IGF-1R/MDM2 relationship confers enhanced sensitivity to RITA in

Author Manuscript Published OnlineFirst on March 28, 2012; DOI: 10.1158/1535-7163.MCT-11-0913
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IGF-1R/MDM2 relationship confers enhanced sensitivity to RITA in Ewing’s
sarcoma cells
Giusy Di Conza1,2, Marianna Buttarelli1,3, Olimpia Monti1, Marsha Pellegrino1,4,
Francesca Mancini1,4, Alfredo Pontecorvi4, Katia Scotlandi5, Fabiola Moretti1
1
2
Cell Biology and Neurobiology Institute-CNR /Fondazione Santa Lucia, Roma
Department of Experimental-Clinical Medicine and Pharmacology, University of
Messina, Messina
3
Department of Surgery, Breast Unit, Catholic University of Rome, Roma
4
Division of Endocrinology, Catholic University of Rome, Roma
5
CRS Development of Biomolecular Therapies, Lab Experimental Oncology,
Orthopaedic Rizzoli Institute, Bologna, Italy
Running Title: Ewing’s sarcoma sensitivity to RITA
Key words: Ewing’s Sarcoma, RITA, MDM2, IGF-1R, p53
Financial Support: This work was supported by a grant of Ministero della Salute
(RF-IOR-2006-422755) to K.Scotlandi, and F.Moretti, and Integrato Oncologia
2009/1536 to K. Scotlandi and of AIRC (Italian Association for Cancer Research) to
F. Moretti and K. Scotlandi. O.Monti is recipient of a FIRC fellowship. G. Di Conza is
recipient of a PhD fellowship from University of Messina.
Corresponding author: Dr. Fabiola Moretti, Cell Biology and Neurobiology Institute,
CNR, Via del Fosso di Fiorano 64, 00143 Tel:+39 06 501703242; fax :+39 06
501703313. E-mail address: mailto:[email protected]
Conflict of interest: No potential conflicts of interest were disclosed
Word Count and Figures/Tables Number: 4195 words (abstract excluded) and 6
figures
1
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Abstract
Ewing’s sarcoma is one of the most frequent bone cancers in adolescence. Although
multi-disciplinary therapy has improved the survival rate for localized tumors, a
critical step is the development of new drugs to improve the long term outcome of
recurrent and metastatic disease and to reduce side-effects of conventional therapy.
Here, we demonstrate that the small molecule RITA (Reactivation of p53 and
Induction of Tumor cell Apoptosis, NSC652287) is highly effective in reducing growth
and tumorigenic potential of Ewing’s sarcoma cell lines. These effects occur both in
the presence of wt-p53 as well as of mutant or truncated forms of p53, or in its
absence, suggesting the presence of additional targets in this tumor histotype.
Further experiments provided evidence that RITA modulates an important oncogenic
mark of these cell lines, insulin like growth factor receptor 1 (IGF-1R). Particularly,
RITA causes downregulation of IGF-1R protein levels. MDM2 degradative activity is
involved in this phenomenon. Indeed, inhibition of MDM2 function by genetic or
pharmacologic approaches reduces RITA sensitivity of Ewing’s sarcoma cell lines.
Overall, these data suggest that in the cell context of Ewing’s sarcoma, RITA may
adopt additional mechanism of action besides targeting p53, expanding its field of
application. Noteworthy, these results envisage the promising utilization of RITA or
its derivative as a potential treatment for Ewing’s sarcomas.
2
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Introduction
Ewing’s sarcoma is the second most common tumor of bone in children and
young adults. Besides surgery, its treatment includes highly intensive chemo- and/or
radiation therapies, which are associated with significant short and long term sideeffects, of particular relevance in consideration of the young age of patients (1, 2).
The potential application of newly developed strategies or the identification of new
targeted therapies is a field of intense study in the treatment of Ewing’s sarcoma.
New anticancer therapies are mostly residing on the development of small chemical
entities against specific oncogenic targets (3). Ewing’s sarcoma are molecularly
characterized by the presence of a somatic translocation that involves chromosome
11 resulting in an oncogenic fusion protein that encompasses the EWS gene (the
more abundant translocation gives rise to EWS-FLI-1 chimera) (4). Therefore,
approaches have been developed to inhibit this oncogene, achieved mainly through
the inhibition of its transcript product, and have demonstrated their efficacy to limit
cell growth and tumorigenicity (5-8). Strictly related to EWS-FLI-1, a crucial
oncogenic pathway in Ewing’s sarcoma is that one mediated by Insulin like growth
factor receptor 1 (IGF-1R) (9-12). Indeed, activation of this receptor is necessary for
EWS-FLI-1 induced transformation of human cells (9). As confirmation of the
relevance of IGF-1R pathway, different therapeutic strategies are showing their
efficacy in preclinical models of the disease, and in Ewing’s sarcoma patients in I or
II phase clinical trials (2, 12). At the same time, increasing evidence suggests that
combination of IGF-1R inhibitory molecules with other treatments may further
improve the efficacy of the first (13).
Reactivation of the oncosuppressor p53 function is a goal of different therapeutic
approaches. These approaches can be grossly divided in two groups: i) one aiming
3
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to correct the activity of mutant p53 in those tumors bearing a mutated gene; ii) a
second one aiming to reactivate p53 function in those tumors where it is still
preserved (14). TP53 gene is rarely altered in Ewing’s sarcoma (12) and therefore
can be a potential target of the second type of approach. Among strategies for p53
reactivation, a promising molecule is the furanic compound RITA (Reactivation of
p53 and Induction of Tumor cell Apoptosis, also NSC652287) (15, 16). At its
discovery, RITA has been identified as a molecule able to bind and activate p53 by
dissociating it from its inhibitor MDM2 (16). Later on, other studies have suggested
the possibility of RITA to bind additional targets, as MDM2 itself and mutant p53 (1719).
In this work, we investigated the sensitivity to RITA of different cell lines derived from
Ewing’s sarcoma and the biological response caused by this molecule. To define the
role of p53 in RITA sensitivity, we used cell lines characterized by a different status
of p53. Our data show that RITA is highly effective in suppressing growth and
tumorigenic properties of Ewing’s sarcoma cell lines independently of the status of
p53. At the molecular levels, we observed a decrease of IGF-1R protein at least in
part mediated by MDM2 degradative activity. Overall, these data indicate the ability
of RITA to affect additional molecules other than p53 and its potential application in
Ewing’s sarcoma.
4
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Materials and Methods
Cell cultures, transfections and treatments. All ES cell lines were obtained
by Orthopaedic Rizzoli Institute Bologna, Italy in 2006. Previously, SK-N-MC
cell line was obtained from the American Type Culture Collection (Rockville,
MD, USA), TC-71 and 6647 cell lines were a generous gift from TJ. Triche
(Childrens Hospital, Los Angeles, CA, USA), WE-68 cell line was established
and kindly provided by F. van Valen (20), LAP-35 and IOR-BRZ71 cell lines
were established in Orthopaedic Rizzoli Institute (21). In all cell lines but WE68 the presence of EWS-FLI1 has been verified by western blot (WB). All cell
lines have been tested for micoplasma contamination and verified to be
mycoplasma free (last control April 2011). SK-N-MC, TC-71 and WE-68 have
been further authenticated by DNA fingerprinting in 2008, using genes MPX-2
and MPX-3 kits (22). The status of TP53 gene in all cell lines, but SK-N-MC,
was confirmed by sequencing in 2008. Genetic analysis of the TP53 gene
was performed by PCR amplification of exons 1-12 with flanking intronic
primers, followed by sequencing. Genomic DNA was extracted from cell lines
according to standard procedures. Cells were cultured in Iscove’s modified
Dulbecco’s medium (IMDM) (Gibco), supplemented with 100 units/ml
penicillin, 100 μg/ml streptomycin and 10%
fetal bovine serum (FBS)
(Invitrogen) and maintained at 37°C in a humidified 5% CO2 atmosphere.
SK-N-MC
cells
were
stably
transfected
with
pCMV-MDM2,
pCMV-
MDM2C438L or pCDNA3.1B using Lipofectamine Plus Reagent (Invitrogen)
and were then maintained in medium supplemented with G418 700 g/ml.
5
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Cells were treated with RITA 1M or 3M, -amanitin 10 g/ml, MG132 10
M or 15 M and with sempervirin 0.5 g/ml.
Cell viability and TUNEL assay. Cell viability was assessed by collecting
both floating and adherent cells counted in a hemocytometer after the addition
of trypan blue. Alternatively cell viability was determined using Cell Titer Blue
colorimetric assay according to manufacturer’s instruction (Promega). Growth
curves were previously set up with increasing cell number (4000, 6000, 8000
cells/well) for each cell line.
Apoptotic DNA fragmentation was performed by TUNEL assays with
fluorescent In Situ Cell Death Detection kit (Roche) following the
manufacturer’s instructions. Alternatively apoptotic cells were determined
using ApoAlert Annexin V kit (Clontech) according to manufacturer’s
instruction.
Immunoprecipitation and WB. For immunoprecipitation experiments, cells
were lysed in Saito’s modified buffer (50mM Tris–HCl, pH 7.4, 0.15M NaCl,
0.5% Triton-X100, 5mM EDTA) contained a cocktail of protease inhibitors
(Boehringer). Immunoprecipitations were performed by preincubation lysates
with protein G-sepharose (Pierce) and then with the indicated antibody, under
gentle rocking at 4°C overnight. For WB, cells were lysed in RIPA buffer
(50mM Tris–Cl, pH 7.5, 150mM NaCl, 1% Nonidet P-40, 0.5% Na
desoxicholate, 0.1% SDS, 1mM EDTA) supplemented with a cocktail of
protease inhibitors (Boehringer).
6
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The following primary antibody were used: rabbit anti-IGF-1R polyclonal
antibody C-20 (Santa Cruz), goat anti-p53 polyclonal antibody FL393 (Santa
Cruz), mouse anti-MDM2 monoclonal antibody 2A10 and Ab1 (Calbiochem),
mouse anti-α-tubulin monoclonal antibody DM1A (Sigma), rabbit anti-FLI-1
polyclonal antibody C-19 (Santa Cruz), mouse anti-PARP1 monoclonal
antibody C2-10 (Pharmigen), mouse anti-Hsp70 monoclonal antibody SPA820 (StressGen), mouse anti-GFP monoclonal antibody 7.1 and 13.1 (Roche)
and mouse anti-actin monoclonal antibody C-40 (Sigma).
Cell cycle analysis. Cell cycle profiles were evaluated by fixing 5x105 cells in
70% ethanol for 1 hour on ice and staining DNA for 30 minutes at room
temperature with 50 μg/mL propidium iodide in PBS containing 1 mg/mL
RNase A. Cell percentages in the different phases of the cycle were
measured by flow cytometric analysis of propidium iodide–stained nuclei
using Multicycle Software (Phoenix Flow System) Epics XL (Coulter,
Fullerton, CA).
Soft-agar foci formation assay. The anchorage-independent growth of LAP35, TC-71 and SK-N-MC cell lines was monitored by the soft agar colony
formation assay. In 60mm dishes 5 ml of hard agar layer, 0.7% agar-noble
(Difco) reconstituted in serum-containing medium, were used as substrate for
the Ewing’s sarcomas cell lines that were plate on a 2.0 ml layer of 0.35%
warm agar in serum-containing medium. Cells were then cultured at 37°C, 5%
CO2 for three weeks and RITA treatment was renewed every 3 days.
7
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Small interfering RNA. MDM2 siRNA and control siRNA were generated by
Invitrogen (Stealth RNAi) and consisted of a mix of three different siRNA.
Cells were transfected using RNAiMAX reagent (Invitrogen) following the
manufacturer’s instructions.
Real Time PCR. Total RNA extraction was carried out with TRIzol reagent
(Invitrogen). For quantitative reverse transcription-PCR (Q-RT-PCR), total
RNA (1 μg) was reverse transcribed using Gene Amp kit (Applied Biosystems)
and subjected to PCR amplification using SYBR Green PCR Master Mix
(Applied Biosystems). The following primers were used:
upper IGF-1R 5-CCATTCTCATGCCTTGGTCT-3´;
lower IGF-1R 5-TGCAAGTTCTGGTTGTCGAG-3´;
upper IGF-1 5-GGTGGATGCTCTTCAGTTCGT-3´;
lower IGF-1 5-CCACGATGCCTGTCTGAGG-3;
upperIGF-BP3 5-TGTGGCCATGACTGAGGAAA-3;
lower IGf-BP3 5-TGCCAGACCTTCTTGGGTTT-3.
upper hTBP 5-GAACATCATGGATCAGAACAACA-3;
lower hTBP 5-ATAGGGATTCCGGGAGTCAT-3.
Each tissue sample mRNA was normalized relative to the GAPDH mRNA.
Samples underwent 40 amplification cycles, monitored by an ABI Prism 7900
sequence detector (Applied Biosystems). All amplification reactions were
conducted at least in duplicate and averages of threshold cycles were used to
interpolate standard curves and calculate transcript amount using the
software SDS version 2.3 (Applied Biosystems).
8
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Results
RITA inhibits growth and tumorigenic potential of Ewing’s sarcoma cell lines
independently of p53 status
To ascertain RITA efficacy in Ewing’s sarcoma, we used cell lines
characterized by the same chromosomal translocation, t(11;22)(q24;q12), that
generates the oncogenic protein EWS/FLI-1. To verify the relevance of p53 in RITA
sensitivity we used cell lines characterized by a different p53 status: LAP-35 and
WE-68 with wild-type p53, TC-71 and IOR-BRZ71 with a truncated p53 lacking the
tetramerization and DNA-binding domains (p53-213STOP), 6647 with a point mutant
p53 (p53-S241F) and SK-N-MC lacking p53 expression (23). The status of TP53
gene in all cell lines, but SK-N-MC, was confirmed by sequencing (data not shown).
At first, we analyzed cell growth of all cell lines in the presence or absence of RITA.
Since analysis of NCI-60 cell panel has evidenced a mean IC50>3,5μM for RITA in
mutant p53 carrying cancer cell lines (24), we treated Ewing’s sarcoma cell lines
with 1 and 3 μM RITA. Unexpectedly, analysis of cell viability evidenced the ability of
RITA to reduce this parameter in all tested cell lines, independently of the presence
and of the status of p53 (Figures 1A-F). In wt-p53 cell lines, cell viability was strongly
reduced in WE68 cells, whereas in LAP-35 it was similar to that observed in mutant
p53 cell lines or even lower in comparison to null p53 SK-N-MC. Cell growth
reduction was enhanced by increased doses (1 or 3 μM) and treatment times of
RITA, although with a different kinetics among cell lines. To confirm these data and
ascertain the specificity of the observed effects, we measured viability by analyzing
cell metabolic activity (by reduction of resazurin) and compared the dose of RITA
able to reduce this parameter to 50% in Ewing cell lines and in H1299, a lung tumor
cell line lacking p53. Although not overlapping with the results obtained by Trypan
9
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Blue uptake, RITA confirmed its efficacy in all Ewing’s cells in comparison to H1299
cells that resulted unaffected by the used doses of RITA (Figure 1G). Particularly,
the p53-null SK-N-MC confirmed the highest sensitivity whereas wt-p53 LAP35
showed a sensitivity similar to that of truncated-p53 IOR-BRZ71. HCT116-/-, a colon
cancer cell line lacking p53 too, and insensitive to the drug (16), showed results
similar to those observed in H1299 (data not shown). These data overall confirm the
specific sensitivity of Ewing’s sarcoma cell lines to RITA.
FACS analysis evidenced a strong alteration of cell cycle caused by RITA as
early as 16 hours after drug treatment in three representative cell lines (Figure 2A-C)
and in the other cell lines (Supplementary Figure 1A-C). In all cell lines it was
evident a progressive accumulation of cells in the S/G2 phases of cell cycle, in
agreement to what previously reported (15, 18); in TC-71 and especially in SK-NMC, it was also evident a sub-G1 population indicative of cell death (Figure 2A-C).
LAP-35, although containing wt-p53, showed a modest increase of sub-G1 at the
latest time point (36 hours). To further characterize the loss of viability evidenced by
FACS analysis, a TUNEL assay was performed. The experiments confirmed a
strong increase of apoptotic cells in TC-71 and SK-N-MC cells upon RITA 3 μM
treatment whereas only a mild increase was observed in LAP-35 in agreement to
FACS data (Figure 2D).
Overall, these data indicate that RITA is effective in suppressing cell growth
of Ewing’s sarcoma cell lines and that its effects are mediated by a strong growth
suppressive and/or apoptotic activity. Noteworthy, these effects appear to be not
related to the status of p53.
RITA exerts a strong anti-tumor activity in tumor cell lines (16, 25). To verify
whether RITA is able to affect tumorigenic properties of Ewing’s sarcoma cells too,
10
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we analyzed their anchorage-independent growth by a soft agar assay using RITA 1
and 3 μM. Different amounts of cells were plated (20.000 or 100.000 cells/60mm
dish) and after 48 hours treated with vehicle (CTR) or RITA, repeating the
administration every three days. After 3 weeks, all CTR cells have formed marked
colonies, more evident in TC-71 and SK-N-MC, less evident in LAP-35 in agreement
with the reduced tumorigenicity of this cell line (26) (Figure 3A, CTR row). Strikingly,
both doses of RITA were able to completely abolish anchorage-independent growth
independently of the number of seeded cells (Figure 3A and data not shown)
indicating that RITA is highly effective in reducing in vitro tumorigenic potential of
these cells. Again these effects are irrespective of p53 status suggesting that RITA
may affect additional pathways in this tumor histotype.
RITA downregulates IGF-1R levels
Anchorage-independent growth requires a combination of proliferation and
survival signaling strongly affected by the presence of a functional IGF-1R pathway
(13, 27). In order to investigate RITA activity in Ewing’s sarcoma cells, the levels of
IGF-1R were analyzed in two cell lines characterized by a null and a truncated p53,
SK-NMC
and
TC-71
respectively.
RITA
treatment
caused
a
progressive
downregulation of IGF-1R levels in both cell lines (Figure 4A-B). IGF-1R decrease
appeared earlier and more evident in cells that undergo apoptotic response
evidenced also by the progressive appearance of PARP cleavage product (Figure
4A-B). Under the same conditions, MDM2 levels decreased at the later time points
as previously reported (Figure 4A-B) (28, 29). Similar results were observed also in
the other cell lines (Supplementary Figure 2 and 3). P53, wt as well as mutant
S241F forms, were not substantially altered or slightly increased in LAP35 and 6647
(Supplementary Figure 3). Conversely, a strong decrease of wt-p53 coexistent with
11
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IGF-1R was observed in WE68 (Supplementary Figure 2), suggesting a specific
susceptibility of this cell line to RITA.
IGF-1R has been previously described as a transcriptional target of RITAmediated p53 transcriptional activity (30, 31). Although, in the cell lines here used,
but LAP-35 and WE68, p53 is transcriptionally inactive or absent, we tested whether
IGF-1R downregulation is correlated to a decrease of its mRNA. Indeed, the levels
of IGF-1R mRNA were not significantly altered in both cell lines (Figure 4C-D). We
therefore tested whether the downregulation of IGF-1R was mediated by increased
protein turnover. IGF-1R degradation is mediated by proteasome and lysosome (32).
To
analyze
the
involvement
of
proteasome
in
RITA-mediated
IGF-1R
downregulation, TC-71 and SK-NM-C cell lines were treated with the proteasome
inhibitor, MG132, or RITA or both drugs. MG132 treatment per se increased the
levels of IGF-1R, confirming the proteasome-mediated degradation of the receptor in
these cell lines. Noteworthy, addition of MG132 to RITA-treated cells was able to
recover the decrease of IGF-1R levels (Figure 4E-F) indicating that RITA-induced
downregulation was indeed caused by degradation of the receptor.
It has been reported that IGF-1R levels are downregulated by insulin-like
growth factor binding protein 3 (IGF-3BP) (33), a protein acting as inhibitor of IGF1R activity by sequestering its ligand IGF-1. IGF-BP3 is a transcriptional target of
EWS-FLI-1, the oncogenic mark of Ewing’s sarcoma (34). Particularly, IGF-BP3 is
repressed by EWS-FLI-1. Since it has been shown that RITA downregulates various
key oncogenic pathways (30), we investigated whether IGF-1R decrease was
related to modification of EWS-FLI-1 levels and/or activity. Nor substantial
modification of EWS-FLI-1 protein levels (Supplementary Figure 4A-C) or of its
transcriptional targets IGF-1 and IGF-BP3 mRNA (Supplementary Figure 4D) was
12
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observed upon RITA treatment in SK-N-MC and TC-71, suggesting that
downregulation of IGF-1R by RITA is not mediated by alteration of EWS-FLI-1.
Conversely, in LAP-35 RITA caused a decrease of EWS-FLI1 (Supplementary
Figure 4C), supporting the p53-mediated downregulation of this oncogenic pathway
too (30).
MDM2 mediates RITA-induced IGF-1R degradation
Degradation of IGF-1R is mediated by two RING ubiquitin ligases, c-Cbl and
MDM2 (35, 36). Although p53 is the only known target of RITA, MDM2 has been
reported as an important mediator of RITA activity (28, 37). We therefore
investigated whether MDM2 may be involved in IGF-1R degradation. We interfered
MDM2 expression by siRNA and analyzed IGF-1R levels of siMDM2 interfered cells
in comparison to control cells interfered with scramble siRNA (siCTR) both in the
absence and in the presence of RITA. Interference of MDM2 expression was indeed
able to counteract RITA-mediated IGF-1R downregulation (Figures 5A), supporting
the potential involvement of MDM2 in RITA-mediated IGF-1R degradation. To further
ascertain whether MDM2 activity is involved in RITA sensitivity, cell viability was
analysed by Annexin staining. Indeed, cell apoptosis induced by RITA was reduced
in siMDM2 in comparison to siCTR cells, paralleling IGF-1R recovery. These effects
were more pronounced in SK-N-MC cells in agreement with the increased apoptosis
observed in these cells.
To ascertain whether MDM2-induced degradation of IGF-1R is mediated by
transcriptional activation of other cofactors by RITA, we induced transcriptional block
by pre-treating cells with the inhibitor of RNA polymerase II, α-amanitin (38, 39). In
the absence of RITA, α-amanitin does not affect IGF-1R levels, in agreement with
the long half life of the receptor (24 hours, as reported in 40). Importantly, the
13
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presence of α-amanitin does not alter RITA-induced decrease of IGF-1R levels
(Figures 5C-D), suggesting that transcription of other factors is not required for
RITA-mediated IGF-1R downregulation. Functionality of α-amanitin was evidenced
by the downregulation of Pol II levels, as previously reported (39).
MDM2-mediated degradation of IGF-1R is mediated by direct association
between the two proteins (35). To support the activity of MDM2 towards IGF-1R, we
analyzed the binding between IGF-1R and MDM2 in the presence or absence of
RITA after few hours of RITA treatment. In the absence of RITA, association
between the two proteins was barely detectable. Conversely, after 4 hours of RITA
treatment, an evident increase in the association between the two proteins was
observed in both SK-N-MC and TC-71 (Figures 5E-F). This early association is in
agreement with the observation that MDM2-mediated ubiquitination of IGF-1R
precedes subsequent internalization and degradation of the receptor (36) and
supports the hypothesis that RITA treatment increases the association between
MDM2 and IGF-1R, leading to IGF-1R degradation.
MDM2-degradation of IGF-1R mediates RITA sensitivity
To confirm the role of MDM2 activity and of MDM2/IGF-1R relationship in
RITA sensitivity, we interfered with MDM2 ubiquitination function. It has been
recently described the ability of sempervirine, a plant alkaloid with well known
anticancer activities (41), to inhibit ubiquitination and degradative activity of MDM2
(42). We therefore impaired MDM2 function by sempervirine and analyzed RITA
effects. A dose of 0.5 μg/ml of sempervirine demonstrated its efficacy to inhibit
MDM2 degradative function both in TC-71 and in SK-N-MC cells, after 8 or 4 hours
of treatment respectively (data not shown). Simultaneous administration of RITA and
sempervirine was able to antagonize downregulation of IGF-1R levels in both TC-71
14
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and SK-N-MC (Figures 6A and C). Most importantly, these effects correlate with an
impairment of cell death induced by RITA (Figures 6B and D), confirming that
MDM2-mediated IGF-1R degradation is an important step of RITA activity and
supporting the role of MDM2/IGF-1R relationship in RITA sensitivity of these tumor
cells.
To confirm the role of MDM2 and of its ubiquitinating activity in RITA
sensitivity, we stably overexpressed wt-MDM2 and a mutant MDM2, MDM2-C438L,
in SK-N-MC. This mutation disrupts the RING region where ubiquitin ligase activity
of MDM2 resides and therefore abolishes it (43). Upon 15 hours of RITA treatment,
the overexpression of wt-MDM2 accelerated IGF-1R downregulation whereas this
was poorly observed in the pcDNA3.1 transfected (CTR) and the MDM2-C438Lexpressing cell populations (Figure 6E). Of note, these molecular data correlate with
altered survival of SK-N-MC. Indeed, the expression of wt-MDM2 potentiates the
effects of RITA whereas the mutant MDM2-C438L does not exhibit such activity and
rather slightly reduced cell sensitivity to the small molecule (Figure 6F), supporting
the role of MDM2 degradative activity in RITA sensitivity of Ewing’s sarcoma cells.
15
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Discussion
The aim of this study was to evaluate the efficacy of the furanic compound
RITA in Ewing’s sarcoma. Indeed, the requirement of new therapeutic tools for this
type of tumor is a field of intense study (2).
The canonical target of RITA is wt-p53 although additional molecules have
been hypothesized (16-18). Here, we compared the effects of RITA among different
cell lines characterized by wt, mutant, truncated and null p53. The first result was
that RITA strongly affects the growth and tumorigenic properties of these cell lines
independently of p53, suggesting a specific sensitivity of this tumor type to RITA.
Particularly, RITA reduced to a similar extent cell viability of wt-p53 LAP-35, null p53
SK-N-MC, and IOR-BRZ71 cells expressing a truncated form p53-213STOP that
resembles a null condition. Additionally, a significant cell death was observed in wtp53 WE-68 cells as well as in p53-213STOP TC-71 and null-p53 SK-N-MC cells.
Conversely, a reduced cell death was evident in the wt-p53 LAP-35 cell line.
Although, this may be attributed to the reduced tumorigenicity and growth rate of this
cell line, the presence of additional factors affecting RITA sensitivity could contribute
as well (28).
RITA has been identified through a cell-based drug screening strategy (16)
based on its biological effects on target function, specifically p53. The possibility that
it can affect additional pathways cannot be excluded. Indeed, RITA activates a DNA
damage response (44) and affects wt p53-related off-targets (28). In addition, cell
lines carrying mutant p53 have been reported to be sensitive to RITA, although at
higher doses in comparison to cell lines carrying wt-p53 (24, 45).
In fact, accumulation of cells in the S/G2 phases of cell cycle, as observed in
Ewing’s sarcoma cell lines, has been previously reported both in wild-type as well as
16
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in mutant and null p53-carrying cancer cell lines (16, 18) suggesting that RITAmediated alteration of the cell cycle may depend from additional factors besides p53.
The hallmarks of Ewing’s sarcoma are the presence of oncogenic protein EWS-FLI1 and the autocrine-activation of IGF-1R. We did not observe substantial
modification of EWS-FLI-1 levels. Conversely, we observed a consistent decrease of
IGF-1R levels upon RITA treatment. IGF-1R is a crucial pathway for Ewing’s
sarcoma survival. Molecular and translational studies have demonstrated that
inhibition of this pathway suppresses tumor growth (reviewed in 12) and in fact
clinical trials with IGF-1R inhibitors are under evaluation (2). The data here
presented indicate that IGF-1R may be indirectly affected by a targeted therapy too,
opening a new potential field of anti-IGF-1R therapies.
Our experiments indicate that downregulation of IGF-1R protein levels is
mediated at least in part by the E3 ubiquitin ligase, MDM2. Various studies have
shown the ability of MDM2 to control IGF-1R levels (35, 46) although it is not
completely clear what activates and controls this MDM2 function. Moreover, it has
been demonstrated that MDM2 counteracts the IGF-1R survival function,
independently of p53 (47). Our data indicate that in Ewing’s sarcoma cell lines, RITA
activates MDM2 function. How this happens, it is not clear. Cell fractionation
experiments did not show any difference in MDM2 localization in the presence or
absence of RITA, excluding a delocalization of MDM2 by RITA treatment (data not
shown). A study of docking simulation indicated that RITA can bind MDM2 (17)
although with an affinity
15000 less than p53. The possibility that MDM2
modifications alter RITA affinity to MDM2 has not been tested. However, the
selective sensitivity of Ewing’s sarcoma to RITA suggests that additional specific
factors mediate and direct MDM2 activity towards IGF-1R in this tumor type.
17
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Interestingly, a recent study reported the overexpression in Ewing’s sarcoma of a
protein involved in ubiquitin ligase activity, CDT2, raising the hypothesis that
deregulation of protein ubiquitin machinery may be of relevance in this tumor type
(48). Finally, downregulation of MDM2 upon RITA treatment as observed in this work
and reported by other authors (28, 29), but yet its role in RITA function, may appear
to counteract its role in RITA sensitivity. However, the crucial role of MDM2
degradative activity in RITA sensitivity has been previously reported by other authors
(28, 37), indicating that MDM2 is important at least for the early response to RITA.
It remains to be ascertained whether MDM2-mediated IGF-1R downregulation
is the main determinant of RITA activity. Unfortunately, experiments of rescue of
RITA effects by overexpression of IGF-1R failed due to the inability of two different
IGF-1R coding plasmids to substantially overexpress IGF-1R levels in the Ewing’s
sarcoma cell lines here studied (data not shown). Of note, MDM2 is able to alter cell
cycle progression and different studies have reported the ability of MDM2 to induce
a G0/G1 arrest in specific cell lines, independently of p53 (49). Therefore, growthcontrol activities of MDM2 too might be involved in RITA activity in Ewing’s sarcoma.
However, the complete understanding of the mechanism by which RITA is effective
in this tumor type is a point of relevance before any consideration of this drug as
therapeutic agent in Ewing’s sarcoma.
Overall, these data expand the spectrum of anti-tumor RITA activity showing
its efficacy in Ewing’s sarcoma. Interestingly, p53 mutation, rare in Ewing’s sarcoma
(12), is increased in a subset characterized by high aggressive behavior and poor
chemoresponse (50) suggesting the useful application of RITA in those cases
escaping standard therapies. Given the low toxicity of this molecule in normal cells
18
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(16), these results pave the way to the potential application of a new therapeutic tool
to Ewing’s sarcoma.
19
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Acknowledgments
We thank Dr.ssa Igea D’Agnano, Dr.ssa Francesca Siepi and Dr.ssa Manuela Natoli
for their assistance in FACS analysis. We are also grateful to Prof. R Baserga and
Prof. A. Belfiore for providing IGF-1R expressing plasmids. We are especially
grateful to Dr.ssa Ada Sacchi and Prof. F. Trimarchi for their invaluable support and
helpful discussion.
Supplementary material is provided
20
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Figure Legends
Figure 1. RITA effects on cell growth. RITA reduces cell viability in A, LAP-35, B,
WE-68, C, TC-71, D, IOR-BRZ71, E, 6647 and F, SK-N-MC. Cells were treated with
RITA at the indicated concentrations for 24h or 48h and then stained with Trypan
blue for immediate count analysis. Cell growth was calculated as number of Trypan
blue negative cells. Percentage of cell viability was obtained by setting 100% the cell
growth of control vehicle treated cells (CTR) at each time point and calculating the
relative cell growth of treated cells. Data are representative of at least 3 different
experiments. G, Cells were plated in 96 well and treated in octuplicate with
increasing concentration of RITA (0.5-1-2-3-6μM) for 24 or 48 hours (*). Cell viability
was assessed by Cell Titer Blue. The dose reported in the graph is the dose able to
reduce cell viability to 50% in comparison to untreated cells.
Figure 2. RITA causes G2 arrest and cell apoptosis. FACS analysis of A, LAP-35,
B, TC-71 and C, SK-N-MC performed at 12h, 24h or 30h after treatment with 3μM
RITA. Cells were grown in the presence or absence of RITA and then stained with PI
for the immediate cell cycle analysis. Each panel reports the percentage of cells in
each cell cycle phase. Sub-G1 region (as indicated) has been independently
evaluated relative to the total population. These analyses are representative of at
least two different experiments. D, TUNEL analysis of indicated cells at 16 or 24 h
after treatment with 3μM RITA. The percentage of apoptotic cells was measured as
TUNEL-positive cells relative to total cells. The data report the mean of three
independent counts.
28
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Figure 3. RITA inhibits anchorage-independent growth. 100.00 cells/60mm dish
were plated in soft agar and 48h after plating, RITA was added at the indicated
concentrations. Photos here reported are representative of 2 independent
experiments performed in duplicate.
Figure 4. RITA downregulates IGF-1R protein levels. A, SK-N-MC and B, TC-71
cells were incubated with RITA 1 or 3μM for 16 or 24 hours. Whole-cells extracts
were prepared and analyzed by western blot (WB) for the indicated proteins. Actin
was used as loading control. C and D, qReal-time PCR of IGF-1R in SK-N-MC and
TC-71 cells respectively, treated as in A and B. E, SK-N-MC were incubated with
3μM RITA and after 14 hours 10 μM MG132 was added. After additional 4 hours,
cells were collected for WB analysis. F, TC-71 were incubated with 3μM RITA and
after 20h, 15 μM MG132 was added. After additional 4 hours, cells were collected for
WB analysis.
Figure 5. MDM2 is involved in RITA-mediated IGF-1R degradation. A, SK-N-MC
and TC-71 cells were transiently transfected with siMDM2 or CTR oligos (siCTR).
After 10 hours, interfered cells were treated with 3μM RITA and after additional 24
hours collected. Whole-cells extracts were prepared and analyzed by WB for the
indicated proteins. B, Cell death was assessed by FACS analysis of Annexin positive
cells (upper panel). Lower panel reports the WB analysis of a fraction of cells
subjected to FACS analysis. C, SK-N-MC and D, TC-71 cells were treated with 3μM
RITA for 24 hours or with 10μg/ml -amanitin for 27 hours or both. Whole-cells
extracts were prepared and analyzed by WB for the indicated proteins. E, SK-N-MC
and F, TC-71 cells were treated with RITA at the indicated concentration or treated
29
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with vehicle (CTR) and after 4 or 8 hours whole-cells extracts collected. MDM2
coimmunocomplexes were analyzed after immunoprecipitation of 800μg of WCE
with α-MDM2 2A10-Ab1antibodies. WCEs indicates analysis of 1/25 of whole cell
extracts.
Figure 6. RITA-sensitivity is affected by MDM2 function. A WB analysis of SK-N-MC
cells treated with vehicle, 3μM RITA for 22 hours or 3μM RITA plus 0,5g/ml
sempervirine added 4 hours before cell lysate collection; B, percentage of cell death
after treatment of indicated cells as in A, C, WB analysis of TC-71 cells treated with
vehicle, 3μM RITA for 24 hours or 3μM RITA plus 0,5g/ml sempervirine added 8
hours before cell lysate collection; D, percentage of cell death after treatment of
indicated cells as in C. The percentage of cell death was calculated as number of
dead /total cell number. Columns, average of three independent experiments. Bars,
SD; E, WB analysis of SK-N-MC stably transfected with pcDNA 3.1 (CTR), wt-MDM2
or MDM2-C438L. Cells were treated with 3μM RITA and after 15 hours collected for
WB analysis of indicated proteins. F, cell death of previously indicated SK-N-MC
populations treated as in E. Cell death was calculated as fold of increase of cell
death of RITA treated cells/untreated cells. Columns, average of three independent
experiments. Bars, SD
30
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IGF-1R/MDM2 relationship confers enhanced sensitivity to RITA
in Ewing's sarcoma cells
Giusy Di Conza, Marianna Buttarelli, Olimpia Monti, et al.
Mol Cancer Ther Published OnlineFirst March 28, 2012.
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