O-GlcNAcylation regulates FoxM1 levels via SIRT1 in breast cancer
Jenna M. Marinock1, Christina M. Ferrer1, Mauricio J. Reginato1
1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA
Hypothesis
Abstract
Cancer cells are known for exhibiting marked increases in glucose uptake.
This leads to an altered metabolic state unique to cancer cells known as
the Warburg effect in which cancer cells upregulate glycolysis even in the
presence of sufficient oxygen. A portion of the glucose obtained will enter
the hexosamine biosynthetic pathway (HBP), however the role this
pathway plays in cancer progression is not well understood. Glucose flux
into the HBP pathway leads to an increase in the modification of O-linkedN-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc Transferase
(OGT). Previously our lab reported that OGT is upregulated in breast
cancer cells and inhibiting OGT reduces cancer cell invasion and the
oncogenic transcription factor FoxM1. SIRT1, a member of the NAD+dependent deacetylase family of Sirtuin proteins involved in longevity, can
function as a tumor suppressor and has been implicated in regulating
breast cancer invasion and metastasis. Indeed SIRT1 has been found to
be downregulated in many cancers including breast, colon and liver
cancer. Recently our lab has shown reducing OGT expression or activity
increases levels of SIRT1 and this increase is required for OGT-mediated
effect on cellular invasion. Here, we demonstrate that the SIRT1 can
regulate FOXM1 levels as MDA-MB-231 and SUM159 breast cancer cells
expressing SIRT1 RNAi increase FoxM1 protein levels. Thus, we identify a
novel pathway in which SIRT1 regulates FoxM1 and contributes to OGlcNAc regulation of invasion in breast cancer cells.
O-GlcNAcylation modulates FoxM1 levels through a SIRT1-dependent
mechanism.
MDA-MB-231
A) Hexosamine Biosynthetic Pathway (HBP) and OGT
Carcinoma
RW
P
N EPT 1
XPC 153
2
3
PC
3M
D
U L
14
5
Normal
O-GlcNAc
shControl shSIRT1
A)
shControl
l
o
sh
r
t
T
n
OG
Co
MDA-MB-157
shControl
shSIRT1
l
l
h
o
o
sh
r
s
r
t
T
t
T
n
n
G
o
o
OG
O
C
C
B)
shSIRT1
l
sh
ro
t
T
n
OG
Co
SIRT1
SIRT1
OGT
OGT
FOXM1
FOXM1
Ac-p53
Ac-p53
Actin
Actin
MDA-MB-231
B)
ls h h
lsh h
o
o
r
Ts ntr GTs
nt
G
o
C
O
O
Co
MDA-MB-468
A)
Results
DMSO
EX-527
MDA-MB-231
h
lsh h
EX-527
h
o
n
T s ontr GT s
o
G
C
O
C
OGT
CC OGT
O
ls
DMSO
o
r
t
SIRT1
OGT
OGT
FOXM1
FOXM1
Ac-p53
MMP2
Figure 3: OGT regulation of FOXM1 through SIRT1 in other Triple
Negative Breast Cancer cell lines
Actin
MMP9
The reduction of SIRT1 levels using a stably expressed SIRT1 shRNA in MDA-MB468 (A) and MDA-MB-157 cells (B) induces the upregulation of FOXM1 in control
and OGT knockdown (KD) as seen previously in MDA-MB-231 mammary
adenocarcinoma cells (Figure 1B).
Ac-p53
SIRT1
Actin
Figure 1: OGT Modulates FOXM1 levels in a SIRT1-dependent manner
The reduction of SIRT1 levels using a stably expressed SIRT1 shRNA (A) or using the
SIRT1 inhibitor EX-527 (B) induces the upregulation of FOXM1 in control and OGT
knockdown (KD) MDA-MB-231 mammary adenocarcinoma cells (A,B).
Background
B) Cancer cells contain elevated
levels of OGT and O-GlcNAc
Results
A)
MDA-MB-231
shControl
shFOXM1
l
sh trol
sh
ro
1
t
1
T
n
n
R
RT
I
Co
Co
SI
S
B)
3D Culture: MDA-MB-231
Controlsh
SIRT1sh
C)
MDA-MB-231
ΔKen FOXM1
WT FOXM1
S
DM
SIRT1
O
T1
R
S
72
M)
u
1
0(
D
O
MS
S
1
RT
72
M)
u
1
0(
WT
FOXM1
FOXM1
Human Breast Cancer Samples:
Inverse Relationship Between FoxM1 and Sirt1
FOXM1sh
ΔKen
FOXM1sh/SIRT1sh
MMP9
Ac-p53
MMP2
Actin
MMP9
OGT
Controlsh
Actin
1.0
SIRT1sh
0.48
0.99
0.95
Skp2
Actin
C) Downregulation of SIRT1 expression in breast
cancer tissues vs. normal breast tissue
O
G
50
µM Ti
+
SR OG
T7 Ti
20
FOXM1
Breast Tissue and Tumor IHC: SIRT1
O-GlcNAc
Normal
Modified from: Wang et al, Cancer Cell (2008)
Cancer
D) SIRT1 Deacetylase: Longevity Protein,
Nutrient Sensor and Tumor Suppressor
Conclusion and Model
50
µM
SR
T7
FOXM1sh/SIRT1sh
DM
FOXM1sh
SO
D)
20
MDA-MB-231
• OGT regulation of FOXM1 is dependent on SIRT1
• OGT regulates FOXM1 through SIRT1 to promote invasion
• OGT regulates FOXM1 in TNBC cell lines such as MDA-MB-231, MDAMB-157, as well as MDA-MB-468.
b
E) Targeting
OGT Inhibits
Cancer Cell
Invasion via
regulation of
oncogenic
transcription
factor
FOXM1.
OGT
Actin
cancer cell
AMPKa
?
g
SIRT1
?
Ac
invasion/metastasis?
FOXM1
P
M
M
P2
ECM Proteins
Figure 2: OGT and SIRT1 cooperate to promote invasion in TNBCs
The reduction of SIRT1 levels using stably expressed SIRT1 shRNA induces the
upregulation of FOXM1 in MDA-MB-231 adenocarcinoma cells (A). 3D culture of MDAMB-231 cells stably expressing SIRT1 and FOXM1 shRNA and fluorescently stained
with DAPI (blue) and Green fluorescence protein (GFP, green) showing that SIRT1 is
required for FOXM1 regulation of 3D growth (B). The upregulation of SIRT1 in
response to the SIRT1 activator SRT720 induces the downregulation of FOXM1 WT
when compared to DMSO treated and no downregulation in a mutant version of
FOXM1 (ΔKEN) (C). Combined treatment of OGT inhibitor with SIRT1 activator
(SRT1720) synergizes to reduce FOXM1 levels in MDA-MB-231 cells (D).
Caldwell et al, Oncogene (2009)
Lynch, TP, Ferrer, CM, et al, J. Biol Chem (2012)
Future Directions
• Determine whether OGT regulation of SIRT1 promotes breast cancer
cell metastasis (in progress)
• Determine whether FOXM1 is directly deacetylated by SIRT1, if so,
map-out acetylation sites
• Determine whether SIRT1 regulates FOXM1 phosphorylation state