1. The role of LIMK in cancer metastasis: Inhibition of LIMK inhibits Cancer Growth Juliana Antonipillai

2. 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

4. 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

5. P P STIMULI P P P cofilin Activation of LIMK1 PAK1 PAK1 ROCK ROCK PAK4P LIM Kinase Rac Cdc42 Rho Receptor

6. P Inhibition of LIMK1

7. 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

8. Yoshioka et al., 2003 NIH3T3 (ras) PC3 MDA-MB231 olf Cos-7 293T NIH3T3 NIH-3T3 (Ras) MCF-7 LnCap PC3 MDA-MB231 AP LIMK1 expression and activity are increased in cancer cell lines

9. 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

10. 1 1.32 0.7 cofilin LIMK1 P-cofilin actin pBABE-LIMK1 pBABE pBABE-DN-LIMK1 Li et al., 2012 Inhibition of LIMK activity suppresses cell proliferation and migration in vitro and in mice a

11. 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

12. 0 0.5 1 1.5 2 2.5 pBABEpBABE-LIMK1pBABE-DN-LIMK1 * * * 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 pBABEpBABE-LIMK1pBABE-DN-LIMK1 0M0M 1M1M 5M5M BMS3 * * * 4T1.2-LIMK1 4T1.2-vector BMS3 0  m BMS3 1  m BMS3 5  m DAPI F-actin 2D-proliferation3D-proliferation assays Li et al., 2012 Pharmacological inhibition of LIMK activity affects tumour growth and invasion

13. Li et al., 2012 Inhibition of LIMK activity on growth and invasion

14. Inhibition of LIMK activity by another LIMK inhibitor, Pyr1 Prudent et al., 2012

15. 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

16. Inhibition of LIMK activity by another LIMK inhibitor, 22j (Lexicon) MCF-7 DMSO 5 10 15 20  M 22j p-cofilin cofilin P-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-LIMK1 - + + + + + + + + + Lexicon(nM) 0 0 10 50 250 500 1000 5000 10000

17. 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

18. 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

19. 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?

20. Acknowledgments St. Vincent Institute & Walter and Eliza Hall Institute Dr. Ora Bernard (SVI) Dr. Rong Li Dr. Karla Acevedo Dr. Victoria Foletta Miss. 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