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

Li et al., 2012

Inhibition of LIMK activity on growth and invasion

Inhibition of LIMK activity by another LIMK inhibitor, Pyr1

Prudent et al., 2012

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