Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 12, 18

Growth Factor receptor-Src-mediated Suppression of GRK6 Dysregulates CXCR4 Signaling and Promotes Medulloblastoma Migration

Affiliations

Growth Factor receptor-Src-mediated Suppression of GRK6 Dysregulates CXCR4 Signaling and Promotes Medulloblastoma Migration

Liangping Yuan et al. Mol Cancer.

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.

Figures

Figure 1
Figure 1
PDGFR activity is required for optimal CXCR4 signaling in SHH MB cells and GRK6 is downregulated and differentially expressed in MB. (A) Left panel: Daoy cells were starved and treated with or without 1 μg/ml anti-PDGFRβ blocking antibody for 24 h, then stimulated by 10 ng/ml PDGF-BB or 100 ng/ml CXCL12 (SDF-1α) for 15 min. Western blot of P-ERK shows PDGFR function is required for optimal CXCR4 signaling; Right panel: Quantitative analysis of Western blot shows CXCL12-induced P-ERK is significantly decreased by PDGFRβ blocking antibody (P < 0.05, lane 3, 1.53 ± 0.12 vs. lane 6, 1.09 ± 0.15, three independent experiments). (B) 29 human MB specimens were collected and used for microarray analysis. Average expression level of GRK6 mRNA is lowest among the detectable GRKs, (P < 0.01). (C) Microarray database of 9 human metastatic and 14 non-metastatic MB showed that the percentage of tumors with detectable GRK6 mRNA was notably decreased in metastatic MB (M+, 22%), compared to non-metastatic MB (M0, 43%). However, the percentage of tumors with detectable GRK5 mRNA was not appreciably different in M + (22%), vs. M0 (29%).
Figure 2
Figure 2
GRK6 expression is negatively regulated by GF/PDGFR. (A) Quantitative real time RT-PCR was performed to determine the expression of GRK6 mRNA in medulloblastoma cells with normal vs. downregulated PDGFR expression. (A) GRK6 mRNA is increased 1.5 folds in B9 (PDGFRβ down-regulated by shRNA in D556, shown below left panel) and 1.5 folds in A4 (PDGFRβ down-regulated by shRNA in Daoy, shown below right panel) respectively, compared to the parental cells (D556 NC1 or Daoy NC1), P < 0.05 (N = 3). (B) Protein level of GRK6 is increased in D556 (B9) and Daoy (A4) with or without CXCL12 treatment. (C) D556 or Daoy was cultured in EMEM medium containing 10% serum (GF+) or serum-free EMEM (GF-) for 24 h, then added 10% serum to the cells cultured in serum-free medium (GF add-back) and cells grown for 24-72 h. Cell lysates were harvested for Western Blot. GF withdrawal results in robust increase of GRK6 in D556 and Daoy (left and middle panels). In right panel, Daoy cells were starved for 24 h and then treated with 10 ng/ml PDGF-BB for 24 h. GRK6 level determined by Western blot shows marked decrease with PDGF treatment.
Figure 3
Figure 3
Growth factor treatment of medulloblastoma cells induces degradation of GRK6 via the proteasomal pathway. (A) D556 or (B) Daoy was cultured in EMEM medium with GF + or serum-free EMEM (GF-), treated with100μg/ml CHX or without CHX at indicated time points. GRK6 level was determined by Western blot. Growth factor withdrawal increases stability of GRK6 (C) Daoy or D556 was treated with CHX alone or CHX plus 10 μM MG132 for 8 h, then GRK6 protein level was examined by Western blot. GRK6 stability is maintained in the presence of proteasomal inhibitor MG132.
Figure 4
Figure 4
GRK6 expression is negatively regulated in a Src-dependent manner. MB cells were transfected with control siRNA or Src siRNA, and then the cells were harvested at 48 h (D556) or 96 h (Daoy) after transfection and examined by real time RT-PCR or Western Blot. (A) Real time RT-PCR shows expression of GRK6 mRNA is significantly increased 1.5 folds in D556 and 3 folds in Daoy by Src siRNA transfection, P < 0.05, respectively. Shown is the representative of three experiments. (B) Western blot shows that the protein level of GRK6 is increased by Src siRNA. (C) Retro-X Tet on Src-inducible stable cell line was treated by 500 ng/ml Dox for 48 h, and then, Src and GRK6 expression were examined by Western blot. Src- induced expression results in decreased GRK6 protein level.
Figure 5
Figure 5
CXCR4 signaling and MB cell migration is inhibited by GRK6. (A) D556 or Daoy was transfected with control siRNA or GRK6 siRNA. The cells were starved for 24 h after transfection and then stimulated by 100 ng/ml CXCL12 (SDF-1α) for 15 min. The results show that down-regulation of GRK6 causes increased CXCL12-induced P-ERK in both cell lines. (B) Daoy was transfected with control siRNA or GRK6 siRNA and then starved for 24 h prior to examination by Boyden chamber migration assay. Results show serum-mediated migration is significantly increased in the cells transfected with GRK6 siRNA (P < 0.01).
Figure 6
Figure 6
Overexpression of GRK6 inhibits CXCR4 signaling and cell migration. (A) Left panel: Lentiviral FUW-Cherry (control) or FUW-Cherry-GRK6 (FUW-GRK6)- infected Daoy cells were serum-starved and treated with AMD3100, an antagonist of CXCR4, for 24 h prior to CXCL12 (SDF-1α) stimulation (100 ng/ml for 15 min). The results show that overexpression of GRK6 inhibits CXCL12-induced P-ERK and CXCL12-induced P-ERK is completely abolished by AMD3100 at 2.5 μg/ml in Daoy-FUW-GRK6 cells, not in Daoy-FUW-Cherry cells, that potentiates the CXCR4 inhibitory effect of AMD3100. GRK5 serves as internal control (no change) to demonstrate that effects are GRK6-specific; Right panel: Quantitative analysis of Western blot shows CXCL12-induced P-ERK is significantly inhibited by AMD3100 treatment in Daoy with overexpression of GRK6 [P < 0.05, lane 3, 0.59 ± 0.1 vs. lane 6, 0.31 ± 0.03, the ratio of P-ERK/ERK in lane 1 is equal to 1.00 (100%), three independent experiments]. (B) Boyden chamber migration assay shows that serum-mediated migration is significantly decreased in cells with FUW-GRK6 overexpression (P < 0.01), compared to FUW-Cherry control cells. (C) xCELLigence was used to monitor real-time cell migration and proliferation. In the left panel, the results show that serum-mediated cell migration is significantly decreased in the cells with FUW-GRK6 overexpression (the upper two lines represent the migration of either FUW-Cherry or FUW-GRK6 in serum-containing medium. The bottom two lines represent the migration of either FUW-Cherry or FUW-GRK6 in serum-free medium). In the right panel, the results show that overexpression of GRK6 has little effect on cell proliferation either at high cell density (2 × 104/well, 20 K) or low cell density (5 × 103/well, 5 K).

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

References

    1. von Hoff K, Rutkowski S. Medulloblastoma. Curr Treat Options Neurol. 2012;14:416–426. doi: 10.1007/s11940-012-0183-8. - DOI - PubMed
    1. Ellison DW, Kocak M, Dalton J, Megahed H, Lusher ME, Ryan SL, Zhao W, Nicholson SL, Taylor RE, Bailey S, Clifford SC. Definition of disease-risk stratification groups in childhood medulloblastoma using combined clinical, pathologic, and molecular variables. J Clin Oncol. 2011;29:1400–1407. doi: 10.1200/JCO.2010.30.2810. - DOI - PMC - PubMed
    1. Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, Bouffet E, Clifford SC, Hawkins CE, French P, Rutka JT, Pfister S, Taylor MD. Medulloblastoma comprises four distinct molecular variants. J Clin Oncol. 2011;29:1408–1414. doi: 10.1200/JCO.2009.27.4324. - DOI - PMC - PubMed
    1. Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S, Gajjar A, Ellison DW, Lichter P, Gilbertson RJ, Pomeroy SL, Kool M, Pfister SM. Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol. 2012;123:465–472. doi: 10.1007/s00401-011-0922-z. - DOI - PMC - PubMed
    1. MacDonald TJ, Brown KM, LaFleur B, Peterson K, Lawlor C, Chen Y, Packer RJ, Cogen P, Stephan DA. Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat Genet. 2001;29:143–152. doi: 10.1038/ng731. - DOI - PubMed

Publication types

MeSH terms

Feedback