Non-canonical EphA2 activation underpins PTEN-mediated metastatic migration and poor clinical outcome in prostate cancer

Abstract

Background: The key process of mesenchymal to amoeboid transition (MAT), which enables prostate cancer (PCa) transendothelial migration and subsequent development of metastases in red bone marrow stroma, is driven by phosphorylation of EphA2^S897 by pAkt, which is induced by the omega-6 polyunsaturated fatty acid arachidonic acid. Here we investigate the influence of EphA2 signalling in PCa progression and long-term survival.

Methods: The mechanisms underpinning metastatic biopotential of altered EphA2 signalling in relation to PTEN status were assessed in vitro using canonical (EphA2^D739N) and non-canonical (EphA2^S897G) PC3-M mutants, interrogation of publicly available PTEN-stratified databases and clinical validation using a PCa TMA (n = 177) with long-term follow-up data. Spatial heterogeneity of EphA2 was assessed using a radical prostatectomy cohort (n = 67).

Results: Non-canonical EphA2 signalling via pEphA2^S897 is required for PCa transendothelial invasion of bone marrow endothelium. High expression of EphA2 or pEphA2^S897 in a PTEN^low background is associated with poor overall survival. Expression of EphA2, pEphA2^S897 and the associated MAT marker pMLC2 are spatially regulated with the highest levels found within lesion areas within 500 µm of the prostate margin.

Conclusion: EphA2 MAT-related signalling confers transendothelial invasion. This is associated with a substantially worse prognosis in PTEN-deficient PCa.

Fig. 1. Upregulated Ephrin signalling in PTEN-deficient PCa correlates with poor disease-free survival.

a cBioportal oncoprints of upregulated Ephrin signalling and PTEN status in TCGA-PRAD (n = 494); and MSKCC prostate cancer cohorts (n = 240). b Kaplan–Meier disease-free survival analysis for patients with available survival data in TCGA-PRAD (n = 334) and MSKCC (n = 112) cohorts stratified by either Ephrin signalling alone or in combination with PTEN status.

Fig. 2. Arachidonic acid-induced transendothelial invasion requires pAkt dependent non-canonical pEphA2^S897 signalling.

a Real-time invasion assay towards a 20 µM AA substrate at 37 °C 5% CO₂ through a Matrigel and BMEC barrier. b Diagrammatic representation of the non-canonical pathway initiated by arachidonic acid. c Western blot images showing temporal alteration in expression of total pEphA2^S897, pEphA2^Y772, EphA2, pAkt^S473 and Akt after stimulation with 20 µM AA. d Temporal expression of pEphA2^Y772 (canonical signalling) and pEphA2^S897 (non-canonical signalling) in PTEN⁻ and PTEN⁺ PC3 cells after stimulation with 20 µM AA. e Endpoint invasion assay measuring invasion of wild-type PC3-PTEN⁻ or PC3-PTEN⁺ cells through a Matrigel and BMEC barrier towards 20 µM AA. Fluorescence was captured after 18-h incubation at 37 °C, 5% CO₂.

Fig. 3. EphA2^hi and pEphA2^S897hi status are associated with poor overall survival in an unselected PCa TMA cohort.

a Composite multiplex fluorescence images of representative TMA cores based upon EphA2 (red), pEphA2^S897 (yellow) and PTEN (green) expression status. b Kaplan–Meier overall survival curves stratified by either EphA2 status alone or EphA2 and PTEN status. c Kaplan–Meier overall survival curves stratified by either pEphA2^S897 status alone or pEphA2^S897 and PTEN status. The black dotted line represents median survival. P value denotes the result of the log-rank test.

Fig. 4. EphA2, pEphA2^S897 and pMLC2 expression are spatially regulated within tumour lesions.

a Representative multispectral image of a radical prostatectomy section stained for PTEN (green), EphA2 (red), pEphA2^S897 (gold), pMLC2 (cyan), Cytokeratin (white) and DAPI (blue). Zone x, y and z are serial 500-µm wide zones from the prostate margin. A region of normal-adjacent glandular architecture (n) has been defined by the dotted white line. b Scaled intensity (95% confidence interval) of marker expression across the tissue section depicted in a. c Scaled intensity marker expression within each zone for PTEN, EphA2, pEphA2^S897 and pMLC2 for the radical prostate cohort (n = 67). P values denote the result of Kruskal–Wallis test.