Journal of Pathology J Pathol 2014; 233: 238–246 Published online 21 May 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4356
ORIGINAL PAPER
Mutant p53 accumulation in human breast cancer is not an intrinsic property or dependent on structural or functional disruption but is regulated by exogenous stress and receptor status Pavla Bouchalova,1 Rudolf Nenutil,1 Petr Muller,1 Roman Hrstka,1 M Virginia Appleyard,2 Karen Murray,2 Lee B Jordan,3 Colin A Purdie,3 Philip Quinlan,2 Alastair M Thompson,2,4 Borivoj Vojtesek1 and Philip J Coates5* 1 2 3 4 5
Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic Clinical Research Centre, Dundee Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK Department of Pathology, Ninewells Hospital and Medical School, Dundee, UK Department of Surgical Oncology, MD Anderson Cancer Center, Houston, TX, USA Tayside Tissue Bank, Jacqui Wood Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK
*Correspondence to: PJ Coates, Tayside Tissue Bank, Jacqui Wood Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. E-mail: [email protected]
Abstract Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure–function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: p53; mutation; breast cancer; immunohistochemistry; therapy
Received 3 January 2014; Revised 13 March 2014; Accepted 21 March 2014
No conflicts of interest were declared.
Introduction TP53 gene mutations are common in human tumours and many studies have attempted to correlate p53 mutation status and prognosis, with disappointing results [1–4]. Tumours containing mutant p53 are usually considered as a single class and are compared to tumours that retain wild-type (wt) p53. However, individual TP53 mutations differentially influence the overall functional properties of the mutant protein and certain missense p53 mutant proteins have oncogenic functions, in contrast to the tumour-suppressor function of wtp53 or the loss of function caused by nonsense mutations or TP53 Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
loss [3,4]. Clinically, discriminating between specific TP53 mutations or mutation types provides prognostic information in certain human malignancies [5–10]. One consequence of a missense mutation in TP53 is accumulation of the mutant protein, allowing mutation detection by immunohistochemical staining; tumours showing a high percentage of p53-positive cells are classified as containing mutant p53. This simple approach is inaccurate because of the highly variable numbers of cells that stain for p53 in tumours with wtp53 or mutant p53, confounding the results of many studies [1,11–14]. On the other hand, immunohistochemical studies suggest that missense mutant p53 is not always inherently J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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stabilized in tumour cells, and the extent of protein accumulation may therefore reflect the specific type of mutation or some other aspect of the tumour’s pathobiology. Simple explanations for these observations would be that the immunohistochemical data are due to artifacts of fixation and processing, loss of the mutant allele from some tumour cells, or gain of mutation as a later event during clonal evolution. These explanations are unlikely, because heterogeneity of staining is not localized to specific areas of the tumour. More plausible explanations are that staining patterns reflect the intrinsic nature of the mutation, where some mutant proteins are inherently more stable than others, or where some mutations are particularly disruptive to function and the mutant proteins are consequently less able to induce their own destruction thorough transcriptional regulation of Mdm2 [15]. Indeed, the site and nature of the mutation in p53 results in proteins with native (folded) or denatured (unfolded) structures that have stabilization and degradation pathways different from those of wtp53 [16–18]. TP53 mutations have also been classified as disruptive or non-disruptive to function, depending on mutation site and type [8], and can be given a severity score from structural/functional calculations [19]. Therefore, variations in mutant p53 levels may relate to the inherent stability/activity of the individual mutant, which would allow information regarding the class of mutation by immunohistochemical staining. A more recent hypothesis derived from experimental studies of transgenic mice and zebrafish is that tumour-specific factors are responsible for mutant p53 stabilization [20–24]. In mice containing only a mutant Tp53 gene, p53 accumulation is not seen in non-tumour cells under normal conditions, but occurs in spontaneously developing tumours [20]. Factors that stabilize wtp53 (DNA damage, lack of Mdm2 and oncogenic stress) also stabilize mutant p53 in non-tumour cells. Thus, levels of mutant p53 in human tumours may be symptomatic of specific aspects of tumour pathobiology, such as genetic instability or the precise oncogenic events that have occurred. These hypotheses are not mutually exclusive and the extent of mutant p53 stabilization may represent a combination of intrinsic properties together with endogenous or exogenous stresses. To clarify the reasons for variability of mutant p53 stabilization, we investigated the relationships between specific mutations, cellular stress and p53 levels in a panel of cell lines and primary human breast cancers containing different TP53 missense mutations.
Materials and methods Tissue culture Human cell lines with known p53 mutational status were obtained from ATCC (Table 1) and cultured in the recommended medium containing 10% fetal calf serum (FCS), 0.3 mg/ml L-glutamine and 1% penicillin–streptomycin (Invitrogen, Life Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Table 1. Immunohistochemical and structural analysis of 17 human cell lines for TP53 mutation classification Cell line Mutation HOS BT549 SKUT-1 A431 HT-29 DLD-1 MB231 SW837 BT20 MB468 CAMA-1 RD HT3 SKBR3 MOLT4 BT474 T47D
R156P R249S R175H R273H R273H S241F R280K R248W K132Q R273H R282T R282W G245V R175H R248Q E285K L194F
Staining (%)a
X-rayb
NMRb
99.3 98.3 97.0 96.8 93.2 92.8 92.4 91.6 87.5 86.5 86.3 86.0 86.2 80.5 62.4 62.0 53.4
U (1.84) ? (0.67) n.a. F (0.00) F (0.00) F (0.06) F (0.34) F (0.23) U (1.82) F (0.00) ? (0.61) F (−1.11) ? (0.51) n.a. F (0.21) U (1.21) F (−1.15)
U (1.64) U (1.50) U (0.97) F (−0.04) F (−0.04) F (0.52) n.a. ? (0.64) U (1.29) F (−0.04) U (0.30) F (−0.72) F (1.45) U (0.97) F (0.02) U (1.16) ? (0.56)
Disruptivec Severityd No Yes No No No Yes No Yes No No No No Yes No Yes No No
71.75 95.70 92.55 87.26 87.26 82.44 71.45 83.31 75.40 87.26 94.99 92.37 111.3 92.55 62.49 74.72 66.05
a
The mean percentage of p53-positive cells. The folding scores of mutant p53 predicted from the X-ray crystallography or NMR structures. A higher score indicates a more unfolded structure. U, predicted to be a largely unfolded protein (score > 0.7); F, predicted to adopt a folded structure (score < 0.5); n.a., not available from the structure; ?, no prediction made (score 0.5–0.7). c Classification according to whether or not the mutation disrupts function. d Mutation severity score. A cut-off value of 60.18 discriminates severe from non-severe mutations; all mutations are classed as severe. b
Technologies, Paisley, UK) at 37∘ C and 10% CO2 . Cells were exposed to UV light (40 J/m2 using a Stratalinker 1800) or Roscovitine (10 μM; Sigma-Aldrich, Dorset UK) and collected 16 h later [25]. T47D, BT474 and SKBR3 cells were also treated with the Mdm2 inhibitor Nutlin-3 (10 μM; Sigma-Aldrich) for 24 h. To produce cell pellets, cells were fixed in 4% neutral buffered formalin, resuspended in 1.5% molten agarose and processed into paraffin wax using standard histological procedures. Alternatively, cells grown directly onto glass slides were fixed in methanol:acetone (50:50) at −20∘ C for 10 min, air-dried overnight and stored at −80∘ C. For colony formation, cells were dissociated into single cells through 21g needles, checked for the absence of doublets by microscopy before plating at cloning density on glass slides, and fixed after colonies of > 20 cells had formed (7–10 days).
Human tumour samples A series of 240 untreated primary breast cancers taken at a resectional surgery in Dundee were collected. Portions of tissue were used for diagnostic histopathology and separate tumour pieces were dissected by a specialist breast pathologist and stored frozen for analysis of TP53 DNA sequence, using the Roche Amplichip (Roche Diagnostics). Patient details have been reported previously [26]. A second cohort of 55 triple-negative breast cancers was examined by direct Sanger sequencing and p53 immunohistochemistry. Microarrays were prepared in the Tayside Tissue Bank from diagnostic histopathology blocks, using six 0.6 mmm cores/cancer. The use of the tissues was approved by local ethical J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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review and all patients provided informed consent for the collection and use of their tumour material for research. For this study, only those tumours identified as containing mutant p53 were analysed.
Immunohistochemistry and analysis of staining patterns Sections and cells were stained for p53 using rabbit polyclonal CM1 antiserum and the mouse monoclonal antibody DO-1. Immunoreactivity was detected by avidin–biotin complex peroxidase staining (Elite ABC Kit, Vector Laboratories, Peterborough, UK) or by EnVision+ System–HRP (Dako Denmark A/S) with diaminobenzidine as chromogen, as described [11,27]. For cell lines, the percentage of cells showing p53 immunostaining was calculated by examination of at least 10 independent microscope fields. p53 staining patterns in tumours were described as showing ‘heterogeneous’ or ‘homogeneous’ staining by visual examination of the images by two investigators with experience of p53 immunohistochemistry (RN and PJC). Homogeneous staining was defined as > 90% of tumour cells positive for nuclear p53 staining, whereas heterogeneous staining was defined by > 10% of nuclei showing an absence of p53 staining. Variable intensity of staining in individual cells is not taken into account when considering heterogeneity – a simple positive and negative staining for each tumour cell was used. In practice, tumours either show a very small number of negative cells ( 20% negative cells, so that the discrimination of homogeneity and heterogeneity is easily achieved. Breast cancer microarrays were also stained for Ki67 (Dako), Cdkn1a/p21 and Mdm2 [11,27]. The latter two were scored by the QuickScore method, where intensity of staining is recorded on a scale of 0–3 and percentages of positive cells are scored on a scale of 0–6, and the two values multiplied to give scores in the range 0–18 [28]. Only the percentage scale is recorded for Ki67. Oestrogen receptor (ER), progesterone receptor (PR) and the HER2 status of breast cancers were assessed by immunohistochemistry and in situ hybridization, as previously reported [29,30].
Subclassification of p53 mutation types by sequence data Using the nucleotide sequence data from each cell line and tumour, we classified TP53 mutations using four different approaches. (a) We predicted the effect of the mutation on the relative ability of the protein to fold into a wild-type conformation using the wtp53 structures derived from X-ray crystallography and (b) those derived from NMR data, calculating the structural effects of individual mutations with PoPMuSic [31]. This algorithm produces a score for folding and allows classification as either unfolded or folded. The data are applicable only to the core regions of p53 and predictions are lacking for some individual amino acid residues in either the NMR-derived structure or the X-ray Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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structure. Because the NMR and X-ray data sometimes provide different folding predictions, we analysed each method separately. Non-sense mutations (stop codon or frame-shift) that lead to an absence of protein are not classified as folded or unfolded. (c) Mutations were classified as disruptive or non-disruptive, based on the degree of disturbance to the structure of p53 in complex with DNA [8]. In brief, disruptive mutations produce a non-similar amino acid within the L2 and L3 loops of p53 or introduce a stop codon into the coding sequence. This scheme classifies some TP53 hotspot mutations as non-disruptive, due to similarity between wild-type and mutant amino acids, eg the common R175H and R273H mutations [8]. (d) The fourth system uses 12 weighted parameters related to structure, stability and activity to assess the effect of the mutation through the PREDMUT algorithm [19] at: http://www.ifm.liu.se/bioinfo. The combined effects are reported as a severity score and classified as severe if the score is > 60.18 [19].
Statistics For cell line data, Pearson correlation coefficients were used for comparing staining patterns with PoPMuSiC or PREDMUT scores and non-paired two-way t-tests, assuming unequal variance for disruptive versus non-disruptive mutants. Statistical significance of associations of clinical data with staining patterns, folded state, disruptive nature or severity was assessed using two-tailed p values (Fisher’s exact test), using the Inspire programme [32], or Kaplan–Meier for survival. Pearson coefficients or ANOVA were used for continuous variables (folding and severity scores).
Results p53 staining patterns in human cancer cell lines Immunostaining for p53 was optimized to detect a low level of p53 in normal tissues and cell lines with wtp53 [11,27], such that an absence of staining indicates non-sense or frame-shift TP53 mutation or loss of both alleles. In 17 human cell lines that contain p53 missense point mutations, the percentage of p53-positive cells varied between 99.3% and 53.4% (Figure 1, Table 1). Increasing the antibody concentration two-fold did not decrease the proportion of negatively-stained cells. Scoring p53 in multiple different colonies of T47D, SKBR3 and BT474 cells derived from low-density plating showed that each colony contained a mixture of positively stained and unstained cells in the same proportions as in bulk cultures. Cell lines were classified as expressing p53 mutants that adopt folded or unfolded conformations, on the basis of X-ray or NMR-derived structures of the p53 core domain. The percentages of p53-positive cells did not correlate with either the folding state or the folding scores of the mutations derived from the NMR- (r2 = 0.082) or X-ray (r2 = 0.016) -derived J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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Figure 1. Variable degrees of mutant p53 stabilization in human cancer cell lines. Two examples of immunostaining for p53 in cells lines containing missense TP53 mutations are shown in the photomicrographs and the percentages of p53-positive cells in each cell line used in this study is presented graphically (mean ± SD)
structures (Pearson coefficient of determination). Mutations were also classified as disruptive/non-disruptive or as severe/non-severe, including the severity score. The percentage of positive cells is independent of the disruptive or non-disruptive nature of the mutation. All mutations are classified as severe and percentages of positive cells correlate weakly with severity score in the different cell lines (r2 = 0.30) (Table 1).
The effects of genotoxic and non-genotoxic agents on mutant p53 stabilization To investigate the mechanisms involved in stabilizing mutant p53, we examined the effect of agents that stabilize wtp53. UV and roscovitine, a cyclin-dependent kinase inhibitor that stabilizes wtp53 [25] and mutant p53 in zebrafish [23], increased the percentage of p53-positive cells in T47D, BT474 and SKBR3 breast cancer cell lines that express different p53 mutant proteins and show a heterogeneous staining pattern (Figure 2). Treatment of BT549, HOS, MDA-MB-231 and MDA-MB-468 cells did not lead to a significant increase in the percentage of cells staining for p53 (these cells show homogeneous staining prior to treatment) (Figure 2 and data not shown). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Figure 2. Stabilization of mutant p53 by genotoxic and non-genotoxic stress. The indicated cells were grown without treatment (control) or were exposed to UV or Roscovitine. The y axis shows the mean percentages of p53-positive cells (±SD)
Staining patterns and mutation classification in human breast cancers The TP53 coding sequences of 240 untreated breast cancers were analysed by array-based sequencing (see supplementary material, Table S1, for sequence details of each tumour, including polymorphisms) and 62 (25.8%) cancers were identified with TP53 mutation. One cancer (no. 429) contained two separate mutations and was J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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not included in subsequent analyses. In the remaining 61 patients, mutations were classified as producing folded or unfolded proteins, based on either X-rayor NMR-derived structures; as causing a disruptive or non-disruptive mutation, and their severity scores were calculated. Since all mutations are classified as severe, cancers were divided into groups with severity scores above the median value (81.63) or ≤ this value. There were 12 frame-shift or nonsense mutations that could not be classified as folded or unfolded mutations or given a severity score but were all classed as disruptive mutations. One mutation in exon 10 could not be given a severity score and is not included in the structural data, and individual mutations do not have data for their structural effect using X-ray crystal or NMR data. Immunohistochemical staining patterns for p53 could not be classified in three cancers, due to lack of sufficient tumour tissue in the microarray, and the 12 nonsense/frame-shift mutations were all unstained for p53, as expected, leaving a group of 46 cancers that contain missense TP53 mutations for classification of staining pattern (31 with heterogeneous p53 staining and 15 with homogeneous staining; see Figure 3 for examples of staining patterns). In those cancers with heterogeneous p53 staining, all cores showed a similar pattern, where positively stained tumour cells were mixed with negatively stained tumour cells in each tissue core (six cores/cancer were used, taken from different areas of the tumour).
Association of mutation types with clinicopathological data Clinicopathological data include HER2, ER, PR, number of nodes containing tumour, invasive grade, tumour size, disease-free survival and immunohistochemcal scores for Ki67, Mdm2 and p21. The associations of p53 mutation classifications are summarized in Table 2 (see supplementary material, Table S2, for full details of each cancer). There were no associations between the predicted classes of missense mutations in terms of protein folding, functional disruption or severity score within this tumour set, and p53 immunohistochemical staining patterns were independent of the mutation type, based on these sequence-based classifications (Table 2). Mutations predicted to adopt a folded structure associated with higher expression of p21 but not with any other clinicopathological variable. Using the severity score as a continuous variable, or after classification into two groups based on the median score, revealed no associations with clinicopathological factors. The homogeneous staining pattern associated with higher Ki67 score (p = 0.0079), lower Mdm2 levels, PR-negative cancers (p = 0.0003) and ER-negative cancers (p = 0.03). None of eight grade 2 tumours showed a homogeneous staining pattern compared to 15 of 37 grade 3 tumours (p = 0.038), and tumours with heterogeneous staining showed improved survival by Kaplan–Meier analysis (p = 0.036; log rank; Mantel–Cox). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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The association of mutant p53 staining pattern with ER/PR status was confirmed in a separate group of triple-negative breast cancers. Of 27 patients identified with TP53 mutations (details given in Table S3, see supplementary material) from 55 tumours sequenced, four contained non-sense or frame-shift mutation and 21of the 23 tumours with missense mutation showed homogeneous staining (91%). Thus, from the combined analysis of all patients included in this study, 35 of 48 PR− cancers with missense TP53 mutations showed homogeneous staining, whereas 19 of 20 PR+ tumours with missense TP53 mutations showed heterogeneous staining (p = 2.44 × 10−7 ). We also noticed that three of four triple-negative cell lines showed homogeneous mutant p53 accumulation, whilst all four breast cancer cell lines that expressed ER, PR or amplified HER2 showed heterogeneous p53 staining (see supplementary material, Table S4).
Mdm2 influences mutant p53 stabilization in some but not all breast cancer cell lines In view of the correlation between high levels of Mdm2 and the heterogeneity of mutant p53 accumulation seen in human breast cancer samples, we treated three breast cancer cell lines that showed heterogeneous p53 staining with Nutlin, an inhibitor of Mdm2 [33]. Nutlin treatment increased the percentage of cells containing stabilized mutant p53 in the two steroid receptor-positive cell lines, T47D and BT474 (p < 0.01 for each), but not in SKBR3 HER2-amplified cells (Figure 4).
Discussion Numerous studies have attempted to employ TP53 mutation as a predictive or prognostic biomarker in human cancer, with generally disappointing results. However, subclassification of TP53 mutations may provide more accurate prediction than a simple mutant versus non-mutant classification [5–8]. In breast cancer, a large phase 3 trial showed that TP53 status was prognostic for overall survival but did not predict taxane sensitivity [34], which may relate to different prognostic and predictive impacts in luminal and triple-negative cancers [35,36]. Thus, the emerging picture is of variable effects of TP53 status, depending on mutation type and tumour type. In immunohistochemical studies, mutant p53 stabilization is often heterogeneous within an individual tumour, for unknown reasons [11–14,37]. We demonstrated a similarly variable accumulation of mutant p53 in cell lines that contained different TP53 missense mutations. The finding of stable staining patterns in cell lines indicates that different mutant p53 stabilization patterns in primary tumours are not an immunohistochemical artifact of fixation or processing. We also showed that individual colonies of heterogeneously stained cell lines retained the same J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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Figure 3. Variable stabilization of mutant p53 in human breast cancers. Two examples of homogeneous staining and two with heterogeneous staining in tumours with missense TP53 mutations are shown. One tumour with a non-sense mutation is shown for comparison
heterogeneity of mutant p53 stabilization in each colony, arguing against a lack of staining in individual cells being caused by loss of the mutant allele. Therefore, these cell lines showed similarities to primary tumours and are appropriate model systems to investigate the underlying reasons for differential stabilization. We hypothesized that variable p53 stabilization may be due to the precise missense TP53 mutation that alters the inherent stability of the mutant protein. However, the extent of p53 stabilization did not associate with predicted ability to adopt a folded conformation. In addition, classification of mutations as disrupting function, or using a complex set of structural and functional characteristics to calculate a severity score, did not correlate with the extent of accumulation. In themselves, these data indicate that mutant p53 stabilization in cancer cells is not an inherent property of the protein, a notion that is supported by the increase in mutant p53 stabilization in all cell lines tested following treatment with UV irradiation or a Cdk inhibitor, agents that also induce wtp53. These data support findings of mutant p53 stabilization in non-tumour cells in transgenic mice and zebrafish in response to factors such as genomic damage or oncogenic stress [20,22–24]. In primary breast cancers, unfolded mutant p53 proteins associated with a decreased level of the p53 target, Cdkn1a/p21, demonstrating that different mutation Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
types may associate with functional properties in primary tumours. Sequence-based classifications did not correlate with clinicopathological parameters, whereas the staining pattern of missense mutant p53 correlated with proliferation, steroid receptor expression and overall survival. Similar to the primary cancers, homogeneity of p53 stabilization was not seen in steroid receptor-positive and HER2-positive breast cancer cell lines, suggesting a direct association of p53 stabilization with breast cancer subtype. This over-representation of homogeneous mutant p53 accumulation in ER− PR− tumours is the main contributory factor to the relatively poor survival of this group of breast cancer patients, and homogeneous staining versus heterogeneous is not an independent prognostic factor. Regarding the underlying reasons for the differences in stabilization in the two groups of breast cancer, it is unlikely that our results can be explained by genetic differences such as, perhaps, a clonal dominance in triple-negative cancers as a consequence of TP53 mutation, occurring as an early event in this specific subtype and occurring as a later event in ER- or HER2-positive cancers. Such a scenario would be expected to produce distinct areas of positivity and negativity during subclonal expansion, whereas our data indicate that p53-positive and -negative cells do not form discrete cellular groups within an individual cancer. Moreover, heterogeneity of stabilization in cell lines cannot be J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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Table 2. Clinical and pathological correlations with immunohistocemical and structural properties of 61 primary human breast cancers with TP53 mutation DO-1a Variable (total cases) Crystal F (13) Crystal U (31) Disruptive Y (16) Disruptive N (45) Severity Y (23) Severity N (24) Ki67 0–4 (43) Ki67 5–6 (15) P21 0–3 (38) P21 4–18 (21) Mdm2 0–10 (30) Mdm2 12–18 (29) PR + (24) PR – (36) Grade 1–2 (10) Grade 3 (50) Dead (26) Alive (26)
Crystalb
Hom
Het
pe
2 12 0 15 7 7 8 7 10 5 12 3 1 14 0 15 9 4
11 16 4 27 13 17 28 3 17 14 14 17 19 11 8 22 9 16
0.156 0.288 0.752 0.0079 0.533 0.031 0.0003 0.038 0.087
U
1 30 14 17 22 7 21 8 18 11 13 18 6 25 11 15
F
2 11 8 5 11 2 4 9 5 8 4 8 1 11 5 4
Disruptivec p
Severityd
Y
N
p
Y
N
p
2 1
11 30
0.204
8 14 2 21
5 17 2 22
0.51
17 5 15 7 11 11 8 14 4 19 8 10
19 4 12 11 14 9 12 12 3 20 9 11
0.204 0.51 0.695 0.018 0.192 0.735 0.652 0.700
2 2 10 5 12 4 6 10 7 9 3 13 7 6
21 22 33 10 26 17 24 19 17 27 7 37 18 20
1.00
1.00 0.502 0.370 0.251 0.771 1.00 0.755
0.722 0.365 0.554 0.388 1.00 1.00
a Classification
of p53 immunohistochemistry with the DO-1 monoclonal antibody into homogeneous (Hom) or heterogeneous (Het) staining of tumour nuclei. X-ray-derived crystal structure of p53 was used to classify mutations as adopting an unfolded (U) or folded (F) structure. Mutations were classified as disruptive (Y) or non-disruptive (N). d Mutations were classified as having a severity score above the median value for this series (Y) or not above the median value (N). e Two-tailed p values (Fisher’s exact test). b The c
Figure 4. Stabilization of mutant p53 by Nutlin. The indicated cells were exposed or not exposed (Con) to Nutlin. The y axis shows the mean percentages of p53-positive cells (±SD)
explained by differences in cell genetics, necessitating the consideration of other mechanisms. It has been proposed previously that mutant p53 stabilization in tumours is caused by exogenous or endogenous stress in cancer cells [20–24]. If this notion is correct, stabilization differences between breast cancer subtypes may relate to the distinctive induction patterns of wtp53 in luminal versus basal breast epithelial cells following genotoxic damage in vitro and in vivo [27,38]. Different mutant p53 stabilization patterns in ER-positive and -negative cancers can also help to explain why the p53 gene signature is different and has different prognostic and predictive value in the two groups [35]. In our study, tumours with homogeneous p53 staining had higher proliferative rates, which may relate to the ability of mutant p53 to drive proliferation [4]. Conversely, rapid proliferation may cause p53 Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
stabilization, due to increased levels of replication-associated DNA damage and/or enhanced Hsp90 activity [39], so that this association may be cause or effect, or a mixture of the two. We also identified an association between higher Mdm2 levels and heterogeneous missense mutant p53 staining, in keeping with previous experimental data suggesting that mutant p53 is subject to degradation by Mdm2 [15,20]. Unlike UV and roscovitine, which increase p53 in all cells tested, Mdm2 inhibition increased the percentages of p53-positive cells in two steroid receptor-positive breast cancer cell lines, but not in HER2-amplified SKBR3 cells. Thus, Mdm2 seems to play an important role in regulating mutant p53 stability in some, but not all, cancers. Similarly, clinical samples showed an incomplete association between Mdm2 and mutant p53, indicating that other pathways are important in specific tumours, such as the major role of Hsp90 co-chaperones in regulating mutant, but not wild-type, p53 stability [16,17,39]. One important implication of our findings is that wtp53 stabilizing agents also stabilize mutant p53 in human breast cancer cells, implying that conventional genotoxic therapies will paradoxically increase p53 gain-of-function oncogenic activity [22,40]. However, therapy-induced stabilization will not be a significant issue for those tumours that already show homogeneous accumulation, and enhanced gain-of-function mutant p53 is therefore relevant only for the specific group of cancers that show heterogeneous accumulation. In conclusion, mutant p53 stability in breast cancer is a complex and variable process that is not related to the particular type of mutation present but reflects specific aspects of the individual tumour biology. The simple assessment of p53 staining patterns, combined with J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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p53 pathway analysis for mutant and wtp53 tumours [11,37,41], may have benefit in identifying p53 functionality in human cancer and for identifying patients in whom therapy may inadvertently cause activation of mutant p53 oncogenic function.
Acknowledgements We thank all of the women who kindly donated tissue to the Tayside Tissue Bank for research purposes, and the nurses, clinicians and other staff involved. We are grateful to the Tayside Tissue Bank for preparing TMAs and for immunostaining. This study was supported by the Czech Republic (Grant No. IGA MZ CR NT/13794-4/2012), the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO; Grant Nos CZ.1.05/2.1.00/03.0101 and MH CZ-DRO MMCI 00209805) and Breast Cancer Research (Scotland).
Author contributions PB carried out cell culture and immunohistochemical analysis of cell lines; RN analysed p53 staining in primary breast cancer samples, performed statistical analysis in cell lines and primary material and was involved in the study concept and design; PM performed structural predictions of mutant p53 folding and was involved in the design of the study; RH was involved in interpretation of cell line data and participated in study design; MVA and KM performed cell culture experiments and analysis of p53 stabilization; LBJ and CAP selected tissues for TMA construction and analysis of TP53 mutation and performed immunohistochemical analysis on breast cancer material; PQ collated clinicopathological and immunostaining data and performed statistical analyses; AMT was involved in project design and coordination and writing the manuscript; BV was involved in the concept of the study, study design and coordination, and writing the manuscript; and PJC was involved in study concept, design and coordination, analysis of p53 immunostaining, categorization of mutant types and writing the manuscript. All authors read and approved the final manuscript.
Abbreviations ER, oestrogen receptor; HER2, human epidermal growth factor receptor-2; NMR, nuclear magnetic resonance; PR, progesterone receptor; wtp53, wild-type p53
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34. Bonnefoi H, Piccart M, Bogaerts J, et al. TP53 status for prediction of sensitivity to taxane versus non-taxane neoadjuvant chemotherapy in breast cancer (EORTC 10994/BIG 1–00): a randomised phase 3 trial. Lancet Oncol 2011; 12: 527–539. 35. Coutant C, Rouzier R, Qi Y, et al. Distinct p53 gene signatures are needed to predict prognosis and response to chemotherapy in ER-positive and ER-negative breast cancers. Clin Cancer Res 2011; 17: 2591–2601. 36. De Laurentiis M. Optimum chemotherapy regimen for early breast cancer. Lancet Oncol 2011; 12: 514–515. 37. Abdel-Fatah TM, Powe DG, Agboola J, et al. The biological, clinical and prognostic implications of p53 transcriptional pathways in breast cancers. J Pathol 2010; 220: 419–434. 38. Huper G, Marks JR. Isogenic normal basal and luminal mammary epithelial isolated by a novel method show a differential response to ionizing radiation. Cancer Res 2007; 67: 2990–3001. 39. Muller P, Ruckova E, Halada P, et al. C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances. Oncogene 2013; 32: 3101–3110. 40. Prives C, White E. Does control of mutant p53 by Mdm2 complicate cancer therapy? Genes Dev 2008; 22: 1259–1264. 41. Thompson AM, Lane DP. p53 transcriptional pathways in breast cancer: the good, the bad and the complex. J Pathol 2010; 220: 401–403.
SUPPLEMENTARY MATERIAL ON THE INTERNET The following supplementary material may be found in the online version of this article: Table S1. TP53 sequence data of 240 breast cancers Table S2. Detailed clinicopathological information for 61 breast cancers with TP53 mutations Table S3. Immunohistocemical classification of 27 triple-negative breast cancers with TP53 mutations Table S4. Receptor status and p53 immunohistochemical stabilization patterns of eight breast cancer cell lines
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
ORIGINAL PAPER
Mutant p53 accumulation in human breast cancer is not an intrinsic property or dependent on structural or functional disruption but is regulated by exogenous stress and receptor status Pavla Bouchalova,1 Rudolf Nenutil,1 Petr Muller,1 Roman Hrstka,1 M Virginia Appleyard,2 Karen Murray,2 Lee B Jordan,3 Colin A Purdie,3 Philip Quinlan,2 Alastair M Thompson,2,4 Borivoj Vojtesek1 and Philip J Coates5* 1 2 3 4 5
Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic Clinical Research Centre, Dundee Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK Department of Pathology, Ninewells Hospital and Medical School, Dundee, UK Department of Surgical Oncology, MD Anderson Cancer Center, Houston, TX, USA Tayside Tissue Bank, Jacqui Wood Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK
*Correspondence to: PJ Coates, Tayside Tissue Bank, Jacqui Wood Cancer Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. E-mail: [email protected]
Abstract Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure–function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: p53; mutation; breast cancer; immunohistochemistry; therapy
Received 3 January 2014; Revised 13 March 2014; Accepted 21 March 2014
No conflicts of interest were declared.
Introduction TP53 gene mutations are common in human tumours and many studies have attempted to correlate p53 mutation status and prognosis, with disappointing results [1–4]. Tumours containing mutant p53 are usually considered as a single class and are compared to tumours that retain wild-type (wt) p53. However, individual TP53 mutations differentially influence the overall functional properties of the mutant protein and certain missense p53 mutant proteins have oncogenic functions, in contrast to the tumour-suppressor function of wtp53 or the loss of function caused by nonsense mutations or TP53 Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
loss [3,4]. Clinically, discriminating between specific TP53 mutations or mutation types provides prognostic information in certain human malignancies [5–10]. One consequence of a missense mutation in TP53 is accumulation of the mutant protein, allowing mutation detection by immunohistochemical staining; tumours showing a high percentage of p53-positive cells are classified as containing mutant p53. This simple approach is inaccurate because of the highly variable numbers of cells that stain for p53 in tumours with wtp53 or mutant p53, confounding the results of many studies [1,11–14]. On the other hand, immunohistochemical studies suggest that missense mutant p53 is not always inherently J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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stabilized in tumour cells, and the extent of protein accumulation may therefore reflect the specific type of mutation or some other aspect of the tumour’s pathobiology. Simple explanations for these observations would be that the immunohistochemical data are due to artifacts of fixation and processing, loss of the mutant allele from some tumour cells, or gain of mutation as a later event during clonal evolution. These explanations are unlikely, because heterogeneity of staining is not localized to specific areas of the tumour. More plausible explanations are that staining patterns reflect the intrinsic nature of the mutation, where some mutant proteins are inherently more stable than others, or where some mutations are particularly disruptive to function and the mutant proteins are consequently less able to induce their own destruction thorough transcriptional regulation of Mdm2 [15]. Indeed, the site and nature of the mutation in p53 results in proteins with native (folded) or denatured (unfolded) structures that have stabilization and degradation pathways different from those of wtp53 [16–18]. TP53 mutations have also been classified as disruptive or non-disruptive to function, depending on mutation site and type [8], and can be given a severity score from structural/functional calculations [19]. Therefore, variations in mutant p53 levels may relate to the inherent stability/activity of the individual mutant, which would allow information regarding the class of mutation by immunohistochemical staining. A more recent hypothesis derived from experimental studies of transgenic mice and zebrafish is that tumour-specific factors are responsible for mutant p53 stabilization [20–24]. In mice containing only a mutant Tp53 gene, p53 accumulation is not seen in non-tumour cells under normal conditions, but occurs in spontaneously developing tumours [20]. Factors that stabilize wtp53 (DNA damage, lack of Mdm2 and oncogenic stress) also stabilize mutant p53 in non-tumour cells. Thus, levels of mutant p53 in human tumours may be symptomatic of specific aspects of tumour pathobiology, such as genetic instability or the precise oncogenic events that have occurred. These hypotheses are not mutually exclusive and the extent of mutant p53 stabilization may represent a combination of intrinsic properties together with endogenous or exogenous stresses. To clarify the reasons for variability of mutant p53 stabilization, we investigated the relationships between specific mutations, cellular stress and p53 levels in a panel of cell lines and primary human breast cancers containing different TP53 missense mutations.
Materials and methods Tissue culture Human cell lines with known p53 mutational status were obtained from ATCC (Table 1) and cultured in the recommended medium containing 10% fetal calf serum (FCS), 0.3 mg/ml L-glutamine and 1% penicillin–streptomycin (Invitrogen, Life Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Table 1. Immunohistochemical and structural analysis of 17 human cell lines for TP53 mutation classification Cell line Mutation HOS BT549 SKUT-1 A431 HT-29 DLD-1 MB231 SW837 BT20 MB468 CAMA-1 RD HT3 SKBR3 MOLT4 BT474 T47D
R156P R249S R175H R273H R273H S241F R280K R248W K132Q R273H R282T R282W G245V R175H R248Q E285K L194F
Staining (%)a
X-rayb
NMRb
99.3 98.3 97.0 96.8 93.2 92.8 92.4 91.6 87.5 86.5 86.3 86.0 86.2 80.5 62.4 62.0 53.4
U (1.84) ? (0.67) n.a. F (0.00) F (0.00) F (0.06) F (0.34) F (0.23) U (1.82) F (0.00) ? (0.61) F (−1.11) ? (0.51) n.a. F (0.21) U (1.21) F (−1.15)
U (1.64) U (1.50) U (0.97) F (−0.04) F (−0.04) F (0.52) n.a. ? (0.64) U (1.29) F (−0.04) U (0.30) F (−0.72) F (1.45) U (0.97) F (0.02) U (1.16) ? (0.56)
Disruptivec Severityd No Yes No No No Yes No Yes No No No No Yes No Yes No No
71.75 95.70 92.55 87.26 87.26 82.44 71.45 83.31 75.40 87.26 94.99 92.37 111.3 92.55 62.49 74.72 66.05
a
The mean percentage of p53-positive cells. The folding scores of mutant p53 predicted from the X-ray crystallography or NMR structures. A higher score indicates a more unfolded structure. U, predicted to be a largely unfolded protein (score > 0.7); F, predicted to adopt a folded structure (score < 0.5); n.a., not available from the structure; ?, no prediction made (score 0.5–0.7). c Classification according to whether or not the mutation disrupts function. d Mutation severity score. A cut-off value of 60.18 discriminates severe from non-severe mutations; all mutations are classed as severe. b
Technologies, Paisley, UK) at 37∘ C and 10% CO2 . Cells were exposed to UV light (40 J/m2 using a Stratalinker 1800) or Roscovitine (10 μM; Sigma-Aldrich, Dorset UK) and collected 16 h later [25]. T47D, BT474 and SKBR3 cells were also treated with the Mdm2 inhibitor Nutlin-3 (10 μM; Sigma-Aldrich) for 24 h. To produce cell pellets, cells were fixed in 4% neutral buffered formalin, resuspended in 1.5% molten agarose and processed into paraffin wax using standard histological procedures. Alternatively, cells grown directly onto glass slides were fixed in methanol:acetone (50:50) at −20∘ C for 10 min, air-dried overnight and stored at −80∘ C. For colony formation, cells were dissociated into single cells through 21g needles, checked for the absence of doublets by microscopy before plating at cloning density on glass slides, and fixed after colonies of > 20 cells had formed (7–10 days).
Human tumour samples A series of 240 untreated primary breast cancers taken at a resectional surgery in Dundee were collected. Portions of tissue were used for diagnostic histopathology and separate tumour pieces were dissected by a specialist breast pathologist and stored frozen for analysis of TP53 DNA sequence, using the Roche Amplichip (Roche Diagnostics). Patient details have been reported previously [26]. A second cohort of 55 triple-negative breast cancers was examined by direct Sanger sequencing and p53 immunohistochemistry. Microarrays were prepared in the Tayside Tissue Bank from diagnostic histopathology blocks, using six 0.6 mmm cores/cancer. The use of the tissues was approved by local ethical J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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review and all patients provided informed consent for the collection and use of their tumour material for research. For this study, only those tumours identified as containing mutant p53 were analysed.
Immunohistochemistry and analysis of staining patterns Sections and cells were stained for p53 using rabbit polyclonal CM1 antiserum and the mouse monoclonal antibody DO-1. Immunoreactivity was detected by avidin–biotin complex peroxidase staining (Elite ABC Kit, Vector Laboratories, Peterborough, UK) or by EnVision+ System–HRP (Dako Denmark A/S) with diaminobenzidine as chromogen, as described [11,27]. For cell lines, the percentage of cells showing p53 immunostaining was calculated by examination of at least 10 independent microscope fields. p53 staining patterns in tumours were described as showing ‘heterogeneous’ or ‘homogeneous’ staining by visual examination of the images by two investigators with experience of p53 immunohistochemistry (RN and PJC). Homogeneous staining was defined as > 90% of tumour cells positive for nuclear p53 staining, whereas heterogeneous staining was defined by > 10% of nuclei showing an absence of p53 staining. Variable intensity of staining in individual cells is not taken into account when considering heterogeneity – a simple positive and negative staining for each tumour cell was used. In practice, tumours either show a very small number of negative cells ( 20% negative cells, so that the discrimination of homogeneity and heterogeneity is easily achieved. Breast cancer microarrays were also stained for Ki67 (Dako), Cdkn1a/p21 and Mdm2 [11,27]. The latter two were scored by the QuickScore method, where intensity of staining is recorded on a scale of 0–3 and percentages of positive cells are scored on a scale of 0–6, and the two values multiplied to give scores in the range 0–18 [28]. Only the percentage scale is recorded for Ki67. Oestrogen receptor (ER), progesterone receptor (PR) and the HER2 status of breast cancers were assessed by immunohistochemistry and in situ hybridization, as previously reported [29,30].
Subclassification of p53 mutation types by sequence data Using the nucleotide sequence data from each cell line and tumour, we classified TP53 mutations using four different approaches. (a) We predicted the effect of the mutation on the relative ability of the protein to fold into a wild-type conformation using the wtp53 structures derived from X-ray crystallography and (b) those derived from NMR data, calculating the structural effects of individual mutations with PoPMuSic [31]. This algorithm produces a score for folding and allows classification as either unfolded or folded. The data are applicable only to the core regions of p53 and predictions are lacking for some individual amino acid residues in either the NMR-derived structure or the X-ray Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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structure. Because the NMR and X-ray data sometimes provide different folding predictions, we analysed each method separately. Non-sense mutations (stop codon or frame-shift) that lead to an absence of protein are not classified as folded or unfolded. (c) Mutations were classified as disruptive or non-disruptive, based on the degree of disturbance to the structure of p53 in complex with DNA [8]. In brief, disruptive mutations produce a non-similar amino acid within the L2 and L3 loops of p53 or introduce a stop codon into the coding sequence. This scheme classifies some TP53 hotspot mutations as non-disruptive, due to similarity between wild-type and mutant amino acids, eg the common R175H and R273H mutations [8]. (d) The fourth system uses 12 weighted parameters related to structure, stability and activity to assess the effect of the mutation through the PREDMUT algorithm [19] at: http://www.ifm.liu.se/bioinfo. The combined effects are reported as a severity score and classified as severe if the score is > 60.18 [19].
Statistics For cell line data, Pearson correlation coefficients were used for comparing staining patterns with PoPMuSiC or PREDMUT scores and non-paired two-way t-tests, assuming unequal variance for disruptive versus non-disruptive mutants. Statistical significance of associations of clinical data with staining patterns, folded state, disruptive nature or severity was assessed using two-tailed p values (Fisher’s exact test), using the Inspire programme [32], or Kaplan–Meier for survival. Pearson coefficients or ANOVA were used for continuous variables (folding and severity scores).
Results p53 staining patterns in human cancer cell lines Immunostaining for p53 was optimized to detect a low level of p53 in normal tissues and cell lines with wtp53 [11,27], such that an absence of staining indicates non-sense or frame-shift TP53 mutation or loss of both alleles. In 17 human cell lines that contain p53 missense point mutations, the percentage of p53-positive cells varied between 99.3% and 53.4% (Figure 1, Table 1). Increasing the antibody concentration two-fold did not decrease the proportion of negatively-stained cells. Scoring p53 in multiple different colonies of T47D, SKBR3 and BT474 cells derived from low-density plating showed that each colony contained a mixture of positively stained and unstained cells in the same proportions as in bulk cultures. Cell lines were classified as expressing p53 mutants that adopt folded or unfolded conformations, on the basis of X-ray or NMR-derived structures of the p53 core domain. The percentages of p53-positive cells did not correlate with either the folding state or the folding scores of the mutations derived from the NMR- (r2 = 0.082) or X-ray (r2 = 0.016) -derived J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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Figure 1. Variable degrees of mutant p53 stabilization in human cancer cell lines. Two examples of immunostaining for p53 in cells lines containing missense TP53 mutations are shown in the photomicrographs and the percentages of p53-positive cells in each cell line used in this study is presented graphically (mean ± SD)
structures (Pearson coefficient of determination). Mutations were also classified as disruptive/non-disruptive or as severe/non-severe, including the severity score. The percentage of positive cells is independent of the disruptive or non-disruptive nature of the mutation. All mutations are classified as severe and percentages of positive cells correlate weakly with severity score in the different cell lines (r2 = 0.30) (Table 1).
The effects of genotoxic and non-genotoxic agents on mutant p53 stabilization To investigate the mechanisms involved in stabilizing mutant p53, we examined the effect of agents that stabilize wtp53. UV and roscovitine, a cyclin-dependent kinase inhibitor that stabilizes wtp53 [25] and mutant p53 in zebrafish [23], increased the percentage of p53-positive cells in T47D, BT474 and SKBR3 breast cancer cell lines that express different p53 mutant proteins and show a heterogeneous staining pattern (Figure 2). Treatment of BT549, HOS, MDA-MB-231 and MDA-MB-468 cells did not lead to a significant increase in the percentage of cells staining for p53 (these cells show homogeneous staining prior to treatment) (Figure 2 and data not shown). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Figure 2. Stabilization of mutant p53 by genotoxic and non-genotoxic stress. The indicated cells were grown without treatment (control) or were exposed to UV or Roscovitine. The y axis shows the mean percentages of p53-positive cells (±SD)
Staining patterns and mutation classification in human breast cancers The TP53 coding sequences of 240 untreated breast cancers were analysed by array-based sequencing (see supplementary material, Table S1, for sequence details of each tumour, including polymorphisms) and 62 (25.8%) cancers were identified with TP53 mutation. One cancer (no. 429) contained two separate mutations and was J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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not included in subsequent analyses. In the remaining 61 patients, mutations were classified as producing folded or unfolded proteins, based on either X-rayor NMR-derived structures; as causing a disruptive or non-disruptive mutation, and their severity scores were calculated. Since all mutations are classified as severe, cancers were divided into groups with severity scores above the median value (81.63) or ≤ this value. There were 12 frame-shift or nonsense mutations that could not be classified as folded or unfolded mutations or given a severity score but were all classed as disruptive mutations. One mutation in exon 10 could not be given a severity score and is not included in the structural data, and individual mutations do not have data for their structural effect using X-ray crystal or NMR data. Immunohistochemical staining patterns for p53 could not be classified in three cancers, due to lack of sufficient tumour tissue in the microarray, and the 12 nonsense/frame-shift mutations were all unstained for p53, as expected, leaving a group of 46 cancers that contain missense TP53 mutations for classification of staining pattern (31 with heterogeneous p53 staining and 15 with homogeneous staining; see Figure 3 for examples of staining patterns). In those cancers with heterogeneous p53 staining, all cores showed a similar pattern, where positively stained tumour cells were mixed with negatively stained tumour cells in each tissue core (six cores/cancer were used, taken from different areas of the tumour).
Association of mutation types with clinicopathological data Clinicopathological data include HER2, ER, PR, number of nodes containing tumour, invasive grade, tumour size, disease-free survival and immunohistochemcal scores for Ki67, Mdm2 and p21. The associations of p53 mutation classifications are summarized in Table 2 (see supplementary material, Table S2, for full details of each cancer). There were no associations between the predicted classes of missense mutations in terms of protein folding, functional disruption or severity score within this tumour set, and p53 immunohistochemical staining patterns were independent of the mutation type, based on these sequence-based classifications (Table 2). Mutations predicted to adopt a folded structure associated with higher expression of p21 but not with any other clinicopathological variable. Using the severity score as a continuous variable, or after classification into two groups based on the median score, revealed no associations with clinicopathological factors. The homogeneous staining pattern associated with higher Ki67 score (p = 0.0079), lower Mdm2 levels, PR-negative cancers (p = 0.0003) and ER-negative cancers (p = 0.03). None of eight grade 2 tumours showed a homogeneous staining pattern compared to 15 of 37 grade 3 tumours (p = 0.038), and tumours with heterogeneous staining showed improved survival by Kaplan–Meier analysis (p = 0.036; log rank; Mantel–Cox). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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The association of mutant p53 staining pattern with ER/PR status was confirmed in a separate group of triple-negative breast cancers. Of 27 patients identified with TP53 mutations (details given in Table S3, see supplementary material) from 55 tumours sequenced, four contained non-sense or frame-shift mutation and 21of the 23 tumours with missense mutation showed homogeneous staining (91%). Thus, from the combined analysis of all patients included in this study, 35 of 48 PR− cancers with missense TP53 mutations showed homogeneous staining, whereas 19 of 20 PR+ tumours with missense TP53 mutations showed heterogeneous staining (p = 2.44 × 10−7 ). We also noticed that three of four triple-negative cell lines showed homogeneous mutant p53 accumulation, whilst all four breast cancer cell lines that expressed ER, PR or amplified HER2 showed heterogeneous p53 staining (see supplementary material, Table S4).
Mdm2 influences mutant p53 stabilization in some but not all breast cancer cell lines In view of the correlation between high levels of Mdm2 and the heterogeneity of mutant p53 accumulation seen in human breast cancer samples, we treated three breast cancer cell lines that showed heterogeneous p53 staining with Nutlin, an inhibitor of Mdm2 [33]. Nutlin treatment increased the percentage of cells containing stabilized mutant p53 in the two steroid receptor-positive cell lines, T47D and BT474 (p < 0.01 for each), but not in SKBR3 HER2-amplified cells (Figure 4).
Discussion Numerous studies have attempted to employ TP53 mutation as a predictive or prognostic biomarker in human cancer, with generally disappointing results. However, subclassification of TP53 mutations may provide more accurate prediction than a simple mutant versus non-mutant classification [5–8]. In breast cancer, a large phase 3 trial showed that TP53 status was prognostic for overall survival but did not predict taxane sensitivity [34], which may relate to different prognostic and predictive impacts in luminal and triple-negative cancers [35,36]. Thus, the emerging picture is of variable effects of TP53 status, depending on mutation type and tumour type. In immunohistochemical studies, mutant p53 stabilization is often heterogeneous within an individual tumour, for unknown reasons [11–14,37]. We demonstrated a similarly variable accumulation of mutant p53 in cell lines that contained different TP53 missense mutations. The finding of stable staining patterns in cell lines indicates that different mutant p53 stabilization patterns in primary tumours are not an immunohistochemical artifact of fixation or processing. We also showed that individual colonies of heterogeneously stained cell lines retained the same J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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Figure 3. Variable stabilization of mutant p53 in human breast cancers. Two examples of homogeneous staining and two with heterogeneous staining in tumours with missense TP53 mutations are shown. One tumour with a non-sense mutation is shown for comparison
heterogeneity of mutant p53 stabilization in each colony, arguing against a lack of staining in individual cells being caused by loss of the mutant allele. Therefore, these cell lines showed similarities to primary tumours and are appropriate model systems to investigate the underlying reasons for differential stabilization. We hypothesized that variable p53 stabilization may be due to the precise missense TP53 mutation that alters the inherent stability of the mutant protein. However, the extent of p53 stabilization did not associate with predicted ability to adopt a folded conformation. In addition, classification of mutations as disrupting function, or using a complex set of structural and functional characteristics to calculate a severity score, did not correlate with the extent of accumulation. In themselves, these data indicate that mutant p53 stabilization in cancer cells is not an inherent property of the protein, a notion that is supported by the increase in mutant p53 stabilization in all cell lines tested following treatment with UV irradiation or a Cdk inhibitor, agents that also induce wtp53. These data support findings of mutant p53 stabilization in non-tumour cells in transgenic mice and zebrafish in response to factors such as genomic damage or oncogenic stress [20,22–24]. In primary breast cancers, unfolded mutant p53 proteins associated with a decreased level of the p53 target, Cdkn1a/p21, demonstrating that different mutation Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
types may associate with functional properties in primary tumours. Sequence-based classifications did not correlate with clinicopathological parameters, whereas the staining pattern of missense mutant p53 correlated with proliferation, steroid receptor expression and overall survival. Similar to the primary cancers, homogeneity of p53 stabilization was not seen in steroid receptor-positive and HER2-positive breast cancer cell lines, suggesting a direct association of p53 stabilization with breast cancer subtype. This over-representation of homogeneous mutant p53 accumulation in ER− PR− tumours is the main contributory factor to the relatively poor survival of this group of breast cancer patients, and homogeneous staining versus heterogeneous is not an independent prognostic factor. Regarding the underlying reasons for the differences in stabilization in the two groups of breast cancer, it is unlikely that our results can be explained by genetic differences such as, perhaps, a clonal dominance in triple-negative cancers as a consequence of TP53 mutation, occurring as an early event in this specific subtype and occurring as a later event in ER- or HER2-positive cancers. Such a scenario would be expected to produce distinct areas of positivity and negativity during subclonal expansion, whereas our data indicate that p53-positive and -negative cells do not form discrete cellular groups within an individual cancer. Moreover, heterogeneity of stabilization in cell lines cannot be J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
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Table 2. Clinical and pathological correlations with immunohistocemical and structural properties of 61 primary human breast cancers with TP53 mutation DO-1a Variable (total cases) Crystal F (13) Crystal U (31) Disruptive Y (16) Disruptive N (45) Severity Y (23) Severity N (24) Ki67 0–4 (43) Ki67 5–6 (15) P21 0–3 (38) P21 4–18 (21) Mdm2 0–10 (30) Mdm2 12–18 (29) PR + (24) PR – (36) Grade 1–2 (10) Grade 3 (50) Dead (26) Alive (26)
Crystalb
Hom
Het
pe
2 12 0 15 7 7 8 7 10 5 12 3 1 14 0 15 9 4
11 16 4 27 13 17 28 3 17 14 14 17 19 11 8 22 9 16
0.156 0.288 0.752 0.0079 0.533 0.031 0.0003 0.038 0.087
U
1 30 14 17 22 7 21 8 18 11 13 18 6 25 11 15
F
2 11 8 5 11 2 4 9 5 8 4 8 1 11 5 4
Disruptivec p
Severityd
Y
N
p
Y
N
p
2 1
11 30
0.204
8 14 2 21
5 17 2 22
0.51
17 5 15 7 11 11 8 14 4 19 8 10
19 4 12 11 14 9 12 12 3 20 9 11
0.204 0.51 0.695 0.018 0.192 0.735 0.652 0.700
2 2 10 5 12 4 6 10 7 9 3 13 7 6
21 22 33 10 26 17 24 19 17 27 7 37 18 20
1.00
1.00 0.502 0.370 0.251 0.771 1.00 0.755
0.722 0.365 0.554 0.388 1.00 1.00
a Classification
of p53 immunohistochemistry with the DO-1 monoclonal antibody into homogeneous (Hom) or heterogeneous (Het) staining of tumour nuclei. X-ray-derived crystal structure of p53 was used to classify mutations as adopting an unfolded (U) or folded (F) structure. Mutations were classified as disruptive (Y) or non-disruptive (N). d Mutations were classified as having a severity score above the median value for this series (Y) or not above the median value (N). e Two-tailed p values (Fisher’s exact test). b The c
Figure 4. Stabilization of mutant p53 by Nutlin. The indicated cells were exposed or not exposed (Con) to Nutlin. The y axis shows the mean percentages of p53-positive cells (±SD)
explained by differences in cell genetics, necessitating the consideration of other mechanisms. It has been proposed previously that mutant p53 stabilization in tumours is caused by exogenous or endogenous stress in cancer cells [20–24]. If this notion is correct, stabilization differences between breast cancer subtypes may relate to the distinctive induction patterns of wtp53 in luminal versus basal breast epithelial cells following genotoxic damage in vitro and in vivo [27,38]. Different mutant p53 stabilization patterns in ER-positive and -negative cancers can also help to explain why the p53 gene signature is different and has different prognostic and predictive value in the two groups [35]. In our study, tumours with homogeneous p53 staining had higher proliferative rates, which may relate to the ability of mutant p53 to drive proliferation [4]. Conversely, rapid proliferation may cause p53 Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
stabilization, due to increased levels of replication-associated DNA damage and/or enhanced Hsp90 activity [39], so that this association may be cause or effect, or a mixture of the two. We also identified an association between higher Mdm2 levels and heterogeneous missense mutant p53 staining, in keeping with previous experimental data suggesting that mutant p53 is subject to degradation by Mdm2 [15,20]. Unlike UV and roscovitine, which increase p53 in all cells tested, Mdm2 inhibition increased the percentages of p53-positive cells in two steroid receptor-positive breast cancer cell lines, but not in HER2-amplified SKBR3 cells. Thus, Mdm2 seems to play an important role in regulating mutant p53 stability in some, but not all, cancers. Similarly, clinical samples showed an incomplete association between Mdm2 and mutant p53, indicating that other pathways are important in specific tumours, such as the major role of Hsp90 co-chaperones in regulating mutant, but not wild-type, p53 stability [16,17,39]. One important implication of our findings is that wtp53 stabilizing agents also stabilize mutant p53 in human breast cancer cells, implying that conventional genotoxic therapies will paradoxically increase p53 gain-of-function oncogenic activity [22,40]. However, therapy-induced stabilization will not be a significant issue for those tumours that already show homogeneous accumulation, and enhanced gain-of-function mutant p53 is therefore relevant only for the specific group of cancers that show heterogeneous accumulation. In conclusion, mutant p53 stability in breast cancer is a complex and variable process that is not related to the particular type of mutation present but reflects specific aspects of the individual tumour biology. The simple assessment of p53 staining patterns, combined with J Pathol 2014; 233: 238–246 www.thejournalofpathology.com
Mutant p53 stabilization in breast cancer
p53 pathway analysis for mutant and wtp53 tumours [11,37,41], may have benefit in identifying p53 functionality in human cancer and for identifying patients in whom therapy may inadvertently cause activation of mutant p53 oncogenic function.
Acknowledgements We thank all of the women who kindly donated tissue to the Tayside Tissue Bank for research purposes, and the nurses, clinicians and other staff involved. We are grateful to the Tayside Tissue Bank for preparing TMAs and for immunostaining. This study was supported by the Czech Republic (Grant No. IGA MZ CR NT/13794-4/2012), the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO; Grant Nos CZ.1.05/2.1.00/03.0101 and MH CZ-DRO MMCI 00209805) and Breast Cancer Research (Scotland).
Author contributions PB carried out cell culture and immunohistochemical analysis of cell lines; RN analysed p53 staining in primary breast cancer samples, performed statistical analysis in cell lines and primary material and was involved in the study concept and design; PM performed structural predictions of mutant p53 folding and was involved in the design of the study; RH was involved in interpretation of cell line data and participated in study design; MVA and KM performed cell culture experiments and analysis of p53 stabilization; LBJ and CAP selected tissues for TMA construction and analysis of TP53 mutation and performed immunohistochemical analysis on breast cancer material; PQ collated clinicopathological and immunostaining data and performed statistical analyses; AMT was involved in project design and coordination and writing the manuscript; BV was involved in the concept of the study, study design and coordination, and writing the manuscript; and PJC was involved in study concept, design and coordination, analysis of p53 immunostaining, categorization of mutant types and writing the manuscript. All authors read and approved the final manuscript.
Abbreviations ER, oestrogen receptor; HER2, human epidermal growth factor receptor-2; NMR, nuclear magnetic resonance; PR, progesterone receptor; wtp53, wild-type p53
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SUPPLEMENTARY MATERIAL ON THE INTERNET The following supplementary material may be found in the online version of this article: Table S1. TP53 sequence data of 240 breast cancers Table S2. Detailed clinicopathological information for 61 breast cancers with TP53 mutations Table S3. Immunohistocemical classification of 27 triple-negative breast cancers with TP53 mutations Table S4. Receptor status and p53 immunohistochemical stabilization patterns of eight breast cancer cell lines
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