2015 - Raynal Et Al - Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer

Published OnlineFirst December 30, 2015; DOI: 10.1158/0008-5472.
CAN-14-2391
Cancer
Therapeutics, Targets, and Chemical Biology Research
Targeting Calcium Signaling Induces Epigenetic

Reactivation of Tumor Suppressor Genes in
Cancer
Noe€ l J.-M. Raynal1,2, Justin T. Lee1, Youjun Wang3, Annie Beaudry2, Priyanka Madireddi1,
Judith Garriga1, Gabriel G. Malouf4, Sarah Dumont4, Elisha J. Dettman4,
Vazganush Gharibyan4, Saira Ahmed4, Woonbok Chung1, Wayne E. Childers5,
Magid Abou-Gharbia5, Ryan A. Henry6, Andrew J. Andrews6, Jaroslav Jelinek1,
Ying Cui7, Stephen B. Baylin7, Donald L. Gill8, and Jean-Pierre J. Issa1
Abstract
Targeting epigenetic pathways is a promising approach for prominently cardiac glycosides, did not change DNA methyl-
cancer therapy. Here, we report on the unexpected finding that ation locally or histone modifications globally. Instead, all 11
targeting calcium signaling can reverse epigenetic silencing of drugs altered calcium signaling and triggered calcium-calmod-
tumor suppressor genes (TSG). In a screen for drugs that ulin kinase (CamK) activity, leading to MeCP2 nuclear exclu-
reactivate silenced gene expression in colon cancer cells, we sion. Blocking CamK activity abolished gene reactivation and
found three classical epigenetic targeted drugs (DNA methyl- cancer cell killing by these drugs, showing that triggering
ation and histone deacetylase inhibitors) and 11 other drugs calcium fluxes is an essential component of their epigenetic
that induced methylated and silenced CpG island promoters mechanism of action. Our data identify calcium signaling as a
driving a reporter gene (GFP) as well as endogenous TSGs in new pathway that can be targeted to reactivate TSGs in cancer.
multiple cancer cell lines. These newly identified drugs, most Cancer Res; 76(6); 1494–505. 2015 AACR.
Introduction novo methylation in cytosine of CpG dinucleotide at the promoter

region of TSG results in stable gene silencing through direct
In cancer, the epigenome is aberrantly reprogrammed, leading
inhibition of transcription factor binding or by recruitment of
to a wide range of heritable changes in gene expression such as
methyl-binding domain (MBD) proteins such as MeCP2 (1, 2).
silencing of tumor suppressor genes (TSG; ref. 1). The most
These MBDs are associated with other repressor complexes includ-
studied epigenetic aberrations in cancer involve DNA methylation
ing histone deacetylases (HDAC) that are responsible for global
and histone posttranslational modifications. Acquisition of de
loss of histone acetylation, resulting in gene silencing and het-
erochromatin formation (2).
1
As these epigenetic modifications are reversible, one goal of
Fels Institute for Cancer Research and Molecular Biology, Temple
University School of Medicine, Philadelphia, Pennsylvania.
epigenetic therapy of cancer is to reverse these alterations and
2
De partement de pharmacologie, Universite de Montre al and induce TSG reactivation, leading to cancer cell differentiation and
al, Que
Sainte-Justine University Hospital Research Center, Montre bec, cancer cell death (3). Clinical efficacy of epigenetic drugs led to
3
Canada. Beijing Key Laboratory of Gene Resources and Molecular their approval for the treatment of hematologic malignancies and
Development College of Life Sciences, Beijing Normal University,
Beijing, P.R. China. 4Department of Leukemia, The University of Texas occasional proof-of-principle responses can be seen in solid
MD Anderson Cancer Center, Houston, Texas. 5Moulder Center for tumors (2, 4). However, treatment options are limited to a small
Drug Discovery Research, Philadelphia, Pennsylvania. 6Department number of epigenetic drugs approved in the clinic with two DNA
of Cancer Biology, Fox Chase Cancer Center, Philadelphia, Pennsyl-
vania. 7The Sidney Kimmel Comprehensive Cancer Center at Johns methylation inhibitors (decitabine and azacitidine) and two
Hopkins, Baltimore, Maryland. 8Department of Cellular and Molecular HDAC inhibitors (vorinostat and depsipeptide). There is a need
Physiology, The Pennsylvania State University College of Medicine, to discover new candidate epigenetic drugs, including some that
The Milton S. Hershey Medical Center, Hershey, Pennsylvania.
work through other mechanisms of action. Drug discovery initia-
Note: Supplementary data for this article are available at Cancer Research tives are underway in rare and specific cancer types with well-
Online (http://cancerres.aacrjournals.org/).
defined mutations in epigenetic effectors. However, these efforts
^pital de la Pitie
Current address for G.G. Malouf: G.G. Malouf, Ho -Salpe
trie
^re, may take years before approval and may have limited effects
Medical Oncology, Paris, France. outside of a restricted patient population (5).
Corresponding Author: Jean-Pierre J. Issa, Fels Institute for Cancer Research To discover new epigenetic drugs that can be rapidly tested in
and Molecular Biology, Temple University School of Medicine, 3307 North Broad the clinic, we performed an unbiased epigenetic drug screen
Street, Rm 154 – PAHB, Philadelphia, PA 19140. Phone: 215-707-4307; using FDA-approved drug libraries. The rationale is that pos-
Fax: 215-707-1454; E-mail: jpissa@temple.edu
itive hits can be rapidly repositioned for cancer treatment
doi: 10.1158/0008-5472.CAN-14-2391 because these drugs have known safety, pharmacodynamics,
2015 American Association for Cancer Research. and pharmacokinetics (6). As a platform for epigenetic drug
1494 Cancer Res; 76(6) March 15, 2016
Downloaded from cancerres.aacrjournals.org on June 17, 2016. © 2016 American Association for Cancer Research.
Published OnlineFirst December 30, 2015; DOI: 10.1158/0008-5472.CAN-14-2391
Targeting Calcium Signaling Produces Epigenetic Reactivation
Figure 1.
Epigenetic drug screening reveals
candidate epigenetic drugs among
FDA-approved libraries. A, scheme
showing CMV-GFP locus in YB5
cells. CMV promoter is DNA
hypermethylated and marked by
repressive chromatin. Epigenetic
drugs induced GFP reactivation as
shown by GFP fluorescence after
decitabine treatment (50 nmol/L,
72 hours). B, illustration of dose
schedules selected for drug screening.
C, drug screening results after
treatment with library of drugs at 10
mmol/L for 72 hours. Twenty-three
positive hits were identified (n 1). D,
drug screening results after treatment
with the same library at 50 mmol/L for
24 hours. Seventy-seven positive hits
were identified (n 1). E, Venn
diagram showing the number of
positive hits specific or shared to each
dose schedule and the validation
results. F, positive hits and screening
values of GFP-expressing cells (n 1).
screening, we used the well-characterized YB5 cell-based sys- reactivate silenced TSGs, we used the YB5 system as a cell-based
tem, which is derived from the human colon cancer cell line, assay for epigenetic drug screening.
SW48 (7, 8). YB5 cells contain a single insertion of cytomeg- Here, our screen revealed a new mechanism where targeting
alovirus (CMV) promoter driving GFP gene. GFP expression is calcium signaling can reactivate TSGs silenced by epigenetic
silenced in >99.9% of YB5 cells by epigenetic mechanisms mechanism in cancer cells. The alteration in calcium signaling
because its CMV promoter has DNA hypermethylation and is results in activated calcium-calmodulin kinase (CamK), which
embedded in repressive chromatin with histone deacetylation played a central role in TSG reactivation and cancer cell killing.
and histone methylation marks (Fig. 1A; ref. 7). In YB5 cells,
GFP behaves similarly to endogenous TSGs silenced by epige-
netic mechanisms and it can be reactivated by treatment with
Materials and Methods
DNA methylation inhibitors and/or HDAC inhibitors (7, 8). Cell culture
We previously demonstrated that GFP reactivation induced by Human colon cancer cell line YB5 and its parental cell line
decitabine is characterized by both DNA demethylation and SW48 were cultured in L-15 medium. Human colon cancer cell
chromatin resetting at the CMV promoter region (7). Moreover, line HCT116 was cultured in McCoy's medium. In HCT116
we also showed that HDAC inhibitors induced GFP reactivation human colon cancer cells, GFP sequence was inserted in exon 2
through chromatin resetting at the CMV promoter with an of the TSG secreted frizzled-related protein 1 (SFRP1) making the
increase in histone acetylation without any changes in DNA HCT116 SFRP1-GFP cell line. This locus is silenced by promoter
methylation (8). As the goal of the epigenetic therapy is to DNA hypermethylation in HCT116 (9). GFP expression detected
www.aacrjournals.org Cancer Res; 76(6) March 15, 2016 1495
Raynal et al.
by flow cytometry corresponded to the activity of SFRP1 promoter bleaches into GFP channel. Drugs that showed more than
and was measured in less than 10% of the cells. Normal colon 8% of the cells in the double positive quadrant were discarded.
cells, CRL-1831, were grown in DMEM F-12 medium supplemen- This threshold value was calculated as the sum of the average
ted with 10 ng/mL cholera toxin, 0.005 mg/mL insulin, 0.005 plus one SD of the percentage of cells detected in the double
mg/mL transferrin, 100 ng/mL hydrocortisone, and 100 mmol/L positive quadrant in the entire screen dataset. Second, the
HEPES. Leukemia cell lines, K562 and HL-60, were cultured in lowest detection threshold for GFP reactivation was set at
RPMI1640. HEK293T were grown in DMEM. All cell culture 2.2% of the cells present in the GFPþ quadrant. This value was
media were supplemented with 10% FBS. Colon cancer and established experimentally and corresponded to the lowest GFP
leukemic cells were grown in log phase in 1% and 5% CO2 value obtained in the screen that was confirmed during our
atmosphere, respectively. SW48, K562, and HL-60 cell lines were validation step with drugs purchased from independent
obtained from ATCC (2005–2007) and were authenticated at MD suppliers.
Anderson Cancer Center genomic core facility by DNA finger-
printing in 2011. CRL-1831 cell line was obtained from ATCC in RNA extraction, cDNA synthesis, qPCR
2012 and used within 2 months from the purchase. HCT116 and Total RNA (2 mg) was extracted using TRIzol (Invitrogen) and
HEK293T were obtained from the laboratories of S.B. Baylin and cDNA was synthesized using High-Capacity cDNA Kit (Applied
D.L. Gill and were genetically engineered cell lines that were not Biosystems). Quantification of cDNA was done by qPCR with the
further authenticated. Universal PCR Master Mix (Bio-Rad) using ABI Prism 7900HT
system. Results were obtained from at least three independent
Drugs and treatments experiments where each sample was analyzed in triplicate. 18S
Drug libraries of FDA-approved drugs were obtained from NCI- was used as a reference gene. All primers have been described
Developmental Therapeutics Program (Combo Plate 3948/99 previously (7, 8).
containing 77 drugs, NCI Oncology Drug set 4640/34 containing
52 drugs, NCI Oncology Drug set 4641/34 with 37 drugs) and DNA extraction and DNA methylation analysis
from commercially available U.S. Drug collection library with DNA extraction, bisulfite conversion, and pyrosequencing were
1,040 drugs (MS Discovery). A total of 1,118 unique FDA- carried out as previously described (7, 8).
approved drugs were screened. Log-phase growing YB5 cells were
treated with drug libraries (n 1) using two different schedules. Histone extraction and Western blots
The first schedule consisted of a 72-hour treatment at 10 mmol/L For acid histone extraction, cells were harvested and washed
where drugs and media were replaced every day. Cells were twice with ice-cold PBS supplemented with 10 mmol/L sodium
incubated for an additional 24 hours without drugs before anal- butyrate to retain levels of histone acetylation. Cell pellets were
ysis. This schedule was designed to discover putative new hypo- resuspended cells in Triton Extraction Buffer [TEB:PBS containing
methylating agents as the induction of DNA demethylation may 0.5% Triton X100 (v/v), 2 mmol/L phenylmethylsulfonylfluor-
require several cell divisions to become detectable (2). The second ide, 0.02% (w/v) NaN3, complete protease inhibitor cocktail, 10
schedule consisted of a 24-hour treatment at 50 mmol/L. Cells mmol/L sodium butyrate]. Cell lysis was induced after 10 minutes
were immediately analyzed after the treatment. This schedule was incubation on ice with gentle stirring. Lysates were centrifuged at
designed to discover drugs that would reactivate GFP by chroma- 6,500 g for 10 minutes at 4 C. Supernatant was discarded. Cells
tin remodeling because, in this cell line, HDAC inhibitors can were washed in half the volume of TEB and centrifuged as before.
reactivate GFP in 24 hours without any changes in DNA meth- Pellets were resuspended in 0.2 N HCl. Histones were acid
ylation at the CMV promoter. For validation purposes and phar- extracted overnight at 4 C. Samples were centrifuged at 6,500
macologic (leptomycin L2913) modulation, drugs were pur- g for 10 minutes at 4 C. Histone proteins contained in the
chased at Sigma-Aldrich and dissolved either in DMSO, ethanol, supernatant were saved and protein concentration was deter-
or sterile PBS according to the manufacturer's recommendations mined using the Bradford assay. Extracted histones were stored
and stocks were kept frozen at 80 C. CamK inhibitors were at 80 C. Histone extracts were run in 12% SDS–PAGE precast
purchased at EMD Millipore (Kn-93, 422712 and Kn-92, gels (Bio-Rad, 345-0118). Proteins were transferred to polyviny-
422709). lidene difluoride (PVDF) membranes in 10 mmol/L CAPS (pH
11) containing 10% methanol and detected by using specific
Flow cytometry primary antibodies and horseradish peroxidase–conjugated sec-
After treatment, cells were trypsinized and resuspended in ondary antibodies (GE Healthcare) and Enhanced Chemilumi-
cell culture media with propidium iodide (PI) to stain dead nescence reagent (Pierce). PVDF membranes were incubated with
cells. For epigenetic drug screening with YB5 cells, GFP and PI specific primary antibodies. Commercially available antibodies
fluorescence were measured using BD LSR II flow cytometer. were purchased for H3K4-3me (Active motif, 39159), H3K9-3me
Validations were performed using Millipore Guava flow cyt- (Abcam, AB 8898), H3K14-Ac (Active motif, 39697), H3K9-Ac
ometer with YB5 or SFRP1-GFP HCT116 cells. Flow cytometry (Active motif, 39917), H3K27-3me (Active motif, 39155),
analysis was also used prior to any type of extractions (RNA, H3K27-Ac (Upstate Biotechnology, 07360), H3 (Active motif,
DNA, and proteins) to ensure a similar level of GFP reactivation 39763), and MeCP2 (Abcam, AB2828).
following drug treatments for each biologic replicate. Flow
cytometry results were plotted as GFP fluorescence on the x- Statistical analysis
axis and PI on the y-axis (Supplementary Fig. S1A–S1D). Differences between groups were assessed using a one-way
Positive hits were designated using two selection criteria. First, ANOVA. The P value was evaluated by the Tukey–Kramer multiple
we excluded all autofluorescent drugs (like antimalarials) comparison test. Statistics and graphical representations were
because autofluorescence creates a false positive signal that performed using GraphPad Prism software.
1496 Cancer Res; 76(6) March 15, 2016 Cancer Research
Results endogenous TSGs silenced by epigenetic mechanism in YB5 cells

(CDH13, RIL, TIMP-3) as well as the stress response gene P21
Epigenetic drug screening and positive hits validation
(CDKN1A; Supplementary Fig. S3A–S3D). In addition, we mea-
We screened over 1,100 FDA-approved drugs obtained from
sured SFRP1 reactivation, another DNA hypermethylated TSG in
the NCI and commercial sources using two dose schedules (Fig.
HCT116 colon cancer cells (9, 10). In this cell line, GFP sequence
1B). The first schedule (10 mmol/L for 72 hours followed by 24
was inserted in exon 2 of SFRP1 making its activation detectable
hours without drug) was designed to discover epigenetic drugs
by flow cytometry (9). Similar to YB5 cells, all drugs reactivated
that require several cell cycles to induce robust gene reactiva-
SFRP1 gene in the HCT116 SFRP1-GFP system (Fig. 3B). To
tion, as observed with DNA methylation inhibitors. This sched-
identify a mechanism of action, we asked whether candidate
ule induced significant DNA demethylation and was associated
epigenetic drugs induced gene reactivation through known epi-
with gene reactivation in YB5 cells (7). The second schedule (50
genetic mechanism such as DNA demethylation or histone acet-
mmol/L for 24 hours) was selected to discover epigenetic drugs
ylation. First, we measured DNA methylation locally in the
that produce rapid gene reactivation, as seen with HDAC
promoter regions of reactivated genes (CMV and endogenous
inhibitors. On the basis of the very low background in the
TSGs) and at LINE-1 repeats to assess global DNA methylation
YB5 system (less than 0.1% of the cells express detectable levels
levels. Using bisulfite pyrosequencing assays designed in the
of GFP; Supplementary Fig. S1A) and activity seen in negative
promoter regions close to the transcription start site (8), we found
controls, positive hits in the screen were defined as those non-
that none of the drugs reduced DNA methylation at the CMV (Fig.
autofluorescent drugs inducing more than 2.2% of GFPþ cells
3C) or TIMP-3 (Supplementary Fig. S4A) promoters or globally at
(Supplementary Fig. S1B–S1E).
LINE-1 repeats (Fig. 3D), except for decitabine (a well-known
The drug screen revealed 23 (2%) and 77 (7%) positive hits at
hypomethylating drug). We then measured quantitatively histone
10 mmol/L (for 72 hours) and at 50 mmol/L (for 24 hours)
acetylation levels by mass spectrometry on 9 residues in histone 3
respectively, whereas 12 drugs (1%) were identified in both
(H3) and histone 4 (H4). No significant changes were observed,
schedules (Fig. 1C–E). The highest inducers of GFP expression
except with vorinostat, a well-known HDAC inhibitor that
were the DNA methylation inhibitors decitabine (at 10 mmol/L,
increased histone acetylation levels on lysine 9, 14, 18, and 23
for 72 hours) and azacitidine (at 50 mmol/L, for 24 hours)
of histone 3 and on lysine 8, 12, and 16 of histone H4 (Fig. 3E and
producing, respectively, 30% and 16% GFPþ cells (Supplemen-
Supplementary Fig. S4B). A similar lack of global effects was
tary Tables S1 and S2). Azacitidine induced GFP reactivation after
observed by Western blots for histone-activating marks (H3K4-
a short treatment of 24 hours, which can be explained by the
me3, H3K9-ac, and H3K27-ac) or histone-inactivating marks
number of YB5 cells in S-phase during the treatment (2). The
(H3K9-me3 and H3K27-me3; Supplementary Fig. S4C).
HDAC inhibitor vorinostat was also identified in this screen
producing 2.6% GFPþ cells (Supplementary Table S2). Thus, all Intracellular Ca2þ fluxes induced by the newly identified drugs
three known epigenetic drugs represented in the libraries were As neither DNA demethylation nor global chromatin modifi-
rediscovered in this blinded screen, validating the approach. cations explained TSG reactivation by the newly identified drugs,
Because of the unexpected large number of positive hits, we we searched for alternative mechanisms. Surprisingly, when
focused on the 12 drugs identified in both dose schedules (Fig. 1E). searching for a mechanism of action of the newly identified drugs,
For validation, compounds were purchased from separate suppliers we noted that most of the (9/11) drugs were known to increase
and tested for GFP reactivation in YB5 cells. Of these 12 hits, all intracellular calcium (Ca2þ) levels (11–14). No data were found
except tilmicosin (which was low positive) were validated. These 11 regarding the three known epigenetic drugs and calcium signaling.
validated drugs fell into three groups (Fig. 1F). The first one Interestingly, it was reported in neurons that cytosolic increase in
included the known epigenetic drugs decitabine, azacitidine, and Ca2þ levels activate CamK, which releases the methyl-binding
vorinostat. The second group encompassed cardiac glycosides such protein MeCP2 from silenced promoters, leading to gene reacti-
as ouabain, lanatoside C, digoxin, digitoxin, and proscillaridin A. vation (15, 16).
All cardiac glycosides represented in the libraries reactivated GFP, To test whether this mechanism could explain TSGs reactiva-
suggesting a drug class effect. The last group was labeled as anti- tion in cancer cells by the newly identified drugs, we first char-
biotics/others and contained oxyquinoline (antibiotic), disulfiram acterized Ca2þ channels in YB5 cells. Using Ca2þ sensitive dye
(a drug used for alcoholism treatment), and arsenic trioxide (anti- (fura-2), we determined that YB5 cells do not express functional
cancer drug; Fig. 1F). In the validation studies, GFP reactivation was L-type voltage-operated Ca2þ channels (Supplementary Fig. S5A
tested at multiple doses (Fig. 2A–F). All cardiac glycosides induced and S5B). Instead, YB5 cells have functional plasma membrane
dose-dependent GFP reactivation in the nanomolar range at clin- store-operated Ca2þ entry (SOCe) channels, which transport
ically relevant concentrations, with proscillaridin A being the most extracellular Ca2þ into the cytosol in response to a decrease in
potent (Supplementary Fig. S2A and S2B). We also validated three Ca2þ level in the endoplasmic reticulum (ER) store. The activity of
additional antibiotics (pyrithion zinc, cycloheximide, and thiram) these plasma membrane channels was demonstrated by the effect
in the antibiotic/others subgroup that were identified in the screen of SOCe inhibitors reducing cytosolic Ca2þ entry triggered by the
at 50 mmol/L only but were positive during the validation using ER Ca2þ pump blocker, thapsigargin (Supplementary Fig. S5C;
both schedules. Therefore, we validated a total of 14 FDA-approved refs. 17, 18).
drugs encompassing three well-known epigenetic drugs and 11 Then, we characterized the effects of drug treatments in YB5
candidate epigenetic drugs. cells on cytosolic Ca2þ levels at rest, ER Ca2þ levels and SOCe
activity in response to Ca2þ ionophore, ionomycin or thapsigar-
No changes in classical epigenetic pathways gin followed by the addition of 1 mmol/L Ca2þ in the media (a
We confirmed GFP mRNA reactivation after 24 hours treatment cation-safe solution). Interestingly, known epigenetic drugs did
by qPCR (Fig. 3A). We also detected gene reactivation in three not alter Ca2þ homeostasis (Fig. 4A). However, cardiac glycosides
Raynal et al.
Figure 2.
Validation of positive hits with known epigenetic drugs, cardiac glycosides, and antibiotics/others subgroups. Dose–response curves were generated measuring GFP
expression in a validation step in YB5 cells treated for 24 hours (left) or 72 hours (right) with known epigenetic drugs (A and B), cardiac glycosides
(C and D), and antibiotics/others (n 3; E and F).
and arsenic trioxide reduced ER Ca2þ levels (first peak) while Ca2þ levels in the ER (first peak; Fig. 4C). Thus, most of the
strongly inhibiting SOCe-mediated Ca2þ entry (second peak; Fig. candidate epigenetic drugs induce measurable intracellular Ca2þ
4B and Supplementary Fig. S5D). Even though ER Ca2þ levels changes in YB5 cells by different mechanisms of action, which
were significantly reduced, we could not measure an increase in result in cytosolic Ca2þ sparks or sustained increased levels. It is
cytosolic Ca2þ, suggesting a transient spark in cytosolic Ca2þ after noteworthy that cardiac glycosides are well-known Naþ/Kþ-
treatment with digitalis compounds (19). In another model of ATPase pump blockers used in heart failure treatment (11). To
HEK293 cells expressing the genetically encoded calcium sensors assess the impact of Naþ/Kþ-ATPase pump inhibition on GFP
(D1ER cameleon system) specifically expressed in the ER, we reactivation in YB5 cells, we treated YB5 cells with orthovanadate,
confirmed digitoxin-induced reduction in ER Ca2þ (Supplemen- an inorganic Naþ/Kþ-ATPase inhibitor and did not detect GFP
tary Fig. S5E; 20). Finally, we could detect an increased cytosolic reactivation. These data suggest that the epigenetic activity of
Ca2þ level (higher baseline Ca2þ signal) after only pyrithion zinc cardiac glycosides might be independent of Naþ/Kþ-ATPase (data
treatment whereas both pyrithion zinc and disulfiram increased not shown).
Figure 3.
Gene reactivation by newly identified drugs is independent of DNA demethylation and chromatin modifications. A, GFP reactivation in YB5 cells treated for 24 hours,
measured by qPCR (n ¼ 3). B, SFRP1, TSG is reactivated after treatment detected by GFP fluorescence in HCT116 SFRP1-GFP cell line. Cells were treated
either 72 hours with decitabine (followed by 24 hours without drug) or 24 hours with the identified hits at doses indicated on the graph (n ¼ 3). C and D, CMV promoter
(C) and LINE-1 (D) DNA methylation levels measured by bisulfite-pyrosequencing in YB5 cells treated with drugs and doses indicated on the graph (n ¼ 3).
(Cyclohex., cycloheximide; Oxyquin., oxyquinoline; Prosc. A, proscillaridin A; Pyrith. Z., pyrithion zinc.), E, mass spectrometry showing histone acetylation levels at
several residues in YB5 cells treated for either 72 hours or 24 hours with drugs and doses indicated on the graph (n ¼ 3).
We further explored the mechanism of SOCe inhibition not caused by changes in cytosolic pH (Supplementary Fig.
induced by cardiac glycosides. SOCe is mediated by the highly S5H; refs. 20, 24, 25). Finally, digitoxin specifically inhibited
Ca2þ-selective Orai1 channels, which are activated by STIM pro- Ca2þ influx through Orai1 in HEK293 cells expressing either
teins in response to a decrease in ER Ca2þ levels. STIM translocates constitutively active STIM1 mutant (STIM1D76A) or constitu-
into ER-plasma membrane junctions to tether and activate Orai1 tively active Orai1 mutant (O1-V102C; Fig. 4E and F; 26). In
channels (21, 22). For these experiments, we used a well-estab- these experiments, we used the antihistamine fexofenadine as a
lished model of HEK293 cells overexpressing Orai1-CFP and control for specificity of action as this drug treatment does not
STIM1-YFP (23). We found that digitoxin did not affect STIM1- induce GFP reactivation. Together, the data demonstrate that
Orai1 coupling as maximal FRET signal between STIM1 and cardiac glycosides altered Ca2þ levels by causing potent SOCe
Orai1, obtained after ionomycin treatment, was unaltered by inhibition.
digitoxin (Supplementary Fig. S5F). Wild-type and overexpressing
STIM1-Orai1 HEK293 cells showed similar inhibition of SOCe Ca2þ signaling through CamK is essential for gene reactivation
and reduction in ER Ca2þ size by digitoxin (Fig. 4D and Supple- CamK, a multifunctional serine/threonine kinase, is a central
mentary Fig. S5G). Importantly, these effects on Ca2þ levels were molecule that is activated by an increase in cytosolic Ca2þ, leading
Raynal et al.
Figure 4.
2þ 2þ
Newly identified drugs induce changes in intracellular Ca levels. Intracellular changes in Ca levels in YB5 cells pretreated for 8 hours with known epigenetic drugs
2þ 2þ
(A), cardiac glycosides (B), pyrithion zinc and disulfiram (C) in response to ionomycin, extracellular Ca , and thapsigargin. D, time-lapse intracellular Ca
2þ
changes in HEK293 cells overexpressing STIM1-Orai1 proteins pretreated for 8 hours with digitoxin in response to ionomycin and extracellular Ca . Time-lapse
2þ
intracellular Ca changes in HEK293 cells transfected with constitutively active STIM1-mutant protein (D67A; E) or with constitutively active Orai-mutant
2þ 2þ
channel (V102C; F) in response to extracellular Ca exposure and SOCe inhibitor (2-APB). The arrows indicate the addition of Ca into the extracellular media while
2þ 2þ
a line indicates the time of Ca exposure prior to its removal by washes of Ca -free solution. Fexofenadine was used as a control as this drug does not
induce GFP reactivation.
to gene expression changes in part caused by the unbinding of We next hypothesized that the epigenetic effects of CamK
chromatin repressors such as the methyl-binding protein MeCP2 activation by Ca2þ signaling relate to unbinding of MeCP2 from
from their silenced promoters (15, 27–29). Except for known repressed promoters, as previously reported in normal neuronal
epigenetic drugs, all candidate epigenetic drugs induce intracel- cells (15, 16, 28, 29). Using ChIP-qPCR following cardiac glyco-
lular Ca2þ changes. Thus, we tested the role of CamK in YB5 cells side treatment (Lanatoside C), we found that MeCP2 occupancy
treated with known and candidate epigenetic drugs. Drug- was reduced at several DNA hypermethylated promoters (Sup-
induced reactivation of GFP and endogenous TSGs (TIMP-3 and plementary Fig. S7), suggesting that drug-induced dissociation of
WIF-1) was significantly reduced by pharmacologic inhibition of MeCP2 binding is associated with gene reactivation. Most inter-
CamKII using Kn-93 whereas Kn-92, its weaker analogue, had a estingly, using confocal microscopy, we observed nuclear versus
much smaller effect (Fig. 5A and Supplementary Fig. S6A and S6B; cytoplasmic redistribution of MeCP2 after proscillaridin treat-
ref. 30). As expected, GFP expression induced by azacitidine was ment (Fig. 5B). Following 24-hour and 48-hour treatment with
not sensitive to Kn-93 inhibition as this hypomethylating drug proscillaridin at 500 nmol/L, MeCP2 staining was enriched in the
did not alter Ca2þ fluxes (Fig. 5A). cytoplasm in most of the cells (Fig. 5B). In untreated cells, MeCP2
Figure 5.
Drug-induced gene reactivation is dependent on CamK activity. A, GFP reactivation by flow cytometry in YB5 cells pretreated (4 hours) with CamK inhibitor Kn-93
and its weaker analogue Kn-92 and treated for 24 hours. B, fluorescent immunochemistry in SW48 cells (YB5 parental cells) treated for 24 hours and
48 hours with proscillaridin at 500 nmol/L stained with DAPI, MeCP2 (RFP) antibody. White arrows, MeCP2 signal in the cytoplasm post-proscillaridin A treatment.
C, percentage of apoptotic cells in YB5 cells pretreated (4 hours) with CamK inhibition and treated for 48 hours.
staining was mainly concentrated in the nucleus and a weak signal Western blot experiments and confocal microscopy on YB5
was detected in the cytoplasm as already observed by others (31). untreated and treated with proscillaridin A) and HEK293T cells
MeCP2 antibody specificity was verified by using siRNA (in overexpressing MeCP2 (Supplementary Figs. S8 and S9). Finally,
Raynal et al.
we showed that these effects of the positive hits identified in the Ca2þ signaling also occurred in our cancer cell screening system
screen may relate to export of regulatory proteins from the nucleus (YB5 cells) and only after treatment with the newly epigenetic
to the cytoplasm. Blocking the exportin pathway of protein drugs but not with well-known epigenetic drugs such as DNA
transport from the nucleus to the cytoplasm by leptomycin methylation and HDAC inhibitors. This difference suggested a
significantly reduced drug-induced GFP reactivation in YB5 cells separate mechanism of action. Our data showed that reactivation
(Supplementary Fig. S10). of endogenous epigenetically silenced TSGs was linked to Ca2þ
As CamKII appeared to mediate gene reactivation, we next signaling and CamK activation. Mechanistically, our data suggest
tested whether the selective cancer cell killing was also CamKII- that gene reactivation may involve gene derepression by inducing
dependent. Percentages of apoptotic cells, viable cells, and dead MeCP2 eviction from repressed promoters to the cytoplasm
cells were measured in colon cancer (YB5 and its parent cell line through the exportin pathway (Supplementary Fig. S14). Inter-
SW48) and leukemia (K562 and HL-60) cell lines following 48- estingly, the anticancer activity could also be modulated by CamK
hour drug treatment alone or in combination with CamKII pharmacologic inhibition.
inhibitor, Kn-93 and its less active derivative, Kn-92. For all cell Consistent with our work, other studies have demonstrated a
lines, drugs significantly induced cytotoxicity whereas, as expected CamK-dependent mechanism producing MeCP2 release from
for this brief treatment period, hypomethylating drugs did not. silenced promoters, leading to gene reactivation (15, 28, 29).
Kn-93 rescued almost completely the drug-induced toxic effects Interestingly, our data further demonstrate redistribution of
(Fig. 5C and Supplementary Figs. S11–S13). These data suggest MeCP2 to the cytoplasm after drug-induced CamK activation.
that CamK activation is a key effector of the anticancer activity of The relationship between MeCP2 binding to methylated-CpGs
these FDA-approved drugs. and Ca2þ has been described in neurons where MeCP2 phos-
phorylation is involved in intracellular localization during neu-
Anticancer activity and cancer selectivity ronal differentiation (31, 32). Phosphorylation sites of MeCP2 are
We next assessed the anticancer activity of the newly identified known to modify its binding to other epigenetic cofactors but
candidate epigenetic drugs in YB5 cells. Anchorage-dependent more studies are needed to clearly understand the role of CamK
colony formation assays were performed for long-term cytotox- and MeCP2 phosphorylation sites. Nevertheless, our study shows
icity evaluation. All drugs reduced cancer cell survival in a dose that the CamK–MeCP2 interaction previously linked only to
and time-dependent manner (Fig. 6A–C). Anticancer activity was differentiation can be exploited against cancer cells to reactivate
stronger for the newly identified drugs compared with known silenced TSG. These data also demonstrate a key role for calcium
epigenetic drugs. Anchorage-independent colony assays were fluxes in epigenetic silencing in neoplasia.
then performed by gauging colony formation in soft agar. All Interestingly, we identified a class effect for the cardiac glycoside
drugs produced a significant dose-dependent reduction in the subgroup where all these heart failure drugs present in the libraries
number of colonies; with proscillaridin A being the most potent scored positive in the screen and also separately validated. These
(Fig. 6D). To address the question of cancer cell selectivity, we drugs are relevant for cancer treatment because epidemiologic
compared the viability of normal colon cells (CRL-1831) versus studies have shown that patients treated with cardiac glycosides
colon cancer cells (SW48). We used YB5's parental cell line SW48 for heart failure exhibit a reduced cancer rate and less aggressive
for this because GFP expression could affect viability measure- cancers (11, 33, 34). Moreover, in vitro studies have previously
ments performed by flow cytometry. After 48-hour treatment at shown selective cancer killing for some of these cardiac glycosides,
doses producing the strongest GFP reactivation in YB5 cells, cell which we now relate to Ca2þ signaling and TSG reactivation (35).
viability was reduced in cancer cells but not in normal cells (Fig. Our data suggest that their chemopreventive or chemotherapeutic
6E) demonstrating the cancer selectivity of these drugs. activity may be in part due to their ability to target Ca2þ fluxes and
reactivate epigenetically silenced TSGs. Another drug we identi-
fied as having effects on gene silencing is arsenic trioxide, which
Discussion has marked activity in acute promyelocytic leukemia (36), a
There is a need to discover new epigenetic targets and drugs to disease characterized by a block in cell differentiation (37). Our
induce TSG reactivation in cancer cells. The arsenal of epigenetic data suggest that epigenetic modulation may be part of its
drugs approved in the clinic is currently limited to only four drugs antineoplastic mechanism of action. Interestingly, cytotoxic drugs
targeting only two separate mechanisms. On the basis of the high or targeted drugs (e.g., tyrosine kinase inhibitors) used in cancer
number of epigenetic targets in cancer, there is a great interest in chemotherapy did not reactivate GFP, pointing to the specificity of
discovering new targets that can be used to reactivate TSGs and the identified hits. This was verified separately from the screen for
reprogram cancer cells to eradicate their clonogenic potential and many cytotoxic drugs (38). Thus, the mechanism of action of the
induce their differentiation. We used our live cell-based assay positive hits is likely to be specific disruption of epigenetic path-
where we measured GFP reactivation as surrogate for TSG reac- ways involved in gene silencing rather than nonspecific effects
tivation. Overall, we identified 14 hits including 3 well-known seen with many antiproliferatives.
epigenetic drugs and 11 other FDA-approved drugs encompassing Finally, it is worth noting that Ca2þ signaling is essential to
cardiac glycosides and some antibiotics. initiating epigenetic reprogramming in early embryogenesis (39)
To identify the mechanism responsible for GFP and endoge- and our data extend these findings to cancer therapy. Also, the
nous TSGs reactivation, we first analyzed promoter DNA meth- mechanism of epigenetic action of these drugs is unlikely to be
ylation and histone modifications. No changes were detected after traced to a single chromatin regulator; rather, Ca2þ signaling
treatment with the cardiac glycosides or the antibiotics while TSG through CamK activation may have effects on multiple epigenetic
reactivation was detected. Surprisingly, we noticed that most of acting proteins simultaneously. It will be interesting to determine
the newly identified drugs are known to induce alterations in Ca2þ what targets, other than MeCP2, might be phosphorylated by
signaling in normal cells. We demonstrated that these changes in CamK activation.
Figure 6.
Anticancer efficacy and selectivity of candidate epigenetic drugs. Anchorage-dependent toxicity assays were performed in YB5 cells treated for 72 hours
(top) or 24 hours (bottom) with known epigenetic drugs (A), cardiac glycosides (B), and antibiotics/others (n ¼ 3; C). D, anchorage-independent clonogenic assays
in soft agar were performed after a 24-hour treatment at the doses indicated on the graph (n ¼ 3). E, cell viability assays using normal colon cells (CRL-1831)
and SW48 (YB5's parental cell line) after a 48-hour treatment, measured by flow cytometry. Drugs and doses are indicated on the graph (n ¼ 3). Cyclohex,
cycloheximide; Oxiquin, oxiquinoline; pyrithion, Pyrition zinc.
Raynal et al.
In summary, our data identify Ca2þ signaling as a new pathway Writing, review, and/or revision of the manuscript: N.J.-M. Raynal, J.T. Lee,
that can be targeted to reactivate TSGs in cancer. This study suggest Y. Wang, J. Garriga, S. Dumont, W.E. Childers, R.A. Henry, J. Jelinek, S.B. Baylin,
D.L. Gill, J.-P.J. Issa
that cardiac glycosides and other FDA-approved drugs such as
Administrative, technical, or material support (i.e., reporting or organizing
some antibiotics represent interesting drugs for cancer epigenetic data, constructing databases): N.J.-M. Raynal, J.T. Lee, W. Chung, Y. Cui
chemotherapy aiming to reactivate epigenetically silenced TSGs. Study supervision: S.B. Baylin, J.-P.J. Issa
Other (perform experiments): S. Ahmed
Disclosure of Potential Conflicts of Interest Other (assisted with selection of compounds tested in this study):
No potential conflicts of interest were disclosed. W.E. Childers
Other (collaboration by applying HTS using the Center's screening capabil-
ities and compound library): M. Abou-Gharbia
Authors' Contributions
Conception and design: N.J.-M. Raynal, Y. Wang, S.B. Baylin, D.L. Gill,
J.-P.J. Issa Grant Support
Development of methodology: N.J.-M. Raynal, Y. Wang, P. Madireddi, J.-P.J. Issa is an American Cancer Society Clinical Research professor sup-
J. Garriga, G.G. Malouf, E.J. Dettman, V. Gharibyan, W. Chung, M. Abou- ported by a generous gift from the F.M. Kirby Foundation. This work was
Gharbia, D.L. Gill supported by NIH grants CA100632 and CA046939 (J.-P.J. Issa).
Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the
provided facilities, etc.): N.J.-M. Raynal, J.T. Lee, Y. Wang, A. Beaudry, G. payment of page charges. This article must therefore be hereby marked
Malouf, S. Dumont, E.J. Dettman, R.A. Henry, S.B. Baylin advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, this fact.
computational analysis): N.J.-M. Raynal, Y. Wang, G.G. Malouf, S. Dumont,
E.J. Dettman, V. Gharibyan, W. Chung, W.E. Childers, R.A. Henry, A.J. Andrews, Received August 13, 2014; revised December 3, 2015; accepted December 18,
J. Jelinek, S.B. Baylin, D.L. Gill, J.-P.J. Issa 2015; published OnlineFirst December 30, 2015.
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Targeting Calcium Signaling Induces Epigenetic Reactivation of

Tumor Suppressor Genes in Cancer
Noël J.-M. Raynal, Justin T. Lee, Youjun Wang, et al.
Cancer Res 2016;76:1494-1505. Published OnlineFirst December 30, 2015.
Updated version Access the most recent version of this article at:
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Published OnlineFirst December 30, 2015; DOI: 10.1158/0008-5472.

Targeting Calcium Signaling Induces Epigenetic

Introduction novo methylation in cytosine of CpG dinucleotide at the promoter

1494 Cancer Res; 76(6) March 15, 2016

Targeting Calcium Signaling Produces Epigenetic Reactivation

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Targeting Calcium Signaling Produces Epigenetic Reactivation

Results endogenous TSGs silenced by epigenetic mechanism in YB5 cells

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Targeting Calcium Signaling Produces Epigenetic Reactivation

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Targeting Calcium Signaling Produces Epigenetic Reactivation

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1502 Cancer Res; 76(6) March 15, 2016 Cancer Research

Targeting Calcium Signaling Produces Epigenetic Reactivation

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1504 Cancer Res; 76(6) March 15, 2016 Cancer Research

Targeting Calcium Signaling Produces Epigenetic Reactivation

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Targeting Calcium Signaling Induces Epigenetic Reactivation of

Cancer Res 2016;76:1494-1505. Published OnlineFirst December 30, 2015.

Supplementary Access the most recent supplemental material at:

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