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AFAP1L1 accelerates growth of sarcoma cells in vivo through the association with protein complex in invadopodia. Ryo Takahashi 1,2 , Yoichiro Kajita 1 , Tomohisa Kato 1 , Moritoshi Furu 1 , Masaya Ikegawa 3 , Satoshi Nagayama 4 , Yoshiharu Sakai 2 and Junya Toguchida 1

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  1. AFAP1L1 accelerates growth of sarcoma cellsin vivo through the association with protein complex in invadopodia Ryo Takahashi1,2, Yoichiro Kajita1, Tomohisa Kato1, Moritoshi Furu1, Masaya Ikegawa3, Satoshi Nagayama4, Yoshiharu Sakai2 and Junya Toguchida1 1 Dept. Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto Univ. 2 Dept. Surgery, Graduate School of Medicine, Kyoto Univ. 3 Dept. Genomic Medical Sciences, Graduate School of Medical Sciences, Kyoto Prefectural Univ. of Medicine 4 Gastroenterology Center, The Cancer Institute Hospital of Japanese Foundation of Cancer Research

  2. Outline Background: AFAP1L1 : Actin filament-associated protein 1-like 1 - isolated as a metastasis-related gene in soft tissue spindle cell sarcomas (STSs) - involved in the invasion of sarcoma cells in vitro. Research question: - What is the molecular mechanism of AFAP1L1 in the invasive or metastatic phenotype?

  3. Background: AFAP1L1 as a metastasis-related gene in spindle cell sarcomas MFH LMS SS 65 STS resected specimens metastasis (+) n=29 (-) n=36 Human mesenchymal stem cell (reference) UDS PLS MPNST cDNA microarray Genes involved in distant metastasis Isolation of AFAP1L1 (Furu et al. Oncogene 2011)

  4. Background: AFAP1L1 as a metastasis-related gene in spindle cell sarcomas STS cases with metastases * * * * * * * * * MFH 100 LMS 10 SS LS 0 MPNST Relative expression of AFAP1L1 UDS * Relative value > 100 STS cases without metastases (Furu et al. Oncogene 2011) * * 100 10 0 High incidence of distant metastasis in AFAP1L1-positive tumors irrespective of type of tumors.

  5. Background: • AFAP1L1 expression is significantly • associated with distant metastases • Cox’s proportional hazards model <Endpoint > The occurrence of distant metastases (Furu et al. Oncogene 2011)

  6. AFAP1L1 as a prognostic marker for STSs Background: P = 0.0093 1000 100 AFAP1L1 (-) Relative expression of AFAP1L1 (-) (±) P = 0.0058 (+) (++) Metastasis-free fraction (%) 10 AFAP1L1 (+) (n=23) (n=18) (Furu et al. Oncogene 2011) Follow-up period (years) 1 Mets (-) Mets (+)

  7. AFAP1L1 expression positivelycorrelates with cell invasiveness in vitro Background: Gelatin zymogram (Furu et al. Oncogene 2011) AFAP1L1 may promote matrix invasion via MMP-9 secretion.

  8. Research question What is the molecular mechanism of AFAP1L1 in the invasive or metastatic phenotype?

  9. Methods Immunocytochemistry - Change of morphology by AFAP1L1 - Subcellular localization of AFAP1L1 Xenografting - Effect of AFAP1L1 expression in vivo Immunoprecipitation - Associating protein

  10. AFAP1L1 induces actin-rich dots,and localizes in the structure F-actin AFAP1L1 F-actin / DAPI Mock AFAP1L1 AFAP1L1 10um 10um Merge

  11. Structure of invadopodia SaOS2 /AFAP1L1 10um F-actin Vinculin Actin rich core Merge Adhesive ring (Upper: Yamaguchi et al. Biochem Biophys Acta 2007) (Lower: Yilmaz et al. Cancer Metastasis Rev 2009)

  12. AFAP1L1 induces gelatin-degrading invadopodia Fluorescence-conjugated gelatin F-actin Gelatin-AF488 Merge Mock AFAP1L1 20um X-Z Defect

  13. AFAP1L1 localizes in the ring structure of invadopodia. AFAP1L1 Vinculin Merge 10µm SaOS2 / AFAP1L1 5um

  14. AFAP1L1 accelerates tumor growth of sarcoma cells (Furu et al. Oncogene 2011)

  15. AFAP1L1 accelerates tumor growth of carcinoma cells RKO pLenti6 / AFAP1L1 pLenti6 / LacZ Clone A2 Parental Bulk AFAP1L1 * P = 0.008 β-actin AFAP1L1 β-actin * Tumor Volume (mm3) Cell Numbers (×106) Week Day

  16. siRNA local administration suppresses tumor growth Control siRNA AFAP1L1 siRNA Tumor Volume (mm3) P = 0.009 RKO/AFAP1L1 A2 clone (Approx. 100mm3) Day P = 0.002 siRNA administration Untreated Control siRNA AFAP1L1 siRNA Tumor Weight (mg) AFAP1L1 β-actin

  17. * AFAP1L1 regulates cellular morphology and motility *** ** *** RKO *P<0.05; **P<0.01; ***P<0.001 AFAP1L1 (Bulk) AFAP1L1 (Clone A2) (µm / min) Parental LacZ 80 50µm Mean Speed % Rounded Cells 40 80 0 80 -80 ** (µm) -80 80 Control siRNA AFAP1L1 siRNA-3 Untreated 40 % Rounded Cells 0 A2 Untreated 50µm AFAP1L1 β-actin

  18. Rounded cells form fewer focal adhesions in RKO cells F-actin /Vinculin /DAPI Flattened Elongated Round RKO 10µm Block MR. et al. Eur J Cell Biol. (2008)

  19. Vinculin regulates cell shape and motility • F9 (mouse embryonic carcinoma cells) Round phenotype Smaller focal adhesion complex Cell motility ↑ (on 2D substrate) Vinculin -/- Rodriguez Fernandez et al. J Cell Biol. (1992) • MEF (mouse embryonic fibroblast) Cell-matrix adhesion ↓ Cell motility ↑ (on 2D substrate) Vinculin -/- Xu et al. J Cell Sci. (1998)

  20. Hypothesis AFAP1L1 Negative modulation ? Association ? Mature focal adhesion complex Cell-matrix adhesion Flattened or elongated phenotype Vinculin

  21. AFAP1L1 associates with vinculin RKO / (Flag)3-AFAP1L1 + (HA)3-Vinculin LoVo IP: HA IP: AFAP1L1 WCL IP: Vinculin IgG kDa IgG kDa 150 - 150 - (Flag)3-AFAP1L1 Vinculin 100 - 100 - 75 - IB: Flag 75 - IB: Vinculin 50 - 50 - 150 - IB: HA 100 - IB: AFAP1L1 100 -

  22. Vinculin regulates anoikis Vinculin↓ →anoikis↓ (Subauste et al. J Cell Biol 2004) Anoikis: apoptosis induced by lack of correct cell-matrix attachment

  23. AFAP1L1-transduced cells are resistant to anoikis RKO HEMA coating (2-Hydroxyethyl methacrylate) AFAP1L1 Parental LacZ (kDa) 150 - Full length PARP 100- Cleaved PARP 75- Suspension culture AFAP1L1 ACTB possibly the cause of accelerated tumor growth in vivo

  24. Conclusion What is the molecular mechanism of AFAP1L1 in the invasive or metastatic phenotype? #AFAP1L1 modulates cell-matrix interaction; 1. assembly of invadopodia, 2. cell shape and motility, # AFAP1L1 plays a role in the inhibition of anoikis, presumably through the interaction with vinculin.

  25. Acknowledgement Toguchida-lab Makoto Ikeya YongHui Jin Akira Nasu Kazuo Hayakawa Sakura Tamaki Elalaf Hassan Kyosuke Kobayashi Yoshihisa Matsumoto Koji Yokoyama Makoto Fukuda Sho Hineno Naoko Takahara Chie Koizumi Michiko Ueda Yukiko Kobayashi Hisayo Yasuda Marie Yoshino Institute for Medical Science, University of Tokyo ToyomasaKatagiri Yusuke Nakamura SeiyaImoto Hidetoshi Shimohira Department of Orthopaedic Surgery, Kyoto University Koichi Nishijo Tomoki Aoyama

  26. Conclusion What is the molecular mechanism of AFAP1L1 in the invasive or metastatic phenotype? #AFAP1L1 modulates cell-matrix interaction; 1. assembly of invadopodia, 2. cell shape and motility, # AFAP1L1 plays a role in the inhibition of anoikis, presumably through the interaction with vinculin.

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