title: Growth factor receptor-Src-mediated suppression of GRK6 dysregulates CXCR4 signaling and promotes medulloblastoma migration.
abstract: BACKGROUND
  Metastasis in medulloblastoma (MB) is associated with poor survival. Recent genetic studies revealed MB to comprise distinct molecular subgroups, including the sonic hedgehog (SHH) subgroup that exhibits a relatively high rate of progression. To identify targeted therapeutics against metastasis, a better understanding of the regulation of MB cell migration is needed. G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through their regulation of G-protein coupled receptors (GPCRs) involved in growth factor (GF)-mediated cell migration. However, the specific roles and regulation of GRKs in MB have not been investigated.
  
  
  METHODS
  Microarray mRNA analysis was performed for GRKs, GPCRs, and GFs in 29 human MB, and real time RT-PCR was used to detect GRK6 expression in MB cells. Lenti- or retro-virus infection, and siRNA or shRNA transfection, of MB cells was used to overexpress and knockdown target genes, respectively. Western blot was used to confirm altered expression of proteins. The effect of altered target protein on cell migration was determined by Boyden chamber assay and xCELLigence migration assays.
  
  
  RESULTS
  We observed co-overexpression of PDGFRA, CXCR4, and CXCL12 in the SHH MB subtype compared to non-SHH MB (5, 7, and 5-fold higher, respectively). GRK6, which typically acts as a negative regulator of CXCR4 signaling, is downregulated in MB, relative to other GRKs, while the percentage of GRK6 expression is lower in MB tumors with metastasis (22%), compared to those without metastasis (43%). In SHH-responsive MB cells, functional blockade of PDGFR abolished CXCR4-mediated signaling. shPDGFR transfected MB cells demonstrated increased GRK6 expression, while PDGF or 10% FBS treatment of native MB cells reduced the stability of GRK6 by inducing its proteosomal degradation. Overexpression or downregulation of Src, a key mediator of GF receptor/PDGFR signaling, similarly inhibited or induced GRK6 expression, respectively. siRNA downregulation of GRK6 enhanced CXCR4 signaling and promoted MB migration, while lentiviral-GRK6 overexpression suppressed CXCR4 signaling, potentiated the effect of AMD3100, a CXCR4 antagonist, and impaired migration.
  
  
  CONCLUSIONS
  Our findings demonstrate a novel mechanism of GF receptor/PDGFR-Src-mediated dysregulation of CXCR4 signaling that promotes MB cell migration, which could potentially be exploited for therapeutic targeting in SHH MB.
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Emory University School of Medicine, 2015 Uppergate Drive NE, Atlanta, GA 30322, USA.
authors.forename: Liangping
authors.initials: L
authors.lastname: Yuan
authors.forename: Hongying
authors.initials: H
authors.lastname: Zhang
authors.forename: Jingbo
authors.initials: J
authors.lastname: Liu
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Rubin
authors.forename: Yoon-Jae
authors.initials: YJ
authors.lastname: Cho
authors.forename: Hui Kuo
authors.initials: HK
authors.lastname: Shu
authors.forename: Matthew
authors.initials: M
authors.lastname: Schniederjan
authors.forename: Tobey J
authors.initials: TJ
authors.lastname: MacDonald
mesh_terms: D001596:Benzylamines; D045744:Cell Line, Tumor; D002465:Cell Movement; D002528:Cerebellar Neoplasms / Q000378:metabolism* / Q000473:pathology; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism; D000080027:Cyclams; D054768:G-Protein-Coupled Receptor Kinases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D053823:Hedgehog Proteins / Q000502:physiology; D006571:Heterocyclic Compounds / Q000494:pharmacology; D006801:Humans; D008527:Medulloblastoma / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000502:physiology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D001596:Benzylamines; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D000080027:Cyclams; D053823:Hedgehog Proteins; D006571:Heterocyclic Compounds; D010982:Platelet-Derived Growth Factor; D019718:Receptors, CXCR4; C509239:SHH protein, human; D020796:Receptor, Platelet-Derived Growth Factor alpha; D019061:src-Family Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D046988:Proteasome Endopeptidase Complex; C088327:plerixafor