alpha-interferon/retinoic acid combination inhibits growth ... · 2/4/2012 · fax:...

Alpha-interferon/retinoic acid combination inhibits growth and promotes

apoptosis in mantle cell lymphoma through Akt-dependent modulation of

critical targets

Jessica Dal Col,1 Katy Mastorci,1 Damiana Antonia Faè,1 Elena Muraro,1 Debora Martorelli,1

Giorgio Inghirami,2 and Riccardo Dolcetti.1

1Cancer Bio-Immunotherapy Unit, Dept. of Medical Oncology, Centro di Riferimento Oncologico,

IRCCS - National Cancer Institute, Aviano (PN), Italy. 2Department of Pathology and CeRMS,

University of Torino, Torino, Italy.

Running title: Effects of RA/alpha-IFN in mantle cell lymphoma

Keywords: Mantle cell lymphoma; Akt; cyclin D1; retinoids; interferon; Noxa

Corresponding author: Riccardo Dolcetti, Cancer Bio-Immunotherapy Unit, C.R.O. - IRCCS,

National Cancer Institute, Via F. Gallini 2, 33081 - Aviano (PN), Italy. Phone: 39-0434-659660;

Fax: 39-0434-659196; E-mail: [email protected]

Conflict-of-interest disclosure: The authors declare no competing financial interests

Word count: Abstract: 199; Text 5050. Tables: 1; Figures: 6

on April 16, 2021. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 6, 2012; DOI: 10.1158/0008-5472.CAN-11-2505

http://cancerres.aacrjournals.org/

Effects of RA/alpha-IFN in mantle cell lymphoma

2

Abstract

Mantle cell lymphoma (MCL) is characterized by a profound deregulation of the mechanisms

controlling cell cycle progression and survival. We herein demonstrate that the combination of 9-

cis-retinoic acid (RA) and interferon-alpha (IFN-α) induces marked anti-proliferative and pro-

apoptotic effects in MCL cells through the modulation of critical targets. Particularly, IFN-α

enhances RA-mediated G0/G1 cell accumulation by down-regulating cyclin D1 and increasing

p27Kip1 and p21WAF1/Cip1 protein levels. Furthermore, RA/IFN-α combination also induces apoptosis

by triggering both caspase-8 and caspase-9 resulting in Bax and Bak activation. In particular,

RA/IFN-α treatment down-regulates the anti-apoptotic Bcl-xL and Bfl-1 proteins and up-regulates

the pro-apoptotic BH3-only Noxa protein. Sequestration of Mcl-1 and Bfl-1 by up-regulated Noxa

results in the activation of Bid, and the consequent induction of apoptosis is inhibited by Noxa

silencing. Noxa up-regulation is associated with nuclear translocation of the FOXO3a transcription

factor as consequence of RA/IFN-α-induced Akt inhibition. Pharmacological suppression of Akt,

but not of TORC1, increases Noxa protein levels and down-regulates Bfl-1 protein supporting the

conclusion that the inhibition of the Akt pathway, the resulting FOXO3a activation and Noxa up-

regulation are critical molecular mechanisms underlying RA/IFN-α-dependent MCL cell apoptosis.

These results support the potential therapeutic value of RA/IFN-α combination in MCL

management.





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Introduction

MCL is a distinct CD5+ B-cell non-Hodgkin’s lymphoma (NHL) characterized by the

t(11;14)(q13;q32) translocation that leads to over-expression of cyclin D1 and subsequent cyclin

D/Rb pathway deregulation (1, 2). Nevertheless, other genetic changes such as c-myc over-

expression, loss of the ATM gene, low levels of the CDK inhibitor p27Kip1, and p53 deregulation (3-

5) are probably required for MCL development. Gene expression profiling of MCL cells have

recently demonstrated the dysregulation of several genes/proteins involved in NF-κB, PI3-K/Akt,

and mTOR signalling pathways, which are all constitutively activated in MCL cells (6-9). These

findings suggest that a profound alteration of pathways regulating cell survival is likely responsible

for the aggressive clinical behaviour of MCL, which usually show poor response to conventional

therapeutic regimens and a very unfavourable prognosis, even when the disease is treated with high-

dose therapy and hematopoietic stem cell transplantation (1). Therefore, new therapeutic options

need to be explored in patients affected by these lymphomas.

We have recently demonstrated that 9-cis-RA induces marked anti-proliferative responses in

MCL cells by interfering with G1/S transition through the inhibition of ubiquitination and

proteasome-dependent degradation of p27Kip1. RA-up-regulated p27Kip1 binds to cyclin D1/CDK4

complexes, resulting in decreased CDK4 kinase activity and pRb hypophosphorylation. Notably,

RA also inhibited the growth-promoting effect induced in primary MCL cells by micro-

environmental factors such as CD40 triggering and IL-4 (10). These findings make these

compounds highly attractive in terms of potential clinical efficacy in this setting. Since RA alone

does not induce relevant apoptotic responses in MCLs, we investigated the ability of RA to

cooperate with interferons (IFNs) in inducing apoptosis in MCL cells. Indeed, RA/IFN combination

was previously shown to exert synergistic anti-proliferative and pro-apoptotic effects in different

cancer cell systems (11, 12). With particular regard to the pro-apoptotic activity, these compounds





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can variably promote both the down-regulation of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and

Mcl-1 (12, 13), often overexpressed in B-NHLs, including MCL (14-18) and/or the induction of

pro-apoptotic proteins, including Bax, Noxa, and XAF1 (12, 19-23). Intriguingly, recent studies

demonstrated a critical role of Noxa/Mcl-1 interaction in regulating MCL cell survival. In fact,

both Noxa-up-regulating and BH3-mimetic drugs were shown to induce significant apoptotic

responses in MCL (24-26). Noxa belongs to the BH3-only subfamily of proteins with pro-apoptotic

activity (27), which may directly or indirectly promote Bax and Bak activation. A crucial event in

this process is the selective interaction with and the consequent inactivation of the pro-survival

members of the Bcl-2 family, which, specifically for Noxa, are represented by Mcl-1 and A1/Bfl-1

(28).

On these grounds, we have investigated the ability of RA/IFN-α treatment to induce apoptotic

responses in MCL cells and, particularly, to shift the critical balance between anti- and pro-

apoptotic regulators in favor of apoptotic machinery activation. Moreover, taking into account our

previous findings indicating that the PI3-K/Akt pathway is critical for MCL cell survival, and that

Akt, but not mTOR, inhibition induces apoptotic responses in MCL (9), we also investigated the

effects of RA/IFN-α treatment on the inherent PI3-K/Akt activation.

Materials and Methods

Patient samples

Four patients with MCL were identified on the basis of morphological, immunophenotypic and

molecular criteria according to W.H.O. lymphoma classification (Table 1). The study was

performed in accordance with protocols approved by the local IRB, and all patients gave their

informed consent. Mononuclear cells were isolated from unicellular suspension obtained from

mechanically minced lymph nodes or spleen. Enriched MCL samples (>70% MCL) were





5

cryopreserved in 10% DMSO until further study. Before use, samples were re-suspended in RPMI

1640 medium (Lonza) containing 10% fetal calf serum (FCS) and antibiotics.

Cell lines

Mino, SP-53, and Jeko-1 cell lines were generously contributed by Dr. Raymond Lai, Canada.

Granta 519 was purchased by DSMZ (Braunschweig, Germany) (29). Cell lines were authenticated

by fingerprinting (Power Plex 1.2, Promega) in January 2011. Cells were cultured as previously

described (10).

Proliferation assay, apoptosis and caspase activity detection.

Cell proliferation was evaluated by 3H-thymidine uptake (10) and apoptosis by Annexin

V/Propidium iodide (PI) stains and/or by active/cleaved caspase 3. Caspase 8, 9, and 3 activation

was evaluated using a fluorimetric commercial kit (Immunochemistry Technologies). All flow

cytometric analyses were performed on a FC500 flow cytometer (Beckman Coulter).

Antibodies and reagents

Bcl-2 antibody was from Calbiochem, Bcl-xL, A1/Bfl-1, Mcl-1, Bax, Bid (Rabbit), Puma, Phospho-

Akt (Ser473), phospho-S6rp (Ser235/236), phospho-GSK-3α/β (S21/9), phospho-FOXO3a

(S318/321), Akt, and active-caspase 3 (D175) antibodies were from Cell Signaling Technology;

Noxa, Mcl-1 (Y37), Bcl-2A1 (EP517Y) antibodies were from Abcam, p27Kip1 and p21Waf1 from BD

Transduction Laboratories, PARP (F2), cyclin D1 (DCS-6), p57Kip2, p45Skp2 and Cks1 from Santa

Cruz Biotechnology, Bid (Mouse) antibody from SoutherBiothec, and Bak (NT) and FOXO3a

antibodies from Millipore. Vital nuclear dye DRAQ5, SH5 (Akt inhibitor), and LY294002 were

purchased from Alexis Biochemicals, rapamycin, G418. 9-cis-RA (Sigma) and IFN-α (IntronA, SP

Europe) were used at 1 μM and 1000U/ml, respectively. Ro 406055 (RARα agonist) was kindly





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provided by Dr. W. Bollag ( Basel, Switzerland) and SR11237 (RXR agonist) was a kind gift of Dr.

H. Gronemeyer ( Strasbourg, France).

Extracts preparation, immunoprecipitation and immunoblotting

Whole cell lysate extracts and immunoprecipitated samples were prepared as previously described

(10). Proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membranes.

Immunoblotting was performed using the enhanced chemiluminescence plus detection system

(PerkinElmer).

Intracellular flow cytometry

Cells (106) were fixed with 2% of paraformaldehyde, permeabilized with 500 μL of cold methanol

and incubated with the primary antibodies for 1 hour at room temperature. After two washes with

PBS/0.5% BSA, cells were incubated for 30 minutes in ice with PE-anti-Rabbit secondary antibody

and analyzed by flow cytometry.

Bid-Mcl-1 and Bid-A1/Bfl-1 co-localization and FOXO3a nuclear internalization

Cells (106 per sample) were fixed, permeabilized and labelled as described above. Samples were

acquired with the ImageStreamX (Amnis) using the INSPIRE software. For co-localization

experiments, samples were labelled with Mcl-1 or A1/Bfl-1 antibodies and Bid antibody and

DRAQ5. Then, cells double-positive for Mcl-1 and Bid or A1/Bfl-1 and Bid were selected and

compared using an algorithm of the IDEAS software which calculates the specificity and the degree

of the fluorescence signals co-localization through the Similarity Bright Details Score (SBDS) (see

supplementary methods) (30). For the analysis of FOXO3a nuclear internalization, samples were

labelled with an antibody against FOXO3a (1:100) at 4°C overnight. Then, cells were stained with

the PE-anti Rabbit secondary antibody and DRAQ5. Using an algorithm of the IDEAS software, the

Similarity Score (SS) (31) between FOXO3a and DRAQ5 staining was calculated for each sample.





7

Noxa silencing

Two different shRNA PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1) constructs

were obtained by sub-cloning the double-stranded 64-mer oligonucleotide containing the PMAIP1

target sequences (A: 5�-AAACTGAACTTCCGGCAG-3�; B: 5�-TCTGATATCCAAACTCT-3�)

into the pSUPER.retro.neo+GFP vector (pSUPER; OligoEngine). Infectious supernatant from

pSUPER and pSUPER.retro-shPMAIP1 retrovirally transfected Phoenix cells were collected after

48 hours and used for three cycles of infections (32). Upon infection, cells were selected with G418

(1 mg/mL) and the infection efficiency was checked by flow cytometry (97% GFP-positive cells).

Different clones of infected cells were then obtained after seeding cells in 96-well plate at an initial

density of 25 cells/well in 200 μL of medium supplemented with G418.





8

Results

Interferon-α significantly enhances the anti-proliferative activity exerted by retinoic acid in

MCL cells.

9-cis-RA is the isomer with the strongest antiproliferative activity against MCL cells (10).

Treatment of SP53 cells with 9-cis-RA (1μM) in combination with IFN-α (1000U/mL) for 2, 4, and

7 days resulted in a more pronounced inhibition of MCL cell proliferation as compared with cells

treated with RA alone, showing an additive effect (Figure 1A). Consistently, 9-cis-RA/IFN-α

combination increased the number of MCL cells in G0/G1 (not shown), suggesting a likely

involvement of regulators of G1 to S phase transition. Immunoblotting analysis showed that IFN-�

enhances the up-regulation of the p27Kip1 protein induced by 9-cis-RA as a result of a more

pronounced inhibition of p45Skp2 and Cks1, two SCFSkp2 complex components that are required for

proteasome-dependent p27Kip1 degradation (Figure 1B). Furthermore, 9-cis-RA/IFN-α combination

also induces p21WAF1/Cip1 up-regulation, whereas the levels of p57Kip2 were unaffected (Figure 1B).

Notably, p21WAF1/Cip1 up-regulation was detected in MCL cell lines with either wild-type p53

(Granta-519, SP53) or mutated p53 (Jeko-1,Mino), excluding a p53-only-dependent effect (Figure

1C). Quantitative real-time PCR experiments showed no or low effects on the mRNA levels of the

factors studied (Supplemental Figure1), consistently with a mainly post-translational modulation

induced by the treatment. More interestingly, after 5 days of 9-cis-RA/IFN-α treatment , SP53 and

Mino cells showed a marked down-regulation of cyclin D1 protein levels, an effect that was not

observed in cells exposed to each drug alone (Figure 1D). These findings indicate that IFN-α

enhances the anti-proliferative activity exerted by RA in MCL cells by decreasing the protein levels

of cyclin D1 and further up-regulating p27Kip1 and p21WAF1/Cip1.





9

9-cis-RA sensitizes MCL cells to the pro-apoptotic effect of IFN-α through both RARα and

RXRs.

We previously demonstrated that RA alone does not induce relevant apoptotic responses in MCL

(10). Given the ability of IFN-α to cooperate with RA in inhibiting MCL cell growth, we also

explored the possible induction of pro-apoptotic effects. To this end, SP53 and Mino cells were

treated with each drug alone or in combination for 3 and 5 days, and apoptosis was evaluated using

Annexin V/PI staining. RA/IFN-α combination induced more pronounced apoptotic effects in both

MCL cell lines as compared with single treatments (Figure 2A). In particular, sequential treatment

experiments indicated that a 24-hour pre-treatment with 9-cis-RA sensitizes MCL cells to the pro-

apoptotic effect of IFN-α, whereas the reverse induced only modest effects (Figure 2B).

Considering that the pleiotropic effects of retinoids are mainly mediated by two classes of nuclear

receptors, the RARs and RXRs (33, 34), and that 9-cis-RA is a pan-RAR and -RXR agonist, the

relative contribution of distinct retinoic receptors was assessed using synthetic selective agonists for

RARα (Ro 40-6055) and RXR (SR11237). As shown in Figure 2C, both RARα and RXR agonists

sensitize MCL cells to IFN-α-induced apoptosis, although the relative contribution of RARα is

markedly higher. These findings are clinically relevant, since RARα agonists are associated with

less pronounced side effects than the pan-RAR/RXR agonist 9-cis-RA when used in vivo.

9-cis-RA/IFN-α-dependent MCL cell apoptosis involves multiple caspase activation and

modulation of anti- and pro-apoptotic proteins.

The involvement of initiator and effectors caspases in 9-cis-RA/IFN-α-induced apoptosis was

investigated in SP53 and Mino cells using specific fluorimetric caspase assays. Time-course

experiments showed that both caspases 8 and 9 are activated, almost simultaneously, after 36 hours

of 9-cis-RA/IFN-α treatment; nevertheless, at later time points, their activity differs (Figure 3A). In

fact, caspase 8 activation increases progressively, whereas the caspase 9 activity, after the initial





10

triggering, increases more slowly, showing a down-modulation at 60 hours and a subsequent

increase at 72 hours (Figure 3A). Given the involvement of the mitochondrial pathway, the role of

Bak and Bax proteins was analyzed by flow cytometry using antibodies specific for the N-terminal

domains of these proteins that are exposed only upon Bak and Bax activation. As shown in Figure

3B, the conformational changes of Bak and Bax were detectable only in cells exposed to 9-cis-

RA/IFN-α (3 and 5 days) concomitantly with the presence of cleaved caspase 3, as an indicator of

ongoing apoptosis.

Given that the interactions between pro-apoptotic and anti-apoptotic members of the Bcl-2

superfamily are essential for mitochondrial integrity, the expression levels of several proteins of the

Bcl-2 and BH3-only families were analyzed by immunoblotting in SP53 cells treated with 9-cis-

RA, IFN-� or their combination. While no change in Bcl-2 and Mcl-1 expression levels was

observed in the different conditions, a marked down-regulation of the Bcl-xL and A1/Bfl-1 anti-

apoptotic proteins was induced by 9-cis-RA/IFN-� (Figure 3C). Although this treatment induced

conformational changes indicating Bax and Bak activation, the expression levels of these proteins in

untreated and treated cells were comparable. Analysis of BH3-only proteins disclosed a marked

increase of Noxa protein levels, especially in 9-cis-RA/IFN-�-treated cells, whereas the expression

of Puma did not appreciably change. The levels of the full-length Bid protein decreased

significantly as likely consequence of its activation by caspase-dependent cleavage (Figure 3C).

Notably, 9-cis-RA/IFN-α treatment was also able to significantly up-regulate Noxa concomitantly

with enhanced caspase 3 activation in all 4 primary MCL cultures investigated (Figure 3D). This

effect was apparently specific for lymphoma cells, since 9-cis-RA/IFN-α did not up-regulate Noxa,

nor exerted any pro-apoptotic activity in normal B lymphocytes obtained from 2 different donors

(not shown). Taken together, these results indicate that 9-cis-RA/IFN-α combination triggers both

mitochondrial/intrinsic and death receptor/extrinsic apoptotic pathways and promotes the shift of

the critical balance between anti- and pro-apoptotic proteins in favor of apoptotic machinery

activation.





11

Noxa is a critical mediator of 9-cis-RA/IFN-α-induced apoptosis.

The BH3-only protein Noxa was shown to be one of the critical players of the apoptotic responses

induced in MCL cells by different drugs, such as bortezomib and the MDM2 inhibitor nutlin 3 (24,

25). Interestingly, immunoblotting experiments demonstrated that Noxa up-regulation was induced

by 9-cis-RA and at higher levels by 9-cis-RA/IFN-α co-treatment. These effects are probably

mediated by transcriptional regulation, as shown by microarray-based expression profiling carried

out in SP53 cells (not shown). To assess the role of Noxa in 9-cis-RA/IFN-α-dependent apoptosis,

we knocked-down Noxa expression by retrovirally infecting Mino cells with a pSUPER.retro-

GFP/neo vector (pSUPER) carrying two different short hairpin RNAs (shRNAs), corresponding to

nucleotides 106-124 and 132-148 of PMAIP1 consensus coding sequence, and GFP

(pSUPER.retro-shPMAIP1). Silencing of Noxa prevents the 9-cis-RA/IFN-α-induced up-regulation

of the protein and consequently reduces the extent of treatment-dependent apoptosis in Mino cells

assessed by cleaved caspase 3 and PARP (Figure 4A). Considering the ability of Noxa to

specifically bind and consequently inactivate the anti-apoptotic Mcl-1 and A1/Bfl-1 proteins, the

interactions between Noxa and these two Bcl-2 family members were investigated. Most of 9-cis-

RA/IFN-α-induced Noxa co-immunoprecipitated with Mcl-1, the remaining amount being

associated to A1/Bfl-1 (Figure 4B). Furthermore, the sequestration of Mcl-1 by up-regulated Noxa

results in the displacement of the full-length Bid protein from Bid-Mcl-1 complexes (Figure 4C),

allowing thus the consequent Bid activation through enzyme-cleavage. The resulting truncated-Bid

may thus directly contributes to the activation of the Bak and Bax apoptotic effectors. Taking

advantage from multi-spectral imaging flow cytometry, we analyzed the co-localization between

Bid and Mcl-1 and A1/Bfl-1 also in vivo. To this end, we set up a protocol in which the cells were

stained with specific antibodies to Mcl-1 or A1/Bfl-1 and Bid proteins and then the Bid-Mcl-1 and

Bid-A1/Bfl-1 co-localization was analyzed only in double-positive live cells. As shown in Figures

4D and 4E, the SBDS detected in untreated samples was 2.48 ± 0.42 for Bid-Mcl-1 and 2.45 ± 0.49

for Bid-A1/Bfl-1 and in both cases the score significantly decreased when the cells were treated for





12

3 days with RA/IFN-α. Moreover, in treated samples, the percentage of cells showing a significant

co-localization of the two proteins (with SBDS � 2,25) was reduced from 72% to 19.2% for Bid-

Mcl-1, and from 66.4% to 17.2% for Bid-A1/Bfl-1. These results indicate that the treatment induces

the displacement of Bid from Bid-Mcl-1 and Bid-A1/Bfl-1 complexes through Noxa up-regulation

and this event precedes and promotes the apoptotic process.

9-cis-RA/IFN-α-dependent MCL cell apoptosis is mediated by inhibition of the PI3-K/Akt

pathway

We and others recently demonstrated that the PI3-K/Akt pathway is constitutively activated in MCL

resulting in a strong deregulation of both cell proliferation and survival (8, 9). Particularly,

pharmacologic inhibition of PI3-K or Akt induced significant levels of apoptosis in both established

cell lines and primary MCL cultures, whereas the TORC1 inhibitor rapamycin induced no or only

modest effects on cell survival (9). On these grounds, we investigated the ability of 9-cis-RA/IFN-α

to interfere with the constitutive activation of these kinases. In particular, SP53 cells were treated

for 3 days with 9-cis-RA, IFN-α or their combination and analyzed for the presence of the

phosphorylated form of Akt and of its substrates GSK-3β and FOXO3a. In addition, TORC1

activation was investigated by analyzing the phosphorylation status of one of its main substrates,

the S6 ribosomal protein. Interestingly, 9-cis-RA inhibited Akt and mTOR activation, as shown by

the down-regulation of phospho-(S473)-Akt, of its substrates phospho-(Ser21)-GSK-3β and

phospho-(Ser318/321)-FOXO3a, and of phospho-(S235/236)-S6RP, and more importantly, IFN-α

significantly enhanced the inhibitory effects exerted by RA on these kinases (Figure 5A). In

particular, 9-cis-RA/IFN-α-induced Akt inhibition was associated with Noxa up-regulation and the

decrease of A1/Bfl-1 protein levels (Figure 5B). Furthermore, the PI3-k/Akt inhibitor LY294002

induced a marked up-regulation of Noxa and a complete depletion of A1/Bfl-1, whereas rapamycin

did not affect the levels of these proteins (Figure 5C), consistently with our previous demonstration





13

that rapamycin is unable to induce apoptosis in MCL cells (9). Notably, Noxa up-regulation induced

by the treatment was observed in MCL cell lines carrying either wild-type (SP53) or mutant p53

(Jeko-1) (Figure 5B), supporting the hypothesis that a different transcription factor is involved in

this phenomenon. Interestingly, FOXO3a activates the transcription of several genes, including

PMAIP1, which encodes for the Noxa protein (35). FOXO3a transcriptional activity is regulated by

the control of its intracellular localization through the phosphorylation/dephosphorylation of

different Serine/Threonine residues. In particular, Akt-dependent phosphorylation on Thr32, Ser

318/321, and Ser253 abolishes its nuclear translocation. Given the ability of 9-cis-RA/IFN-α to

inhibit Akt-dependent FOXO3a phosphorylation, using multispectral imaging flow cytometry, we

evaluated FOXO3a intracellular localization after 48 hour exposure to 9-cis-RA/IFN-α, SH5 (10

μM), or rapamycin (0.1 μM). As shown in Figure 5D, FOXO3a protein is clearly retained in the

cytoplasm of untreated cells, whereas in 9-cis-RA/IFN-α- and SH5-treated cells, the protein is also

detectable within the nucleus in 58.7% and 64.1% of cells, respectively. In contrast, rapamycin did

not affect FOXO3a intracellular localization, confirming that this transcription factor is Akt- but not

TORC1-dependent. Notably, the analysis were performed excluding apoptotic cells, given that

FOXO3a nuclear internalization and consequent Noxa up-regulation are two events occurring in the

first steps of the apoptotic process. These results are consistent with a role of FOXO3a as a

molecular mediator of the 9-cis-RA/IFN-α-induced Noxa up-regulation. Moreover, these findings

indicate that the 9-cis-RA/IFN-α combination induces MCL cell apoptosis through inhibition of the

inherent Akt activation, and the consequent increment of FOXO3a activity and, in turn, of Noxa

expression (Figure 6AB).





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Discussion

As our understanding of the biology of MCL advances, novel agents rationally designed to

target the key pathogenic mechanisms of MCL, such as cyclin D1, cyclin/CDK inhibitors, pro-

apoptotic proteins, mTOR, and proteasome, continue to emerge. Previously, we demonstrated that

the constitutive PI3-K/Akt/mTOR activation contributes to the stability of cyclin D1 and p27Kip1 in

MCL cells (9, 36), suggesting that this signalling pathway may be a crucial therapeutic target.

While the therapeutic potential of TORC1 inhibitors is being extensively studied in patients with

relapsed or refractory MCL (37), specific inhibitors of the upstream kinase Akt are still under

evaluation in phase I clinical trials (38). Nevertheless, the Akt kinase may constitute a more

effective target in MCL compared to TORC1, since Akt inhibition not only reduces proliferation,

but also induces significant apoptotic responses (9).

Herein, we demonstrate that RA/IFN-α co-treatment has significant effects on both

proliferation and cell survival by affecting key molecular targets of MCL, such as cyclin D1,

p27Kip1, Akt, and Noxa (Figure 6AB). In particular, IFN-α enhances the anti-proliferative activity

exerted by 9-cis-RA by inducing a down-regulation of cyclin D1, which is strongly over-expressed

in most MCLs. Nevertheless, the observation that cyclin D1 over-expression alone is not sufficient

for MCL development and that its down-regulation has only limited effects on MCL cell

proliferation and survival (39) indicates that additional targets should be affected in order to obtain

clinically relevant therapeutic efficacy. Intriguingly, the anti-proliferative activity of RA/IFN-α

involves also the increased expression of the p27Kip1 and p21WAF1/Cip1 cell cycle inhibitors as a

consequence of enhanced protein stability. This is particularly relevant for the p27Kip1 protein,

which shows an abnormally short half-life in most of MCLs (5). Furthermore, p21WAF1/Cip1 up-

regulation is induced irrespective of the p53 mutational status of the cells, thus excluding a p53-

only-dependent effect and suggesting that this drug combination could be efficient also in cases

showing deregulations in this critical pathway. This is particularly intriguing in the light of the

observation that ≈25% of MCLs shows a deregulated p53 (40, 41), a characteristic that could





15

promote the resistance to novel drugs targeting the HDM2/p53 pathway, such as the MDM2

antagonist Nutlin-3 (25) and MI-63 (42).

More relevant in a therapeutic perspective is the demonstration that, unlike RA alone (10), the

RA/IFN-α combination induces significant levels of apoptosis in both established cell lines and

primary MCL cultures. The exposure of MCL cells to 9-cis-RA for 24 hours and the following

addition of IFN-α indicate this sequential treatment as the most effective combination, providing

thus the rationale for the design of appropriate treatment schedules. Moreover, we also show that 9-

cis-RA ability to sensitize MCL cells to IFN-α-dependent pro-apoptotic effect involves both RARα

and RXRs. These findings are of potential clinical relevance, since RARα agonists are associated

with less pronounced side effects than the pan-RAR/RXR agonist 9-cis-RA when used in vivo.

RA/IFN-α combination triggers both the death receptor/extrinsic and the mitochondrial/intrinsic

apoptotic pathways and promotes the activation of the pro-apoptotic effectors Bak and Bax.

Moreover, RA/IFN-α treatment induced up-regulation of Noxa and the concomitant Mcl-1 and

A1/Bfl-1 inactivation as a result of protein-protein interactions. (24-26). In contrast to other

compounds inducing Noxa-dependent MCL cell apoptosis, RA/IFN-α combination does not

increase Mcl-1 protein levels, and even down-regulates A1/Bfl-1. Intriguingly, our results

demonstrate that, under normal conditions, both Mcl-1 and A1/Bfl-1 can be bound to the full-length

form of Bid, thus preventing its cleavage and repressing its activation. MCL cells exposure to

RA/IFN-α combination relieves this repression through a competitive inhibition exerted by Noxa,

which favors Bid displacement from pro-apoptotic/anti-apoptotic proteins complexes and its

subsequent activation.

Notably, we took advantage of multispectral imaging flow cytometry to selectively analyze

Mcl-1-Bid and A1/Bfl-1-Bid co-localization in cells with morphometric features of early apoptosis,

a distinction that is not usually feasible in co-immunoprecipitation experiments. This

methodological approach is particularly relevant if we consider that the binding of Mcl-1 or A1/Bfl-





16

1 to Bid abolishes its pro-apoptotic activity and that RA/IFN-�-induced Bid displacement from

these complexes is an early event in the activation of apoptotic machinery.

Noxa was initially identified as transcriptional target of p53 (43), but now it is well known

that it could be induced also by other factors, as it occurs in neuronal cells where it is induced by

the activation of the FOXO3a transcription factor, a downstream target of the Akt kinase (35). We

previously demonstrated that the PI3-K/Akt pathway is crucial for MCL cells survival, and that the

inhibition of Akt, but not of TORC1, induced apoptosis in MCL cells, suggesting that an Akt

substrate different from TORC1 is likely involved in mediating these effects. The results presented

herein support the conclusion that the inhibition of the Akt pathway by RA/IFN-α, resulting in

FOXO3a de-phosphorylation/activation and its subsequent nuclear internalization followed by

Noxa up-regulation, is one of the main molecular mechanisms underlying RA/IFN-α-dependent

MCL cell death.

Our findings are also consistent with a relevant role of RA/IFN-α-induced Akt inhibition in

mediating MCL cell growth inhibition (9, 36). In particular, this drugs combination mimics the

effects induced in MCL cells by pharmacologic inhibition of Akt, which results in cyclin D1 down-

regulation and p27Kip1 over-expression.

Overall, the results of the present study demonstrate how the combination 9-cis-RA/IFN-α

affects critical molecular targets of MCL thus providing a strong rationale for the clinical

application of known and relatively cheap drugs in the management of this aggressive and poorly

responsive lymphoma.





17

Grant support

The study was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro

(contract 10301 to R.D. and to Special Program Molecular Clinical Oncology 5-per-mille to G.I.)

and Fondazione Federica per la Vita.





18

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21

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22

Table 1. MCL cases

Case No. Sex/Age Malignant cells (%)

Type Cyclin D1 p27Kip1 Sample analyzed

MCL4 F/72 95% Classical + NA Lymph node biopsy

MCL5 M/50 86% Classical + low Lymph node biopsy

MCL6 M/64 95% Classical + low Lymph node biopsy

MCL7 M/72 96% Classical + low Spleen

biopsy

The expression of cyclin D1 and p27Kip1 proteins was detected by immunohistochemistry. (NA: not

available).





23

Figure legends

Figure 1.

A) IFN-α enhances the anti-proliferative activity exerted by 9-cis-RA in MCL cells. DNA synthesis

was assessed in SP53 cells by [3H] thymidine incorporation after 6 hours. Points, mean from

triplicate wells; bars, SD. The results are representative of one of three experiments. B) RA/IFN-α

up-regulate p27Kip1 and p21WAF1/Cip1 in SP53 cells (3 days) C) RA/IFN-α-induced p21WAF1/Cip1 up-

regulation is not a p53-only-dependent event, being detected in MCL cell lines with wild-type

(SP53, Granta 519) and mutated p53 (Mino, Jeko-1) (3 days). D) RA/IFN-α combination down-

regulates cyclin D1 expression in SP53 and Mino cells (5 days).

Figure 2.

A) Pro-apoptotic effects induced by RA/IFN-α in SP53 and Mino cells. Data are representative of

one of three independent experiments. B) 9-cis-RA sensitizes MCL cells to the apoptosis triggered

by IFN-α. SP53 cells were sequentially treated with 9-cis-RA (or IFN-α) for 24 hours and then

IFN-α (or 9-cis-RA) was added. Apoptosis was evaluated after 3 days. C) RA/IFN-α-dependent

apoptosis involves both RARα and RXR. SP53 cells were cultured with 1 μM 9-cis-RA, 1 μM RO

40-6055 (RARα agonist), or 1 μM SR11237 (RXR agonist) in the absence or presence of IFN-α for

3 days. Results in B) and C) are reported as percentage of increment relative to the control

(untreated sample). Bars, mean from three independent experiments; error bars, SD.

Figure 3.

A) RA/IFN-α-induced apoptosis is caspase-dependent. Mino cells were cultured in absence or

presence of 9-cis-RA/IFN-α and the activation of caspases 8, 9, and 3 was evaluated at the

indicated time points. B) RA/IFN-α treatment induces Bak and Bax activation. Mino and SP53 cells

were treated or not with 9-cis-RA/IFN-α for 3 and 5 days and stained with antibodies specific for





24

the N-terminus domain of Bak or Bax, and for active caspase 3. Data reported in A) and B) are

representative of one of three independent experiments. C) RA/IFN-α treatment modulates Bcl-2

family members and BH3-only proteins expression. Total-cell lysates (100 μg) 3 days treatment. D)

RA/IFN-α combination induces Noxa up-regulation associated with caspase 3 activation in primary

MCL cultures. Purified primary lymphoma cells from 4 different MCL patients were treated with 9-

cis-RA/IFN-α for 48 hours. Total-cell lysates (30 μg) were analyzed by immunoblotting for Noxa

and cleaved caspase 3. The extent of RA/IFN-α-induced Noxa up-regulation is indicated in

arbitrary units assigning to each untreated sample the value of 1.00.

Figure 4.

A) Noxa knock-down reduces RA/IFN-α-dependent apoptosis. Mino cells infected with empty

vector (pSuper) and 2 different clones (CL.a and CL.b) of cells infected with vector containing

shPMAIP1 sequence A and 1 clone (CL.c) with the sequence B (see methods) were treated with 9-

cis-RA/IFN-α for 3 days. Total-cell lysates (50 μg) were analyzed by immunoblotting for Noxa,

cleaved caspase 3, and PARP. B) RA/IFN-α combination promotes the formation of Noxa-Mcl-1

and Noxa-A1/Bfl-1 complexes. SP53 cells were cultured in absence or presence of 9-cis-RA/IFN-α

for 3 days. Mcl-1 (left) and A1/Bfl-1 (right) were immunoprecipitated from 500 μg of total proteins.

Immunoprecipitated (IP; bound) and non-immunoprecipitated (unbound) fractions were analyzed

by immunoblotting for Noxa, Mcl-1, and A1/Bfl-1 proteins. C) RA/IFN-α-induced Noxa favors Bid

displacement from Bid-Mcl-1 complexes in SP53 cells (3 days). Mcl-1 was immunoprecipitated

from 500 μg of total proteins followed by immunoblotting with antibodies against Noxa, full-length

Bid, and Mcl-1. Data depicted in B) and C) are representative of three independent experiments. D)

RA/IFN-α treatment inhibits Bid-Mcl-1 and Bid-Bfl-1 interactions in vivo. Mino cells were cultured

in absence (untreated) or presence of 9-cis-RA/IFN-α for 3 days, then 106 cells per sample were

labeled with primary antibodies against Bid (1:50) and Mcl-1 (1:100) or E) A1/Bfl-1 (1:75). The





25

vital nuclear dye DRAQ5 was added to each sample. 20x103 cells were acquired with the

ImageStream X and analyzed with a specific algorithm for the SBDS calculation. Data are

representative of one of three independent experiments.

Figure 5.

A) and B) RA/IFN-α combination inhibits the inherent PI3-K/Akt pathway activation in SP53 and

Jeko-1 cells (72 hours). Total-cell lysates, (100 μg) were subjected to immunoblotting using

phosphospecific antibodies. C) Inhibition of Akt, but not of TORC1, is associated with Noxa up-

regulation and A1/Bfl-1 depletion. SP53 cells were untreated (C) or treated with 50 μM LY294002

(LY) or 0.1 μM rapamycin (rapa) for 48 hours and total-cell lysates analyzed for the expression of

phospho-Akt, Noxa, and A1/Bfl-1 proteins. D) RA/IFN-α treatment promotes FOXO3a nuclear

localization. Mino cells were untreated or treated with 9-cis-RA/IFN-α, SH5 (10 μM), or rapamycin

(0.1 μM) for 48 hours and labeled with antibody against FOXO3a (1:100) and the vital nuclear dye

DRAQ5. Cells (20x103) were acquired with ImageStream X and analyzed with the IDEAS software.

FOXO3a nuclear localization was calculated as Similarity Score between FOXO3a and DRAQ5

intensities. Data are representative of one of three independent experiments.

Figure 6.

Anti-proliferative and pro-apoptotic effects induced by RA/IFN-� in MCL cells. A) untreated MCL

cell; B) modulation of critical regulators by RA/IFN-�.




Untreated - + - + IFN-α- - + +

SP53 9-cis-RA

A BSP53

1000

10000

100000

1000000

pora

tion

(cpm

)

UntreatedIFN-a9-cis-RAIFN-a + 9-cis-RA

p27KIP1

(short exposure)

p21CIP1

p27KIP1

IFN α

1

10

100

1000

Thym

idin

e in

corp

p45Skp2

Cks1

p57KIP2

tubulin2 days 4 days 7 days

9-cis-RA- - + +SP53Jeko-1

- - + +- - + +Mino

- - + +Granta 519

tubulin

C

IFN-αp21CIP1

- + - + + +

- + - +

mtp53 wtp53

- - + +- + - +

mtp53

- + - +

wtp53tubulin

mtp53 wtp53mtp53 wtp53

D Mino- - + + - - + +

SP53

- + - + - + - +9-cis-RAIFN-α

tubulin

cyclin D1

Figure 1




SP53untreated IFN-α 9-cis-RA

9-cis-RA+IFN-α

A Minountreated IFN-α 9-cis-RA

9-cis-RA+IFN-α

Ann

exin

-V

10.4%6.9% 4.5% 6.1%8.4% 15.0%12.0% 6.4%

5.5% 2.5% 8.2% 4.8% 13.5% 6.4% 26.3% 13.8% 3.2% 3.3% 6.6% 6.7% 6.0% 3.6% 18.0% 23.3%

4.0% 3.7% 6.2% 6.2% 6.3% 6.8% 11.9% 10.2%

ays

3 da

ys5

da

Propidium iodide

B

%) C

%)

SP53

ntof

apop

tosi

s(% SP53

ntof

apop

tosi

s(%

Incr

eme

Incr

eme

Figure 2




A

ive

ase

8

0.3% 2.5% 5.9% 32.4% 35.1% 78.4%

0.5% 2.3% 5.6% 13.6% 6.2% 55.9%

acti

casp

aac

tive

aspa

se9

43.6%19.6%30.9%7.9%5.9%1.2%

cac

tive

casp

ase

3

12h 24h 36h 48h 60h 72h

B

k-N

T

Mino SP53

Bcl 2- + - +

RA- - + +IFN-α

SP53C

Ba

Bax

-NT Bcl-xl

A1/Bfl-1

Mcl-1

Bcl-2

Cle

aved

Cas

pase

3

3 days 3 days 5 days5 days

D MCL6 MCL7MCL4 MCL5

Bid

Puma

B k

Noxa

NOXA

cleaved casp.3tubulin

D MCL6 MCL7

1.00 1.37 1.00 1.45 1.00 4.22 1.00 2.79

MCL4 MCL5

Bax

Bak

tubulin

- + - + RA+IFN-α- + - +

Figure 3




NOXA

pSupershPMAIP1

CL.1shPMAIP1

CL.2pSuper shPMAIP1CL.3

A

PARP

cleaved casp.3

tubulin- + + - + + - + + - + + - + + IFN-α 1000U/ml

NOXA

C

- + - - + - - + -- - + - - + - - +

- + - - + -- - + - - +

9-cis-RA 1μM9-cis-RA 0.1μM

NOXA

bound unboundIP: A1/Bfl-1

NOXA

bound unboundIP: Mcl-1B

bound unboundIP: Mcl-1

Bid

IgG

NOXA

Mcl-1

A1/Bfl-1IgG

NOXA

- + - + RA+IFN-αIgGMcl-1

NOXA

- + - +

- - + + - - + + - + - + - + - +

RAIFN-α

2614

4

y

72 0%

untreated

ency

D3

y 66 4%

untreated

ncy

E647

3838

235

711

SBDS 2.49 ± 0.42

High Co-localizatiomHigh Co-localizatiomHigh Co-localizatiomHigh Co-localizatiomHigh Co-localizatiomHigh Co-localizatiom

31 420

3

1

2

0

q

72.0%

Nor

mal

ized

Freq

ue

Mcl-1/Bid Bright Detail Similarity Score

High Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localization

1 3 420

1

2q

66.4%

Nor

mal

ized

Freq

uen

Bfl-1/Bid Bright Detail Similarity Score

4911

Ch01 Ch02 Ch03 Ch02/Ch03

1562

1305

SBDS 2.45 ± 0.48SBDS 2.49 ± 0.42

High Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localization

5

4

2

3

qy

19.2%

9-cis-RA+IFN-α

edFr

eque

ncy

High Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localizationHigh Co-localization2

3

4

17.2%

9-cis-RA+IFN-α

edFr

eque

ncy

g y SBDS 2.45 ± 0.48

1 2 30 40

1

2

Mcl-1/Bid Bright Detail Similarity Score

Nor

mal

ize

SBDS 1.92 ± 0.370 1 432

0

1

Nor

mal

ize

Bfl-1/Bid Bright Detail Similarity Score SBDS 2.13 ± 0.42

Figure 4




C LY Rapa

SP53

pAkt

C

Ak

B

- + - + - - + +

SP53 Jeko-1RAIFN-αpAktAkt

- + - + - - + +

pAktAkt

RAIFN-α

A- - + +- + - +

SP53

NOXAA1/Bfl1tubulin

Akt

NOXA A1/Bfl1

Akt

tubulin

pFOXO3aAktpGSK3

pS6rp

GSK3pFOXO3a

S6rptubulin

Untreated

ncyD

5424

1462

6463High SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh Similarity

2

4

3

1

qy

17.5%

orm

aliz

edFr

eque

n

SS -0.58 ± 0.69 -1 0 2 4-3 -2 310

FOXO3a/DRAQ5_Similarity Score

N

3

58 7%

9-cis-RA+IFN-α3

2qy

64 1%

4

3

10 8%

SH5 10μM Rapamycin 0.1μM

Freq

uenc

y

High SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh Similarity

-1 0 2 4-3 -2 310

1

2 58.7%High SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh Similarity

-1 0 2 4-3 -2 310

2

1

q 64.1%

High SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh SimilarityHigh Similarity

-1 0 2 4-3 -2 310

1

210.8%

FOXO3a/DRAQ5_Similarity Score

Nor

mal

ized

F

981

946

35

18

791

751

1251

SS +0.16 ± 0.82

53

SS +0.27 ± 0.30

2570

SS -0.78 ± 0.37

Figure 5




A

B

Figure 6




Published OnlineFirst February 6, 2012.Cancer Res Jessica Dal Col, Katy Mastorci, Damiana Antonia Faè, et al. Akt-dependent modulation of critical targetspromotes apoptosis in mantle cell lymphoma through Alpha-interferon/retinoic acid combination inhibits growth and

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