the role of limk in cancer metastasis: inhibition of limk inhibits cancer growth juliana...
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The role of LIMK in cancer metastasis: Inhibition of LIMK
inhibits Cancer Growth
Juliana Antonipillai
LIMK
• LIMK domain structure
• Splice variant: LIMK1-s
• The LIMK protein is expressed in all mouse tissues examined
(high levels in brain, kidney, lung, stomach and testes)
• The LIMK family of serine protein kinases includes LIMK1 and LIMK2. These proteins have identical structure and overall 50% amino acid homology with 70% in their kinase domain
Regulation of actin dynamics
• Actin is a highly dynamic protein involved in many cellular functions
• The dynamics of the actin cytoskeleton is tightly regulated by actin binding proteins
• LIM kinase (LIMK) regulates the actin dynamics process by controlling the binding affinity of ADF/cofilin towards actin
PP
PP
STIMULISTIMULI
PPPP
PP
cofilincofilin
Activation of LIMK1Activation of LIMK1
PAK1PAK1 ROCKROCK PAK4PAK4
PPLIM KinaseLIM Kinase
RacRac Cdc42Cdc42RhoRho
ReceptorReceptor
PP
Inhibition of LIMK1Inhibition of LIMK1
PP
STIMULISTIMULI
PPPP
PP
cofilincofilin
BMPRIIBMPRII
Protein kinaseProtein kinase
SSHSSH
ReceptorReceptor
PPLIM KinaseLIM Kinase
LIMKs
LIMK1 and Metastasis
LIMK1 is highly expressed in metastatic cancers
Overexpression of LIMK1 increases the invasiveness of non-metastatic tumour cells
Expression of dominant-negative LIMK1 inhibits the invasiveness of highly invasive breast cancer cell lines
Yoshioka et al., 2003
NIH
3T3
(ras
)P
C3
MD
A-M
B2
31
olf
Cos
-729
3T
NIH
3T3
NIH
-3T
3 (R
as)
MC
F-7
LnC
apP
C3
MD
A-M
B2
31
AP
LIMK1 expression and activity are increased in cancer cell lines
•Decrease LIMK activity was judged by decreased p-cofilin levels using Kinase assays in vitro and Kinase binding assays and in cells (western blots with anti-p-cofilin)
•Observed inhibition of cell migration, invasion and cell proliferation
Inhibition of LIMK activity by kinase inhibitors
1 1.32 0.7
cofilin
LIMK1
P-cofilin
actin
pBABE-LIM
K1
pBABEpBABE-D
N-LIM
K1
Li et al., 2012
Inhibition of LIMK activity suppresses cell proliferation and migration in vitro and in mice
a
LIMK inhibitors used in Dr.Bernard’s lab
1. CTX (CRC) (Not published)
2. BMS3 (Bristol Myers Squibb)
3 Pyr1 (French, Grenoble)
4) 22j (Lexicon)
All inhibitors inhibit LIMK1 and LIMK2 activity in vitro
0
0.5
1
1.5
2
2.5
pBABE pBABE-LIMK1 pBABE-DN-LIMK1
*
*
*
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
pBABE pBABE-LIMK1 pBABE-DN-LIMK1
0M1M5M
BMS3
*
*
*
4T1.2-LIMK1 4T1.2-vector
BMS3 0 m
BMS3 1 m
BMS3 5 m
DAPI F-actin
2D-proliferation 3D-proliferation assays Li et al., 2012
Pharmacological inhibition of LIMK activity affects tumour growth and invasion
Effects of BMS3 and Pyr1 on LIMK activity in MDA-MB-231
p-cofilin
cofilin
Detyro-tubulin
Tubulin
p25
0 1 3 5 25 M O/N BMS3 Pyr1
LIMK1
actin
Inhibition of LIMK activity by another LIMK inhibitor, 22j (Lexicon)
MCF-7DMSO 5 10 15 20 M 22j
p-cofilin
cofilin
P-GST-cofilin
GST-cofilin
GST-cofilin + + + + + + + + + + GST-LIMK2 - + + + + + + + + + Lexicon( nM) 0 0 10 50 250 500 1000 5000 10000
PI
Coomassie
P-GST-cofilin
GST-cofilin
GST-cofilin + + + + + + + + + + GST-LIMK1 - + + + + + + + + + Lexicon( nM) 0 0 10 50 250 500 1000 5000 10000
LIMK inhibitors used in Dr.Bernard’s lab
1. CTX (CRC) (Not published)
2. BMS3 (Bristol Myers Squibb)
3 Pyr1 (French, Grenoble)
4) 22j (Lexicon)
All inhibitors inhibit LIMK1 and LIMK2 activity in vitro
Effects of Pyr1 on tumor growth in mice.
Prudent R et al. Cancer Res 2012;72:4429-4439
©2012 by American Association for Cancer Research
Summary
• LIMK is highly expressed in invasive cell lines
• LIMK activity is high in invasive cell lines
• LIMK activity is important for the invasiveness and migration of cancer cells
• Inhibition of LIMK activity by small drug molecules inhibits tumour cell growth and invasiveness in vitro
• LIMK inhibitors are potential anti-metastatic drugs?
Acknowledgments
St. Vincent Institute &Walter and Eliza Hall Institute
Dr. Ora Bernard (SVI)Dr. Rong LiDr. Karla AcevedoDr. Victoria FolettaMiss. Natalie Mussie
Collaborators
1) Dr Laurence Lafanechere and her laboratory members. Institut Albert Bonniot, CRI INSERM/UJF U823, Team 3, "Polarity, Development and Cancer",Rond-point de la Chantourne, 38706 La Tronche Cedex, France.
2) Dr Robin L. Anderson and her laboratory members. The Sir Peter MacCallum Institute, Department of Oncology and the University of Melbourne, Parkville, Melbourne, VIC 3010, Australia
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