Biochemical Pharmacology 147 (2018) 93–103

Contents lists available at ScienceDirect

Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

FoxO-1 contributes to the efficacy of the combination of the XPO1

inhibitor selinexor and cisplatin in ovarian carcinoma preclinical models
Cristina Corno a,1, Simone Stucchi a,1, Michelandrea De Cesare a, Nives Carenini a, Serena Stamatakos a,
Emilio Ciusani b, Lucia Minoli c,d, Eugenio Scanziani c,d, Christian Argueta e, Yosef Landesman e,
Nadia Zaffaroni a, Laura Gatti a, Paola Perego a,⇑
a
Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo 42, 20133 Milan, Italy
b
Laboratory of Clinical Pathology and Medical Genetics, Fondazione IRCCS Istituto Neurologico C. Besta, via Celoria 11, 20133 Milan, Italy
c
Department of Veterinary Medicine, Università degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy
d
Mouse and Animal Pathology Laboratory, Fondazione Filarete, viale Ortles 22/4, 20139 Milan, Italy
e
Karyopharm Therapeutics, 85 Wells Ave., Newton, MA 02459, USA

a r t i c l e i n f o a b s t r a c t

Article history: The XPO1/CRM1 inhibitor selinexor (KPT-330), is currently being evaluated in multiple clinical trials as
Received 5 October 2017 an anticancer agent. XPO1 participates in the nuclear export of FoxO-1, which we previously found to
Accepted 14 November 2017 be decreased in platinum-resistant ovarian carcinoma. The aim of this study was to determine whether
Available online 16 November 2017
enriching FoxO-1 nuclear localization using selinexor would increase ovarian cancer cell sensitivity to
cisplatin. Selinexor, as a single agent, displayed a striking antiproliferative effect in different ovarian car-
Chemical compounds: cinoma cell lines. A schedule-dependent synergistic effect of selinexor in combination with cisplatin was
Cisplatin (PubChem CID: 2767)
found in cisplatin-sensitive IGROV-1, the combination efficacy being more evident in sensitive than in the
Selinexor (PubChem CID: 71481097)
resistant cells. In IGROV-1 cells, the combination was more effective when selinexor followed cisplatin
Keywords: exposure. A modulation of proteins involved in apoptosis (p53, Bax) and in cell cycle progression
XPO1/CRM1 inhibitors (p21WAF1) was found by Western blotting. Selinexor-treated cells exhibited enriched FoxO-1 nuclear
Cisplatin staining. Knock-down experiments with RNA interference indicated that FOXO1-silenced cells displayed
Ovarian carcinoma a reduced sensitivity to selinexor. FOXO1 silencing also tended to reduce the efficacy of the drug
combination at selected cisplatin concentrations. Selinexor significantly inhibited tumor growth, induced
FoxO-1 nuclear localization and improved the efficacy of cisplatin in IGROV-1 xenografts. Taken together,
our results support FoxO-1 as one of the key factors promoting sensitivity towards selinexor and the syn-
ergistic interaction between cisplatin and selinexor in ovarian carcinoma cells with selected molecular
backgrounds, highlighting the need for treatment regimens tailored to the molecular tumor features.
Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction cancer patients involves the surgical removal of tumor followed

by platinum-based chemotherapy. Consistently, late diagnosis
Ovarian carcinoma is the most lethal gynecological disease and and resistance to platinum-based chemotherapy in advanced dis-
the seventh most common cancer in women [1]. As with most ease account for a poor survival rate [2]. Although there has been
cancers, early detection increases the probability of a favorable an improvement in the understanding of the molecular features
prognosis. However, due to the lack of symptoms associated with of ovarian cancer owing to pathological and genomic findings [3],
ovarian cancer development and the absence of a reliable screen- the acquired knowledge has not been fully translated into substan-
ing regimen, most patients are diagnosed at later times making tial changes to disease treatment. Thus, despite the availability of
the disease hard to treat. The current standard of care for ovarian various effective second-line treatments, and of poly ADP ribose
polymerase (PARP) inhibitors in a specific subset of patients
harbouring BRCA1/2 mutations, there is a need for novel therapeu-
⇑ Corresponding author at: Molecular Pharmacology Unit, Fondazione IRCCS tic approaches.
Istituto Nazionale dei Tumori, via Amadeo 42, 20133 Milano, Italy. Mechanistic studies have clearly defined resistance as a multi-
E-mail address: [email protected] (P. Perego). factorial phenomenon, in part explained by evolutionary models
1
Equal contribution.

https://doi.org/10.1016/j.bcp.2017.11.009
0006-2952/Ó 2017 Elsevier Inc. All rights reserved.
94 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103

of clonal selection [4]. It is unclear how clonal heterogeneity 2.2. Cell growth inhibition and drug interaction analyses
impacts resistance in ovarian carcinoma, but the major pathways
accounting for reduced drug efficacy have been defined. In partic- Cells were seeded in 12-well plates and 24 h later exposed for
ular, cellular resistance to platinum compounds can involve activa- 72 h to increasing concentrations of cisplatin, selinexor or to the
tion of cell survival and inhibition of cell death pathways [5]. In combination. For combination studies, the cells were treated
this context, the export of nuclear proteins into the cytoplasm according to different schedules: a) 72 h concomitant exposure
has been proposed to play a role in evading apoptosis and promot- (simultaneous); b) selinexor 24 h treatment, followed by 48 h cis-
ing tumor growth [6]. The karyopherin exportin-1 (XPO1)/Chromo- platin exposure (pre-incubation with selinexor); c) pre-incubation
some Region Maintenance-1 (CRM1), a major nuclear export with cisplatin for 24 h prior to addition of selinexor for 48 h (pre-
receptor, participates in the nuclear export of more than 230 car- incubation with cisplatin). At the end of treatment, cell growth
gos, including tumor suppressor proteins and cell cycle regulating inhibition was evaluated by counting cells (Z2 Particle Counter,
proteins, such as p53, p21WAF1, and FoxO family proteins [7]. Con- Beckman Coulter, Milan, Italy). All experiments were performed
sistently, XPO1 has been shown to regulate multiple cellular func- at least three times. IC50 is defined as the concentration of a drug
tions, including cell cycle progression, proliferation and apoptosis inhibiting 50% of cell growth. Drug interaction was evaluated
[8]. In cancer, XPO1 expression is frequently up-regulated in according to the Chou–Talalay method, assigning a combination
different solid tumors (e.g., gynecological cancers) [9,10] and index (CI) value to each drug combination using the Calcusyn soft-
hematological malignancies [8], suggesting that its broad range ware (Biosoft, Cambridge, United Kingdom). CI values lower than
of cellular functions is critical for malignant progression. Thus, tar- 0.85–0.90 indicate synergistic drug interactions, whereas CI values
geting XPO1 and restoring the cellular localization of XPO1 cargos, higher than 1.20–1.45 or around 1 stand for antagonism or additive
which are critical for their biological activities, have emerged as an effect, respectively.
attractive therapeutic approach [11]. Incidentally, activation of
FoxO transcriptional properties has been implicated in resistance 2.3. Western blot analyses
to treatment [12]. Specifically, FOXO1 has been shown to be
down-regulated in cells resistant to cisplatin and target-specific Western blot analysis was carried out as previously described
agents [13,14]. [21]. Cells were lysed using 0.125 M Tris HCl pH 6.8 (Sigma-
Selinexor (KPT-330) is the first in class selective inhibitor of Aldrich), 5% sodium dodecyl sulfate (SDS, Lonza) and protease/
nuclear export (SINE) targeting XPO1. This orally available small phosphatase inhibitors (all purchased from Sigma-Aldrich) and a
molecule has demonstrated clinical activity in different tumor cell scraper was used to harvest cell lysate. When tumors from
types as single agent or in combination with other treatments mice were processed, protein lysates were obtained from frozen
[15–17]. Selinexor has also been shown to restore platinum tumor samples pulverized by a Mikro-Dismembrator II (B. Brown
sensitivity in p53-dependent and independent manner in ovarian Biotech International, Melsungen, Germany) in lysis buffer as indi-
carcinoma preclinical models [18]. In addition to p53, other mech- cated above. Lysed samples were boiled for 5 min, sonicated for 25
anisms have been proposed as the basis for the potent anti-cancer s at 10% amplitude (Branson Digital SonifierÒ S-250D, Emerson
activity exhibited by selinexor, such as the deactivation of NF-kB or Electric Co, Ferguson, Missouri) and quantified using the bicin-
the transcriptional down-regulation of survivin [19,20]. Another choninic acid assay method (Pierce, Thermo Fisher Scientific, Wal-
pathway involves the transcriptional activity of FoxO-1 protein tham, Massachusetts, USA). Lysates were fractionated by SDS-
family, which has been implicated in the resistance to platinum- polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were
based treatment [12] and has been shown to be down-regulated transferred to nitrocellulose membranes using a wet transfer sys-
in an ovarian carcinoma variant resistant to cisplatin and in tem (Trans-BlotÒ TurboTM Transfert System, BIO-RAD, Milan, Italy).
lapatinib-resistant gastric cancer cells [13,14]. Because XPO1 par- Binding of secondary antibodies to membranes was detected by
ticipates in the nuclear export of FoxO-1, which we previously chemiluminescence (ECL, GE Healthcare, Little Chalfont, United
found to be decreased in ovarian carcinoma platinum-resistant Kingdom). Western blot analyses were performed at least three
cells, we hypothesized that FoxO-1 may contribute to the efficacy times using independent biological cell line samples or tumors
of the selinexor-cisplatin combination. Thus, the aim of this study from different mice. Vinculin and actin were used as loading con-
was to examine the possibility to exploit the selinexor-induced trols. FoxO-1 (C29H4; #2880) and Bax (# 2772) antibodies were
enrichment of FoxO-1 nuclear localization to increase cisplatin purchased from Cell Signaling Technologies (Danvers, Mas-
sensitivity in ovarian carcinoma cells. sachusetts). Actin (# A2066) and vinculin (# V9131) antibodies
were purchased from Sigma-Aldrich. XPO1 (ab3459), antibody
2. Materials and methods was purchased from Abcam (Cambridge, United Kingdom). p53
(clone DO-7; # 544294) and p21WAF1 (clone 70/Cip1/WAF1; #
2.1. Cell lines and drugs 610234) antibodies were purchased from BD Biosciences (New Jer-
sey, USA). Secondary antibodies were obtained from GE Health-
The parental IGROV-1, cisplatin-resistant IGROV-1/Pt1, A2780 care. Gel quantification was performed taking advantage of
and OVCAR-5 ovarian carcinoma cell lines were obtained as previ- Image Studio Lite developed by LI-COR Biosciences (Bad Homburg
ously described [13]. The TOV21G (# CRL-11730) and TOV112D vor der Höhe, Germany).
(# CRL-11731) cell lines were from ATCC. All ovarian carcinoma
cell lines were grown in RPMI-1640 medium (Lonza, Basel, 2.4. Analysis of apoptosis
Switzerland), supplemented with 10% FBS (Gibco, Life Technolo-
gies, Carlsbad, California). Cells were routinely checked for Apoptosis was evaluated by Annexin V-binding assay
mycoplasma contamination (Lonza), used within 20 passages from (Immunostep, Salamanca, Spain) in IGROV-1 cells treated for 48
thawing from a frozen stock, and authenticated using the Stem and 72 h with cisplatin (24 h pre-treatment) alone or in combina-
Elite ID System (Promega). For in vitro studies, cisplatin (Accord tion with selinexor and in tumors harvested from mice xenografted
Healthcare Italia, Milan, Italy) was diluted in saline and selinexor with IGROV-1 cells. Cells or tumor specimens, minced and
(Karyopharm Therapeutics, Inc., Newton, MA, USA) dissolved in mechanically dissociated on a 100 lm nylon cell strainer (BD Fal-
dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, Missouri). Final con, Milan, Italy), were washed with cold PBS. Cell suspensions
DMSO concentration in medium never exceeded 0.25%. were resuspended in binding buffer (10 mM HEPES-NaOH, pH
 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103 95

7.4, 2.5 mM CaCl2, and 140 mM NaCl, Immunostep). A fraction of cDNA Reverse Transcription Kit according to manufacturer proto-
105 cells were incubated in binding buffer at room temperature col (Applied Biosystems, Foster City, CA, USA). Gene expression
in the dark for 15 min with 5 lL of FITC-conjugated Annexin V was determined by quantitative Real-Time PCR (qRT-PCR)
and 10 lL of 2.5 lg/mL propidium iodide (Immunostep). Annexin using TaqMan assays (FOXO1, Hs01054576_m1; GAPDH,
V binding was detected by flow cytometry. At least 104 events/ Hs02758991_g1; Applied Biosystems). Technical triplicate reac-
sample were acquired and analyzed using a specific software (Cell- tions were carried out in 10 ll containing 2.5 ll cDNA, 5 ll master
QuestPro, Becton Dickinson). mix (TaqMan Universal Fast PCR Master Mix, Applied Biosystems),
0.5 ll of the specific assay. Reactions were performed using a
2.5. Cell cycle analysis 7900HT Fast Real-Time PCR System (Applied Biosystems) equipped
with SDS (Sequence Detection Systems) 2.4 software (Applied
Floating and adherent IGROV-1 cells were harvested and Biosystems). Data analysis was performed with RQ manager
washed with saline following 48 or 72 h of treatment. Cells were software (Applied Biosystems). Relative levels of cDNA were
pre-incubated with 0.3 lM cisplatin for 24 h prior to addition of determined as previously described [21], through the relative
0.03 lM selinexor. Cells were fixed in 70% cold ethanol and incu- quantification (RQ) method. Untransfected cells were chosen as
bated for 1 h at 4 °C with PBS containing 50 lg/ml propidium calibrator.
iodide (Sigma-Aldrich) and 1 mg/ml RNase A (Sigma-Aldrich). Cell
cycle perturbations were measured using flow cytometry. At least 2.9. Antitumor activity studies
2
105 cells were collected and evaluated for DNA content. Cell
cycle distribution was analyzed using FlowJo 10. All experiments were carried out using 8–10 week-old female
athymic CD-1 nude mice, (Charles River, Calco, Italy). Mice were
2.6. Immunofluorescence analysis maintained in laminar flow rooms at constant temperature and
humidity with free access to food and water. Experiments were
Cells were seeded in 12-well plates containing circular cover- authorized by the Italian Ministry of Health according to the
slips slides. Twenty-four hours later, cells were treated with cis- national law in compliance with international policies and guide-
platin and, after 24 h, selinexor was added for further 24 h. Cells lines. Selinexor was dissolved in Pluronic F-68 and PVP K-29/32
were fixed in 3.7% paraformaldehyde (MERCK, Darmstadt, Ger- following manufacturer’s instructions; cisplatin was diluted in sal-
many) in PBS for 15 min at room temperature. After washing in ine. The compounds were delivered in a volume of 10 ml/kg of
PBS, cells were incubated for 1 h in PBS containing 5% foetal bovine body weight.
serum and 0.3% Triton X-100 (Fluka-Sigma-Aldrich). The coverslips IGROV-1 and IGROV-1/Pt1 human ovarian carcinoma xeno-
slides were incubated overnight at 4 °C with the primary antibody grafts were used in the study. Exponentially growing cells (107-
against FoxO-1 (1:100, Cell Signaling). The slides were then /mouse) were subcutaneously injected into the right flank on
washed in PBS and incubated for 1 h at room temperature with day 0. Tumor diameter growth was observed biweekly using a
the secondary antibody conjugated with AlexaFluor 488 (1:500, Vernier caliper. Tumor volume (TV) was calculated according
Molecular Probes, Thermo Fisher). Samples were counterstained to the formula: TV (mm3) = d2
D/2, where d and D are the
with Hoechst 33342 for 2 min and mounted with Prolong Gold shortest and the longest diameters, respectively. Treatment
AntiFade Reagent (Life Technologies). Images were collected using started 5–6 days after cell inoculum, when tumors were estab-
a fluorescence microscope (Leica Microsystems, Milan, Italy) with a lished (around 90 mm3 TV). As single treatment, 10 mg/kg seli-
Spot Insight digital camera (Delta Sistemi) equipped with a system nexor was delivered orally every 3–4 days a week for 4 weeks
of image analysis (IAS 2000, DeltaSistemi). Cells in three fields of and 4.5 mg/kg cisplatin i.v. every week for 3 weeks. In combina-
the coverslips slides were counted to examine the number of tion studies, selinexor at a dose of 5 mg/kg was delivered 24 h
FoxO-1 positive or negative nuclei. after cisplatin. Student’s t test (two-tailed) was used for statisti-
cal comparison of TV in mice. Alternatively, IGROV-1 cells from
2.7. FOXO1 loss of function studies cultures (107/mouse) were i.p. injected. For ethical reasons, the
animals were inspected and weighed daily, and were sacrificed
IGROV-1 cells were plated in 6-well plates (25,000 cells/cm2), at the appearance of ascites. The days of disease onset were
and 24 h later, cells were transfected using Opti-MEM transfection recorded and the median day (considered as median survival
medium (Gibco by Life Technologies) and Lipofectamine RNAiMAX time, MST) was calculated. Treatment started 4 days after cell
(Thermo Fisher Scientific) with 30 nM of small interfering RNA inoculum orally delivering 10 mg/kg selinexor every 3–4 days
(siRNA) directed against FOXO1 (SilencerÒ Select s5259, Life Tech- for 6 times. The ascitis take, i.e. the ratio between the number
nologies, Thermo Fisher) or negative control siRNA (Silencer Select of mice developing ascitis over the number of cell-injected mice,
Negative Control #2 siRNA, Life Technologies). Cells were incu- was employed to assess treatment efficacy. T/C%, i.e. the ratio of
bated with transfection mix for 5 h and then the transfection med- MST in treated over control mice x 100 was calculated. A careful
ium (Opti-MEM) was replaced with complete medium. necropsy was performed to evaluate the ovarian tumor take and
Transfection efficiency was evaluated by qRT-PCR as indicated, spread in the abdominal cavity. Solid masses were gently
72 and 144 h after transfection start. Cells were harvested 72 h detached from organs and abdominal walls, removed and
after transfection and were re-seeded in 12-well plates at a density weighed to calculate the percentage of tumor weight inhibition
of 104 cells/cm2 for cell growth inhibition assays, performed after (TWI%) in mice. For statistical analysis two-sided Student’s t test
the treatment with cisplatin, selinexor or their combination (24 h was used to compare i.p. tumor weights in mice. Percent of
pre-incubation with cisplatin followed by addition of selinexor disease-free mice over time was estimated by the Kaplan-
for 48 h). Meier product method and compared by the Log-rank test.

2.8. Quantitative real time PCR 2.10. Immunohistochemical evaluation of FoxO-1

RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Hil- IGROV-1 subcutaneous tumors were removed from mice 24 h
den, Germany). Reverse transcription was carried out using 1 lg after the last treatment (n = 8 for control group; n = 7 for selinexor
RNA in the presence of RNAse inhibitors, using the High Capacity group), fixed in 10% buffered formalin, and paraffin-embedded.
96 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103

Sections (4 lm thick) were stained with haematoxylin-eosin for Table 1

histopathological examination. Serial sections were stained Sensitivity of ovarian carcinoma cell lines to cisplatin and to the XPO1 inhibitor
selinexor.1
immunohistochemically for FoxO-1. After heat-induced epitope
retrieval, sections were incubated for one hour at room tempera- Cell line Cisplatin Selinexor
ture with the primary antibody anti-FoxO-1 (1:100, rabbit mono- IC50 (lM)2
clonal from Cell Signaling Technologies). Then incubated with a A2780 0.4 ± 0.1 0.0173 ± 0.028
biotinylated secondary antibody (anti-rabbit IgG, Vinci Biochem) OVCAR-5 2.27 ± 0.7 0.2 ± 0.075
TOV21G 0.035 ± 0.01 0.058 ± 0.007
and labeled by the avidin-biotin-peroxidase (ABC) procedure with TOV112D 1.41 ± 0.1 0.068 ± 0.007
a commercial immunoperoxidase kit (Vectastain Standard Elite, IGROV-1 0.33 ± 0.1 0.0278 ± 0.01
Vector Laboratories, Burlingame, CA). The immunoreaction was IGROV-1/Pt1 3.4 ± 1.3 0.0575 ± 0.02
revealed with 3,30 -diaminobenzidine substrate (Vector Laborato- 1
Sensitivity to cisplatin and to the inhibitor of XPO1 was assessed by cell growth
ries, Burlingame, CA) for 5 min and sections were counterstained inhibition assays. Cells were seeded and 24 h later exposed to the compounds for
with Mayer’s haematoxylin. Three microscopic fields were evalu- 72 h. Cells were counted using a cell counter.
2
ated for each sample at 400
, to assess the number of cells in IC50 is defined as the concentration inhibiting cell growth by 50%. The reported
values are the mean ± SD of at least three independent experiments.
which the FoxO-1 protein was expressed in the nucleus. Histolog-
ical and immunohistochemical analyses were performed in a blind
fashion. Results were expressed as percent of cells with nuclear
The effect of the drug combination was not synergistic in the
expression. Statistical analysis was performed by the Mann Whit-
IGROV-1/Pt1 cells using the simultaneous schedule of treatment
ney test.
when employing 0.03 lM selinexor (Fig. 1C). Also CI values
obtained with 0.01 lM selinexor, which ranged between 0.72
2.11. Statistical analysis
(with 0.03 lM cisplatin) and 4.26 (with 1 lM cisplatin), indicated
a less favorable effect of the combination in the resistant variant.
Statistical analyses were performed using the GraphPad
Additional treatment schedules were tested in IGROV-1 cells to
PrismTM software (GraphPad Software, San Diego, CA) as detailed
further optimize the effect of the drug combination. Specifically,
in each paragraph. In addition, Student’s t test (two tailed) was
we tested the effect of pre-incubation with selinexor prior to the
used for statistical analysis of western blots when comparing band
addition of cisplatin, and vice versa.
intensities, after normalization with loading controls.
In IGROV-1 cells pre-incubated with selinexor for 24 h prior to a
48 h co-incubation with cisplatin, the CI mean values ranged
3. Results
between 0.9 and 2.03, when the selinexor concentration was
0.03 lM (Fig. 2A). The CI values were always >1 in combination
3.1. Selinexor inhibits cell growth in ovarian carcinoma cell lines with
with 0.01 lM selinexor (data not shown). Conversely, a 24 h incu-
variable FoxO-1 protein levels
bation with cisplatin prior to the co-incubation of the two drugs
resulted in a synergistic effect evidenced at cisplatin concentra-
Sensitivity to the XPO1 inhibitor selinexor was examined using
tions below or near the IC50 values combined with 0.03 lM seli-
growth-inhibition assays after 72 h drug exposure in the following
nexor (Fig. 2B). This schedule appeared to be the most effective
ovarian carcinoma cell lines: A2780, OVCAR-5, TOV21G, TOV112D,
as CI values lower than 0.7 were obtained at 3 different cisplatin
IGROV-1 and the cisplatin-resistant variant IGROV-1/Pt1 (Table 1).
concentrations. When cisplatin was combined with 0.01 lM seli-
In all tested cell lines, the IC50 values were in the submicromolar
nexor, the most favorable CI value was 0.91 ± 0.16, obtained in
range. A striking antiproliferative effect was also observed in cell
combination with 0.3 lM cisplatin.
lines characterized by intrinsic (i.e., OVCAR-5) or acquired cisplatin
The effect of the drug combination was also tested in an addi-
resistance (IGROV-1/Pt1).
tional cell model, the TOV21G cell line, characterized by a remark-
Western blot analyses indicated that the different cell lines all
able FoxO-1 protein level. In cells pre-incubated with cisplatin
expressed the target of selinexor and variable levels of FoxO-1
before selinexor-cisplatin incubation, CI values below 0.7 were
were displayed by the cell lines (Fig. 1A), with decreased FoxO-1
observed when 0.03 lM selinexor was combined with 0.03 and
in IGROV-1/Pt versus IGROV-1 cells (P < 0.05 by Student’s t test,
0.1 lM cisplatin (Fig. 3).
n = 3).

3.2. Selinexor displays a schedule-dependent interaction with cisplatin 3.3. Analysis of cell response to the drug combination in IGROV-1 cells
in FOXO1-expressing ovarian carcinoma cells
Cell response to the drug combination was examined with ref-
The transcription factor FoxO-1 has been reported to be a cargo erence to proteins involved in cell cycle progression and apoptosis.
of XPO1 [8]. We previously found that FOXO1 is down-regulated in We found that selinexor alone (0.03 lM) or in combination with
the cisplatin-resistant variant IGROV-1/Pt1 [13]. IGROV-1 and cisplatin (0.3 lM) tended to induce an increase in p21WAF1 (Stu-
IGROV-1/Pt1 cells were selected for drug combination studies, dent’s t test – 48 h: P = 0.05 for selinexor versus control; P < 0.05
based on their differential expression of FoxO-1. for combination versus control; 72 h: P = 0.08 for selinexor versus
IGROV-1 cells were simultaneously exposed to increasing cis- control; P < 0.05 for combination versus control; n = 3) and p53
platin and selinexor (0.01 and 0.03 mM) concentrations as single (Student’s t test – 48 h: P = 0.06 for selinexor versus control; P =
agents or in combination. The cells were exposed to selinexor 0.06 for combination versus control; 72 h: P = 0.08 for selinexor
concentrations of 0.01 and 0.03 mM, which represent subtoxic versus control; P < 0.05 for combination versus control, n = 3) in
and near IC50 concentrations, respectively (Fig. 1B). According to IGROV-1 cells (Fig. 4A). Bax up-modulation upon treatment was
the CI values calculated with the Chou and Talalay method, seli- also observed (P < 0.05 for selinexor/combination versus control
nexor tended to synergize with cisplatin when 0.03 lM selinexor at 48 and 72 h by Student’s t test, n = 3). In contrast, a negligible
was combined with cisplatin concentrations ranging between 0.3 modulation of FoxO-1 levels was observed in treated as compared
and 0.03 lM, which are at or below the IC50 value of cisplatin. CI to control cells. The pattern of p21WAF1 and Bax modulation was
values obtained with 0.01 lM selinexor ranged between 0.85 (with somehow similar when using a lower cisplatin concentration
0.03 lM cisplatin) and 2.39 (with 3 lM cisplatin). (0.1 lM) (data not shown). Selinexor (0.03 lM) alone and in
 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103 97

Fig. 1. Western blot analysis of XPO1 and FoxO-1 levels in ovarian carcinoma cell lines and effect of the simultaneous combination of cisplatin and selinexor. (A) Western blot
analysis was carried out in ovarian carcinoma exponentially growing cells. Control loading is shown by actin. One experiment representative of 3 is reported. Band intensities
were quantified (mean ± SE, n = 3) using ImageJ, normalized to actin and referred to IGROV-1 cells (set to 1). *, P < 0.05 by two tailed unpaired Student’s t test. Cell sensitivity
to drugs was assessed by cell growth inhibition assays in IGROV-1 (B) and IGROV-1/Pt1 (C) cells. Cells were seeded and 24 h later exposed to cisplatin and selinexor for 72 h.
Cells were then counted using a cell counter. The combination index values obtained with the 0.03 lM selinexor concentration combined with different cisplatin
concentrations are shown. The reported values are the mean ± SE of at least three independent experiments.

combination with cisplatin (0.3 lM) promoted FoxO-1 nuclear tion induced the accumulation of cells in G2/M phase at 48 h and
accumulation as shown by immunofluorescence analysis carried 72 h of exposure (P < 0.05 of cells treated with versus control
out 48 h after cisplatin exposure start (Fig. 4B). Analysis of cell cells/single agents treated cells by unpaired Student’s t test). More-
cycle perturbations indicated that cells in G1 phase increased after over, the drug combination induced apoptosis in IGROV-1 cells
48 h selinexor treatment (P < 0.05 of selinexor-treated versus con- pre-treated with cisplatin for 24 h and co-incubated with selinexor
trol cells by unpaired Student’s t test, Fig. 4C). The drug combina- for additional 48 h, as shown by Annexin V-binding assay (Fig. 4D).
98 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103

Fig. 2. Effect of the combination of cisplatin and selinexor in IGROV-1 cells according to different schedule treatments. Sensitivity to drugs was assessed by cell growth
inhibition assays in IGROV-1 cells. (A) Cells were pretreated with selinexor for 24 h before coincubation with cisplatin for 48 h or (B) pre-incubated with cisplatin for 24 h
before concomitant exposure to cisplatin-selinexor for 48 h. Cells were then counted using a cell counter. The combination index values obtained with the 0.03 lM selinexor
concentration combined with different cisplatin concentrations are shown. The reported values are the mean ± SE of at least three independent experiments.

3.4. Effect of FOXO1 silencing on the cell response to the drug Selinexor significantly impaired tumor growth in both models pro-
combination in IGROV-1 cells ducing TVI of 78 and 74% in IGROV-1 and IGROV-1/Pt1, respectively
(Fig. 6A and Table 2). Immunohistochemical analysis of the intracel-
We examined the effect of knocking down FOXO1 expression in lular localization of FoxO-1 in vehicle and selinexor-treated IGROV-1
IGROV-1 cells to clarify its contribution to the efficacy of the drug tumors revealed that selinexor induced a nuclear enrichment of
combination. Transient transfection of siRNA duplexes targeting FoxO-1, as shown by the percent of cells with nuclear FoxO-1 expres-
FOXO1 mRNA markedly reduced the levels of FoxO-1 mRNA and sion whose median value was 0.45 (confidence interval 0.17–0.77, n
protein (Fig. 5A and B, for western blot analysis at 72 h: P < 0.05 = 8) in control and 8.30 (confidence interval 2.71–14.43, n = 7) in
for comparison of negative control transfected cells versus siRNA treated samples (P < 0.001, Mann Whitney’s test, Fig. 6B).
transfected cells by Student’s t test, n = 3). FOXO1 silencing resulted The effects of oral selinexor were also evaluated on the IGROV-1
in decreased sensitivity to selinexor (IC50 > 0.3 lM in FOXO1 knock- model system growing as i.p. carcinomatosis. Solvent-treated mice
down cells and 0.05 ± 0.001 lM in untransfected and negative presented evidence of ascites by 17 days after cell injection and
control-transfected cells) and the cisplatin-selinexor combination fluid and tumor masses in the peritoneal space at necropsy
tended to be slightly less effective as indicated by CI values at 0.3 (Fig. 6C). In contrast, selinexor-treated mice did not exhibit ascites
and 1 lM obtained with 24 h pre-incubation with cisplatin fol- on the day of sacrifice (day 23, T/C > 164%, P = 0.0002), i.e., 6 days
lowed by additional 48 h exposure to 0.03 lM selinexor (Fig. 5C). after the last disease appearance in the controls. Moreover, at
necropsy, the animals receiving selinexor presented a reduced i.
3.5. Antitumor activity of selinexor in ovarian carcinoma in vivo p. tumor load with respect to controls (TWI = 75%, P = 0.0003).
models Specifically, the tumor weights were 0.224 ± 0.146 and 0.914 ± 0.
303 g in treated and control groups, respectively. Therefore, the
The antitumor activity of oral selinexor was assayed in nude mice XPO1 inhibitor could impair the growth of the IGROV-1 carcinoma,
subcutaneously bearing the IGROV-1 and IGROV-1/Pt1 carcinomas. even when this was xenografted in the abdominal cavity.
 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103 99

proteins promoting apoptosis induction [24,25]. Thus, the interfer-

ence with XPO1 to improve FoxO-1 nuclear localization may be
exploited to enhance cisplatin efficacy and represents a novel
effective therapeutic strategy in this disease.
In this study, we used ovarian carcinoma cell lines representing
different ovarian carcinoma histologies [26]. All cell lines displayed
a marked sensitivity to selinexor which inhibited cell growth at
submicromolar concentrations, and a variable sensitivity to cis-
platin, not necessarily explained by FoxO-1 levels, but likely mul-
tifactorial, as A2780 cells similarly sensitive to cisplatin as
IGROV-1 cells exhibited lower FoxO-1 levels than IGROV-1 cells.
A marked sensitivity to the antiproliferative effect of selinexor
was also observed in IGROV-1/Pt1 cells. The therapeutic potential
of selinexor has been recently highlighted by the work of Chen
et al., [18] in which treatment efficacy was evaluated in an
in vivo platinum-resistant ovarian carcinoma model as well as in
in vitro models in which synergism was found in a cisplatin-
resistant variant derived from A2780 cells, suggesting that the type
of drug interaction may be dependent on the tumor molecular
background. Previous studies have also pointed out the relevance
of XPO1 inhibition in ovarian carcinoma, showing the growth inhi-
bitory and pro-apoptotic effect of leptomycin B, the first discovered
inhibitor of XPO1 mediated nuclear export, which proved inade-
quate for drug development due to in vivo toxicity [8]. Western
blot analyses suggest that the sensitivity to the compound is not
solely dependent on the XPO1 expression levels.
Given a genome-wide expression analysis [27] which identified
FOXO1 as down-regulated in platinum-resistant ovarian carci-
noma cells [12] and the known susceptibility to drug-induced
apoptosis of IGROV-1 cells [12,28], this model was considered
the most suitable for evaluation of the effect of the cisplatin-
Fig. 3. Effect of the combination of cisplatin and selinexor in TOV21G cells. Cell selinexor combination. The drug combination effect proved to be
sensitivity to drugs was evaluated by growth-inhibition assays. TOV21G cells were
schedule dependent, as the best synergism was achieved in
pre-incubated with cisplatin for 24 h before concomitant exposure to cisplatin-
selinexor for 48 h. Cells were then counted using a cell counter. The combination
IGROV-1 cells when selinexor was added 24 h after cisplatin expo-
index values obtained with the 0.03 lM selinexor concentration combined with sure. A favorable effect of the drug combination, although less
different cisplatin concentrations are shown. The reported values are the mean ± SE marked than in IGROV-1 cells, was observed in the TOV21G cell
of at least three independent experiments. line, likely due to the different features as reported based on an
integrated analysis of immunohistochemical markers, copy num-
The effects of the combination of selinexor and cisplatin were ber variations and mutations [29].
also tested. Cisplatin and selinexor were sequentially delivered at To define the mechanism underlying the drug interaction in
suboptimal doses to mice bearing the subcutaneous IGROV-1 IGROV-1 cells, we first examined the modulation of proteins
tumor (Fig. 6D). Despite negligible activity of single agents, the involved in cell cycle arrest and apoptosis. The cell cycle protein
combination induced 65% TVI (P < 0.001 versus control mice and p21WAF1, undetectable in vehicle-treated cells, was markedly up-
P < 0.05 versus cisplatin-treated mice), the effect being similar to regulated in cells exposed to the selinexor-cisplatin combination.
that achieved by selinexor at its optimal dose and schedule, i.e. a The combination-treated cells also exhibited higher levels of
TVI of 70% on day 16 (Table 2). All treatments were well tolerated. p21WAF1 when compared to single agent-treated cells (similar to
An analysis of apoptosis in tumors harvested 24 h after the last what was found in in vivo studies). A similar pattern was observed
treatment indicated that the drug combination induced the highest with p53, as expected based on the well-known transcriptional
level of apoptosis (P < 0.01, drug combination versus selinexor by regulation of p21WAF1 by p53. The effect on p21WAF1 levels is likely
Student’s t test) (Fig. 6E). Treatment with the drug combination p53-dependent in keeping with the presence of functional p53 in
tended to increase p21WAF1 protein levels as compared to the effect IGROV-1 cells [28]. Moreover, the pro-apoptotic protein Bax was
of selinexor alone (Student’s t test: P = 0.07 for selinexor versus up-regulated 48 and 72 h following every treatment, thereby indi-
combination, Fig. 6F, n = 3). A slight up-modulation of p53 was cating that the apoptotic pathway was triggered by all treatments.
observed under the same conditions, although it was not signifi- However, none of the treatments induced an up-regulation of
cant. FoxO-1 protein levels were not modulated. FoxO-1 protein levels in total cell lysates. A quantitative analysis
of drug-induced apoptosis by Annexin V-binding assay indicated
4. Discussion the occurrence of apoptosis after exposure to each agent, although
the effect was remarkable only upon exposure to the drug combi-
Cellular response to antitumor treatments involves multiple nation. Moreover, cell cycle perturbations were observed in treated
factors including components of apoptotic pathways. Tumor cell cells with G1 accumulation after 48 h exposure to selinexor and
resistance to clinically available agents (e.g., cisplatin) is often time-dependent G2/M accumulation after exposure to the drug
associated with reduced susceptibility to drug-induced apoptosis, combination.
which can be related to reduced expression of the FoxO-1 tran- Fluorescence microscopy experiments in cells stained with an
scription factor, a regulator of pro-apoptotic genes like PUMA anti FoxO-1 antibody indicated that selinexor could promote
and TRAIL [12,22,23]. XPO1, a nuclear export protein, contributes nuclear localization of FoxO-1 in IGROV-1 cells, suggesting that
to the dynamic subcellular localization of FoxO-1, among other under our experimental conditions XPO1-mediated nuclear export
100 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103

Fig. 4. Cell response to the combination of selinexor and cisplatin in IGROV-1 cells. (A) Western blot analysis was carried out in cells incubated with 0.3 lM cisplatin, 0.03 lM
selinexor or their combination adding selinexor 24 h after cisplatin. Protein lysates were prepared from cells harvested 48 h or 72 h after cisplatin exposure start. Control
loading is shown by vinculin. For each protein band intensity was quantified using ImageJ (mean ± SE, n = 3), normalized to loading control and referred to the levels of
control cells (set to 1). 1, control; 2, cisplatin; 3, selinexor; 4, combination. *, P < 0.05 versus control by two tailed unpaired Student’s t test (n = 3). (B) Immunofluorescence
analysis of FoxO-1 (green) after cell treatment with 0.03 lM selinexor, 0.3 lM cisplatin or their combination adding selinexor 24 h after cisplatin. Cells were observed 24 h
after selinexor exposure start. Nuclei were visualized by staining with Hoechst 33342. Cells were scored by fluorescence microscopy for FoxO-1 localization. Counts from
three independent fields/group are shown. (C) Cell cycle analysis of cells incubated with cisplatin, selinexor or their combination as described in A. Cells were stained with
propidium iodide and analyzed by flow cytometry. (D) Analysis of apoptosis in IGROV-1 cells exposed to cisplatin, selinexor and their combination adding selinexor 24 h after
cisplatin. Apoptosis was assessed by Annexin V-binding assay 48 h after selinexor treatment start. *, P < 0.005 by two tailed unpaired Student’s t test.

of FoxO-1 is inhibited; the effect was maintained in cells treated subcutaneous xenografts. Although cellular studies indicated that
with the combination of cisplatin and selinexor. knockdown of FOXO1 results in reduced selinexor growth inhibi-
The interest of selinexor for the treatment of ovarian carcinoma tory activity, a significant tumor growth inhibition was found
is supported by our antitumor efficacy studies, indicating a striking in vivo also in IGROV-1/Pt1, consistently with the capability of
activity of the compound both in platinum-sensitive and resistant selinexor to regulate the localization of multiple proteins. It should
 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103 101

A B 72h 144h

FoxO-1
Actin

Fig. 5. Efficacy of cisplatin, selinexor and their combination upon FOXO1 knockdown. (A) qRT-PCR of FOXO1 levels in IGROV-1 cells at different times after siRNA
transfection; untransfected cells were used as calibrator and GAPDH as housekeeping. RQ values (± SD) from triplicates are shown. (B) Western blot analysis of FoxO-1 levels
in IGROV-1 cells at different times after siRNA transfection. The band intensity was quantified using ImageJ, normalized to loading control and referred to the levels of
untransfected cells (set to 1). *, P < 0.05 FOXO1 siRNA- versus negative control siRNA transfected cells by two tailed unpaired Student’s t test (n = 3). (C) Sensitivity of IGROV-1
cells to selinexor and effect of the selinexor-cisplatin combination. Cells were transfected with siRNA and 48 h later harvested and seeded for growth inhibition assays. Cells
were incubated with cisplatin and 24 h later selinexor was added for 48 h. Cells were counted using a cell counter. The combination index values obtained with the 0.03 lM
selinexor concentration combined with different cisplatin concentrations are shown. The reported values are the mean ± SE of at least three independent experiments.

be noted that orally administered selinexor exhibited a marked once per week. Such a schedule has been shown to preserve nor-
capability to reduce intraperitoneal tumor growth in cell-injected mal immune functioning [31]. Thus, our findings may be useful
mice, in keeping with previous results obtained in orthotopic peri- in an attempt to optimize clinical dosing. Of note, a phase I clinical
toneal mesothelioma model [30]. Moreover, selinexor-treated mice study has provided promising results in platinum-resistant ovarian
did not develop ascites, a clinically relevant observation. When cancer patients [18].
selinexor and cisplatin were combined according to the best sched- In conclusion, our findings show that FoxO-1 finely modulates
ule identified in in vitro studies, a therapeutic advantage in terms the effect of selinexor and marginally of the combination with
of TVI was found as compared to single agents. These results are cisplatin providing preclinical evidence of potential clinical impact
consistent with those by Chen et al., although we used a much on ovarian carcinoma with selected molecular backgrounds. These
lower selinexor dose [18]. In fact, drug combination experiments results highlight the interest of combination therapies designed to
were carried out using a low dose of selinexor (5 mg/kg) given only increase cell death in tumor cells. However, given that XPO1 can
102 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103

Fig. 6. Antitumor activity studies and analysis of apoptosis and protein modulation in tumors from mice treated with selinexor, cisplatin and their combination (combo). (A)
Effects of oral selinexor, 10 mg/kg q3-4d/wx4w, on the growth of the IGROV-1 and IGROV-1/Pt1 carcinoma cells s.c. injected into the right flank of female nude mice (n = 8;
107 cells/mouse) on day 0. Treatment started on day 5–6. Mean values of tumor volumes (± SD) are shown. (B) IGROV-1 tumors from control and treated mice harvested 24 h
after last treatment were processed for immunohistochemical analysis of FoxO-1. Cells with different pattern of expression of FoxO-1 with cytoplasmic positivity (top) and
with both cytoplasmic and nuclear (arrowhead) positivity (bottom) are shown at 400x. (C) Effects of oral selinexor, 10 mg/kg q3-4dx6, on the ascites onset in mice i.p.
injected with the IGROV-1 carcinoma cells (n = 7; 107 cells/mouse) on day 0. Treatment started on day 4. Kaplan-Meier plot of the percentage of disease-free animals. P by
Log-rank test. (D) Effects of oral 5 mg/kg selinexor on the antitumor activity of 4.5 mg/kg i.v. cisplatin against the growth of the IGROV-1 carcinoma cells s.c. injected into the
right flank of female nude mice (n = 7; 107 cells/mouse) on day 0. Cisplatin was given on day 5, 12 and 19, selinexor on day 6, 13 and 20. Mean values of tumor volumes (± SD)
are shown. **P < 0.001 by Student’s t test versus control mice. (E) Samples from control and treated mice were processed for apoptosis analysis by Annexin V binding assay 24
h after the last selinexor treatment. The columns indicate late apoptotic (Annexin V-PI positive cells) cells. The values represent the mean ± SD. **, P < 0.01 by two-tailed
unpaired Student’s t test. (F) Tumors from control and treated mice were processed for western blot analyses to examine levels of the indicated proteins. Samples obtained
from 3 mice/group 24 h after the last selinexor treatment were fractionated by SDS-PAGE. Control loading is shown by actin. Mean values with respect to control mean value
arbitrarily set to 1 are shown in the histograms (n = 3).
 C. Corno et al. / Biochemical Pharmacology 147 (2018) 93–103 103

Table 2
Antitumor activity of oral selinexor alone or in combination with cisplatin in nude mice s.c. bearing human ovarian carcinomas.1

Model Drug Days of treatment Dose (mg/kg) TVI% (day)2 BWL%3 Tox4
* *
IGROV-1/Pt1 Selinexor 5, 8, 12, 16, 20, 23, 26, 29 10 69 (16) 74 (20) 5 0/8
IGROV-1 6, 9, 13, 16, 19, 22, 26, 29 1 70** (16) 78** (26) 2 0/8
6, 13, 20 5 31 (16) 0 0/7
Cisplatin 5, 12, 19 4.5 32 (16) 7 0/7
Cisplatin ? 5, 12, 19 4.5 65*° (16) 7 0/7
Selinexor 6, 13, 20 5
1
Tumor cells (107/mouse) were s.c. inoculated into the right flank on day 0. Treatment started when tumors were established.
2
Tumor volume inhibition (TVI) percentage in treated over control mice, in parentheses the day on which it was assessed.
3
Body weight loss (BWL) percentage induced by treatment; the highest value is reported.
4
Dead/treated mice.
*
P < 0.001 by Student’s t test versus control mice.
**
P < 0.0001 by Student’s t test versus control mice.
°
P < 0.05 by Student’s t test versus cisplatin-treated mice.

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Conflict of interest statement [17] J.G. Turner, T. Kashyap, J.L. Dawson, J. Gomez, A.A. Bauer, S. Grant, et al., XPO1
inhibitor combination therapy with bortezomib or carfilzomib induces nuclear
Christian Argueta and Yosef Landesman are employees of Kar- localization of IkappaBalpha and overcomes acquired proteasome inhibitor
resistance in human multiple myeloma, Oncotarget 7 (2016) 78896–78909.
yopharm Therapeutics. No other conflicts of interest to disclose. [18] Y. Chen, S.C. Camacho, T.R. Silvers, A.R. Razak, N.Y. Gabrail, J.F. Gerecitano,
et al., Inhibition of the nuclear export receptor XPO1 as a therapeutic target for
Acknowledgements platinum-resistant ovarian cancer, Clin. Cancer Res. 23 (2017) 1552–1563.
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Mohammad, et al., Selinexor, a selective inhibitor of nuclear export (SINE)
The study was supported by Associazione Italiana per la Ricerca compound, acts through NF-kappaB deactivation and combines with
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