Prostate-specific membrane antigen associates with anaphase-promoting complex and induces chromosomal instability

Abstract

Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathologic consequence of elevated PSMA expression in not known. Here, we report that PSMA is localized to a membrane compartment in the vicinity of mitotic spindle poles and associates with the anaphase-promoting complex (APC). PSMA-expressing cells prematurely degrade cyclin B and exit mitosis due to increased APC activity and incomplete inactivation of APC by the spindle assembly checkpoint. Further, expression of PSMA in a karyotypically stable cell line induces aneuploidy. Thus, these findings provide the first evidence that PSMA has a causal role in the induction of aneuploidy and might play an etiologic role in the progression of prostate cancer.

Figure 1

Localization of PSMA at the mitotic spindle poles. Note distinct colocalization of PSMA (green) with centrosomes (red) at the mitotic spindle poles in PC3-PSMA, MDCK-PSMA-1, and MDCK-PSMA-Δ103-750 cells but not in MDCK-PSMA-ΔCD cells. Scale bars, 8 μm. PSMA, prostate-specific membrane antigen.

Figure 2

Immunoelectron microscopy showing PSMA localization around the centrosomes. A, Shows a cartoon of the electron micrograph shown in B. A, B, The arrows indicate centriolar cylinders and the asterisks indicate the gold particles (10 nm) decorating PSMA. One of the spindle poles is shown in this image. The condensed chromatin from metaphase chromosomes is indicated by an arrowhead in this mitotic cell. Insets in B are magnified images of regions corresponding to arrows in B. In C the magnification facilitates visualization of gold particles on membrane like structures indicated by arrows. Bars in A-C, 0.5 μm. PSMA, prostate-specific membrane antigen.

Figure 3

Analysis of the cell cycle progression in PSMA expressing cells after release of the mitotic block. Cells were blocked in mitosis as described in Materials and Methods. The mitotic block was released for 60, 90, and 120 minutes for PC3 clones (A, B) and 2, 4, and 8 hours for MDCK clones (C-G) and analyzed for cell cycle progression. Profiles shown are representative of the data shown in Table 1.

Figure 4

Cyclin B₁ levels and APC activation and association with PSMA. A, Immunoblot analysis of cyclin B₁ in PC3 and PC3-PSMA cells after release of the mitotic block. Data represent the means ± SD of two independent experiments. B, In vitro APC ubiquitin ligase activity assay in PC3 and PC3-PSMA cells after 60 and 120 min release from nocodazole block.

Figure 5

Association of PSMA with APC. A, Immunoblot analysis of Mad2, BubR1, Cdc20, and Cdc27 in interphase (I) and Mitotic (M) cells. B, Co-immunoprecipitation of Mad2, BubR1, and Cdc20 with Cdc27 in PC3 and PC3-PSMA cells. Equal amounts of Cdc 27 used in the immunoprecipitation (IP) was confirmed by immunoblotting. C, Co-immunoprecipitation of PSMA with Cdc27 (left top) and Cdc27 co-immunoprecipitating PSMA (left bottom) in PC3-PSMA and MDCK-PSMA cells. Note Cdc27 does not co-immunoprecipitate with PSMA-ΔCD. Affinity precipitation of Cdc27 by GST-PSMA from PC3 cell lysate (right). D, Co-immunoprecipitation of Cdc27 with PSMA in prostate cancer tissues (top). PSMA levels in same tissues are shown (bottom).

Figure 6

PSMA expressing human cells are genetically unstable. HCT-PSMA and HCT-GFP cells were analyzed by multicolor FISH with Chromosome 3 labeled with Spectrum Red (CEP3), Chromosome 7 with Spectrum Green (CEP7), Chromosome 17 with Spectrum Aqua (CEP 17), and 9p21 region (p16 gene) with Spectrum Gold. Control HCT-GFP cells at passage 45 (A), and HCT-PSMA cells at passage 2 (B), are diploid whereas HCT-PSMA cells at passage 15 (C), 34 (D), and 45 (E) show aneuploidy. Micronucleus formation (E, arrow and F) and abnormal metaphase, (G) in HCT-PSMA cells at passage 45 are shown.