PP2A methylesterase PME-1 suppresses anoikis and is associated with therapy relapse of PTEN-deficient prostate cancers

Abstract

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.

Keywords: LAP2A/B; PP2A-C methylation; PPME1; anoikis; integrin; nuclear lamina.

Fig. 1

PME‐1 overexpression associates with PTEN loss and therapy relapse of PTEN‐negative prostate cancer patients. (A) PME‐1 protein expression in PCa tissue microarray material from 358 patients treated primarily with radical prostatectomy in the Helsinki University Hospital between 1983 and 1998 was assessed by immunohistochemical stainings. PME‐1 protein content was scored using 4‐tier scale; negative (not shown), low, intermediate and strong expression. In subsequent clinical analysis negative‐intermediate immunopositivity of PME‐1 was considered as PME‐1 Low, whereas strong PME‐1 immunopositivity was considered as PME‐1 High. Scale bar 1 mm. (B) Kaplan‐Meyer analysis of time to secondary therapies after primary treatment based on PME‐1 status alone. P‐value by log rank test. (C) Assessment of the status of PME‐1 and PTEN in the patient populations and the putative effect on PP2A activity. (D, E) Kaplan‐Meyer analysis of time to secondary therapies after primary treatment based on PME‐1 status in combination with PTEN deletion (D) and AR expression (E). P‐values by log rank test.

Fig. 2

PME‐1 promotes anchorage‐independent growth of prostate cancer cells. (A) The effect of PME‐1 depletion, using two independent siRNAs, was investigated by colony formation assays (10 days of growth) in two PTEN‐deficient PCa cells lines PC‐3 and DU‐145. (Left) Representative images of the wells. (Right) Bar graphs depicting the quantified data, mean ± SD of one (PC‐3) or two (DU‐145) independent experiments. (B) The effect of PME‐1 knock‐down on anchorage‐independent growth in soft agar assays (14 days of growth) in both PC‐3 and DU‐145 cells. (Left) Representative images depicting the colonies. (Right) Bar graphs displaying the quantified data, mean ± SD of two independent experiments. *P < 0.05, Welch's t‐test.

Fig. 3

PME‐1 promotes anoikis resistance in PTEN‐deficient prostate cancer cells. (A) siCtrl‐ and siPME‐1‐transfected PC‐3 cells were plated 72 h post‐transfection on normal cell culture or low‐attachment plates for 24 h, before collection and lysis, and subsequently analyzed by western blotting for cleaved PARP‐1 (cPARP (p25)). Shown is a representative result from six independent experiments. (B) Apoptosis induction, as measured by PARP cleavage, in CRISPR/Cas9 generated PC‐3 cells, with and without non‐targeting gRNA (lanes 1–3), a pool of PME‐1 gRNA transfected cells (lane 4), and a single cell subclone of PME‐1 targeted cells (lane 5), after 24 h detachment. Shown is a representative result from three independent experiments. (C) The effect of stable PME‐1 overexpression in mouse embryonic fibroblasts (MEFs) from a PTEN/p53KO mouse model, after 24 h of detachment, was assessed by western blotting for cleaved PARP. Shown is a representative result from two independent experiments. (D) Mechanosensitive PARP cleavage in PME‐1‐depleted PC‐3 cells. The cells were cultured on soft (0.5 kPa) or stiff (50 kPa) polyacrylamide hydrogel for 24 h before being scraped into PBS, spun down, lyzed and analyzed for protein expression. Shown is a representative result from four independent experiments. (E) The number of Annexin V positive objects, a marker for apoptosis, in cultures of siCtrl‐ and siPME‐1‐transfected PC‐3 cells upon varying degrees of cell‐matrix interaction. The cells were grown on PEG (Poly(ethylene glycol))‐coated surfaces with 0–20% biotinylated compound bound to integrin ligand, fibronectin fragment FNIII(7–10). STS = staurosporine. Mean ± SD of three independent experiments. (F) Annexin V positive objects in PC‐3 cultures with no or high levels of integrin ligand, after 60 h of incubation. Mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one‐way ANOVA with Bonferroni's multiple‐comparison test.

Fig. 4

PME‐1 supports in vivo anoikis resistance and survival of prostate cancer cells in circulation. (A) The effect of PME‐1 on anchorage‐independent growth of PC‐3 cells on chick chorioallantoic membrane (CAM). PC‐3 cells were transiently transfected with either control siRNA (siCtrl) or PME‐1‐targeting siRNA (siPME‐1), and 24 h post‐transfection placed on the CAM. Growth of tumours was followed for 3–5 days. Shown are representative examples from three replicate experiments. (B) Immunohistological staining of dissected tumours using antibodies for PME‐1, Vimentin, TUNEL and Ki67. Shown are representative images from three (Ki67) or two (TUNEL) replicate experiments. Scale bar 50 μm for PME‐1 and Vimentin stainings and 100 μm for TUNEL and Ki67. (C) Quantification of TUNEL‐ and Ki‐67‐positive nuclei in the excised CAM tumours. Mean ± SD from a representative experiment with four eggs per condition, *P < 0.05, Mann–Whitney test. (D) The survival of siCtrl‐ and siPME‐1‐transfected PC‐3 cells in circulation. The cells were microinjected into the common cardinal veins of zebrafish embryos 72 h post‐transfection. After overnight incubation the embryos were imaged by fluorescence stereomicroscopy. Scale bar 500 μm. (E) The number of surviving fluorescent tumour cells per embryo from (D). Box plots depicting the range, 25th, 50th and 75th percentiles of the data overlaid with the individual data points combined from three independent experiments. **P < 0.01, Mann–Whitney test.

Fig. 5

The potential mechanisms for PME‐1‐mediated anoikis resistance. (A) Immunofluorescence images (left) and quantification (right) depicting phosphorylated FAK (Y397) and early endosomal marker EEA1 in control and PME‐1‐depleted PC‐3 cells grown on soft (0.5 kPa) hydrogels for 24 h. Scale bar, 10 μm (main), 2 μm (inset). Mean ± SD from a representative of two independent experiments. (B) Fluorescence images depicting filamentous actin in control and PME‐1 KO PC‐3 cells treated with DMSO or increasing concentrations of Cytochalasin D (0.1–10 μm). Scale bar, 20 μm. Shown is a representative of two independent experiments. (C) Western blot (top) and quantification (bottom) displaying PARP cleavage in PC‐3 cells upon actin cytoskeleton disruption with Cytochalasin D. Mean ± SD of two independent experiments. (D, E) Images (D) and quantification (E) showing nuclear deformation in PC‐3 cells subjected to micropipette aspiration, as described in [46], over the course of 2 min. Mean ± SD of three independent experiments. *P < 0.05, Welch's t‐test. (F, G) Quantification of GFP‐NLS‐positive cells with cytoplasmic GFP signal (F) and the percentage of transfected cells with visible cGAS‐mCherry foci (G), respectively, in siCtrl‐ and siPME‐1‐treated cells grown on soft (0.5 kPa) substrate. Both readouts serve as a surrogate measure of compromised nuclear envelope. Mean ± SD of two independent experiments. **P < 0.01, Welch's t‐test (H–I). Immunofluorescence images and quantification depicting H3K9me3 (H) and H3K27me3 (I) levels in transiently PME‐1‐depleted and control PC‐3 cells, 48 h post‐transfection and 24 h after seeding on soft (0.5 kPa) substrate. Scale bar, 10 μm. Mean ± SD, representative of two independent experiments. ***P < 0.001, Mann–Whitney test. (J) Schematic representation of a putative model for including PME‐1 and its role in anoikis sensitivity/resistance in PCa diagnostics and therapy decision.