Research.
Ther; 14(7); 1728–37. 2015 AACR.
1728 Mol Cancer Ther; 14(7) July 2015
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Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
Nrf2 and Aromatase Inhibitor Drug Resistance
for Cul3/Rbx1-mediated degradation of Nrf2 (12). In response to
chemical/drug/radiation including antioxidant tert-butyl hydroquinone (t-BHQ)-induced oxidative/electrophilic stress, Nrf2 is
switched on (separation from INrf2 and stabilization of Nrf2) and
then off (ubiquitination and degradation of Nrf2) by distinct early
and delayed mechanisms (12). Oxidative/electrophilic modifications of INrf2 cysteine151 and/or PKC phosphorylation of Nrf2
serine40 result in the escape or release of Nrf2 from INrf2 (12).
Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with small Maf or Jun proteins, and binds antioxidant
response elements resulting in coordinated activation of gene
expression (12). Indeed, in vivo evidence has demonstrated the
importance of Nrf2 in protecting cells from the toxic and carcinogenic effects of many environmental insults. Nrf2-knockout
mice were susceptible to acute damages induced by acetaminophen, ovalbumin, cigarette smoke, and pentachlorophenol and
had increased tumor formation when exposed to carcinogens
such as benzo[a]pyrene, diesel exhaust, and N-nitrosobutyl (4hydroxybutyl) amine (19–22). Therefore, Nrf2 appears to play a
significant role in cytoprotection and cell survival (12). In addition, Nrf2 plays significant role in prevention of cancer metastasis
(23–25).
Studies have also described the detrimental effects of Nrf2 (26–
30). Persistent stabilization and nuclear accumulation of Nrf2 is
suggested to play a role in survival of cancer cells and drug
resistance. Increase in Nrf2 due to inactivating mutations in INrf2
has been reported in lung cancer (26, 27). Although Nrf2 is
thought to contribute to drug resistance by inducing cytoprotective proteins (28, 29), its role in resistance of breast cancer to
aromatase inhibitors remains unknown.
The studies in this report showed that aromatase inhibitor–
resistant breast cancer cells contain lower INrf2 and higher Nrf2
levels, as compared with drug-sensitive cells. Studies also revealed
that higher Nrf2 was due to decreased INrf2, lower ubiquitination,
and slower degradation of Nrf2 in aromatase inhibitor–resistant
cells. Higher Nrf2-mediated increase in biotransformation
enzymes, drug transporters, and antiapoptotic proteins contributed to reduced efficacy of drugs and prevention of apoptosis that
led to drug resistance. Interestingly, LTLT Ca cells deficient in Nrf2
(LTLTCa-Nrf2KD) showed reduced levels of aldehyde dehydrogenase (ALDH), a marker of tumor-initiating cells (TIC), significantly decreased mammosphere formation and increased sensitivity to exemestane and doxorubicin, as compared with parental
LTLTCa cells expressing higher levels of Nrf2. These results collectively suggest that persistent aromatase inhibitor treatment
downregulated INrf2 leading to higher Nrf2 and downstream
cytoprotective proteins that resulted in increased aromatase
inhibitor drug resistance.
(cat. no. 3473) for mammospheres was obtained from Corning.
DCFDA Cellular ROS Detection Assay Kit (cat. no. ab113851) and
g-glutamylcysteine synthetase (GCLC, ab40929) antibody were
obtained from Abcam. Anti-LDH (cat. no. 3558) from Cell
Signaling Technology, anti-MRP4 (cat. no.ALX-801-038) from
Enzo Life Sciences, anti-BCRP (cat. no. OP191-200UL), Ku80
(cat. no. NA54), and proteasome inhibitor MG-132 (cat. no.
474790) from Millipore were purchased for Western blotting.
Aldefluor assay kit was obtained from Stem Cell Technologies.
Aromatase inhibitors (letrozole and anastrozole) were provided
by Dr. Brodie's laboratory.
Cells and cell culture conditions
Aromatase inhibitor–sensitive cells (MCF-7Ca and AC1) and
aromatase inhibitor–resistant cells (LTLTCa and AnaR) have been
described previously (31–33). Briefly, human breast cancer MCF7 cells were stably transfected with the human aromatase gene to
generate MCF-7Ca cells (32). Letrozole-resistant LTLTCa cells
were isolated from MCF-7Ca mouse xenograft tumors treated
with letrozole for 56 weeks. The lower expression of ERa in
aromatase inhibitor–resistant cells (LTLTCa and AnaR) as compared with aromatase inhibitor–sensitive cells was previously
described (34). However, we did observe as is already published
(31) that LTLTCa cells express significantly less ERa than MCF7Ca cells. Similar to MCF-7Ca and LTLTCa cells, AC1 cells were
generated from the MCF-7 cells by stable transfection with human
aromatase gene. AnaR cells were anastrozole-resistant cells isolated form AC1 mouse xenograft tumors treated with anastrozole
for 14 weeks (31). MCF-7Ca cells were grown in DMEM containing 700 mg/mL G418 sulfate and 5% FBS. DMEM with 700 mg/mL
G418 sulfate and 10% FBS were used to culture AC1 cells. LTLTCa
cells were maintained in phenol red–free modified Improved
Minimum Essential Medium (IMEM) containing 700 mg/mL
G418 sulfate, 1 mmol/L letrozole and 5% charcoal-stripped FBS.
AnaR cells were grown in modified IMEM with 700 mg/mL G418
sulfate, 20 mmol/L anastrozole, and 10% charcoal-stripped FBS.
The LTLTCa-Nrf2 knock down (LTLTCa-Nrf2KD) cells were cultured in modified IMEM medium supplemented with 700 mg/mL
G418 and 5% charcoal-stripped FBS. MCF-7Ca, LTLTCa, AC1, and
AnaR cells were grown in monolayer in medium containing 1%
penicillin/streptomycin in an incubator at 37 C with 95% air and
5% CO2.
Generation of stable LTLTCa cells expressing Nrf2 shRNA
LTLTCa cells were transduced with Nrf2 shRNA or control
shRNA lentiviral particles and cells stably expressing Nrf2 shRNA
or control shRNA were selected in the presence of 10 mg/mL
puromycin and designated as LTLTCa-Nrf2 knockdown (LTLTCaNrf2KD) and LTLTCa-Vector control (LTLTCa-V), respectively.
Materials and Methods
Chemicals and reagents
Puromycin dihydrochloride (sc-108071), control shRNA lentiviral particles-A (sc-108080), Nrf2 shRNA (sc-37030-V), antiNrf2 (sc-13032), anti-Keap1 (sc-15246), anti-HO-1 (sc-10789),
anti-NQO1 (sc-32793), anti-Bcl-2 (sc-492), anti-Bcl-xL (sc-8392),
anti-Mcl-1 (sc-819), anti-Lamin B (sc-6217), anti-Mdr-1 (sc8318), anti-MRP1 (sc-13960), anti-HER2 (sc-284), anti-Ub (sc8017), anti-Ku70 (sc-17789) antibodies were from Santa Cruz
Biotechnology. Glutathione assay kit (item No. 703002) was
from Cayman Chemical. Ultra-low attachment of 24-well plate
www.aacrjournals.org
Western blotting
Cells were untreated or treated with proteasome inhibitors MG132 or epoxomicin or DMSO vehicle control. The cells were
washed with cold PBS and lysed in radioimmunoprecipitation
assay (RIPA) buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl,
0.2 mmol/L EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate) supplemented with 1 protease inhibitor (Roche Applied
Science). Subcellular fractionation of the cells was performed
according to manufacturer's protocol (Active Motif). Proteins
were quantified using Bio-Rad protein assay. The cell lysates
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Khatri et al.
(30–50 mg) were separated on SDS-PAGE and transferred to
nitrocellulose membranes. The membranes after blocking in
5% nonfat milk solution in Tris buffered Saline Tween-20 (TBST)
were incubated with the primary antibodies overnight at 4 C and
washed four times with TBST. This was followed by incubation
with secondary antibody at room temperature for 1 hour and
washed four times with TBST. The protein bands were visualized
using chemiluminescence (ECL) system (Thermo Scientific, product no. 32209). ImageJ software (NIH, Bethesda, MD) was used to
quantify the intensity of proteins bands. The protein bands were
normalized against loading controls.
Degradation assay
Cells were treated with 25 mg/mL cycloheximide for the indicated time points, washed twice with ice-cold 1 PBS, and lysed in
RIPA buffer with protease inhibitors. Thirty micrograms of total
cell lysate was loaded per well of 10% SDS-PAGE gel, transferred,
and immunoblotted with Nrf2 and b-actin antibodies. Nrf2 band
intensity was quantified and normalized to b-actin. The relative
levels of Nrf2 from sample with zero (0) minute was considered as
initial level. The graphs represent the natural logarithm of the
relative levels of the Nrf2 protein as a function of the cycloheximide chase time. The half-life of protein was determined in the
linear range of the degradation curve.
Immunoprecipitation and ubiquitination assay
For ubiquitination assay, cells were treated with 2 mmol/L of
MG-132 for 16 hours and lysed in RIPA buffer. One milligram
of whole-cell lysate was immunoprecipitated with 1 mg of
rabbit IgG or Nrf2 antibody by incubating the reaction mixture
overnight in RIPA buffer supplemented with 0.1% SDS at 4 C.
After adding 20 mL of washed protein A/G plus beads (Santa
Cruz Biotechnology), the mixture was incubated for 2 hours at
4 C and centrifuged at 4,000 rpm for 1 minute. The beads were
washed twice with RIPA buffer. Thirty microliters of SDS
sample dye was added to each tube and boiled for 5 minutes
and immunoprecipitated Nrf2 was separated by 8% SDS-PAGE
and immunoblotted with anti-ubiquitin antibody and the
same blot was reprobed for Nrf2.
Cell survival assay
MCF-7Ca, LTLTCa, and LTLTCa-Nrf2KD cells were seeded at
the density of 10,000; 20,000 and 20,000 cells per well, respectively, in 24-well plates. After 24-hour incubation, cells were
treated with different concentrations of exemestane (viz. 0, 5,
10, 20, and 30 mmol/L) for 72 hours. The cells were incubated with
freshly prepared MTT dye (200 mL/well of 5 mg/mL MTT dye in
PBS) for 2 hours. MTT dye is reduced by mitochondria aldehyde
dehydrogenase to form insoluble formazan crystals. The amount
of formazan produced is proportional to viable cells. After dissolving formazan crystals in DMSO, absorbance was recorded
spectrophotometrically at 570 nm. Cell viability was calculated
from absorbance and normalized to the value of the corresponding vehicle control cells. Each data point represents a mean SD
from three independent experiments.
Aldefluor staining
ALDEFLUOR assay (Stem Cell Technologies) was performed
according to the manufacturer's instructions. MCF-7Ca and
LTLTCa cells and LTLTCa-V and LTLTCa-Nrf2KD cells expressing
1730 Mol Cancer Ther; 14(7) July 2015
ALDH were stained with Aldefluor reagent and identified by
comparing the same sample with and without the ALDH inhibitor
diethylaminobenzaldehyde (DEAB). Cells were acquired using
FACSCanto and analyzed using FlowJo software (BD Biosciences). Dead cells were excluded on the basis of light scatter
characteristics and using viability dye (propidium iodide) gating
parameters.
Isolation of TICs using Aldefluor staining
Aldefluor assay/Aldehyde dehydrogenase assay (Stem Cell
Technologies) was performed according to the manufacturer's
instructions. Briefly, LTLTCa cells were stained with Adlefluor
reagent along with the inhibitor of ALDH, DEAB, and sorted using
FACS ARIA (BD Biosciences). All cells showing differential ALDHstaining pattern were sorted and designated as ALDH-high and
ALDH-low cells based on highest and lowest expression of ALDH
enzyme, respectively.
Mammosphere assay
Mammosphere assay was performed using reagents from Stem
Cell Technologies, as per manufacturer's instructions. Briefly,
LTLTCa-V and LTLTCa-Nrf2KD cells were suspended in complete
Mammocult media and 10,000 cells per well were plated in ultralow attachment 24-well plates. Mammospheres were counted
after 3 weeks. Stabilized spheres with a colony count of at least
50 cells were considered as mammospheres (34).
Gene expression analysis
Total RNA was isolated from the untreated cells and cells
treated with DMSO or t-BHQ for the indicated time periods
using RNeasy mini kit, following manufacturer's protocol.
cDNA was synthesized from 1 mg of total RNA as template
and the cDNA was used to determine the target gene expression
by quantitative real-time PCR using TaqMan gene expression
assays.
ROS detection
DCFDA Cellular ROS detection assay kit was used to measure
the cellular levels of ROS. Cells were trypsinized and washed with
PBS. The cells were suspended in 20 ,70 -dichlorofluorescein diacetate (DCFDA) and incubated at 37 C for 30 minutes in the dark
and washed with 1 buffer. A total of 106 DCFDA-stained cells
were suspended in 1 mL of 1 supplemented buffer and 105 cells
in 100 mL of the cell suspension were added to each well of 96-well
black plate. A total of 50 mmol/L of t-butyl hydroperoxide was
added and the cells were incubated at 37 C for 3 hours to generate
ROS as positive control. Using TECAN Infinite M1000 PRO plate
reader, ROS-mediated fluorescence intensity was recorded with
excitation wavelength at 485 nm and emission wavelength at
535 nm.
Glutathione quantification
Total glutathione content was determined spectrophotometrically using Cayman's glutathione assay kit following the manufacturer's protocol. Briefly, the cells were seeded on 6-well plates
on day 1 and harvested on day 3 and lysed in 1 buffer supplied in
Glutathione detection kit and oxidized and reduced form of
glutathione was quantified following the kit protocol using
TECAN plate reader (405 nm). Glutathione content is expressed
as mmol/L/mg protein.
Molecular Cancer Therapeutics
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Nrf2 and Aromatase Inhibitor Drug Resistance
0
0 1.0 0.9
LDH
1
1.2
2.1
5.2
BCRP
2.9
1
0.8
β-Actin
6.2
β-Actin
1
2.0
1
0.9
β-Actin
1
F
2.7
Bcl-xL
1
1
1
Mcl-1
1
HO-1
E
1
MRP4
1
1 1.1 0 0
1.8
LTLTCa
0.2
β-Actin
0
*,P ≤ 0.003
LTLTCa
Lamin B
1
1.6
1.0 2.4
2.5
INrf2
1
β-Actin
Nrf2
1
1
MRP1
GCLC
MCF-7Ca
4.8
3.2
MCF-7Ca
Nrf2
LTLTCa
*
LTLTCa
MCF-7Ca
LTLTCa
MCF-7Ca
Fluorescence
intensity (×1,000)
6.4
Nuclear
LTLTCa
D
Cytosol
MCF-7Ca
C
1
AnaR
B
MCF-7Ca
A
AC1
Aromatase inhibitor–resistant cells contain higher ROS, lower
INrf2, and higher Nrf2 protein levels, as compared with
sensitive cells
Letrozole-sensitive MCF-7Ca and -resistant LTLTCa cells were
analyzed for ROS and immunoblotted for Nrf2, INrf2, Nrf2regulated proteins, and actin (Fig. 1). The results demonstrated
that drug-resistant LTLTCa cells contain higher ROS, lower INrf2,
and higher Nrf2 levels, as compared with drug-sensitive MCF-7Ca
cells (Fig. 1A and B). Subcellular fractionation followed by
immunoblotting analysis revealed that nuclear Nrf2 was significantly higher in LTLTCa cells, as compared with MCF-7Ca cells
(Fig. 1C). In the same experiment, the cytosolic fraction did not
show Nrf2 in either LTLTCa or MCF-7Ca cells (Fig. 1C). The
resistant LTLTCa cells also demonstrated significantly increased
Nrf2-regulated GCLC (catalytic subunit of glutathione synthesizing enzyme g-GCS), heme oxygenase-1 (HO-1), drug transporters
LTLTCa
Results
MCF-7Ca
(MRP-1, MRP-4, and BCRP), and antiapoptotic (Bcl-xL and Mcl1) proteins, as compared with sensitive MCF-7Ca cells (Fig. 1D).
Further analysis of letrozole-sensitive and -resistant cells demonstrated an increase in total and reduced glutathione in resistant
LTLTCa cells, as compared with sensitive MCF-7Ca cells (Fig. 1E).
In similar experiments, a second cell line AnaR that is resistant to
another aromatase inhibitor anastrozole, also showed lower
INrf2, higher Nrf2, and GCLC levels, as compared with drugsensitive AC1 cells (Fig. 1F). In addition, the AnaR cells showed
increased expression of Nrf2 downstream genes encoding detoxifying enzymes (GCLC, HO-1), as compared with drug-sensitive
AC1 cells (Fig. 1F). Together, these results indicate that persistent
treatment of cells with aromatase inhibitors increase ROS,
decreases INrf2, increases nuclear Nrf2, increases expression of
Nrf2-regulated genes and levels of reduced glutathione, and
suggest that Nrf2 and Nrf2-regulated genes play a role in aromatase inhibitor resistance. Interestingly, both letrozole-resistant
LTLTCa and anastrozole-resistant AnaR cells containing higher
levels of Nrf2 showed downregulation of the Nrf2 downstream
gene NQO1, as compared with sensitive cells (Supplementary
Fig. S2). The reasons for downregulation of NQO1 gene expression in aromatase inhibitor–resistant cells remain unknown. It is
noteworthy that the lack of induction of NQO1 gene in letrozoletreated Hepa 1c1c7 cells was observed earlier (35).
Statistical analyses
Data from cell survival, cell death assay, and real-time PCR were
analyzed using a two-tailed Student test. Data were presented as
the mean SD. Two datasets with P < 0.05 were considered as
statistically significant.
1
5.0
1
0.2
1
1.3
1
3.3
1
1.1
Nrf2
Glutathione
(μmol/L/μg protein)
3.00
P ≤ 0.01
MCF-7Ca
P ≤ 0.003
INrf2
2.50
LTLTCa
2.00
GCLC
1.50
1.00
HO-1
0.50
0.00
β-Actin
Reduced
GSH (μmol/L)
GSSG (μmol/L)
Total GSH
(μmol/L)
Figure 1.
Aromatase inhibitor–resistant cells generated increased levels of ROS and expressed lower levels of INrf2, higher levels of Nrf2, and Nrf2 downstream gene
expression. A, cellular ROS-mediated fluorescence in letrozole-sensitive (MCF-7Ca) and letrozole-resistant (LTLTCa) cells. Cells were incubated with DCFDA and
cellular ROS–mediated fluorescence intensity was recorded using microplate reader. B, D, and F, MCF-7Ca and LTLTCa cells were lysed and analyzed by
Western blot analysis. C, cytosolic and nuclear fractions from MCF-7Ca and LTLTCa cells were immunobloted. E, total and reduced glutathione in MCF-7CA and
LTLTCa cells was determined spectrophotometrically at 570 nm. F, Western blot analysis of the relative levels of Nrf2, INrf2 (Keap1), and Nrf2 target genes
anastrozole-sensitive AC1 and anastrozole-resistant AnaR cells.
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cells (Fig. 2C). Furthermore, the Nrf2 levels in LTLTCa-Nrf2KD
cells were similar to MCF-7Ca cells (Fig. 2A) and their sensitivities
to exemestane were not significantly different (Fig. 2C; P >
0.7758). In other words, knockdown of Nrf2 in LTLTCa-Nrf2KD
cells sensitized cells to exemestane to a similar extent as observed
with MCF-7Ca cells. Interestingly, LTLTCa-Nrf2KD cells also
showed increased sensitivity to genotoxic antitumor drugs doxorubicin and etoposide as compared with LTLTCa cells (Supplementary Fig. S3). Together, these results suggested a role for Nrf2
in aromatase inhibitor drug resistance. It is noteworthy that
LTLTCa cells contained significantly lower ERa, as compared with
MCF-7Ca cells and shRNA inhibition of Nrf2 in LTLTCa cells had
more or less no effect on ERa level in LTLTCa cells (Supplementary Fig. S4), indicating that Nrf2 does not regulate ERa expression
in resistant cells.
Letrozole-resistant LTLTCa cells show lower Nrf2
ubiquitination levels and a decreased rate of Nrf2 degradation
when compared with sensitive MCF-7Ca cells
LTLTCa cells demonstrated decreased Nrf2 ubiquitination and
degradation, as compared with MCF-7Ca cells (Fig. 3A). In related
experiments, the rate of degradation of Nrf2 was significantly
lower in LTLTCa cells, compared with MCF-7Ca cells (Fig. 3B).
These results collectively suggested that higher levels of Nrf2 in
LTLTCa cells are due to decreased ubiquitination and degradation
of Nrf2. Notably INrf2, which functions as an adaptor protein for
Cul3-Rbx1–mediated ubiquitination and degradation of Nrf2, is
downregulated in aromatase inhibitor–resistant LTLTCa cells
(Figs. 1B and 3C). Therefore, it is reasonable to conclude that
lower INrf2 levels were responsible for the reduced ubiquitination
and degradation of Nrf2 in LTLTCa cells. We also determined
whether downregulation of INrf2 in LTLTCa cells is due to
degradation and/or decreased transcript levels of the INrf2 gene,
as compared with MCF-7Ca cells (Fig. 3C–E). The treatment of
C
LTLTCaNrf2KD
A
LTLTCa-V
MCF-7Ca
shRNA inhibition of Nrf2 in LTLTCa cells decreased Nrf2
downstream gene expression and increased sensitivity to
exemestane
LTLTCa cells were transduced with either lentiviral vector
(control) or Nrf2shRNA viral particles and positive clones selected
in puromycin. MCF-7Ca, LTLTCa-V (vector control), and LTLTCaNrf2KD (Nrf2 knockdown) cells were immunoblotted for Nrf2
and INrf2; detoxifying proteins GCLC and NQO1; membrane
transporters MRP1, MRP4, and BCRP; and antiapoptotic proteins
Mcl-1, Bcl-xL, and actin (Fig. 2A and B). shRNA silencing of Nrf2
significantly reduced the levels of Nrf2, GCLC, NQO1, MRP4, BclxL, and Mcl-1 (Fig 2A and B). Nrf2KD cells also showed downregulation of MRP1 but the change was insignificant. This is
presumably due to relatively lower contribution of Nrf2, as
compared with other factors including NF-kB and c-Jun that
regulate expression of MRP1 in LTLTCa cells (15). Previous
studies have suggested the option of using steroidal aromatase
inhibitor exemestane to treat HER2-negative, hormonal receptor–
positive, postmenopausal metastatic breast cancer patients with
resistance to nonsteroidal aromatase inhibitor (reviewed in
ref. 36). Therefore, one of the aims of the experiment was to
evaluate exemestane sensitivity of aromatase inhibitor–resistant
LTLTCa cells. The MCF-7Ca, LTLTCa, and LTLTCa-Nrf2KD cells
were compared for exemestane sensitivity (Fig. 2C). Interestingly,
the treatment of LTLTCa cells (expressing higher Nrf2 compared
with MCF-7Ca cells) with exemestane showed some degree of
sensitivity that increased with increasing concentration of exemestane (Fig. 2C). However, MCF-7Ca cells containing lower
Nrf2 showed significant sensitivity to 20 and 30 mmol/L exemestane, as compared with LTLTCa cells containing higher Nrf2
(Fig. 2C). Intriguingly, shRNA inhibition of Nrf2 significantly
sensitized LTLTCa-Nrf2KD cells to exemestane (Fig. 2C). The 20
and 30 mmol/L exemestane concentrations significantly decreased
cell survival in LTLTCa-Nrf2KD cells as compared with LTLTCa
MCF-7Ca
Nrf2
1
2.5 1.1
140
LTLTCa
1
0.4 0.4
120
LTLTCa-Nrf2KD
1
3.6 2.8
100
1
1
0.5
1
1
1
INrf2
% Cell survival
GCLC
NQO1
1
1.7 0.8
LTLT-CaNrf2KD
LTLTCa-V
2 1.9
MRP-1
MCF-7Ca
1
LTLT-CaNrf2KD
LTLTCa-V
B
MCF-7Ca
Actin
Mcl-1
MRP-4
1.5 0.7
BCRP
60
40
20
0
0
5
10
20
Exemestane (μmol/L)
Bcl-xL
1
80
1
1.9 1.3
1
1 1.1
30
Figure 2.
LTLTCa-Nrf2KD cells were more sensitive to
exemestane. A and B, Western blot analysis
of Nrf2, INrf2, and Nrf2 downstream proteins
in MCF-7Ca, LTLTCa, and LTLTCaNrf2KD
cells. C, comparative sensitivities of MCF7Ca, LTLTCa, and LTLTCa-Nrf2KD cells to
exemestane. The cells were exposed to 10%
ethanol in PBS (vehicle control represented
by 0 exemestane) and varying
concentrations of exemestane (1, 2.5, 5.0. 10,
20, and 30 mmol/L) for 72 hours and
analyzed for cell survival by MTT assay. Cell
survival obtained at 1 and 2.5 mmol/L has
been excluded from the graph as the results
were similar to those obtained from
5 mmol/L. Each data point represents a mean
SD from three independent experiments.
Actin
1
4.2 2.1
1732 Mol Cancer Ther; 14(7) July 2015
Molecular Cancer Therapeutics
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Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
Nrf2 and Aromatase Inhibitor Drug Resistance
Input
B
CHX(min)
0
30
60
Ln [Relative Nrf2]
0.3
0.5
IP: Nrf2
0.7 0.2
WB:Nrf2
Nrf2
β-Actin
IgG
2
12
Nrf2
Epoxomicin
DMSO (100 nmol/L/12 h)
LTLTCa
MCF-7Ca
1
D
1
1
8
8
Nrf2
11
INrf2
INrf2
1
0.6
0.6
0.3
Actin
60
90
1
0.9
0.7
1
1
30
60
90
Half-life of Nrf2
MCF-7Ca 28.9 min
LTLTCa52.1 min
−0.6
−1.2
MCF-7Ca
LTLT-Ca
−1.8
MCF-7Ca
LTLTCa
MG132
(2 μmol/L/16 h)
MCF-7Ca
DMSO
1
LTLTCa
0.9
MCF-7Ca
0.8
LTLTCa
1
30
0.0
Ubiquitin
1
1
0
0.7
E
1.2
MCF-7Ca
LTLTCa
1
Relative quantification
of mRNA of INr2
(Keap1)
3.2
INrf2
0
Time (min)
WB:
2. 2.3
90
Actin
IP: Nrf2
1
LTLTCa
Nrf2
Nrf2
C
MCF-7Ca
LTLTCa
MCF-7Ca
LTLTCa
MCF-7Ca
MG132
DMSO 2 μmol/L/16 h
IgG
LTLTCa
LTLTCa
MCF-7Ca
DMSO
IP
MG132
2 μmol/L/16 h
MCF-7Ca
A
0.8
P ≤ 0.003
0.6
0.4
0.2
Actin
0
1
1
1
1
1
1
Figure 3.
LTLTCa cells showed decreased ubiquitination and degradation of Nrf2 compared with MCF-7Ca cells. A, 1 mg of total cell lysate from MG-132–treated cells was
immunoprecipitated with 1 mg of rabbit IgG or Nrf2 antibody. The immunoprecipitated Nrf2 was immunoblotted for ubiquitin and Nrf2. B, cells were treated
with 25 mg/mL cycloheximide (CHX) for the indicated time points and 30 mg of total cells lysate was immunoblotted with Nrf2 and b-actin antibodies. The graphs
represent the natural logarithm of the relative levels of the Nrf2 protein versus the cycloheximide chase time and the half-life of Nrf2 was determined using
the linear part of the degradation curve. LTLTCa cells showed no difference in rate of INrf2 protein degradation but demonstrated lower levels of INrf2 transcripts,
as compared with MCF-7Ca cells. Cells treated with MG-132 (C) and with epoxomicin (D) were lysed and immunoblotted. E, total RNA was isolated from the
cells and cDNA was synthesized from 1 mg of total RNA and the cDNA was used to quantify the INrf2 gene transcripts at basal level.
LTLTCa cells with proteasome inhibitors MG132 (Fig. 3C) or
epoxomicin (Fig. 3D) failed to stabilize INrf2 indicating that the
lower INrf2 level in LTLTCa cells is not due to degradation of
INrf2. We also performed experiments to investigate whether
epigenetic and/or autophagy mechanisms contribute to lower
levels of INrf2 in LTLTCa cells. Epigenetic regulation of INrf2
(Keap1) was investigated by treating LTLTCa cells with an inhibitor of DNA methyltransferase and inhibitors of histone deacetylase. Treatment with those epigenetic modulators failed to
restore the levels of INrf2. We also utilized inhibitors of autophagy to examine the possibility of INrf2 degradation by autophagy (37). Even though we observed higher level of autophagy in
LTLTCa cells as compared with MCF-7Ca cells, the inhibition of
autophagy did not increase the levels of INrf2. It is noteworthy
that negative data on epigenetic and autophagy regulation of
INrf2 are not included. Interestingly, RT-PCR analysis of INrf2
RNA demonstrated significantly lower INrf2 RNA transcripts in
aromatase inhibitor–resistant LTLTCa cells, as compared with
drug-sensitive MCF-7Ca cells (Fig. 3E). This result suggested that
www.aacrjournals.org
INrf2 gene expression is downregulated in aromatase inhibitor–
resistant cells. RT-PCR analysis also showed a marginal increase in
Nrf2 gene expression in LTLTCa cells, as compared with sensitive
MCF-7Ca cells that might also have contributed to higher Nrf2 in
resistant cells (Supplementary Fig. S3).
In TICs from LTLTCa cells, lower INrf2 and higher Nrf2
expression levels lead to expression of Nrf2 targets, GCLC,
DNA repair proteins, and HER2
Recent studies have implicated mammary TICs in resistance to
chemotherapy and radiation (38, 39). TICs are immature, poorly
differentiated, and highly tumorigenic (40–42). TICs have a
decreased ability to undergo apoptosis and a higher ability for
DNA repair, making them more resistant to cancer therapy,
compared with differentiated counterparts (43, 44). We isolated
TICs expressing ALDH from LTLTCa cell culture. It has been
reported that chemoresistant cancer stem cells have high ALDH
activity (45) and ALDH is considered as a marker of normal and
malignant human mammary stem cells (46). MCF-7Ca, LTLTCa,
Mol Cancer Ther; 14(7) July 2015
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Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
LTLTCa-low ALDH
LTLTCa-high ALDH
Khatri et al.
1.0 4.8 6.8
8.0
1.0 1.2
1.6
2.1
1.0 1.0 1.0
0.9
1.0 0.9 0.8
0.5
1.0 2.1 2.4
2.9
LTLTCa-V
MCF-7Ca
*
HER-2
Nrf2
Actin
INrf2
GCLC
Ku80
1.0 1.1 1.6
1.9
1.0 0.7 1.8
2.3
Ku70
Actin
1.0 1.0 1.0
1.1
Figure 4.
TICs isolated from LTLTCa cells expressed lower levels of INrf2 and higher
levels of Nrf2. LTLTCa cells were subjected to Aldefluor staining (Aldefluor
Staining Kit, Stem Cell Technologies) and the cells expressing ALDH were
further gated into ALDH-high and ALDH-low cells. Cells were sorted as ALDHhigh (TIC) and ALDH-low (non-TIC) fractions. MCF-7Ca and LTLTCa cells also
received the same treatment as LTLTCa-low ALDH and LTLTCa-high ALDH
cells. MCF-7Ca and LTLTCa cells were used as control cells. The cells were
lysed and total cell lysate was immumoblotted with indicated antibodies.
, Control live cells were sorted by propidium iodide exclusion.
LTLTCa-low ALDH, and LTLTCa-high ALDH cells were lysed and
immunoblotted for Nrf2, INrf2, GCLC, DNA repair proteins, and
HER2 (Fig. 4). Results revealed that TICs expressed lower levels of
INrf2 and higher levels of Nrf2 and Nrf2 downstream GCLC gene
expression. TICs also expressed higher levels of HER2. Intriguingly, TICs with high ALDH levels (stem cells) expressed significantly higher levels of nonhomologous end-joining (NHEJ)
DNA repair proteins Ku80 and Ku70, as compared with MCF7Ca and LTLT-Ca cells. This observation has high significance as
TICs are believed to contribute to drug resistance.
shRNA inhibition of Nrf2 in LTLTCa cells significantly decreases
TICs and mammosphere formation
MCF-7Ca and LTLTCa cells were immunoblotted for Nrf2
(Fig. 5A) and in separate experiments were stained to assess the
expression of stem cell marker ALDH in the absence and presence
of ALDH inhibitor DEAB (Fig. 5B). As expected, the levels of Nrf2
and ALDH were significantly higher in LTLTCa cells compared
with MCF-7Ca cells (Fig. 5A and B). This indicated the presence of
an increased stem cell-like population in LTLTCa cells containing
higher levels of Nrf2, as compared with MCF-7Ca cells with lower
Nrf2 protein. In related experiments, LTLTCa-V (vector control),
1734 Mol Cancer Ther; 14(7) July 2015
LTLTCa-Nrf2KD clone #2 and clone #1 cells were immunoblotted
for Nrf2 and actin (Fig. 5C). The results demonstrated that
LTLTCa-V cells showed highest expression of Nrf2, which was
followed by clone #2 and clone #1 cells LTLTCa-V and the two
clones of LTLTCa-Nrf2KD cells were stained for assessing the
expression of stem cell marker ALDH in the absence and presence
of ALDH inhibitor DEAB (Fig. 5D). Results demonstrated a direct
correlation between Nrf2 expression levels and the magnitude of
ALDH expression. LTLTCa-V cells expressing the highest levels of
Nrf2 led the highest percentage of cells in a gated region R3 that
stained positive for ALDH. Moreover, clone #2, containing lower
expression levels of Nrf2 demonstrated significantly decreased
ALDH. Notably, shRNA downregulation of Nrf2 in LTLTCa (clone
#1) containing lowest level of Nrf2 also showed the least staining
for ALDH. These results showed that shRNA inhibition of Nrf2 led
to an Nrf2-dependent decrease in ALDH-positive TICs. It is
noteworthy that ALDH is an Nrf2 downstream gene and its
expression is regulated by Nrf2 (47–49). Therefore, our observation of a relationship between Nrf2 and ALDH is strengthened by
previous reports (47–49). In related experiments, LTLTCa and
both clones of LTLTCa-Nrf2KD cells were also analyzed for
mammosphere formation (Fig. 6). The results (compare Figs.
5C and 6) revealed a direct correlation between Nrf2 and mammosphere formation. shRNA inhibition of Nrf2 in LTLTCa cells
led to Nrf2 concentration–dependent decrease in mammosphere
formation. Together, the results suggest a direct correlation
between Nrf2, ALDH-positive TICs and mammosphere formation
with implications in aromatase inhibitor drug resistance that
warrant further studies.
Discussion
This is the first report demonstrating a role for Nrf2 in aromatase inhibitor resistance in breast cancer. Letrozole-resistant
LTLTCa cells generated significantly higher ROS levels, as compared with letrozole-sensitive MCF-7Ca cells. This increase in ROS
was more significant considering that reduced glutathione that
scavenges ROS was also increased. We believe that long-term
treatment of letrozole could result in continuous generation of
ROS, which is known to activate Nrf2. However, Nrf2-mediated
antioxidant gene expression might not be sufficient to lower the
levels of ROS leading to higher levels of ROS in drug-resistant
LTLTCa cells, as compared with drug-sensitive MCF-7Ca cells.
LTLTCa cells also showed lower INrf2 and higher expression levels
of Nrf2, as compared with MCF-7Ca cells. Similar results were also
observed in anastrozole-resistant AnaR cells. The increase in ROS
and decrease in INrf2 led to decreased ubiquitination and degradation of Nrf2 leading to the stabilization and nuclear translocation of Nrf2. High levels of Nrf2 in the nucleus led to
coordinated induction of Nrf2 downstream cytoprotective proteins including detoxifying enzymes, membrane transporters, and
antiapoptotic proteins. On the basis of the documented role of
Nrf2-activated cytoprotective proteins in drug resistance in other
systems (28), our results strongly suggest that Nrf2 plays a role in
aromatase inhibitor resistance. This was further supported by
studies showing that inhibition of Nrf2-sensitized LTLTCa cells
to the aromatase inhibitor exemestane.
Our studies also revealed that lower levels of INrf2 in drugresistant LTLTCa cells is not due to instability of INrf2 protein as
inhibitors of proteasomes failed to increase the level of INrf2.
Furthermore, the lower levels of INrf2 in LTLTCa cells are also not
Molecular Cancer Therapeutics
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Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
Nrf2 and Aromatase Inhibitor Drug Resistance
LTLTCa
1.0
2.4
LTLTCa
0.3%
5.19%
0.01%
0.45%
ALDH
Actin
LTLTCa-Nrf2KD # 1
C
LTLTCa-Nrf2KD # 2
DEAB
D
1
0.5 0.3
LTLTCa-V LTLTCa-Nrf2KD # 2 LTLTCa-Nrf2KD #1
2.27%
1.58%
0.05%
0.33%
0.23%
0.01%
ALDH
Nrf2
DEAB
Actin
due to epigenetic modulation or autophagy as inhibitors of DNAmethyl transferase, histone deacetylation, and autophagy all
failed to increase INrf2 levels in aromatase inhibitor–resistant
LTLTCa cells. Additional studies demonstrated that downregulation of INrf2 in letrozole-resistant LTLTCa cells is due to a
significant decrease in INrf2 transcripts. This raises an intriguing
question regarding the mechanism of aromatase inhibitor–mediated downregulation of INrf2 gene expression and is a subject of
future studies.
Aromatase inhibitor–resistant cells showed higher population
of TIC cells that are believed to contribute to resistance to
chemotherapy and radiation (34, 39). The TICs showed lower
INrf2 and higher Nrf2. TICs also showed higher expression of Nrf2
downstream cytoprotective proteins and higher levels of Ku70
and Ku80 proteins that participate in the NHEJ pathway that is
known to be active in repairing DNA double-strand breaks, one of
the most lethal forms of DNA damage. NHEJ is known to be active
in G0–G1 phase of the cell cycle in which many "stem" cells reside
(50). These observations suggested that higher Nrf2 in TICs
contributed to aromatase inhibitor drug resistance. While a previous publication found Ku proteins to be reduced in LTLTCa cells
(51), their high expression levels in TICs are significant. NHEJ may
participate in drug resistance in "stem"-like TICs by increasing
repair of DNA damage, leading to cell survival. This conclusion
was further strengthened from following observations. First,
shRNA inhibition of Nrf2 led to Nrf2 concentration–dependent
reduced survival of TICs. In other words, Nrf2 enhances survival of
TICs in the presence of drugs that contribute to resistance. It is
noteworthy that a role of Nrf2 in cell survival by inhibiting
apoptosis is well established in other systems (16). Second, in
related experiments, inhibition of Nrf2 led to significant decrease
in mammosphere formation. This signifies the importance of
www.aacrjournals.org
MCF-7Ca
Nrf2
LTLTCa-V
Figure 5.
Increased Nrf2 in LTLTCa cells is directly
associated with higher ALDH. A and B,
LTLTCa cells containing higher Nrf2 showed
increased ALDH. A, Western blot analysis.
MCF-7Ca and LTLTCa cells were
immunoblotted for Nrf2 and b-actin. B,
ALDH measurement. MCF-7Ca and LTLTCa
cells were stained with Aldefluor in absence
and presence of ALDH inhibitor DEAB and
the percentage of cells expressing ALDH
was determined. C and D, Nrf2-knocked
down LTLTCa cells expressed lower levels of
ALDH. Two clones of LTLTCa cells
expressing Nrf2shRNA were selected. A,
LTLTCa-V and LTLTCa-Nrf2KD cells were
lysed and immunoblotted with Nrf2 to
confirm reduced levels of Nrf2. B, all the cells
were subjected to Aldefluor staining and the
percentage of cells expressing ALDH was
determined by comparing the same sample
with and without the ALDH inhibitor DEAB.
LTLTCa-Nrf2KD cells contained lower
percentage of cells expressing ALDH. Data
were acquired using FACScanto.
B
MCF-7Ca
A
Nrf2 in mammosphere development. Therefore, it is reasonable
to conclude that higher Nrf2 levels in TICs contributed to aromatase inhibitor resistance.
Collectively, the results led to the hypothesis (Supplementary Fig.
S5) that aromatase inhibitor downregulates INrf2 transcripts that,
combined with higher ROS, leads to increased Nrf2 and Nrf2
downstream cytoprotective proteins including detoxifying enzymes,
antioxidants, and efflux pumps. In addition, higher Nrf2-mediated
increased expression of antiapoptotic proteins that led to reduced
apoptosis. Finally, higher Nrf2 increased TIC survival and mammosphere formation. Together, all these Nrf2-dependent processes
contributed to aromatase inhibitor drug resistance.
Previous studies have shown increased signaling through HER2
receptors and increased MAPK activity as probable causes of
endocrine drug resistance (reviewed in refs. 7 and 52). In addition,
breast cancer cells receiving long-term antiestrogen treatment
appear to have increased ROS and disruption of reversible redox
signaling that involves redox-sensitive factors including protein
phosphatases, protein kinases such as ERK, and transcription
factors AP-1 and NF-kB that contribute to drug resistance
(reviewed in ref. 53). Furthermore, PI3K–Akt–mTOR pathway
has also been implicated in endocrine drug resistance (54). The
INrf2:Nrf2 system identified in the current report is a novel
mechanism of aromatase inhibitor drug resistance but is also
related to the abovementioned mechanisms. This is because Nrf2
is a transcription factor that controls redox homeostasis (reviewed
in ref. 12). In addition, Nrf2 itself is regulated by PI3K–Akt–
mTOR, MAPKs, and redox-sensitive factors including AP1
(reviewed in ref. 12). Further studies are required to explore the
exact relationship between these factors and pathways leading to
aromatase inhibitor drug resistance and possible therapeutic
intervention.
Mol Cancer Ther; 14(7) July 2015
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Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
Khatri et al.
LTLTCa-Nrf2KD # 2
LTLTCa(CTRL)
LTLTCa-Nrf2KD #1
Mammosphere
numbers
175
150
P ≤ 0.02
125
P ≤ 0.001
100
Figure 6.
Nrf2-knocked down LTLTCa cells form fewer
4
mammospheres. A total of 10 LTLTCa-V
and LTLT-Nrf2KD cells were seeded in
complete Mammocult media per well in
ultra-low attachment 24-well plates.
Mammospheres were counted after 3 weeks
and photographed.
75
50
25
0
LTLTCa
LTLTCa-Nrf2KD
#2
LTLTCa-Nrf2KD
#1
Recent studies have shown some benefit in using steroidal
aromatase inhibitor exemestane in treating HER2-negative,
hormonal receptor–positive, and postmenopausal metastatic
breast cancer patients with resistance to nonsteroidal aromatase
inhibitor (36). The studies in this report suggest that it might be
possible to increase the efficacy of exemestane in the patients
with endocrine drug resistance by inhibiting Nrf2. This assumption is based on observation that shRNA inhibition of Nrf2 in
letrozole-resistant cells significantly increased the sensitivity to
exemestane.
In conclusion, the current studies present strong evidence for a
role of INrf2:Nrf2 in aromatase inhibitor drug resistance in breast
cancer. The mechanism(s) involving Nrf2 to combat drug resistance is especially interesting as it is estrogen independent. The
studies also suggest that Nrf2 inhibitors from natural sources
could be explored for use as adjuvants with aromatase inhibitor
drugs to treat aromatase inhibitor–resistant breast cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Development of methodology: P. Shah, A. Brodie
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): P. Shah, R. Guha, A. Brodie
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): R. Khatri, R. Guha, A. Brodie, A.K. Jaiswal
Writing, review, and/or revision of the manuscript: R. Khatri, P. Shah, R. Guha,
F.V. Rassool, A.E. Tomkinson, A. Brodie, A.K. Jaiswal
Study supervision: A.K. Jaiswal
Acknowledgments
The authors thank their colleagues at the University of Maryland School of
Medicine (Baltimore, MD) for helpful discussions.
Grant Support
This work was supported by NIH grant RO1 ES012265 (to A.K. Jaiswal), RO1
ES021483 (to A.K. Jaiswal), RO1 CA62483 (to A. Brodie), RO1 GM047466 (to
A.K. Jaiswal), and a grant from V-Foundation and Endow Funds (to A.K.
Jaiswal).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Authors' Contributions
Conception and design: R. Khatri, R. Guha, A.E. Tomkinson, A. Brodie,
A.K. Jaiswal
Received August 7, 2014; revised April 1, 2015; accepted May 5, 2015;
published OnlineFirst May 14, 2015.
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Mol Cancer Ther; 14(7) July 2015
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1737
Published OnlineFirst May 14, 2015; DOI: 10.1158/1535-7163.MCT-14-0672
Aromatase Inhibitor−Mediated Downregulation of INrf2 (Keap1)
Leads to Increased Nrf2 and Resistance in Breast Cancer
Raju Khatri, Preeti Shah, Rupa Guha, et al.
Mol Cancer Ther 2015;14:1728-1737. Published OnlineFirst May 14, 2015.
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