1. The c-Met receptor contributes to motility and invasion in high grade STS; a potential therapeutic target Sarah E. Myers, Theresa G. Nguyen, Quan-Sheng Zhu, Alexander J.F. Lazar, Raphael E. Pollock, Dina C. Lev Presented by Gonzalo Lopez, MS Sarcoma Research Laboratory MD Anderson Cancer Center Houston, TX

2. Complex karyotype: Leiomyosarcoma MFH/UPS MPNST Generally poor prognosis Sarcomas are heterogeneous

3. Migration and invasion critical for STS metastasis Primary tumor Proliferation/ angiogenesis Migration /invasion Embolism/circulation Transport Arrest in organs Adherence to vessel wall Extravasation Establishment of a microenvironment Proliferation/ angiogenesis Metastasis What are the molecular drivers of these processes???

4. Candidate mechanisms: receptor tyrosine kinases (RTK) RTKs: c-Met, EGFR, VEGFR, c-Kit, etc.RTKs: c-Met, EGFR, VEGFR, c-Kit, etc. RTKs: central components of signaling networks (embryogenesis, cell proliferation, apoptosis)RTKs: central components of signaling networks (embryogenesis, cell proliferation, apoptosis) RTK deregulation: diabetes, developmental defects, and cancerRTK deregulation: diabetes, developmental defects, and cancer Activated RTKs: induce pathways of cell migration and invasion

5. c-Met receptor: relevant biology Found in mesenchymal and epithelial tissues Found in mesenchymal and epithelial tissues Ligand HGF (hepatocyte growth factor) Ligand HGF (hepatocyte growth factor) Involved in embryogenesis, wound healing Involved in embryogenesis, wound healing Broad range of downstream effects Broad range of downstream effects

6. c-Met receptor WG Jiang et al. 2005

7. Experimental goal To evaluate the impact of c-Met activation and inhibition on complex karyotype STS migration and invasion

8. c-Met is expressed in human STS tissue Leiomyo/UPS TMA MPNST TMA

9. Phospho c-Met HGF Total c-Met Phosphorylated c-Met correlates with dismal outcome (HR 2.32; p=0.012)

10. c-Met is expressed in human STS cell lines c-Met SKLMS1 HT1080 A204 RD MES-SA SW684 SW872 NHF  -actin c-Met p c-Met HT1080SKLMS1A204 - + - + - + HGF

11. Birchmeier,C. Nature Reviews. 2003

12. HGF activates ERK and AKT p c-Met c-Met β-actin p AKT AKT p ERK ERK SKLMS1A204 - + HGF

13. A204 SKLMS1 HGF induces migration HGF induces migration HGF - + Avg. # of cells/HPF HGF (-) HGF (+) SKLMS1

14. HGF induces invasion HGF induces invasion A204 SKLMS1 HGF - + HGF (-) HGF (+) Avg. # of cells/HPF SKLMS1

15. Small molecule inhibitor (PHA-665752, Pfizer) Anti c-Met siRNA Inhibition of c-Met

16. PHA inhibits c-Met activation and downstream signaling c-Met p c-Met ERK pERK pAKT AKT - + + + + HGF - - + + + PHA 1 uM 2.5 uM 5 uM

17. MockNTsiRNA - + - + - + c-Met siRNA effect on downstream signaling c-Met p c-Met ERK pERK pAKT AKT HGF

18. DMSOPHA HGF - + DMSODMSO + HGFPHA PHA + HGF c-Met blockade abrogates HGF induced invasion Avg. # of cells/HPF

19. - HGF + HGF FN1 ITGB5 LAMB1 LAMA5 MMP2 GAPDH - + HGF LAMB1 LAMA5 ITGB5 FN1 HGF induces migration and invasion genes

20. Conclusions Human STS samples highly express c-Met, HGF, and activated c-MetHuman STS samples highly express c-Met, HGF, and activated c-Met Activated c-Met expression correlates with dismal outcome in MPNSTActivated c-Met expression correlates with dismal outcome in MPNST Activation of c-Met receptor on STS inducesActivation of c-Met receptor on STS induces migration and invasion migration and invasion c-Met blockade abrogates these processesc-Met blockade abrogates these processes

21. Acknowledgements Sarcoma Research Laboratory at UTMDACC  Dina C. Lev, MD  Raphael E. Pollock, MD/PhD  Alex J.F. Lazar, MD/PhD  Sarah E. Myers, BS  Wenhong Ren, MD  Colleagues and Staff