Background
Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy derived from mature B cells, characterized by a rapid clinical evolution and a poor response to current therapies [
1]. The first oncogenic hit for tumor development is the translocation t(11;14)(q13;q32) which juxtaposes activating sequences from the
IGH gene promoter upstream of the
CCND1 gene. This translocation leads to the constant expression of cyclin D1 protein and in turn, abnormalities of cell cycle, and compromises the G1-S checkpoint [
1]. This initial oncogenic event is followed by various chromosomal alterations targeting DNA damage response (DDR), survival pathways, NOTCH and NF-κB pathways, and chromatin modification machinery [
2] as well as reprograming metabolism [
3].
ATM (Ataxia telangectasia mutant) and ATR (ATM and Rad3-related) act as apical kinases and key regulators of DDR. Following double-strand DNA breaks (DSBs), ATM/ATR phosphorylate downstream effectors including checkpoint kinases (CHK1/CHK2), DNA repairing factors and transcriptional regulators such as p53 [
4]. Next, depending on the cellular context, cells initiate cell cycle arrest, DNA repair through two main mechanisms: homologous recombination (HR) or non-homologous end joining (NHEJ), and/or apoptosis.
ATM alterations are very common in MCL patients, mutations and deletions occurring in up to half of cases [
5]. Genetic alterations of
TP53 are also very common (30% of cases) and concurrent alterations of
ATM and
TP53 are found in almost 10% of patients [
6]. Defaults in responding intracellular and extracellular genotoxic stresses could explain why MCL is the B-cell malignancy with the highest degree of genomic instability [
7].
Abnormalities of the ubiquitin-proteasome pathway are also recognized in MCL cells. They could account for defaults in the DDR and resistance towards genotoxic drugs that are used in clinics such as cyclophosphamide, doxorubicin and chlorambucil [
8]. For example, MCL cells show frequent deletion within the
FBXO25 gene located at 8p23.3 [
9].
FBXO25 encodes a F-box containing protein, part of the Skp1/Cullin/F-box containing protein or SCF
FBXO25 complex that targets the prosurvival HAX1 mitochondrial protein. The monoallelic loss of
FBXO25 and thus, the disruption of the PRKCD (a protein kinase C)/FBXO25/HAX1 axis promotes survival of MCL cells. A high percentage of MCL tumors (20%) have mutations within the
UBR5 gene [
10]. UBR5 encodes an E3 ubiquitin ligase that targets KATNA1 (katanin p60), TOPBP1 (DNA topoisomease 2-binding protein 1) and PAIP2 (polyadenylate-binding protein-interacting protein 2) proteins whose functions are not fully known. The human double minute(HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover. The gene is located within the 12q13 locus which is amplified in MCL [
11]. This accounts for elevated HDM2 expression and prevention of both p53 transcriptional activity and degradation. Thus, the response of MCL cells to DNA damaging agents is impaired through various mechanisms.
Studying a set of MCL cell lines, we noticed that REC1 cells were particularly resistant to genotoxic stresses. Looking for cellular mechanisms that could sustain this resistance, we observed that the ubiquitin/proteasome degradation pathway was inefficient. We ruled out a default of β-transducin repeat containing protein (βTrCP), the E3 ubiquitin ligase of the SCFβTrCP complex which was a good candidate. We further used fluorescent probes to study specifically the 26S proteasome activity and observed that this activity was specifically down-regulated in REC1 cells compared to other MCL cell lines.
Methods
Cell cultures, treatments and cell proliferation determination
MCL cell lines were provided by Gaël Roué (IDIBAPS, Barcelona, Spain) except Granta519 cells which were purchased from DSMZ (ACC-342). MCL cell lines were maintained in culture as described [
12]. Cell authentication was done by STR profiling (IdentiCell, Aarhus, Denmark). Cell proliferation was analyzed using the CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega, Charbonnières, France) according to the supplier. MCL cells were treated with vehicle (0.01% DMSO) or 1–40 μg/ml etoposide (Sigma-Aldrich, St Louis, MO) for 24–72 h depending on the experiment. For co-treatment with MG132, the cells were incubated with 5 μM MG132 (Sigma-Aldrich) together with 4 μg/ml etoposide for 24 h.
Quantification of apoptotic and senescent cells, cell cycle analysis
MCL cells exposed to vehicle or etoposide were stained with an Apo 2.7 PE-conjugated antibody (Ab, Beckman Coulter, Villepinte, France). The APO2.7-stained cells were analyzed by flow cytometry (Gallios, Beckman Coulter) and data were processed with the Kaluza software (Beckman Coulter). At least, 2 × 104 cells were analyzed for each culture condition, for each experiment.
For cell cycle experiments, the cells were washed with PBS and fixed in 70% ethanol/PBS at −20 °C for 30 min. After washing, the cells were then incubated with PBS containing 10 mg/ml of propidium iodide (PI) and 100 mg/ml of RNAse A. At least, 2 × 104 cells were analyzed by flow cytometry for each experimental condition.
To assess the presence of senescent cells after etoposide treatment (4 μg/ml for 24 h), we used a cytometry-based assay after staining of living cells with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C
12FDG) as described previously [
13]. A shift of the mean fluorescence intensity (MFI) is representative of an enrichment of senescent cells in the whole population.
Indirect immunofluorescence and confocal fluorescence microscopy analysis
Cells (105 cells per spot) were cytospun on Superfrost glass slides, at 500 x g for 3 min, then fixed in 4% paraformaldehyde (PFA) for 15 min, and permeabilized by incubation with 0.5% Triton-X100 (v/v) for 5 min. The slides were then incubated with 3% BSA in PBS for 30 min at room temperature and, next with Abs anti-γH2AX (p-S139, final dilution 1/2000) (GTX61796 from GeneTex Inc., Irvine, CA) or anti-cyclin D1 (sc-718, Santa Cruz Tech., final dilution 1/50) for overnight in the dark at 4 °C. Goat anti-rabbit IgG (H + L) polyclonal Alexa Fluor® 546 (Life Technologies) served as secondary Ab. Following staining steps, cells were mounted with VECTASHIELD® with DAPI (Vector Lab.). The slides were analyzed with the Fluoview FV 1000 confocal microscope and Fluoview Viewer software (Olympus, Rungis, France).
Western blotting
Whole cell lysates and western blotting were prepared as previously described [
12]. Using the Cell Fractionation Kit (#9038, Cell Signaling Tech.), we separated cells into cytoplasmic (c), membrane/organelle (m), and nuclear/cytoskeletal (n) fractions and prepared the corresponding protein extracts according to the manufacturer’s instructions. We used primary Abs against β-actin (sc-47,778, final dilution 1/1000), cyclin D1 (sc-718, final dilution 1/400), cyclin D2 (sc-593, final dilution 1/200), p53 (sc-126, final dilution 1/400), p16
INK4A (sc-468, final dilution 1/100), and p21
CIP1 (sc-397, final dilution 1/400) from Santa Cruz Biotech. (Santa Cruz, CA). We purchased Abs against MCL1 (#4572, final dilution 1/500), p-T256-cyclin D1 (#3300, final dilution 1/1000), CHK2 (#6334, final dilution 1/200), p-T68-CHK2 (#2197, final dilution 1/1000), p-S15-p53 (#9286, final dilution 1/1000), βTrCP1 ((#4394, final dilution 1/1000) from Cell Signaling Tech. (Danvers, MA). An Ab against BCL2 (clone 124, M0887, final dilution 1/200) was purchased from Dako (Glostrup, Denmark); an Ab against γH2AX (GTX61796, final dilution 1/1000) from Genetex (Irvine, CA); an Ab against GAPDH (clone 6C5, final dilution 1/2000) from Ambion (Thermo Fischer Scientific, Waltham, MA). We used ImmunoPure goat anti-rabbit or rabbit anti-mouse IgG peroxidase-conjugated as secondary Abs (Pierce, Thermo Fisher Scientific). For densitometric analyses, images were captured with a ChemiDoc™ XRS+ molecular imager and analyzed using Image Lab™ software (Bio-Rad). The background of each image was subtracted from the bands of interest, then the densities of each protein of interest were normalized against the density of control housekeeping proteins.
Proteasome function assays
Our procedure was adapted from Vlashi et al. [
14]. Cells were washed with PBS, pelleted and lysed in a homogenization buffer (25 mM Tris pH 7.5, 100 mM NaCl, 5 mM ATP, 0.2% (vol/vol) NP-40 and 20% glycerol). Cell debris were removed by centrifugation at 4 °C. Protein concentration in the resulting crude cellular extracts was determined. To measure 26S proteasome activity, 100 μg of protein of each sample were diluted with buffer I (50 mM Tris pH 7.4, 2 mM dithiothreitol, 5 mM MgCl
2, 2 mM ATP) to a final volume of 1 ml and assayed in triplicate. The fluorogenic proteasome substrates Suc-LLVY-AMC (chymotryptic substrate; Enzo Life Sciences, Villeurbanne, France), Z-ARR-AMC (tryptic substrate; Calbiochem, Molsheim, France), and Z-LLE-AMC (caspase-like substrate; Enzo Life Sci.) were dissolved in DMSO and added to a final concentration of 80 μM. Proteolytic activities were continuously monitored for 120 min by measuring the release of the fluorescent group, 7-amido-4-methylcoumarin (AMC), with the use of a fluorescence plate reader (VICTOR X4 multilabel plate reader, Perkin Elmer) at 37 °C, at excitation and emission wavelengths of 380 and 460 nm, respectively. For analyzing the effects of proteasome/protease inhibitors on proteasome activities, cells were treated for 4 h with MG-132 (500 nM), bortezomib (5 nM) or leupeptin (20 μM) before the purification of whole cell extracts.
RNA extraction and real-time polymerase chain reaction
Total RNAs were extracted using RNAeasy® Mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instructions and quantified using a Smartspec™ 3000 spectrometer (Bio-Rad, Hercules, CA) from cultured MCL cells. cDNAs were synthesized using 2 μg of RNA and M-MuLV-reverse transcriptase as recommended by the supplier (Invitrogen, Thermo Fisher Scientific). SYBR Green real-time polymerase chain reaction (RT-PCR, Applied Biosystems, Thermo Fisher Scientific) was performed on cDNAs with primers for
BTRC and
FBXW11 previously described [
15], using a StepOnePlus real-time PCR System (Applied Biosystems). Data were analyzed with the Step One software V2.2.2 (Applied Biosystems). Gene expression was determined by real-time RT-PCR and quantified using
GAPDH expression as internal standard. Relative gene expression was evaluated by the 2
-ΔΔCt method.
Statistical analysis
The Student’s t-test was used to determine the significance of differences between two experimental groups. Data were analyzed by two-sided tests, with p < 0.05 (*) considered to be significant.
Discussion
Although therapeutic strategies for MCL have evolved these last years, the disease remains largely incurable. MCL patients develop
de novo resistance or acquire resistance to frontline drugs [
37]. There is a great need to overcome resistance in MCL patients and to improve their clinical outcome. This can be achieved through a better knowledge of the mechanisms of resistance. We studied the response of a panel of MCL cell lines to genotoxic stress and observed a heterogeneous response. REC1 cells are the most resistant and JeKo1 cells the most sensitive to etoposide, a Top 2 inhibitor that generates DSBs. We observed that three main actors of cell cycle arrest, senescence and apoptosis, namely cyclin D1, MCL1 and CDC25A that are enrolled after a genotoxic stress, fail to be degraded in response to etoposide. We finally provided evidence for a lowered 26S proteasome activity that could sustain the accumulation of these proteins and in turn, the resistance of MCL cells.
Defective proteolysis have been reported in MCL cells. Indeed, mutations of
CCND1 gene at the N-terminus increases cyclin D1 protein stability through the attenuation of threonine 286 phosphorylation and its nuclear retention [
38]. The phosphorylation of cyclin D1 is mandatory for protein degradation by the UPS. We have shown that cyclin D1 is correctly phosphorylated in resistant REC1 cells after etoposide treatment and exported to the cytoplasmic compartment. Although E36K, Y44D, and C47S
CCND1 mutations have not been reported for REC1 cells, we can rule out such a mechanism of resistance. Importantly, in MCL cell lines,
CCND1 mutations promote resistance to ibrutinib, an inhibitor of the Bruton tyrosine kinase (BTK) involved in the B-cell receptor (BCR) signaling pathway. Recent studies have provided some clues about ibrutinib resistance including activation of the NF-κB, ERK1/2 and AKT, alteration of the BCR signaling pathways [
39‐
41]. These studies suggest that multiple mechanisms contribute to ibrutinib resistance. Moreover, a same mechanism, i.e. the accumulation of nuclear cyclin D1, contributes to resistance to several drugs: ibrutinib [
38], lenalidomide [
12], and etoposide (the present study), drugs that target different pathways. According to these data, an increased stability of cyclin D1 is a major mechanism for MCL cells resistance.
REC1 cells are resistant to proteasome inhibitors: bortezomib (Additional file
3: Figure S2C, upper panel), MG132, and carfilzomib (data not shown)). However, REC1 cells enter apoptosis when treated with KNK-437, an inhibitor of heat-shock factor 1 (HSF1). HSF1 is the master transcription factor for heat shock proteins (HSPs) encoding genes. The inhibition of HSF1 downregulates simultaneously the transcription of
HSPB1 and
HSPA4 coding for HSP27 and HSP70, respectively. Acosta-Alvear and colleagues reported recently that proteostasis factors such as chaperones and HSPs controlled the response to proteasome inhibitors [
42]. In particular, the knockdown of HSF1 sensitizes cells to carfilzomib in agreement with our observation. Moreover, the fact that REC1 cells respond to a HSF1 inhibitor suggests that the UPS although downregulated can be rearmed. Moreover, combining HSP inhibitors and proteasome inhibitors could be efficient therapies for MCL patients resistant to bortezomib/carfilzomib. Clinical trials have demonstrated such efficacy for bortezomib associated with several types of HSP90 [
43‐
45] or HSF1 [
46‐
48] inhibitors for multiple myeloma (MM) patients.
REC1 cells are sensitive to sn38, the metabolically active form of irinotecan, an inhibitor of Top 1, (Additional file
3: Figure S2C, lower panel). Importantly, the IC
50 for sn38 is similar for REC1 and NCEB1 and smaller than JeKo1 cells. sn38 selectively targets Top 1-DNA cleavage complexes which form at the vicinity of replication and transcription complexes to unwind DNA. The stabilization of Top 1-DNA cleavage complexes leads to DNA damage at the time of DNA replication or transcription, and finally to DSBs [
49]. DSBs either generated by sn38 or etoposide elicit the same DNA repair response. We showed in this study that DDR was activated in REC1 cells but that DNA repair mechanisms were deficient. This data is confirmed by the triggering of apoptosis after sn38-treatment in REC1 cells, in which the apoptotic machinery is fully functional. There is no cross-resistance in REC1 cells to Top 1 and Top 2 inhibitors. Moreover, the sensitivity/resistance pattern of MCL tumor cells towards drugs is multifactorial and largely dependent on the cell context.
Two studies reported recently a paradoxical resistance of multiple myeloma tumor cells to proteasome inhibitors by decreased levels of 19S proteasome regulatory sub-units [
42,
50]. These are ATPase subunits as well as non-ATPase subunits and seem specific for a cell line. A downregulation of 19S sub-units could sustain the global decreased proteasome activity observed in REC1 cells and could be key determinant of resistance to proteasome inhibitors and other drugs in MCL tumor cells. However, the demonstration that the reduced trypsin-like activity bore by the 20S core complex and, in particular, the β2 subunit (or PSMB7), highlights another type of proteasome subunit abnormality. To our knowledge, this is the first report of a putative role of the β2 subunit in a resistance process. In contrast, the involvement of the β5 subunit in the resistance of various hemopathies is well described. For example, in a myelomonocytic THP1 cell line, selected for acquired resistance to bortezomib, the
PSMB5 gene coding for PSMB5, the β5 subunit, is mutated and the corresponding protein overexpressed [
51]. In MM tumor cells resistant to bortezomib, no such mutations were found [
52], rather a constitutive activation of the STAT3 signaling pathway and in turn, the upregulation of the β5 subunit [
53]. However, the
PSMB7 gene coding for β2 subunit is overexpressed in a large variety of solid cancers and myeloid leukemias [
54]. A survey of public available data bases (COSMIC, canSAR, Cancer Cell Line Ecyclopedia) indicated that
PSMB5 and
PSMB7 genes are not mutated, deleted nor amplified in REC1 cells. Further experiments should address these points.
Cancer stem cells (CSCs) or cancer-initiating cells (CICs) belong to a population of self-renewing cells that sustain the long-term clonal maintenance of the tumor [
55]. Strong evidences support a link between stemness and resistance to drugs. CSCs/CICs have develop plethora of strategies to resist anticancer therapies including elevated activity for DNA damage detection and repair, increased ability for xenobiotic efflux, unbalance between the anti- vs. pro-apoptotic mechanisms, reduced production of free radicals etc. Interestingly, a low proteasome activity has been reported as a marker for breast cancer and head and neck CSCs/CICs [
56,
57].
Several recurrent somatic mutations are described in tumor cells of MCL patients [
58]. Among them, mutations of
ATM,
CCND1,
TP53,
MLL2,
TRAF2 and
NOTCH1 genes are frequently encountered and account for resistance to drugs. Most of them target the BCR and NF-κB signaling pathways and define actionable gene targets. However, deletion of
FBXO25 [
9], mutation of
UBR5 [
10] or, as suggested here, a decreased of global 26S proteasome activity modify the UPS and in turn, the sensitivity to drugs and the clinical response of MCL patients. Since, the resistance to proteasome inhibitor and/or other drugs is convoyed by defaults of UPS, the restoration of its activity seems determinant to bypass resistance and to achieve a full response towards treatments.
Acknowledgements
We thank Anne Barbaras for technical assistance, Daniele Guardavaccaro (Hubrecht Institute, Utrecht, The Netherlands) for the gift of βTrCP1/2 expression plasmids, and Olivier Coqueret (Centre de Recherche en Cancérologie, Angers, France) for the gift of sn38. We are grateful to Véronique Baldin (Centre de Recherche en Biologie Moléculaire, Montpellier, France), Sébastien Léon (Université Paris Diderot, Institut Jacques Monod, Paris, France), Manuel Rodriguez (Institut des Technologies Avancées en Sciences du Vivant, CNRS USR3505, Toulouse, France), Chann Lagadec (Université de Lille 1, INSERM U908, Lille, France) and Hervé Mittre (Laboratoire de génétique, Centre Hospitalo-Universitaire, Caen, France) for helpful discussions, and Gaël Roué (IDIBAPS, Barcelona, Spain) and Arthur Vincent-Coves for reading the manuscript.