Journal of Pathology J Pathol 2014; 233: 51–60 Published online 27 January 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4319
ORIGINAL PAPER
Proline-rich acidic protein 1 (PRAP1) is a novel interacting partner of MAD1 and has a suppressive role in mitotic checkpoint signalling in hepatocellular carcinoma Karen Man-Fong Sze,1,2 Glanice Kin-Yan Chu,1,2 Queenie Ho-Yan Mak,1,2 Joyce Man-Fong Lee1,2 and Irene Oi-Lin Ng1,2* 1 2
State Key Laboratory for Liver Research, University of Hong Kong Department of Pathology, University of Hong Kong
*Correspondence to: IOL Ng, Room 127B, University Pathology Building, Department of Pathology, University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong. E-mail: [email protected]
Abstract Loss of mitotic checkpoint of cells contributes to chromosomal instability and leads to carcinogenesis. Mitotic arrest deficient 1 (MAD1) is a key component in mitotic checkpoint signalling. In this study, we identified a novel MAD1 interacting partner, proline-rich acidic protein 1 (PRAP1), using yeast-two hybrid screening, and investigated its role in mitotic checkpoint signalling in hepatocellular carcinoma (HCC). We demonstrated the physical interaction of PRAP1 with MAD1 and of PRAP1 with MAD1 isoform MAD1β, using a coimmunoprecipitation assay. Moreover, stable expression of PRAP1 in mitotic checkpoint-competent HCC cells, BEL-7402 and SMMC-7721, induced impairment of the mitotic checkpoint (p < 0.01), formation of chromosome bridges (p < 0.01) and aberrant chromosome numbers (p < 0.001). Interestingly, ectopic expression PRAP1 in HCC cells led to significant under-expression of MAD1. In human HCC tumours, 40.4% (23/57) of HCCs showed under-expression of PRAP1 protein as compared with their corresponding non-tumorous livers; up-regulation of MAD1 protein was significantly associated with down-regulation of PRAP1 (p = 0.030). Our data revealed that PRAP1 is a protein interacting partner of MAD1 and that PRAP1 is able to down-regulate MAD1 and suppress mitotic checkpoint signalling in HCC. Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: hepatocellular carcinoma; proline-rich acidic protein 1; MAD1; mitotic checkpoint; chromosomal instability; protein interactions
Received 7 August 2013; Revised 11 December 2013; Accepted 17 December 2013
No conflicts of interest were declared.
Introduction Accurate chromosome segregation is important for cells to inherit its genetic material to its daughter cells during cell duplication. Mitotic checkpoint signalling is activated to prevent the onset of anaphase until all kinetochores have been attached to microtubules [1]. However, DNA aneuploidy or chromosomal instability is commonly observed in major types of human cancers, including hepatocellular carcinoma (HCC) [2]. Defective mitotic checkpoint signalling is one of the leading causes of chromosomal instability in eukaryotic cells. In recent decades, more than 10 protein components playing differential roles in the mitotic checkpoint control have been identified in eukaryotic cells, including budding uninhibited by benzimidazole (BUB) proteins, BUB1-3, mitotic arrest-deficient (MAD) proteins (MAD1–3), cell division cycle 20 (CDC 20), monopolar spindle 1 (MPS1), Mis 12, ZW10-interacting protein 1 (ZWINT-1), highly expressed in cancer 1 (HEC1), Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Nuf2, spindle pole body component (SPC) protein 24 (SPC24) and SPC25 [3–11]. At prometaphase, when a single kinetochore is unattached to the microtubules, the MAD1–MAD2 complex will remain localized to the kinetochore, and this will enhance the recruitment of MAD2 to the APC/Ccdc20 complex in the presence of MPS1 activation [12–14], resulting in the formation of mitotic checkpoint complex (MCC), which consists of BUBR1, CDC20, BUB3 and MAD2 and activates mitotic checkpoint signalling to prevent the onset of chromosome segregation [15–19]. When all kinetochores have been attached to the microtubules, this signals the onset of anaphase by ubiquitination of CDC20 and subsequent dissociation of checkpoint proteins from the APC–Ccdc20 complex [20,21]. MAD1 is a key component in mitotic checkpoint signalling. Depletion of MAD1 protein by RNA interference leads to failure of MAD2 to localize to the kinetochore, resulting in impairment of the mitotic checkpoint and aneuploidy in human cells [12,22]. In addition, haplo-insufficiency of MAD1 in J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
52
heterozygous knockout mice promotes genomic instability and enhances the development of various cancers, including lung carcinoma and HCC [23]. Expression of viral oncoprotein, such as Tax, in human T cell leukaemia virus type 1 (HTLV-1) can inactivate mitotic checkpoint signalling via targeting MAD1 [24]. However, mutations of MAD1 in human cancers are uncommon [25–27]. We previously identified a novel isoform of MAD1, which we named MAD1β [28]. The MAD1β isoform has deletion of exon 4 and is derived from alternative splicing of pre-mRNA. Ectopic expression of the MAD1β protein induced mitotic checkpoint impairment via altering the level of the endogenous MAD2 in HCC cell lines [28]. In the present study we identified a novel MAD1 binding partner, proline-rich acidic protein 1 (PRAP1), using yeast-two hybrid screening. Mechanistically, stable expression of PRAP1 in HCC cell lines resulted in a deranged mitotic checkpoint and led to chromosomal instability, with the formation of chromosome bridges and aberrant chromosome numbers. Furthermore, in human HCC tumours, up-regulation of MAD1 protein was associated with down-expression of PRAP1. We propose that PRAP1 may have suppressive roles in mitotic checkpoint signalling in HCC cells, possibly involving down-regulation of MAD1.
KM-F Sze et al
Full-length PRAP1 cDNA was amplified from normal liver cDNA and subcloned into pEFFP-N1 vectors. Cloning of full-length MAD1 and MAD1β cDNA were described previously [28].
Yeast two-hybrid library screening and binding assay The detailed protocol was described previously [29]. The library screening was done by co-transformation of yeast strain Y190 with 1–249 MAD1β–pAS2-1 plasmid and pACT2 fused with normal human liver cDNA library that contained 3.5 × 106 independent clones (Clontech Laboratories, CA, USA).
Generation of PRAP1-stably-expressing cell lines BEL-7402 and SMMC-7721 cell lines were transfected with PRAP1–pEGFP-N1 and pEGFP-N1 vector, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Stably expressing cells were selected with G418 at 800 µg/ml (for BEL-7402 cells) or 400 µg/ml (for SMMC-7721 cells) for 14 days. These selected stably-expressing clones were maintained in growth medium with G418 at 100 µg/ml.
Measurement of mitotic index Materials and methods Patients and samples Fifty-seven pairs of human HCCs and their corresponding non-tumorous liver tissues from patients with surgical resection for HCC between 1992 and 2001 at Queen Mary Hospital and the University of Hong Kong were randomly selected for study. The patients were aged 24–74 years. All specimens were collected at the time of surgical resection, snap-frozen in liquid nitrogen and kept at −80 ◦ C. Use of human samples was approved by the Institutional Review Board (IRB) of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB).
Cell lines Human HCC cell lines, BEL-7402 and SMMC-7721, were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. They were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high-glucose; GIBCO-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). HEK293 cells were maintained in DMEM high-glucose supplemented with 10% FBS.
Plasmids The N-terminus of MAD1β [1–249 amino acids (aa)] was amplified from full-length MAD1β–Flag –pcDNA3.1+ vector and subcloned into pAS2-1 vector to generate the 1–313 MAD1β–pAS2-1 plasmid. Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
The detailed protocol was described previously [30]. Mitotic indices were determined by evaluating the percentage of cells with chromosomal condensation per total number of viable cells at 24 h after treatment with nocodazole or colcemid [28]. At least 300 cells were counted in each experiment and the experiment was done at least three times.
Cell synchronization and DNA morphological analysis in mitotic cells Cells were enriched at mitotic phase with overnight serum starvation and incubation in culture medium containing 5 mM thymidine for 16 h, followed by replacement with normal culture medium for 12–14 h. The cells were fixed according to the protocol previously described [30]. The number of cells showing chromosome bridges and micronuclei was assessed in at least 200 mitotic cells for each cell line.
Metaphase spread and chromosome counting Cells were treated with 100 ng/ml colcemid for 4 h before harvest. The cells were then harvested by trypsinization, treated with 75 mM KCl at 37◦ C for 20 min and fixed in ice-cooled fixative (methanol:acetic acid, 1:1 v/v). A drop of fixed cells was allowed to fall from a height of around 20 cm onto glass slide. The air-dried slides were then submerged in Giemsa solution diluted with MilliQ water, followed by brief washing once, and allowed to air-dry before microscopic observation. Chromosome counting was done on at least 40 cells for each cell line. J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
Western blot analysis and co-immunoprecipitation (Co-IP) assay For western blot analysis, cells were lysed in 50 mM Tris, 150 mM NaCl, 5 mM EDTA (NET) buffer with 1% NP-40 and complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany), and separated in SDS–PAGE gel for western blot analysis. Immunodetection was performed using anti-Flag (Sigma, St Louis, MO, USA), anti-α-tubulin (Sigma), anti-β-actin (Sigma), anti-PRAP1 (Sigma), anti-MAD1 (Santa Cruz Biotechnology, CA, USA), anti-GFP (Santa Cruz) and anti-MAD2 (Transduction Laboratories, CA, USA) antibodies. For co-IP assay, HEK293 cells were transfected with plasmids containing cDNA of Flag-tagged and EGFP-tagged fusion proteins, respectively. The cells were lysed in NET buffer supplemented with 0.1% Triton X-100 and protease inhibitor cocktail. The cell lysate was incubated with 1 µg anti-Flag (Sigma) antibody with the addition of protein G sepharose. Unbound protein was washed away from the beads before electrophoresis. The anti-GFP and anti-Flag antibodies were used as primary antibodies for western blot analysis.
53
A
B
Immunohistochemical staining for PRAP1 in human HCC samples Immunohistochemistry was performed on formalinfixed, paraffin-embedded (FFPE) sections, as described [31], using anti-PRAP1 monoclonal antibody (Sigma) at 1:500 dilution.
Clinicopathological correlation and statistical analysis The clinicopathological features of HCC patients, including tumour size, cellular differentiation according to the Edmondson grading, venous invasion into portal or hepatic venules, direct liver invasion, tumour microsatellite formation, tumour encapsulation and number of tumour nodules, were analysed using SPSS 20 for Windows (SPSS, Chicago, IL, USA). Fisher’s exact test was used for analysis of categorical data. The results were considered significant at p < 0.05.
Results Identification of PRAP1 as a novel binding partner of MAD1 Previously, we had identified a novel MAD1 isoform, which we named MAD1β [28]. MAD1β has exon 4 deleted at the N-terminal part of the MAD1 protein. In addition, MAD1β displays differential subcellular localization as well as functional roles, as compared with the MAD1 protein [28]. To gain more insight into the molecular basis of these two isoforms, we attempted to search for their potential binding partners of the N-terminal parts of MAD1 and MAD1β Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Figure 1. Identification of PRAP1 as a novel MAD1 binding partner by yeast two-hybrid screening. (A) Result of positive colony lift filter β-galactosidase assay using 1–249 aa MAD1/pAS2-1 as bait. The agar plate showed the positive colonies of co-transformed bait with human liver cDNA fragment in pACT2 plasmid together with MAD1/pAS2-1 plasmid. The filter paper contained the replicates of the yeast colonies from the agar plate, lysed by freeze–thaw cycles and subjected to colony lift filter β-galactosidase assay. The grey spots in the picture represented the positive result from the β-galactosidase assay. With subsequent extraction of yeast DNA and DNA sequencing of the cDNA cloned in pACT2 plasmid, PRAP1 was identified to be one of the interacting partners of 1–249 aa MAD1β protein. (B) Physical binding of Flag-tagged full-length MAD1 and MAD1β with EGFP-tagged PRAP1 protein was demonstrated by co-IP assay. Both MAD1 and MAD1β proteins were co-immunoprecipitated with PRAP1.
proteins. We performed yeast two-hybrid screening of a human liver cDNA library using the 1–249 aa of MAD1β as bait. It was estimated that 1 × 107 transformants were screened, and positive clones obtained were confirmed by colony-lift filter β-galactosidase activity assay. With sequence analysis of 32 positive colonies, one of the clones (clone 50) ligated with incomplete cDNA of PRAP1 (NM_145202; Figure 1A). According to the online database regarding mRNA expression of PRAP1 in various tissue types, PRAP1 is predominantly expressed in liver and kidney [32]. Furthermore, when we cloned the full-length PRAP1 into the pEGFP–N1 vector, we were able to confirm its interaction with both MAD1 and MAD1β by co-IP J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
54
KM-F Sze et al
A
B
Figure 2. Characterization of PRAP1-stably-expressing BEL-7402 and SMMC-7721 HCC cells. (A) The protein levels of exogenous EGFPtagged PRAP1 protein in BEL-7402 and SMMC-7721 cell lines were determined by western blot analysis with anti-GFP antibody. (B) After treatment with either colcemid or nocodazole, the PRAP1-stably-expressing BEL-7402 and SMMC-7721 cell lines showed significantly lower mitotic indices as compared to the corresponding vector control cells.
assay. The result showed that the full-length PRAP1 physically interacted with both MAD1 and MAD1β (Figure 1B).
Ectopic expression of PRAP1 led to mitotic checkpoint impairment The functional role of PRAP1 in mitotic checkpoint signalling was largely unknown. Therefore, we investigated the role of PRAP1 in the mitotic checkpoint in BEL-7042 and SMMC-7721 HCC cell lines. These two cell lines have relatively low mRNA expression of PRAP1 as compared with the immortalized normal liver cell line MIHA (see supplementary material, Figure S1). In addition, these two cell lines have a competent mitotic checkpoint, as we previously reported [30]. The GFP-tagged PRAP1 plasmid was stably transfected into these two HCC cell lines and the level of over-expression was confirmed by western Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
blot analysis, using anti-GFP antibody (Figure 2A). After treatment with nocodazole and colcemid, respectively, the PRAP1-stably-expressing BEL-7402 and SMMC-7721 clones showed a significant reduction of mitotic indices from 58.8–61.1% to 23.3–31.3% in BEL-7402 cells (p = 0.007 and p = 0.008, respectively) and from 59.5–60.5% to 24–24.1% in SMMC7721 cells (p = 0.003 and p < 0.001, respectively), as compared with the corresponding vector control cells (Figure 2B). This indicates a significant degree of loss of mitotic checkpoint competence induced by PRAP1. In addition, formation of chromosome bridges, which is a reflection of mitotic checkpoint defect, was significantly more frequently observed in both PRAP1stably-expressing BEL-7402 and SMMC-7721 cells, as compared with the corresponding vector control cells (p = 0.006 and p = 0.011, respectively) (Figure 3A). Furthermore, micronuclei formation was enhanced in both PRAP1-stably-expressing BEL-7402 and SMMC J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
55
A
B
C
Figure 3. Ectopic expression of PRAP1 led to abnormal chromosome segregation and aberrant chromosomal numbers. (A) In EGFP-tagged PRAP1-stably-expressing cells, 11.5–15% of mitotic cells exhibited chromosome bridge formation (white arrowheads) during mitosis. Chromosome bridge formation was more frequent in the PRAP1-stably-expressing cells as compared with corresponding vector control in both BEL-7402 and SMMC-7721 cell lines (p = 0.006 and 0.011, respectively). (B) Micronuclei formation was more frequent in the PRAP1-stably-expressing cells as compared with the corresponding vector control in both BEL-7402 (8.8% versus 1.3%) and SMMC7721 (12.7% versus 2.1%) cell lines. White arrows indicate the micronuclei formed in cells. (C) With metaphase spreading analysis, PRAP1-stably-expressing cell lines had chromosome numbers ranging widely between 20 and 101, as compared with vector control.
7721 cells, as compared with the corresponding vector control cells (Figure 3B).
Ectopic expression of PRAP1 resulted in aberrant chromosomal numbers To assess the role of PRAP1 in chromosomal instability, the chromosome numbers of individual cells in the PRAP1-stably-expressing BEL-7402 and SMMC7721 cells were compared with the corresponding vector cells. The BEL-7402 vector control cells had chromosome numbers in the range 44–62. In contrast, PRAP1-over-expressing BEL-7402 cells had widely varying chromosome numbers in the range 20–54 (p < 0.001, Levene’s test) (Figure 3C). Similarly, Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
SMMC-7721 vector control cells had chromosome numbers mainly in the range 63–94, whereas the PRAP1-over-expressing SMMC-7721 cells had widely varying chromosome numbers in the range 40–101 (p < 0.001, Levene’s test) (Figure 3C). The results indicate that PRAP1 expression induces a significant change of chromosome numbers, suggestive of chromosomal instability.
PRAP1 expression resulted in reduced MAD1 protein in HCC cells It is known that the MAD1–MAD2 protein complex localizes in the nucleus, binds to unattached kinetochores and activates the mitotic spindle checkpoint J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
56
KM-F Sze et al
A
B
Figure 4. PRAP1 reduced MAD1 protein level. (A) Western blot analysis showed significant reduction of MAD1 protein level in the MAD1 and PRAP1 co-transfected BEL-7402 and SMMC-7721 cells, as compared with the corresponding EGFP co-expressing control cells. However, similar down-regulation of exogenous MAD1β was not seen in MAD1β and PRAP1 co-transfected cells as compared with MAD1β and EGFP co-expressing control cells. (B) Western blot analysis showed significant reduction of endogenous MAD1 protein level in PRAP1-stably-expressing BEL-7402 and SMMC-7721 cells. No reduction of endogenous MAD2 protein level was seen in both PRAP1-expressing BEL-7402 and SMMC-7721 cells.
[24,33]. In this study, we demonstrated that PRAP1 is a protein binding partner of both MAD1 and MAD1β, using a co-IP assay. To dissect the possible mechanism by which PRAP1 led to mitotic checkpoint defect, we examined whether PRAP1 could affect the MAD1 and MAD1β proteins. We transiently co-expressed EGFP-tagged PRAP1 with either Flag-tagged MAD1 or Flag-tagged MAD1β in BEL-7402 and SMMC7721 HCC cells and studied the effect on MAD1 protein. Interestingly, the protein level of MAD1 was dramatically reduced when it was co-expressed with PRAP1, as compared with EGFP protein control in Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
both BEL-7402 and SMMC-7721 cells (Figure 4A). Surprisingly, although the PRAP1 could bind with MAD1β, as shown in co-IP assay, PRAP1 did not alter the protein level of MAD1β as compared with the EGFP controls in both BEL-7402 and SMMC-7721 cells (Figure 4A). To further verify the effects of PRAP1 in altering the MAD1 protein levels, we studied its effect in PRAP1-stably-expressing BEL-7402 and SMMC-7721 cells. Similar to the results from experiments using transient transfection, there was observable down-regulation of protein expression of endogenous MAD1 in PRAP1-stably-expressing cells J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
57
Table 1. Summary of the findings of clinicopathological correlation PRAP1 expression Clinicopathological features Sex Tumour size Cellular differentiation (Edmondson’s grading) Encapsulation Tumour microsatellite formation Number of tumour nodules Direct liver invasion Venous invasion Background liver disease
Under-expression
Normal or over-expression
p
17 6 6 17 7 16 15 8 18 15 26 7 14 8 10 13 12 11
28 6 9 24 18 16 20 13 15 19 27 7 19 11 16 18 18 16
0.517
Male Female ≤ 5 cm > 5 cm I–II III–IV Absent Present Absent Present 1 ≥2 Absent Present Absent Present Normal and chronic hepatitis Cirrhosis
as compared with corresponding vector control cells in both BEL-7402 and SMMC-7721 (Figure 4B). Moreover, the protein levels of endogenous MAD2 in the PRAP1-stably-expressing cells remained more or less unchanged and similar to the corresponding vector control in either BEL-7402 or SMMC-7721 (Figure 4B).
Inverse correlation between the protein levels of PRAP1 and MAD1 in human HCCs In this study, we observed that PRAP1 expression was associated with reduced MAD1 protein in HCC cell lines. We further examined the PRAP1 expression in human HCCs by immunohistochemistry in 57 pairs of human HCCs and their corresponding nontumorous livers and assessed its clinicopathological correlation. PRAP1 was quite abundantly expressed in the non-tumorous livers; 40.4% (23/57) of HCCs showed under-expression of PRAP1 protein as compared with their corresponding non-tumorous livers; 33.3% (19/57) showed over-expression of PRAP1; and 26.3% (15/57) showed similar PRAP1 protein expression. However, no association between the clinicopathological features and PRAP1 under-expression was observed (Table 1). In addition, we performed western blot analysis to detect endogenous MAD1 protein expression in 22 pairs of HCCs randomly selected from these 57 cases of human HCCs with immunohistochemistry for PRAP1 performed. In these 22 cases, relatively low expression of MAD1 protein was seen in the nontumorous liver tissues. The protein levels of PRAP1 and MAD1 correlated inversely with one another and up-regulation of MAD1 protein (by two-fold) was significantly associated with down-regulation of PRAP1 in human HCC tissue (p = 0.030) (Figure 5). Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
1.000 0.110 0.785 0.586 0.532 1.000 1.000 1.000
Discussion In mammalian cells, the MAD1–MAD2 protein complex localizes in the nucleus. It binds to the unattached kinetochores and activates the mitotic spindle checkpoint, thus preventing cells from undergoing improper chromosomal segregation [24,33]. In this study, with yeast two-hybrid screening, we identified PRAP1 as a novel protein interacting partner of MAD1. PRAP1 could physically bind with MAD1 and MAD1β. We further characterized the functional role of PRAP1 in mitotic checkpoint control by stably over-expressing it in two mitotic checkpoint-competent HCC cell lines, BEL-7402 and SMMC-7721 [30], and observed a significant reduction of mitotic indices and severe chromosome aberrations when PRAP1 was over-expressed in these cells. These results indicate that PRAP1 induces mitotic checkpoint incompetence and chromosomal instability. We showed that both transient and stable expression of PRAP1 led to reduced levels of endogenous MAD1 protein in cells. Our results indicate that PRAP1 physically binds to MAD1 and reduces the endogenous level of MAD1. Of note, we observed that in human HCCs the protein levels of PRAP1 and MAD1 correlated inversely with one another and that down-regulation of PRAP1 was significantly associated with up-regulation of MAD1 protein. In mammalian cells, MAD1 and MAD2 have been shown to localize at nuclear pore complexes at interphase and at unattached kinetochores during prometaphase [34], and reduction of MAD2 protein expression was associated with mitotic checkpoint incompetence in HCC and other cancer cell lines [30,35,36]. However, in PRAP1-stably-expressing HCC cells, the protein level of MAD2 remained unchanged. Moreover, using the proteasome inhibitor MG132 to block proteosomedependent degradation in PRAP1-stably-expressing J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
58
KM-F Sze et al
A
B
Figure 5. MAD1 protein expression inversely correlated with PRAP1 expression in human HCCs. (A) Representative cases showing protein expression of PRAP1 in human HCCs by immunohistochemistry. Cases 309 and 343 showed under-expression of PRAP1 in tumorous tissue as compared with the corresponding non-tumorous liver tissues, while Case 202 showed over-expression of PRAP1 in the tumour. (B) Representative cases showing protein expression of MAD1 in human HCCs by western blot analysis. Cases 309 and 343 showed over-expression of MAD1 in tumorous tissue as compared with corresponding non-tumorous liver tissue. Table 1 shows the correlation between PRAP1 and MAD1 protein expression in 22 pairs of human HCC tissues.
cells, our result has shown that this reduction in MAD1 protein level may not involve proteosome-dependent degradation (see supplementary material, Figure S2A). However, with cycloheximide treatment, MAD1 protein level was reduced in the presence of PRAP1, but not EGFP, in BEL-7402 cells (see supplementary material, Figure S2B). Thus, PRAP1 may down-regulate MAD1 in cells and exert a suppressive role in mitotic checkpoint signalling. So far, the exact mechanism of MAD1 stability in cells is largely unknown and further investigation is needed. There are two hypotheses in relation to the transcriptional regulation of PRAP1, one of which is via p53 to switch on the transcription of PRAP1 upon genotoxic stress treatment [37], whereas the other is through epigenetic regulation to suppress PRAP1 mRNA transcription [38]. Our preliminary data on p53 expression and mutation status in human HCC tumours with PRAP1 under-expression showed that most of the tumour samples show p53 mRNA expression. Furthermore, with direct sequencing of the p53 PCR product, Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
only one missense mutation was found in the p53coding region, suggesting that the p53 status might not be related to the loss of PRAP1 expression in HCC (see supplementary material, Figure S3A). In addition, we preliminarily performed methylation-sensitive PCR on the PRAP1 promoter region to assess whether loss of PRAP1 expression was due to PRAP1 promoter hypermethylation. We observed that seven of 10 cases with PRAP1 under-expression in human HCCs showed PRAP1 partial promoter methylation at the specific locus, –46 nt before the transcriptional start site of the PRAP1 gene (see supplementary material, Figure S3B). Taken together, promoter hypermethylation might play an important role in suppressing PRAP1 gene expression. However, the regulation of PRAP1 gene expression in HCC needs further investigations. Over-expression of MAD1 mRNA and protein has been reported in human breast and liver cancers [28,39]. The expression of MAD1 can be activated by gain-of-function p53 mutant, induction of cellular J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
proliferation [40,41] and gain of chromosome region 7p22.3, as in small-cell lung cancer [42]. On the other hand, partial down-regulation of MAD1 protein can result in spindle checkpoint inactivation, aneuploidy in cell and enhanced tumour formation in MAD1 heterozygous knock-out mice [22,23]. In recent studies, over-expression of MAD1 and its constitutive targeting to kinetochores could result in chromosomal instability in mammalian cells [43,44]. In addition, it has been suggested that the ratio between MAD1 and MAD2 protein expression level is important in mitotic checkpoint activation [45]. These results suggest that steady amounts of MAD1 and other mitotic checkpoint proteins are important for spindle checkpoint control in cells. It is established that accumulation of genetic alterations occurs along the multistep process of hepatocarcinogenesis [46]; however, whether the expression of PRAP1 is an early or late event and its importance in hepatocarcinogenesis are still largely unknown. Taken together, our data show that PRAP1 is a novel interacting partner of MAD1 and indicate that PRAP1 reduces the endogenous protein level of MAD1, possibly by enhanced degradation, and suppresses mitotic checkpoint signalling in HCC.
Acknowledgements This study was supported by the Hong Kong Research Grants Council General Research Fund (Grant No. HKU 772608) and Collaborative Research Fund (Grant No. HKU 7/CRF/09), and HKU Small Project Fund (Grant No. HKU 201109176118). IOL Ng is Loke Yew Professor in Pathology.
Author contributions KMFS and IOLN designed the study; KMFS, QHYM, GKYC and JMFL designed and performed the experiments; and KMFS and IOLN analysed the data, performed the clinicopathological analysis and wrote the manuscript.
References 1. Rieder CL, Cole RW, Khodjakov A, et al. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J Cell Biol 1995; 130: 941–948. 2. Ng IO, Lai EC, Ho JC, et al. Flow cytometric analysis of DNA ploidy in hepatocellular carcinoma. Am J Clin Pathol 1994; 102: 80–86. 3. Abrieu A, Magnaghi-Jaulin L, Kahana JA, et al. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell 2001; 106: 83–93. 4. Visintin R, Prinz S, Amon A. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 1997; 278: 460–463. Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
59
5. Martin-Lluesma S, Stucke VM, Nigg EA. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 2002; 297: 2267–2270. 6. DeLuca JG, Howell BJ, Canman JC, et al. Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores. Curr Biol 2003; 13: 2103–2109. 7. Obuse C, Iwasaki O, Kiyomitsu T, et al. A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1. Nat Cell Biol 2004; 6: 1135–1141. 8. Li R, Murray AW. Feedback control of mitosis in budding yeast. Cell 1991; 66: 519–531. 9. Hoyt MA, Totis L, Roberts BT. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 1991; 66: 507–517. 10. DeLuca JG, Gall WE, Ciferri C, et al. Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 2006; 127: 969–982. 11. Bharadwaj R, Qi W, Yu H. Identification of two novel components of the human NDC80 kinetochore complex. J Biol Chem 2004; 279: 13076–13085. 12. Luo X, Tang Z, Rizo J, et al. The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol Cell 2002; 9: 59–71. 13. Hewitt L, Tighe A, Santaguida S, et al. Sustained Mps1 activity is required in mitosis to recruit O–Mad2 to the Mad1–C–Mad2 core complex. J Cell Biol 2010; 190: 25–34. 14. Lad L, Lichtsteiner S, Hartman JJ, et al. Kinetic analysis of Mad2–Cdc20 formation: conformational changes in Mad2 are catalyzed by a C–Mad2–ligand complex. Biochemistry 2009; 48: 9503–9515. 15. Fang G, Yu H, Kirschner MW. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev 1998; 12: 1871–1883. 16. Chen RH. BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1. J Cell Biol 2002; 158: 487–496. 17. Fraschini R, Beretta A, Sironi L, et al. Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. Embo J 2001; 20: 6648–6659. 18. Hardwick KG, Johnston RC, Smith DL, et al. MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p. J Cell Biol 2000; 148: 871–882. 19. Braunstein I, Miniowitz S, Moshe Y, et al. Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint. Proc Natl Acad Sci USA 2007; 104: 4870–4875. 20. Stegmeier F, Rape M, Draviam VM, et al. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 2007; 446: 876–881. 21. Howell BJ, Hoffman DB, Fang G, et al. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. J Cell Biol 2000; 150: 1233–1250. 22. Kienitz A, Vogel C, Morales I, et al. Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol. Oncogene 2005; 24: 4301–4310. 23. Iwanaga Y, Chi YH, Miyazato A, et al. Heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1) increases the incidence of tumors in mice. Cancer Res 2007; 67: 160–166. 24. Jin DY, Spencer F, Jeang KT. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 1998; 93: 81–91. 25. Tsukasaki K, Miller CW, Greenspun E, et al. Mutations in the mitotic check point gene, MAD1L1, in human cancers. Oncogene 2001; 20: 3301–3305. J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
60
26. Cahill DP, da Costa LT, Carson-Walter EB, et al. Characterization of MAD2B and other mitotic spindle checkpoint genes. Genomics 1999; 58: 181–187. 27. Nomoto S, Haruki N, Takahashi T, et al. Search for in vivo somatic mutations in the mitotic checkpoint gene, hMAD1, in human lung cancers. Oncogene 1999; 18: 7180–7183. 28. Sze KM, Ching YP, Jin DY, et al. Role of a novel splice variant of mitotic arrest deficient 1 (MAD1), MAD1β, in mitotic checkpoint control in liver cancer. Cancer Res 2008; 68: 9194–9201. 29. Yam JW, Ko FC, Chan CY, et al. Interaction of deleted in liver cancer 1 with tensin 2 in caveolae and implications in tumor suppression. Cancer Res 2006; 66: 8367–8372. 30. Sze KM, Ching YP, Jin DY, et al. Association of MAD2 expression with mitotic checkpoint competence in hepatoma cells. J Biomed Sci 2004; 11: 920–927. 31. Wong CM, Fan ST, Ng IO. beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance. Cancer 2001; 92: 136–145. 32. Yanai I, Benjamin H, Shmoish M, et al. Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification. Bioinformatics 2005; 21: 650–659. 33. Chen RH, Shevchenko A, Mann M, et al. Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J Cell Biol 1998; 143: 283–295. 34. Campbell MS, Chan GK, Yen TJ. Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase. J Cell Sci 2001; 114: 953–963. 35. Wang X, Jin DY, Wong YC, et al. Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells. Carcinogenesis 2000; 21: 2293–2297. 36. Wang X, Jin DY, Ng RW, et al. Significance of MAD2 expression to mitotic checkpoint control in ovarian cancer cells. Cancer Res 2002; 62: 1662–1668.
KM-F Sze et al
37. Huang BH, Zhuo JL, Leung CH, et al. PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage. Cell Death Dis 2012; 3: e442. 38. Zhang J, Wong H, Ramanan S, et al. The proline-rich acidic protein is epigenetically regulated and inhibits growth of cancer cell lines. Cancer Res 2003; 63: 6658–6665. 39. Yuan B, Xu Y, Woo J-H, et al. Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability. Clin Cancer Res 2006; 12: 405–410. 40. Iwanaga Y, Jeang KT. Expression of mitotic spindle checkpoint protein hsMAD1 correlates with cellular proliferation and is activated by a gain-of-function p53 mutant. Cancer Res 2002; 62: 2618–2624. 41. Gualberto A, Aldape K, Kozakiewicz K, et al. An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control. Proc Natl Acad Sci USA 1998; 95: 5166–5171. 42. Coe BP, Lee EH, Chi B, et al. Gain of a region on 7p22.3, containing MAD1L1, is the most frequent event in small-cell lung cancer cell lines. Genes Chromosomes Cancer 2006; 45: 11–19. 43. Maldonado M, Kapoor TM. Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation. Nat Cell Biol 2011; 13: 475–482. 44. Ryan SD, Britigan EM, Zasadil LM, et al. Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons. Proc Natl Acad Sci USA 2012; 109: E2205–2214. 45. Schuyler SC, Wu YF, Kuan VJ. The Mad1–Mad2 balancing act – a damaged spindle checkpoint in chromosome instability and cancer. J Cell Sci 2012; 125: 4197–4206. 46. Lee JM, Wong CM, Ng IO. Hepatitis B virus-associated multistep hepatocarcinogenesis: a stepwise increase in allelic alterations. Cancer Res 2008; 68: 5988–5996.
SUPPLEMENTARY MATERIAL ON THE INTERNET The following supplementary material may be found in the online version of this article: Figure S1. mRNA expression of PRAP1 in human HCC cell lines. Figure S2. (A) No alteration of MAD1 protein upon MG-132 treatment in PRAP1-stably-expressing HCC cells. (B) With cycloheximide treatment, MAD1 protein level was reduced in the presence of PRAP1, but not EGFP, in BEL-7402 cells. Figure S3. Transcriptional regulation of PRAP1 in HCC.
Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
ORIGINAL PAPER
Proline-rich acidic protein 1 (PRAP1) is a novel interacting partner of MAD1 and has a suppressive role in mitotic checkpoint signalling in hepatocellular carcinoma Karen Man-Fong Sze,1,2 Glanice Kin-Yan Chu,1,2 Queenie Ho-Yan Mak,1,2 Joyce Man-Fong Lee1,2 and Irene Oi-Lin Ng1,2* 1 2
State Key Laboratory for Liver Research, University of Hong Kong Department of Pathology, University of Hong Kong
*Correspondence to: IOL Ng, Room 127B, University Pathology Building, Department of Pathology, University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong. E-mail: [email protected]
Abstract Loss of mitotic checkpoint of cells contributes to chromosomal instability and leads to carcinogenesis. Mitotic arrest deficient 1 (MAD1) is a key component in mitotic checkpoint signalling. In this study, we identified a novel MAD1 interacting partner, proline-rich acidic protein 1 (PRAP1), using yeast-two hybrid screening, and investigated its role in mitotic checkpoint signalling in hepatocellular carcinoma (HCC). We demonstrated the physical interaction of PRAP1 with MAD1 and of PRAP1 with MAD1 isoform MAD1β, using a coimmunoprecipitation assay. Moreover, stable expression of PRAP1 in mitotic checkpoint-competent HCC cells, BEL-7402 and SMMC-7721, induced impairment of the mitotic checkpoint (p < 0.01), formation of chromosome bridges (p < 0.01) and aberrant chromosome numbers (p < 0.001). Interestingly, ectopic expression PRAP1 in HCC cells led to significant under-expression of MAD1. In human HCC tumours, 40.4% (23/57) of HCCs showed under-expression of PRAP1 protein as compared with their corresponding non-tumorous livers; up-regulation of MAD1 protein was significantly associated with down-regulation of PRAP1 (p = 0.030). Our data revealed that PRAP1 is a protein interacting partner of MAD1 and that PRAP1 is able to down-regulate MAD1 and suppress mitotic checkpoint signalling in HCC. Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: hepatocellular carcinoma; proline-rich acidic protein 1; MAD1; mitotic checkpoint; chromosomal instability; protein interactions
Received 7 August 2013; Revised 11 December 2013; Accepted 17 December 2013
No conflicts of interest were declared.
Introduction Accurate chromosome segregation is important for cells to inherit its genetic material to its daughter cells during cell duplication. Mitotic checkpoint signalling is activated to prevent the onset of anaphase until all kinetochores have been attached to microtubules [1]. However, DNA aneuploidy or chromosomal instability is commonly observed in major types of human cancers, including hepatocellular carcinoma (HCC) [2]. Defective mitotic checkpoint signalling is one of the leading causes of chromosomal instability in eukaryotic cells. In recent decades, more than 10 protein components playing differential roles in the mitotic checkpoint control have been identified in eukaryotic cells, including budding uninhibited by benzimidazole (BUB) proteins, BUB1-3, mitotic arrest-deficient (MAD) proteins (MAD1–3), cell division cycle 20 (CDC 20), monopolar spindle 1 (MPS1), Mis 12, ZW10-interacting protein 1 (ZWINT-1), highly expressed in cancer 1 (HEC1), Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Nuf2, spindle pole body component (SPC) protein 24 (SPC24) and SPC25 [3–11]. At prometaphase, when a single kinetochore is unattached to the microtubules, the MAD1–MAD2 complex will remain localized to the kinetochore, and this will enhance the recruitment of MAD2 to the APC/Ccdc20 complex in the presence of MPS1 activation [12–14], resulting in the formation of mitotic checkpoint complex (MCC), which consists of BUBR1, CDC20, BUB3 and MAD2 and activates mitotic checkpoint signalling to prevent the onset of chromosome segregation [15–19]. When all kinetochores have been attached to the microtubules, this signals the onset of anaphase by ubiquitination of CDC20 and subsequent dissociation of checkpoint proteins from the APC–Ccdc20 complex [20,21]. MAD1 is a key component in mitotic checkpoint signalling. Depletion of MAD1 protein by RNA interference leads to failure of MAD2 to localize to the kinetochore, resulting in impairment of the mitotic checkpoint and aneuploidy in human cells [12,22]. In addition, haplo-insufficiency of MAD1 in J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
52
heterozygous knockout mice promotes genomic instability and enhances the development of various cancers, including lung carcinoma and HCC [23]. Expression of viral oncoprotein, such as Tax, in human T cell leukaemia virus type 1 (HTLV-1) can inactivate mitotic checkpoint signalling via targeting MAD1 [24]. However, mutations of MAD1 in human cancers are uncommon [25–27]. We previously identified a novel isoform of MAD1, which we named MAD1β [28]. The MAD1β isoform has deletion of exon 4 and is derived from alternative splicing of pre-mRNA. Ectopic expression of the MAD1β protein induced mitotic checkpoint impairment via altering the level of the endogenous MAD2 in HCC cell lines [28]. In the present study we identified a novel MAD1 binding partner, proline-rich acidic protein 1 (PRAP1), using yeast-two hybrid screening. Mechanistically, stable expression of PRAP1 in HCC cell lines resulted in a deranged mitotic checkpoint and led to chromosomal instability, with the formation of chromosome bridges and aberrant chromosome numbers. Furthermore, in human HCC tumours, up-regulation of MAD1 protein was associated with down-expression of PRAP1. We propose that PRAP1 may have suppressive roles in mitotic checkpoint signalling in HCC cells, possibly involving down-regulation of MAD1.
KM-F Sze et al
Full-length PRAP1 cDNA was amplified from normal liver cDNA and subcloned into pEFFP-N1 vectors. Cloning of full-length MAD1 and MAD1β cDNA were described previously [28].
Yeast two-hybrid library screening and binding assay The detailed protocol was described previously [29]. The library screening was done by co-transformation of yeast strain Y190 with 1–249 MAD1β–pAS2-1 plasmid and pACT2 fused with normal human liver cDNA library that contained 3.5 × 106 independent clones (Clontech Laboratories, CA, USA).
Generation of PRAP1-stably-expressing cell lines BEL-7402 and SMMC-7721 cell lines were transfected with PRAP1–pEGFP-N1 and pEGFP-N1 vector, using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Stably expressing cells were selected with G418 at 800 µg/ml (for BEL-7402 cells) or 400 µg/ml (for SMMC-7721 cells) for 14 days. These selected stably-expressing clones were maintained in growth medium with G418 at 100 µg/ml.
Measurement of mitotic index Materials and methods Patients and samples Fifty-seven pairs of human HCCs and their corresponding non-tumorous liver tissues from patients with surgical resection for HCC between 1992 and 2001 at Queen Mary Hospital and the University of Hong Kong were randomly selected for study. The patients were aged 24–74 years. All specimens were collected at the time of surgical resection, snap-frozen in liquid nitrogen and kept at −80 ◦ C. Use of human samples was approved by the Institutional Review Board (IRB) of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB).
Cell lines Human HCC cell lines, BEL-7402 and SMMC-7721, were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. They were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high-glucose; GIBCO-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). HEK293 cells were maintained in DMEM high-glucose supplemented with 10% FBS.
Plasmids The N-terminus of MAD1β [1–249 amino acids (aa)] was amplified from full-length MAD1β–Flag –pcDNA3.1+ vector and subcloned into pAS2-1 vector to generate the 1–313 MAD1β–pAS2-1 plasmid. Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
The detailed protocol was described previously [30]. Mitotic indices were determined by evaluating the percentage of cells with chromosomal condensation per total number of viable cells at 24 h after treatment with nocodazole or colcemid [28]. At least 300 cells were counted in each experiment and the experiment was done at least three times.
Cell synchronization and DNA morphological analysis in mitotic cells Cells were enriched at mitotic phase with overnight serum starvation and incubation in culture medium containing 5 mM thymidine for 16 h, followed by replacement with normal culture medium for 12–14 h. The cells were fixed according to the protocol previously described [30]. The number of cells showing chromosome bridges and micronuclei was assessed in at least 200 mitotic cells for each cell line.
Metaphase spread and chromosome counting Cells were treated with 100 ng/ml colcemid for 4 h before harvest. The cells were then harvested by trypsinization, treated with 75 mM KCl at 37◦ C for 20 min and fixed in ice-cooled fixative (methanol:acetic acid, 1:1 v/v). A drop of fixed cells was allowed to fall from a height of around 20 cm onto glass slide. The air-dried slides were then submerged in Giemsa solution diluted with MilliQ water, followed by brief washing once, and allowed to air-dry before microscopic observation. Chromosome counting was done on at least 40 cells for each cell line. J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
Western blot analysis and co-immunoprecipitation (Co-IP) assay For western blot analysis, cells were lysed in 50 mM Tris, 150 mM NaCl, 5 mM EDTA (NET) buffer with 1% NP-40 and complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany), and separated in SDS–PAGE gel for western blot analysis. Immunodetection was performed using anti-Flag (Sigma, St Louis, MO, USA), anti-α-tubulin (Sigma), anti-β-actin (Sigma), anti-PRAP1 (Sigma), anti-MAD1 (Santa Cruz Biotechnology, CA, USA), anti-GFP (Santa Cruz) and anti-MAD2 (Transduction Laboratories, CA, USA) antibodies. For co-IP assay, HEK293 cells were transfected with plasmids containing cDNA of Flag-tagged and EGFP-tagged fusion proteins, respectively. The cells were lysed in NET buffer supplemented with 0.1% Triton X-100 and protease inhibitor cocktail. The cell lysate was incubated with 1 µg anti-Flag (Sigma) antibody with the addition of protein G sepharose. Unbound protein was washed away from the beads before electrophoresis. The anti-GFP and anti-Flag antibodies were used as primary antibodies for western blot analysis.
53
A
B
Immunohistochemical staining for PRAP1 in human HCC samples Immunohistochemistry was performed on formalinfixed, paraffin-embedded (FFPE) sections, as described [31], using anti-PRAP1 monoclonal antibody (Sigma) at 1:500 dilution.
Clinicopathological correlation and statistical analysis The clinicopathological features of HCC patients, including tumour size, cellular differentiation according to the Edmondson grading, venous invasion into portal or hepatic venules, direct liver invasion, tumour microsatellite formation, tumour encapsulation and number of tumour nodules, were analysed using SPSS 20 for Windows (SPSS, Chicago, IL, USA). Fisher’s exact test was used for analysis of categorical data. The results were considered significant at p < 0.05.
Results Identification of PRAP1 as a novel binding partner of MAD1 Previously, we had identified a novel MAD1 isoform, which we named MAD1β [28]. MAD1β has exon 4 deleted at the N-terminal part of the MAD1 protein. In addition, MAD1β displays differential subcellular localization as well as functional roles, as compared with the MAD1 protein [28]. To gain more insight into the molecular basis of these two isoforms, we attempted to search for their potential binding partners of the N-terminal parts of MAD1 and MAD1β Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Figure 1. Identification of PRAP1 as a novel MAD1 binding partner by yeast two-hybrid screening. (A) Result of positive colony lift filter β-galactosidase assay using 1–249 aa MAD1/pAS2-1 as bait. The agar plate showed the positive colonies of co-transformed bait with human liver cDNA fragment in pACT2 plasmid together with MAD1/pAS2-1 plasmid. The filter paper contained the replicates of the yeast colonies from the agar plate, lysed by freeze–thaw cycles and subjected to colony lift filter β-galactosidase assay. The grey spots in the picture represented the positive result from the β-galactosidase assay. With subsequent extraction of yeast DNA and DNA sequencing of the cDNA cloned in pACT2 plasmid, PRAP1 was identified to be one of the interacting partners of 1–249 aa MAD1β protein. (B) Physical binding of Flag-tagged full-length MAD1 and MAD1β with EGFP-tagged PRAP1 protein was demonstrated by co-IP assay. Both MAD1 and MAD1β proteins were co-immunoprecipitated with PRAP1.
proteins. We performed yeast two-hybrid screening of a human liver cDNA library using the 1–249 aa of MAD1β as bait. It was estimated that 1 × 107 transformants were screened, and positive clones obtained were confirmed by colony-lift filter β-galactosidase activity assay. With sequence analysis of 32 positive colonies, one of the clones (clone 50) ligated with incomplete cDNA of PRAP1 (NM_145202; Figure 1A). According to the online database regarding mRNA expression of PRAP1 in various tissue types, PRAP1 is predominantly expressed in liver and kidney [32]. Furthermore, when we cloned the full-length PRAP1 into the pEGFP–N1 vector, we were able to confirm its interaction with both MAD1 and MAD1β by co-IP J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
54
KM-F Sze et al
A
B
Figure 2. Characterization of PRAP1-stably-expressing BEL-7402 and SMMC-7721 HCC cells. (A) The protein levels of exogenous EGFPtagged PRAP1 protein in BEL-7402 and SMMC-7721 cell lines were determined by western blot analysis with anti-GFP antibody. (B) After treatment with either colcemid or nocodazole, the PRAP1-stably-expressing BEL-7402 and SMMC-7721 cell lines showed significantly lower mitotic indices as compared to the corresponding vector control cells.
assay. The result showed that the full-length PRAP1 physically interacted with both MAD1 and MAD1β (Figure 1B).
Ectopic expression of PRAP1 led to mitotic checkpoint impairment The functional role of PRAP1 in mitotic checkpoint signalling was largely unknown. Therefore, we investigated the role of PRAP1 in the mitotic checkpoint in BEL-7042 and SMMC-7721 HCC cell lines. These two cell lines have relatively low mRNA expression of PRAP1 as compared with the immortalized normal liver cell line MIHA (see supplementary material, Figure S1). In addition, these two cell lines have a competent mitotic checkpoint, as we previously reported [30]. The GFP-tagged PRAP1 plasmid was stably transfected into these two HCC cell lines and the level of over-expression was confirmed by western Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
blot analysis, using anti-GFP antibody (Figure 2A). After treatment with nocodazole and colcemid, respectively, the PRAP1-stably-expressing BEL-7402 and SMMC-7721 clones showed a significant reduction of mitotic indices from 58.8–61.1% to 23.3–31.3% in BEL-7402 cells (p = 0.007 and p = 0.008, respectively) and from 59.5–60.5% to 24–24.1% in SMMC7721 cells (p = 0.003 and p < 0.001, respectively), as compared with the corresponding vector control cells (Figure 2B). This indicates a significant degree of loss of mitotic checkpoint competence induced by PRAP1. In addition, formation of chromosome bridges, which is a reflection of mitotic checkpoint defect, was significantly more frequently observed in both PRAP1stably-expressing BEL-7402 and SMMC-7721 cells, as compared with the corresponding vector control cells (p = 0.006 and p = 0.011, respectively) (Figure 3A). Furthermore, micronuclei formation was enhanced in both PRAP1-stably-expressing BEL-7402 and SMMC J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
55
A
B
C
Figure 3. Ectopic expression of PRAP1 led to abnormal chromosome segregation and aberrant chromosomal numbers. (A) In EGFP-tagged PRAP1-stably-expressing cells, 11.5–15% of mitotic cells exhibited chromosome bridge formation (white arrowheads) during mitosis. Chromosome bridge formation was more frequent in the PRAP1-stably-expressing cells as compared with corresponding vector control in both BEL-7402 and SMMC-7721 cell lines (p = 0.006 and 0.011, respectively). (B) Micronuclei formation was more frequent in the PRAP1-stably-expressing cells as compared with the corresponding vector control in both BEL-7402 (8.8% versus 1.3%) and SMMC7721 (12.7% versus 2.1%) cell lines. White arrows indicate the micronuclei formed in cells. (C) With metaphase spreading analysis, PRAP1-stably-expressing cell lines had chromosome numbers ranging widely between 20 and 101, as compared with vector control.
7721 cells, as compared with the corresponding vector control cells (Figure 3B).
Ectopic expression of PRAP1 resulted in aberrant chromosomal numbers To assess the role of PRAP1 in chromosomal instability, the chromosome numbers of individual cells in the PRAP1-stably-expressing BEL-7402 and SMMC7721 cells were compared with the corresponding vector cells. The BEL-7402 vector control cells had chromosome numbers in the range 44–62. In contrast, PRAP1-over-expressing BEL-7402 cells had widely varying chromosome numbers in the range 20–54 (p < 0.001, Levene’s test) (Figure 3C). Similarly, Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
SMMC-7721 vector control cells had chromosome numbers mainly in the range 63–94, whereas the PRAP1-over-expressing SMMC-7721 cells had widely varying chromosome numbers in the range 40–101 (p < 0.001, Levene’s test) (Figure 3C). The results indicate that PRAP1 expression induces a significant change of chromosome numbers, suggestive of chromosomal instability.
PRAP1 expression resulted in reduced MAD1 protein in HCC cells It is known that the MAD1–MAD2 protein complex localizes in the nucleus, binds to unattached kinetochores and activates the mitotic spindle checkpoint J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
56
KM-F Sze et al
A
B
Figure 4. PRAP1 reduced MAD1 protein level. (A) Western blot analysis showed significant reduction of MAD1 protein level in the MAD1 and PRAP1 co-transfected BEL-7402 and SMMC-7721 cells, as compared with the corresponding EGFP co-expressing control cells. However, similar down-regulation of exogenous MAD1β was not seen in MAD1β and PRAP1 co-transfected cells as compared with MAD1β and EGFP co-expressing control cells. (B) Western blot analysis showed significant reduction of endogenous MAD1 protein level in PRAP1-stably-expressing BEL-7402 and SMMC-7721 cells. No reduction of endogenous MAD2 protein level was seen in both PRAP1-expressing BEL-7402 and SMMC-7721 cells.
[24,33]. In this study, we demonstrated that PRAP1 is a protein binding partner of both MAD1 and MAD1β, using a co-IP assay. To dissect the possible mechanism by which PRAP1 led to mitotic checkpoint defect, we examined whether PRAP1 could affect the MAD1 and MAD1β proteins. We transiently co-expressed EGFP-tagged PRAP1 with either Flag-tagged MAD1 or Flag-tagged MAD1β in BEL-7402 and SMMC7721 HCC cells and studied the effect on MAD1 protein. Interestingly, the protein level of MAD1 was dramatically reduced when it was co-expressed with PRAP1, as compared with EGFP protein control in Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
both BEL-7402 and SMMC-7721 cells (Figure 4A). Surprisingly, although the PRAP1 could bind with MAD1β, as shown in co-IP assay, PRAP1 did not alter the protein level of MAD1β as compared with the EGFP controls in both BEL-7402 and SMMC-7721 cells (Figure 4A). To further verify the effects of PRAP1 in altering the MAD1 protein levels, we studied its effect in PRAP1-stably-expressing BEL-7402 and SMMC-7721 cells. Similar to the results from experiments using transient transfection, there was observable down-regulation of protein expression of endogenous MAD1 in PRAP1-stably-expressing cells J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
57
Table 1. Summary of the findings of clinicopathological correlation PRAP1 expression Clinicopathological features Sex Tumour size Cellular differentiation (Edmondson’s grading) Encapsulation Tumour microsatellite formation Number of tumour nodules Direct liver invasion Venous invasion Background liver disease
Under-expression
Normal or over-expression
p
17 6 6 17 7 16 15 8 18 15 26 7 14 8 10 13 12 11
28 6 9 24 18 16 20 13 15 19 27 7 19 11 16 18 18 16
0.517
Male Female ≤ 5 cm > 5 cm I–II III–IV Absent Present Absent Present 1 ≥2 Absent Present Absent Present Normal and chronic hepatitis Cirrhosis
as compared with corresponding vector control cells in both BEL-7402 and SMMC-7721 (Figure 4B). Moreover, the protein levels of endogenous MAD2 in the PRAP1-stably-expressing cells remained more or less unchanged and similar to the corresponding vector control in either BEL-7402 or SMMC-7721 (Figure 4B).
Inverse correlation between the protein levels of PRAP1 and MAD1 in human HCCs In this study, we observed that PRAP1 expression was associated with reduced MAD1 protein in HCC cell lines. We further examined the PRAP1 expression in human HCCs by immunohistochemistry in 57 pairs of human HCCs and their corresponding nontumorous livers and assessed its clinicopathological correlation. PRAP1 was quite abundantly expressed in the non-tumorous livers; 40.4% (23/57) of HCCs showed under-expression of PRAP1 protein as compared with their corresponding non-tumorous livers; 33.3% (19/57) showed over-expression of PRAP1; and 26.3% (15/57) showed similar PRAP1 protein expression. However, no association between the clinicopathological features and PRAP1 under-expression was observed (Table 1). In addition, we performed western blot analysis to detect endogenous MAD1 protein expression in 22 pairs of HCCs randomly selected from these 57 cases of human HCCs with immunohistochemistry for PRAP1 performed. In these 22 cases, relatively low expression of MAD1 protein was seen in the nontumorous liver tissues. The protein levels of PRAP1 and MAD1 correlated inversely with one another and up-regulation of MAD1 protein (by two-fold) was significantly associated with down-regulation of PRAP1 in human HCC tissue (p = 0.030) (Figure 5). Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
1.000 0.110 0.785 0.586 0.532 1.000 1.000 1.000
Discussion In mammalian cells, the MAD1–MAD2 protein complex localizes in the nucleus. It binds to the unattached kinetochores and activates the mitotic spindle checkpoint, thus preventing cells from undergoing improper chromosomal segregation [24,33]. In this study, with yeast two-hybrid screening, we identified PRAP1 as a novel protein interacting partner of MAD1. PRAP1 could physically bind with MAD1 and MAD1β. We further characterized the functional role of PRAP1 in mitotic checkpoint control by stably over-expressing it in two mitotic checkpoint-competent HCC cell lines, BEL-7402 and SMMC-7721 [30], and observed a significant reduction of mitotic indices and severe chromosome aberrations when PRAP1 was over-expressed in these cells. These results indicate that PRAP1 induces mitotic checkpoint incompetence and chromosomal instability. We showed that both transient and stable expression of PRAP1 led to reduced levels of endogenous MAD1 protein in cells. Our results indicate that PRAP1 physically binds to MAD1 and reduces the endogenous level of MAD1. Of note, we observed that in human HCCs the protein levels of PRAP1 and MAD1 correlated inversely with one another and that down-regulation of PRAP1 was significantly associated with up-regulation of MAD1 protein. In mammalian cells, MAD1 and MAD2 have been shown to localize at nuclear pore complexes at interphase and at unattached kinetochores during prometaphase [34], and reduction of MAD2 protein expression was associated with mitotic checkpoint incompetence in HCC and other cancer cell lines [30,35,36]. However, in PRAP1-stably-expressing HCC cells, the protein level of MAD2 remained unchanged. Moreover, using the proteasome inhibitor MG132 to block proteosomedependent degradation in PRAP1-stably-expressing J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
58
KM-F Sze et al
A
B
Figure 5. MAD1 protein expression inversely correlated with PRAP1 expression in human HCCs. (A) Representative cases showing protein expression of PRAP1 in human HCCs by immunohistochemistry. Cases 309 and 343 showed under-expression of PRAP1 in tumorous tissue as compared with the corresponding non-tumorous liver tissues, while Case 202 showed over-expression of PRAP1 in the tumour. (B) Representative cases showing protein expression of MAD1 in human HCCs by western blot analysis. Cases 309 and 343 showed over-expression of MAD1 in tumorous tissue as compared with corresponding non-tumorous liver tissue. Table 1 shows the correlation between PRAP1 and MAD1 protein expression in 22 pairs of human HCC tissues.
cells, our result has shown that this reduction in MAD1 protein level may not involve proteosome-dependent degradation (see supplementary material, Figure S2A). However, with cycloheximide treatment, MAD1 protein level was reduced in the presence of PRAP1, but not EGFP, in BEL-7402 cells (see supplementary material, Figure S2B). Thus, PRAP1 may down-regulate MAD1 in cells and exert a suppressive role in mitotic checkpoint signalling. So far, the exact mechanism of MAD1 stability in cells is largely unknown and further investigation is needed. There are two hypotheses in relation to the transcriptional regulation of PRAP1, one of which is via p53 to switch on the transcription of PRAP1 upon genotoxic stress treatment [37], whereas the other is through epigenetic regulation to suppress PRAP1 mRNA transcription [38]. Our preliminary data on p53 expression and mutation status in human HCC tumours with PRAP1 under-expression showed that most of the tumour samples show p53 mRNA expression. Furthermore, with direct sequencing of the p53 PCR product, Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
only one missense mutation was found in the p53coding region, suggesting that the p53 status might not be related to the loss of PRAP1 expression in HCC (see supplementary material, Figure S3A). In addition, we preliminarily performed methylation-sensitive PCR on the PRAP1 promoter region to assess whether loss of PRAP1 expression was due to PRAP1 promoter hypermethylation. We observed that seven of 10 cases with PRAP1 under-expression in human HCCs showed PRAP1 partial promoter methylation at the specific locus, –46 nt before the transcriptional start site of the PRAP1 gene (see supplementary material, Figure S3B). Taken together, promoter hypermethylation might play an important role in suppressing PRAP1 gene expression. However, the regulation of PRAP1 gene expression in HCC needs further investigations. Over-expression of MAD1 mRNA and protein has been reported in human breast and liver cancers [28,39]. The expression of MAD1 can be activated by gain-of-function p53 mutant, induction of cellular J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
Novel MAD1 interacting partner PRAP1
proliferation [40,41] and gain of chromosome region 7p22.3, as in small-cell lung cancer [42]. On the other hand, partial down-regulation of MAD1 protein can result in spindle checkpoint inactivation, aneuploidy in cell and enhanced tumour formation in MAD1 heterozygous knock-out mice [22,23]. In recent studies, over-expression of MAD1 and its constitutive targeting to kinetochores could result in chromosomal instability in mammalian cells [43,44]. In addition, it has been suggested that the ratio between MAD1 and MAD2 protein expression level is important in mitotic checkpoint activation [45]. These results suggest that steady amounts of MAD1 and other mitotic checkpoint proteins are important for spindle checkpoint control in cells. It is established that accumulation of genetic alterations occurs along the multistep process of hepatocarcinogenesis [46]; however, whether the expression of PRAP1 is an early or late event and its importance in hepatocarcinogenesis are still largely unknown. Taken together, our data show that PRAP1 is a novel interacting partner of MAD1 and indicate that PRAP1 reduces the endogenous protein level of MAD1, possibly by enhanced degradation, and suppresses mitotic checkpoint signalling in HCC.
Acknowledgements This study was supported by the Hong Kong Research Grants Council General Research Fund (Grant No. HKU 772608) and Collaborative Research Fund (Grant No. HKU 7/CRF/09), and HKU Small Project Fund (Grant No. HKU 201109176118). IOL Ng is Loke Yew Professor in Pathology.
Author contributions KMFS and IOLN designed the study; KMFS, QHYM, GKYC and JMFL designed and performed the experiments; and KMFS and IOLN analysed the data, performed the clinicopathological analysis and wrote the manuscript.
References 1. Rieder CL, Cole RW, Khodjakov A, et al. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J Cell Biol 1995; 130: 941–948. 2. Ng IO, Lai EC, Ho JC, et al. Flow cytometric analysis of DNA ploidy in hepatocellular carcinoma. Am J Clin Pathol 1994; 102: 80–86. 3. Abrieu A, Magnaghi-Jaulin L, Kahana JA, et al. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell 2001; 106: 83–93. 4. Visintin R, Prinz S, Amon A. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 1997; 278: 460–463. Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
59
5. Martin-Lluesma S, Stucke VM, Nigg EA. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 2002; 297: 2267–2270. 6. DeLuca JG, Howell BJ, Canman JC, et al. Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores. Curr Biol 2003; 13: 2103–2109. 7. Obuse C, Iwasaki O, Kiyomitsu T, et al. A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1. Nat Cell Biol 2004; 6: 1135–1141. 8. Li R, Murray AW. Feedback control of mitosis in budding yeast. Cell 1991; 66: 519–531. 9. Hoyt MA, Totis L, Roberts BT. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 1991; 66: 507–517. 10. DeLuca JG, Gall WE, Ciferri C, et al. Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 2006; 127: 969–982. 11. Bharadwaj R, Qi W, Yu H. Identification of two novel components of the human NDC80 kinetochore complex. J Biol Chem 2004; 279: 13076–13085. 12. Luo X, Tang Z, Rizo J, et al. The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol Cell 2002; 9: 59–71. 13. Hewitt L, Tighe A, Santaguida S, et al. Sustained Mps1 activity is required in mitosis to recruit O–Mad2 to the Mad1–C–Mad2 core complex. J Cell Biol 2010; 190: 25–34. 14. Lad L, Lichtsteiner S, Hartman JJ, et al. Kinetic analysis of Mad2–Cdc20 formation: conformational changes in Mad2 are catalyzed by a C–Mad2–ligand complex. Biochemistry 2009; 48: 9503–9515. 15. Fang G, Yu H, Kirschner MW. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev 1998; 12: 1871–1883. 16. Chen RH. BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1. J Cell Biol 2002; 158: 487–496. 17. Fraschini R, Beretta A, Sironi L, et al. Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores. Embo J 2001; 20: 6648–6659. 18. Hardwick KG, Johnston RC, Smith DL, et al. MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p. J Cell Biol 2000; 148: 871–882. 19. Braunstein I, Miniowitz S, Moshe Y, et al. Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint. Proc Natl Acad Sci USA 2007; 104: 4870–4875. 20. Stegmeier F, Rape M, Draviam VM, et al. Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 2007; 446: 876–881. 21. Howell BJ, Hoffman DB, Fang G, et al. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. J Cell Biol 2000; 150: 1233–1250. 22. Kienitz A, Vogel C, Morales I, et al. Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol. Oncogene 2005; 24: 4301–4310. 23. Iwanaga Y, Chi YH, Miyazato A, et al. Heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1) increases the incidence of tumors in mice. Cancer Res 2007; 67: 160–166. 24. Jin DY, Spencer F, Jeang KT. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 1998; 93: 81–91. 25. Tsukasaki K, Miller CW, Greenspun E, et al. Mutations in the mitotic check point gene, MAD1L1, in human cancers. Oncogene 2001; 20: 3301–3305. J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
60
26. Cahill DP, da Costa LT, Carson-Walter EB, et al. Characterization of MAD2B and other mitotic spindle checkpoint genes. Genomics 1999; 58: 181–187. 27. Nomoto S, Haruki N, Takahashi T, et al. Search for in vivo somatic mutations in the mitotic checkpoint gene, hMAD1, in human lung cancers. Oncogene 1999; 18: 7180–7183. 28. Sze KM, Ching YP, Jin DY, et al. Role of a novel splice variant of mitotic arrest deficient 1 (MAD1), MAD1β, in mitotic checkpoint control in liver cancer. Cancer Res 2008; 68: 9194–9201. 29. Yam JW, Ko FC, Chan CY, et al. Interaction of deleted in liver cancer 1 with tensin 2 in caveolae and implications in tumor suppression. Cancer Res 2006; 66: 8367–8372. 30. Sze KM, Ching YP, Jin DY, et al. Association of MAD2 expression with mitotic checkpoint competence in hepatoma cells. J Biomed Sci 2004; 11: 920–927. 31. Wong CM, Fan ST, Ng IO. beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance. Cancer 2001; 92: 136–145. 32. Yanai I, Benjamin H, Shmoish M, et al. Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification. Bioinformatics 2005; 21: 650–659. 33. Chen RH, Shevchenko A, Mann M, et al. Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores. J Cell Biol 1998; 143: 283–295. 34. Campbell MS, Chan GK, Yen TJ. Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase. J Cell Sci 2001; 114: 953–963. 35. Wang X, Jin DY, Wong YC, et al. Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells. Carcinogenesis 2000; 21: 2293–2297. 36. Wang X, Jin DY, Ng RW, et al. Significance of MAD2 expression to mitotic checkpoint control in ovarian cancer cells. Cancer Res 2002; 62: 1662–1668.
KM-F Sze et al
37. Huang BH, Zhuo JL, Leung CH, et al. PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage. Cell Death Dis 2012; 3: e442. 38. Zhang J, Wong H, Ramanan S, et al. The proline-rich acidic protein is epigenetically regulated and inhibits growth of cancer cell lines. Cancer Res 2003; 63: 6658–6665. 39. Yuan B, Xu Y, Woo J-H, et al. Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability. Clin Cancer Res 2006; 12: 405–410. 40. Iwanaga Y, Jeang KT. Expression of mitotic spindle checkpoint protein hsMAD1 correlates with cellular proliferation and is activated by a gain-of-function p53 mutant. Cancer Res 2002; 62: 2618–2624. 41. Gualberto A, Aldape K, Kozakiewicz K, et al. An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control. Proc Natl Acad Sci USA 1998; 95: 5166–5171. 42. Coe BP, Lee EH, Chi B, et al. Gain of a region on 7p22.3, containing MAD1L1, is the most frequent event in small-cell lung cancer cell lines. Genes Chromosomes Cancer 2006; 45: 11–19. 43. Maldonado M, Kapoor TM. Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation. Nat Cell Biol 2011; 13: 475–482. 44. Ryan SD, Britigan EM, Zasadil LM, et al. Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons. Proc Natl Acad Sci USA 2012; 109: E2205–2214. 45. Schuyler SC, Wu YF, Kuan VJ. The Mad1–Mad2 balancing act – a damaged spindle checkpoint in chromosome instability and cancer. J Cell Sci 2012; 125: 4197–4206. 46. Lee JM, Wong CM, Ng IO. Hepatitis B virus-associated multistep hepatocarcinogenesis: a stepwise increase in allelic alterations. Cancer Res 2008; 68: 5988–5996.
SUPPLEMENTARY MATERIAL ON THE INTERNET The following supplementary material may be found in the online version of this article: Figure S1. mRNA expression of PRAP1 in human HCC cell lines. Figure S2. (A) No alteration of MAD1 protein upon MG-132 treatment in PRAP1-stably-expressing HCC cells. (B) With cycloheximide treatment, MAD1 protein level was reduced in the presence of PRAP1, but not EGFP, in BEL-7402 cells. Figure S3. Transcriptional regulation of PRAP1 in HCC.
Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2014; 233: 51–60 www.thejournalofpathology.com
