Tumor Biol. (2015) 36:6067–6074 DOI 10.1007/s13277-015-3284-7
RESEARCH ARTICLE
Cancerous inhibitor of protein phosphatase 2A promotes premature chromosome segregation and aneuploidy in prostate cancer cells through association with shugoshin Rajash Pallai & Aishwarya Bhaskar & Natalie Barnett-Bernodat & Christina Gallo-Ebert & Joseph T. Nickels Jr. & Lyndi M. Rice
Received: 13 November 2014 / Accepted: 18 February 2015 / Published online: 4 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Yeast two-hybrid (Y2H) studies have shown that cancerous Inhibitor of protein phosphatase 2A (CIP2A) interacted with several proteins, including leucine-rich repeat-containing protein 59 (LRRC59), suggesting that CIP2A may interact with the chromosome maintenance protein, shugoshin (Sgol1). We previously showed that LRRC59 interacted with CIP2A, which was required for CIP2A nuclear localization. Thus, we predicted that CIP2A and Sgol1 may also interact. Sgol1 is a nuclear protein that regulates chromosome segregation during cell division via protection of cohesin ring proteins. Here, we demonstrated that Sgol1 and the C-terminus of CIP2A interact in prostate carcinoma cell lines in a protein phosphatase 2A (PP2A)-dependent manner. Moreover, we demonstrated that depletion of CIP2A in PC-3 cells decreases premature chromosome segregation, whereas overexpression of CIP2A in an immortalized prostate cell line increases premature chromosome segregation. Importantly,
Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3284-7) contains supplementary material, which is available to authorized users. R. Pallai : A. Bhaskar : L. M. Rice (*) Oncoveda, Cancer Signaling and Cell Cycle Team, Medical Diagnostic Laboratories, LLC, Genesis Biotechnology Group, LLC, 1000 Waterview Drive, Hamilton, NJ 08691, USA e-mail:
[email protected] N. Barnett-Bernodat : C. Gallo-Ebert : J. T. Nickels Jr. Institute of Metabolic Disorders, Genesis Biotechnology Group, LLC, 1000 Waterview Drive, Hamilton, NJ 08691, USA Present Address: N. Barnett-Bernodat Femeris, Medical Diagnostic Laboratories, LLC, Genesis Biotechnology Group, LLC, 2349 Kuser Road, Hamilton, NJ 08690, USA
we further showed that CIP2A depletion decreases the incidence of aneuploidy and stabilizes cohesin complex proteins, while overexpression of CIP2A destabilizes Sgol1. Thus, our findings strongly suggest that CIP2A promotes cell cycle progression, premature chromosome segregation, and aneuploidy, possibly through a novel interaction with Sgol1. Keywords CIP2A . Shugoshin . LRRC59 . PP2A . Prostate cancer . Aneuploidy
Introduction Protein phosphatase 2A (PP2A) is a heterotrimeric serine/ threonine phosphatase implicated in several tumor suppressor roles (see reviews [1–3]). Cancerous inhibitor of PP2A (CIP2A) was first identified to inhibit PP2A and promote cell proliferation through stabilization of c-Myc [4]. CIP2A is primarily a cytoplasmic protein and is overexpressed in a variety of cancers, including prostate cancer [5–7]; high expression correlates with poor prognosis [8–10]. Interestingly, depletion of CIP2A has recently been suggested to sensitize castrationresistant prostate cancer to cabazitaxel [5]. Previous work has led to the identification of novel protein partners [11, 12] that suggested CIP2A acts in a PP2A-independent manner within the nucleus. We have described a novel protein interaction between leucine-rich repeat-containing protein 59 (LRRC59) and CIP2A (manuscript submitted); this interaction is required for CIP2A to enter the nucleus. Cancer exhibits higher proliferation rates and mitotic defects, resulting in aneuploidy, a characteristic of tumor cells [13] that leads to chromosomal instability. Shugoshin (Sgol1), a nuclear protein that protects cohesin—a protein complex that tethers replicated chromosomes together until
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anaphase—inhibits premature chromosome segregation (PCS) and aneuploidy [14, 15]. Sgol1 recruits PP2A to cohesin counteracting Plk1-dependent phosphorylation [16, 17], thereby stabilizing chromatid pairs. We determined that CIP2A interacts with LRRC59 protein, which is known to be important for nuclear import of fetal growth factor [18] in yeast two-hybrid (Y2H) studies (manuscript submitted). This interaction was confirmed in prostate cancer cell lines and was shown to mediate CIP2A translocation into the nucleus. LRRC59 and Sgol1 have previously been reported to interact [16]. CIP2A, primarily cytoplasmic, enters the nucleus in a cell cycle-dependent manner [12], and interacts with Plk1 [12] and NEK2 [11], causing mutant phenotypes. We hypothesized that CIP2A enters the nucleus through its interaction with LRRC59 and destabilizes cohesin through the CIP2A-Sgol1 interaction, promoting PCS and aneuploidy.
Materials and methods
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Fluorescent in situ hybridization (FISH) DU145 cells were treated with siRNA, harvested, and washed twice in PBS. Cells were swelled and fixed as above. The FISH digestion kit (Keratech) was used per manufacturer’s recommendation. The centromere 8 (D8Z1) (Kreatech) satellite enumeration (SE) probe PlatinumBright 495 was used, and microscopy was conducted as above. Cell synchronization and immunofluorescence microscopy A double thymidine or thymidine-nocodazole treatment was performed to arrest cells at G1/S phase or G2/M, respectively, as in Supplementary Fig. S1. Cells were harvested, washed in PBS, and fixed in 4 % paraformaldehyde, prior to washing and permeabilization. Cells were blocked and treated with appropriate antibodies. A list of antibodies and conditions is available in Supplementary Materials. Immunoprecipitation, immunodepletion, and Western blot analysis
Complete information can be found in the supplementary materials.
Protein extraction and immunoblotting were performed as previously described [6]. Antibodies and conditions are available in Supplementary Materials.
Cell culture
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Cell lines were purchased from ATCC (ATCC ethical standards for procurement of human material can be found at www.ATCC.org) and grown per recommendation and were authenticated in late 2013 via STR analysis conducted at Genetica DNA Laboratories, Inc. (www.genetica.com). An amount of 100 nM of siRNA(s) and Lipofectamine RNAiMax (Invitrogen) was used for transient transfection (Table S1). PC-3 stable cells were established using pGIPZ shRNAmir lentiviral vectors (Open Biosystems) and selected with puromycin (InvivoGen). RWPE-1 stable cells were transfected with SparQ dual promoter lentivectors (System Biosciences) and selected with puromycin.
Chromosome spread analysis PC-3 and RWPE-1 cells were treated with siRNA and incubated with KaryoMAX Colcemid (Invitrogen). Cells were harvested, washed with PBS and were swelled in hypotonic solution, centrifuged, and washed in a fixative three times, then suspended in 200 μl of fixative. A volume of 50 μl of cells were dropped on a slide, dried, and stained with DAPI (Cell Signaling), sealed with VECTASHIELD Mounting Medium (Vector Labs), and viewed at ×100 with the Leica DMI6000 B microscope (Leica, Germany).
Extraction of RNA and qRT-PCR were performed as previously described [6]. Primer information is available in Supplementary Table S2.
Results CIP2A interacts with Sgol1 in the nucleus Using N-terminal and C-terminal portions of CIP2A as bait, we identified multiple protein interactions for CIP2A using Y2H that suggested it has multiple roles in cell cycle regulation. One such interaction, between CIP2A and LRRC59, was confirmed in prostate cancer cell lines (manuscript submitted). Since LRRC59 and shugoshin (Sgol1) interact [13], we tested if CIP2A also interacted with Sgol1. Based on the proteins identified to interact with CIP2A, we asked whether the interaction of CIP2A with Sgol1 promoted premature chromosome segregation (PCS) and aneuploidy. We reasoned that since the CIP2APP2A interaction is well-established and PP2A inhibits chromatid segregation through interactions with Sgol1 and cohesin proteins [17, 19–21] that a CIP2A-Sgol1 interaction may promote chromosome missegregation through inhibition of Sgol1. We examined these hypotheses in prostate cancer cells, which exhibit high levels of aneuploidy [22, 23].
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We first confirmed that LRRC59 and Sgol1 interact in PC3 prostate cancer cells. Immunoprecipitation (IP) of GFPSgol1 in asynchronous whole cell extracts resulted in the detection of FLAG-LRRC59 via Western blot analysis (Fig. 1a, compare GFP IP to Input for α-FLAG western) thus confirming an interaction. We next demonstrated that endogenous Sgol1 interacted with CIP2A in the nucleus of PC-3 cells (Supplementary Fig. S2). To confirm, we determined that GFP-tagged Sgol1
interacted with full-length FLAG-tagged CIP2A, and we found that that the C-terminal portion of CIP2A (amino acids # 450–905) was required (Fig. 1b). We next asked if CIP2A and Sgol1 interacted within the nucleus. Cells were arrested at G2/M using thymidine-nocodazole treatment to stimulate CIP2A translocation to the nucleus during G2/M (Fig. S1 and Materials and methods). Immunofluorescence (IF) microscopy showed that both CIP2A and Sgol1 were localized to the nucleus during G2/M, forming punctate structures
Fig. 1 CIP2A interacts with Sgol1 in prostate cancer cells. a GFP-Sgol1 and FLAG-LRRC59 or corresponding empty vectors were co-expressed in PC-3 cells, and Sgol1 was immunoprecipitated (IP) with α-GFP. Rabbit α-Sgol1 and α-FLAG were used to detect Sgol1 and LRRC59, respectively, via Western blot analysis. b GFP-Sgol1 and constructs of FLAG-CIP2A or corresponding empty vectors (−) were co-transfected into PC-3 cells. α-GFP was used to IP Sgol1, and CIP2A constructs were detected with α-FLAG via Western blot analysis. FL=full-length CIP2A, #1=CIP2A aa 1–450, #2=CIP2A aa 450–905. c Immunofluorescent microscopy of metaphase-arrested PC-3 cells was conducted to determine CIP2A and SGOL1 localization when cells were arrested at G2/M with thymidine-nocodazole treatment (see schematic in
Supplementary Fig. S1). DAPI is a nuclear stain (blue) and immunofluorescence analysis was conducted using antibodies specific for CIP2A (green) or Sgol1 (red). d Protein lysates from PC-3 coexpressing GFP-Sgol1 and FLAG-CIP2A or corresponding empty vectors were immunodepleted with antibodies against PP2A subunits, Ppp2ca, and PR65/A, prior to immunoprecipitation with α-GFP. A GFP IP was completed followed by Western blot analysis, as described in the text (middle panels). The far-right panel shows successful immunodepletion of the a and c subunits of PP2A. For all IP studies, inputs represent 10 % of protein used in the IP (left panel), and experiments were completed at least twice with similar results
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(Fig. 1c). Merged images show areas of co-localization (Fig. 1c, arrows). Due to the known interactions among CIP2A, PP2A, and Sgol1, we determined if the assembly of the CIP2A-Sgol1 interaction required PP2A. Protein extracts from PC-3 cells expressing FLAG-CIP2A and GFP-Sgol1 or the corresponding empty vectors were immunodepleted with antibodies for Ppp2ca and PR65/A, the catalytic and scaffolding subunits of PP2A, respectively (Fig. 1d). Immunodepletion of Ppp2ca and PR65/A (Fig. 1d, right panels) caused a marked reduction in FLAG-CIP2A binding when GFP-Sgol1 was immunoprecipitated (Fig. 1d, middle panels). CIP2A promotes premature chromatid segregation Since CIP2A and Sgol1 interacted (Fig. 1b), we next tested if CIP2A regulated chromosomal segregation in the nucleus through modulating Sgol1 stability. The role of CIP2A in PCS in PC-3 cells was determined using metaphase spread
Fig. 2 CIP2A promotes premature chromatid segregation. Cells were arrested in metaphase via colcemid treatment, and metaphase spreads were prepared and stained with DAPI. At least 75 nuclei were counted per condition. a Four chromosome phenotypes were counted and were grouped into one of two categories, unseparated and separated. Unseparated chromosomes included zipped chromosomes (i) and butterfly chromosomes (ii). Separated chromosomes included the Bloss of centromere^ phenotype (iii) and completely separated chromosomes (iv). The white bar represents 10 μm. b PC-3 cells were treated with non-
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analyses. Phenotypes were organized into two categories [17]: Bunseparated^ and Bseparated^ chromosomes (Fig. 2a). At metaphase, chromosomes are connected along the entire length (i.e., zipped, Fig. 2ai) or exist in a butterfly conformation (Fig. 2a ii ) and are characterized as Bunseparated chromosomes.^ Separated chromosomes include the Bloss of centromere^ phenotype depicted in Fig. 2aiii, where chromosomes are aligned but are not attached at the centromere or completely separated chromosomes (Fig. 2aiv); both phenotypes indicate PCS. Control siRNA-treated PC-3 cells exhibited ∼20 % separated chromosomes. Depletion of CIP2A led to a significant two-fold decrease in PCS (Fig. 2b). To confirm a role for CIP2A in PCS, we overexpressed CIP2A in the immortalized prostate cell line, RWPE-1. We found that RWPE-1 cells have a reduced basal level of PCS (20 %, compare Fig. 2c vs. Fig. 2b). Overexpression of CIP2A caused a five-fold increase in PCS in RWPE-1 cells
targeting (control) siRNA or siRNA-targeting CIP2A (CIP2A) prior to arrest and completion of metaphase spreads. c RWPE-1 immortalized prostate cells transfected with an empty vector (empty) or CIP2A overexpression plasmid (+CIP2A) prior to metaphase arrest. b, c The percentage of unseparated chromosomes (white bars) and separated chromosomes (blue bars) is shown for each condition, and Western blot analysis (inset) confirms appropriate expression of CIP2A. Statistical significance was determined using the Student’s t test, where **p3 copies). Statistical significance was determined using the oneway ANOVA, where **p
