PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55α/PP2A reconstitution that triggers centrosome destabilization

Abstract

The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.

Fig. 1. PPP2R2A DNA copy number and mRNA expression are reduced in prostate tumors and this correlates with worse tumor stage and poorer prognosis.

a TCGA and SU2C database mining for DNA copy number in PCa. b, c Oncoprints show increased frequency of PPP2R2A hemizygous loss with higher AJCC tumor stage (TCGA data) and prostate cancer metastases (SU2C). See legend for genetic alterations. d Loss of PPP2R2A gene copy number correlates with poorer prognosis of PCa patients (p = 0.0466). e cBioportal analysis from TCGA PanCancer Atlas PCa data: PPP2R2A gene copy number alterations are associated with decreased mRNA expression in matched prostate tumors (p = 6.6814E−11, t-test). The same association is not seen with NKX3-1 (p = 0.739622967). f B55α expression is scored medium in normal prostate as compared to other tissues (see Suppl. Fig. 1G). B55α expression is low or negative in more than 60% of prostate tumors (Tissues were stained with anti-B55α (100C1) rabbit mAb).

Fig. 2. Complete loss of PPP2R2A reduces proliferation by slowing cell cycle progression.

a B55α is not expressed in VCaP cells and is expressed at relatively low levels in PC3 and DU145 cells. Expression of B55α, PP2A/C and PP2A/A was determined by western blot. b, c Elimination of the remaining allele of PPP2R2A in PC3 cells via CRISPR knockout does not increase proliferation or transformation potential. b Western blot showing expression of B55α, PP2A/C and PP2A/A in PC3 wild-type, PPP2R2A-KO clones A6 and B9 and a control clone (B7) that did not exhibit deletion of PPP2R2A. Clonogenic assay and corresponding histogram showing colony count of these cell lines in triplicate. c Anchorage independent growth assay and corresponding histogram showing colony count of these cell lines in triplicate. Data represent mean ± SD. *p < 0.05; **p < 0.01; ns p > 0.05, t-test. d PC3 wild-type and PPP2R2A-KO clones A6 and B9 were arrested in G1 with palbociclib, released and allowed to progress to mitosis in the presence of nocodazole, collecting cells at times indicated in the timeline scheme. Cell cycle progression was analyzed by FACS/propidium iodide staining and the expression of indicated cyclins determined by immunoblot with juxtaposing images. e PC3 wild-type and PPP2R2A-KO clones A6 and B9 were synchronized via thymidine block followed by RO3306 treatment, released and collected at times indicated in the timeline scheme. Cell cycle progression was analyzed by FACS/phospho-Histone H3 (Ser10)/propidium iodide staining and cyclin B1 expression was determined by immunoblot. f Prostate Adenocarcinomas (TCGA Provisional) with hemizygous but not homozygous PPP2R2A alterations exhibit upregulation of many cell cycle genes as determined via STRING analysis. Experiments shown are representative of two independent experiments unless indicated.

Fig. 3. Reconstitution of B55α is toxic in PC3 and DU145 cells and inhibits transformation and tumorigenicity in SCID mice by inducing mitotic arrest.

a-d Limited ectopic expression of B55α via lentiviral transduction a blocks proliferation (green dashed line shows end of Puromycin selection), b induces cell death and apoptosis, c results in a senescence-like flat cell morphology with large nuclei, and d suppresses tumor xenograft growth in SCID mice (mean tumor size 56 days post injection was 2117.37 +/− 462.14 mm³ [mean + /− SEM] in untreated cells and not detectable for Dox-treated cells). e Limited ectopic expression of B55α causes G2/M arrest and euploidy (PI/BrdU staining). PC3 cells are near triploid. G2/M = triploid G2/M cell population. Eu G1 = hexaploid G1 cells. Eu G2/M = hexaploid G2/M cells. f–h Inducible expression of B55α in PC3 and DU145 but not immortalized normal BJ-hTERT cells induces mitotic arrest. Dox inducible F-B55α PC-3 (f), DU145 (g), and BJ-hTERT cells (h) were generated by transduction with lentiviral Tet-on vectors and selected with zeocin or puromycin in tetracycline-free media. f–h Cells were induced with Dox and collected at the indicated times. DNA content was determined by PI/FACS analysis in at least two independent experiments. Expression of Flag-B55α, B55α, cyclin B1 and loading controls was determined via western blot analysis, quantitated and represented by histogram. Western blot analyses are representative of at least two independent experiments.

Fig. 4. Reconstitution of B55α in PC3 and DU145 iB55α cells blocks mitotic exit as a result of extended mitotic checkpoint and chromosome segregation failure.

a, b PC3 iB55α cells were synchronized by Nocodazole, shaken off, and reseeded as scheme shown. a B55α reconstitution slowed cell cycle progression through G2/M. b Expression of F-B55α, B55α, cyclin B1 and the indicated proteins was determined via western blot analysis. c, d Mitotic defects resulting from B55α reconstitution in DU145-iB55α-EGFP-H2B cells were determined by live imaging using confocal microscopy. Three fields of each treatment were imaged in at least three independent experiments. c Untreated cells went through normal mitosis in about 60 min. d Dox induced B55α caused an extended metaphase checkpoint followed by chromosome segregation failure and apoptosis. e Co-expression of EGFP-H2B and mRFP-α-tubulin in DU145-iB55α cells allowed visualization of bipolar spindles prior to centrosome collapse when B55α is reconstituted. Lens: ×20. f DU145-iB55α cells were synchronized by nocodazole as in a, reseeded and collected at the indicated time points. Cell cycle analysis was determined via PI/FACS in at least two independent experiments.

Fig. 5. Mitotic defects in B55α-reconstituted cells are linked to dephosphorylation of centrosomal proteins.

a, b SILAC/Phosphoproteome analysis was performed with PC3-iB55α cells treated with or without Dox. The consensus amino acid sequence of the top downregulated phosphopeptides was analyzed with (a) Icelogo and (b) the KEA2 algorithm, which predicts potential upstream kinases. c Ingenuity pathway analysis of phosphoproteome data shows that B55α reconstitution in PC3 cells reduces phosphorylation of multiple centriolar and PCM proteins. The cartoon displays the distribution of centriolar and PCM proteins. d Western blot analysis showed that B55α reconstitution in DU145 cells prevents phosphorylation of HAUS6 and NEDD1 in late G2/early mitosis (differently migrating protein species are indicated). e Purified GST, GST-HAUS6, GST-NEDD1, GST-CEP170 were incubated with 293T lysate and pull-downs analyzed by western blot for B55α. f Purified GST-HAUS6 was phosphorylated with PLK1 and/or CDK1/CYCLIN B in presence of γ-³²P-ATP and dephosphorylated with purified trimeric B55α/PP2A complex (Suppl. Fig. 4B). Samples were resolved by SDS-PAGE, stained with Coomassie blue (lower panel) and analyzed by autoradiography (upper panel). Western blot and kinase/phosphatase analyses are representative of at least two independent experiments.

Fig. 6. B55α reconstitution in DU145 cells promotes numerical centrosomal aberrations via PCM fragmentation and centriole overduplication.

Immunofluorescent co-staining of (a) Pericentrin, (b) CDK5RAP2, or (c) Centrin1 with α-tubulin in control and Dox induced DU145-iB55α cells using specific antibodies. The number of (a) Pericentrin, (b) CDK5RAP2, and (c) Centrin1 bodies per cell were counted and expressed as bar graphs (on the right). Control cells show one Pericentrin, CDK5RAP2 or Centrin foci in interphase (G1/S/G2) (left panels). Dox-induced B55α expression results are consistent with centrosome overduplication, PCM fragmentation and disrupted spindles (right panels). DAPI was used to stain chromatin. Lens: ×63. d Diagram representing different mechanisms of centrosome disruption.

Fig. 7. Pharmacological inhibition of the G2/M transition, centrosomal separation, cyclin B degradation or metaphase checkpoint activation in B55α reconstituted cells indicate that dephosphorylation of mitotic substrates leads to centrosomal amplification and/or weakening, causing its disruption following extended checkpoints.

a (right panels) Images were taken after 20 h treatment of BI2536, RO-3306 or reversine, respectively, on thymidine synchronized DU145-iB55α-EGFP-H2B cells with or without Dox treatment, as the timeline scheme shown in upper left panel. Cells were counted and scored to the specified categories according to their chromatin state (Histone H2B-EGFP) (lower left panel) Lens: ×20. b Western blot analysis of cell lysates prepared from cells treated as in Fig. 7a. c DNA content was determined in cells treated as in Fig. 7a by PI/FACS analysis (upper panel) and immunofluorescence staining with DAPI, CDK5RAP2 and acetylated-α-tubulin (lower panel). d, e As shown in the timeline scheme, thymidine-nocodazole synchronized DU145-iB55α-EGFP-H2B cells treated (d) without or (e) with Dox were treated with 10 µM MG132 and images were taken at 0, 4, and 8 h respectively after treatment (middle panels). Lens: ×20. Chromatin states were scored via EGFP-H2B microscopic visualization, counted and represented as bar graphs (right panels). Cells collected at indicated times were also analyzed with PI/FACS (left panel).