KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2
Cocco et al. Clinical Epigenetics
KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2
Elisa Cocco 0 4
Manuela Leo 3
Claudia Canzonetta 2
Serena Di Vito 1
Antonello Mai 6
Dante Rotili 6
Arianna Di Napoli 0 4
Andrea Vecchione 0 4
Cosimo De Nunzio 5
Patrizia Filetici 1
Antonella Stoppacciaro 0 4
0 Surgical Pathology Units, Department of Clinical and Molecular Medicine, Ospedale Sant'Andrea, La Sapienza University , Rome , Italy
1 Institute of Molecular Biology and Pathology-CNR, La Sapienza University of Rome , P.le, A. Moro 5, Rome , Italy
2 Department of Immunology, IRCCS Bambino Gesù Children's Hospital , Rome , Italy
3 Department of Biology and Biotechnology “C. Darwin”, La Sapienza University of Rome , Rome , Italy
4 Surgical Pathology Units, Department of Clinical and Molecular Medicine, Ospedale Sant'Andrea, La Sapienza University , Rome , Italy
5 Urology Unit, Department of Clinical and Molecular Medicine, Ospedale Sant'Andrea, La Sapienza University , Rome , Italy
6 Department of Drug Chemistry and Technology, Istituto Pasteur Italia - Fondazione Cenci Bolognetti, La Sapienza University , P.le Aldo Moro, 5, 00185 Rome , Italy
Background: Kidney cancer and clear cell renal carcinoma (ccRCC) are the 16th most common cause of death worldwide. ccRCC is often metastasized at diagnosis, and surgery remains the main treatment; therefore, early diagnosis and new therapeutic strategies are highly desirable. KAT inhibitor CPTH2 lowers histone H3 acetylation and induces apoptosis in colon cancer and cultured cerebellar granule neurons. In this study, we have evaluated the effects of CPTH2 on ccRCC 786-O cell line and analyzed drug targets expressed in ccRCC tumor tissues at different grade. Results: CPTH2 decreases cell viability, adhesion, and invasiveness in ccRCC cell line 786-O. It shows preferential inhibition for KAT3B-p300 with hypoacetilating effects on histone H3 at specific H3-K18. Immunohistochemical analysis of 70 ccRCC tumor tissues compared with peritumoral normal epithelium showed a statistical significant reduction of p300/H3AcK18 paralleled by an increase of H3AcK14 in G1 grade and an opposed trend during tumor progression to worst grades. In this study, we demonstrate that these marks are CPTH2 targets and significative prognosticators of low-grade ccRCC tumor. Conclusions: ccRCC is substantially insensitive to current therapies, and the efficacy of clinical treatment is dependent on the dissemination stage of the tumor. The present study shows that CPTH2 is able to induce apoptosis and decrease the invasiveness of a ccRCC cell line through the inhibition of KAT3B. In a tumor tissue analysis, we identified new prognosticator marks in grade G1 ccRCC tumors. Low KAT3B/H3AcK18 vs. high H3AcK14 were found in G1 while an opposed trend characterized tumor progression to worst grades. Our collected results suggest that CPTH2 reducing KAT3B and H3AcK18 can be considered a promising candidate for counteracting the progression of ccRCC tumors.
CPTH2; KAT3B-p300; ccRCC; Tumor tissues; H3-AcK18
Kidney cancer is classified as the 16th most common cause
of death from cancer worldwide . The majority of kidney
cancers (70%) are classified as clear cell renal carcinoma
(ccRCC), at average age of diagnosis (60–64 years) . It is
often metastasized ; therefore, identification of new
therapeutic strategies is highly desirable. ccRCC is
associated with VHL loss of function with stabilization of hypoxia
inducible factors (HIF-1α and HIF-2α) in both sporadic and
familial forms . Recent studies highlighted major roles for
epigenetic regulation in the development and progression of
the disease. Several epigenetic regulators such as SWI/SNF
polybromo PBRM1, histone deubiquitinase BAP1,
methyltransferase SETD2, and acetyltransferase MYST1 (KAT8)
are significantly altered in ccRCC [5–8]. Noteworthy, global
levels of histone H3 acetylation has been correlated with
disease progression [9, 10] suggesting that histone
modifications are tightly linked to ccRCC. The K-histone acetyl
transferase KAT3B (p300) could play a role , and its
overexpression is detected in the most aggressive cases of
hepatocellular carcinoma . In prostate cancer, KAT3B
promotes tumor growth and activation of androgen
receptor  weakens invasiveness in melanoma, breast, and
prostate cancer cell lines [14–16]. Its nuclear localization is
linked to pro-tumoral effects while cytoplasmic to a less
severe outcome . Epigenetic drugs are potential tools for
pharmacological research and therapeutic applications.
HDAC inhibitors have been extensively studied as potential
anticancer treatment; however, they exhibit different effects
across various renal cell lines . On the other hand, there
is little information on the use of K-acetyltransferase
inhibitors (KATi). Nonetheless, some of them were shown to
prevent growth in a broad panel of cancer cells lines [19, 20]
such as neuroblastoma and glioma [21, 22]. In the past, we
identified a novel KAT inhibitor,
cyclopentylidene-[4-(4chlorophenyl)thiazol-2-yl)hydrazone (CPTH2), in yeast .
CPTH2 was tested in different experimental models with
effects on axon outgrowth , adenovirus infection ,
expression of superoxide dismutase in human monocytes
, and impaired antibody response in B lymphocytes
. In colon adenocarcinoma, CPTH2 lowers cancer
growth, decreasing GCN5 activity regulated by cMyc/E2F1
. In the presented study, we assessed the effects of
CPTH2 on K1 papillary thyroid and ccRCC 786-O cell
lines. CPTH2 lowered KAT activity of nuclear cell extracts
showing specificity for KAT3B. Importantly, it lowered cell
viability, impaired invasiveness and migration, and modified
cell adhesion with a global effect on cytoskeleton
organization. We also analyzed the effects of the drug on
the acetylation of histone H3 globally and at selected
residues, and we found a hypoacetilating effects at specific
H3AcK18. The results obtained by treating the cells with
CPTH2 were comparable with the effects induced by
silencing KAT3B, thus confirming the inhibitory selectivity of
CPTH2 for KAT3B in 786-O cell line. The collected
experimental results on cell lines were paralleled by a detailed
analysis of KAT3B broad distribution and global levels of
histone H3AcK14 and H3AcK18 in 70 ccRCC patients.
Tumor and normal tissues from kidney specimens at
different grades and stages were compared. Notably, we found a
sudden increase of H3AcK18 and KAT3B in the switch
from G1 to G2 tumor grade. Surprisingly, this effect was
paired by a progressive decrease of H3AcK14 levels. This
analysis suggests a novel approach and identifies promising
prognosticators in clear cell carcinoma.
Cell cultures and treatment with CPTH2
786-O human ccRCC cell line (ATCC, Manassas, VA) was
grown in RPMI 1640 plus 10% FBS, 2 mM L-glutamine,
25 U/ml penicillin, and 25 U/ml streptomycin (Gibco,
Thermo Fisher Scientific, Waltham, MA). K1 human
thyroid carcinoma cell line (ECACC, Sigma-Aldrich, St. Louis,
MO) was cultured in DMEM:Hamm’s F12:MCDB 105
medium (2:1:1) plus 10% FBS, 2 mM L-glutamine, 25 U/ml
penicillin, and 25 U/ml streptomycin (Gibco, Thermo
Fisher Scientific). Cells were maintained in a humidified
atmosphere of 5% CO2 at 37 °C.
Cyclopentylidene-[4-(4chlorophenyl) thiazol-2-yl)hydrazone (CPTH2) was dissolved in
100 mM/L dimethyl sulfoxide (DMSO; Sigma-Aldrich) and
diluted to the final concentrations in complete medium.
For all the experiments, cells were treated with 1% DMSO
as control. After 24 h from seeding, exponentially growing
786-O and K1 tumor cells were treated with CPTH2 at
concentrations ranging from 1 to 200 μmol/L for 24 to
120 h; 24–48 h culture and 100 mM/L were then chosen as
CPTH2 concentration giving the strongest growing
inhibitory effects without signs of cell damage or exhaustion of
the drug function .
2.9 mg of CPTH2 (MW 291.80) have been dissolved in 0.
3 ml of DMSO and then diluted with PBS buffer (pH = 7.
4) to 2 ml (5 mM). Then, the resulting solution was
incubated at 37 °C on a heating block and checked for purity
after different times (30 min, 1, 2, 12, 24, 36, 48, 72, and
132 h) by Thin Layer Chromatography (TLC) on silica gel
plates eluting with the mixture Ethyl Acetate:Petroleum
Ether (1:2). Over time, no traces of degradation products
were detected by TLC. CPTH2 is stable in PBS buffer at
37 °C since no traces of degradation products can be
detected by TLC up to 132 h of incubation.
In vitro HAT assay
Histone acetyltransferase activity was measured with
fluorescent in vitro HAT Assay Kit (Active Motif, CA) in
nuclear extracts (7 μg) prepared with EpiQuik™ Nuclear
Extraction Kit I (Epigentek, NY) treated 24 or 48 h with
CPTH2 100 μM or solvent DMSO. Twenty-five
micrograms of recombinant proteins p300 (Active Motif ),
GCN5 (Active Motif ), and PCAF (Biomol, DE) were
incubated with Anacardic Acid 15 μM, CPTH2 400 μM,
and 600 μM or in DMSO. HAT activity was measured in
tubes in presence of Ac-CoA (0.5 mM) and histone H3
(50 μM). Resulting fluorescence was measured with
GloMax® (Promega, WI) after conjugation between
developer and free sulfhydryl groups on CoA-SH.
Cell viability and apoptosis assay
Percentage of viable cells was evaluated by trypan blue dye
exclusion method. After monolayer cell trypsinization (0.
25 mg/ml trypsin, Gibco, Thermo Fisher Scientific), cells
were stained with trypan blue (0.04%, Gibco) for 2 min, and
vital, unstained cells were counted with emacytometer. The
percentage of unstained cells was obtained as mean ± SD of
four independent experiments. 786-O cell apoptosis was
assayed with MuseAnnexinV and Dead Cell kit (Millipore,
Darmstadt, DE); 1 × 105 cells from untreated, DMSO and
CPTH2 samples were centrifuged (2000 rpm, 5 min),
washed in PBS, and resuspended in PBS plus 1% BSA
(Sigma-Aldrich) 1% FBS, with 100 μL of Dead Cell reagent
containing AnnexinV and 7-aminoactinomycin D (7-AAD).
After 20 min RT incubation at room temperature in the
dark, cells were applied to a Muse Cell Analyzer (Millipore)
, and the results are expressed as percentage of apoptotic
cells ± SD.
In vitro migration, invasiveness, and adhesion
Migratory and invasive capacities of 786-O cells were
evaluated using the BioCoat Invasion Chamber system
(BD Biosciences, San Jose, CA). Matrigel invasion
chambers, containing an 8-μm-pore-size PET membrane,
were treated with Matrigel Basement Membrane Matrix
(invasion test; BD Bioscience) or with BSA (migration
test; Sigma); ~ 1.5 × 105 cells diluted in 0.2%
FBSDMEM were added to upper compartment and 2.5 ml
10% FBS-DMEM to the lower compartment. Migration
assay was performed for 24 and 48 h in a humidified
tissue culture incubator at 37 °C in a 5% CO2 atmosphere.
After incubation, no migrating cells were removed by
scrubbing the upper face of the membrane, and migrated
cells present on the lower surface of the membrane were
stained with Diff-Quick; cells present in 10,400×
enlargement field were counted in each filter. The data are
given as mean ± SD of triplicate filters. For adhesion test,
a 96-well plate was coated with BSA (5 mg/ml,
SigmaAldrich), fibronectin (5 μg/ml, bovine, Calbiochem),
matrigel (3 mg/ml, standard matrigel matrix, BD
Biosciences), and collagen type IV (5 μmg/ml, bovine,
SigmaAldrich). Cells treated for 24 and 48 h with CPTH2 or
DMSO were plated 5 × 104 on each coated well in
triplicate and incubated to allow adhesion at 37 °C for
1 h. After PBS washing, cells were fixed in ice-cold
acetone/ethanol (1:1, Sigma-Aldrich) for 10 min and washed
with 20% methanol. Cells stained with crystal violet
solution (0.5% w/v in 20% methanol, Sigma-Aldrich) were
measured in a spectrophotometer at 540 nm (Multiskan
spectrum, Thermo) after color solubilization with 0.1 M
sodium citrate pH 4.2 (50% EtOH, Sigma-Aldrich).
Cell migration was tested with “wound healing” assay
. Briefly, 786-O cells were seeded in a 6-well plate
and cultured until confluence, scraped with a 200-μl
micropipette tip, then incubated with CPTH2 (100 μM),
DMSO, or RPMI; the growth was photographed at 0 and
48 h with an inverted microscope (Nikon Eclipse
TE2000-S) and digital camera (Nikon Coolpix S4, 6.0
Mpix, 10× zoom). Wound area was measured and
quantified with TScratch Software .
18-20 h before transfection, 786-O were plated in 6-well
plates in complete growth medium; at 60% of
confluency, cells were placed in OptiMEM (serum-and
antibiotics-free medium; Thermo Fisher Scientific) and
transfected with 30 nM of p300 small interfering RNA
(HSC.RNAII.N001429.12.1, IDT, San Jose, CA) or
Negative Control 1 (IDT) using Lipofectamine 2000
according to the manufacturer (Invitrogen, Thermo Fisher). Six
hours after transfection, the medium was changed to full
growth conditions, and cells were harvested at 6, 12, 24,
and 48 h post-transfection. p300si efficiency was
assessed by real-time PCR transcript analysis of p300
786-O cells were seeded on glass coverslips in 35 mm
Petri dishes and cultured until 50% confluence. They
were treated with CPTH2 (100 μM) for 18 h or
transfected with 30 nM si-p300 for 24 h, washed three times
with PBS and fixed with 4% paraformaldehyde (PFA;
Sigma-Aldrich) in PBS, permeabilized with 0.2% Triton
X-100 (Sigma-Aldrich), and blocked with 1% BSA. Then,
they were incubated with rhodamine–phalloidin (1:1000,
Thermo Fisher Scientific) in 2% BSA in PBS for 1 h,
washed with PBS, and stained with DAPI (1:10000,
1 mg/mL stock solution, Roche, Basel, CH). Images were
acquired with the Nikon fluorescent microscopy, and
stress fibers were counted by analyzing 100 cells in
different fields for each experimental point. The data are
given as mean ± SEM of stress fiber numbers per cell.
RNA isolation and real-time PCR analysis
Total RNA was isolated from 786-O cell line with TRIzol
reagent (Ambion, Thermo Fisher), quantified with
Age ± SD
64.21 ± 11.24
64.63 ± 11,84
63.42 ± 10.18
Tumor grade 
G1 = 20
G2 = 26
G3 = 24
G1 = 15
G2 = 16
G3 = 15
Nanodrop 2000 (Thermo Fisher). Two hundred fifty
nanograms of total RNA were reverse transcribed with
HighCapacity RNAtocDNA Reverse Trascription kit (Applied
Biosystems, Thermo Fisher). Real-time PCR was performed
in Stratagene Mx3005P (Agilent Technologies, Santa Clara,
CA) with TaqMan2X Universal Master Mix (Applied
Biosystems) in 20 μl mixture. Each sample assayed in triplicate
was performed with PCR cycles: (10 min) at 95 °C and
60 cycles of (15 s) at 95 °C and a final (1 min) at 60 °C. The
primers and probes of the following transcripts were EP300
(Hs00914223_m1), AKT-1 (Hs00178289_m1), TGF-b2
(Hs00234244_m1), HIF-1a (Hs00153153_m1), CD44
(Hs01075864_m1), ITGb1 (Hs01127536_m1), ITGb3
(Hs01001469_m1), ITGa5 (Hs01547673_m1), and ITGa6
(Hs01041011_m1) (Applied Biosystems). The fold change
of gene expression was calculated using the 2-ΔΔCT
method, and all values were normalized to endogenous
control ACTB (Hs 99999903_m1, Applied Biosystems) and
expressed in arbitrary units.
Bulk histone preparations and western blot analysis
Cells were seeded at 200,000 per well, after 24 h were
treated with CPTH2, DMSO, p300si, NC1 and incubated
at 37 °C for 12, 24, and 48 h in a humidified atmosphere
of 5% CO2. Total protein extracts were resuspended in
Laemmli buffer (Bio-Rad, CA) and heated 5 min at 90 °
C. Protein extracts were run on 15% SDS-PAGE, blotted
onto nitrocellulose (GE Healthcare Life Sciences, UK)
and hybridized with anti-H3AcK18 (Abcam, UK),
antiH3Ac (Merck, Germany), and anti-GAPDH (Santa-Cruz,
TX) antibodies. Fluorescence detected by Long Lasting
Chemilumiscent Substrate (EuroClone, Italy) was
visualized by ChemiDoc™ MP Imaging System (Bio-Rad).
Tissue samples and immunohistochemistry
Clear cell RCC tissues and matched normal adjacent
tissues were collected from 70 patients (listed in Table 1)
with primary ccRCC between January 2008 and December
2014, who underwent kidney tumor radical surgery at the
Sant’Andrea Hospital of Roma “La Sapienza” University.
The use of the histological material was authorized by
personal patient consensus according to S. Andrea Hospital
policy form. Patient medical records including tumor
staging, pathological diagnosis, and surgical data were
reviewed and classified according to the American Joint
Committee on Cancer . Formalin-fixed and
paraffinembedded ccRCC tissue blocks were sectioned,
deparaffinized in xylene, and rehydrated through a graded ethanol
series and then subjected to antigen retrieval by boiling in
0.01 M sodium citrate buffer (pH 6) 10 min in microwave.
Endogenous peroxidase was blocked for 10 min in 3%
hydrogen peroxide in methanol, incubated 1 h RT with
primary antibody diluted to anti-p300 rabbit polyclonal
antibody 1:1000 (Bethyl Laboratories), anti-H3AcK18 1:
2000 (Abcam), and anti-H3AcK14 1:2000 (Abcam).
Reactions were followed with DAB detection kit (Dako).
Immunostaining results were recorded as percentage of
positive cells in increments of 10% regardless of the
intensity of the staining. Cases were considered as negative if <
5% of tumor cells were positive. Immunohistochemical
and morphological analyses were evaluated by
pathologists (AS and AV).
104 si-p300 cells untreated and treated in DMSO w/w
CPTH2 were spotted on glass slide using cytocentrifuge
(Cytospin3 Seongkohn traders, Korea) at 700 rpm for
5 min; slides were fixed with cold acetone for 15 min,
allowed to dry, and stored in PBS at 4 °C. The cell spots
were incubated with primary antibody as previously
Experiments were performed in triplicate and results
recorded. Cell line data were presented as mean ± standard
deviation (SD). Statistical analysis was performed using
the S-PSS 12.0 software. Evaluation of data distribution
showed a non-normal distribution of the study data set.
Differences between groups of patients in medians for
quantitative variables and differences in distributions for
categorical variables were tested with the Kruskal–Wallis
one-way analysis of variance and chi-square test,
respectively. Using multiple logistic regression with the
enter method, variables as assessed in the univariate
Tumor stage 
Stage 1 = 35Stage 2 = 16Stage 3 = 16Stage 4 = 1
Stage I = 23Stage II = 9Stage III =12Stage IV = 1
Stage I = 12Stage II = 7Stage III = 4Stage IV = 0
analysis were entered and investigated as predictors of
ccRCC G2-G3 versus G1 and in a separate model
predictors of high stage (stages II–III) versus low stage
(stage I) were compared. The logistic regression analysis
was carried out using the data from patients for whom
complete data were available. The variables considered
for entry into the model included age, p300, H3AcK14,
and H3AcK18. An alpha value of 5% was considered as
the threshold for significance. The data are presented as
mean ± standard deviation (SD); in vitro HAT assay,
RTPCR, and western blot analysis were presented as mean
± SEM. Student t test was calculated, and p value ≤ 0.05
was considered significant. Odds ratios and 95% CIs
were calculated for the parameters in each group using
ccRCC G1 and ccRCC stage I as a reference group.
CPTH2 inhibits HAT activity and decreases tumor cell viability through apoptosis
Papillary thyroid (K1) and clear cell Renal Cell
Carcinoma (ccRCC-786-O) cell lines were incubated with
CPTH2 at the most effective concentration of 100 μM
in comparison with untreated and excipient (DMSO)
controls (Fig. 1). In vitro HAT activity was measured in
24 h K1 and 48 h 786-O treated cells, respectively,
depending on the responsiveness of individual cell lines.
Despite intrinsic levels of HAT activity been higher in
K1 than in 786-O, CPTH2 treatment caused a
comparable drop of the activity in both lines (Fig. 1a), according
to a direct enzyme’s inhibition of the drug. To
investigate the capacity of CPTH2 to affect cell proliferation,
cells were treated in DMSO w/w CPTH2 for 12, 24, and
48 h and washed and the number of cells adjusted by
day exclusion and cultured in medium. CPTH2
treatment caused a decrease in cell proliferation after as early
as 12 h with a further significant reduction after 48 h
stimulation. K1 cell line, which is derived from papillary
thyroid carcinoma and is responsive to chemotherapy
and apoptotic drugs, showed a reduction of 80% after
48 h. ccRCC-786-O, which is from renal clear cell
carcinoma and is much less sensitive to anti-proliferative
drugs, presented a significant, but less pronounced,
decrease, 40% (Fig. 1b). In order to demonstrate whether
CPTH2 affected the cell cycle FACS analysis was
performed confirming that no relevant defects in cell cycle
progression were induced in ccRCC 786-O cells after
48 h treatment with CPTH2 (Fig. 1c). CPTH2 was also
unable to change cell cycle progression in ccRCC 786-O
and K1 papillary thyroid cells treated with prolonged
treatment (Additional file 1). Accordingly, no substantial
differences were obtained when 786-O cells were treated
for 24 and 48 h in DMSO w/w CPTH2 as shown in the
immunostaining with Ki67 and Cyclin D1 antibodies
(Additional file 2). Annexin-V FACS analysis of K1 and
786-O cell lines showed in addition that CPTH2
treatment produced a drastic increase in apoptotic/dead cell
population after 48 h quantified as a percentage of total
apoptotic cells (Fig. 1d and Additional file 1), suggesting
that CPTH2 treatment leads to cell death rather than
cell cycle arrest. Collectively, these results indicate that
the KAT inhibitor CPTH2 is active on both thyroid
papillary K1 and 786-O cell lines, inhibits in vitro HAT
activity in nuclear extracts, and lowers cell viability after
48 h treatments, thus leading to cell death.
CPTH2 changes the cytoskeleton organization of ccRCC786-O cells and decreases invasiveness and migration
The capability of a novel compound to counteract
invasion and metastatic growth is a fundamental feature to be
considered in the development of new potential antitumor
drugs. We therefore analyzed the effects of CPTH2
against the invasive properties of 786-O cell line. We
treated cells at increasing time with CPTH2 (100 μM);
after 24 and 48 h, we observed an evident reduction of cell
volume and a gross rearrangement of cytoskeleton
organization with conglobation and disaggregation of the
actin stress fibers and retraction of organized phila as
evidenced by the phalloidin-TRITC staining (Fig. 2a). The
number of visible actin stress fibers was already strongly
lowered after 18 h of drug treatment suggesting a
modulation of cell adhesion (Fig. 2b). We next assayed the
adhesion capabilities of 786-O cells grown on different
substrates treated with CPTH2 for 12 and 24 h.
Interestingly, CPTH2 was able to modulate cell adhesion only
when 786-O cells were plated on the complex synthetic
matrix Matrigel, while on single components such as
fibronectin and collagen, it was substantially unaffected
(Fig. 2c). This effect was evident after 48 h treatment
hinting that the reduced adhesiveness of 786-O tumor cells is
likely dependent on the concerted modulation of several
different adhesion molecules. We next asked whether
genes known to be involved in adhesion and migration
such as integrins were affected at transcriptional level.
mRNA expression profiles of selected marker genes such
as the transmembrane glycoprotein CD44, involved in
lymphocyte migration and metastasis  and integrins
ITGb3, ITGb1, ITGa5, and ITGa6  that were
comparatively analyzed by RT-qPCR (Fig. 2d). The summary
panel shows an overall coordinated upregulation after 24
and 48 h of treatment, indicating a clear deregulation of
adhesion genes and a coordinated behavior induced by
drug treatment. This result is in agreement with effects in
the deregulation of integrins. It is known, in fact, that their
up or downregulation [35, 36] is responsible to modulate
cell invasion and migration in cancer microenvironment.
We finally confirmed the inhibitory activity of CPTH2 on
the cell invasiveness properties, performing an in vitro
wound healing scratch tests. 786-O cells were scraped and
allowed to grow and migrate in CPTH2 versus DMSO
(Fig. 2e). Results indicate that after 48 h, it is clearly seen
that while cells in DMSO were actively migrating and able
to fill the gap in the presence of CPTH2 migration was
severely inhibited. Migration and Matrigel invasion were
tested in Boyden chambers. Figure 2f shows results,
migration (upper panel), and invasion (lower panel) were
evaluated after 24 and 48 h in DMSO w/w CPTH2
showing a drastic decrease after 48 h of treatment. Collectively,
our data demonstrate that CPTH2 is capable to
counteract invasion and migration of 786-O cells in culture.
CPTH2 shows selectivity for KAT3B-p300
In order to evaluate the selectivity of CPTH2 in the
inhibition of individual KATs, we carried out an in vitro HAT
assay with recombinant p300, GCN5, and PCAF in the
presence of increasing concentrations of CPTH2 (400 and
600 μΜ) and the known KAT inhibitor anacardic acid [20,
37]. CPTH2 preferentially inhibited p300 compared to
GCN5 while it did not affect PCAF (Fig. 3a). Starting from
this evidence, we first analyzed the effects of CPTH2 on
p300 mRNA expression in 786-O cell line, and RT-qPCR in
6 to 48 h time lap cultures showed that p300 mRNA
expression was unaffected by CPTH2 treatment at
transcriptional level (Fig. 3b). We next asked whether CPTH2 may
act at the protein level. Since p300 can be expressed both in
the cell nucleus and cytoplasm, immunocytochemistry was
performed using anti-p300 antibodies on 786-O (Fig. 3c)
and K1 thyroid cultures (Additional file 3) in untreated,
DMSO control, and after 72 h of CPTH2 treatment, to
better depict the eventual subcellular change of expression.
Surprisingly, p300 staining decreased progressively to reach
an almost complete clearing after 72 h of CPTH2
stimulation in both cell lines. Our analysis also revealed that
CPTH2 lowered p300 protein levels on both its nuclear and
cytoplasmatic localization. We wanted to assay the effects
of CPTH2 on p300 in presence of proteasomal degradation.
Cells were pretreated with MG 132 for 1 h and then grown
in DMSO w/w CPTH2; no relevant differences in the
number of apoptotic cells nor in immunostaining intensity of
p300 were obtained. These results suggest that the
proteasome has not a relevant function in the decrease of p300
upon CPTH2 treatment. These results suggested that, even
on live cells, CPTH2 may exert its main inhibitory activity
on KAT3B-p300 and is able to lower the protein level
independently from its cellular localization. On the basis of
CPTH2 selectivity for KAT3B, we decided to investigate the
effects of p300 silencing in 786-O cells. Figure 3d shows
that KAT3B-p300 mRNA interference (left panel) was
followed by an early drop of 786-O cell viability (right
panel) comparable in value to the effect induced by
treatment with CPTH2 100 μM (Fig. 1b vs. Fig. 3d).
Furthermore, p300 silencing after 18 h resulted in a remarkable
cytoskeleton rearrangement with clear drop in the number
of stress fibers per cell (Fig. 3e). To provide a further
demonstration that CPTH2 and p300si act through the same
target, 786-O cells transfected with control (nc) and si-p300
RNA were grown in DMSO and treated with CPTH2 for
48 h (Fig. 3f). We found that cell proliferation, number of
stress fibers, and migration showed the same values in
controls and si-p300 cells, thus demonstrating full overlap
between CPTH2 activity and silencing of p300. Collectively,
all these results demonstrate the selectivity of CPTH2 in
the inhibition of KAT3B-p300.
CPTH2 treatment and p300si show similar effects on histone H3K18 acetylation and expression of selected cancer markers
Increased expression of KAT3B correlates with higher
acetylation of histone H3K18 in the axonal regeneration
of optic nerve  while its and CREBB knockdown led
to a global decrease of H3AcK18 in human embryonic
stem cells . In budding yeast, we showed a selective
acetylating activity of KAT2A on H3K18 . Following
this line of results, we next asked whether CPTH2 might
similarly affect acetylation of histone H3 and at selected
H3K18 in 786-O cells. Western blot analysis of bulk
histone preparations from 786-O cells were serially
hybridized with anti-AcH3 and anti-H3AcK18 antibodies after
a 48-h time course incubation with CPTH2 showing a
reduced acetylation of both global AcH3 histone and
H3AcK18 (Fig. 4a). Next, based on the matching
between CPTH2 treatment and p300 silencing, we tested
the global levels of AcH3 and H3AcK18 at different time
points during p300si treatment in 786-O confirming the
decrease of AcH3 and H3AcK18 after 24 h (Fig. 4b).
Immunohistochemical staining of 786-O for specific H3
lysines, H3AcK14, and H3AcK18 was then performed in
cells treated for 48 h with CPTH2 (Fig. 4c). Strikingly,
while the staining pattern of H3AcK14 was unaltered by
CPTH2 treatment, the staining of H3AcK18 was
drastically weakened in comparison to control and DMSO
cells, suggesting that the inhibitory activity of CPTH2
was highly selective for histone H3K18 residue with
respect to H3K14. The immunohistochemical analysis on
p300si 786-O cells with anti-p300, as a control,
antiH3AcK14, and H3AcK18 compared to untreated nc cells
(Fig. 4d) showed substantially the same result. This
unbiased result confirms that silencing of p300 and CPTH2
treatment induce similar effects lowering the degree of
global histone H3AcK18 in a p300 dependent way. We
then added a control experiment by comparing the
activity of the previously described specific KAT3B
inhibitor, C646  with CPTH2. Western blot analysis
showed that the effects of C646 and CPTH2 on
inhibition of bulk AcH3, H3AcK14, and H3AcK18 were fully
comparable at increasing times (Fig. 4e). To reinforce
this point, we finally compared the transcriptional effects
of p300si and CPTH2 treatment on the mRNA expression
of three oncogenes: AKT1, TGFb2, and HIF-1a at
different time points (Fig. 4f ). The summary diagram of
collected results showed convincingly that the expression
trend of each gene in time is highly comparable in the two
experimental conditions providing an additional
conclusive demonstration on the convergent effects obtained by
treatment of CPTH2 and silencing p300.
Expression of p300, H3AcK18, and H3AcK14 of ccRCC ex vivo carcinoma
To analyze whether ccRCC cells selected acetylated forms
of histone H3K18 and H3K14, bulk preparations of
histone nuclear proteins were extracted from 11 ccRCC
specimens and from normal adjacent tissues and tested by
western blot. The results were normalized to the amount
of unmodified H3 and expressed as arbitrary units (Fig.
5a). As shown, the amount of H3AcK18 was generally
much lower than H3AcK14, and overall, the acetylation of
both was extremely variable among cases. Interestingly,
however, the respective acetylation of tumor versus
normal paired tissue was higher for H3AcK18 and lower for
H3AcK14. Furthermore, G3-stage II (nos. 6 and 7),
G2stage I (nos. 13 and 23), and G3-stage I (nos. 17 and 18)
[32, 41] were characterized by higher content of H3AcK18
and lower content of H3AcK14 in tumor with respect to
normal paired tissues. Taken together, these data
evidenced a global heterogeneity in the levels of H3AcK14
and H3AcK18 among individuals but hinted of an
opposite influence of H3AcK18 and H3AcK14 residues on
tumor growth. To test the hypothesis of a reciprocal
behavior between H3AcK14 and H3AcK18 and the possible
correlation between p300 and H3AcK18 expression as
found in vitro on 786-O cell line, we decided to extend
the analysis to tumor specimen from ccRCC patients after
a careful tumors reevaluation. Overall, 70 ccRCC patients
were entered in the study: 46 (65%) males and 24 females
(35%). The mean age was 64.2 ± 11.2 years. Twenty
patients (29%) present a grade 1 and 50 grades 2–3 (71%);
35 patients a stage I (50%) and 35 at stages II–III (50%)
[32, 41] (Table 1). Sections from 2 to 4 different blocks for
each case of ccRCC, everyone comprising normal
peritumoral renal parenchyma, were immunostained with
H3AcK18, H3AcK14, and KAT3B-p300 antibodies, and
the results were reported as the mean percentage of
stained nuclei. The choice of antibody experimental
settings and parameters were chosen in order to exclude any
cytoplasm staining, thus avoiding misleading results. More
importantly, to avoid differential conditions in
experimental setting, the cases were stained altogether in a single
experiment in order to obtain a fully comparable result.
Although the number of the cases analyzed in this study is
limited, their distribution for sex, age, grade, and stage is
representative of usual ccRCC epidemiology. As expected,
expression of H3AcK18 and H3AcK14 was limited to the
cell nucleus of both clear renal cell carcinoma and
epithelial cells of normal kidney (podocytes and epithelial cells
of the proximal and distal tubules), whereas KAT3B-p300
antibody stained both the nucleus and the cytoplasm in
42/70 ccRCC cases and 67/69 epithelial cell of the
peritumoral normal kidneys. Our results have taken into
consideration only the normal kidney or neoplastic epithelial
cells whereas endothelial and inflammatory intra and
perineoplastic cells, also expressing the three proteins, were
carefully excluded. The percentage of tumor or normal
epithelial cells expressing the three antibodies was highly
variable among the different cases (Additional file 4).
Patients’ characteristics according to p300, H3AcK14, and
H3AcK18 expression are summarized in Table 2. Overall,
there was no significant difference in the frequency of
positive cells between the tumor and adjacent normal
kidney tissues for all the three antibodies used. The frequency
of H3AcK18, H3AcK14, and p300 positive epithelial cells
in normal tissues, although different among cases, showed
similar mean + SD values independently from the sex, age,
grade, and stage of the hosted tumor. Surprisingly, only
the G1 tumors showed a significant variation of the three
parameters with respect to their normal counterpart,
suggesting that G1 tumors may show different pathway of
transformation or progression (Fig. 5b). As shown in Fig.
5b and Table 2, G2-G3 ccRCC presented a significant
higher expression of p300 and H3AcK18 and a lower
expression of H3AcK14 compared to G1 ccRCC (p < 0.001).
Furthermore, high-stage ccRCCs presented a significant
lower expression of H3AcK14 (Table 2). Interestingly, in
all the tumor cases, the expression of p300 is directly
correlated with H3AcK18 and inversely to H3AcK14, Fig. 5c
shows two significant cases. On the multivariable analysis,
H3AcK14 and p300 were found to be independent
predictors of high-grade ccRCC (Table 3). Particularly, p300
increased the risk of high-grade ccRCC by 7.6% per unit
(OR 1.076, IC 1.029–1.236, p = 0.001) and H3AcK14
reduced the risk of high-grade ccRCC by 3% per unit (OR 0.
971, IC 0.943–0.999, p = 0.0041). H3AcK14 was also
independent predictors of high-stage ccRCC. Particularly,
H3AcK14 expression reduced the risk of stages II–III by
2.5% per unit (OR 0.975, IC 0.953–0.999, p = 0.041).
In the last decades, it has been demonstrated that genes
aberrantly regulated in human cancer play a fundamental
role in tumor onset and progression. Overall, epigenetic
regulation tuning cell differentiation in response to
environmental stimuli can be considered a driving alteration of
tumor progression and a response sign to therapy.
Aberrant patterns of post-translational modifications of
histones and cellular proteins lead to alteration of the
epigenetic landscape that characterizes human diseases,
from cancer to inflammatory and neurological disorders
[42, 43]. K-histone acetyltransferases (KATs) are indeed
aberrantly expressed in cancer and contribute to
oncogenic transformation, thus representing, more
importantly, potential targets for therapeutic intervention.
KAT3B-p300 is a critical regulator of hematopoiesis, and
its heightened expression is recurrent in human
malignancies such as prostate , liver [12, 45], and breast cancer
and is predictive of worse prognosis. Accordingly, the
activation of several oncogenes which directly sustain cancer
proliferation such as STAT3, NF-κB, and HIF1α are
subjected to acetylation. Furthermore, it has been highlighted
the role of p300 as a coactivator in the induction of
superenhancers, master hub coordinating the expression of
cluster of transcriptional enhancers controlling
fundamental gene circuitries responsible for cell identity . p300
is engaged not only in the acetylation of nuclear histones
but also of non-histone proteins such as transcription
factors involved in autophagy , motility, and metastatic
processes . Collectively, these reports, as part of a vast
literature, suggest many reasons why p300 represents a
promising therapeutic target in the treatment of refractory
cancer types . Although the mechanisms that regulate
p300 activity have not been yet fully highlighted, the
importance of p300 intracellular localization for its activity
[50–53] has acquired relevance. Lysine acetylation
coregulates several cellular functions through large
macromolecular complexes involved in chromatin
remodeling, splicing, nuclear transport, and actin nucleation
. In pancreatic cancer, a nuclear signaling between Src
and p300, with a Src-dependent phosphorylation of p300,
regulates gene promoters of AT-hook (HMGA)2, SET, and
SMYD3 with effects on cell migration and invasiveness of
tumor cells . In the cytoplasm, acetylation increases
the stability of actin fibers of the cytoskeleton  while
opposed deacetylation leads to their destabilization with
consequent-reduced migration and motility of cells. In
renal clear cell carcinoma 786-O cell line and in papillary
thyroid K1, we have demonstrated that KAT inhibitor
CPTH2  increases cell death and apoptosis, changes
adhesion and cytoskeletal organization, and decreases cell
invasiveness and migration. Interestingly, we showed that
CPTH2 acts primarily on KAT3B-p300. Although CPTH2
was shown to inhibit GCN5 in human cells, it is also to be
underlined that KAT2A-GCN5 expression in renal normal
cells and in ccRCC is very low (according to Human
Protein Atlas and our unpublished results). Administration of
CPTH2 and silencing of p300 showed identical effects in
treated cells. We also report that CPTH2 lowers the
concentration of p300 at protein level both in the nucleus and
in the cytoplasm suggesting that it may carry out several
functions in the cell. In the cytoplasm, it may affect actin
cytoskeleton stability lowering cell motility while, in the
nucleus, it inhibits global levels of histone H3 acetylation
and p300 dependent H3AcK18  with several
regulatory effects, such as regulation of the expression of genes
and oncogenes such as AKT1, TGFb2, and HIF1a.
Alteration of post-translational modifications at selected
histone N-terminal residues is an emerging novel tool for
early diagnosis and prognosis. Oxidative stress induced by
malignant transformation of renal tubular epithelium
changes global acetylation of histones H3-K9, K18, K27,
Table 3 Odds ratios (OR) and 95% confidence interval (CI) for
predicting G2-G3 ccRCC and stage II–III ccRCC
Risk of G2-G3 ccRCC
OR 95% CI
and K14 . Hypoacetylation of H3AcK18 is associated
to prostate carcinoma with poor prognosis , and
increase of H3AcK18 caused by absence of SIRT7 at sites of
DNA damage affects the maintenance of genome integrity
. Lower levels of H3K3me2 and H3AcK18 are in fact
predictive for higher recurrence in prostate, lung, and
kidney cancer patients and can be used for distinguishing
clinical outcomes of patients with substantially similar
clinico-pathological variables . On the basis of the
effects of CPTH2 in lowering p300 and H3AcK18 and the
vast literature on the relevance of this histone mark as
prognosticator in cancer patients, we wanted to extend
the analysis of histone H3 acetylation at K18 and K14
along with p300 in 70 cases of ccRCC tumor patients
(listed in Table 1). In the presented study, we started from
western blot analysis on global levels of H3AcK18 and
H3AcK14 performed by comparison of nuclear extracts
from tumor tissue and peritumoral normal epithelium of
each single patient, which revealed a great heterogeneity
in the global levels of H3AcK14 and H3AcK18 with
respect to unmodified H3 among patients. This observation
motivated us to extend our study to 70 ccRCC cases
grouped for tumor grade. To be sure to exclude from our
tissue analysis immunostaining due to non-specific
cytoplasm background, we used high dilutions of H3AcK18,
H3AcK14 antibodies (methods), and this allowed us to
stain, exclusively, cell nuclei and exclude false positive and
cross reaction (Fig. 4c, d and Additional file 3). Statistical
analysis of our collected results showed that p300 and
H3AcK18 levels gave identical profiles supporting
selectivity of p300 for H3AcK18 also in ccRCC specimens.
Importantly, the analysis showed an identical pattern of
p300, H3AcK18, and H3AcK14 in the normal peritumoral
tissue of all patients confirming the relevance of analyzing
normal epithelium in our screening. Lower p300/
H3AcK18 opposed to higher H3AcK14 in low-grade
ccRCC, G1 cases with respect to normal epithelium was
observed. In addition, with tumor worsening to G2-G3
grade, while p300/H3AcK18 come back to levels found in
normal tissues, H3AcK14 showed a significant,
progressive decrease (Fig. 5b) as summarized in Table 2. The
opposed degree of H3AcK14 vs H3AcK18/p300 expression
is significatively restricted to G1 tumors and may
therefore represent an important epigenetic signature of
lowgrade ccRCC. Notably, conflicting results have been
reported on the expression levels of histone H3
acetylation and specific H3AcK18 and H3AcK14 in cancer.
While conflicting results reported the relevance of the
acetylation of H3, H3K18 and H3K14 in ccRCC
progression [9, 61] warrant are issued for a more thorough study
of ccRCC from different and relevant casuistic and
analytical procedures. Collectively, the presented study reports
the identification of a novel epigenetic signature for
tracing ccRCC tumor tissues based on low-p300/H3AcK18
vs high-H3AcK14 ratio in global histone H3 acetylation,
distinctive of low-grade G1 tumors, and prognosticators
for tumor aggressiveness. Further molecular analysis for
cancer markers such as expression of oncogenes and
oncosuppressors or noncoding RNAs can be developed to
identify additional characterizing features for the
classification of high- or low-H3K18/K14 ratio found in ccRCC
low-grade G1 tumors.
The p300 inhibitor CPTH2 lowers cell invasiveness and
viability in ccRCC 786-O cell line. It is a promising compound
for counteracting the increase of p300 and H3AcK18 found
in higher grade G2, G3 ccRCC tumor tissues. Finally, the
opposed ratio of p300-H3AcK18 vs H3AcK14 represents a
novel prognosticator signature of low grade, and G1 ccRCC
tumors and CPTH2 may efficiently counteract the
increasing of both p300 and H3AcK18 in tumor progression.
Additional file 1: Cell cycle progression is not affected by treatment of
ccRCC 786-O cell line with CPTH2. FACS analysis of ccRCC 786-O cell line
treated with DMSO w/w CPTH2 (100 μM) at increasing times. Apoptotic
profiles of ccRCC 786-O and papillary thyroid K1 cell lines untreated and
grown in DMSO w/w CPTH2 at increasing times. (TIFF 24906 kb)
Additional file 2: 786-O cells grown in DMSO w/w CPTH2 for 24 and
48 h and immunostained with anti-Ki67 and anti-cyclin D1 show that
CPTH2 treatment does not affect cell cycle progression. (TIFF 24905 kb)
Additional file 3: Immunostaining of K1 papyllary thyroid cells with
p300 antibody after 72 h of treatment with CPTH2 (100 μM) compared to
untreated and DMSO controls. Apoptotic percentage of 786-O cells
treated with proteasome inhibitor MG-132 (1 h) and then incubated in
DMSO w/w CPTH2 shows no significative changes of the apoptotic
profiles compared to the untreated controls. p300 immunostaining of 786-O
cells were pretreated for 1 h with proteasome inhibitor MG132, then
grow in DMSO w/w CPTH2 for 18 h suggest that there is no significative
proteolysis of p300 upon inhibition of the proteasome. (TIFF 30444 kb)
Additional file 4: Immunostaining of tissue sections from ccRCC tumor
and normal tissues with p300, H3AcK18, and H3AcK14 antibodies. Two
opposite cases are shown, patient no. 1 with low p300/H3AcK18 vs. high
H3AcK14. Patient no. 41, the opposite, high p300/H3AcK18 vs. low
H3AcK14. (TIFF 37242 kb)
C646: KAT3B-p300 inhibitor; ccRCC: Clear cell renal carcinoma;
K1: Papillary thyroid cancer cells; KAT: K-Histone acetyltransferase;
KAT2A: GCN5; KAT3B: p300
The authors wish to thank Giuseppe Pisaneschi for technical assistance
to P. Filetici.
E.Cocco was supported by the AV7-PRIN 2011 (2010W4J4RM_004). M.Leo was
a recipient of PhD grant in the Cell Biology and Development, La Sapienza
Rome University. The work was financed by the A.Vecchione-AIRC 2016
(IG16862) and A. Mai-PRIN 2016 (2TE5PK), A. Mai-AIRC 2016 (19162).
Availability of data and materials
All data generated during this study are included in the publication and in
figures (text and additional files).
AS and PF contributed equally to this work as co-corresponding authors, principal
investigators, planning experimental strategy, interpretation of data, writing, and
revision of the manuscript with conclusions. AV assisted in discussing the results
and ms revision. EC, ML, CC, SDV, and ADN contributed to the experimental part.
AM and DR were responsible for the synthesis and characterization of the CPTH2
compound. AS was responsible for the immunohistochemical analysis of the
tissues. PF was responsible for the epigenetic interpretation of the results and
analysis of histone PTMs. CDN provided the kidney biopsies and statistical analysis
of patient tissues. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The use of the histological material was authorized by personal patient
consensus according to S. Andrea Hospital policy form.
Consent for publication
All authors approved the publication of the submitted paper.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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