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. 2020 Nov:93:10-24.
doi: 10.1016/j.matbio.2020.03.009. Epub 2020 May 15.

Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

Simone Buraschi  1 , Thomas Neill  1 , Shi-Qiong Xu  2 , Chiara Palladino  2 , Antonino Belfiore  3 , Renato V Iozzo  4 , Andrea Morrione  5
Affiliations

Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

Simone Buraschi et al. Matrix Biol. 2020 Nov.

Abstract

The growth factor progranulin plays a critical role in bladder cancer by modulating tumor cell motility and invasion. Progranulin regulates remodeling of the actin cytoskeleton by interacting with drebrin, an actin binding protein that regulates tumor growth. We previously discovered that progranulin depletion inhibits epithelial-to-mesenchymal transition and markedly reduces in vivo tumor growth. Moreover, progranulin depletion sensitizes urothelial cancer cells to cisplatin treatment, further substantiating a pro-survival function of progranulin. Until recently, the progranulin signaling receptor remained unidentified, precluding a full understanding of progranulin action in tumor cell biology. We recently identified EphA2, a member of a large family of receptor tyrosine-kinases, as the functional receptor for progranulin. However, it is not established whether EphA2 plays an oncogenic role in bladder cancer. Here we demonstrate that progranulin, and not ephrin-A1, the canonical ligand for EphA2, is the predominant EphA2 ligand in bladder cancer. Progranulin evoked Akt- and Erk1/2-mediated EphA2 phosphorylation at Ser897, which could drive bladder tumorigenesis. We discovered that EphA2 depletion severely blunted progranulin-dependent motility and anchorage-independent growth, and sensitized bladder cancer cells to cisplatin treatment. We further defined the mechanisms of progranulin/EphA2-dependent motility by identifying liprin-α1 as a novel progranulin-dependent EphA2 interacting protein and establishing its critical role in cell motility. The discovery of EphA2 as the functional signaling receptor for progranulin and the identification of novel downstream effectors offer a new avenue for understanding the underlying mechanism of progranulin action and may constitute novel clinical and therapeutic targets in bladder cancer.

Keywords: Bladder cancer; EphA2; Liprinα-1; Motility; Progranulin.

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Conflict of interest statement

Disclosure

The authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.
Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.
Fig. 2.
Fig. 2.
Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.
Fig. 3.
Fig. 3.
EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.
Fig. 4.
Fig. 4.
EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.
Fig. 5.
Fig. 5.
Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.
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