Northwestern University Feinberg School of Medicine ...€¦ · ELMO1 Src PI3K CrkII SHP-2 Cell...
Transcript of Northwestern University Feinberg School of Medicine ...€¦ · ELMO1 Src PI3K CrkII SHP-2 Cell...
Northwestern University
Feinberg School of Medicine
Northwestern Brain Tumor Institute
Lurie Comprehensive Cancer Center
Dysregulated Signaling and Epigenetics in Glioblastoma
Shi-Yuan Cheng Ph.D.
Department of Neurology
Therapeutic targeting cancer
hallmarks
Douglas Hanahan and Robert A. Weinberg. Hallmarks of Cancer: The Next Generation, Cell, March 2011
Non-coding RNAAutophagy
Epigenetics
RTK
Glioblastoma
• Grade IV astrocytomas (Glioblastoma multiforme, GBM).
• Overall survival 14-16 months, 2-year survival rates <10%.
• Most patients develop resistant to radiation, chemotherapy and molecular targeted therapies.
T1 weighted MRI
2005
Stupp R. et al NEJM
2014
Chinot OL et al NEJM
Genetic Pathways to Primary and Secondary Glioblastoma
Ohgaki H , and Kleihues P Clin Cancer Res 2013; 19:764-772
The Cancer Genome Atlas Research Network Nature , 2008
Oncogenic Signaling Pathways Are Activated in GBMs
PDGFR Signaling Promotes GBM Growth and Invasion
Liu, KW, 2011, JCI; Feng, H. et al, 2011, JCI,
2012, Oncogene, 2015, Neuro-Onc
TK
Ig
Ig
Ig
Ig
TK
Ig
JM
pTyrs
pTyrs
Glioma
CellsPDGFPDGF
PDGFR
Rac1
Dock1
Cell
Migration
ELMO1
Src
CrkIIPI3K
SHP-2
Cell
Growth
Dyn2
PKA
Andrae, J., Gallini, R. & Betsholts, C., 2008,
Genes & Dev
Kun-wei Liu, PhD Haizhong Feng,
PhD
EGFR Signaling Promotes Glioma Growth and Invasion
TK
Ig
Ig
Ig
Ig
TK
Ig
JM
pTyrs
pTyrs
Glioma
CellsEGF
EGF
EGFR
Rac1
Cell
Migration
Survival
ELMO1
Src PKA
Dock1
Feng, H. et al, 2012, PNAS;
2013, Oncogene
Haizhong Feng,
PhD
EGFR Phosphorylation of DCBLD2 Recruits TRAF6 andStimulates Akt-promoted Tumorigenesis
DCBLD2
DCBLD2 DCBLD2
EGFR
EGFR EGFR
EGF EGF
TRAF6
TRAF6 TRAF6
Akt
AktAkt Akt
Akt
EGF
P
DCBLD2 EGFR
EGF
TRAF6Akt
P
P
P
P
T308
S473
Ub
UbUb
UbUb
Feng, H. et al., JCI , 2014
Haizhong Feng,
PhD
GBM Subtypes/Heterogeneity
Verhaak, R.G.W., et al., 2010, Cancer Cell, 17:98-110
Other papers
Targeting Mature GBM Cells and Glioma Stem Cells
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Nduom, E.K-
E., et al,
2012, The
Cancer JBiochem Pharmacol. 2010 80(5): 654–6Cancer Cell 21, 2012, SnapShot
Two Distinct Subtypes of Glioma Stem Cells (GSC)
Derived from Clinical Glioma Tumor Tissues
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Mao, P. et al, 2013, PNAS
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Gene Profile Analyses Identify GBM Subtypes of GSCs
Mao, P. et al, 2013, PNAS
MicroRNA (miR) Biogenesis
Tianzhi Huang,
PhD
Differentially Expressed miRs Classify PN and MES GBM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
PNMES
miR-
Top 20 miRNAs
22a
let-7g126b
let-7dlet-7f
116130
99a354b138b
31
193a-5p
112
3
83
52
8
81
6
84
AC
20
17
71
8
1303711
193a-3p
630
517b
Rank in Ordered Dataset
0.8
0.6
0.4
0.2
0
0.25
0
0.50 100 200 300 400 500
“MES” (negatively
correlated)
Ran
k lis
t M
etr
icE
nri
chm
en
t S
core
Enrichment plot: Top 20 PN Upegulated
P=0.03808
TCGA dataset
Re
lative
exp
ressio
n le
ve
l
MES PN
0
4
6
8
2
112
38
33
02
02
81
11
71
92
33
415
78
4
**
miR-125b
50
25
151050
11
23
83
30
20
267
302
17
19
23
34
157
84
11
MES PN
**
miR-20b
Re
lative
exp
ressio
n le
ve
lHuang, TZ et al, 2016,
Nat. Comms.
miRs Regulate GBM Tumorigenicity
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
10
20
30
40
23.5
6.0
22.5
5.5
****
Ctrl + - + -miR - + - +
83 1123
Fre
qu
en
cy o
f sp
here
form
ing
cells
(%
)
23.2
7.5
21.8
6.60
10
20
30
40 ****
+ - + - - + - +
1000 cells/well 500 cells/well 200 cells/well
100 cells/well 50 cells/well
83 1123 125b 20b
MES
83
11
23
Ctrl 125b
**
T
um
or
siz
e (
mm
3)
0
10
20
30
Ctrl + - + -125b - + - +
83 1123
**
MES
MES
Fre
que
ncy o
f sp
he
refo
rmin
g c
ells
(%
)
0
20
40
8.5
12.0
10
14.9
**
0
10
20
30
8.5 10 1012.3
**
1000 cells/well 500 cells/well 200 cells/well
100 cells/well 50 cells/well
Ctrl + - + -Zip - + - +
157 84
+ - + - - + - +
157 84 125b 20b
PN
0
10
20
30
T
um
or
siz
e (
mm
3)
Ctrl + - + -125bZip - + - +
157 84
** **
PN
15
78
4
Ctrl 125b Zip
PN
Huang, TZ et al, 2016,
Nat Comms.
Canonical Wnt Signaling Pathway: Off and On
Anastas, JN and Moon, RT, Nat Rev Cancer, 13, 11-26 (2013)
miR-Wnt Regulates GBM Phenotypes through FDZ6
+ - + -- + - +
83
FZD6
Ctrl 20b
APC
β-actin
1123
+ - -- + - +
157-Zip+
FZD6
Ctrl 20b
APC
β-actin
+ - + -- + - +
83 1123Ctrl
125b
+ - -- + - +
84-Zip+ Ctrl
125b
MES
PN
FZD6 mRNA
miR-20b
5' ...UAUUGUGAUAUUUUAGCACUUUG...3'
GAUGGACGUGAUACUCGUGAAAC
FZD6 mRNA
miR-125b
CTTGTTCACCCGGTTTCAGGAG...3'
AGUGUUCAAUCCCAGAGUCCCU
5' ...
0
20
40
60
Ctrl 20b Zip 125b Zip
0
5
10
15
Rela
tive e
xp
ressio
n level
FZD6PN84PN157
****
* ***
**
* *APC FZD6APC
831123
sh-Ctrl sh-FZD6
MES
APCAxin
GSK3bpβ-catenin
β-catenin miR-20b miR-125b
Nucleus
Self-renewalCell growth
PN GBM
Positivefeedback loop
PP2A
Destruction complex
ALDH1A3
FZD6
cell growth
self-renewal
TCF4 LEF
APC
Re
lative
lu
cife
rase
activity
T F T F T F
Ctrl
0
1
2
3
4
** ***
**
Ctrl20b125b20b+125b
ABC
Ctrl 125b
20b
125b
+20b
β-actin
TCF4
BIOWnt3A
Huang, TZ et al, 2016,
Nat. Comms
miR-Wnt Regulates GBM Phenotypes through FDZ6
FZD6β-actin
23Ctrl
FZD6 - + TCF4ABC
p-Stat3Stat3
p65p-p65
17+ -- +
TAK1p-TAK1
NLK
Ctrl FZD6
*
**
Rela
tive e
xpre
ssio
n level
**
+ -
12
8
4
0
ALD
H1A
3
CD44
Olig
2
Sox
2
1.2
0.8
0.4
0
PNPN23
Rela
tive
expre
ssio
n leve
l
Ctrl
KN93
5z-7
-oxo
sh-N
LK
FZD6
KN93
+FZD
6
5z-7
-oxo
+FZD
6
sh-N
LK+F
ZD6
3
2
1
0
20b 125b
***
*
LEF1
*
*
ID2
*
*
*
0
1
2
3
4
01
2
3
4
Ctrl
FZD6
KN93
5z-7
-oxo
+FZD6
sh-N
LK+F
ZD6
KN93
+FZD
6
5z-7
-oxo
sh-N
LK Ctrl
FZD6
KN93
5z-7
-oxo
+FZD
6
sh-N
LK+F
ZD6
KN93
+FZD
6
5z-7
-oxo
sh-N
LK
Rela
tive e
xpre
ssio
n levelPN23 PN23
Ctr
lF
ZD
6
PN23
FZD6
KN93CaMKII
TAK1
NLK
Nucleus
Self-
5Z-7-oxo
MES GBM
miR-20b miR-125b x
NF-kB
Stat3
b-catenin
TCF4
cell growth
self-renewal
LEF
Huang, TZ et al, 2016,
Nat Comms.
Inhibition of GBM Subtypes by Pathway-Specific Inhibitors
Ctrl
LGK974
BAY-117082
MES83
BAY-117082 LGK974Ctrl
Tu
mo
r vo
lum
es (
mm
3)
0
400
800
1200
0 5 8 12 14 17 19
CtrlLGK974BAY-117082
***
Days
BAY-117082
Enriched in
MES GBM
Ctrl
LGK974
BAY-117082
PN23
BAY-117082LGK974Ctrl
Tu
mo
r vo
lum
es (
mm
3)
Days
0
400
800
1200 BAY-117082LGK974Ctrl
40 50 60 70
***
0
LGK974
Enriched in
PN GBM
Huang, TZ et al, 2016,
Nat Comms.
miRs-Wnt Signaling establish Regulatory Circuits
Controlling GBM Phenotypes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20APC
AxinGSK3βp
β-catenin
β-catenin miR-20b miR-125b
NucleusSelf-renewal
KN93/KDCaMKII
TAK1
NLK
Nucleus
Self-
5Z-7-oxo/KD
Cell growth
MES GBMPN GBM
miR-20b miR-125b
Positive
feedback loop
PP2A
ALDH1A3
FZD6
x
Wnt/β-Catenin
NF-kB
Stat3
β-catenin
cell growth
self-renewal
TCF4 LEF TCF4
APC
cell growth
self-renewal
miR-125b/20b
FZD6
LEF
KD
FZD6
PORC
LGK974
Wnt
Wnt
CBP
ICG-001
Indo/KD
KD
LRP6
FZD
Huang, TZ et al, 2016,
Nat. Comms
Jones, A.P.. et al., 2012, Nat Rev Genet.
CpG Sites in Chromatin and Their Roles in Gene Expression
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
DNA Methylation Regulates Gene Expression
Pangeni RP. et al, 2016,
in submission
CCCGTTTCGTAGAGCGCGTGT
CH3
GCCCGTTTCGTAGAGCGCGTGT
TTTGCGCGGGCGTGGCCTTTCGTAGAGTTCGTGG
CH3 CH3 CH3
GeneNOTexpressedPromoter
TTTGCGCGGGCGTGGCCTTTCGTAGAGTTCGTGG
CH3 CH3
Normalcell
Cancercell
Promoter
Geneexpressed
mRNA Protein
TSS
CpGisland
CpGisland
Intragenicregion
Intragenicregion
Unmethylated Methylated
Hypermethylated Hypomethylated(demethylated)
(Genesilencing) (Genomicdysregula on
A)
B)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Epigenetic Determinant for GBM Phenotypes
Pangeni RP. et al, 2016,
in submission
Rajendra Pangeni,
PhD
DNA Methylation Profiling Identifies Subtype-
associated Methylation landscapes in GBM and GSCs
GSC GBM GSC GBM
MES N CL PN PN U1 U2
Hypermethylated
Hypomethylated
0
25
50
0
3
6
0
0.6
1.2
0
2
4
MES
0
8
16
0
10
20
0
12
24
0
12
24
No of genes (x100) No of genes (x100)
603
16
34
528
84
157
19
67
92
46
608
1123
83
30
600
17
83
586
42
44
59
543
576
PN MESU2 U1
Pangeni RP. et al, 2016,
in submission
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Subtypes-specific Methylation Signatures in
GBM and GSCs
Pangeni RP. et al, 2016,
in submission
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Epigenetic Dysregulation Correlate to Patients Prognosis
PathwaysassociatedwithMES-methylatedsignaturegenesinGSCs
PathwaysassociatedwithPN-methylatedsignaturegenesinGSCs
High, n=271Low, n=271CFLAR
GBM (TCGA)
Months
p=0.009
0.4
0.8
0.0
6633 99 132
PrognosisofPN-methylatedgenes
High, n=103Low, n=103TMCC1
Glioma (TCGA)
Months
0.4
0.8
0.0
6633 99 132 198165
P=0.003
PrognosisofMES-methylatedgenes
Pangeni RP. et al, 2016,
in submission
Cheng Laboratory At Northwestern Univ.
Current Funding:NIH CA158911, NS095634, NS093843 & CA209345;
James McDonnell Foundation (PI: Hu); Northwestern Brain Tumor Institute
Zell Scholar Fund & NU Department of Neurology
AcknowledgementUSA and Europe
Webster Cavenee, Frank Furnari, Ludwig Inst for Cancer Res & UCSD, CA
Hai Yan, Duke Univ., NC; Jenny Grandis, UCSF, CA
Philip Auron, Duquesne Univ. Pittsburgh, PA
Kristiina Vuori, Sanford Burnham Med Res Institute, La Jolla CA
Xinghau Lu, R. Hamilton, Univ. of Pittsburgh, PA
Ichiro Nakano, Univ. of Alabama, AL
Wei Zhang, C. David James, Craig Horbinsky, Jeffery Raizer, John Kessler,
Northwestern Univ. Chicago, IL
Hui-Kuan Lin, Wake Forest Univ. NC
Susan Keezer, Cell Signaling Technology, Danvers, MA
Cameron Brenann, Sloan Kettering Cancer Center, NY
Erik Sulman, MD Anderson Cancer Center, Houston, TX
Gaetano Finocchiaro, Istituto Neurologico Besta, Via Celoria, Italy
Ming Tan, Robert Sobol, Univ of South Alabama, AL,Jan Sarkaria, Mayo Clinic, MN
JapanRyo Nishikawa, Saitama Medical Univ.Motoo Nagane, Kyorin Univ.
ChinaHaizhong, Feng, Wei-Qiang Gao, Shanghai Jiao Tong Univ.& Ren Ji Stem Cell CtrTao Cheng, Yanxin Li, Chinese Academy of Med Sci, Tainjin
Wnt Signaling is active in PN GBM but not in MES GBM
TCF4
ABCβ-actin
APC
FZD6
19
23
84
15
71
73
03
26
83
11
23
PN MES
Re
lative
TO
P/F
OP
expre
ssio
n
83
11
23
17
84
15
7
PN MES
0
5
10
15
*
**
*
Huang, TZ et al, 2016,
Nat Comm in revision
Wnt Signaling Induces miRs in GBM
Huang, TZ et al, 2016,
Nat Comm in revision
Correlative expressions of miRs, TCF-4 and FDZ6 in GBM
0
0.5
1
1.5
2
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
T25
T26
T27
T28
T29
T30
T31
T32
T33
T34
T35
T36
T37
T38
T39
20b 125b
Re
lative
exp
ressi
on
leve
l
TCF4FZD6
b-actin
100
50
00 20 40 P
erc
en
t su
rviv
al
Months
20b high 20b low P=0.0407
TCF4 highTCF4 low
100
50
00 40 80
Months
Pe
rce
nt
surv
iva
l
FZD6 highFZD6 low
P=0.0047 P=0.0013
100
50
0
125b high125b low P=0.0348
Pe
rce
nt su
rviv
al
0 20 40
Months
0 40 80Months
Pe
rce
nt
surv
iva
l
100
50
0
TCF4 high/FZD6 low (n=8)
TCF4 low/FZD6 high (n=15)
P=0.0057
100
50
00 20 40 60 80
months
Pe
rce
nt
su
rviv
al
Huang, TZ et al, 2016,
Nat Comm in revision
PDGFR Signaling Promotes Glioma Cell Growth and Invasion
TK
Ig
Ig
Ig
Ig
TK
Ig
JM
pTyrs
pTyrs
Glioma
CellsPDGF
PDGF
PDGFR
PI3K
SHP-2
Cell
Growth
Dyn2
Liu, K. et al., 2011, JCI; Feng, H. et al, 2011: JCI, 2012:
Oncogene; 2014: Neuro-Oncol.
Rac1
Dock1
Cell
Migration
ELMO1
Src
CrkII
PKA
Haizhong Feng,
PhD
Unconventional Guanine Nucleotide
Exchange Factors (GEFs)
DHR-1 DHR-2
ELMO1 PIP3 Rac1 CrkII
SH3 PxxP
MicroRNAs Targeting the Hallmarks of Cancer
Iorio, M.V. and Croce, C.M., 2012, EMBO Mol Med
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20
Methylation Profiling (MIDN) in PN and Mes GSC
GSC GBMA
B C
0
0.4
0.8
MU
β-
valu
e
MES PN
0
0.4
0.8
MES PN
MU
PN MES
15
7
U C
Sa
m D
NA
U C
84
U C
19
U C
17
U C
54
3
U C
16
U C
34
U C
83
U C
30
U C60
0U C
11
23
U C
** *
*
*
52
8
U C
5 1084
30
83
1123
14
PN
ME
S
CpG1 CpG2 CpG3 CpG4
* Methylated, U: Uncut control, C: Cut by enzyme
UnmethylatedMethylated
Pangeni RP. et al, 2016,
in submission