TRAF6 Silencing with Interfering RNA to
the C-Terminal TRAF Domain Inhibits
Proliferation and Enhances Apoptosis of
Multiple Myeloma Cells
Haiming Chen, MD, PhD
Institute for Myeloma & Bone Cancer
Research
West Hollywood, California
Tumor-Necrosis Factor (TNF) Receptor-
Associated Factor (TRAF)
Some TRAFs are adaptor proteins that link the cell-surface
receptors:
-TNF receptor (TNFR) super family
-Interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR)
Some TRAF proteins mediate signaling through both:
-NF-B pathways
-JNK pathways
Structure of the Different TRAFs
TRAF1
416
Ring Finger
Zn Finger
TRAF-N
TRAF-C
TRAF2
501
TRAF3
467
TRAF4
470
TRAF5
557
TRAF6
511
(Aggarwal [2003] Nature Reviews Immunology 3: 745)
·TRAF1 and TRAF3 are not linked to NF-B or AP1 signaling
·TRAF4 has no known function in the signaling of TNF or its family members
Molecular Structure of TRAF Proteins
not Australia
(Ye Hong et al., (2002) Nature 412: 443-447)
Signaling Through TRAF6 Stimulates NF-B and JNK Pathways
IL-1/CD40
IL1R CD40R
IRAK
TRAF6
TRAF6 siRNA
TAB2
TAB1
TAK1
MKK3,4,6,7
IB Kinase (IKK)
JNK
NF-B
AP-1
Design of TRAF6 siRNA Constructs
RING Finger
Zn-Finger
TRAF-N
TRAF-C
5'-AAACTGTGAAAACAGCTGTGG-3'
(TRAF6 nt 233-253)
5'- AAGTATGAATGCCCCATCTGC - 3'
(TRAF6 nt 420-440)
dRNA
sRNA sRNA
siTRAF6 Blocks TRAF6
but not TRAF2 or TRAF5 Protein Expression
TRAF6
TRAF2
TRAF5
GAPDH
0
50
100
siTRAF6C concentration (nM)
Western Blot Analysis of Phosphorylated IB, NF-B,
SEK1/MKK4 and AP-1 Proteins Following IL-1 Stimulation:
TRAF6 Silencing Inhibits NF-B and AP-1 Activation
Vector
TRAF6C siRNA
Phospho-IB (Ser32)
Phospho-NF-B (Ser536)
Phospho-SEK1/MKK4 (Ser80)
Phospho-cJun (AP-1, Ser63/67)
0
15
30
25nM
50nM
100nM
IL-1 stimulation (min)
TRAF6C siRNA concentration
(IL-1 stimulation 30 min)
·RPMI8226 cells were transfected with TRAF6C siRNA or vector alone
·Transfected cells were stimulated with IL-1
·Phosphorylated proteins were detected by Western blot
TRAF6 Silencing Inhibits IL-1-Induced NF-B Activation
RPMI8226 cells were
transfected with
pNF-B-Luc reporter
or pTAL control
vectors.
TRAF6C siRNA was
introduced into the
transfectants.
Co-transfected cells
were stimulated with
IL-1.
NF-B activity was
assayed by
luminometry.
Silencing TRAF6 with TRAF6C siRNA
Inhibits Proliferation of GFP-Expressing RPMI8226 MM Cells
siTRAF6C
siTRAF6Zn-finger
siGAPDH
0
50
100
siRNA concentration (nM)
TRAF6 Silencing Inhibits RPMI8226 Growth
TRAF6C
4
4
Zn-Finger
4 3.5
0^
GAPDH
3
1 2.5
l
s X
2
*
cel 1.5
l
e
1
*
ab
vi viable cells x 10 0.5
0
0.107374034
0
0.120792522
25
0.30899128
50
0.062772074
100
siRNA Concentration (nM)
RPMI8226 MM cells were transfected with varying concentrations of
TRAF6C, TRAF6Zn-finger or control siRNA and proliferation assayed
by MTT. *Denotes statistically significant growth inhibition (p <0.01).
TRAF6 Silencing Inhibits Growth
of Fresh MM Tumor Cells
TRAF6C
TRAF6C
3.5
GAPDH
3.5
GAPDH
3
3
4
2.5
4
10
2.5
2
x 10
l
l
s
x
2
ls
*
1.5
1.5
l
e
ce
le cel
*
ab
1
viab
1
vi
0.5
0.5
0
0
0
25
50
100
0
25
50
100
Pt # 1034
siRNA concentration (nM)
Pt # 1141 siRNA concentration (nM)
Mononuclear cells were isolated from myeloma patients' BM aspirates,
transfected with varying concentrations of TRAF6C or control siRNA, and cell
proliferation assayed by MTT. BM aspirates contained >95% plasma cells.
*Denotes statistically significant growth inhibition (p < 0.05).
TRAF6 Silencing Enhances Apoptosis
of RPMI8226 Cells
TRAF6C
30
Zn-finger
25
*
GAPDH
l
s
20
*
15
t
i
c
MM cel
t
o
p
o 10
ap
%
5
0
0
25
50
100
siRNA concentration (nM)
RPMI8226 cells were transfected with varying concentrations of
TRAF6C, TRAF6 Zn-finger or control siRNA and apoptosis
assayed by AnnexinV staining and flow cytometry.
*Denotes a statistically significant increase in apoptosis (p < 0.01).
Assessment of TRAF6 Silencing on MM Growth In Vivo
8226/GFP MM cells
Irradiated mice
106 cells
TRAF6C siRNA
injected into
or
the mouse
GAPDH siRNA
femoral BM
8226/GFP MM cells
Harvest after 1 week
Assay proliferation by flow cytometry
TRAF6 Silencing Inhibits MM Proliferation In Vivo
180
160
140
cells
lls4
e 120
c
+
P 100
F
r
G 80
e
b
*
60
m
u
n 40
20
number GFP+ cells/10
0
mouse BM
mouse BM
8226/siGAPDH
8226/siGAPDH
8226/siTRAF6C
8226/siTRAF6C
Flow cytometry showed the growth of RPMI8226 MM cells in mouse
bone marrow were inhibited by TRAF6 but not GAPDH siRNA.
*Denotes statistically significant growth inhibition (p < 0.05).
Summary
·
TRAF6C siRNA reduced TRAF6 protein but did not affect TRAF2 or TRAF5 protein
expression
·
Inhibition of TRAF6 with TRAF6 siRNA interfered with NF-B and AP-1 activation and
NF-B-mediated transcription
·
TRAF6 silencing by TRAF6C siRNA
­ Decreased MM proliferation in vitro
­ Enhanced MM apoptosis in vitro
­ Reduced MM cell number in vivo
·
These studies on the effects of TRAF6 siRNA on MM
­ may provide important clues about the role of this signaling protein in MM cell
growth and apoptosis
­ may lead to future clinical applications of this novel therapeutic strategy
Acknowledgements
Institute for Myeloma & Bone Cancer Research
James Berenson, MD
Melinda Gordon, PhD
Richard Campbell
Kim Burkhardt
Cathy Wang
UCLA
Myeloma Research Fund
Benjamin Bonavida, PhD
Annenberg Foundation
Kramer Foundation
Skirball Foundation