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A novel extracellular Hsp90 mediated co-receptor function for LRP1 regulates EphA2 dependent glioblastoma cell invasion.

Gopal U, Bohonowych JE, Lema-Tome C, Liu A, Garrett-Mayer E, Wang B, Isaacs JS - PLoS ONE (2011)

Bottom Line: Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function.We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT

Background: Extracellular Hsp90 protein (eHsp90) potentiates cancer cell motility and invasion through a poorly understood mechanism involving ligand mediated function with its cognate receptor LRP1. Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal brain cancers. The receptor tyrosine kinase EphA2 is overexpressed in the majority of GBM specimens and is a critical mediator of GBM invasiveness through its AKT dependent activation of EphA2 at S897 (P-EphA2(S897)). We explored whether eHsp90 may confer invasive properties to GBM via regulation of EphA2 mediated signaling.

Principal findings: We find that eHsp90 signaling is essential for sustaining AKT activation, P-EphA2(S897), lamellipodia formation, and concomitant GBM cell motility and invasion. Furthermore, eHsp90 promotes the recruitment of LRP1 to EphA2 in an AKT dependent manner. A finding supported by biochemical methodology and the dual expression of LRP1 and P-EphA2(S897) in primary and recurrent GBM tumor specimens. Moreover, hypoxia mediated facilitation of GBM motility and invasion is dependent upon eHsp90-LRP1 signaling. Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.

Significance: We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function. We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2. Taken together, our results demonstrate activation of the eHsp90-LRP1 signaling axis as an obligate step in the initiation and maintenance of AKT signaling and EphA2 activation, thereby implicating this pathway as an integral component contributing to the aggressive nature of GBM.

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eHsp90-LRP1 signaling regulates activated EphA2 (phospho-S897), its association with AKT, and lamellipodia formation.(A) Control or LRP1 silenced G48a cells were treated with NPGA, ephrin A1, or the combination, and the effects upon P-AKTS473 and P-EphA2S897 were evaluated. The effect of src silencing was included for relative comparison. (B) Interference with eHsp90 signaling (NPGA or LRP1 silencing), or AKT activation (src silencing or treatment with ephrin A1) disrupts EphA2-AKT protein complexes. (C) G48a cells grown were fixed 4 hr after wounding, followed by immunostaining with the indicated antibodies. Arrows indicate the leading edge localization of P-EphA2S897 (a–c). Stimulation of cells with ephrinA1 was included as positive control for suppression of EphA2S897 phosphorylation (d–f). Lamellipodia formation and concomitant localization of P-EphA2S897 is similarly suppressed by NPGA (g–i) or LRP1 silencing (j–l), or by src silencing (m–o). Scale bars 25 µm.
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pone-0017649-g002: eHsp90-LRP1 signaling regulates activated EphA2 (phospho-S897), its association with AKT, and lamellipodia formation.(A) Control or LRP1 silenced G48a cells were treated with NPGA, ephrin A1, or the combination, and the effects upon P-AKTS473 and P-EphA2S897 were evaluated. The effect of src silencing was included for relative comparison. (B) Interference with eHsp90 signaling (NPGA or LRP1 silencing), or AKT activation (src silencing or treatment with ephrin A1) disrupts EphA2-AKT protein complexes. (C) G48a cells grown were fixed 4 hr after wounding, followed by immunostaining with the indicated antibodies. Arrows indicate the leading edge localization of P-EphA2S897 (a–c). Stimulation of cells with ephrinA1 was included as positive control for suppression of EphA2S897 phosphorylation (d–f). Lamellipodia formation and concomitant localization of P-EphA2S897 is similarly suppressed by NPGA (g–i) or LRP1 silencing (j–l), or by src silencing (m–o). Scale bars 25 µm.

Mentions: To further solidify the possibility of pathway crosstalk between eHsp90 signaling and EphA2, we evaluated whether NPGA specifically impacted upon EphA2 activation. To explore this, we determined whether eHsp90 modulated the phosphorylation status of EphA2S897. Perturbation of eHsp90 signaling by either NPGA or LRP1 silencing effectively suppressed P-EphA2S897 (Figure 2A). EphA2 was recently identified as a substrate for AKT in GBM, wherein AKT-directed phosphorylation of EphA2 at S897 is required for EphA2 dependent cell motility [10]. NPGA markedly suppressed, and LRP1 silencing abrogated, AKT activation, demonstrating that perturbation of eHsp90 signaling suppresses AKT activation and concomitant AKT mediated phosphorylation of EphA2. AKT phosphorylation can be induced by src [33], and we found that NPGA or LRP1 silencing suppressed src activation (Figure S2E). To define the potential role of src in modulating P-EphA2S897, src was silenced (Figure S2F), which dramatically suppressed both P-AKTS473 and P-EphA2S897 (Figure 2A). This suggests that eHsp90-LRP1 mediated src signaling is a prerequisite for subsequent serine phopshorylation of AKT and EphA2.


A novel extracellular Hsp90 mediated co-receptor function for LRP1 regulates EphA2 dependent glioblastoma cell invasion.

Gopal U, Bohonowych JE, Lema-Tome C, Liu A, Garrett-Mayer E, Wang B, Isaacs JS - PLoS ONE (2011)

eHsp90-LRP1 signaling regulates activated EphA2 (phospho-S897), its association with AKT, and lamellipodia formation.(A) Control or LRP1 silenced G48a cells were treated with NPGA, ephrin A1, or the combination, and the effects upon P-AKTS473 and P-EphA2S897 were evaluated. The effect of src silencing was included for relative comparison. (B) Interference with eHsp90 signaling (NPGA or LRP1 silencing), or AKT activation (src silencing or treatment with ephrin A1) disrupts EphA2-AKT protein complexes. (C) G48a cells grown were fixed 4 hr after wounding, followed by immunostaining with the indicated antibodies. Arrows indicate the leading edge localization of P-EphA2S897 (a–c). Stimulation of cells with ephrinA1 was included as positive control for suppression of EphA2S897 phosphorylation (d–f). Lamellipodia formation and concomitant localization of P-EphA2S897 is similarly suppressed by NPGA (g–i) or LRP1 silencing (j–l), or by src silencing (m–o). Scale bars 25 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050925&req=5

pone-0017649-g002: eHsp90-LRP1 signaling regulates activated EphA2 (phospho-S897), its association with AKT, and lamellipodia formation.(A) Control or LRP1 silenced G48a cells were treated with NPGA, ephrin A1, or the combination, and the effects upon P-AKTS473 and P-EphA2S897 were evaluated. The effect of src silencing was included for relative comparison. (B) Interference with eHsp90 signaling (NPGA or LRP1 silencing), or AKT activation (src silencing or treatment with ephrin A1) disrupts EphA2-AKT protein complexes. (C) G48a cells grown were fixed 4 hr after wounding, followed by immunostaining with the indicated antibodies. Arrows indicate the leading edge localization of P-EphA2S897 (a–c). Stimulation of cells with ephrinA1 was included as positive control for suppression of EphA2S897 phosphorylation (d–f). Lamellipodia formation and concomitant localization of P-EphA2S897 is similarly suppressed by NPGA (g–i) or LRP1 silencing (j–l), or by src silencing (m–o). Scale bars 25 µm.
Mentions: To further solidify the possibility of pathway crosstalk between eHsp90 signaling and EphA2, we evaluated whether NPGA specifically impacted upon EphA2 activation. To explore this, we determined whether eHsp90 modulated the phosphorylation status of EphA2S897. Perturbation of eHsp90 signaling by either NPGA or LRP1 silencing effectively suppressed P-EphA2S897 (Figure 2A). EphA2 was recently identified as a substrate for AKT in GBM, wherein AKT-directed phosphorylation of EphA2 at S897 is required for EphA2 dependent cell motility [10]. NPGA markedly suppressed, and LRP1 silencing abrogated, AKT activation, demonstrating that perturbation of eHsp90 signaling suppresses AKT activation and concomitant AKT mediated phosphorylation of EphA2. AKT phosphorylation can be induced by src [33], and we found that NPGA or LRP1 silencing suppressed src activation (Figure S2E). To define the potential role of src in modulating P-EphA2S897, src was silenced (Figure S2F), which dramatically suppressed both P-AKTS473 and P-EphA2S897 (Figure 2A). This suggests that eHsp90-LRP1 mediated src signaling is a prerequisite for subsequent serine phopshorylation of AKT and EphA2.

Bottom Line: Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function.We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina, United States of America.

ABSTRACT

Background: Extracellular Hsp90 protein (eHsp90) potentiates cancer cell motility and invasion through a poorly understood mechanism involving ligand mediated function with its cognate receptor LRP1. Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal brain cancers. The receptor tyrosine kinase EphA2 is overexpressed in the majority of GBM specimens and is a critical mediator of GBM invasiveness through its AKT dependent activation of EphA2 at S897 (P-EphA2(S897)). We explored whether eHsp90 may confer invasive properties to GBM via regulation of EphA2 mediated signaling.

Principal findings: We find that eHsp90 signaling is essential for sustaining AKT activation, P-EphA2(S897), lamellipodia formation, and concomitant GBM cell motility and invasion. Furthermore, eHsp90 promotes the recruitment of LRP1 to EphA2 in an AKT dependent manner. A finding supported by biochemical methodology and the dual expression of LRP1 and P-EphA2(S897) in primary and recurrent GBM tumor specimens. Moreover, hypoxia mediated facilitation of GBM motility and invasion is dependent upon eHsp90-LRP1 signaling. Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.

Significance: We herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function. We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2. Taken together, our results demonstrate activation of the eHsp90-LRP1 signaling axis as an obligate step in the initiation and maintenance of AKT signaling and EphA2 activation, thereby implicating this pathway as an integral component contributing to the aggressive nature of GBM.

Show MeSH
Related in: MedlinePlus