Background
The
CCND1 gene encoding cyclin D1 is the second most frequently amplified locus in solid cancers [
1]. Moreover, cyclin D1 is overexpressed in human cancers, including malignant hemopathies, after genetic alterations, such as chromosomal translocation, but also in the absence of any detectable genetic alteration [
2]. Tumor cells with high cyclin D1 levels have higher proliferation rate and lower nutrient requirements that tumor cells that do not express cyclin D1. This is consistent with the well known function of cyclin D1 in cell cycle regulation through cyclin-dependent kinase 4/6 activation [
3]. However, the role of cyclin D1 in oncogenesis might not be limited to the increase in proliferation. Indeed, depending on its subcellular distribution (nuclear, cytoplasmic, at the outer mitochondrial membrane) and partners (transcription factors, chromatin-modifying enzymes, cytosolic proteins), cyclin D1 can regulate transcriptional regulation [
4], DNA damage response [
5,
6], centrosome duplication [
7], chromosomal instability [
8], senescence [
9], mitochondrial function [
10] and migration [
11-
13]. All these processes, if left uncontrolled, can initiate or/and maintain transformation processes.
In 15% of patients with multiple myeloma (MM), a hematological disease characterized by the accumulation of malignant plasma cells in the bone marrow, cyclin D1 is aberrantly expressed as a result of the t(11;14)(q13;q32) translocation in [
14]. Moreover, biallelic cyclin D1 expression is detected in 40% of MM cases, most displaying hyperdiploidy [
15]. Consistent with its role in cell cycle regulation, cyclin D1 has been shown to regulate MM cell proliferation [
16]. Paradoxically, MM patients with cyclin D1-expressing tumor cells have a good prognosis and a longer overall survival [
17]. The possibility of additional functions for cyclin D1 in MM cells is key issue that has been little addressed. We investigated this possibility, by generating stable MM cell line-derived clones expressing a cyclin D1-green fluorescent protein (GFP) fusion protein (D1-GFP) or GFP alone. We used arrays to investigate gene expression in D1-GFP- and GFP-expressing cells. We found that the presence of cyclin D1 altered the expression of genes involved in metabolism, membrane trafficking, adhesion/migration, cell proliferation, inflammation, and cell death/apoptosis. We also found that cyclin D1 expression was sufficient to sensitize MM cells to the induction of apoptosis by bortezomib. This greater sensitivity of cyclin D1-expressing cells was mediated by the activation of the unfolded protein response (UPR) pathway and endoplasmic reticulum (ER)-stress signaling, triggering apoptosis. Our data reveal a novel molecular mechanism by which cyclin D1 expression directly targets the UPR, enhancing the response to bortezomib in MM tumor cells, as highlighting by clinical observations.
Methods
Chemicals
Bortezomib and Z-LEVD [Z-LE(OMe)VD(OMe)-FMK], a caspase 4 inhibitor, were purchased from Euromedex. Q-VD-OPh [quinoyl-valyl-O-methylaspartyl-(2,6-difluoro-phenoxy)-methyl ketone], a pancaspase inhibitor, was purchased from Sigma-Aldrich. Q-VD-OPh and Z-LEVD were dissolved in dimethyl-sulfoxide (DMSO) (Sigma-Aldrich) and bortezomib was dissolved in 0.9% NaCl. For controls, vehicle (DMSO or NaCl) was added at the same final concentration.
Generation of cyclin D1-expressing cell lines
RPMI 8226 cells (referred to here as 8226 cells) were purchased from DSMZ (ACC-402). LP1 cells were generously provided by R Bataille (Centre de recherche en cancérologie Nantes-Angers, Nantes, France). U266 (ACC-9) and KMS-12-PE (ACC-606), from DSMZ, were used as positive control for cyclin D1 expression in immunocytochemistry analysis. Human myeloma cell lines (HMCLs) were maintained in RPMI 1640 medium (Lonza) supplemented with 2 mM L-glutamine (Lonza), 10% fetal calf serum (FCS, PAA Laboratories) and antibiotics (Lonza).
The pEGFP-N1 plasmid was purchased from Clontech Laboratories Inc. and the p-cyclin D1-EGFP plasmid was kindly provided by D. Salomon (USCF School of Medicine, San Francisco, CA, USA). This plasmid was sequenced to check the integrity of the coding sequence, amplified and purified with the QIAGEN maxi kit (Qiagen). We electroporated (250 V, 950 μF, Gene Pulser II, Bio Rad) 107 8226 or LP1 cells with 10 μg pEGF-N1 or p-cyclin D1-EGFP plasmids in RPMI 1640 medium without FCS. After incubation for 24 h, the cells were transferred to complete medium supplemented with 500 μg/mL G418 (PAA Laboratories). They were cloned by limiting dilution methods. Clones were maintained under selective pressure and selected on the basis of their FL1-fluorescence by flow cytometry (Gallios, Beckman Coulter). Data were analyzed with the Kaluza software (Beckman Coulter).
Cell viability measurement
Cell proliferation was determined by directly counting the cells after trypan blue staining. Cell viability was quantified with the CellTiter 96® AQueous One Solution (MTT [(3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Promega), according to the manufacturer’s instructions.
Apoptosis determination by APO2.7 staining and cytometry sorting
Drug-exposed HCMLs and control cells were stained with the APO2.7-PE-conjugated antibody (Ab), as described by the manufacturer (Beckman Coulter), and analyzed by flow cytometry. We analyzed at least 104 cells for each set of culture conditions, and each experiment was carried out three times.
Indirect immunofluorescence and confocal microscopy analysis
Cells (2 × 10
5 per spot) were cytopsun on Superfrost slides at 500 g for 3 min, then fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X100 for 5 min. Slides were processed as previously described [
10], with an anti-cyclin D1 (sc-718, Santa Cruz Biotech., Santa Cruz CA) as primary Ab and AlexaFluor 633-labeled anti-rabbit IgG (Molecular Probes) as secondary Ab. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI). Slides were analyzed with a Fluoview FV1000 confocal microscope and Fluoview Viewer software (Olympus).
Western blotting
Whole-cell lysates were obtained with M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, Illinois, USA), and western blotting was carried out as described elsewhere [
18]. We used primary Abs against poly(ADP-ribosyl)transferase (or PARP, #9542), caspase 9 (#9508), XBP1 (#7160), eIF2α (#9722), phospho(p)-eIF2α (#9721), and BiP (#3183) from Cell Signaling Tech. (Danvers, MA). The Abs against MCL1 (S-19); caspase 3 (H-277), caspase 8 (H-134), cyclin D1 (M-20), cyclin D2 (M-20), XBP1 (M-186), and β-actin (C4) were obtained from Santa Cruz Biotech. The Ab against GAPDH (clone 6C5) was obtained from Life Technologies; that against caspase 4 (M029-3) was obtained from MBL and the Ab against BCL2 (M0887) was obtained from Dako. The secondary Abs used were goat anti-rabbit or anti-mouse peroxidase-conjugated IgGs (Abcam).
RNA was extracted with the RNAeasy® Mini kit (Qiagen), according to the manufacturer’s instructions, and quantified with a Smartspec™ 3000 spectrometer (Bio-Rad). We generated cDNAs from 2 μg of RNA with M-MuLV-reverse transcriptase, according to the manufacturer’s instructions (Invitrogen, Life Technologies). The cDNA was then subjected to real-time PCR with the SYBR Green system (Applied Biosystems, Life Technologies) and primers for
RPLP0,
GAPDH,
CXCR3,
BTBD3,
MCL1,
BCL2L1,
RND3 and
CXCL10 (Additional file
1).
BiP,
GRP94 and
CHOP cDNAs were amplified as previously described [
19]. We used the StepOnePlus real-time PCR System (Applied Biosystems). Data were analyzed with Step One software V2.2.2 (Applied Biosystems). Gene expression was quantified by the ΔΔ
Ct method, with
RPLP0 used as the internal standard. The fold change in expression (Fc) was calculated as 2
-ΔΔCt.
Whole-genome expression analysis
Total RNA was purified from four independent cultures of 8226 GFP (Cl1), RPMI 8226 D1-GFP (Cl2), LP1 GFP (Cl4) and LP1 D1-GFP (Cl3) cells, with the RNAeasy® Mini kit (Qiagen). RNA was quantified with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.). RNA integrity was assessed with a Bioanalyzer 2100 machine and the RNA6000 Nano kit (Agilent Technologies Inc.). All samples had a RNA integrity number of at least 7.00. No signs of DNA contamination were detected. We used 400 ng of total RNA per sample. We used the Illumina Total Prep RNA Amplification Kit (Ambion, Life Technologies) to generate biotinylated, amplified cRNA, according to the manufacturer’s recommendations. Hybridization on Illumina HumanHT-12 v4 Expression BeadChips, and the staining and detection of cRNAs with the I-Scan system were performed according to the manufacturer’s recommendations (Illumina Inc., San Diego, USA). The HumanHT-12 v4 Expression BeadChip assesses a total of 47,231 marker probes, 28,688 of which are NM coding transcripts; and 11,121 are XM coding transcripts (RefSeq Content, Build 33.1, Release 5). It also contains 3,461 experimentally confirmed mRNA sequences that have been aligned with EST clusters. GenomeStudio 2011.1 software (Illumina Inc.) and its Gene Expression Analysis Module (version 1.9.0) were used for signal extraction and quantile normalization. Processed probe data were then filtered according to the following criteria: minimal signal intensity fold-change of 1.50 across all samples; minimal absolute change in probe signal intensity of 150 across all samples, based on the variance of gene expression, with a 10% threshold. The filtered data were then log-transformed and exported to appropriate software for secondary analyses. Omics Explorer 2.2 software (Qlucore, Lund, Sweden [
20]) was used for hierarchical clustering and analyses of differential expression. An adjusted
p-value (
q-value) of 0.01 was considered as significant in the differential expression analyses. The primary probe data were subjected to quantile normalization. Genes were considered to be differentially expressed if the absolute fold-change (Fc) in mean expression values between D1-GFP-expressing cells and GFP-expressing cells was at least 1.5.
Statistical analysis
Student’s t-test was used to determine the significance of differences between two experimental groups. Data were analyzed in two-tailed tests, with p < 0.05 (*) considered significant and p < 0.01 (**) considered highly significant.
Discussion
MM is relatively homogeneous in terms of its histological, clinical and biologic properties, but it is characterized by genetic and phenotypic complexities with implications for treatments. Gene expression profiling assays have confirmed that MM patients from the CD-1/2 group (MM expressing cyclin D1) have a better prognosis than those from the MS and MF groups (MM expressing cyclin D2) [
17]. We investigated the molecular basis of this behavior, by expressing cyclin D1 in HMCLs belonging to groups of MM patients with a poor prognosis. We studied the 8226 and LP1 cell lines because they produce little or no cyclin D1. The constitutive expression of cyclin D1 is cytotoxic in B cells [
24]. We therefore obtained cell clones producing only moderate to low levels of cyclin D1. Nevertheless, cyclin D1 expression was found to be associated with higher levels of cell proliferation and the downregulation of cyclin D2, as previously reported [
16,
28]. These observations are consistent with previous reports that MM of the CD-1/2 group respond rapidly to treatment, and that their response rates are high. However, the duration of complete responses has been shown to be shorter for this group of tumors than for other types of MM [
39], possibly due to the higher rates of proliferation of cyclin D1-expressing tumor cells.
Comparison of the transcriptional profiles of 8226 and LP1 with and without cyclin D1 expression showed that many genes were up- or down-regulated in the presence of cyclin D1. However, only a small number of genes were coordinately regulated by cyclin D1 in both cell types (Additional file
4). We then focused on the cell functions controlled by cyclin D1. The genes differentially expressed in both cell types were enriched in genes associated with apoptosis/cell death and inflammation. We then investigated the potential mechanism by which cyclin D1 might modulate the apoptotic response.
Cyclin D1 expression significantly increased the apoptosis of 8226 and LP1 cells after a bortezomib treatment. This finding is consistent with those obtained by Kuroda
et al. for RPMI 8226 cells [
28] and indicates that the expression of cyclin D1 amplifies the response when the apoptotic machinery is activated.
We found that cyclin D1 affected at least two of the three branches of UPR and enhanced an ER stress-induced apoptosis, regardless of genetic background of the HMCL. Plasma cells are terminally differentiated B lymphocytes that secrete immunoglobulins and have a high demand for protein synthesis and exocytosis [
40]. The survival of MM cells is highly dependent on the UPR, and inefficient or prolonged UPR activation generates ER stress that can signal apoptosis [
41,
42]. In MM cells, the three arms of the ER stress program are activated, but the CHOP response is limited. Moreover, MM cells can increase the ER stress response further, with ATF4 and ATF6 coordinately inducing
CHOP transcription. This causes a shift in the ER stress from a protective to a destructive mechanism. Bortezomib, a potent antimyeloma compound, takes advantage of this property and upregulates PERK activity [
36]. In the cell models we developed, cyclin D1 expression disrupted the UPR balance, favoring death and amplifying the effects of bortezomib on the PERK/CHOP axis.
The mechanism by which cyclin D1 modifies the UPR has not yet been determined. ATP, Ca
2+ and an oxidizing environment are required for correct protein folding [
43]. We previously reported that cyclin D1 inhibits mitochondrial activity in B-cell lymphoma, by competing with hexokinase 2 for binding to the voltage-dependent anion channel [
10]. Cyclin D1 binding modifies the flux of ATP/ADP/Pi metabolites, with a potentially major impact on protein synthesis and folding. Further experiments are required to confirm this hypothesis in our cell models.
Pro- and antiapoptotic members of the BCL2 family are crucial regulators of apoptosis in plasma cells. Several studies have demonstrated that BCL2 and MCL1 are essential for the survival of plasma cell survival [
44,
45]. Consistent with these findings, BCL2 and MCL1 levels rapidly decrease in cyclin D1-expressing cells (
i.e. in cells programmed for death). The MCL1 and BCL2 proteins regulate the apoptotic pathway mediated by mitochondria, but they are also involved in ER stress-mediated apoptosis. Indeed, chemical treatments or changes in the microenvironment lead to severe ER stress, initiating apoptosis. This process is mediated by the induction of CHOP, which eliminates BCL2, thereby counteracting the anti-apoptotic function of this protein [
37]. It has recently been shown, in MM cells, that the UPR and, more specifically, the PERK-eIF2α-ATF4 branch of this pathway, regulates MCL1 levels [
46,
47]. Thus, both the apoptotic and UPR pathways are involved in the degradation of BCL2 and MCL1. We found that caspase activation was correlated with the decrease in BCL2 and MCL1 levels after bortezomib-treatment. Bortezomib inhibits the proteasome, so the degradation of BCL2 and MCL1 may be essentially due to caspase-mediated cleavage, as previously suggested [
48-
50], rather than degradation by the proteasome [
51]. This would be consistent with a master role for caspases in the downregulation of BCL2 and MCL1 after bortezomib treatment.
Acknowledgements
We would like to thank A Barbaras for technical assistance with cell cultures, D Lambert for whole-genome expression analysis, S Body for immunocytochemistry assays, F Baran-Marszak, C Pellat-Deceunynck and F Gouilleux for critical reading of manuscript, D Solomon (UCSF, School of Medicine, San Francisco, CA) for the p-cyclin D1-EGFP plasmid, and the technical platforms for flow cytometry and microscopy (SFR ICORE, Université de Caen Basse-Normandie). This work received financial support from the Fondation de France (Engt n°201200029144) and Comité de l’Eure de la Ligue contre le Cancer (to BS). SB was financially supported by the Ministère de l’Enseignement Supérieur et de la Recherche, JC by the Conseil Régional de Basse-Normandie.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SB conceived and designed the study, carried out most of the experiments, interpreted the data and drafted the manuscript. JC participated in study design, experiments and data analysis. PG carried out the transcriptomic and statistical analyses. BS conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.