Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer
Chaoyang Li, … , Wei Xin, Man-Sun Sy
Chaoyang Li, … , Wei Xin, Man-Sun Sy
Published August 17, 2009
Citation Information: J Clin Invest. 2009;119(9):2725-2736. https://doi.org/10.1172/JCI39542.
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Research Article Oncology

Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer

Abstract

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A–binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.

Authors

Chaoyang Li, Shuiliang Yu, Fumihiko Nakamura, Shaoman Yin, Jinghua Xu, Amber A. Petrolla, Neena Singh, Alan Tartakoff, Derek W. Abbott, Wei Xin, Man-Sun Sy

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Figure 3

FLNa binds to the GPI-PSS of pro-PrP.

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FLNa binds to the GPI-PSS of pro-PrP. (A) A silver-stained gel shows a b...
(A) A silver-stained gel shows a band with MW of 280 kDa (*) is coimmunoprecipitated with anti-PrP mAb 8B4 but not with control mAb D7C7. (B) Immunoblots show the copurification of FLNa with PrP and vice versa. (C) Confocal microscopic images show colocalization of FLNa (green) and PrP (red) in PDAC cell lines. Arrows show area of colocalization. Original magnification, ×1,000. (D) Immunoblots show PrP and FLNa are present in similar fractions after centrifugation in sucrose gradient. (E) An in vitro pull-down experiment shows much stronger binding of full-length FLNa1–24 to a PrP GPI-PSS GST fusion protein than to a GST protein without the GPI-PSS. A FLNa1–23 monomer did not bind PrP GPI-PSS GST fusion protein. Immune complexes were pulled down with GST binding beads and immunoblotted with an anti-FLAG mAb to detect FLNa. (F) Immunoblots show binding of FLNa1–24 to recombinant pro-PrP23–253 but not mature recombinant PrP23–231. Anti-PrP mAb 8H4 was used to pull down the immune complexes. The immunoblot was done either with an anti-Flag mAb or anti-PrP mAb 8H4. (G) Immunoblots show competition of binding of FLNa to pro-PrP by a PrP-GPI-PSS synthetic peptide. Copurification of PrP and FLNa in the PDAC cell lysates was carried out in the presence of different concentrations of either a synthetic peptide corresponding to the GPI-PSS or a control synthetic peptide. Anti-PrP mAb 8B4 coimmunoprecipitated proteins were then immunoblotted with an anti-FLNa mAb. Con, control peptide.