Journal of Pathology J Pathol 2015; 237: 460–471 Published online 19 August 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4592
ORIGINAL PAPER
PBRM1 (BAF180) protein is functionally regulated by p53-induced protein degradation in renal cell carcinomas Stephan Macher-Goeppinger,1,2* Martina Keith,1,2 Katrin E Tagscherer,1,2 Stephan Singer,1 Juliane Winkler,1 Thomas G Hofmann,3 Sascha Pahernik,4 Stefan Duensing,5 Markus Hohenfellner,4 Juergen Kopitz,6 Peter Schirmacher1 and Wilfried Roth1,2 1 2 3 4 5 6
Institute of Pathology, University Hospital Heidelberg, Germany Molecular Tumour Pathology, German Cancer Research Centre (DKFZ), Heidelberg, Germany Cellular Senescence, German Cancer Research Centre (DKFZ), DKFZ–ZMBH Alliance, Heidelberg, Germany Department of Urology, University Hospital Heidelberg, Germany Molecular Uro-oncology, Department of Urology, University Hospital Heidelberg, Germany Department of Applied Tumour Biology, Institute of Pathology, University Hospital Heidelberg, Germany
*Correspondence to: S Macher-Goeppinger, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany. E-mail: [email protected]
Abstract About 40% of clear-cell renal cell carcinomas (ccRCC) harbour mutations in Polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex. This qualifies PBRM1 as a major cancer gene in ccRCC. The PBRM1 protein alters chromatin structure and its known functions include transcriptional regulation by controlling the accessibility of DNA and influencing p53 transcriptional activity. Since little is known about the regulation of PBRM1, we studied possible mechanisms and interaction partners involved in the regulation of PBRM1 expression. Activation of p53 in RCC cells resulted in a marked decrease of PBRM1 protein levels. This effect was abolished by siRNA-mediated down-regulation of p53, and transcriptional activity was not crucial for p53-dependent PBRM1 regulation. Pulse-chase experiments determined post-translational protein degradation to be the underlying mechanism for p53-dependent PBRM1 regulation, which was accordingly inhibited by proteasome inhibitors. The effects of p53 activation on PBRM1 expression were confirmed in RCC tissue ex vivo. Our results demonstrate that PBRM1 is a target of p53-induced proteasomal protein degradation and provide further evidence for the influence of PBRM1 on p53 function in RCC tumour cells. Considering the paramount role of p53 in carcinogenesis and the presumptive impact of PBRM1 in RCC development, this novel regulation mechanism might be therapeutically exploited in the future. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: renal cell carcinoma; kidney; PBRM1; TP53; protein degradation
Received 15 December 2014; Revised 23 June 2015; Accepted 13 July 2015
No conflicts of interest were declared.
Introduction Von Hippel–Lindau disease is an autosomal dominant disorder predisposing individuals to benign and malignant tumours, including clear-cell renal cell carcinoma (ccRCC). In 1993, Latif et al identified the von-Hippel–Lindau tumour suppressor gene (VHL) on chromosome 3p25.3 [1]. Somatic inactivation of VHL occurs in about 70% of sporadic ccRCCs [2], which comprise the majority of malignant tumours arising in the kidneys. This qualifies VHL loss as a driving force in ccRCC pathogenesis. Mutations in oncogenes or tumour suppressor genes that are frequently mutated in other epithelial tumour entities, such as RAS, RB or TP53, do not seem to contribute substantially to ccRCC genesis. Nevertheless, the p53 pathway is compromised in kidney cancer by a still unknown mechanism, as Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
shown in RCC-derived cell lines [3], and increased expression of p53, independent of mutations, is associated with reduced patient survival and disease progression [4,5]. This indicates the clinical relevance of p53 in RCC. Although loss of VHL function is a pivotal step in the development of ccRCC, additional genetic alterations have to accumulate to finally give rise to ccRCC [6]. However, until recently knowledge about further somatic mutations in ccRCC was limited. Based on technical improvements in DNA sequencing, the level of knowledge about sporadic mutations in ccRCC is increasing rapidly and substantial genetic heterogeneity has been reported [7]. Varela et al have shown that about 40% of ccRCCs harbour mutations in polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex (PBAF) [8]. The J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
PBRM1 is regulated by p53 in renal cell carcinomas
PBRM1 protein contributes to transcriptional regulation by altering chromatin structure and controlling the accessibility of DNA [9]. Interestingly, besides PBRM1, several other genes influencing chromatin organization, such as BAP1, SETD2, KDM5C and KDM6A, have been identified to be mutated in ccRCC [7,10,11]. This highlights defects of chromatin remodelling and histone methylation as crucial steps in ccRCC development. PBRM1 regulates p53 function by influencing p53 transcriptional activity and is required for p53-induced senescence and proper p21 expression [12,13]. Burrows et al [12] showed that PBRM1 together with BRD7 (another subunit of the SWI/SNF chromatin remodelling complex) is required for p53 transcriptional activity towards multiple target genes, and suggested that disruption of PBAF compromises p53 function. Moreover, Xia et al [13] described BAF180 as a physiological mediator of p21 expression. However, little is known about the regulatory mechanisms governing PBRM1 expression and function in RCC. Here, we have characterized the regulation of PBRM1 and identified an unforeseen regulatory mechanism driven by p53.
Patients, materials and methods Materials A list of primary antibodies and inhibitors is provided in Table S1 (see supplementary material).
Patients Tissue samples from 932 patients with primary RCC treated at the Department of Urology at the University of Heidelberg between 1987 and 2005 were collected. The human tissue samples were provided by the Tissue Bank of the National Centre for Tumour Diseases (NCT), Heidelberg, after approval by the ethics committee of the University of Heidelberg. Clinical follow-up was available for 912 cases. Patient treatment and evaluation was performed as described previously [14]. Survival was calculated from the date of surgery until the last visit or death. All tissue samples were reviewed by at least two pathologists experienced in urological pathology (SMG and WR). Tumour classification and grading were performed according to the World Health Organization [2]; for staging, the 7th edition of the TNM classification (TNM 2009) was used. The study focuses on 767 patients with clear-cell renal cell carcinoma. The clinical and pathological features are summarized in Table S2 (see supplementary material). Median follow-up time was 58 (mean 69) months.
Tissue microarrays and immunohistochemistry A series of tissue microarrays containing 932 primary tumour and corresponding normal tissue samples were created as described previously. Slide preparation and Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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semi-automated staining were performed as indicated previously [14]. Primary antibodies were used as follows: polyclonal rabbit anti-PBRM1 antiserum (1:30; Sigma; HPA015629) for 45 min; monoclonal mouse anti-p53 antibody (1:50, M7001, DakoCytomation) for 25 min. PBRM1 expression was independently scored by two experienced pathologists (SMG and WR) blinded to tissue annotations and patient outcomes.
Cell lines ACHN, A704, 769-P, 786-0 and HepG2 (purchased from ATCC, Rockville, USA); HuH6 (purchased from JCRB Cell Bank, Japan); HCT-116 and HCT-116p53 –/– (kindly provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD, USA); SW-480 and RKO were authenticated by SNP-based profiling [15], which was performed in the core facility of the DKFZ; HK-2 (kindly provided by I. Hofmann, Division of Vascular Oncology and Metastasis, DKFZ, Heidelberg, Germany); FuDDLS and T449 have been characterized previously [16–18]. All cell lines were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS), 1 mM glutamine, 25 mM glucose and 1% penicillin–streptomycin (Life Technologies) and cultured at 37 ∘ C in a 5% CO2 atmosphere. For the experiments described here, the cells were thawed and cultured for no more than 3 months. Cell lines were regularly tested for contamination by multiplex PCR [19], which was performed in the core facility of the DKFZ. For preparation of cell pellets, cells were fixed and embedded in paraffin as follows: briefly, exponentially growing cells were harvested using trypsin and resuspended and washed in phosphate-buffered saline (PBS) and fixed in 4% formalin, followed by another wash step; then the cells were transferred into 100% alcohol and precipitated with 30% bovine serum albumin (BSA). Paraffin embedding was done according to standard protocols also used for tissue samples.
Immunoblot analysis Cells were rinsed twice with ice-cold PBS and lysed on ice with 1× cell lysis buffer (Cell Signaling, cat. no. 9803) containing 1× protease/phosphatase inhibitor cocktail (Cell Signaling, cat. no. 5872). After 15 min on ice, the lysates were centrifuged at 13 000 × g for 20 min at 4 ∘ C. The total protein concentration of the lysates was measured using the Bradford assay (Bio-Rad Protein Assay, Bio-Rad, Munich, Germany); 25–50 μg protein/lane were separated on 6–12% polyacrylamide gels and blotted onto nitrocellulose membrane, using standard procedures. The membranes were blocked and incubated overnight with primary antibody at 4 ∘ C, followed by another extended washing procedure and incubation with the appropriate secondary antibody. Bound antibodies were visualized by an enhanced chemiluminescence detection system (Western Lightning Plus-ECL, Perkin Elmer, Hamburg, Germany). J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Densitometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). PBRM1 and p21 expression levels were quantified in relation to vinculin and normalized to control cells.
siRNA-mediated gene knock-down Transient transfection was used to introduce short interfering RNA (siRNA) against PBRM1 (Santa Cruz, sc-76075), MDM-2 (Dharmacon/Thermo Scientific, Lafayett, USA; J-003279-12-0005) and GSK-3 (Dharmacon/Thermo Scientific, J-003010-12-0005), in accordance with the manufacturers’ instructions. Knock-down of p53 and p21 was performed as described previously [20].
Adenoviral transduction Recombinant Ad5–CMV–PBRM1 and Ad5-empty (referred to as Ad5–PBRM1 or Ad5-empty) were obtained from Sirion Biotech (Martinsried, Germany). For adenoviral transduction, cells were incubated with the AdVs directly after seeding, using a multiplicity of infection of 5 for A704 and 100 for ACHN. The culture medium was replaced 24 h post-transduction.
Quantitative PCR (qPCR) analysis qPCR was performed as described previously [20]. The following primer pairs were used: pbrm1, forward 5′ -CCAAAGCGAAGAAATCAACC-3′ , reverse 5′ -CTGGAAGTCAGCAGTCAGCA-3′ ; actin, forward 5′ -CCTAAAAGCCACCCCACTTCTC-3′ , reverse 5′ -A TGCTATCACCTCCCCTGTGTG-3′ . Primer pairs against p21, 14–3–3σ, ATF3 and PUMA have been described previously [20,21].
Colony-forming assay After adenoviral transduction, cells were plated at variable densities (500, 2000, 5000)/well and allowed to grow for 10 days to form colonies, which were then stained with crystal violet (5 g/l; ACROS Organics, Geel, Belgium). The plates were photographed and the entire plate areas were counted using ClonoCounter densitometric software, with a threshold of 120 [22].
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incubated in 20 μM (tumours 1 and 3) or 40 μM (tumour 2) Nutlin-3 for 24 h. After treatment, the tissue slices were fixed in formalin and embedded in paraffin. The approval of the local ethics committee of the University Hospital of Heidelberg, Germany, was acquired for usage of tumour tissue for research purposes. All data were analysed anonymously.
Pulse-chase experiments These were performed as described previously [24]. The cells were washed with PBS and starved in methionine/cysteine-free Dulbecco’s modified Eagle’s medium (DMEM) for 30 min, then labelled by adding 500 μCi [35 S]-cysteine/methionine (Met-[35 S]-label, Hartmann Analytic, Braunschweig, Germany)/plate. After labelling, the cells were washed with PBS and chased with complete DMEM for the indicated times. Then the radiolabelled cells were sonicated in lysis buffer (30 mM Tris–HCl, pH 7.4, 120 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM KCl, 10% Triton X-100; AppliChem, Darmstadt, Germany, cat. no. 4975.0500) and incubated for 30 min at 4 ∘ C. Supernatants were collected by a 30-min centrifugation at 4 ∘ C and adjusted to the same protein content with lysis buffer. Lysates were incubated with anti-PBRM1 (1:50) for at least 1 h at 4 ∘ C. Preparation of recombinant-Protein G-Sepharose 4B conjugate (Invitrogen, Darmstadt, Germany, cat. no. 101241): 30 μl (=15 μg) of well-mixed Sepharose beads/sample were treated five times with ice-cold 1× PBS and collected by centrifugation (1 min, 4000 rpm). The pellet was dispersed in 30 μl sterile 1× PBS and added to the lysate–antibody mixture. After an overnight incubation at 4 ∘ C, the beads were collected by 10 min of centrifugation at 4000 rpm (4 ∘ C), washed five times with buffer for IP (containing 1% Triton X-100), followed by 2 min of 2500 rpm centrifugation at 4 ∘ C. Proteins were released from the beads by incubation with 0.5 M NaOH at 37 ∘ C for 1 h. Finally, protein samples were mixed with Ultima Gold™ scintillation cocktail (PerkinElmer) and counted for radioactivity in a liquid scintillation counter with automatic quench correction (Perkin Elmer). Non-specific binding to agarose beads was determined by conducting the whole immunoprecipitation procedure without PBRM1 antibody. All measurements were corrected for non-specific binding.
Ex vivo tissue slice technique This was performed as described previously [23]. Fresh human renal cell carcinoma tissue samples were obtained from the Tissue Bank of NCT Heidelberg, directly after surgery. They were maintained in RPMI medium on ice and cut into 300 μm-thick slices (Leica VT1200 S vibrating blade microtome; Leica, Wetzlar, Germany). Tissue slices were then placed onto porous filter membranes, suspended in six-well plates and cultured in RPMI supplemented with 10% fetal calf serum (FCS) and 1% penicillin–streptomycin mixture in a conventional CO2 incubator. The slices were then Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Statistical methods Data were analysed using the R software package, v. 2.5.1 (http://www.rproject.org). For count data, Fisher’s exact test (two-sided) was used. The Kaplan–Meier method was applied to calculate survival probabilities for both progression-free and cancer-specific overall survival. For multivariate analysis, the Cox proportional hazards regression model was used. Univariate survival data were tested for significance using the Mantel–Haenszel log-rank test. p < 0.05 was considered significant. J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
PBRM1 is regulated by p53 in renal cell carcinomas
Results PBRM1 expression in RCCs and comparison with clinical and pathological features We first examined endogenous PBRM1 expression in different RCC cell lines by immunoblot analysis. As depicted in Figure 1A, PBRM1 protein was detected in all cell lines except A-704 (a cell line with loss of PBRM1) [8]. To compare immunoblot and immunohistochemical analyses, we next performed PBRM1 immunocytochemistry on cell block sections, revealing, as expected, negative A704 cells, in contrast to HK-2 cells with prominent nuclear staining (Figure 1B). These results demonstrated that the antibody used recognized PBRM1 in formalin-fixed, paraffin-embedded (FFPE) cells and in immunoblots. Next, we examined human tumour tissue, using a tissue microarray containing tumour samples of 767 patients with clear-cell carcinoma; 122 cases had to be excluded from further analyses, due to either insufficient tumour tissue, fixation artifacts interfering with PBRM1 immunohistochemistry or missing patient information, leaving 645 cases for evaluation; 395 (61%) tumours showed nuclear PBRM1 positivity in variable quantity and intensity; the remaining 250 (39%) were negative (Figure 1C–E). When tumours were grouped according to PBRM1 expression, univariate survival analysis revealed a (statistically non-significant) tendency to reduced cancer-specific survival in patients affected by tumours, with loss of PBRM1 expression (Figure 1F). In multivariate analysis using the Cox proportional hazards model, including grade of malignancy, tumour extent, distant and regional lymph node metastasis, gender and performance status, PBRM1 hazards for cancer-specific death did not reach statistical significance (see supplementary material, Table S3). Comparison of PBRM1 expression with clinical and pathological parameters revealed that loss of PBRM1 expression was significantly associated with higher tumour extent (see supplementary material, Table S4). This finding is in line with results described by Pawlowski et al [25]. No other significant association was found for PBRM1 expression and other clinical or pathological parameters. Additionally, we quantitatively analysed PBRM1-positive RCCs in more detail by assessing the percentage of positive tumour cell nuclei. Figure S1A, B (see supplementary material) depicts Kaplan–Meier curves and bar graphs based on survival analysis and distribution according to clinical and pathological parameters. However, this additional statistical analysis did not reveal further statistical significant differences or prognostic information.
PBRM1 regulates p53 function in renal cell carcinoma Varela et al [8] reported that silencing of PBRM1 increased proliferation of RCC cell lines in vitro, enhanced colony formation and stimulated cell migration. We consistently found that adenoviral transduction Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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of PBRM1 into A704 cells (harbouring a homozygous PBRM1-truncating mutation) resulted in diminished colony formation (Figure 2A, B). To investigate whether PBRM1 alters p53 function, we examined the effects of PBRM1 transduction on p21, a well-known transcriptional target of p53. Furthermore, we silenced PBRM1 expression by siRNA knock-down, and activated p53 by Nutlin-3 treatment. As demonstrated in Figure 2C, over-expression of PBRM1 by adenoviral transduction results in a moderately higher p21 transcription, whereas the Nutlin-3-dependent activation of p21 was diminished upon siRNA-mediated down-regulation of PBRM1. The effect of p53-induced p21 activation on PBRM1 was also confirmed at the protein level (Figure 2D). Whereas transcription of p21 is inhibited after PBRM1 knock-down, mRNA levels of 14–3–3σ, another p53 target [21], are increased; in contrast, mRNA levels of PUMA and ATF3 are not substantially influenced by PBRM1 knock-down (see supplementary material, Figure S2A). This finding suggests that PBRM1 does not necessarily repress, but rather modulates, the transcriptional activity of p53. To further elucidate the relationship between PBRM1 and p21, we took advantage of the the Cancer Genome Atlas (TCGA) data on renal clear cell carcinoma [26]. In Figure S2B (see supplementary material), data are depicted as boxplots of reverse-phase protein array (RPPA) analysis, using the cBioPortal for Cancer Genomics [27,28], which shows significant reduction of p21 protein levels (p = 0.001) in ccRCCs with mutation in PBRM1. Of note, only one of 424 ccRCCs harboured mutations in both TP53 and PBRM1 (see supplementary material, Figure S2C). These findings support a PBRM1-dependent modification of p53 transcriptional activity in RCC cells.
PBRM1 expression is influenced by p53 activation Importantly, we observed a substantial decrease of PBRM1 protein levels after Nutlin-3 treatment (Figure 2D), indicating a dynamic regulation of PBRM1 protein levels. Time-course experiments substantiated this finding and showed that PBRM1 is down-regulated with a delay of about 12 h upon p53-dependent p21 induction (Figure 3A). Additionally, radiation-induced p53 activation also resulted in diminished PBRM1 levels (Figure 3B). To exclude that these effects were a cell-line specific phenomenon, we repeated the experiments using different cell lines, including cell lines derived from RCC, colon carcinoma and hepatoblastoma (Figure 3C). All cell lines expressing functional p53 (ACHN, 769-P, HCT116, RKO, HepG2, HUH-6) showed a strong down-regulation of PBRM1 after Nutlin-3-mediated p53 activation, whereas cell lines with impaired p53 function, such as HCT-116 p53 –/– , SW-480 or 786-O, exhibited no effect on PBRM1 protein levels after Nutlin-3 treatment. Therefore, functional p53 is required for Nutlin-3- and radiation-induced decrease of PBRM1. To corroborate these cell line-derived in vitro results J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 1. Expression of PBRM1 in human RCCs and association with patient survival. (A) Immunoblot analysis of A704 (harbouring a homozygous PBRM1 truncating mutation), Caki-2, ACHN, 786-O, 796-P and HK-2 cells. (B) Immunohistochemistry of FFPE cell pellets. (C–E) Immunohistochemistry on human tumour samples (ccRCCs); black arrowhead, tumour nuclei; empty arrowhead, nuclei of endothelial cells (intrinsic positive control); scale bar = 20 μm; (C) complete loss of PBRM1; (D) loss of PBRM1 in only a fraction of tumour cells; (E) uniformly positive nuclei in all tumour cells. (F) Cancer-specific survival depending on PBRM1 expression, depicted as Kaplan–Meier curves Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Figure 2. Over-expression of PBRM1 reduces colony formation and loss of PBRM1 alters p53 function. (A) Colony formation assay of A704 cells transduced with PBRM1, empty vector and wild-type as control; colonies were stained with crystal violet, photographed and counted using densitometric software. (B) Immunoblot analysis of transduced A704 cells. (C) Quantitative real-time PCR assaying relative p21 mRNA expression of A704 and ACHN cells upon adenoviral transduction and ACHN cells 48 h after siRNA transfection and consecutive Nutlin-3 (10 μM) treatment for 24 h (total time span, 72 h). Results are the mean of three individual measurements; error bars indicate ± SD. (D) Immunoblot analysis of ACHN cells 48 h after siRNA transfection and consecutive Nutlin-3 (10 μM) treatment for 24 h (total time span, 72 h)
in a less artificial model, we used an ex vivo RCC tissue slice culture technique to examine p53-dependent PBRM1 expression in primary human RCC tissue. As demonstrated in Figure 4A, the extent of p53 activation is mirrored by a respective down-regulation of PBRM1 in the tissue. Besides, the heterogeneity of PBRM1 staining in HK-2 cells (Figure 1B) might be caused by a substantial heterogeneity in p53 expression levels, as depicted in Figure S3A (see supplementary Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
material). Furthermore, silencing of p53 in ACHN cells reduced the effects of Nutlin-3 or radiation on PBRM1, confirming the p53-dependent regulation of PBRM1 expression (Figure 4B; see also supplementary material, Figure S3B). Time course experiments with siRNA-mediated down-regulation of p21 and concomitant treatment with Nutlin-3 resulted in delayed and diminished degradation of PBRM1, indicating that p21 is functionally involved in p53-mediated PBRM1 J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 3. PBRM1 is repressed upon p53 activation in cell lines of different origin. (A) Immunoblot analysis of ACHN cells treated with Nutlin-3 (10 μM); PBRM1 and p21 expression levels were densitometrically quantified using ImageJ software. (B) Immunoblot analysis of ACHN cells upon ionizing radiation. (C) Effects of Nutlin-3 treatment (24 h, 10 μM) on cell lines of different origin (kidney, ACHN, 786-O and 769-P; colon, HCT116, SW-480 and RKO; liver, HepG2 and HUH-6). Of these, 786-O, HCT116-p53 –/– and SW-480 had impaired p53 function
down-regulation (Figure 4C). Together, these results indicate that p53 activation mediates depletion of PBRM1.
p53 induces PBRM1 protein degradation To clarify the mechanism of p53-dependent PBRM1 regulation, we performed RT–PCR analysis to examine whether p53 alters PBRM1 expression at the transcriptional level. Although a moderate reduction of PBRM1 mRNA levels was observed after treatment with Nutlin-3 (Figure 5A), the marked decrease of protein levels, as observed in Figures 2D and 3A, was not explained solely by this effect. Besides transcriptional regulation of p53 target genes, other mechanisms, such as protein degradation, have been described [29,30]. Therefore, we performed a pulse-chase experiment that showed a substantial degradation of PBRM1 upon p53 activation (Figure 5B; see also supplementary material, Figure S3C), whereas general protein stability was not affected (see supplementary material, Figure S3D). Together, these data indicate that protein degradation is crucial for p53-dependent repression of PBRM1. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
PBRM1 degradation is proteasome-dependent Protein degradation can occur via lysosome- or proteasome-dependent pathways. To determine which process was responsible for PBRM1 degradation, we treated RCC cells with proteasome and lysosome inhibitors. Co-treatment with the proteasome inhibitor MG-132 rescued PBRM1 degradation upon Nutlin-3-induced p53 activation in ACHN and HCT-116 cells (Figure 5C). In contrast, co-treatment with Z-VAD (a caspase inhibitor) did not influence PBRM1 protein levels. Similar results were found upon treatment with PS-341 (Bortezomib), another proteasome inhibitor (Figure 5D). No substantial effect on PBRM1 degradation was observed upon lysosome inhibition (see supplementary material, Figure S4A, B). P53-dependent proteasomal degradation can be mediated by MDM-2, a p53-regulated E3 ubiquitin ligase [31]. Therefore, we examined the effects of MDM-2 on PBRM1 protein levels by using MDM-2-specific siRNA. Cell lines derived from dedifferentiated liposarcomas and well differentiated liposarcomas with endogenous MDM-2 over-expression (MDM-2 amplification is a crucial step in the J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 4. Regulation of PBRM1 protein expression levels by p53 and p21 in vitro and ex vivo. (A) Nutlin-3 treatment depletes PBRM1 in ex vivo RCC tissue samples: immunohistochemical analysis of PBRM1 and p53 expression after treatment with 20 μM (tumours 1 and 3) or 40 μM (tumour 2) Nutlin-3 for 24 h; representative images are shown; scale bar = 50 μm. (B) Immunoblot analysis of ACHN cells after gene silencing by various siRNAs (siPB1, 50 nM; siP53 1, 25 nM; siP53 2, 25 nM; 48 h) and subsequent 24 h treatment with Nutlin-3 (10 μM). (C) Immunoblot analysis of ACHN cells after gene silencing of p21 by siRNA for 24 h and subsequent treatment with Nutlin-3 (10 μM)
development of these tumour entities) showed a substantial decrease of PBRM1 protein upon siRNA-mediated down-regulation of MDM-2, with consequent p53 activation, as indicated by the increased p21 levels (Figure 6A). MDM2 silencing in ACHN RCC cells resulted in similar but less marked effects (see supplementary material, Figure S4C). Hence, MDM-2 is most likely not required for PBRM1 degradation. Another protein that has been linked to p53-induced protein degradation is GSK-3 [32]. Treatment with LiCl, an Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
inhibitor of GSK3, resulted in reduced degradation of PBRM1 (Figure 6B). Nevertheless, siRNA-mediated down-regulation of GSK3 revealed that the LiCl effect is independent of GSK3 (Figure 6C). This finding is in line with previous reports describing GSK3-independent direct inhibition of proteasomal activity by LiCl [33]. However, co-treatment with Nutlin-3 and PYR-41, a specific inhibitor of ubiquitin-activating enzyme (E1) [34], abolished PBRM1 degradation, indicating that ubiquitin-dependent proteasomal protein degradation J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 5. p53-dependent PBRM1 regulation; transcriptional repression and protein degradation. (A) Quantitative real-time PCR assaying relative p21 and PBRM1 mRNA expression of different cell lines upon 48 h of 10 μM Nutlin-3 treatment; results are the means of three individual measurements; error bars indicate ± SD. (B) Immunoprecipitation of PBRM1 and pulse-chase analysis of ACHN cells after Nutlin-3 (10 μM) treatment for 1, 2, 4, 8 and 24 h; results are the means (± SD) of three independent measurements. (C) Immunoblot analysis of ACHN, HCT-116 and HCT-116 p53 –/– cells after (co-)treatment with Nutlin-3 (10 μM), MG-132 (20 μM) and Z-VAD (40 μM) for 16 h. (D) ACHN cells pretreated with PS-341 (48 nM) for 24 h and then co-treated with Nutlin-3 (10 μM) for 24 h; total time span, 48 h
is a pivotal mechanism in p53-mediated regulation of PBRM1 (Figure 6D).
Discussion Clear-cell renal cell carcinomas are a VHL-associated tumour entity. Not only hereditary carcinomas in the context of autosomal dominant inherited von-Hippel– Lindau syndrome, but also the vast majority of non-familial ccRCCs display loss of VHL function, due to either deletions, mutations or epigenetic mechanisms [2]. However, it was presumed that additional mutations are required for the development of malignant tumours [6]. Recent advances in DNA-sequencing techniques have revealed that genes coding for proteins participating in the regulation of Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
chromatin remodelling and histone methylation are commonly mutated in ccRCCs [7,8,10,11]. Among these is PBRM1, which encodes the BAF180 subunit of a SWI–SNF complex designated PBAF [35,36]. PBRM1 is not exclusively inactivated in ccRCC but also in other tumour entities, with loss of heterozygosity in 48% of breast cancers [13], mutations in 17% of intrahepatic cholangiocarcinomas [37] and recurrent truncating insertions and deletions (indels) in pancreatic cancers [38]. This gave rise to the assumption that PBRM1 functions as a tumour suppressor gene. Truncating mutations of PBRM1 in up to 40% of ccRCCs have been reported, qualifying PBRM1 as a major ccRCC cancer gene [8]. Our immunohistochemistry studies revealed complete loss of PBRM1 expression in up to 39% of ccRCCs; however, a heterogeneous staining pattern was observed in a substantial fraction J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 6. Mechanisms of p53-induced protein degradation of PBRM1. Immunoblot analysis of: (A) FuDDLS and T442 cells after gene silencing of MDM-2 by siRNA (*20 nM;**50 nM). (B) ACHN, HCT116 and HCT-116 p53 –/– cells after treatment with LiCl (ACHN 75 nM, HCT116 5, 10 or 25 nM) and/or 10 μM Nutlin-3 for 24 h. (C) ACHN cells after gene silencing of GSK3β by siRNA (70 nM; 24, 48 and 72 h) and subsequent treatment with 10 μM Nutlin-3 for 24 h. (D) ACHN cells treated with PYR-41 (20 μM) for 4 h and then co-treated with Nutlin-3 (20 h, 10 μM); total time span, 24 h
of tumours, favouring functional regulation of protein levels in RCC without loss of PBRM1. Little is known about PBRM1. However, so far an influence on p53 function has been described in primary fibroblasts and breast cancer cell lines with decreased p53-dependent p21-expression after silencing of PBRM1 [12,13]. Our results demonstrate similar effects in ccRCC-derived cell lines. Interestingly, although the majority of RCCs possess wild-type TP53, impairment of the p53 pathway is observed. An unknown dominant mechanism, acting in RCCs and normal kidney tissue, has been proposed [3]. Recent findings indicating aberrant chromatin remodelling in ccRCCs [7,10,11] may illuminate this Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
so far puzzling finding. On the other hand, PBRM1 mutations disturb p53-dependent chromatin regulation, which may contribute to ccRCC development by enabling escape from p53-mediated tumour surveillance. Considering PBRM1 as a co-factor for proper p53 function, PBRM1 mutations would be more relevant in p53 wild-type tumours. Interestingly, a query [24,25] on the data provided by the TCGA [23] revealed that only one of 424 ccRCCs harboured concurrent mutations in TP53 and PBRM1, whereas the remaining 160 ccRCCs with PBRM1 mutation expressed wild-type TP53, implying a tendency toward mutual exclusivity (p = 0.1). J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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We demonstrated that re-expression of PBRM1 in A704 cells (devoid of PBRM1) results in diminished colony formation, which is in line with previous reports showing that knock-down of PBRM1 enhanced colony formation in p53-deficient SN12C cells [8]. However, knock-down of PBRM1 in ACHN RCC cells with wild-type p53 did not result in increased colony formation (data not shown). These varying results may reflect cell line- and context-dependent functions of PBRM1. In addition, we observed that PBRM1 itself is subject to p53-dependent post-translational protein degradation, as shown by pulse-chase assays. Treatment with proteasome inhibitors abrogated this effect and our results obtained with PYR-41 suggest E1-enzyme-dependent targeting of PBRM1 to the proteasome. However, the siRNA studies revealed that this process is independent of GSK3β and MDM-2, which have been shown to have the potential to facilitate p53-dependent protein degradation [32,39,40]. p53 has an essential role in maintaining genomic integrity and targeted chromatin remodelling [41]. p53-dependent depletion of BAF180 may be another way in which p53 regulates chromatin structure to control transcriptional activity. In addition, p53 also regulates other subunits of PBAF, including BRG1 (see supplementary material, Figure S4D). This suggests a profound effect of p53 not only on this SWI/SNF complex, but also on BAF (Brg1-associated factors). At first glance, it is unexpected that the tumour suppressor p53, often classified as a gatekeeper for growth and division [42], represses PBRM1. However, recent studies have identified several pro-survival pathways engaged by transcriptional activity of p53 [43]. In addition, p53 has been linked to stimulus-dependent, pro-survival, anti-apoptotic functions. For instance, p53 drives proteasome-dependent degradation of the apoptotic kinase HIPK2 through up-regulation of the E3 ligase Siah1, which facilitates cellular recovery upon mild genotoxic stress [44]. Besides, at present the knowledge of PBRM1 function is limited. Given that chromatin modification results in widespread alteration of gene expression and defines cell behaviour and fate [45,46], it is likely that PBRM1 may have additional functions. In summary, our data describe a close interrelation between p53 and PBRM1 in RCCs: PBRM1 influences transcriptional activity of p53 and p53 induces protein degradation of PBRM1. For now it remains unclear whether this functional interaction contributes to tumour surveillance and whether disruption of this interaction by PBRM1 mutation facilitates RCC development or progression. Future studies, for example detailed monitoring of chronological activation of p53 targets with reference to PBRM1 status, will be needed to clarify the precise biological significance. However, given the paramount role of p53 in tumour biology and PBRM1 being a major cancer gene in RCCs, our studies may point to a molecular connection with a pivotal impact on the cellular and molecular biology of renal cell carcinomas. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Acknowledgements We thank David Jansen for excellent technical assistance and Hildegard Jakobi for excellent help with the patient data and the Tissue Bank of the National Centre for Tumour Diseases, Heidelberg. The results shown here are based in part upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/
Author contributions SMG, WK, MH, SD, SP and PS developed the study design; SMG and WR conceived experiments; SMG, MK, KET, SS and JW carried out experiments; and SMG, WK, TGH, KET and JK analysed and interpreted data. All authors were involved in writing the paper and had final approval of the submitted version.
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SUPPLEMENTARY MATERIAL ON THE INTERNET The following supplementary material may be found in the online version of this article: Figure S1. Graphical presentation of survival analysis and distribution of clinical and pathological parameters in PBRM1-positive ccRCCs Figure S2. qPCR analysis of additional transcriptional targets of p53 and TCGA data demonstrating reduction of p21 protein levels in ccRCCs with mutation in PBRM1 Figure S3. Expression of p53 in HK-2 cells Figure S4. Effect of lysosome inhibition and MDM2 silencing on PBRM1 protein levels and impact of Nutlin-3 treatment on other subunits of the PBAF complex Table S1. List of antibodies and inhibitors Table S2. Clinico-pathological characteristics of the study population Table S3. Uni- and multivariate analyses of prognostic factors influencing cancer-specific survival (CSS) Table S4. Correlation of PBRM1 expression in clear-cell renal cell carcinomas with clinico-pathological characteristics
Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
ORIGINAL PAPER
PBRM1 (BAF180) protein is functionally regulated by p53-induced protein degradation in renal cell carcinomas Stephan Macher-Goeppinger,1,2* Martina Keith,1,2 Katrin E Tagscherer,1,2 Stephan Singer,1 Juliane Winkler,1 Thomas G Hofmann,3 Sascha Pahernik,4 Stefan Duensing,5 Markus Hohenfellner,4 Juergen Kopitz,6 Peter Schirmacher1 and Wilfried Roth1,2 1 2 3 4 5 6
Institute of Pathology, University Hospital Heidelberg, Germany Molecular Tumour Pathology, German Cancer Research Centre (DKFZ), Heidelberg, Germany Cellular Senescence, German Cancer Research Centre (DKFZ), DKFZ–ZMBH Alliance, Heidelberg, Germany Department of Urology, University Hospital Heidelberg, Germany Molecular Uro-oncology, Department of Urology, University Hospital Heidelberg, Germany Department of Applied Tumour Biology, Institute of Pathology, University Hospital Heidelberg, Germany
*Correspondence to: S Macher-Goeppinger, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany. E-mail: [email protected]
Abstract About 40% of clear-cell renal cell carcinomas (ccRCC) harbour mutations in Polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex. This qualifies PBRM1 as a major cancer gene in ccRCC. The PBRM1 protein alters chromatin structure and its known functions include transcriptional regulation by controlling the accessibility of DNA and influencing p53 transcriptional activity. Since little is known about the regulation of PBRM1, we studied possible mechanisms and interaction partners involved in the regulation of PBRM1 expression. Activation of p53 in RCC cells resulted in a marked decrease of PBRM1 protein levels. This effect was abolished by siRNA-mediated down-regulation of p53, and transcriptional activity was not crucial for p53-dependent PBRM1 regulation. Pulse-chase experiments determined post-translational protein degradation to be the underlying mechanism for p53-dependent PBRM1 regulation, which was accordingly inhibited by proteasome inhibitors. The effects of p53 activation on PBRM1 expression were confirmed in RCC tissue ex vivo. Our results demonstrate that PBRM1 is a target of p53-induced proteasomal protein degradation and provide further evidence for the influence of PBRM1 on p53 function in RCC tumour cells. Considering the paramount role of p53 in carcinogenesis and the presumptive impact of PBRM1 in RCC development, this novel regulation mechanism might be therapeutically exploited in the future. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: renal cell carcinoma; kidney; PBRM1; TP53; protein degradation
Received 15 December 2014; Revised 23 June 2015; Accepted 13 July 2015
No conflicts of interest were declared.
Introduction Von Hippel–Lindau disease is an autosomal dominant disorder predisposing individuals to benign and malignant tumours, including clear-cell renal cell carcinoma (ccRCC). In 1993, Latif et al identified the von-Hippel–Lindau tumour suppressor gene (VHL) on chromosome 3p25.3 [1]. Somatic inactivation of VHL occurs in about 70% of sporadic ccRCCs [2], which comprise the majority of malignant tumours arising in the kidneys. This qualifies VHL loss as a driving force in ccRCC pathogenesis. Mutations in oncogenes or tumour suppressor genes that are frequently mutated in other epithelial tumour entities, such as RAS, RB or TP53, do not seem to contribute substantially to ccRCC genesis. Nevertheless, the p53 pathway is compromised in kidney cancer by a still unknown mechanism, as Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
shown in RCC-derived cell lines [3], and increased expression of p53, independent of mutations, is associated with reduced patient survival and disease progression [4,5]. This indicates the clinical relevance of p53 in RCC. Although loss of VHL function is a pivotal step in the development of ccRCC, additional genetic alterations have to accumulate to finally give rise to ccRCC [6]. However, until recently knowledge about further somatic mutations in ccRCC was limited. Based on technical improvements in DNA sequencing, the level of knowledge about sporadic mutations in ccRCC is increasing rapidly and substantial genetic heterogeneity has been reported [7]. Varela et al have shown that about 40% of ccRCCs harbour mutations in polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex (PBAF) [8]. The J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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PBRM1 protein contributes to transcriptional regulation by altering chromatin structure and controlling the accessibility of DNA [9]. Interestingly, besides PBRM1, several other genes influencing chromatin organization, such as BAP1, SETD2, KDM5C and KDM6A, have been identified to be mutated in ccRCC [7,10,11]. This highlights defects of chromatin remodelling and histone methylation as crucial steps in ccRCC development. PBRM1 regulates p53 function by influencing p53 transcriptional activity and is required for p53-induced senescence and proper p21 expression [12,13]. Burrows et al [12] showed that PBRM1 together with BRD7 (another subunit of the SWI/SNF chromatin remodelling complex) is required for p53 transcriptional activity towards multiple target genes, and suggested that disruption of PBAF compromises p53 function. Moreover, Xia et al [13] described BAF180 as a physiological mediator of p21 expression. However, little is known about the regulatory mechanisms governing PBRM1 expression and function in RCC. Here, we have characterized the regulation of PBRM1 and identified an unforeseen regulatory mechanism driven by p53.
Patients, materials and methods Materials A list of primary antibodies and inhibitors is provided in Table S1 (see supplementary material).
Patients Tissue samples from 932 patients with primary RCC treated at the Department of Urology at the University of Heidelberg between 1987 and 2005 were collected. The human tissue samples were provided by the Tissue Bank of the National Centre for Tumour Diseases (NCT), Heidelberg, after approval by the ethics committee of the University of Heidelberg. Clinical follow-up was available for 912 cases. Patient treatment and evaluation was performed as described previously [14]. Survival was calculated from the date of surgery until the last visit or death. All tissue samples were reviewed by at least two pathologists experienced in urological pathology (SMG and WR). Tumour classification and grading were performed according to the World Health Organization [2]; for staging, the 7th edition of the TNM classification (TNM 2009) was used. The study focuses on 767 patients with clear-cell renal cell carcinoma. The clinical and pathological features are summarized in Table S2 (see supplementary material). Median follow-up time was 58 (mean 69) months.
Tissue microarrays and immunohistochemistry A series of tissue microarrays containing 932 primary tumour and corresponding normal tissue samples were created as described previously. Slide preparation and Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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semi-automated staining were performed as indicated previously [14]. Primary antibodies were used as follows: polyclonal rabbit anti-PBRM1 antiserum (1:30; Sigma; HPA015629) for 45 min; monoclonal mouse anti-p53 antibody (1:50, M7001, DakoCytomation) for 25 min. PBRM1 expression was independently scored by two experienced pathologists (SMG and WR) blinded to tissue annotations and patient outcomes.
Cell lines ACHN, A704, 769-P, 786-0 and HepG2 (purchased from ATCC, Rockville, USA); HuH6 (purchased from JCRB Cell Bank, Japan); HCT-116 and HCT-116p53 –/– (kindly provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD, USA); SW-480 and RKO were authenticated by SNP-based profiling [15], which was performed in the core facility of the DKFZ; HK-2 (kindly provided by I. Hofmann, Division of Vascular Oncology and Metastasis, DKFZ, Heidelberg, Germany); FuDDLS and T449 have been characterized previously [16–18]. All cell lines were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS), 1 mM glutamine, 25 mM glucose and 1% penicillin–streptomycin (Life Technologies) and cultured at 37 ∘ C in a 5% CO2 atmosphere. For the experiments described here, the cells were thawed and cultured for no more than 3 months. Cell lines were regularly tested for contamination by multiplex PCR [19], which was performed in the core facility of the DKFZ. For preparation of cell pellets, cells were fixed and embedded in paraffin as follows: briefly, exponentially growing cells were harvested using trypsin and resuspended and washed in phosphate-buffered saline (PBS) and fixed in 4% formalin, followed by another wash step; then the cells were transferred into 100% alcohol and precipitated with 30% bovine serum albumin (BSA). Paraffin embedding was done according to standard protocols also used for tissue samples.
Immunoblot analysis Cells were rinsed twice with ice-cold PBS and lysed on ice with 1× cell lysis buffer (Cell Signaling, cat. no. 9803) containing 1× protease/phosphatase inhibitor cocktail (Cell Signaling, cat. no. 5872). After 15 min on ice, the lysates were centrifuged at 13 000 × g for 20 min at 4 ∘ C. The total protein concentration of the lysates was measured using the Bradford assay (Bio-Rad Protein Assay, Bio-Rad, Munich, Germany); 25–50 μg protein/lane were separated on 6–12% polyacrylamide gels and blotted onto nitrocellulose membrane, using standard procedures. The membranes were blocked and incubated overnight with primary antibody at 4 ∘ C, followed by another extended washing procedure and incubation with the appropriate secondary antibody. Bound antibodies were visualized by an enhanced chemiluminescence detection system (Western Lightning Plus-ECL, Perkin Elmer, Hamburg, Germany). J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Densitometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). PBRM1 and p21 expression levels were quantified in relation to vinculin and normalized to control cells.
siRNA-mediated gene knock-down Transient transfection was used to introduce short interfering RNA (siRNA) against PBRM1 (Santa Cruz, sc-76075), MDM-2 (Dharmacon/Thermo Scientific, Lafayett, USA; J-003279-12-0005) and GSK-3 (Dharmacon/Thermo Scientific, J-003010-12-0005), in accordance with the manufacturers’ instructions. Knock-down of p53 and p21 was performed as described previously [20].
Adenoviral transduction Recombinant Ad5–CMV–PBRM1 and Ad5-empty (referred to as Ad5–PBRM1 or Ad5-empty) were obtained from Sirion Biotech (Martinsried, Germany). For adenoviral transduction, cells were incubated with the AdVs directly after seeding, using a multiplicity of infection of 5 for A704 and 100 for ACHN. The culture medium was replaced 24 h post-transduction.
Quantitative PCR (qPCR) analysis qPCR was performed as described previously [20]. The following primer pairs were used: pbrm1, forward 5′ -CCAAAGCGAAGAAATCAACC-3′ , reverse 5′ -CTGGAAGTCAGCAGTCAGCA-3′ ; actin, forward 5′ -CCTAAAAGCCACCCCACTTCTC-3′ , reverse 5′ -A TGCTATCACCTCCCCTGTGTG-3′ . Primer pairs against p21, 14–3–3σ, ATF3 and PUMA have been described previously [20,21].
Colony-forming assay After adenoviral transduction, cells were plated at variable densities (500, 2000, 5000)/well and allowed to grow for 10 days to form colonies, which were then stained with crystal violet (5 g/l; ACROS Organics, Geel, Belgium). The plates were photographed and the entire plate areas were counted using ClonoCounter densitometric software, with a threshold of 120 [22].
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incubated in 20 μM (tumours 1 and 3) or 40 μM (tumour 2) Nutlin-3 for 24 h. After treatment, the tissue slices were fixed in formalin and embedded in paraffin. The approval of the local ethics committee of the University Hospital of Heidelberg, Germany, was acquired for usage of tumour tissue for research purposes. All data were analysed anonymously.
Pulse-chase experiments These were performed as described previously [24]. The cells were washed with PBS and starved in methionine/cysteine-free Dulbecco’s modified Eagle’s medium (DMEM) for 30 min, then labelled by adding 500 μCi [35 S]-cysteine/methionine (Met-[35 S]-label, Hartmann Analytic, Braunschweig, Germany)/plate. After labelling, the cells were washed with PBS and chased with complete DMEM for the indicated times. Then the radiolabelled cells were sonicated in lysis buffer (30 mM Tris–HCl, pH 7.4, 120 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM KCl, 10% Triton X-100; AppliChem, Darmstadt, Germany, cat. no. 4975.0500) and incubated for 30 min at 4 ∘ C. Supernatants were collected by a 30-min centrifugation at 4 ∘ C and adjusted to the same protein content with lysis buffer. Lysates were incubated with anti-PBRM1 (1:50) for at least 1 h at 4 ∘ C. Preparation of recombinant-Protein G-Sepharose 4B conjugate (Invitrogen, Darmstadt, Germany, cat. no. 101241): 30 μl (=15 μg) of well-mixed Sepharose beads/sample were treated five times with ice-cold 1× PBS and collected by centrifugation (1 min, 4000 rpm). The pellet was dispersed in 30 μl sterile 1× PBS and added to the lysate–antibody mixture. After an overnight incubation at 4 ∘ C, the beads were collected by 10 min of centrifugation at 4000 rpm (4 ∘ C), washed five times with buffer for IP (containing 1% Triton X-100), followed by 2 min of 2500 rpm centrifugation at 4 ∘ C. Proteins were released from the beads by incubation with 0.5 M NaOH at 37 ∘ C for 1 h. Finally, protein samples were mixed with Ultima Gold™ scintillation cocktail (PerkinElmer) and counted for radioactivity in a liquid scintillation counter with automatic quench correction (Perkin Elmer). Non-specific binding to agarose beads was determined by conducting the whole immunoprecipitation procedure without PBRM1 antibody. All measurements were corrected for non-specific binding.
Ex vivo tissue slice technique This was performed as described previously [23]. Fresh human renal cell carcinoma tissue samples were obtained from the Tissue Bank of NCT Heidelberg, directly after surgery. They were maintained in RPMI medium on ice and cut into 300 μm-thick slices (Leica VT1200 S vibrating blade microtome; Leica, Wetzlar, Germany). Tissue slices were then placed onto porous filter membranes, suspended in six-well plates and cultured in RPMI supplemented with 10% fetal calf serum (FCS) and 1% penicillin–streptomycin mixture in a conventional CO2 incubator. The slices were then Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Statistical methods Data were analysed using the R software package, v. 2.5.1 (http://www.rproject.org). For count data, Fisher’s exact test (two-sided) was used. The Kaplan–Meier method was applied to calculate survival probabilities for both progression-free and cancer-specific overall survival. For multivariate analysis, the Cox proportional hazards regression model was used. Univariate survival data were tested for significance using the Mantel–Haenszel log-rank test. p < 0.05 was considered significant. J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Results PBRM1 expression in RCCs and comparison with clinical and pathological features We first examined endogenous PBRM1 expression in different RCC cell lines by immunoblot analysis. As depicted in Figure 1A, PBRM1 protein was detected in all cell lines except A-704 (a cell line with loss of PBRM1) [8]. To compare immunoblot and immunohistochemical analyses, we next performed PBRM1 immunocytochemistry on cell block sections, revealing, as expected, negative A704 cells, in contrast to HK-2 cells with prominent nuclear staining (Figure 1B). These results demonstrated that the antibody used recognized PBRM1 in formalin-fixed, paraffin-embedded (FFPE) cells and in immunoblots. Next, we examined human tumour tissue, using a tissue microarray containing tumour samples of 767 patients with clear-cell carcinoma; 122 cases had to be excluded from further analyses, due to either insufficient tumour tissue, fixation artifacts interfering with PBRM1 immunohistochemistry or missing patient information, leaving 645 cases for evaluation; 395 (61%) tumours showed nuclear PBRM1 positivity in variable quantity and intensity; the remaining 250 (39%) were negative (Figure 1C–E). When tumours were grouped according to PBRM1 expression, univariate survival analysis revealed a (statistically non-significant) tendency to reduced cancer-specific survival in patients affected by tumours, with loss of PBRM1 expression (Figure 1F). In multivariate analysis using the Cox proportional hazards model, including grade of malignancy, tumour extent, distant and regional lymph node metastasis, gender and performance status, PBRM1 hazards for cancer-specific death did not reach statistical significance (see supplementary material, Table S3). Comparison of PBRM1 expression with clinical and pathological parameters revealed that loss of PBRM1 expression was significantly associated with higher tumour extent (see supplementary material, Table S4). This finding is in line with results described by Pawlowski et al [25]. No other significant association was found for PBRM1 expression and other clinical or pathological parameters. Additionally, we quantitatively analysed PBRM1-positive RCCs in more detail by assessing the percentage of positive tumour cell nuclei. Figure S1A, B (see supplementary material) depicts Kaplan–Meier curves and bar graphs based on survival analysis and distribution according to clinical and pathological parameters. However, this additional statistical analysis did not reveal further statistical significant differences or prognostic information.
PBRM1 regulates p53 function in renal cell carcinoma Varela et al [8] reported that silencing of PBRM1 increased proliferation of RCC cell lines in vitro, enhanced colony formation and stimulated cell migration. We consistently found that adenoviral transduction Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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of PBRM1 into A704 cells (harbouring a homozygous PBRM1-truncating mutation) resulted in diminished colony formation (Figure 2A, B). To investigate whether PBRM1 alters p53 function, we examined the effects of PBRM1 transduction on p21, a well-known transcriptional target of p53. Furthermore, we silenced PBRM1 expression by siRNA knock-down, and activated p53 by Nutlin-3 treatment. As demonstrated in Figure 2C, over-expression of PBRM1 by adenoviral transduction results in a moderately higher p21 transcription, whereas the Nutlin-3-dependent activation of p21 was diminished upon siRNA-mediated down-regulation of PBRM1. The effect of p53-induced p21 activation on PBRM1 was also confirmed at the protein level (Figure 2D). Whereas transcription of p21 is inhibited after PBRM1 knock-down, mRNA levels of 14–3–3σ, another p53 target [21], are increased; in contrast, mRNA levels of PUMA and ATF3 are not substantially influenced by PBRM1 knock-down (see supplementary material, Figure S2A). This finding suggests that PBRM1 does not necessarily repress, but rather modulates, the transcriptional activity of p53. To further elucidate the relationship between PBRM1 and p21, we took advantage of the the Cancer Genome Atlas (TCGA) data on renal clear cell carcinoma [26]. In Figure S2B (see supplementary material), data are depicted as boxplots of reverse-phase protein array (RPPA) analysis, using the cBioPortal for Cancer Genomics [27,28], which shows significant reduction of p21 protein levels (p = 0.001) in ccRCCs with mutation in PBRM1. Of note, only one of 424 ccRCCs harboured mutations in both TP53 and PBRM1 (see supplementary material, Figure S2C). These findings support a PBRM1-dependent modification of p53 transcriptional activity in RCC cells.
PBRM1 expression is influenced by p53 activation Importantly, we observed a substantial decrease of PBRM1 protein levels after Nutlin-3 treatment (Figure 2D), indicating a dynamic regulation of PBRM1 protein levels. Time-course experiments substantiated this finding and showed that PBRM1 is down-regulated with a delay of about 12 h upon p53-dependent p21 induction (Figure 3A). Additionally, radiation-induced p53 activation also resulted in diminished PBRM1 levels (Figure 3B). To exclude that these effects were a cell-line specific phenomenon, we repeated the experiments using different cell lines, including cell lines derived from RCC, colon carcinoma and hepatoblastoma (Figure 3C). All cell lines expressing functional p53 (ACHN, 769-P, HCT116, RKO, HepG2, HUH-6) showed a strong down-regulation of PBRM1 after Nutlin-3-mediated p53 activation, whereas cell lines with impaired p53 function, such as HCT-116 p53 –/– , SW-480 or 786-O, exhibited no effect on PBRM1 protein levels after Nutlin-3 treatment. Therefore, functional p53 is required for Nutlin-3- and radiation-induced decrease of PBRM1. To corroborate these cell line-derived in vitro results J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 1. Expression of PBRM1 in human RCCs and association with patient survival. (A) Immunoblot analysis of A704 (harbouring a homozygous PBRM1 truncating mutation), Caki-2, ACHN, 786-O, 796-P and HK-2 cells. (B) Immunohistochemistry of FFPE cell pellets. (C–E) Immunohistochemistry on human tumour samples (ccRCCs); black arrowhead, tumour nuclei; empty arrowhead, nuclei of endothelial cells (intrinsic positive control); scale bar = 20 μm; (C) complete loss of PBRM1; (D) loss of PBRM1 in only a fraction of tumour cells; (E) uniformly positive nuclei in all tumour cells. (F) Cancer-specific survival depending on PBRM1 expression, depicted as Kaplan–Meier curves Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Figure 2. Over-expression of PBRM1 reduces colony formation and loss of PBRM1 alters p53 function. (A) Colony formation assay of A704 cells transduced with PBRM1, empty vector and wild-type as control; colonies were stained with crystal violet, photographed and counted using densitometric software. (B) Immunoblot analysis of transduced A704 cells. (C) Quantitative real-time PCR assaying relative p21 mRNA expression of A704 and ACHN cells upon adenoviral transduction and ACHN cells 48 h after siRNA transfection and consecutive Nutlin-3 (10 μM) treatment for 24 h (total time span, 72 h). Results are the mean of three individual measurements; error bars indicate ± SD. (D) Immunoblot analysis of ACHN cells 48 h after siRNA transfection and consecutive Nutlin-3 (10 μM) treatment for 24 h (total time span, 72 h)
in a less artificial model, we used an ex vivo RCC tissue slice culture technique to examine p53-dependent PBRM1 expression in primary human RCC tissue. As demonstrated in Figure 4A, the extent of p53 activation is mirrored by a respective down-regulation of PBRM1 in the tissue. Besides, the heterogeneity of PBRM1 staining in HK-2 cells (Figure 1B) might be caused by a substantial heterogeneity in p53 expression levels, as depicted in Figure S3A (see supplementary Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
material). Furthermore, silencing of p53 in ACHN cells reduced the effects of Nutlin-3 or radiation on PBRM1, confirming the p53-dependent regulation of PBRM1 expression (Figure 4B; see also supplementary material, Figure S3B). Time course experiments with siRNA-mediated down-regulation of p21 and concomitant treatment with Nutlin-3 resulted in delayed and diminished degradation of PBRM1, indicating that p21 is functionally involved in p53-mediated PBRM1 J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 3. PBRM1 is repressed upon p53 activation in cell lines of different origin. (A) Immunoblot analysis of ACHN cells treated with Nutlin-3 (10 μM); PBRM1 and p21 expression levels were densitometrically quantified using ImageJ software. (B) Immunoblot analysis of ACHN cells upon ionizing radiation. (C) Effects of Nutlin-3 treatment (24 h, 10 μM) on cell lines of different origin (kidney, ACHN, 786-O and 769-P; colon, HCT116, SW-480 and RKO; liver, HepG2 and HUH-6). Of these, 786-O, HCT116-p53 –/– and SW-480 had impaired p53 function
down-regulation (Figure 4C). Together, these results indicate that p53 activation mediates depletion of PBRM1.
p53 induces PBRM1 protein degradation To clarify the mechanism of p53-dependent PBRM1 regulation, we performed RT–PCR analysis to examine whether p53 alters PBRM1 expression at the transcriptional level. Although a moderate reduction of PBRM1 mRNA levels was observed after treatment with Nutlin-3 (Figure 5A), the marked decrease of protein levels, as observed in Figures 2D and 3A, was not explained solely by this effect. Besides transcriptional regulation of p53 target genes, other mechanisms, such as protein degradation, have been described [29,30]. Therefore, we performed a pulse-chase experiment that showed a substantial degradation of PBRM1 upon p53 activation (Figure 5B; see also supplementary material, Figure S3C), whereas general protein stability was not affected (see supplementary material, Figure S3D). Together, these data indicate that protein degradation is crucial for p53-dependent repression of PBRM1. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
PBRM1 degradation is proteasome-dependent Protein degradation can occur via lysosome- or proteasome-dependent pathways. To determine which process was responsible for PBRM1 degradation, we treated RCC cells with proteasome and lysosome inhibitors. Co-treatment with the proteasome inhibitor MG-132 rescued PBRM1 degradation upon Nutlin-3-induced p53 activation in ACHN and HCT-116 cells (Figure 5C). In contrast, co-treatment with Z-VAD (a caspase inhibitor) did not influence PBRM1 protein levels. Similar results were found upon treatment with PS-341 (Bortezomib), another proteasome inhibitor (Figure 5D). No substantial effect on PBRM1 degradation was observed upon lysosome inhibition (see supplementary material, Figure S4A, B). P53-dependent proteasomal degradation can be mediated by MDM-2, a p53-regulated E3 ubiquitin ligase [31]. Therefore, we examined the effects of MDM-2 on PBRM1 protein levels by using MDM-2-specific siRNA. Cell lines derived from dedifferentiated liposarcomas and well differentiated liposarcomas with endogenous MDM-2 over-expression (MDM-2 amplification is a crucial step in the J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 4. Regulation of PBRM1 protein expression levels by p53 and p21 in vitro and ex vivo. (A) Nutlin-3 treatment depletes PBRM1 in ex vivo RCC tissue samples: immunohistochemical analysis of PBRM1 and p53 expression after treatment with 20 μM (tumours 1 and 3) or 40 μM (tumour 2) Nutlin-3 for 24 h; representative images are shown; scale bar = 50 μm. (B) Immunoblot analysis of ACHN cells after gene silencing by various siRNAs (siPB1, 50 nM; siP53 1, 25 nM; siP53 2, 25 nM; 48 h) and subsequent 24 h treatment with Nutlin-3 (10 μM). (C) Immunoblot analysis of ACHN cells after gene silencing of p21 by siRNA for 24 h and subsequent treatment with Nutlin-3 (10 μM)
development of these tumour entities) showed a substantial decrease of PBRM1 protein upon siRNA-mediated down-regulation of MDM-2, with consequent p53 activation, as indicated by the increased p21 levels (Figure 6A). MDM2 silencing in ACHN RCC cells resulted in similar but less marked effects (see supplementary material, Figure S4C). Hence, MDM-2 is most likely not required for PBRM1 degradation. Another protein that has been linked to p53-induced protein degradation is GSK-3 [32]. Treatment with LiCl, an Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
inhibitor of GSK3, resulted in reduced degradation of PBRM1 (Figure 6B). Nevertheless, siRNA-mediated down-regulation of GSK3 revealed that the LiCl effect is independent of GSK3 (Figure 6C). This finding is in line with previous reports describing GSK3-independent direct inhibition of proteasomal activity by LiCl [33]. However, co-treatment with Nutlin-3 and PYR-41, a specific inhibitor of ubiquitin-activating enzyme (E1) [34], abolished PBRM1 degradation, indicating that ubiquitin-dependent proteasomal protein degradation J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 5. p53-dependent PBRM1 regulation; transcriptional repression and protein degradation. (A) Quantitative real-time PCR assaying relative p21 and PBRM1 mRNA expression of different cell lines upon 48 h of 10 μM Nutlin-3 treatment; results are the means of three individual measurements; error bars indicate ± SD. (B) Immunoprecipitation of PBRM1 and pulse-chase analysis of ACHN cells after Nutlin-3 (10 μM) treatment for 1, 2, 4, 8 and 24 h; results are the means (± SD) of three independent measurements. (C) Immunoblot analysis of ACHN, HCT-116 and HCT-116 p53 –/– cells after (co-)treatment with Nutlin-3 (10 μM), MG-132 (20 μM) and Z-VAD (40 μM) for 16 h. (D) ACHN cells pretreated with PS-341 (48 nM) for 24 h and then co-treated with Nutlin-3 (10 μM) for 24 h; total time span, 48 h
is a pivotal mechanism in p53-mediated regulation of PBRM1 (Figure 6D).
Discussion Clear-cell renal cell carcinomas are a VHL-associated tumour entity. Not only hereditary carcinomas in the context of autosomal dominant inherited von-Hippel– Lindau syndrome, but also the vast majority of non-familial ccRCCs display loss of VHL function, due to either deletions, mutations or epigenetic mechanisms [2]. However, it was presumed that additional mutations are required for the development of malignant tumours [6]. Recent advances in DNA-sequencing techniques have revealed that genes coding for proteins participating in the regulation of Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
chromatin remodelling and histone methylation are commonly mutated in ccRCCs [7,8,10,11]. Among these is PBRM1, which encodes the BAF180 subunit of a SWI–SNF complex designated PBAF [35,36]. PBRM1 is not exclusively inactivated in ccRCC but also in other tumour entities, with loss of heterozygosity in 48% of breast cancers [13], mutations in 17% of intrahepatic cholangiocarcinomas [37] and recurrent truncating insertions and deletions (indels) in pancreatic cancers [38]. This gave rise to the assumption that PBRM1 functions as a tumour suppressor gene. Truncating mutations of PBRM1 in up to 40% of ccRCCs have been reported, qualifying PBRM1 as a major ccRCC cancer gene [8]. Our immunohistochemistry studies revealed complete loss of PBRM1 expression in up to 39% of ccRCCs; however, a heterogeneous staining pattern was observed in a substantial fraction J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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Figure 6. Mechanisms of p53-induced protein degradation of PBRM1. Immunoblot analysis of: (A) FuDDLS and T442 cells after gene silencing of MDM-2 by siRNA (*20 nM;**50 nM). (B) ACHN, HCT116 and HCT-116 p53 –/– cells after treatment with LiCl (ACHN 75 nM, HCT116 5, 10 or 25 nM) and/or 10 μM Nutlin-3 for 24 h. (C) ACHN cells after gene silencing of GSK3β by siRNA (70 nM; 24, 48 and 72 h) and subsequent treatment with 10 μM Nutlin-3 for 24 h. (D) ACHN cells treated with PYR-41 (20 μM) for 4 h and then co-treated with Nutlin-3 (20 h, 10 μM); total time span, 24 h
of tumours, favouring functional regulation of protein levels in RCC without loss of PBRM1. Little is known about PBRM1. However, so far an influence on p53 function has been described in primary fibroblasts and breast cancer cell lines with decreased p53-dependent p21-expression after silencing of PBRM1 [12,13]. Our results demonstrate similar effects in ccRCC-derived cell lines. Interestingly, although the majority of RCCs possess wild-type TP53, impairment of the p53 pathway is observed. An unknown dominant mechanism, acting in RCCs and normal kidney tissue, has been proposed [3]. Recent findings indicating aberrant chromatin remodelling in ccRCCs [7,10,11] may illuminate this Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
so far puzzling finding. On the other hand, PBRM1 mutations disturb p53-dependent chromatin regulation, which may contribute to ccRCC development by enabling escape from p53-mediated tumour surveillance. Considering PBRM1 as a co-factor for proper p53 function, PBRM1 mutations would be more relevant in p53 wild-type tumours. Interestingly, a query [24,25] on the data provided by the TCGA [23] revealed that only one of 424 ccRCCs harboured concurrent mutations in TP53 and PBRM1, whereas the remaining 160 ccRCCs with PBRM1 mutation expressed wild-type TP53, implying a tendency toward mutual exclusivity (p = 0.1). J Pathol 2015; 237: 460–471 www.thejournalofpathology.com
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We demonstrated that re-expression of PBRM1 in A704 cells (devoid of PBRM1) results in diminished colony formation, which is in line with previous reports showing that knock-down of PBRM1 enhanced colony formation in p53-deficient SN12C cells [8]. However, knock-down of PBRM1 in ACHN RCC cells with wild-type p53 did not result in increased colony formation (data not shown). These varying results may reflect cell line- and context-dependent functions of PBRM1. In addition, we observed that PBRM1 itself is subject to p53-dependent post-translational protein degradation, as shown by pulse-chase assays. Treatment with proteasome inhibitors abrogated this effect and our results obtained with PYR-41 suggest E1-enzyme-dependent targeting of PBRM1 to the proteasome. However, the siRNA studies revealed that this process is independent of GSK3β and MDM-2, which have been shown to have the potential to facilitate p53-dependent protein degradation [32,39,40]. p53 has an essential role in maintaining genomic integrity and targeted chromatin remodelling [41]. p53-dependent depletion of BAF180 may be another way in which p53 regulates chromatin structure to control transcriptional activity. In addition, p53 also regulates other subunits of PBAF, including BRG1 (see supplementary material, Figure S4D). This suggests a profound effect of p53 not only on this SWI/SNF complex, but also on BAF (Brg1-associated factors). At first glance, it is unexpected that the tumour suppressor p53, often classified as a gatekeeper for growth and division [42], represses PBRM1. However, recent studies have identified several pro-survival pathways engaged by transcriptional activity of p53 [43]. In addition, p53 has been linked to stimulus-dependent, pro-survival, anti-apoptotic functions. For instance, p53 drives proteasome-dependent degradation of the apoptotic kinase HIPK2 through up-regulation of the E3 ligase Siah1, which facilitates cellular recovery upon mild genotoxic stress [44]. Besides, at present the knowledge of PBRM1 function is limited. Given that chromatin modification results in widespread alteration of gene expression and defines cell behaviour and fate [45,46], it is likely that PBRM1 may have additional functions. In summary, our data describe a close interrelation between p53 and PBRM1 in RCCs: PBRM1 influences transcriptional activity of p53 and p53 induces protein degradation of PBRM1. For now it remains unclear whether this functional interaction contributes to tumour surveillance and whether disruption of this interaction by PBRM1 mutation facilitates RCC development or progression. Future studies, for example detailed monitoring of chronological activation of p53 targets with reference to PBRM1 status, will be needed to clarify the precise biological significance. However, given the paramount role of p53 in tumour biology and PBRM1 being a major cancer gene in RCCs, our studies may point to a molecular connection with a pivotal impact on the cellular and molecular biology of renal cell carcinomas. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Acknowledgements We thank David Jansen for excellent technical assistance and Hildegard Jakobi for excellent help with the patient data and the Tissue Bank of the National Centre for Tumour Diseases, Heidelberg. The results shown here are based in part upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/
Author contributions SMG, WK, MH, SD, SP and PS developed the study design; SMG and WR conceived experiments; SMG, MK, KET, SS and JW carried out experiments; and SMG, WK, TGH, KET and JK analysed and interpreted data. All authors were involved in writing the paper and had final approval of the submitted version.
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SUPPLEMENTARY MATERIAL ON THE INTERNET The following supplementary material may be found in the online version of this article: Figure S1. Graphical presentation of survival analysis and distribution of clinical and pathological parameters in PBRM1-positive ccRCCs Figure S2. qPCR analysis of additional transcriptional targets of p53 and TCGA data demonstrating reduction of p21 protein levels in ccRCCs with mutation in PBRM1 Figure S3. Expression of p53 in HK-2 cells Figure S4. Effect of lysosome inhibition and MDM2 silencing on PBRM1 protein levels and impact of Nutlin-3 treatment on other subunits of the PBAF complex Table S1. List of antibodies and inhibitors Table S2. Clinico-pathological characteristics of the study population Table S3. Uni- and multivariate analyses of prognostic factors influencing cancer-specific survival (CSS) Table S4. Correlation of PBRM1 expression in clear-cell renal cell carcinomas with clinico-pathological characteristics
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