Abstract: Chemokine receptor CXCR4 is involved in the maintenance
of stemness property of stem cells, aging and metastasis of cancer. NRF1 is one of
the major transcription factors that controls transcription of CXCR4. In this study, we
have investigated whether transcription regulation of CXCR4 by NRF1 controls
estrogen-induced malignant transformation of breast epithelial cells to breast cancer
stem cells. The functional regulation of transcription of NRF1 target genes has not
been explored in breast cancer. We have previously shown that NRF1 may be
involved in 17 β-estradiol (E2) induced malignant transformation of breast epithelial
cells, however, the mechanism of transcriptional regulation of the NRF1 target
genes, such as CXCR4 remain unknown. In this study we showed that NRF1 and
estrogen jointly contributed in reprogramming of breast epithelial cells to breast
cancer stem cells. Levels of breast cancer stem cell markers
(CD44+CD24+ALDH1+CD133+) were significantly increased by E2 treatment in
NRF1 overexpressing cells compared to cells transfected with vector receiving E2.
E2-induced increases of spheroid formation, cell survival and growth of cancer stem
cells were modulated by functional gain or loss of NRF1. Overexpression of NRF1
promoted the transition of E2-treated breast epithelial MCF-10A cells to
mesenchymal stem cell-like phenotype. The ChIP qPCR, RT-qPCR, Western
blotting and immunofluorescence microscopic assays showed that NRF1 mediated
transcriptional changes of its target genes -CXCR4, BNIP3, and DJ-1 correlated with
malignant phenotypic changes. In summary, our findings for the first time showed
that transcriptional regulation of CXCR4 by NRF1 may contribute in the induction of a
pre-malignant phenotype by estrogen presumably by promoting generation of breast
cancer stem cells. These data suggest NRF1 as an emerging potential target for
therapeutic intervention against breast cancer.
Jayanta Kumar Das1, 2 and Deodutta Roy1,2
1Department of Environmental & Occupational Health, Florida International University, Miami, FL 33199, 2Miami VA Medical Center, 1201 NW 16th St, Miami, FL 33125.
Transcription regulation of chemokine receptor CXCR4 by nuclear respiratory factor 1 (NRF1) controls estrogen-induced malignant
transformation of breast epithelial cells to breast cancer stem cells
Results
AcknowledgmentsThis work was in part supported by a VA MERIT Review (VA BX001463)
grant to Dr. Roy. This work is also in part supported by Phase Holographic
Imaging (PHI),Lund, Sweden by providing a holographic imaging cytometry
platform - HoloMonitorM4.
E2 Increased the Proportion of CD44+CD24+CD133+ALDH+ cels.
Figure 2. (A) & (B).The representative flow sorted data of the cells for CSC markers
CD44+CD24-CD133+ALDH1+. (C) Histogram represents the flow sorted data of
CD44+CD24-CD133+ALDH1+. MCF10A NRF1E2 CSC markers cells were approximately 2
fold by treatment of E2 compare to control of NRF1. Error bars represent the mean ± SD. *
p<0.05 vs. NRF1 at 14D.
NRF1 induced expression of 8-oxo-dG, CXCR4, & BNIP3 as a result of
reactive oxygen species (ROS) production with the treatment of E2.
NRF1–dependent activation of CXCR4 mRNA.
Figure 8. The levels of CXCR4mRNA of NRF1+ cells were 2 fold higher (A) & BNIP3 mRNA of
NRF1+ showed 1 fold increase (B) with E2, . **p<0.01 vs. E2. Statistics by ANOVA; Tukey HSD test.
Summary of Key Findings
NRF1 might influence estrogen-induced breast cancer
risk by generating tumor-initiating breast cancer stem
cells.
NRF1 through regulating CXCR4 seems to contribute
in the estrogen-induced malignant transformation of
MCF10A cells, including anchorage-independent cell
growth, and increased cell migration and invasion.
NRF1-mediated transcription of CXCR4 is dependent
on DNA oxidation and ROS-dependent redox
signaling.
Live imaging by HoloMonitor showed that siRNA of
CXCR4 inhibited NRF1-dependent E2-induced
tumorphere formation.
ReferencesOkoh V., Garba N., Penney R., Das JK., Deoraj A., Singh K., Sarkar S., Felty
Q., Yoo C., Jackson R., Roy D. (2015). Redox Signaling to Nuclear
Regulatory Proteins by Reactive Oxygen Species Contributes to Estrogen-
Induced Growth of Breast Cancer Cells. Br J Cancer; 112, 1687–1702, doi:
10.1038/bjc.2014.586
Das JK, Roy D. (2015). Overexpression of NRF1 leads to the generation of
cancer stem-like cells and resistance to anoikis pathways to anchorage-
independent growth during estrogen-induced malignant transformation,
Cancer Res, 75:803; doi:10.1158/1538-445.AM2015-803
(A) (B)
(A)
Introduction: Treatment of metastatic breast cancer is still
unsuccessful due to development of breast cancer stem cells (BCSCs) which are
resistant to antiestrogen/chemo-/radio-therapy. The chemokine receptor CXCR4
has been found to be a prognostic marker in various types of cancer, including
breast cancer. In this study, we have investigated whether transcription regulation of
CXCR4 by NRF1 controls estrogen-induced malignant transformation of breast
epithelial cells to breast cancer stem cells. We found increased expression level of
breast cancer stem cell markers (CD44+CD24+/24-ALDH1+CD133+) when treated
with E2. The ChIP qPCR, RT-qPCR, Western blotting and immunofluorescence
microscopic assays confirmed that NRF1 mediated transcriptional changes of its
target genes -CXCR4, BNIP3, and DJ-1 .
Ve
cto
rE2
NR
F1
NR
F1
E2
Figure 1. (A) & (B).The
representative flow sorted data of the
cells for co-expression of CSC markers
CD44+CD24+CD133+ALDH1+. (C)
Histogram represents the flow sorted
data of CD44+CD24+CD133+ALDH1+
from MCF10A and MDAMB231 cells
with and without NRF1 exposed to E2
(100 pg/ml). Error bars represent the
mean ± SD. . **p<0.01 or * p<0.05 vs.
NRF1 at 14D. Statistics by ANOVA;
Tukey HSD test.
(B)(A) MCF10A MDA-MB231
0
6
12
VectorE2 NRF1 NRF1E2
% o
f C
D4
4+
CD
24
+C
D1
33
+A
LD
H+
ce
lls
MCF10A
MDAMB231
(C)** *
E2 also Increased the Proportion of CD44+CD24- CD133+ALDH+ cells.
Ve
cto
rE2
NR
F1
NR
F1
E2
MDA-MB231
*
**
*
MCF10A
(c)
0
10
20
Vec
tor
NR
F1+
NR
F1-
CX
CR
4 m
RN
A
Levels
(fo
ld)
- + - + - + E20
10
20
Vec
tor
NR
F1
+
NR
F1
-
BN
IP3 m
RN
A
Levels
(fo
ld)
- + - + - + E2
**
**
Alician Blue
NRF1+
Vector
Chondrogenic
differentiationSmooth muscle
differentiation
α-SMA+/DRAQ5®
Neuron like cell
differentiation
β-tubulin III+/DRAQ5®
Differentiation of Tumor Initiating Stem Cells into Other Cells).
Figure 6. Differentiation of tumor initiating stem cells (TIS or CSC) to chondrocytes,
neurons and smooth muscle cells.
JRK (57 kDa)
ß-actin (42 kDa)
CXCR4 (40 kDa)
ß-actin (42 kDa)
PINK1 (63 kDa)
Parkin/PARK2 (52 kDa)
LC3B (15 kDa)
ß-actin (42 kDa)
Park-7/DJ-1 (21 kDa)
E2
Vector NRF1+ NRF1-
- + - + - +
CD133+
E2
VehicleMCF10A
WT
SOX2+
E2
VehicleMCF10A
NRF1+
Nanog+ Oct4+ Cd49f+ CD44+
E2 induced NRF1 dependent expression of CXCR4,
BNIP3 and DJ1 proteins6h
Figure 4. (A) The representative HoloMonitor images showing the area scratched (wounds)
and the areas healed after 6hrs. The images were captured by holographic live imagining
system of TIS cells. (B) Graph shows the relative changes of healing areas at 6h. Error bars
represent the mean ± SD. **p<0.01 or * p<0.05 vs. vector or NRF1.
HoloMonitoring showed that E2 treatment increased the healing area of
tumor initiating CD44+24+CD133+ALDH1+ MCF10A stem cells .
(A) (B)0h
CSC (E2)
Vector ( WT)
NRF1+CSC
(No treatment)
NRF1+CSC+E2
Wound Heal
0
50
100
Ve
cto
rc
on
trol
Ve
cto
rCS
CE
2
NR
F1
CS
CC
on
trol
NR
F1
CS
CE
2
He
ali
ng
%
***
Figure 3.Increased sizes of
spheroids were
observed in NRF1
with E2 treatment
where as NRF1
mutant 109
showed inhibition
of spheroid
formation at 15D.
200X
Both E2 (100pg/mL) and NRF1 jointly increased spheroid sizes and
Mutant NRF1 109 inhibited the spheroid formation.
Vector NRF1 Mut 109Mut 221
Vehicle
E2
MCF10A
MDA-
MB 231 E2
Vehicle
Vector (WT)
NRF1+ csc
(CD44+24-
133+ALDH1)
Vehicle E2
Tumorpheres (15D)
Figure 7. ChIP assays (A, B) were performed using anti-NRF1 antibodies. CXCR4, & BNIP3 promoter
regions of the NRF1 precipitated chromatin were amplified by real-time PCR using Epitech Chip qPCR
primer assay. Analysis of E2-induced NRF-1 binding to promoters of CXCR4 (A), & BNIP3 (B). **p<0.01
vs. E2. Statistics by ANOVA; Tukey HSD test.
- - - E2+ + + E2
(A)
NRF1 protein binds to the promoter of CXCR4 and BNIP3 genes.
(B)
0
100
200
300
400
500
NR
F1
-N
RF
1+
Vec
tor
NR
F1-
NR
F1+
Vec
tor
NR
F1/C
XC
R4
Pro
mo
ter
(fo
ld)
0
100
200
300
400
500
NR
F1-
NR
F1+
Vec
tor
NR
F1-
NR
F1+
Vec
tor
NR
F1/B
NIP
3
Pro
mo
ter
(fo
ld)
****
****
- - - + + +
- + E2
Vector NRF1+NRF1-
- +
E-cadherin
E2
VehicleMCF10A
WT
N-cadherinVimentin
E2
Vehicle
MCF10A
NRF1+
Expression of Epithelial-Mesenchymal Transition Markers E- and N-
Cadherin, and Vimentin and Pluripotency Markers in Tumor Initiating Stem
Cells.
Figure 10. EMT markers
detected by confocal
microscopy and Western
blotting.
N-cadherin (130 kDa)
Nanog (42 kDa)
Vimentin (55 kDa)
ß-actin (42 kDa)
E-cadherin (120 kDa)
ß-actin (42 kDa)
ALDH1 (56 kDa)
- +
Figure 9.
Immunofluorescence
staining of 8-oxo-dG as
a red color, CXCR4 as
green color and BNIP3
as a blue color in vector,
NRF1 & NRF1- of
MCF10A cells. E2
treatment increased the
expression level of 8-
oxo-dG, BNIP3 &
CXCR4 with increased
ROS level. ROS
scavenger (NAC, Eb, &
Peg-Cat) reduced these
levels.
Photomicrographs were
captured with Nikon
Confocal microscopy.
Scale bar=70um
E2 (367.1 pM)
Vehicle
MCF10A Vector MCF10A NRF1+ MCF10A NRF1-
NAC (1mM)
H2O2(600uM)
NAC+E2
H2O2+PEG-Cat
(500ug/mL)
Eb (20uM)
Eb+E2
CXCR
4
8-Oxo-
dG
BNIP3 CXCR48-Oxo-
dG
BNIP3CXCR48-Oxo-
dGBNIP3
Vector (WT)
NRF1+ csc
(CD44+24-
133+ALDH1)
Scratch assay confirmed the findings of HoloMonitoring sowing that E2 treatment
increases the healing area of NRF1 TIS or CSC (CD44+24-CD133+ALDH1+) MCF10A
cells .0h 6h
(B)(A)
Figure 5. (A) The representative
photomicrographs shows the different groups with
wounds and the areas healed after 6hrs. Wound
healing was higher in both NRF1 as well as
NRF1+E2 treated cancer stem like cells (CSCs).
The images were captured by confocal
microscopy. Magnification 200X. GFP+ cells
showed green fluorescence.(B) Histogram shows
the relative changes of healing areas at 6h. (C)
Histogram shows the migrating cells in the healing
areas. Error bars represent the mean ± SD.
**p<0.01 or * p<0.05 vs. vector or NRF1.
CSC (E2)
Vector ( WT)
NRF1+CSC
(No treatment)
NRF1+CSC+E2
Wound
0
60
120
Vecto
rco
ntro
l
Vecto
rCS
CE
2
NR
F1C
SC
Co
ntro
l
NR
F1C
SC
E2
Healin
g %
0
60
120
Vecto
rco
ntro
l
Vecto
rCS
CE
2
NR
F1C
SC
Co
ntro
l
NR
F1C
SC
E2M
igra
tin
g c
ells
co
un
t
(c)
***
***
Both E2 and NRF1 jointly increased the expression of
mRNA level of CXCR4 & BNIP3, and ROS scavenger (NAC,
Eb, & Peg-Cat) reduced E2 or NRF1-induced the mRNA
levels of CXCR4 in MCF10A.
0
20
40
Vector NRF1+ NRF1-
CX
CR
4 m
RN
A L
eve
ls (F
old
)
Control
E2
H2O2
NAC
NAC+E2
Eb
Eb+E2
H2O2+PegCat
**
(B)
0
20
40
Vector NRF1+ NRF1-
BN
IP3
mR
NA
le
ve
l (f
old
)
Control
E2
H2O2
NAC
NAC+E2
Eb
Eb+E2
H2O2+PegCat
**
Figure11. Histograms represents the mRNA level of CXCR4 & BNIP3 at
24h. **p<0.01 vs. vector. Statistics by ANOVA; Tukey HSD test.
siRNA
CXCR4
Scrambled
RNA
E2Vehicle
NRF1
NRF1+siRNA
CXCR4
NRF1- dependent E2 induced protein expression of CXCR4, BNIP3 and Park-
7/D-J1
E2
CXCR4
+
NRF1+BNIP3 Park-7
E2
VehicleMCF10A
WT
VehicleMCF10A
NRF1+
Tumorpheres (15D)
3D 15D