Nuclear inclusion bodies of mutant and wild-type p53
in cancer
Citation for published version (APA):
De Smet, F., Rubio, M. S., Hompes, D., Naus, E., De Baets, G., Langenberg, T., Hipp, M. S., Houben, B.,
Claes, F., Charbonneau, S., Blanco, J. D., Plaisance, S., Ramkissoon, S., Ramkissoon, L., Simons, C.,
van den Brandt, P., Weijenberg, M., Van Engeland, M., Lambrechts, S., ... Rousseau, F. (2017). Nuclear
inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis
remodelling by p53 aggregation. Journal of Pathology, 242(1), 24-38. https://doi.org/10.1002/path.4872
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DOI:
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Journal of Pathology
J Pathol 2017; 242: 24–38
Published online 23 March 2017 in Wiley Online Library
(wileyonlinelibrary.com)
DOI: 10.1002/path.4872
ORIGINAL PAPER
Nuclear inclusion bodies of mutant and wild-type p53 in cancer:
a hallmark of p53 inactivation and proteostasis remodelling
by p53 aggregation
Frederik De Smet
1,2,3,4†
, Mirian Saiz Rubio
1,2†
, Daphne Hompes
5
, Evelyne Naus
1,2
, Greet De Baets
1,2
,
Tobias Langenberg
1,2
, Mark S Hipp
6
, Bert Houben
1,2
, Filip Claes
1,2
, Sarah Charbonneau
3
, Javier Delgado Blanco
1,2
,
Stephane Plaisance
7
, Shakti Ramkissoon
3,8,9
, Lori Ramkissoon
3
, Colinda Simons
10
, Piet van den Brandt
10
,
Matty Weijenberg
10
, Manon Van England
11
, Sandrina Lambrechts
12
,FredericAmant
12,13
, André D’Hoore
5
,
Keith L Ligon
3,4,8,9,14
, Xavier Sagaert
15
, Joost Schymkowitz
1,2
*
and Frederic Rousseau
1,2
*
1
The Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
2
VIB Center for Brain and Disease Research, Leuven, Belgium
3
Department of Medical Oncology, Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA, USA
4
The Broad Institute, Cambridge, MA, USA
5
Department of Abdominal Surgery, University Hospitals Gasthuisberg, Leuven, Belgium
6
Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
7
Nucleomics Core, Flanders Institute for Biotechnology (VIB), Leuven, Belgium
8
Department of Pathology, Division of Neuropathology, Brigham and Women’s Hospital and Children’s Hospital Boston, Boston, MA, USA
9
Department of Pathology, Harvard Medical School, Boston, MA, USA
10
Department of Epidemiology – GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
11
Department of Pathology – GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
12
Department of Obstetrics and Gynaecology, Division of Gynaecological Oncology, University Hospitals Leuven, KU Leuven, Leuven, Belgium
13
Centre for Gynaecological Oncology Amsterdam, Netherlands Cancer Institute, Amsterdam, The Netherlands
14
Department of Pathology, Children’s Hospital Boston, Boston, MA, USA
15
Translational Cell and Tissue Research, KU Leuven, Leuven, Belgium
*Correspondence to: F Rousseau or J Schymkowitz, The Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leu-
ven, and the VIB Centre for Brain and Disease Research, Leuven, B-3000, Belgium. E-mail: Frederic.rousseau@switch.vib-kuleuven.be;
Joost.Schymkowitz@switch.vib-kuleuven.be
†
Equal contributions.
Abstract
Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer
remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies,
and identied nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most
frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear
aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In
cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most
prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated
with the loss of p53’s transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the
proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which
tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that
was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs
suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a
differential proteostatic expression prole associated with p53-positive nIBs. p53-positive nIBs therefore highlight
a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53
through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic
dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels
between cancer and neurodegeneration.
Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: p53 aggregation; proteostasis; colon cancer; glioblastoma; nuclear inclusion bodies
Received 17 June 2016; Revised 20 December 2016; Accepted 27 December 2016
No conicts of interest were declared.
Copyright © 2016 Pathological Society of Great Britain and Ireland. J Pathol 2017; 242: 24–38
Published by John Wiley & Sons, Ltd. www.pathsoc.org www.thejournalofpathology.com
Nuclear inclusion bodies of p53 in cancer 25
Introduction
The cellular protein quality control (PQC) machinery
of chaperones and proteases ensures protein homeosta-
sis or ‘proteostasis’ [1–3]. Because of ageing, muta-
tion, or (patho-)physiological insults [4], the processing
of misfolded proteins becomes less efcient, and this
can lead to protein accumulation and aggregation, and
vice versa [1–3]. Abnormal protein aggregation causes
well-known misfolding diseases, including neurodegen-
erative and amyloidogenic disorders [1–3], in which
there is typically accumulation of disease-specic pro-
teins in inclusion bodies or extracellular deposits [1–3].
In rare familial cases, germline mutations increase the
aggregation propensity of disease-specic proteins, the
chronic expression of which is believed to initiate a
vicious cycle of proteostatic dysregulation and aggre-
gation. In the more common sporadic conformational
diseases, age-associated erosion of PQC is more likely
to cause wild-type protein aggregation.
The tumour suppressor p53 is the most commonly
mutated gene in cancer [5]. Most mutations occur
in the DNA-binding domain and can be categorized
according to their conformational effect: whereas ‘con-
tact’ mutations alter DNA-binding properties without
disturbing the overall folding, ‘structural’ mutations
disrupt the native fold [6,7]. Inactivity of p53 com-
monly correlates with its aberrant accumulation in can-
cer cells [5,8], which was initially explained by an
impeded human double minute-2 (MDM2) response that
mediates p53 degradation [9]. Recently, we and others
showed that aggregation can also contribute to p53 inac-
tivation, accumulation and gain-of-function activities
[10–18]. Importantly, although mutation often increases
the aggregation propensity of p53 by destabilizing its
structure, wild-type p53 (p53-WT) is itself already ther-
modynamically labile (T
melt
of 42
∘
C) [10,15,16]. As
ageing favours both mutation and proteostatic decline,
the aggregation of both mutant p53 and p53-WT might
be a common and possibly physiology-modifying event.
The accumulation of p53 aggregates has been previously
reported by us and others in tumour lines transiently
overexpressing mutant p53 or in sparse human tumours
[13,14,17,19].
Here, we present a study agglomerating 370 tumour
biopsies investigating the presence of p53 aggregates
in cancer. We found that a large fraction of mutant
and p53-WT-positive tumours contain nuclear inclu-
sion bodies (nIBs) of inactive p53 that co-localize with
known markers for aggregates in neurodegeneration.
The presence of aggregated p53 in cell lines and nIBs
in tumours was associated with a distinctive proteostatic
prole and patient survival.
Materials and methods
Cell lines
Cells were grown in standard conditions [Dulbecco’s
modied Eagle’s medium (DMEM)/10% fetal bovine
serum (FBS); Life Technologies, Gent, Belgium]. Stress
insults included hypoxia (0.5% oxygen), proteostatic
stress (0.5 μM MG132, M7449; Sigma-Aldrich, Over-
ijse, Belgium), hypoglycaemia (DMEM without glu-
cose), and oxidative stress (100 μ
M NiCl
2
).
Sodium dodecyl sulphate (SDS)-gradient blue
native polyacrylamide gel electrophoresis
(BN-PAGE) analysis
Cells were lysed in 150 mM NaCl, 50 mM Tris–HCl
(pH 8), 1% IGEPAL(NP40), 1 × Complete inhibitor
(Roche, Vilvoorde, Belgium), and 1 U/μl Universal
Nuclease (Pierce, Leuven, Belgium), and this was fol-
lowed by incubation with SDS as indicated. BN-PAGE
analysis was performed as described previously [17].
Clinical samples
Stage II/III colon cancer samples (n = 163) were
collected by the Department of Abdominal Surgery
(2004–2006; UZLeuven, Belgium; project #S53472),
and glioblastoma (GBM) samples (n = 58, only isoci-
trate dehydrogenase 1/2
WT
) were collected by the Center
for Molecular Oncologic Pathology [2007–2013;
Dana-Farber Cancer Institute, Boston, MA, USA; Insti-
tutional Review Board protocol 10–417]. Patients were
monitored for tumour recurrence and overall survival
(median follow-up: 50.2 months for colon cancer and
17 months for GBM).
Antibodies
The antibodies used were as follows: anti-p53 DO-1
and FL393, anti-HSC70, anti-HSP90, anti-HSPA6,
anti-DNAJB1, and anti-sigma-1 receptor (Sig-
maR) (Santa Cruz Biotechnology, Heidelberg,
Germany); anti-promyelocytic leukaemia (PML),
anti-nucleolin, anti-SQSTM1, anti-HDAC6, anti-BAG2,
and anti-heat shock transcription factor 1 (HSF1)
(Abcam, Cambridge, UK); anti-HSP70 (Cell Sig-
naling Technology, Leiden, The Netherlands); and
A11(AHB0052), Alexa-labelled secondary antibod-
ies, and 4
′
,6-diamidino-2-phenylindole (DAPI) (Life
Technologies). Proximity ligation was performed with
Duolink (Olink, Uppsala, Sweden) according to the
manufacturer’s instructions.
Statistical analysis
Statistical analyses were performed in R-studio
(v0.97.551) with R (v3.0.1) or GraphPad Prism 6.0.
Kaplan–Meier estimates were used to create survival
curves. The Cox proportional hazard model and the
score log rank test were used to determine statistical
signicance. Pairwise comparisons were performed
by the use of likelihood ratio tests with Bonferroni
corrections. Non-parametric Kruskal–Wallis statistics
with Bonferroni correction were used for pairwise
comparisons of high-content data.
Copyright © 2016 Pathological Society of Great Britain and Ireland. J Pathol 2017; 242: 24–38
Published by John Wiley & Sons, Ltd. www.pathsoc.org www.thejournalofpathology.com
26 FDeSmetet al
Additional methods
See supplementary materials and methods for additional
methods.
Results
nIBs of p53
Aggregation-related diseases are typically character-
ized by the abnormal accumulation and aggregation
of disease-specic proteins [1,3], often observed as
intracellular inclusions and/or extracellular deposits [2].
To search for p53 aggregation phenotypes, we used
immunouorescence staining with sufcient sensitivity
to quantify overall expression levels and also to detect
subcellular inhomogeneities (e.g. inclusion bodies) and
aberrant subcellular localization.
Staining of a cohort of colon cancer samples (n = 163)
revealed various subcellular p53 phenotypes: 71% of the
p53-positive tumours contained p53-positive ‘puncta’
within the diffuse p53 staining pattern of the nucleus in
a subset of tumour cells [p53-positive nIBs (p53-nIBs)],
whereas the remainder showed homogeneous, diffuse
nuclear p53 staining throughout the tumour (Table 1;
Figure 1A, B; supplementary material, Figure S1A–H).
The occurrence of p53-nIBs was often associated
with cytosolic staining of lower intensity at a similar
frequency (Table 1). Occasionally, we observed cyto-
plasmic p53 staining without the presence of nIBs,
and pure cytoplasmic IBs were only observed once
(Figure 1C, D). According to these observations, we
dened ve subcategories: tissue without p53 expres-
sion (NULL); tissue with diffuse, well-dispersed nuclear
p53 (SOLUB); tissue with p53-nIBs in >50% of tumour
cells (NUCINC50); tissue with p53-nIBs in 1–5%
(NUCINC5) of tumour cells; and tissue with diffuse
p53 in the nucleus and cytoplasm (CYTO). Markedly,
this classication could not be made reliably by the use
of 3,3
′
-diaminobenzidine /horseradish peroxidase stain-
ing (supplementary material, Figure S1I–L), explaining
why this had not been described earlier.
Also in biopsies from GBM, colon cancer, ovar-
ian cancer, endometrial cancer, melanoma, and Bar-
rett’s oesophagus, we observed p53-nIBs at a tumour
type-specic frequency in samples originating from all
contributing institutes with both monoclonal and poly-
clonal antibodies (Table 1; Figure 1E; supplementary
material, Figure S1). p53-nIBs therefore constitute a
genuine and widespread, but so-far uncharacterized,
phenotype. Also in lymph nodes containing metastatic
colon cancer cells, the p53 phenotype of the primary
tumour was generally maintained (supplementary mate-
rial, Table S1).
Biopsies containing p53-nIBs were subsequently
co-stained with amyloid dyes [i.e. luminescent
conjugated oligothiophenes (LCOs)] [20] or the
conformational-specic antibody A11, which recog-
nizes oligomeric aggregates [21]. Although we did not
observe specic LCO staining (suggesting that p53 did
not generate textbook amyloids in vivo), p53/p53-nIBs
and A11 did co-localize in ovarian tumours using
proximity ligation (supplementary material, Figure S2),
conrming previous ndings [14] and showing that p53
was primarily present as oligomeric aggregates. How-
ever, the A11 staining was insufciently reliable and
robust for routine screening, forcing us to use p53-nIB
detection as a readout.
nIBs contain transcriptionally inactive mutant p53
or p53-WT
Following p53 genotyping, we observed that, although
favoured by p53 mutation, homogeneous and nIB phe-
notypes were present in both p53-WT and mutant
tumours (Figure 1F, G; supplementary material,
Table S3). In the p53-positive samples, p53 transcrip-
tional activity (as measured by MDM2 mRNA levels)
was also signicantly higher in samples containing
SOLUB-WT than in those containing NUCINC50-WT
(Figure 1H), whereas no difference was found between
the NUCINC50-WT group and the p53-NULL group,
showing severe impairment of p53 activity when it is
present in nIBs. Expression levels of p21 and BAX were
highly variable (supplementary material, Figure S2D,
E), suggesting p53-independent mechanisms [22–24].
TP53 mRNA levels were lower in the NULL samples
than in the p53-positive samples (Figure 1I), suggesting
genetic aberrations.
Mutant p53 accumulates in the nucleus as soluble
oligomeric aggregates
Next, we phenotyped 22 tumour cell lines that endoge-
nously express wild-type, contact or structurally
destabilized mutant p53 (Table 2). When we analysed
thousands of single cells under baseline conditions
by using immunouorescence high-content imag-
ing, p53 was primarily observed in a diffuse pattern
in the nucleus, as is commonly observed [25], and
only 5.1 ± 3.1% of cells showed a small number
of nIBs (<2 nIBs per cell). We also determined
p53’s folding status by immunoprecipitation (IP)
with p53-specic conformational antibodies (i.e.
pAb1620 for native p53 and pAb240 for misfolded
p53) [26]: whereas p53-WT and contact mutants
largely adopted the native/pAb1620-positive confor-
mation, structural mutants predominantly adopted the
misfolded/pAb240-positive conformation (Table 2;
supplementary material, Figure S3A).
Given the overall absence of nIBs in standard cell
culture conditions (in contrast to biopsies) and the A11
positivity of p53 in biopsies, p53 was expected to form
small oligomeric aggregates, similarly to what is seen in
neurodegenerative diseases, where the presence of sol-
uble oligomeric aggregate precursors is often indicative
of pathological activity [1,27]. Previously, we optimized
a BN-PAGE method that discriminates between native
tetrameric p53 and aggregated/oligomeric forms, which
are recognized by a continuous high molecular weight
Copyright © 2016 Pathological Society of Great Britain and Ireland. J Pathol 2017; 242: 24–38
Published by John Wiley & Sons, Ltd. www.pathsoc.org www.thejournalofpathology.com
Nuclear inclusion bodies of p53 in cancer 27
Figure 1. Staining of cancer biopsies reveals the presence p53-nIBs. (A–D) p53 immunouorescence staining of colon cancer biopsies.
(A–D) Overlay of confocal images stained for p53 (red) and with nuclear DAPI staining (blue). (E) Quantication of the number of tumours
that are p53-positive and/or contain p53 inclusions in various tumour types, as indicated. (F, G) Bar chart distribution of the inclusion body
phenotype in p53-WT or mutant (MUT) populations of colon cancer (F) and GBM (G). (H, I) For the transcriptional analysis, patient samples
were divided into four subgroups: NUCINC50-WT (p53-WT nIBs with >50% of tumour cells containing p53-nIBs), MUT (mutant p53 with
nIB or diffuse p53 staining), NULL (without p53 expression), and SOLUB-WT (diffuse, p53-WT), in which MDM2 (H) or p53 (I) mRNA levels
were measured with the n-string method in extracted RNA from 86 colon tumour biopsies. *Statistically signicant.
Copyright © 2016 Pathological Society of Great Britain and Ireland. J Pathol 2017; 242: 24–38
Published by John Wiley & Sons, Ltd. www.pathsoc.org www.thejournalofpathology.com