Abstract
BackgroundMetastasis 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.MethodsMicroarray 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.ResultsWe 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.ConclusionsOur 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.
Highlights
Metastasis in medulloblastoma (MB) is associated with poor survival
Overexpression of CXCL12 and C-X-C chemokine receptor type 4 (CXCR4) correlates with sonic hedgehog (SHH) MB and platelet-derived growth factor receptor (PDGFR) activity is required for optimal CXCR4 signaling in sonic hedgehog-activated MB (SHH MB) cells Because of the dichotomy of clinical outcomes observed for SHH MB, we first investigated whether the expression profiles of CXCL12 and CXCR4 in SHH and non-SHH MB is associated with tumor histology and clinical outcome
Our results show that PDGFR blocking antibody abolished PDGF-induced phosphorylation of ERK (P-ERK), as expected, and CXCL12-induced P-ERK (Figure 1A) [P < 0.05, lane 3, 1.53 ± 0.12 vs. lane 6, 1.09 ± 0.15 by comparing the densitometry of CXCL12-induced P-ERK/ERK between Daoy cells with or without PDGFR blocking antibody treatment, the ratio of P-ERK/ERK in lane 1 is equal to 1.00 (100%), and the relative changes of ratio of P-ERK/ ERK in other lanes were calculated by dividing by the ratio in lane1], indicating that PDGFR activity is required for activation of CXCL12-CXCR4 signaling in SHHresponsive MB cells
Summary
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. 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. Our previous studies described overexpression of the platelet-derived growth factor receptor (PDGFR) in association with metastatic MB, and demonstrated that PDGFR promotes migration through ERK-dependent activation of p21 protein (Cdc42/Rac)-activated kinase 1 (Pak1) signaling [5,6]. Given the prominent association of PDGFR with MB metastasis, we hypothesized that GF receptor signaling, such as that by PDGFR, may dysregulate other GF-mediated pathways as a mechanism to potentiate cell migration. SHH MB with desmoplastic histology has a favorable prognosis, whereas non-desmoplastic SHH MB has higher rates of metastasis and an intermediate prognosis [4] This dichotomy suggests that additional modulators of SHHCXCR4 activity may determine MB clinical behavior
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