Abstract
Over 80% of triple negative breast cancers express mutant p53. Mutant
p53 often gains oncogenic function suggesting that triple negative
breast cancers may be driven by p53 protein type. To determine the
chromatin targets of this gain-of-function mutant p53 we used inducible
knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in
conjunction with stable isotope labeling with amino acids in cell
culture and subcellular fractionation. We sequenced over 70,000 total
peptides for each corresponding reciprocal data set and were able to
identify 3010 unique cytoplasmic fraction proteins and 3403 unique
chromatin fraction proteins. The present proteomics experiment
corroborated our previous experiment-based results that poly ADP-ribose
polymerase has a positive association with mutant p53 on the chromatin.
Here, for the first time we report that the heterohexomeric
minichromosome maintenance complex that participates in DNA replication
initiation ranked as a high mutant p53-chromatin associated pathway.
Enrichment analysis identified the minichromosome maintenance members
2–7. To validate this mutant p53- poly ADP-ribose
polymerase-minichromosome maintenance functional axis, we
experimentally depleted R273H mutant p53 and found a large reduction of
the amount of minichromosome maintenance complex proteins on the
chromatin. Furthermore a mutant p53-minichromosome maintenance 2 direct
interaction was detected. Overexpressed mutant p53, but not wild type
p53, showed a protein-protein interaction with minichromosome
maintenance 2 and minichromosome maintenance 4. To target the mutant
p53- poly ADP-ribose polymerase-minichromosome maintenance axis we
treated cells with the poly ADP-ribose polymerase inhibitor talazoparib
and the alkylating agent temozolomide and detected synergistic
activation of apoptosis only in the presence of mutant p53. Furthermore
when minichromosome maintenance 2–7 activity was inhibited the
synergistic activation of apoptosis was blocked. This mutant p53- poly
ADP-ribose polymerase -minichromosome maintenance axis may be useful
for theranostics.
Personalized medicine: Mutated tumors respond to therapy
Mutations in the p53 tumor suppressor gene could offer a predictive
biomarker of response to certain drugs in triple-negative breast
cancer. Jill Bargonetti from Hunter College in New York, USA, and
colleagues showed that mutant p53, which is expressed in more than 80%
of patients with triple-negative breast cancer, interacts with and
regulates hundreds of proteins, including those found in a complex
needed for DNA replication. Members of this complex, called the
minichromosome maintenance protein complex, interact with mutant
p53—but less with wild-type p53. Bargonetti’s team targeted this
pathway in mutated breast cancer cells with the PARP inhibitor
talazoparib and the chemotherapeutic agent temozolomide. They observed
synergistic cell killing with the two drugs, but only when the
minichromosome maintenance protein complex was working and when p53 was
mutated. These findings point toward a new strategy for personalizing
therapy.
Introduction
Missense mutations in the TP53 gene often results in mutant p53 (mtp53)
protein with gain-of-function (GOF) properties that are associated with
multiple types of cancers, including lung and breast cancer.^[41]1
Mutations in p53 are found in 80% of triple negative breast cancers
(TNBC).^[42]2–[43]4 A number of studies have been carried out to
elucidate the mtp53-associated breast cancer transcriptome but the
mtp53-targeted proteome is less well studied.^[44]5–[45]8 Mtp53 has not
been found to interact with DNA site-specifically but has been found to
interact with cancer cell DNA in association with other cofactors.
Importantly mtp53 modifies chromatin structure to up-regulate vascular
endothelial growth factor receptor 2^[46]9,[47]10 and GOF mtp53
modifies major chromatin pathways by upregulating methyltransferase
chromatin regulatory genes MLL1, MLL2, and the acetyltransferase
MOZ.^[48]11,[49]12 While changes in the transcriptome are a part of the
mechanism of action of GOF mtp53, there are also
transcription-independent mtp53 functions on chromatin that require
further elucidation.
Very few studies have focused on the mtp53-associated proteome but new
work strongly indicates that alternative experimental approaches are
required to understand the complexity of the mtp53
pathway.^[50]7,[51]13 A multiomics approach recently identified the
proteasome machinery as a common target of missense mtp53.^[52]7 We are
the only group to report on the influence of endogenous GOF mtp53 on
the spatial segregation of the cancer cell proteome.^[53]6 The
mtp53-associated cytosolic proteome targets include up-regulation of
cytoplasmic poly ADP-ribose polymerase (PARP) when mtp53 is
depleted^[54]6 and a decrease in the cytosolic mavelonate pathway
enzymes (which is in agreement with previous transcriptome data).^[55]5
During validation of the spatially segregated proteins we discovered
that down-regulation of mtp53 caused a chromatin-segregated decrease of
PARP.^[56]6 We now report on the chromatin-segregated stable isotope in
cell culture (SILAC) screen to identify the spatially restricted
mtp53-targeted proteome of chromatin. We used a bioinformatics approach
to compare the cytoplasmic and chromatin data sets (see Fig. [57]1 for
the work flow). Recent work suggests that a key regulatory role for
mtp53 on chromatin is to regulate transcription by chromatin
remodeling,^[58]12 but we hypothesized that DNA repair and DNA
replication could also be critical targets.
Fig. 1.
[59]Fig. 1
[60]Open in a new tab
SILAC work flow for proteomic targets. Four independent LC-MS/MS
experiments were carried out to compare the proteomes of chromatin and
cytosolic proteomes with mtp53 knockdown. The work flow diagram briefly
details the scientific steps from cell culture conditions to the
identification of unique proteins. See Figs. [61]2 and [62]3 for
identification of chromatin pathway enrichment and the mutant p53
association index for specific proteins and pathways compared for
chromatin and cytosolic fractions
To our knowledge, there has been no direct evidence of GOF mtp53
regulating chromatin-mediated DNA replication and repair. Herein, we
identified a mtp53-PARP-MCM chromatin axis by an unbiased
bioinformatics screen of spatially segregated cytoplasmic vs. chromatin
SILAC data from R273H mtp53 knockdown in TNBC cells. The enzyme PARP1
catalyzes the transfer of ADP-ribose to target proteins and plays a
role in many cellular processes including transcription, DNA
replication, and DNA repair.^[63]14,[64]15 Herein, we validate the
mtp53-PARP-MCM axis and found that blocking PARP1 may be an excellent
therapeutic target for certain mtp53-expressing TNBCs.
Results
Gain-of-function mtp53 influences 3403 chromatin proteins
Stable isotope labeling in cell culture (SILAC) of the MDA-MB-468.shp53
cell line was carried out and mtp53 R273H was depleted by inducible
shRNA expression in two independent reciprocal experiments. A work flow
diagram (Fig. [65]1) shows the experimental approach that included cell
fractionation and LC-MS/MS of heavy and light extract mixed at a 1:1
protein concentration ratio. For one experiment the mtp53 was depleted
in the heavy label conditions (^13C[6] l-Lysine-2HCl and ^13C[6]
^15N[4]l-Arginine-HCl) and for the other mtp53 was depleted in the
light label conditions. Chromatin fractionation was adapted from the
Mendez and Stillman protocol.^[66]6,[67]16 Following gel
electrophoresis we used liquid chromatography and tandem mass
spectrometry (LC-MS/MS) to identify the mtp53 protein targets
associated with the chromatin fraction. We sequenced over 70,000
chromatin-associated peptides and compared the heavy/light ratio
resulting from the depletion of mtp53 to determine how R273H knockdown
reciprocally influenced the 3403 representative proteins. The chromatin
mtp53 SILAC data were examined for gene set enrichment and then
compared to the cytosol mtp53 targets determined in our previously
published results.^[68]6 We carried out a bioinformatics comparison of
the influence of mtp53 depletion on proteins in the cytosol to those
affected on the chromatin.
Gene set enrichment analysis indicates that the hexomeric pre-replicative
MCM2–7 complex is the most highly enriched mtp53-associated chromatin complex
The gene set enrichment analysis was performed using the GSEA software
(GSEA; version 2.0.14)^[69]17 to determine how chromatin associated
proteins were influenced by mtp53. A defined set of genes associated
with the proteins that showed concordant differences between the
biological states of the mtp53 present, vs. the mtp53 absent, was
determined with pathways defined by the Reactome Pathway Database
(version 4.0).^[70]18 The GSEA analysis of the chromatin fraction
revealed a total of 27 Reactome pathways that were positively
associated with mtp53 abundance at a P value < 1%. Interestingly, a key
pathway was the pre-replicative complex, chromatin enriched proteome
pathway, which is a novel finding for mtp53 GOFassociations. The
proteins in this pathway are (in rank order): MCM2, MCM3, MCM6, ORC1,
MCM4, MCM5, MCM7, RPA2 and POLA2 (Fig. [71]2). The GSEA proteomic
chromatin enriched pathway sets are shown in their entirety at the
link:
[72]http://diverge.hunter.cuny.edu/~weigang/silac-chromatin-gsea/. The
first three positive GSEA pathways in the list corresponded with
electron transport, which did not directly correlate with a
chromatin-associated pathway; we hypothesize this resulted from
insoluble cellular components that were associated with the chromatin
pellet. The fourth GSEA pathway associated with generic transcription
pathways, which are currently the focus of many mtp53 GOF
studies.^[73]12 However the fifth pathway identified a very specific
chromatin associated pre-replicative pathway that has not yet been
studied for mtp53 involvement. The GSEA analysis also demonstrated 17
Reactome pathways that were negatively associated with mtp53 abundance
at a P value < 1%. By clicking on enrichment results in html format you
will be directed to the positive association protein sets and negative
association protein sets (Fig. [74]2). We are providing open access to
this powerful data set.
Fig. 2.
[75]Fig. 2
[76]Open in a new tab
Knockdown of mtp53 identifies enrichment of the pre-replicative complex
pathway. GSEA analysis revealed a total of 27 and 17 Reactome pathways
that are positively and negatively, respectively, associated with mtp53
abundance at a nominal P value < 1%. Enrichment of five DNA
replication-related gene sets and pathways including the
pre-replicative complex consisting of 15 genes, shown here with their
enrichment scores (upper panel) and mutant p53 association index (mPAI)
ranks (lower panel). Full GSEA results are available at
[77]http://diverge.hunter.cuny.edu/~weigang/silac-chromatin-gsea/
(chromatin) and
[78]http://diverge.hunter.cuny.edu/Polotskaia_etal_2014/supp-table-s1/
(cytosol)
Distributions of mtp53 associated changes in the cytosol and on the chromatin
indicate that the hexomeric MCM2–7 complex proteins reside in the double
positive quadrant
The chromatin SILAC data were then compared to our previously published
cytosol set
[79]http://diverge.hunter.cuny.edu/Polotskaia_etal_2014/supp-table-s1/.
In order to further summarize and quantify the degree of under- and
over-expression of proteins from the reciprocal knockdown experiments
in the different sub-cellular fractions of the breast cancer cells we
defined a mtp53 association index (mPAI: see “Methods” for the
statistical analysis). Values of mPAI obtained from both the cytosol
and chromatin fractionations were normally distributed with a mean
close to zero and a standard deviation close to one, conforming to the
expectation that abundance of the majority of proteins were indeed not
impacted by mtp53 knockdown (Fig. [80]3). We thereby identified
proteins with mPAI > 1.0 as those displaying significant positive
association with mtp53 abundance and those with mPAI < −1.0 as showing
significant negative association with mtp53 abundance. This was in
agreement with the fact that mtp53 knockdown did not influence the
level of the majority of the proteins in either the cytosol or
chromatin sub-cellular fractions. Moreover, in both sub-cellular
fractions the standard deviation of the mPAI was close to one and mtp53
itself showed an mPAI value of greater than 2.0 (z-score > 2.0). The
mtp53 mPAI index was 3.0 on the chromatin, which was higher than the
positive 2.1 value identified in the cytosol. The mtp53 mPAI served as
excellent internal positive control as its levels necessarily were
reduced by shRNA mediated knockdown. Poly ADP ribose polymerase (PARP)
was associated with the chromatin when mtp53 levels were high and
redistributed to the cytosol when the mtp53 was low as expected and the
mPAI for PARP reflected this as a negative value for the cytosol and a
positive number for the chromatin (Fig. [81]3). In support of our
previous data, we determined that PARP had a positive mPAI on the
chromatin of 1.2 and a negative mPAI in the cytosol of −2.3. Therefore
in addition to providing a new powerful data set we have identified a
potentially important mtp53 protein pathway that is involved in
regulation of DNA replication.
Fig. 3.
Fig. 3
[82]Open in a new tab
Distribution of mutant p53 association index (mPAI) scores in the
cytosol (top) and chromatin (bottom) fractionations. Histograms of mPAI
values (for equation see statistics in the methods) show close fit to
normal curves (in dashed lines) obtained with the same mean (µ = 0.0060
for cytosol, µ= 0.085 for chromatin) and standard deviation (σ = 0.92
for cytosol, σ = 0.96 for chromatin). As expected (and as a negative
control of the experiments and the mPAI statistic), most proteins are
not significantly impacted by mtp53 depletion, showing −1 < mPAI < 1
(shaded in light gray). As a positive control, the mTP53 protein level
(in red) shows significantly high positive mPAI values. As another
positive control, the PARP1 levels (in blue) show contrasting mPAI
values between the two fractionations, consistent with previous
experimental results.^[83]6 The six components of the MCM2–7 complex
(in green) show significant positive association with mtp53 in both
cytosol and nucleus
Comparison of the nuclear and cytosol proteomes displays a double positive
mtp53 influence on the MCM 2-7 hexomeric complex
The mPAI for the entire mtp53-influenced proteome in the cytosol vs.
the mtp53-influenced proteome on the chromatin were graphed as
coordinates of the chromatin proteome on the Y-axis and the cytosol
proteome on the X-axis. This resulted in a scatter plot with four
quadrants demonstrating differentially influenced mtp53 associated
proteins. Figure [84]4 shows a representative image with all the dots
as grey shades, p53 as a prominent red dot, the MCM2–7 helicase
subunits as green dots (zoomed in in upper right), and PARP as a blue
dot. An interactive searchable scatter plot is part of the
[85]Supplementary Data
[86]http://diverge.hunter.cuny.edu/~weigang/mpai-browser/. Each dot
represents a protein and its mPAI in the cytosol and chromatin. The
four p53-influenced quadrants are (a) double positive in the top right,
(b) chromatin positive and cytosol negative in the top left, (c) double
negative in the bottom left, and (d) cytosol positive with chromatin
negative in the bottom right. The mtp53 protein is by definition a
double positive signal and it is highlighted as a red dot (TP53,
Fig. [87]4). The MCM2–7 pre-replication complex proteins are shown in
green and were all situated as double positives (Fig. [88]4). The PARP1
protein appeared in the upper left quadrant and is highlighted as a
blue dot. PARP1 showed a negative association with mtp53 in the cytosol
and a positive association on the chromatin, consistent with our
previous experimental results. The center of the scatter plot
corresponds to proteins that are unchanged by p53 knockdown. While the
majority of proteins are unchanged by the knockdown of mtp53 key
proteins and pathways including those involved in DNA replication and
repair are strongly implicated.
Fig. 4.
Fig. 4
[89]Open in a new tab
A double-positive mtp53 association seen for all MCM2-7 complex
proteins in cytosol (x-axis) and nucleus (y-axis). Each dot (n = 1778)
represents a protein with its position determined by its mPAI values in
the cytosolic (x-axis) and the chromatin fractionations (y-axis). mPAI
values were averaged if multiple peptides of the same protein were
identified. Two side boxplots show the median, the lower and upper
quartiles, and the range of mPAI values. The majority of points fall
inside the x = −1, x = 1, y = −1, and y = 1 lines, indicating that
abundance of most proteins are not significantly impacted by mtp53
knockdown in either fractionation. The mtp53 and the six members of the
MCM2–7 complex fall in the top right quadrant, where protein levels are
positively associated with mtp53 levels in both fractionations. PARP1
falls in the top left quadrant showing negative association with mtp53
in the cytosol but positive association in the nucleus, consistent with
our previous experiment result.^[90]6 A searchable, zoomable, and
clickable scatter plot of mPAI values for 4798 genes and 1330
associated pathways and gene sets is available at
[91]http://diverge.hunter.cuny.edu/~weigang/mpai-browser/
Mutant p53 interacts with members of the MCM hexomeric complex on chromatin
To verify that the R273H mtp53 levels influenced multiple MCM hexomeric
proteins on chromatin in different cells, we reduced GOF mtp53 levels
in MDA-MB-468 cells and HT-29 cells and used Western blot analysis to
examine MCM2, MCM4, and MCM7 (Fig. [92]5). When mtp53 was decreased the
chromatin-associated levels of MCM2, MCM4, and MCM7 were also decreased
(Fig. [93]5a). To further examine this interaction in situ and to
determine if the mtp53 was co-localized with the MCM2–7 we used
proximity ligation assay (PLA) with confocal microscopy
detection.^[94]19 To our knowledge we are the first group to use
antibodies in PLA to detect the interaction of mtp53 and MCM2. Strong
nuclear co-localization of mtp53 and MCM2 was apparent and this signal
was drastically reduced by the knockdown of mtp53 (Fig. [95]5b). The
results from the PLA documented an interaction between mtp53 and MCM2
that was restricted to the subcellular nuclear zone. Our data showed
that mtp53 R273H interacted with MCM2 in the nucleus and made us
interested in seeing if the interaction of missense mtp53 with MCM2 was
a more general phenomenon. In order to address the interaction of other
mtp53 isoforms and wild-type p53 with MCM2 we compared the MDA-MB-468
PLA signal to those seen in a number of other cell lines (Supplemental
Figure 1). The confocal microscope settings were kept constant in order
to have a direct comparison. We observed a strong PLA signal between
R280K mtp53 and MCM2 in MDA-MB-231 cells and this was reduced by mtp53
knockdown. We also observed a strong PLA signal between R248Q mtp53 and
MCM2 in HCC70 cells, which again was reduced by mtp53 knockdown.
Interestingly, we detected some MCM2 interacting with the low level
wtp53 in MCF-7 cells and this reaction was stable. Therefore the high
concentration of different missense mtp53 on the chromatin in cancer
cells corresponds to a strong PLA signal with MCM2, and even low-level
wtp53 can be found in close proximity to MCM2. A previously published
immunoprecipitation screen of mtp53 R175H found an interaction with MCM
proteins that was reported only in the [96]Supplementary Data
section.^[97]20 We found that exogenously expressed mtp53 R175H, and to
a much lesser extent wild-type p53, interacted with both MCM2 and MCM4
(Fig. [98]5c). Mice with the analogous human R175H knockin mutation
(Trp53^R172H/R172H) develop lymphomas.^[99]21,[100]22 We also found
that the mtp53 in these mouse tumors interacted with MCM4
(Fig. [101]5d). In mice with mtp53 R172H, the protein is low in normal
tissue and is only found stable and highly expressed in tumor
tissue.^[102]23 Therefore it is not surprising that there was very
little mtp53 evident in the input, or immunoprecipitation samples from
normal tissue (Fig. [103]5, lanes 1, 2, 7, and 8). The fact that we
observed a stronger interaction between mtp53 R175H and MCM2, and a
weaker interaction between wtp53 and MCM2, corresponds with our
observations for comparative PLA analysis for multiple breast cancer
cell lines (Supplementary Figure [104]1). The MCM4 interaction in the
co-immunoprecipitation was more difficult to assess due to poor
antibody specificity, but nevertheless looked strongest for mtp53. From
these data we conclude that all forms of p53 can be found in close
proximity to MCM proteins but that a higher level of oncogenic mtp53 in
cancer cells corresponds to a much more robust signal for the proximity
interaction with MCM proteins. The fact that we do not see strong
enrichment for co-immunoprecipitation of MCM proteins, but see a strong
proximity interaction suggests that the mtp53–MCM interaction is not
due to a strong direct protein–protein interaction.
Fig. 5.
[105]Fig. 5
[106]Open in a new tab
Mutant p53 associates with MCM2, MCM4 and MCM7 on chromatin. a Protein
levels of MCM2, MCM4, MCM7, mtp53 and fibrillarin in the chromatin
fraction were determined by Western blot analysis in MDA-468.shp53
cells grown in the presence or absence of 8 μg/ml of doxycycline for
12 days, and HT-29 colon cancer cells transfected with p53-siRNA (p53)
or control siRNA (Con). b Analysis of p53/MCM2 complexes (red) by
immunofluorescence microscopy in combination with in situ proximity
ligation assay (PLA) in MDA-468 vector and MDA-468.shp53 cells grown in
the presence of 8 μg/ml of doxycycline for 12 days. DNA was
counterstained with DAPI (blue) and GFP (green) was an indicator of
doxycyline-mediated induction. The z stack confocal maximum intensity
projection images of p53/MCM2 and DAPI, p53/MCM2 and GFP are shown. c
Co-immunoprecipitation (co-IP) of MCM2 and MCM4 with exogenously
expressed mtp53 (R175H) and wtp53 in H1299 cells. Whole cell lystates
of H1299 cells transfected with wtp53 or mtp53 were incubated with
anti-p53 antibody followed by immunoblot with anti-MCM2 or anti-MCM4
antibodies. d Co-IP of MCM4 and mtp53 in thymic lymphomas from mtp53
(Trp53^R172H/R172H) mice. Thymic lymphomas from mtp53 mice and p53−/−
mice as well as normal thymic tissue from mtp53 mice were subjected to
co-IP assays using anti-p53 antibody followed by immunoblot with
anti-MCM4 antibody
Activation of apoptosis and PARP trapping is mitigated by knockdown of mtp53
or inhibition of MCM2–7
We previously saw that the inhibition of PARP was more cytotoxic in the
presence of mtp53 than in its absence.^[107]6 We hypothesized that this
might be due to a mtp53–PARP–MCM interaction at damaged DNA.
Synergistic activity is seen when the PARP inhibitor talazoparib is
used in combination with the DNA damaging agent temozolomide in BRCA1
mutant cells.^[108]24 It has been shown that wtp53 expression decreases
sensitivity of breast cancer cells to PARP inhibition^[109]25 and
ciprofloxacin blocks the MCM2–7 complex.^[110]26 We asked if increased
cytotoxity of PARP inhibition could be detected in the presence of
mtp53 if DNA was damaged by alkylation. We predicted that there would
be synergistic activation of apoptosis of the breast cancer cell lines
with mtp53 in the presence of talazoparib plus temozolomide because
this alkylating agent has been shown to provoke PARP trapping.^[111]27
We found that combination treatment with talazoparib plus temozolomide
induced synergistic activation of apoptosis only in the presence of
mtp53 and only when MCM2–7 processivity was not inhibited by
ciprofloxacin (Fig. [112]6a–c). This was detected by live cell confocal
microscopy scoring for activated caspases 3 and 7 (Fig. [113]6a–c).
PARP inhibition by talazoparib plus DNA damage with temozolomide
resulted in synergistic cell killing of MDA-MB-468 breast cancer cells
that are wild-type BRCA1 and R273H mtp53 (Fig. [114]6a; apoptotic cells
are green stained with active caspase 3 and 7). Moreover, when R273H
mtp53 expression was depleted by siRNA, or MCM2-7 was inhibited by
ciprofloxacin this synergistic activation was blocked (Fig. [115]6b and
[116]6c). In addition, cell viability reduced 59% in talazoparib plus
temozolomide treatment compared to non-treated cells (Fig. [117]6d). We
also found, as predicted, that combination treatment with talazoparib
plus temozolomide increased PARP trapping on the chromatin and this was
mitigated by the knockdown of mtp53 (Fig. [118]6e and [119]6f).
Moreover, depletion of mtp53 reduced the poly-ADP-ribosylated (PAR)
proteins in the combination treatment with talazoparib plus
temozolomide (Fig. [120]6e). Therefore mtp53 R273H and processive
MCM2–7 are required for the higher than additive killing seen when
cells are treated with talazoparib plus temozolomide.
Fig. 6.
[121]Fig. 6
[122]Open in a new tab
Activation of apoptosis and PARP trapping is mitigated by knockdown of
mtp53 or inhibition of MCM2–7. Confocal maximum projection of live-cell
imaging in MDA-468 cells (a, c) or MDA-468 cells transfected with
p53-siRNA or control siRNA (b) treated for 24 h with temozolomide
(Temo), talazoparib (Tal), combination (Temo + Tal) or ciprofloxacin
(Cipro). Apoptotic cells (green) were detected by activated caspase 3/7
green detection reagent and DNA was counterstained with DAPI (blue).
Red fluorescence was Propidium iodide staining. d MTT assay shows
reduction of mitochondrial activity after combination treatment of Temo
plus Tal. e, f PARP trapping and PARylation with and without mtp53
after 4 h treatment with Temo, Tal or combination (Temo + Tal). Protein
levels of PARP, mtp53 and PARylated proteins in the chromatin fraction
were determined by Western blot analysis in MDA-468 vector and
MDA-468.shp53 cells grown in the presence or absence of 8 μg/ml of
doxycycline for 12 days. The Western blot is a representative image.
The histogram is based on signal intensity (quatified using Image J
software) from two independent experiments and normalized to untreated
cells set as one. g Schematic model of the mtp53 dependent synthetic
lethality by the combination of talazoparib plus temozolomide
Discussion
High levels of mtp53 are found in over 50% of all human tumors from
patient samples.^[123]1 Somatic mutations in only three genes occur at
greater than 10% incidence across all different subtypes of breast
cancers, and one of these is mutation in the TP53 gene.^[124]2 More
than 80% of TNBCs contain mtp53 protein.^[125]2–[126]4 As far back as
1984 it was reported that the “oncogene” p53 cooperated with ras to
transform cells;^[127]28,[128]29 however, we still do not use mtp53 as
a diagnostic or treatment-mediated paradigm. TNBCs may serve as an
ideal paradigm for this approach. There are a number of high-occurrence
“hot spot” mutations found in the TP53 gene that result in amino acid
substitutions that inhibit the site-specific DNA binding activity of
p53.^[129]8 Some TP53 mutations contribute to breast cancer metastasis
because of loss of p53 tumor suppressor activity, many missense TP53
mutations cause new-found GOF oncogenic activities that range from
transcriptional activation of target genes that promote tumorigenesis,
to the inhibition of p53 family members p63 and p73.^[130]30 The GOF
mtp53 proteins have a prolonged half-life and are highly expressed in
cancer cells.^[131]1,[132]23 While mtp53-mediated regulation is known
to occur in part by activation and repression of gene
transcription,^[133]8,[134]30–[135]32 mounting evidence including data
from our lab indicates that other biochemical functions exist for
mtp53.^[136]6,[137]13,[138]31 Improved detection of proteomic signal
transduction changes are observed with sub-cellular fractionation
experiments of SILAC followed by LC-MS/MS.^[139]33,[140]34 We are the
only group to study mtp53-proteome interactions in the context of
sub-cellular architecture, which is critical for monitoring stability
of proteins based on location. To carefully analyze the proteomic data
we designed an algorithm to assay four data sets generated by inducible
mtp53 knockdown in conjunction with SILAC and mass spectrometry to rate
the mtp53 association index (mPAI). The mPAI points were graphed in an
interactive four-quadrant map of proteomic changes to compare cytosol
and chromatin targets
[141]http://diverge.hunter.cuny.edu/~weigang/mpai-browser/. The genes
and pathways associated with mtp53 were identified using GSEA and can
be searched with this interactive tool online. When MCM is typed in the
gene search tool you will see that all six members of the hexomeric
complex are found in the double positive quadrant. When the word
“replicative” is typed into the search pathway function the
pre-replicative pathway will appear and when the point is clicked all
six of the MCM proteins will show up again. The strength of this online
analysis tool is that, this is the first time many other mPAI pathways
are presented and they are yet to be validated. Herein, we validate the
mtp53–PARP–MCM pathway. We identified that mtp53 depletion also
depleted chromatin-associated PARP and all members of the MCM2-7
hexomeric complex. We are the first to show that mtp53 influenced MCM
chromatin levels in multiple cancer cell lines and directly associated
with MCM2 in nuclei as seen by confocal microscopy PLA (Fig. [142]5).
TNBCs are resistant to a number of different treatments; temozolomide
is one of the chemotherapeutic drugs TNBCs are resistant to.^[143]35 It
is clear that properties in addition to BRCA1/2 status dictate the
sensitivity to PARP inhibitors^[144]36 and we hypothesize that mtp53
status (with specific hot spot mutations) is a critical determinant.
Synergistic activity is seen with the PARP inhibitor talazoparib in
combination with the DNA modifier temozolomide.^[145]24 PARP is
recruited to DNA damage sites in chromatin to block transcription and
facilitate DNA repair^[146]34 and recently MCM2-7 was also found to
participate in DNA repair.^[147]37 Our study is the first to directly
show that the synergistic activity of temozolomide plus talazoparib is
dependent on the expression of mtp53 and the processivity of MCM2–7
(Fig. [148]6). Interestingly the proteomic study finding BAG2
stabilizes mtp53 also identified MCM proteins interacting with mtp53,
and the proteomic study finding MCM2–7 is involved in DNA repair also
found BAG2 interacting with the complex.^[149]20,[150]37 This suggests
that stable mtp53 may help recruit MCM2–7 and PARP proteins to
chromatin in order to help cancer cells survive during replication
stress. While PARP inhibitors have been used to target breast cancers
with BRCA1 mutations,^[151]38 they have not been approved for use in
cancers that have mutation in the TP53 gene. Breast cancers with BRCA1
mutations include many TNBCs, however PARP inhibitors have not shown a
direct correlation of effectiveness directly related to BRCA1 and BRCA2
mutations in TNBCs.^[152]36 MDA-MB-468 cells and HCC70 cells do not
have BRCA mutations and they are more sensitive to PARP inhibitors than
some breast cancer cell lines that have BRCA mutations. Importantly
both of these TNBC cell lines express high levels of mtp53, however a
correlation between mtp53 status and PARP activity before now had not
been determined. Recent work has shown that the cytotoxicity of PARP
inhibitors requires that the inhibitors trap the PARP enzyme onto the
chromatin.^[153]27,[154]39 Importantly, we found that in the presence
of mtp53, but not in its absence, combination treatment with the PARP
inhibitor talazoparib plus the DNA damaging agent temozolomide resulted
in efficient PARP trapping and apoptosis induction (Fig. [155]6).
It is of interest and important to determine if the temozolomide plus
talazoparib combination strategy works in vivo with a specificity for
tumor cells possessing specific p53 missense mutations. We found that
while mtp53 is highly associated with MCM2 on the chromatin
(Fig. [156]5b and Supplementary [157]Figure 1A), the mutation R273H
associated with the highest level of synergistic killing by the
combination treatment, and the sensitivity for cells with other p53
missense mutations varied (Supplementary [158]Figure 1B). There have
been demonstrations of remarkably synergistic activity of the PARP
inhibition by talazoparib plus temozolomide in a subset of pediatric
Ewing Sarcoma xenografts.^[159]24 The genomic landscape of Ewing
Sarcoma shows an aggressive subtype with TP53 mutations.^[160]40 The
p53 status has been reported for many of the cell lines used for the
Ewing Sarcoma xenograft models, and there is not a direct correlation
between the p53 mutation status and those cell lines that are sensitive
to combination treatment vs. those that are not.^[161]24 Cancer cell
sensitivity to combination treatment may be specific for certain p53
missense mutations in collaboration with other driver or passenger
mutations. We previously documented that the depletion of mtp53 in
MDA-MB-231 cells does not reduce MCM protein on the chromatin.^[162]6
Herein, we saw that there was an interaction between mt53 R280K and
MCM2 in MDA-MB-231 cells; however combination temozolomide plus
talazoparib treatment of MDA-MB-231 cells did not cause synergistic
killing (Supplementary [163]Figure 1B). It is possible that missense
mtp53, in different contexts, influences PARP and MCM in different
ways. Experiments are needed to elucidate the relationship between
different p53 missense mutants, and accessory proteins, for influencing
PARP and MCM2-7 structure and function.
Homologous-recombination-deficient tumors are dependent on
DNA-replication repair mechanisms that are more sensitive to PARP
inhibitors.^[164]41 It is possible the certain missense mutants of p53
block homologous-recombination in humans, as the p53 in C. elegans
inhibits nonhomologous end joining while promoting high fidelity
homologous recombination.^[165]42 Our results describe the close
proximity between mtp53 and the replication initiator mini chromosome
maintenance complex MCM2-7 on replicating DNA. However, they suggest
that each missense mutation has to be evaluated for its specific
activity. Researchers have found a way to reactivate mtp53 to become
wild-type like, but this reactivation is allele specific for
R175H.^[166]43,[167]44 The ability to target a characteristic of
multiple mtp53 proteins will enable using the newfound mtp53 activities
to be used against tumorigenesis. Our results implicate an interaction
of stable mtp53 at replication forks, and with PARP on the chromatin
that can be used to sensitize cancer cells to die. We saw that the
processivity of the MCM2-7 complex was required for synergistic
mtp53-dependent induction of apoptosis by the combination of
talazoparib plus temozolomide (see model in Fig. [168]6g). The MCM2–7
multi-subunit helicase participates in driving DNA replication and
improves replication under stressful conditions. This may be the
connection between mtp53 and MCM2–7 facilitating the synthetic lethal
function of PARP inhibition in treating TNBC. The disruption of p53 by
mutation often allows the subverted protein to interact with normal
partners of wild-type p53 but differentially influences the
outcome.^[169]45 It remains to be determined if PARP and MCM2–7 will be
added to the list of proteins that are influenced by wild-type and
mtp53 in opposing ways or if this is a new paradigm. Our findings
demonstrate a connection between mtp53 expression in TNBC and the
ability to target cells with the combination therapeutic drug protocol
previously intended for BRCA1 mutated cancers. Taken together, our
findings suggest that certain mtp53 missense mutations drive PARP
trapping and then MCM2–7 helps to facilitate the increased cytotoxicity
of PARP inhibitors plus temozolamide. This data also suggests that the
treatment of TNBC, with specific mtp53 proteins, by PARP inhibitors
plus temozolamide may have promising therapeutic effects and therefore
the use of mtp53 status in TNBC may be a predictive marker for
combination PARP-trapping therapy response.
Methods
Statistical analysis
We quantified the degree of under- and over-expression of proteins from
the reciprocal knockdown experiments in the different sub-cellular
fractions of the breast cancer cells by defining a mPAI. The mPAI was
defined as
[MATH: mPAI=log2HLexp1-
log2HLexp2, :MATH]
where
[MATH: HLexp1 :MATH]
is the ratio of peptide abundance in an experiment in which the control
cells were labeled with the heavy isotope and the mtp53 knockdown cells
were labeled with the light isotope, and
[MATH: HLexp2 :MATH]
is the corresponding ratio in the reciprocally labeled experiment. The
use of logarithm with base two converts these ratios to the unit of
fold changes between the control and the knockdown cells. When
abundance of a protein is not affected by mtp53 knockdown, both H/L
ratios are expected to be close to one, resulting in an mPAI ~ 0. For a
protein with abundance increased by the presence of mtp53,
[MATH:
(H<
mi>L)exp1 :MATH]
is expected to be >1 and
[MATH: HLexp2 :MATH]
< 1, resulting in an mPAI > 0. Conversely, mPAI was expected to be <0
for a protein with abundance decreased by the presence of mtp53.
Reagents
Doxycyclin, aprotinin, leupeptin, DTT, temozolomide and ciprofloxacin
were obtained from Sigma, Talazoparib BMN 673 from Selleckchem.
CellEvent Caspase-3/7 Green and ReadyProbes Cell Viability Imaging Kit
Blue/Red for Live Cell Imaging were obtained from Life Technologies.
Duolink in situ red kit goat/rabbit (Sigma) was used for PLA assay.
Cell lines
MDA-MB-468, H1299 and HT-29 cell lines were obtained from ATCC and
cultured in DMEM or McCoy’s (for HT-29) medium (Invitrogen),
supplemented with 10% FBS (Gemini, West Sacramento, CA, USA) and
50 U/ml penicillin and 50 µg/ml streptomycin (Mediatech). Cell lines
with the inducible p53 knockdown were generated and described
previously.^[170]5,[171]46,[172]47 To induce shRNA expression cells
were treated with 8 µg/ml of doxycyclin (Dox) for the time periods
indicated in the figure legends, fresh medium with Dox was supplemented
every 48 h.
Antibodies
Anti-human p53 mouse 1:1:1 mix of hybridoma supernatants pAb421,
pAb240, and pAb1801 (N-terminus, Central and C-terminus regions
respectively), rabbit anti-Actin (Sigma); mouse anti-Fibrillarin
(AbCam), mouse anti-PARP-1 (Santa Cruz), rabbit anti-PAR
(Millipore/Calbiochem), anti-MCM2, MCM4, MCM7 (Cell Signaling),
secondary antibody: anti-mouse and anti-rabbit HRP-conjugated (Sigma).
Sub-cellular fractionation
Cells were harvested and fractionation was performed using the Stillman
protocol.^[173]16 Briefly, cells were scraped from the plates, rinsed
with cold PBS twice and pelleted by centrifugation in 50 ml tubes at
1000 rpm 5 min. Cell pellets were resuspended in buffer A (10 mM HEPES
pH 7.9, 10 mM KCl, 1.5 mM MgCl[2], 0.34 M sucrose, 10% glycerol, 1 mM
DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, 8.5 µg/ml aprotinin) with 0.1%
Triton X-100. After 5 min incubation on ice cells were transferred to
Eppendorf tubes and spun down at 3600 rpm for 5 min at 4 °C. The
supernatant was spun down for an additional 5 min at 13,000 rpm at 4 °C
to clarify (Cytoplasmic Fraction). Pellets were washed two times with
Buffer A by centrifugation at 3600 rpm for 5 min at 4 °C. The nuclear
pellet was resuspended in Buffer B (3 mM EDTA, 0.2 mM EGTA, 0.5 mM
PMSF, 2 µg/ml leupeptin, 8.5 µg/ml aprotinin) and incubated on ice
30 min with vigorous vortexing every 5 min and spun down at 4000 rpm
for 5 min at 4 °C. The supernatant was nuclear soluble proteins. The
pellet enriched in chromatin, was washed two times with Buffer B,
resuspended in buffer B and sonicated three times for 30 s followed by
30 s rest on ice (Chromatin Fraction). Samples were stored at −80 °C.
Gel electrophoresis and immunoblotting
Proteins were separated using 10% SDS-PAGE and transferred to a
nitrocellulose membrane. The membrane was blocked in 5% non-fat milk
solution in PBS/0.1% Tween 20 and probed overnight at 4 °C. Washes were
done with PBS/0.1% Tween 20 solution. Secondary anti-mouse or
anti-rabbit antibody (Sigma) was applied to the membrane for 1 h at
room temperature and the membrane was washed three times. Protein
signal was visualized by chemiluminescence using the Super Signal West
Pico Kit (Pierce) and detected after exposure for autoradiography to
Hyblot CL films (Denville Scientific).
Quantitative proteomics by stable isotope labeling in cell culture SILAC mass
spectrometry
For SILAC mass spectrometry, we used Protein Quantitation Kit—DMEM
(Pierce) with ^13C[6] l-Lysine-2HCl and added to the media a second
amino acid ^13C[6] ^15N[4] l-Arginine-HCl (Pierce) for double labeling.
Cells were passaged for at least five cell doublings by splitting cells
when required and isotope incorporation efficiency was determined by MS
analysis. MDA-468.shp53 R273H depleted and non-depleted cells were
cultured in media containing either non-labeled or labeled amino acids,
harvested, fractionated, cytoplasmic, or chromatin fractions were mixed
at 1:1 ratio, separated by SDS-polyacrylamide gel electrophoresis,
stained with GelCode Blue Stain Reagent (Thermo Scientific), and 15 gel
sections excised with in situ trypsin digestion of polypeptides in each
gel slice performed as described.^[174]48 The tryptic peptides were
desalted using a 2 µl bed volume of Poros 50 R2 (Applied Biosystems,
CA) reversed-phase beads packed in Eppendorf gel-loading tips.^[175]49
The purified peptides were diluted to 0.1% formic acid and each gel
section was analyzed separately by microcapillary liquid chromatography
with tandem mass spectrometry using the NanoAcquity (Waters) with a
100-μm-inner-diameter × 10-cm-length C18 column (1.7 um BEH130, Waters)
configured with a 180-µm × 2-cm trap column coupled to a Q-Exactive
mass spectrometer (Thermo Fisher Scientific). Key parameters for the
mass spectrometer were: AGC 3 E6, resolution 70,000. Tandem mass
spectrometry fragmentation spectra were searched for protein
identification using the Andromeda search engine
([176]http://maxquant.org/) against the reversed and concatenated
IPI_HUMAN protein database (v3.87). One unique peptide was required for
high-confidence protein identifications and a minimum ratio count of
two peptides (one unique and one razor) were required for SILAC ratio
determination. Normalized SILAC ratios (H/L) were used for subsequent
analysis. All MS/MS samples were analyzed using MaxQuant (Max Planck
Institute of Biochemistry, Martinsried, Germany; version 1.3.0.3) at
default settings with a few modifications. The default was used for
first search tolerance and main search tolerance: 20 and 6 ppm,
respectively. Labels were set to Arg10 and Lys6. MaxQuant was set up to
search the reference human proteome database downloaded from Uniprot on
April 2, 2013. Maxquant performed the search assuming trypsin digestion
with up to two missed cleavages. Peptide, Site, and Protein FDR were
all set to 1% with a minimum of 1 peptide needed for identification but
two peptides needed to calculate a protein level ratio. The following
modifications were used as variable modifications for identifications
and included for protein quantification: Oxidation of methionine,
acetylation of the protein N-terminus, phosphorylation of serine,
threonine and tyrosine residues, and propionamide for acrylamide
adducts on cysteine. Raw data files are publicly available via the
Chorus data repository ([177]https://chorusproject.org) with project
I.D. number 1266. Original MaxQuant result files can be provided upon
request.
RNA interference and transfection
For siRNA experiments, HT-29 cells were seeded at 60% confluence in
media without penicillin—streptomycin and allowed to attach overnight.
Cells were transfected with 100 nM of p53 or non-targeted siRNA smart
pool from Dharmacon for 6 h using Lipofectamine 2000 (Invitrogen) as
per manufacturers protocol. At the end of the incubation period equal
volume of McCoy’s media with 40% FBS was added, next morning fresh
media with 10% FBS was added and the cells were allowed to grow for
72 h. Cells were harvested by scraping into the media, washed with PBS
and lysed for chromatin fractionation.
Live cell imaging
Cells were seeded at 2 × 10^5 per well in a 12-well glass bottom plate
(MatTek, Ashland, MA, USA). Detection of apoptotic cells was performed
using the CellEvent™ Caspase 3/7 Green Detection Reagent (Life
Technologies). After treatment, cells were stained with 50 µl CellEvent
Caspase-3/7 green ready probes reagent and 50 µl ReadyProbes Cell
Viability Imaging Kit Blue/Red (Life Technologies) for 15 min at room
temp. z-stack images of stained cells were taken by confocal microscopy
using a Nikon A1 confocal microscope with 20x objective. Active
caspase-3/7: green fluorescence, Propidium iodide: red fluorescence,
Nuclear DNA: blue fluorescence.
In situ PLA
Cells were seeded at 2 × 10^5 per well in a 12-well glass bottom plate
(MatTek). After removing the media, cells were rinsed with cold PBS
three times, fixed in 4% formaldehyde for 15 min and permeabilized in
0.5% Triton x-100 in PBS for 10 min at room temperature. After washing
three times in PBS and one time in distilled water for 2 min, cells
were then carried out PLA assay using Duolink in situ red kit
goat/rabbit (Sigma-Aldrich) according to the manufacturer’s
instructions. Briefly, cells were incubated in the blocking buffer for
30 min at 37 °C in a humidified chamber and then incubated with primary
antibodies diluted in the antibody diluents overnight at room
temperature in a humidified chamber. On the following day, cells were
washed in Buffer A for 5 min three times and incubated with the PLA
probes (anti-rabbit minus and anti-Goat plus) for 60 min at 37 °C in a
humid chamber. This was followed by 5 min wash in Buffer A for two
times. The ligation reaction was carried out at 37 °C for 60 min in a
humid chamber followed by two times 2 min wash in Buffer A. Cells were
then incubated with the amplification mix for 100 min at 37 °C in a
darkened humidified chamber. After washing with 1× Buffer B for 10 min
for two times and a 1 min wash with 0.01× buffer B, cells were mounted
with mounting media containing with 4′,6-diamidino-2-phenylindole
(DAPI). z-stack images were taken using Nikon A1 confocal microscope
with 60× objective oil immersion. The acquisition software is Nikon
elements. The primary antibodies were rabbit anti-p53 (Cat# A300-247A)
and goat anti-MCM2 (Cat# A300-122A) from Bethyl Laboratories.
Immunoprecipitation (IP) assays
IP assays were performed as previously described^[178]50 to determine
mtp53 binding proteins in human cancer cells and mouse tumor tissues.
In brief, 1 × 10^6 p53 null H1299 cells were transfected with
expression vectors of wtp53 or mutp53 (R175H). Cells were collected and
lysed in NP-40 buffer 24 h after transfection for IP experiments by
using anti-p53 antibody (DO-1) (Santa Cruz) to pull down mtp53 and its
binding proteins. For tissues of normal and thymic lymphomas from mtp53
knock-in mice (Trp53R172H/R172H) as well as thymic lymphomas from p53
knockout mice, 1 mg tissue lystates in NP-40 buffer were used for IP
using anti-p53 antibody (FL393) (Santa Cruz).
Electronic supplementary material
[179]Supplementary Figure 1^ (395.4KB, pdf)
[180]Supplementary Data^ (71.5KB, docx)
Acknowledgments