Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects
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Diffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas accounting for approximately a third of non-Hodgkin lymphomas. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins that promote mitochondria homeostasis and consequently cell survival, but biological functions of cytoplasmic PHBs remain largely unknown in DLBCL.
PHB expression was analyzed in 82 DLBCL biopsies and five DLBCL cell lines by immunohistochemistry (IHC) and Western blotting. Pharmacological inhibition of PHB using the synthetic flavagline FL3 was realized in vitro to gain insight PHB cellular functions. Effects of FL3 on DLBCL cell line viability, apoptosis, C-Raf-ERK–MNK–eIF4E signaling pathway and eIF4F complex formation and activity were evaluated by XTT assay, annexin V-FITC/PI dual staining and Western blotting respectively. Subcutaneous DLBCL xenograft model in SCID mice was also performed to determine in vivo FL3 effect.
As in DLBCL cell lines, PHB1 and PHB2 were expressed in germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. In patient samples, high PHB levels were associated with higher serum LDH (PHB1 and PHB2), IPIaa (PHB2), and Ki-67 (PHB2) expression. Higher PHB1 expression tends to be associated with shorter event-free survival (EFS) in patients, especially in male patients. FL3 induced apoptosis of DLBCL cell lines that was associated with inhibition of the ERK-MNK-eIF4E signaling pathway, including aggressive double/triple-hit DLBCL cell lines. This resulted in altered eIF4F complex formation and activity leading to a reduction of Bcl-2 and c-Myc expression levels. Moreover, FL3 strongly downregulated DLBCL cellular levels of Akt protein and AKT mRNA. FL3 antitumor activity was also confirmed in vivo in a murine xenograft model.
Our data indicate that PHB overexpression is associated with markers of tumor aggressiveness in DLBCL, and that targeting PHBs may be a therapeutic option, notably in aggressive subtypes.
KeywordsDLBCL PHB FL3 ERK signaling Akt Apoptosis
Apoptosis activating factor
B cell lymphoma 2
Caspase Recruiting Domain
Cyclophosphamide, doxorubicin, vincristine, prednisone
Diffuse large B-cell lymphoma
Eukaryotic translation initiation factor 4 A
Extracellular signal–regulated kinase
Glyceraldehyde 3-phosphate dehydrogenase
Germinal center B-cell–like
Horse radish peroxidase
Age-adjusted International Prognostic Index
MAPK-interacting Ser/Thr kinase 1
Mammalian target of rapamycine
Nuclear factor kappa-binding
Peripheral blood mononuclear cells
Phosphatase and tensin homolog
Quantitative reverse-transcription polymerase chain reaction
Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, Prednisone
Reactive lymph nodes
Severe combined immune deficiency
World Health Organization
Sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate
Diffuse large B-cell lymphomas (DLBCLs) are highly aggressive non-Hodgkin lymphomas for which the incidence increases with age (median age at diagnosis is 70 years). Gene expression profiling subdivides DLBCLs into two major subtypes, the germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. Among them, patients with GCB DLBCL have a better prognosis [1, 2]. R-CHOP immunochemotherapy (a combination of anti-CD20 antibody, rituximab, with CHOP chemotherapy) provides a significant survival benefit to most patients, but approximately 15–25% of patients exhibit primary refractory disease and 20–30% relapse after initial response to therapy . Furthermore, double-hit (DHL) and more rarely triple-hit (THL) lymphomas are subgroups of very aggressive lymphomas with both MYC and BCL-2 and/or BCL6 gene rearrangements characterized by a rapidly progressing clinical course that is refractory to treatment and poor outcome after standard R-CHOP therapy. Thus, these groups of patients represent a huge therapeutic challenge [4, 5].
Different mechanisms in each DLBCL subtype can activate the PI3K/ Akt/ mTOR pathway to enhance cellular growth and metabolism in DLBCL . In the GCB subtype, the loss of PTEN protein expression correlates with PI3K/Akt/mTOR activation . In contrast, constitutive phosphorylation of Akt was not related with loss of PTEN in ABC DLBCL. The hallmark of the ABC subgroup of DLBCL is the constitutive activation of the nuclear factor κB (NF-κB), which promotes cell survival, proliferation and inhibition of apoptosis. This is largely due to the constitutive activation of the “CBM” signaling complex (formed by CARD11, BCL10 and MALT1) . New therapeutic inhibitors directly targeting PI3K/Akt/mTOR pathway have been developed to treat notably relapsed/refractory DLBCL, some of them being currently under investigation in clinical trials [6, 9].
Several reviews have described the potential importance of therapies that target protein translation in cancer including DLBCL [10, 11]. The translation initiation factor 4F (eIF4F) complex, an important downstream target of the mTOR pathway, plays a critical role in the regulation of cap-dependent translation of mRNAs that mostly encode proteins involved in cellular growth, angiogenesis, survival, and malignancy (e.g. cyclin D1, c-Myc, VEGF, and Bcl-2) . This complex contains a translation initiation factor 4E (eIF4E), a scaffolding protein eIF4G, and the RNA helicase eIF4A. eIF4E has been implicated in tumorigenesis, including lymphomagenesis, and eIF4E phosphorylation upon MNK1/2 activation is required for its oncogenic role [11, 13].
Flavaglines are natural compounds extracted from medicinal plants of the genus Aglaia that display potent anticancer activities [14, 15]. These compounds trigger apoptosis through various pathways and inhibit the proliferation of tumor cells at low concentrations without toxicity to normal cells [14, 15, 16]. Flavaglines exert their activities notably by binding to prohibitins (PHBs) . Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins mainly located in the mitochondria, nucleus and plasma membrane, that elicit multiple functions according to their cellular localization and cell type. These functions include nuclear transcription, plasma membrane lipid scaffold protein, mitochondrial morphogenesis and apoptosis [18, 19, 20, 21]. Prohibitins have been implicated in cancer progression through regulation of key cell signaling pathways known to induce cell proliferation, resistance and metastasis, including the Ras-mediated c-Raf-MEK-ERK signaling pathway [22, 23]. Binding of flavaglines to PHBs specifically inhibits the PHB-c-Raf interaction, thus inhibiting MEK/ERK/MNK signaling, leading to inhibition of eIF4E phosphorylation. Consequently, flavaglines inhibit the cap-dependent synthesis of many proteins involved in cell division and resistance to apoptosis .
PHB protein complex has been recently demonstrated to be required for mitochondrial homeostasis and cell survival included in normal and malignant B cells [24, 25]. However, the clinical significance of PHB expression in DLBCL remains to be determined. Thus, in the present study, we determined the clinical relevance of PHB1 and PHB2 in 82 DLBCL tumor samples and show their potential importance in DLBCL cell survival and proliferation. Furthermore, we also investigated the PHB-mediated cellular signaling pathways in vitro using the PHB-ligand FL3. Our findings on DLBCL cell lines and DLBCL xenograft murine model show PHB involvement in ERK-MNK-eIF4E and Akt pathways and strongly suggest that targeting PHBs using flavaglines may be a potential therapeutic strategy in DLBCL.
Clinical and immunophenotypical characteristics of DLBCL patients
No. of Patients (%)
48 /34 (59/41)
IPI Age adjusted (IPIaa)
Ann Arbor stage
Serum lactate dehydrogenase (>1x normal)
Median follow-up time, months
Isolation of normal B cells
For PHBs analysis, peripheral B lymphocytes from 4 healthy volunteer donors were obtained from blood samples. Briefly, B cells were enriched from ficoll-Paque-separated peripheral blood mononuclear cells (PBMC) by negative magnetic cell sorting of as previously reported .
Cell culture and viability assay
Two GCB-DLBCL lines (SUDHL4 and SUDHL6 - DSMZ) and three ABC-DLBCL lines (OCI-LY3 and OCI-LY10 given by Pr. Feuillard, UMR CNRS 7276, Limoges University, with the kind agreement of the Louis M. Staudt, National Cancer Institute, USA- and U2932 from DSMZ) were used. FL3 was synthesized according to an established procedure . Cells were grown in RPMI 1640 medium (Lonza) supplemented with 10% heat-inactivated fetal calf serum (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). Cell lines were expanded upon receipt and low-passage vials were stored in liquid nitrogen. All experiments were realized within 8 weeks after drawing. The cell lines were routinely tested to confirm the absence of mycoplasma by the MycoAlert Kit from Lonza. For cell viability assays, 5 × 104 cells/well were seeded in flat-bottom 96-well microtiter plates (Starsted, Inc.) and FL3 (0–100 nM) or solvent (DMSO) was added in triplicate. Cell viability was assessed after 48 and 72 h using the XTT assay (Cell Proliferation Kit II, XTT Roche) according to the manufacturer’s instructions. Results are expressed as the relative cell viability (ratio) compared to untreated control cells. Nonlinear dose-response curves were fitted to the data using Graphpad Prism Version 5 software. From these curves, GI50 (the concentration of FL3 at which the fraction of affected cells is 50%) and Emax values (the fraction of affected cells at the maximum effect of FL3) were calculated for each DLBCL cell line tested.
Analysis of apoptosis
DLBCL cells (1 × 106 cells/mL) were treated with FL3 (1–20 nM). After 48 h and 72 h, apoptosis was determined using the propidium iodide (PI)/Annexin V-FITC double staining method (kit Beckman Coulter, Immunotech) as described previously .
For analyzing intracellular trafficking of AIF and PHB1, subcellular fractionation was performed using a nuclear/cytoplasmic fractionation kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. We used GAPDH or Hsp90 (Sigma Aldrich) and Histone 3 antibodies (Santa Cruz) as controls for the cytoplasmic and nuclear fractions, respectively.
Proteins were obtained from whole cell lysates using lysis buffer (20 mM Tris–HCl [pH 7.5], 1% NP-40, 10% glycerol, and 150 mM NaCl supplemented with 1% protease and phosphatase inhibitor mixtures) (Sigma) as previously described . The primary Abs used were: mouse anti-Akt1/2, rabbit anti-phospho-Ser473Akt, rabbit anti-Erk1/2 or anti-phospho-Erk1/2, anti-Mnk1, anti-4E-BP1, anti-AIF, anti-PHB1, anti-PHB2, anti-phospho(Ser/Thr)-Akt substrate, anti-eIF4E, anti-Phospho-eIF4E, anti-C-Raf and anti-phospho-C-Raf (Ser289/296/301) (Cell signaling Technology), mouse anti-PARP, rabbit anti-Bcl-2 and anti-c-Myc (Santa Cruz Biotechnology), and anti-βactin (Sigma). Horseradish peroxidase (HRP)–conjugated secondary antibodies (1:1000) were from DakoCytomation. Blotted proteins were detected and quantified using the Immobilon Western Chemiluminescent HRP Substrate (Millipore) and a bioimaging system (Genesnap; Syngene).
7-Methyl-GTP cap binding assay
5 × 105 cells were washed in PBS and resuspended in lysis buffer. After centrifugation, (16,000 x g for 10 min at 4 °C) 200 μg protein was applied to 20 μL immobilized γ-aminophenyl-7-methyl-guanosine 5′-triphosphate (m7GTP) agarose beads (Jena Bioscience) and incubated for 3 h at 4 °C. The samples were analyzed for eIF4A and eIF4E by western blotting.
Real-time quantitative PCR analysis
Total RNA was isolated using the QIAzol Lysis Reagent from QIAGEN, and 2 μg of total RNA was used as template for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Appleid Biosystems), according to the manufacturer’s instructions. Real-time PCR reactions were conducted in triplicate with TaqMan® Fast Universal PCR Master Mix (Applied Biosystem ThermoFisher Scientific) and AKT1, AKT2, MNK1 or 18S TaqMan® Gene Expression Assays (Primer/Probe Set). Amplification of cDNA was measured using a StepOnePlusTM Real-Time PCR System (Applied Biosystem). Quantitation of results was determined using the delta delta CT method. AKT1, AKT2 and MNK1 mRNA expression in DLBCL cells was normalized to 18S RNA levels (delta CT) and then expressed relative to culture condition control (delta delta CT).
IHC stainings were performed as previously described  using the Envision Kit (Dako Denmark A/S). Rabbit anti-PHB1, anti-PHB2, anti-AIF antibodies were purchased from Cell Signaling Technology and mouse anti-Ki67 was obtained from Dakko. Mouse and rabbit isotypic control antibodies were purchased from Sigma. For PHB expression in biopsy samples, the staining intensity was scored from 0 to 4 as follows: 0, negative; 1, weak; 2, moderate; 3, strong; 4, very strong. All tumor samples were PHB1 positive. For statistical analysis, a staining index score ≥ 3 indicated tumors with high PHB1 or PHB2 expression, and < 3 low PHB1 or PHB2 expression.
In vivo xenografts
All animal studies were conducted in accordance with the guidelines established by the internal Institutional Animal Care and Use Committee (CREEAL N°2-07-2012). Four-week-old SCID female mice (CB17.SCID) were supplied by Janvier Labs. Mice were inoculated with 1 × 107 SUDHL4 cells subcutaneously. After the tumors had become established (~ 6 weeks) i.p. administration of FL3 (15 mg/kg diluted in DMSO/PBS with 5% Cremophor) or vehicle alone (DMSO/PBS with 5% Cremophor) was performed two times per week for 2 weeks. Mice used in the study were monitored every other day. No sign of toxicity in the animals and no unexpected death were observed throughout the experiment. Moreover, no change in body weight of mice was detected (data not shown). The tumor volume (cm3) was estimated by three weekly measurements and calculated using the formula length x width x height. Mice were killed after 13 days of treatment and tumor samples were harvested for analysis.
Analysis of CD20 cell surface expression
Surface expression of CD20 on the ABC cell line U2953 was assessed as compared to SUDHL4 cells by flow cytometry. 106 cells were incubated with anti-CD20-PE specific Ab (Beckman Coulter) or isotypic control Ab (mouse IgG2a-PE, Beckman Coulter) for 30 min at 4 °C. After washing, cells were resuspended in PBS/0.1% PFA and analysed with a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) acquiring 10,000 events gated on light scatter properties (forward vs side: FSC/SSC) to eliminate debris and cellular aggregates.
Statistical analyses were performed using StatView v5.0 software (SAS Institute; Cary, NC). Significance between groups in the in vitro and xenograft studies was determined using the Student’s t-test. For the DLBCL patient studies, correlations between quantitative variables were assessed using the Spearman rank correlation coefficient. A Chi square test (χ2-value) was used to analyze the association between PHB expression and clinicopathological parameters. The duration of overall survival (OS) was calculated from the date of diagnosis to the date of last follow-up or death. Event-free survival (EFS) was defined as time from diagnosis until relapse or progression or death as a result of any cause or to the date on which the study was stopped. Kaplan-Meier survival curves were used to estimate OS and EFS rates, and the log-rank (Mantel-Cox) test was used to assess differences in survival between groups. P values < 5% were considered to be statistically significant.
Expression of PHB1 and PHB2 in DLBCL cell lines and tumor cells from patients
Correlation between immunohistochemical scores and clinical DLBCL patient characteristics
The PHB ligand, FL3, induced apoptosis in DLBCL cell lines associated with nuclear translocation of PHB1 and AIF
Cytotoxicity of FL3 in DLBCL cell lines
FL3 inhibits ERK–MNK–eIF4E signaling pathway in DLBCL cell lines
eIF4F complex formation and activity are altered in DLBCL cell lines treated with FL3
Akt protein level is strongly down-regulated after FL3 treatment in DLBCL cell lines
FL3 activity is maintained in cell line resistant to rituximab
FL3 reduces tumor growth in a DLBCL xenograft model
The present study was designed to evaluate the clinical significance of PHB expression and the potential therapeutic relevance of targeting PHB in DLBCL. We focused on the flavagline FL3 because of its abilities to bind to PHBs and modulate their functions, and its in vivo antitumor activity [30, 35]. PHB1 and PHB2 protein levels were higher in GCB and ABC cell lines compared to normal peripheral B cells suggesting that PHB1 and PHB2 are upregulated during tumorigenesis. Consistent with this, PHB1 and PHB2 were recently shown to be overexpressed in lymphoid and myeloid tumor cell lines compared to normal naive and activated primary human PBMCs . The PHB protein complex promotes mitochondria stabilization and cell survival in these hematologic malignancies. In the present study, data in sample patients strongly suggest that PHBs are mainly localized in mitochondria with AIF, consistent with what we observed in DLBCL cell lines. We found no significant differences between DLBCL subgroups (GCB vs. non-GCB). However, high expression of PHB1 tended to correlate with an aggressive clinical course. Interestingly, when female and male patients were analyzed separately, high PHB1 expression was a bad prognostic indicator for EFS in men but not in women. This result is not due to sex differences in PHB1 and PHB2 expression in patients (Additional file 1: Table S1), as it was also suggested by data on normal peripheral B cells and DLBCL cell lines (Fig. 1a, where SUDHL4, SUDHL6 and OCI-LY3 are derived from males and OCI-LY10 is derived from female patient). Even though the mechanism remains to be determined, these findings could be explained by the discovery of PHBs in mediating sex dimorphic effects, and notably in immune function . While evidence of a role for PHB1 overexpression has been reported in cancer progression [23, 40, 41], higher levels of PHB2 was found in proliferating cells, including neoplastic tissues . Consistent with this, we show here for the first time in DLBCL that high PHB2 expression is associated with high level of Ki-67; moreover PHB1 and PHB2 score expressions were correlated with marker of tumor burden in DLBCL patients (i.e. elevated serum LDH) and PHB2 expression with IPIaa that remains an important prognostic tool for evaluating DLBCL patients. Collectively, these data strongly suggest that PHB proteins may be key factors in DLBCL cell survival and proliferation.
Mitochondrial localization of PHBs was previously shown to be indispensable for mitochondria integrity and function, and thus cell survival, included in normal B lymphocytes and primary leukemia and lymphoma cells [24, 25]. However, in the cytosol and at the inner face of membrane PHB can interact with several key partner proteins involved in cancer progression. PHBs are notably required for the membrane localization and activation of C-Raf by the oncogene Ras, and consequently for the activation of C-Raf-MEK-ERK signaling  that is up-regulated in several cancers, including lymphomas . In this study we used the flavagline FL3 to gain insight into the cytoplasmic role of PHBs in DLBCL cells. Indeed, by binding to PHB, flavaglines were shown to inhibit PHB-mediated signaling pathways, and this interaction did not target mitochondria [36, 43]. As expected, FL3 triggered apoptosis of DLBCL cell lines and also in vivo in a xenograft murine model. Mechanisms involved AIF, as previously reported , and a caspase-dependent pathway demonstrated by PARP cleavage. Of interest, aggressive cell lines as ABC-DLBCL cells or representative models of MYC/BCL-2 DHL and MYC/BCL-2/BCL6 THL DLBCL subtypes were also sensitive to the FL3-induced apoptotic effect. Importantly, flavaglines included FL3 were shown to inhibit the proliferation of tumor cells without any significant toxicity in vivo  and on normal cells, such as human peripheral T, B and bone marrow stem cells [14, 16]. Therefore, our data indicate that targeting PHBs may be a potential therapeutic option for DLBCL notably in aggressive subtypes.
Binding of flavaglines to PHB proteins has been implicated in the inhibition of Raf-MEK-ERK-MNK1-mediated phosphorylation of eIF4E [16, 17, 22]. Our data confirmed reduced activation of c-Raf-ERK1/2-MNK1 pathway in in DLBCL cell lines and for ERK1/2 in the in vivo model. Furthermore, FL3 strongly inhibited eIF4E phosphorylation in GCB cells, which has been implicated in tumorigenesis, including lymphomagenesis. Indeed, eIF4E phosphorylation by MNK1/2 and p38 MAPKs is required for its oncogenic role by translation of a subset of mRNAs encoding proteins involved in survival and tumor invasion [13, 45]. Surprisingly, we found a decrease of total MNK1 level that was associated with a reduced phosphorylation after FL3 exposure. A marked effect was observed on GCB cell lines, which could be explained by the distinct distribution of MNK1 and MNK2 in GCB and ABC DLBCL subtypes respectively . Interestingly, flavaglines have been reported to exert their antitumor activity, including in leukemia and lymphoma mouse models, by binding also to the RNA helicase eIF4A. However this effect may vary between tumor cells. Binding to eIF4A leads to translational inhibition through stabilization of RNA-bound eIF4A in the eIF4F complex, preventing new incorporation of free eIF4A [47, 48, 49, 50]. In DLBCL cell lines, FL3 altered eIF4F complex formation and activity. This was confirmed by reduced expression of Bcl-2 and to a lesser extends c-Myc that are involved in the aggressive clinical course and poor prognosis of patients with DLBCL . In ABC cell lines, c-Myc level was differentially influenced by FL3, which could be related to the constitutive activation of the NF-κB pathway in ABC rather than GCB cell lines . The natural flavagline silvestrol, was previously reported to reduce eIF4A helicase translation activity with a marked reduction of c-Myc and Bcl-2 expression in T-ALL and BCR-activated splenic B cells [48, 53]. Our data strongly suggest that FL3 decreases cap-dependent translation in DLBCL cells by binding both to PHBs and eIF4A, which consequently sensitized cells to programmed cell death. However, further studies would be needed to precise their relative contribution which could vary according the DLBCL subtype. Interestingly, phosphorylation of PHBs by Akt was recently demonstrated to be required for PHB translocation and localization in the mitochondria, as well as for tumor cell proliferation [36, 37]. It was proposed that, by inhibiting Akt/PHBs interaction, FL3 may decrease the PHB localization within mitochondria leading to mitochondria dysfunction and thus apoptosis. Indeed, we observed by confocal microscopy analysis, delocalization of PHB1 from mitochondria in cells exposed to FL3 (data not shown). This effect may be emphasized in DLBCL cells by the global reduction level of Akt induced by FL3, that we demonstrate for the first time in the present study. Consistent with this, indirect analysis of PHB1 phosphorylation strongly argues for a FL3-induced reduction of PHB phosphorylation, and cytoplasmic content of PHB1 was decreased following FL3 exposure with a translocation in part to the nucleus. Interestingly, no effect of FL3 (as for rituximab exposure, Additional file 1: Figure S3b) was observed on PHB1 and PHB2 total cellular levels. Nuclear re-localization of PHBs has been associated to their interaction with transcription factors, such as p53 and Rb, promoting apoptosis and inhibition of transcription [19, 54]. This could explain the down regulation of the AKT1/2 mRNA that we observed in the present work. However, we can’t exclude an apoptosis degradation of AKT mRNA  as we showed for the AKT protein cell levels. Finally, reduced Akt, Mnk1, Bcl-2 and c-Myc protein expression in association with FL3-induced apoptosis was confirmed in a cell line (U2932) characterized by lower surface CD20 which open the possibility to treat rituximab-resistant diseases.
This study revealed that PHB overexpression is associated with clinical and biological markers of tumor aggressiveness in DLBCL, especially in male patients. Furthermore, the PHB-ligand FL3 determined anti-tumor activities in vitro and in vivo that were associated with inhibition of ERK/MNK/eIF4E signaling pathways and AKT expression. These effects were observed also in cell lines representative of aggressive DLBCL subtypes. In conclusion, we propose that targeting PHBs may be a promising tool in DLBCL therapeutic strategies particularly for male patients and patients who do not respond to conventional treatments.
The authors would like to thank Brigitte Fournier (Laboratoire d’Anatomie-Pathologique CHU Limoges, France), Annemilaï Raballand and Lucile Astorgues-Xerri (AFR Oncology), Séverine Mares (EA3842) for technical supports, Dr. Robin Jeannet for reviewing the English version and Pr Jean Feuillard for its critical reading of the manuscript.
DT, HB, LD and AdeG conceived and designed the research; HB, MA, LD, DT, BP and AdeG realized, analyzed and/or interpreted the data; SE and LD synthesized the flavagline FL3; DT and HB wrote the paper including the design of figures; JA and AO provided clinical samples and data; LD, AdeG, MOJ and ER critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.
This research was supported by Cancéropôle GSO (Grand Sud Ouest) and the Ligue Contre le Cancer (Haute-Vienne). We are also grateful to the Libyan Ministry of Higher Education and Scientific Research ANRT for a fellowship to S.E.
Ethics approval and consent to participate
This study was approved the local ethics committee of Limoges Hospital (N° 98–2012-23).
All animal experiments were approved by the internal Institutional Animal Care and Use Committee (CREEAL) of Hospital and University of Limoges, and performed in accordance with the guidelines established by this committee.
Consent for publication
The authors declare that they have no competing interests.
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