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ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration

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Abstract

We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity.

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Acknowledgement

Liangping Yuan, Mariarita Santi and Tobey J. MacDonald are supported by NIH R01 grant CA111835.

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Authors and Affiliations

  1. Emory Children’s Center, Aflac Center for Cancer and Blood Disorders, Emory University School of Medicine, 2015 Uppergate Drive NE, Room 442, Atlanta, GA, 30322, USA

    Liangping Yuan & Tobey J. MacDonald

  2. Department of Pathology, Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA

    Mariarita Santi

  3. Department of Neuropathology, Armed Forces Institute of Pathology, Washington, DC, 20306, USA

    Elisabeth J. Rushing

  4. Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA

    Robert Cornelison

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Correspondence to Tobey J. MacDonald.

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  • Supplementary Fig. 1 PDGF induces Pak1 phosphorylation and siRNA depletion of Pak1 suppresses PDGF-mediated migration of D556 MB cells. Confirmatory assays in D556 MB cells were performed for PDGF-mediated changes in phosphorylation of Pak1 and the effect of changes in phospho-Pak1 on MB migration, as previously described with Daoy cells. a Western blot shows increased levels of Pak1 phosphorylation at the indicated time points following PDGF-BB (10 ng/ml) treatment of serum-deprived D556 cells. b D556 cells transfected with control or Pak1 siRNA show that Pak1-depleted cells have attenuated PDGF-mediated phosphorylation of Pak1. c Boyden chamber cell migration assay confirms that Pak1-depleted D556 cells have significantly suppressed PDGF-mediated cell migration similar to that observed with Daoy MB cells. Each bar represents the S.E. of triplicate wells. (TIFF 792 kb)

  • Supplementary Fig. 2 PDGF-mediated ERK activation is PDGFRβ-dependent in MB cells. Daoy and D556 cells were transfected with control or shRNA PDGFRβ and stable clones for each were established (NC2, Daoy or D556 cells with negative control shRNA; A11, Daoy with shRNA knockdown of PDGFRβ; B7, D556 with shRNA knockdown of PDGFRβ), stimulated with or without PDGF-BB (10 ng/ml for 15 min), and Western blot was performed for the detection of changes in PDGFRβ and ERK phosphorylation. PDGF-mediated phosphorylation of PDGFRβ and ERK were decreased in MB cells with stable downregulation of PDGFRβ in both cell types. Data are representative of at least three separate experiments. (TIFF 677 kb)

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Yuan, L., Santi, M., Rushing, E.J. et al. ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration. Clin Exp Metastasis 27, 481–491 (2010). https://doi.org/10.1007/s10585-010-9337-9

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