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