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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 regulates EphA2 dependent motility and invasion in GBM.Interference with eHsp90-LRP1 signaling inhibits GBM cell motility and invasion. (A, B) Treatment of cells with NPGA (1 µM) or anti-Hsp90 antibodies (20 ug/ml) (A), or suppression of LRP1 (B) similarly impaired G48a cell motility in wound healing assays. NS shRNA represents a nonspecifc shRNA control sequence. Percent migration is normalized to the 16 hr control and values represent the mean ± SD from 3 independent experiments (*p<0.001). (C) Serum starved parental or LRP1 silenced cells were added to top chambers of a Boyden assay and serum induced chemotaxis initiated in the presence of vehicle or NPGA. Cell numbers represent the mean ± SD from five random fields (*p<0.001). (D) The effects of eHsp90 targeting upon cell invasion were assessed by a Matrigel assay in the presence or absence of NPGA. Data is represented as the mean (± SD) of three replicates. *p<0.001. (E–F) Interference with eHsp90 function does not further inhibit cell motility or invasion in tandem with EphA2 silencing. G48a cells were transduced with either nonspecific (NS shRNA) or shEphA2 and effects of NPGA upon cell motility assessed in Boyden (right panel), and invasion assessed by Matrigel (left panel).
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pone-0017649-g001: eHsp90-LRP1 regulates EphA2 dependent motility and invasion in GBM.Interference with eHsp90-LRP1 signaling inhibits GBM cell motility and invasion. (A, B) Treatment of cells with NPGA (1 µM) or anti-Hsp90 antibodies (20 ug/ml) (A), or suppression of LRP1 (B) similarly impaired G48a cell motility in wound healing assays. NS shRNA represents a nonspecifc shRNA control sequence. Percent migration is normalized to the 16 hr control and values represent the mean ± SD from 3 independent experiments (*p<0.001). (C) Serum starved parental or LRP1 silenced cells were added to top chambers of a Boyden assay and serum induced chemotaxis initiated in the presence of vehicle or NPGA. Cell numbers represent the mean ± SD from five random fields (*p<0.001). (D) The effects of eHsp90 targeting upon cell invasion were assessed by a Matrigel assay in the presence or absence of NPGA. Data is represented as the mean (± SD) of three replicates. *p<0.001. (E–F) Interference with eHsp90 function does not further inhibit cell motility or invasion in tandem with EphA2 silencing. G48a cells were transduced with either nonspecific (NS shRNA) or shEphA2 and effects of NPGA upon cell motility assessed in Boyden (right panel), and invasion assessed by Matrigel (left panel).

Mentions: Despite the emerging role for eHsp90 in cancer development, nothing is known about its potential function in GBM. To investigate whether eHsp90 supports GBM aggressiveness, two approaches were utilized to block eHsp90 function. Treatment with DMAG-N-oxide, a non-permeable GA (NPGA) derivative specific for eHsp90 [21], [29] potently inhibited G48a cell motility (70%) (Figure 1A). Alternatively, antibody mediated neutralization of eHsp90 function [21], [22], [30] similarly inhibited motility. These effects were reproducible in other GBM cell lines (data not shown) and highlight a pivotal role for eHsp90 function in supporting GBM motility. eHsp90 signaling is transduced via the multifunctional LRP1 receptor [26], [27] and LRP1 has been implicated in GBM cell motility and invasion [31]. LRP1 silencing [26], (Figure S1A) blocked GBM cell motility in a manner similar to NPGA or Hsp90 antibody treatments, with no further suppression elicited by NPGA (Figure 1B). These results indicate that the pro-motility function of eHsp90 in GBM is mediated through LRP1, a conclusion further strengthened by similar trends obtained with Boyden motility and Matrigel invasion assays (Figure 1C, D, and Figure S1B, C).


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 regulates EphA2 dependent motility and invasion in GBM.Interference with eHsp90-LRP1 signaling inhibits GBM cell motility and invasion. (A, B) Treatment of cells with NPGA (1 µM) or anti-Hsp90 antibodies (20 ug/ml) (A), or suppression of LRP1 (B) similarly impaired G48a cell motility in wound healing assays. NS shRNA represents a nonspecifc shRNA control sequence. Percent migration is normalized to the 16 hr control and values represent the mean ± SD from 3 independent experiments (*p<0.001). (C) Serum starved parental or LRP1 silenced cells were added to top chambers of a Boyden assay and serum induced chemotaxis initiated in the presence of vehicle or NPGA. Cell numbers represent the mean ± SD from five random fields (*p<0.001). (D) The effects of eHsp90 targeting upon cell invasion were assessed by a Matrigel assay in the presence or absence of NPGA. Data is represented as the mean (± SD) of three replicates. *p<0.001. (E–F) Interference with eHsp90 function does not further inhibit cell motility or invasion in tandem with EphA2 silencing. G48a cells were transduced with either nonspecific (NS shRNA) or shEphA2 and effects of NPGA upon cell motility assessed in Boyden (right panel), and invasion assessed by Matrigel (left panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050925&req=5

pone-0017649-g001: eHsp90-LRP1 regulates EphA2 dependent motility and invasion in GBM.Interference with eHsp90-LRP1 signaling inhibits GBM cell motility and invasion. (A, B) Treatment of cells with NPGA (1 µM) or anti-Hsp90 antibodies (20 ug/ml) (A), or suppression of LRP1 (B) similarly impaired G48a cell motility in wound healing assays. NS shRNA represents a nonspecifc shRNA control sequence. Percent migration is normalized to the 16 hr control and values represent the mean ± SD from 3 independent experiments (*p<0.001). (C) Serum starved parental or LRP1 silenced cells were added to top chambers of a Boyden assay and serum induced chemotaxis initiated in the presence of vehicle or NPGA. Cell numbers represent the mean ± SD from five random fields (*p<0.001). (D) The effects of eHsp90 targeting upon cell invasion were assessed by a Matrigel assay in the presence or absence of NPGA. Data is represented as the mean (± SD) of three replicates. *p<0.001. (E–F) Interference with eHsp90 function does not further inhibit cell motility or invasion in tandem with EphA2 silencing. G48a cells were transduced with either nonspecific (NS shRNA) or shEphA2 and effects of NPGA upon cell motility assessed in Boyden (right panel), and invasion assessed by Matrigel (left panel).
Mentions: Despite the emerging role for eHsp90 in cancer development, nothing is known about its potential function in GBM. To investigate whether eHsp90 supports GBM aggressiveness, two approaches were utilized to block eHsp90 function. Treatment with DMAG-N-oxide, a non-permeable GA (NPGA) derivative specific for eHsp90 [21], [29] potently inhibited G48a cell motility (70%) (Figure 1A). Alternatively, antibody mediated neutralization of eHsp90 function [21], [22], [30] similarly inhibited motility. These effects were reproducible in other GBM cell lines (data not shown) and highlight a pivotal role for eHsp90 function in supporting GBM motility. eHsp90 signaling is transduced via the multifunctional LRP1 receptor [26], [27] and LRP1 has been implicated in GBM cell motility and invasion [31]. LRP1 silencing [26], (Figure S1A) blocked GBM cell motility in a manner similar to NPGA or Hsp90 antibody treatments, with no further suppression elicited by NPGA (Figure 1B). These results indicate that the pro-motility function of eHsp90 in GBM is mediated through LRP1, a conclusion further strengthened by similar trends obtained with Boyden motility and Matrigel invasion assays (Figure 1C, D, and Figure S1B, C).

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