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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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Preservation of AKT activation is required for lamellipodia formation, and concomitant cell motility and invasion.(A) Serum starved (8 hr) G48a cells were exposed to either native or Hsp90ΔATPase proteins (3 µg/ml) for 15 min in the presence or absence of NPGA, and the indicated signaling molecules evaluated by immunoblot. The effect of these treatments upon EphA2-AKT interaction was also evaluated. (B) A Matrigel invasion assay was utilized to evaluate the ability of native or Hsp90ΔATPase proteins to sustain cell invasion in the presence of NPGA. Values represent the mean (± SD) of 3 independent experiments. (C) G48a cells stably transduced with the indicated HA-tagged myristolyated AKT constructs were exposed to NPGA and HA immunopurified complexes were evaluated for P-AKT and P-EphA2S897. (D) The pro-motility function of the indicated AKT proteins was evaluated in the presence or absence of NPGA using a scratch wound assay.
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pone-0017649-g003: Preservation of AKT activation is required for lamellipodia formation, and concomitant cell motility and invasion.(A) Serum starved (8 hr) G48a cells were exposed to either native or Hsp90ΔATPase proteins (3 µg/ml) for 15 min in the presence or absence of NPGA, and the indicated signaling molecules evaluated by immunoblot. The effect of these treatments upon EphA2-AKT interaction was also evaluated. (B) A Matrigel invasion assay was utilized to evaluate the ability of native or Hsp90ΔATPase proteins to sustain cell invasion in the presence of NPGA. Values represent the mean (± SD) of 3 independent experiments. (C) G48a cells stably transduced with the indicated HA-tagged myristolyated AKT constructs were exposed to NPGA and HA immunopurified complexes were evaluated for P-AKT and P-EphA2S897. (D) The pro-motility function of the indicated AKT proteins was evaluated in the presence or absence of NPGA using a scratch wound assay.

Mentions: To further strengthen the premise that eHsp90 signaling is essential for P-EphA2S897, we examined whether addition of Hsp90 protein was sufficient as a stimulus to activate this pathway. Addition of exogenous Hsp90α to serum starved G48a cells robustly stimulated phosphorylation of src, AKT and EphA2 (Figure 3A). The N-terminal ATPase domain of Hsp90 is dispensible for its extracellular pro-motility functions [26]. Our data support this notion, as addition of an N-terminally truncated Hsp90 protein (Δ1-235 aa) lacking its ATPase domain (Hsp90ΔATP) [26] activated P-srcY416, P-AKTS473 and P-EphA2S897 to an extent comparable to that of the wild type Hsp90α protein (Figure 3A). To validate the specificity of NPGA's effects upon eHsp90, we utilized NPGA in tandem with Hsp90ΔATP. Since the truncated protein lacks the NPGA binding pocket, this protein should not undergo a drug mediated conformational change and would therefore be expected to be resistant to the effects of drug. In support of this premise, Hsp90ΔATP rescued cells from NPGA's suppressive effects upon src, AKT and EphA2. Moreover, the ability of Hsp90ΔATP to preserve P-AKTS473 upon drug challenge further illustrates the specificity of NPGA for eHsp90, as cell permeable Hsp90 inhibitors suppress AKT activation [36]. We next examined whether the Hsp90ΔATP mediated activation of EphA2 and AKT correlated with its ability to foster association between these proteins. Hsp90ΔATP sustained EphA2-AKT complexes during challenge with NPGA (Figure 3A), and elicited a modest increase in their association, indicating that eHsp90 expression influences the magnitude of downstream signaling and augment the affinity of EphA2 for its binding partners.


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)

Preservation of AKT activation is required for lamellipodia formation, and concomitant cell motility and invasion.(A) Serum starved (8 hr) G48a cells were exposed to either native or Hsp90ΔATPase proteins (3 µg/ml) for 15 min in the presence or absence of NPGA, and the indicated signaling molecules evaluated by immunoblot. The effect of these treatments upon EphA2-AKT interaction was also evaluated. (B) A Matrigel invasion assay was utilized to evaluate the ability of native or Hsp90ΔATPase proteins to sustain cell invasion in the presence of NPGA. Values represent the mean (± SD) of 3 independent experiments. (C) G48a cells stably transduced with the indicated HA-tagged myristolyated AKT constructs were exposed to NPGA and HA immunopurified complexes were evaluated for P-AKT and P-EphA2S897. (D) The pro-motility function of the indicated AKT proteins was evaluated in the presence or absence of NPGA using a scratch wound assay.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050925&req=5

pone-0017649-g003: Preservation of AKT activation is required for lamellipodia formation, and concomitant cell motility and invasion.(A) Serum starved (8 hr) G48a cells were exposed to either native or Hsp90ΔATPase proteins (3 µg/ml) for 15 min in the presence or absence of NPGA, and the indicated signaling molecules evaluated by immunoblot. The effect of these treatments upon EphA2-AKT interaction was also evaluated. (B) A Matrigel invasion assay was utilized to evaluate the ability of native or Hsp90ΔATPase proteins to sustain cell invasion in the presence of NPGA. Values represent the mean (± SD) of 3 independent experiments. (C) G48a cells stably transduced with the indicated HA-tagged myristolyated AKT constructs were exposed to NPGA and HA immunopurified complexes were evaluated for P-AKT and P-EphA2S897. (D) The pro-motility function of the indicated AKT proteins was evaluated in the presence or absence of NPGA using a scratch wound assay.
Mentions: To further strengthen the premise that eHsp90 signaling is essential for P-EphA2S897, we examined whether addition of Hsp90 protein was sufficient as a stimulus to activate this pathway. Addition of exogenous Hsp90α to serum starved G48a cells robustly stimulated phosphorylation of src, AKT and EphA2 (Figure 3A). The N-terminal ATPase domain of Hsp90 is dispensible for its extracellular pro-motility functions [26]. Our data support this notion, as addition of an N-terminally truncated Hsp90 protein (Δ1-235 aa) lacking its ATPase domain (Hsp90ΔATP) [26] activated P-srcY416, P-AKTS473 and P-EphA2S897 to an extent comparable to that of the wild type Hsp90α protein (Figure 3A). To validate the specificity of NPGA's effects upon eHsp90, we utilized NPGA in tandem with Hsp90ΔATP. Since the truncated protein lacks the NPGA binding pocket, this protein should not undergo a drug mediated conformational change and would therefore be expected to be resistant to the effects of drug. In support of this premise, Hsp90ΔATP rescued cells from NPGA's suppressive effects upon src, AKT and EphA2. Moreover, the ability of Hsp90ΔATP to preserve P-AKTS473 upon drug challenge further illustrates the specificity of NPGA for eHsp90, as cell permeable Hsp90 inhibitors suppress AKT activation [36]. We next examined whether the Hsp90ΔATP mediated activation of EphA2 and AKT correlated with its ability to foster association between these proteins. Hsp90ΔATP sustained EphA2-AKT complexes during challenge with NPGA (Figure 3A), and elicited a modest increase in their association, indicating that eHsp90 expression influences the magnitude of downstream signaling and augment the affinity of EphA2 for its binding partners.

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