Cyclin K and cyclin D1b are oncogenic in myeloma cells
© Marsaud et al; licensee BioMed Central Ltd. 2010
Received: 4 September 2009
Accepted: 10 May 2010
Published: 10 May 2010
Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive.
To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis.
Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.
Cyclin D1 is a key actor for the development and progression of various cancers including hematological malignancies. The human CCND1 gene generates two mRNA species by alternative splicing . The two corresponding proteins cyclin D1a and D1b differ only in the last 55 amino acids of the carboxy-terminus. Both isoforms possess the N-terminal domain, necessary for retinoblastoma protein (pRb) binding, the cyclin box, required for cyclin-dependent kinase (CDK) binding and activation and the central region, implicated in transcriptional regulation. The PEST sequence which controls protein turn-over and the threonine 286 (Thr286), the site of phosphorylation by glycogen synthase kinase-3β which promotes the nuclear export of cyclin D1 and its degradation through the proteasome pathway [2, 3], are present only in cyclin D1a. The oncogenic potential of cyclin D1 seems restricted to the isoform b as shown in vitro [4–6]. In transgenic mouse models, inhibition of cyclin D1 proteolysis is the causative factor for mammary carcinomas and B-cell lymphomas [7, 8]. The mechanisms of cyclin D1b-mediated tumorigenesis are not fully understood and could depend on the cellular context and in particular on the concomitant expression of cyclin D1a.
Cyclin K is encoded by Kaposi sarcoma-associated herpes virus (KSHV), a human tumor virus associated with the development of Kaposi sarcoma and lymphoid malignancies in immunocompromised individuals, reviewed in . Cyclin K and cyclin D1 share sequence colinearity and identity. The tumorigenic properties of cyclin K have been demonstrated in transgenic animals in which the lymphocyte compartment has been targeted . In a similar transgenic model, cyclin D1a alone fails to induce leukemogenesis [11, 12].
Mantle cell lymphoma (MCL) and multiple myeloma (MM) are two hematological malignancies for which cyclin D1 expression has been recognized as an oncogenic event [13, 14]. Although cyclin D1a and D1b mRNAs are present in all MCL and MM samples tested, cyclin D1a protein is expressed predominantly [15, 16]. However, a role of cyclin D1b in the leukemogenic process cannot be ruled out. In order to study the oncogenic potential of cyclins D1b and K in the context of mature B cells, we generated several cell clones derived from LP-1 MM cell line, expressing either cyclin D1b, Myc or cyclin K oncogenes. LP-1 cell line was chosen because this MM cell line does not express any cyclin D1 isoform. We report here that cyclin D1b- and cyclin K-expressing LP-1 cells are tumorigenic in vivo in xenograft models. Genome-wide analysis allowed us to describe several mechanisms for cyclin D1b- and K-mediated oncogenesis.
Generation of LP-1-derived clones
LP-1 MM cell line which does not express cyclin D1 was chosen for the generation of stable transfected clones. GRANTA-519 MCL cell line has the t(11;14)(q13;q32) and expresses high level of cyclin D1a. LP-1 and GRANTA-519 cells were maintained in RPMI 1640 containing 10% fetal calf serum (FCS), L-glutamine and antibiotics (Lonza Verviers SPRL, Verviers, Belgium). pcDNA3-flagged cyclin K  (a generous gift of O. Coqueret), pcDNA3-c-Myc (a generous gift of D. Cappellen) and pcDNA3-cyclin D1b  encode for the full-length proteins, respectively. LP-1 cells were transfected by electroporation, selected with 500 μg/ml G418, cloned by limiting dilution in 96-well plates. Single clones were individually tested for exogenous protein expression. After three months in culture without loss of transgene expression, G418 was first reduced and finally removed.
Cell cycle analysis by flow cytometry
Exponentially growing LP-1-derived cells were plated at a density of 5 × 105 cells/ml, harvested 24 h later, fixed in ice-cold EtOH 80% in PBS. Cells were treated with 100 μg/ml RNase A and 20 μg/ml propidium iodide (PI) for 30 min at 37°C. Cells were analyzed with an Epics XL flow cytometer and data with the Expo™ 32 software (Beckman Coulter, Villepinte, France).
Matrigel invasion assay
LP-1-derived cells were suspended in FCS-free RPMI 1640 medium and 2 × 104 cells were placed in the upper chamber of transwell inserts coated with Matrigel (BD BioCoat Matrigel Invasion Chamber, BD Biosciences, Le Pont de Claix, France). In the lower compartment, we added RPMI 1640 medium plus 1% FCS. Plates were incubated for 4 h at 37°C to allow migration of cells. After incubation, inserts were carefully removed, washed, fixed and colored to allow cell counting. Results are expressed as the number of cells that invaded the Matrigel. Statistical analysis between two groups was done with the Student's t test.
The ability of individual cell to grow in semi-solid support was assayed using MethoCult® (StemCell Technologies, Grenoble, France) according to the manufacturer' instructions. Cells were prepared at a density of 3 × 103 cells/ml in Iscove's MDM plus 2% FCS; then added to the same volume (3 ml) of methyl cellulose containing phytohemagglutin-leucocyte conditioned medium (PHA-LCM) as source of growth factor. Cells were dispensed in triplicate in Petri dishes, incubated in humidified atmosphere at 37°C for 10 days. Colonies containing more than 50 cells were counted using inverted microscope and gridded scoring dish.
Methods for protein extraction, SDS-PAGE and immunoblotting were described previously .
In vivo engraftment experiments
Immunohistochemistry of tumor sections
Finefix-fixed paraffin embedded 4 μm-sections were deparaffinized in toluene twice for 5 min and rehydrated by using graded EtOH concentrations. After antigen retrieval in citrate buffer pH 6.2 (5 min, 85°C), immunohistochemical labeling with anti-CD138 or anti-CD34 antibodies (Abs) was performed with the Vector Vectastain Elite kit (Vector Laboratories, Burlingame, CA, USA) and 3',3' Diaminobenzidine (DAB) as chromogen. Sections were counterstained with hemalun.
Microarray hybridization, gene expression data and statistical analyses
For each cell line (LP-1cl1, LP-1K and LP-1D1b), total RNA was extracted from four independent cultures with Trizol reagent (Invitrogen, Cergy Pontoise, France) according to the manufacturer' instructions and used for expression analysis on a 25K human oligonucleotide microarray covering most of the known human transcripts. The 50 mers 5'-amino modified oligonucleotides from the RNG/MRC oligonucleotide collection  (information available at http://www.microarray.fr:8080/merge/index) were diluted to a final concentration of 50 mM in 50% dimethyl sulfoxide, 100 mM potassium phosphate (pH 8.0) and printed onto hydrogel-coated slides (Nexterion H slides, Schott, Jena, Germany) using a microGrid II arrayer (Genomic Solutions, Cambridge, UK). Total RNAs (200 ng) were amplified by linear PCR and labelled with Cy3 using Bioprime Array CGH Genomic Labelling System Kit (Invitrogen). Total RNA from one culture of LP-1cl1 cells was similarly amplified, labelled with Cy5 and used as a reference probe for hybridization. Each Cy3-labelled probe was co-hybridized with the Cy5 reference probe on microarrays in a G2545A oven (Agilent, Massy, France) at 60°C for 18 h. Microarrays were washed (10 min in 6× SSC, 0.005% Triton-X100; 5 min in 0.1× SSC, 0.0025% Triton-X100) and scanned with a G2565B scanner (Agilent). Raw data were extracted from scanned microarray images (.tif) using Feature Extraction Software v9.5 (Agilent) and normalized using the Quantile method adapted to bicolour microarrays. All the protocols used can be obtained by contacting the microarray and sequencing platform of the IGBMC (web site: http://www-microarrays.u-strasbg.fr/). In order to select genes that are differentially expressed among the three biological groups (LP-1cl1, LP-1K and LP-1D1b), we performed an analysis of variance using Cy5/Cy3 log2 ratios. To limit the error due to multiple tests, we used permutation of samples for controlling the false discovery rate . Genes with a p-value less than 0.01 were considered to be significant. Moreover, we filtered out genes with a fold change (FC). The FC between LP-1K and LP-1cl1 was calculated as the median value of the 4 replicates ratios in the LP-1K samples over the median value of the 4 replicates ratios in the LP-1cl1 samples. Three FC were calculated: LP-1K vs. LP-1cl1, LP-1D1b vs. LP-1cl1 and LP-1K vs. LP-1D1b and a threshold equal to 2 was used for selecting three lists of significant genes. To design Venn diagram, we used the VENNY software http://bioinfogp.cnb.csic.es/tools/venny/ and individual gene expression profiles were generated with the TigrMev 4_03 software http://www.tm4.org/mev.html. To determine functional relationships between genes, we used DAVID Bioinformatics Resources http://david.niaid.nih.gov.
Real-time quantitative RT-PCR
To validate the microarray data, we used RNAs previously used for microarray hybridization. Primers for 36B4, CSN2, FGFR3, FHIT, HSP90B1, TUBB2B, TFRC, CD48, LTB, FN1, BCL2, CDK6, GAPDH and UCHL1 genes were designed with the LightCycler® Probe Design software (Roche Diagnostics, Meylan, France). Their sequences are reported in the Additional File 1, Table S1. Q-PCR was carried out in a LightCycler® system (Roche Diagnostics) using the LightCycler® FastStart DNA master SYBR Green I kit (Roche Diagnostics) according to the manufacturer's instructions. Cycles were as follows: a 10 min initial cycle at 95°C, followed by 45 cycles of 10 sec of denaturation at 95°C, 5 sec of annealing at 58°C, and 10 sec of extension at 72°C. The specificity of the fluorescence was verified by the melting curve analysis after each reaction. The relative abundance of each target was normalized to 36B4 expression and the quantification of each mRNA compared to 36B4 was done using the comparative threshold method (Ct).
Tumor engraftment onto chick chorio-allantoic membrane
Fertilized chicken eggs (EARL Morizeau, Dangers, France) were handled as described previously . On embryonic day 10, a plastic ring was placed on chick chorio-allantoic membrane (CAM) and 107 LP-1K or LP-1D1b cells in 30 μl Matrigel (BD Biosciences) were deposited after gentle laceration of the surface. Digital pictures were taken under a stereomicroscope (Nikon SMZ1500) at day 2, 4, 6 of tumor development. Twenty eggs were used for each condition.
Cyclin D1b, cyclin K and c-Myc expressing LP-1-derived clones display tumorigenic properties
Stable LP-1 clones were generated by transfection of cyclin D1b-, cyclin K- or c-Myc-expressing pcDNA3 plasmids or empty pcDNA3 as control. As shown Figure 1a, in the two clones LP-1 D1b (1 and 2), the short isoform b of cyclin D1 was expressed (clone 1) or overexpressed (clone 2) at a level comparable to the one in GRANTA-519 MCL cell line which possesses the t(11;14)(q13;q32) and synthesizes high level of cyclin D1a. Endogenous c-Myc was present in the control LP-1 pcDNA3 clone 1, and exogenous c-Myc was overexpressed (×5) in the two LP-1 c-Myc-expressing clones. In the LP-1 CK clone, cyclin K was detected with the anti-Flag M2 Ab. A representative clone from each series (star in Figure 1a), thereafter referred as LP-1cl1 (control), LP-1K, LP-1 Myc or LP-1D1b was injected s.c. into a first set of five nude mice. Eight weeks after injection, tumors were present at the site of inoculation in 4/5 mice for LP-1K, 5/5 mice for LP-1 Myc and 3/5 mice for LP-1D1b (Figure 1b) but not in mice inoculated with the control clone LP-1cl1. Only one mouse developed a palpable lump (pseudo-tumor, which regresses spontaneously). Macroscopically, tumors were distinguishable from one clone to the other, cyclin D1b-induced tumors being bigger and highly vascularized. After hematoxilin-eosin-safran (HES) staining of fixed tumor sections, histology revealed the presence of typical malignant plasma cells (Figure 1b). In a second series of in vivo experiments, 10 animals per cell line were inoculated. Four weeks after injection, tumors were detected at the site of inoculation in 10/10 mice for LP-1K and 6/10 mice for LP-1D1b (Figure 2a). Five mice from each series were sacrificed and the others monitored for four more weeks. At that time, four more mice in the LP-1D1b series bore tumors. The most striking differences between the two series were the size of the tumors (Figure 2a) and again the rich vascularization of LP-1D1b tumors (data not shown). Immunohistological examination of tumor sections indicated that engrafted tumors contained bona fide myeloma cells expressing CD138 (Figure 2b). Our data show unambiguously that such as c-Myc, cyclin D1b and cyclin K are capable to confer a malignant phenotype to LP-1 MM cells and are oncogenic in vivo.
Cyclin D1b and cyclin K are not mitogenic in LP-1 cells
We used flow cytometry sorting of PI-stained exponentially growing cells to assess the cell proliferation capacities of LP-1-derived clones. As presented in Figure 2c, the overexpression of cyclin D1b, cyclin K or c-Myc did not enhance the percentage of cells within the S phase of the cell cycle. By contrast, both LP-1D1b and LP-1K exhibited spontaneous apoptosis. In LP-1K cells, we observed a concomitant decrease of DNA synthesizing cells. We concluded from these data that the oncogenic properties acquired by LP-1 cells do not rely on an exacerbated proliferation potential.
Cyclin D1b and cyclin K expression alter LP-1 cells transcriptome
Cyclin D1b and cyclin K alter cell cycle and survival genes expression
Real-time quantitative RT-PCR for validation of microarray data
ΔCt (Ct LP-1D1b-Ct LP-1cl1)
ΔCt (Ct LP-1K- Ct LP-1cl1)
Genes coding for cell cycle regulatory molecules displaying altered expression in LP-1 derivatives (|FC|>3)
LP-1D1b vs. cl1
LP-1K vs. cl1
DNA-damage-inducible transcript 3
Cyclin B1 interacting protein 1
Ras association (RalGDS/AP-6) domain family member 5
Cyclin-dependent kinase inhibitor 1A
CDK5 and ABL enzyme substract 1
MAD2 mitotic arrest deficient-like 1
Growth-arrest specific 2
Antigen identified by monoclonal antibody Ki67
PIN2-interacting protein 1
CDC28 protein kinase regulatory subunit 2
Chromatin modifying protein 1A
Cyclin-dependent kinase 6
Cyclin-dependent kinase inhibitor 3
Cyclin-dependent kinase inhibitor 2C (p18)
Cyclin-dependent kinase inhibitor 2B (p15)
Genes coding for signalization molecules displaying altered expression in LP-1 derivatives (|FC|>3)
LP-1D1b vs. cl1
LP-1K vs. cl1
Fibroblast growth factor receptor 3
v-akt oncogene homolog 3
HGFR, Met proto-oncogene
Inositol 1,4,5-triphosphate 3-kinase A
Phosphoinositide 3-kinase gamma
Docking protein 6
MAP kinase 13
Protein kinase D2
Dual specificity phosphatase 6
Src kinase associated phosphoprotein
Spleen tyrosine kinase
MAP kinase 12
B lymphoid tyrosine kinase
The large number of genes and pathways altered by cyclin D1b and/or cyclin K expression precludes a thorough analysis in this manuscript. We focused on two discrete functions of cyclins D-type identified by the microarray analysis and well-known as support for tumorigenic process: cell migration and angiogenesis.
Cyclin K inhibits migration of LP-1-derived clones and enhances its clonogenic capacities
Genes coding for molecules controlling adhesion and movement displaying altered expression in LP-1 derivatives (|FC|>3).
LP-1D1b vs. cl1
LP-1K vs. cl1
Chemokine (C-X-C motif) receptor 1
FXY domain containing ion transport regulator 5
Chemokine (C-X-C motif) ligand 12
RhoGDP dissociation inhibitor (GDI) beta
SUT homolog 3
Intracellular adhesion molecule 3
Laminin beta 3
Contactin associated protein-like 2
Melanoma cell adhesion molecule
Chemokine (C-C motif) ligand 2
Spleen tyrosine kinase
Laminin alpha 3
Erbb2 interacting protein
Neuronal cell adhesion molecule
Integrin beta 2
ADAM metallopeptidase domain 23
Connective tissue growth factor
CD 44 molecule
Contactin associated protein-like 2
Selectin P ligand
Polycystic kidney disease
Sialic acid binding Ig-like lectin 7
L1 cell adhesion molecule
Collagen type XVIII alpha 1
ADAM metallopeptidase domain 15
Sialic acid binding Ig-like lectin 9
Neogenin homolog 1
Integrin alpha 6
Integrin alpha E
Collagen type XXIV alpha 1
Junctional adhesion molecule 3
Junctional adhesion molecule 2
Cyclin D1b allows neo-angiogenesis of engrafted tumors
Genes coding for proangiogenic or antiangiogenic molecules displaying altered expression in LP-1 derivatives (|FC|>3).
LP-1D1b vs. cl1
LP-1K vs. cl1
Fibroblast growth factor receptor 3
Tumor necrosis factor receptor superfamily member 10 B
GATA binding protein 4
Insulin-like growth factor binding protein 3
Endothelial PAS domain protein 1
CXC chemokine ligand 12
SLIT homolog 3
Chemokine ligand 2
CXC chemokine ligand 16
Runt-related transcription factor 1
EGF-like domain multiple 7
Connective tissue growth factor
Tumor necrosis factor superfamily member 13
Serpin peptidase inhibitor clade B member 6
Platelet-derived growth factor beta
CXC chemokine ligand 3
Serpin peptidase inhibitor clade E member 2
Serpin peptidase inhibitor clade B member 1
Matrix metallopeptidase 13
Interferon gamma receptor 1
Heart and neural crest derivatives
Protein tyrosine phosphatase receptor type F
Runt-related transcription factor 1 translocated to 1
Inhibitor of DNA binding 1
Laminin alpha 3
Interferon gamma-inducible protein 16
Zinc finger protein 36
Zinc finger protein 36 C3H type-like 2
Bone morphogenetic protein receptor type 1A
Protein tyrosine phosphatase receprot type M
Jun dimerization protein 2
Insulin-like growth factor 2 binding protein 1
Collagen type IX alpha 1
Collagen type XXIV alpha 1
Death-associated protein kinase 1
Cyclin D1 is overexpressed in a broad range of solid malignancies, expressed in lymphoid tumors such as MM and MCL and not in their normal counterparts. However, in vivo studies failed to reveal a strong oncogenic potential of the conventional cyclin D1, referred to cyclin D1a [11, 12]. By contrast, the cyclin D1 isoform b and the mutant cyclin D1 T286A are capable to transform cells in vitro [4–6] and to induce tumors in vivo [7, 8]. These two forms of cyclin D1 share a strict nuclear localization suggesting that nuclear functions of cyclin D1 are necessary and/or sufficient for tumor formation. Mutations of the CCND1 gene disrupting the phosphorylation at Thr286 and thereby leading to nuclear accumulation of cyclin D1 have been described in endometrial and esophageal carcinomas further reinforcing this notion [25, 26]. However, the molecular mechanisms of cyclin D1b-driven tumorigenesis are not fully elucidated. In cultured cells, cyclin D1b is not capable to activate its catalytic partner CDK4 and in turn, does not regulate positively the cell cycle [5, 18], retains a strong transcriptional co-repressor activity, displays reduced binding to p27Kip1 and does not control cell migration . Here we show that, in the context of MM cells, cyclin D1b confers a full malignant phenotype and allows cells engraftment in immune-compromised mice. The genome-wide analysis of LP-1D1b cells extends our understanding of the biological properties of cyclin D1b. Moreover, we have identified genes regulated by cyclin K, a viral oncogenic homolog of cyclin D1a and confirm the fundamental differences between the two cyclin D1 isoforms.
Cyclin D1b and cyclin K alter LP-1 cells metabolism
The tumorigenic properties of cyclins D1b and K are not conferred by an exacerbated proliferation. LP-1D1b and LP-1K cells display the same proliferation properties and cyclin D1b or cyclin K expressions have no major impact on cell cycle regulation. Conversely, genes involved in metabolism, signal transduction, transport, transcriptional and translational regulations are profoundly altered by cyclin D1b and/or cyclin K. In vivo, cyclin D1 inhibits oxidative glycolysis, lipogenesis, and mitochondrial gene activity in the mammary epithelium [27, 28]. In both LP-1K and LP-1D1b cells, the gene transcription of LDHA (lactate dehydrogenase, FC: -4.37 and -10.78, respectively), GAPDH (glyceraldehyde-3-phosphate dehydrogenase, FC: -4.94 and -3.17, respectively) and ALDOA (aldolase A, FC: -2.69 and -3.73, respectively) is decreased. These enzymes catalyze important energy-yielding steps in carbohydrate metabolism. The expression of genes coding for key enzymes involved in oxidative glycolysis such as pyruvate kinase (PKM2, FC: -3.57), phosphoglycerate kinase 1 (PGK1, FC: -2.10), enolase 1 (ENO1, FC: -2.32) in LP-1D1b cells; enolase 2 (ENO2, FC: -2.82) in LP-1K cells are down-regulated. This suggests a reduction of glycolysis in tumor cells and, therefore, such as in mammary tumor cells, a paradoxical role of cyclin D1 . Indeed, most of tumor cells show an enhanced glycolytic flux . However, only fast growing tumor cells display markedly modified energy metabolism and multiple myeloma cells are considered as accumulating cells rather than proliferating cells.
Cyclin D1b and cyclin K modulate gene transcription and translation within LP-1 cells
Genes coding for transcription factors displaying altered expression in LP-1 derivatives.
LP-1D1b vs. cl1
LP-1K vs. cl1
DNA-damage-inducible transcript 3
Activating transcription factor 3
X-box binding protein 1
Interferon regulatory factor 8
Runt-related transcription factor 1
Transcription factor 4
Microphtalmia-associated transcription factor
Notch homolog 2
Signal transduction and transcription factor 5A
Ets variant 6
v-maf musculoaponeurotic fibrosarcomaa oncogene homolog
v-myc myelocytomatosis viral oncogene homolog
v-ets erythroblastosis virus E26 oncogene homolog
Interferon regulated factor 4
Recombination signal binding protein for Ig kappa region
v-rel reticuloendotheliosis viral onvogene homolog
Runt-related transcription factor related 2
v-myb myeloblastosis viral oncogene homolog-like 2
Activating transcription factor 4
Eukaryotic initiation factors (eIFs) control translation at the limiting step of initiation and several of them have been recognized as major actors in transformation processes . In LP-1D1b cells, several genes coding for eIFs are upregulated (EIF4EBP1, FC: +3.35; EIF3EIP, FC: +2.79; EIF4A2, FC: +2.50; EIF3F, FC: +2.05; EIF1, FC: +2.02). By contrast, in LP-1K, EIF3A and EIF5 are downregulated (FC: -2.28 and -2.92, respectively). A more active translation likely explains the faster growth of LP-1D1b-derived tumors compared to LP-1K tumors.
Cyclin D1b and cyclin K have opposite action on LP-1 cells migration
Clinical observations indicate that cyclin D1 overexpression in human cancers correlate with metastasis. In cyclin D1-/- mouse embryonic fibroblasts, cyclins D1a and b have unique properties with regard to cell migration . Cyclin D1a stabilizes p27Kip1 and inhibits RhoA-induced ROCK kinase activity promoting cell migration while cyclin D1b fails to stabilize p27Kip1 and has no effect on cell migration. Our results confirm that cyclin D1b does not affect LP-1 cells migration. Although cyclin K resembles cyclin D1a in agreement with its known biological functions: binding to CDK4/6, phosphorylation of pRb; one prominent feature of its structure is the impairment of p27Kip1 binding . Accordingly, cyclin K expression in LP-1 is associated with the absence of p27Kip1, the lack of migration capacity and an enhanced clonogenic potential in vitro. Experiments assessing the metastatic potential of LP-1-derived cells in vivo are ongoing.
Cyclin D1b stimulates neoangiogenesis
Cyclin K/D1b-expressing cells, grafted onto the CAM of chicken embryo, generate within a few days tumors whose vascularization is significantly different. Tumors obtained in nude mice after s.c. injection of LP-1-derived cells show the same different vascularization. Indeed, LP-1 MM cells overexpressing cyclin D1b markedly promote tumor angiogenesis. Cyclin D1 regulates vascular endothelial growth factor (VEGF) production and thereby, growth of vascular endothelial cells and tumor . The inhibition of tumor growth after local injection of VEGF siRNA confirmed a major role of VEGF in tumor expesnion. This result was further reinforced by the use of VEGFR inhibitors which could target either the MM tumoral cells or their immediate environment. Cyclins D1b and K induce transcriptional activation/inhibition of proangiogenic/antiangiogenic factors. One striking difference between the two cell lines is the overexpression of FGFR3 in LP-1D1b cells. Activation of the fibroblast growth factor 3 (FGFR3) expressed by myeloma cells and its ligand FGF present in the mouse could sustain in vivo angiogenesis such as in the bone marrow milieu . The expression of 402 angiogenesis-associated genes has been studied in a large series of patients with a MM or a MGUS (monoclonal gammopathy of undetermined significance), considered as the premalignant state of MM, MM cell lines and their normal counterparts . This study concluded that aberrant expression of proangiogenic and downregulation of antiangiogenic genes occur in all MM patients. Interestingly for our purpose, we noted that three genes were silent in MGUS and expressed in MM, namely IL6, FGF9 and FGFR3. It is tempting to speculate that the expression of FGFR3 triggers premalignant cells to enter a malignant state as observed in our model.
Cyclin D1b and cyclin K activate major actors of MM tumorigenesis
Is cyclin D1b involved in MM pathogenesis?
We have previously shown that both isoforms of cyclin D1a and b mRNAs are present in MM cells and their relative levels similar. However, cyclin D1a isoform is predominant both in MM cell lines and primary cells . It has been thought that CCND1 alternative splicing was regulated by a G/A polymorphism at the exon 4/intron 4 boundary . It is now demonstrated that factors associated with chromatin remodelling and translation elongation largely contribute to cyclin D1b accumulation [43, 44]. This indicates that the regulation of cyclin D1b level is complex and only the direct analysis of the cyclin D1b protein could define its impact on disease. In a recent large multiethnic case-control study, Knudsen and his group showed that cyclin D1b is clearly elevated in a significant fraction of primary breast tumors but with a heterogeneous level within specimens and underexpressed in asynchronously proliferating cell lines [45, 46]. They also show unambiguously that cyclin D1b levels are associated with adverse prognostic outcome. Such an analysis of cyclin D1b protein level in MGUS, the primary step of MM and primary MM cells should be conducted in order to definitely conclude on its role in MM pathogenesis.
We thank Anne Barbaras for excellent help with cell culture, Dr J. Cahu for careful reading of the manuscript, Dr D. Cappellen (Friedrich Miescher Institute, Basel, Switzerland) for the gift of c-Myc construct, Dr O. Coqueret, (INSERM U564, CLCC Paul Papin, Angers, France) for the gift of cyclin K construct, Dr F. Bono (Sanofi Aventis, Toulouse, France) for the gift of chemical inhibitors. This work was supported by grants from the Ligue contre le Cancer - Comité du Calvados and Comité de la Manche (to BS). GT received a scholarship form Ligue contre le Cancer - Comité du Calvados and from the Société Française d'Hématologie.
- Betticher DC, Thatcher N, Altermatt HJ, Hoban P, Ryder WD, Heighway J: Alternate splicing produces a novel cyclin D1 transcript. Oncogene. 1995, 11: 1005-1011.PubMedGoogle Scholar
- Diehl JA, Cheng M, Roussel MF, Sherr CJ: Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 1998, 12: 3499-3511. 10.1101/gad.12.22.3499PubMed CentralView ArticlePubMedGoogle Scholar
- Alt JR, Cleveland JL, Hannink M, Diehl JA: Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev. 2000, 15: 3102-3114. 10.1101/gad.854900.View ArticleGoogle Scholar
- Lu F, Gladden AB, Diehl JA: An alternative spliced cyclin D1 isoform, cyclin D1b, is a nuclear oncogene. Cancer Res. 2003, 63: 7056-7061.PubMedGoogle Scholar
- Solomon DA, Wang Y, Fox SR, Lambeck TC, Giesting S, Lan Z, Senderowicz AM, Conti CJ, Knudsen ES: Cyclin D1 splice variants. J Biol Chem. 2003, 278: 30339-30447. 10.1074/jbc.M303969200View ArticlePubMedGoogle Scholar
- Holley SL, Heighway J, Hoban PR: Induced expression of human CCND1 alternative transcripts in mouse Cyl-1 knockout fibroblasts highlights functional differences. Int J Cancer. 2005, 114: 364-370. 10.1002/ijc.20750View ArticlePubMedGoogle Scholar
- Lin DI, Lessie MD, Gladden AB, Bassing CH, Wagner KU, Diehl JA: Disruption of cyclin D1 nuclear export and proteolysis accelerates mammary carcinogenesis. Oncogene. 2008, 27: 1231-1242. 10.1038/sj.onc.1210738PubMed CentralView ArticlePubMedGoogle Scholar
- Gladden AB, Woolery R, Aggarwal P, Wasik MA, Diehl JA: Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma. Oncogene. 2006, 25: 998-1007. 10.1038/sj.onc.1209147PubMed CentralView ArticlePubMedGoogle Scholar
- Javiluoma A, Ojala PM: Cell signaling pathways engaged by KSHV. Biochim Biophys Acta. 2006, 1766: 140-158.Google Scholar
- Verschuren EW, Hodgson JG, Gray JW, Kogan S, Jones N, Evan GI: The role of p53 in suppression of KSHV cyclin-induced lymphomagenesis. Cancer Res. 2004, 64: 581-589. 10.1158/0008-5472.CAN-03-1863View ArticlePubMedGoogle Scholar
- Bodrug SE, Warner BJ, Bath ML, Lindeman GJ, Harris AW, Adams JM: Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene. EMBO J. 1994, 13: 2124-2130.PubMed CentralPubMedGoogle Scholar
- Lovec H, Grzeschiczek A, Kowalski MB, Möröy T: Cyclin D1/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice. EMBO J. 1994, 13: 3487-3495.PubMed CentralPubMedGoogle Scholar
- Bergsagel PL, Kuehl WM: Molecular pathogenesis and a consequent classification of multiple myeloma. J Clin Oncol. 2005, 23: 6333-6338. 10.1200/JCO.2005.05.021View ArticlePubMedGoogle Scholar
- Jares P, Colomer D, Campo E: Genetic and molecular pathogenesis of mantle cell lymphoma: perspectives for new targeted therapeutics. Nat Rev Cancer. 2007, 7: 750-762. 10.1038/nrc2230View ArticlePubMedGoogle Scholar
- Marzec M, Kasprzycka M, Lai R, Gladden AB, Wlodarski P, Tomczak E, Nowell P, Deprimo SE, Sadis S, Eck S, Schuster SJ, Diehl JA, Wasick MA: Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase. Blood. 2006, 108: 1744-1750. 10.1182/blood-2006-04-016634PubMed CentralView ArticlePubMedGoogle Scholar
- Krieger S, Gauduchon J, Roussel M, Troussard X, Sola B: Relevance of cyclin D1b expression and CCND1 polymorphism in the pathogenesis of multiple myeloma and mantle cell lymphoma. BMC Cancer. 2006, 6: 238- 10.1186/1471-2407-6-238PubMed CentralView ArticlePubMedGoogle Scholar
- Lundquist A, Barré B, Bienvenu F, Hermann J, Avril S, Coqueret O: Kaposi sarcoma-associated viral cyclin K overrides cell growth inhibition mediated by oncostatin M through STAT3 inhibition. Blood. 2003, 101: 4070-4077. 10.1182/blood-2002-07-1994View ArticlePubMedGoogle Scholar
- Lévêque C, Marsaud V, Renoir JM, Sola B: Alternative cyclin D1 forms a and b have different biological functions in the cell cycle of B lymphocytes. Exp Cell Res. 2007, 313: 2719-2729. 10.1016/j.yexcr.2007.04.018View ArticlePubMedGoogle Scholar
- Le Brigand K, Russel R, Moreilhon C, Rouillard J-M, Jost B, Amiot F, Magnone V, Bole-Feysot C, Rostagno P, Virolle V, Defamie V, Dessen P, Williams G, Lyons P, Rios G, Mari B, Gulari E, Kastner P, Gidrol X, Freeman TC, Barbry P: An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes. Nucleic Acids Research. 2006, 34: e87- 10.1093/nar/gkl485View ArticlePubMedGoogle Scholar
- Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B. 1995, 57: 289-300.Google Scholar
- Hagedorn M, Zilberberg L, Wilting J, Canron X, Carrabba G, Giussani C, Pluderi M, Bello L, Bikfalvi A: Domain swapping in a COOH-terminal fragment of platelet factor 4 generates potent angiogenesis inhibitors. Cancer Res. 2002, 62: 6884-6890.PubMedGoogle Scholar
- Hurt EM, Wiestner A, Rosenwald A, Schaffer AL, Campo E, Grogan T, Bergsagel PL, Kuehl WM, Staudt LM: Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow. Cancer Cell. 2004, 5: 191-199. 10.1016/S1535-6108(04)00019-4View ArticlePubMedGoogle Scholar
- Li Z, Jiao X, Wang C, Ju X, Lu Y, Yuan L, Lisanti MP, Katiyar S, Pestell RG: Cyclin D1 induction of cellular migration requires p27KIP1. Cancer Res. 2006, 66: 9986-9994. 10.1158/0008-5472.CAN-06-1596View ArticlePubMedGoogle Scholar
- Takei Y, Kadomatsu K, Yuzawa Y, Matsuo S, Muramatsu T: A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res. 2004, 64: 3365-3370. 10.1158/0008-5472.CAN-03-2682View ArticlePubMedGoogle Scholar
- Moreno-Bueno G, Rodríguez-Perales S, Sánchez-Estévez C, Hardisson D, Sarrió D, Prat J, Cigudosa JG, Matias-Guiu X, Palacios J: Cyclin D1 gene (CCND1) mutations in endometrial cancer. Oncogene. 2003, 22: 6115-6118. 10.1038/sj.onc.1206868View ArticlePubMedGoogle Scholar
- Benzeno S, Lu F, Guo M, Barbash O, Zhang F, Herman JG, Klein PS, Rustgi A, Diehl JA: Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1. Oncogene. 2006, 25: 6291-6303. 10.1038/sj.onc.1209644View ArticlePubMedGoogle Scholar
- Sakamaki T, Casimiro MC, Ju X, Quong AA, Katiyar S, Liu M, Jiao X, Li A, Zhang X, Lu Y, Wang C, Byers S, Nicholson R, Link T, Shemluck M, Yang J, Fricke ST, Novikoff PM, Papanikolaou A, Arnold A, Albanese C, Pestell R: Cyclin D1 determines mitochondrial function in vivo. Mol Cell Biol. 2006, 26: 5449-5469. 10.1128/MCB.02074-05PubMed CentralView ArticlePubMedGoogle Scholar
- Wang C, Li Z, Lu Y, Du R, Katiyar S, Yang J, Fu M, Leader JE, Quong A, Novikoff PM, Pestell RG: Cyclin D1 repression of nuclear respiratory factor 1 integrates nuclear DNA synthesis and mitochondrial function. Proc Natl Acad Sci USA. 2006, 31: 11567-11572. 10.1073/pnas.0603363103. 10.1073/pnas.0603363103View ArticleGoogle Scholar
- Gogvadze V, Orreniius S, Zhivotovsky B: Mitochondria in cancer cells: what is so special about them?. Trends Cell Biol. 2008, 18: 165-173. 10.1016/j.tcb.2008.01.006View ArticlePubMedGoogle Scholar
- Fu M, Wang C, Li Z, Sakamaki T, Pestell RG: Cyclin D1: normal and abnormal functions. Endocrinology. 2005, 145: 5439-5447. 10.1210/en.2004-0959.View ArticleGoogle Scholar
- Lamb J, Ramaswamy S, Ford HL, Contreras B, Martinez RV, Kittrell FS, Zahnow CA, Patterson N, Golub TR, Ewen ME: A mechanism of cyclin D1 action encoded in the patterns of gene expression in human cancer. Cell. 2003, 114: 323-334. 10.1016/S0092-8674(03)00570-1View ArticlePubMedGoogle Scholar
- Gandin V, Miluzio A, Barbieri AM, Beugnet A, Kiyokawa H, Marchisio PC, Biffo S: Eukaryotic initiation factor is rate-limiting in translation, growth and transformation. Nature. 2008, 455: 684-688. 10.1038/nature07267PubMed CentralView ArticlePubMedGoogle Scholar
- Li Z, Wang C, Jiao X, Katiyar S, Casimiro MC, Prendergast GC, Powell MJ, Pestell RG: Alternate cyclin D1 mRNA splicing modulates P27KIP1 binding and cell migration. J Biol Chem. 2008, 283: 7007-7015. 10.1074/jbc.M706992200View ArticlePubMedGoogle Scholar
- Baek K, Brown RS, Birrane G, Ladias JA: Crystal structure of human cyclin K, a positive regulator of cyclin-dependent kinase 9. J Mol Biol. 2007, 366: 563-573. 10.1016/j.jmb.2006.11.057PubMed CentralView ArticlePubMedGoogle Scholar
- Yasui M, Yamamoto H, Ngan CY, Damdinsuren B, Sugita Y, Fukunaga H, Gu J, Maeda M, Takemasa I, Ikeda M, Fujio Y, Sekimoto M, Matsuura N, Weinstein IB, Monden M: Antisense to cyclin D1 inhibits vascular endothelial growth factor - stimulated growth of vascular endothelial cells: implication for tumor development. Clin Cancer Res. 2006, 12: 4720-4729. 10.1158/1078-0432.CCR-05-1213View ArticlePubMedGoogle Scholar
- Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC: Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007, 7: 585-598. 10.1038/nrc2189View ArticlePubMedGoogle Scholar
- Hose D, Moreaux J, Meissner T, Seckinger A, Goldschmidt H, Benner A, Mahtouk K, Hillengass J, Rème T, De Vos J, Hundemer M, Condomines M, Bertsch U, Rossi J-F, Jauch A, Klein B, Möhler T: Induction of angiogenesis by normal and malignant plasma cells. Blood. 2009, 114: 128-143. 10.1182/blood-2008-10-184226View ArticlePubMedGoogle Scholar
- Claudio JO, Masih-Khan E, Tang H, Gonçalves J, Voralia M, Li ZH, Nadeem V, Cukerman E, Francisco-Pabalan O, Liew CC, Woodgett JR, Stewart AK: A molecular compendium of genes expressed in multiple myeloma. Blood. 2002, 100: 2175-2186. 10.1182/blood-2002-01-0008View ArticlePubMedGoogle Scholar
- De Vos J, Thykjaer T, Tarte K, Ensslen M, Raynaud P, Requirand G, Pellet F, Pantesco V, Rème T, Jourdan M, Rossi JF, Ørntoft T, Klein B: Comparison of gene expression between malignant and normal plasma cells with oligonucleotide array. Oncogene. 2002, 21: 6848-6857. 10.1038/sj.onc.1205868View ArticlePubMedGoogle Scholar
- Davies FE, Dring AM, Li C, Rawstron AC, Shammas MA, O'Connor SM, Fenton JA, Hideshima T, Chauhan D, Tai IT, Robinson E, Auclair D, Rees K, Gonzalez D, Ashcroft AJ, Dasgupta R, Mitsiades C, Mitsiades N, Chen LB, Wong WH, Munshi NC, Morgan GJ, Anderson KC: Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis. Blood. 2003, 102: 4505-4511. 10.1182/blood-2003-01-0016.View ArticleGoogle Scholar
- Munshi NC, Hideshima T, Carrasco D, Shammas M, Auclair D, Davies F, Mitsiades N, Mitsiades C, Kim RS, Li C, Rajkumar SV, Fonseca R, Bergsagel L, Chauhan D, Anderson KC: Identification of genes modulated in multiple myeloma using genetically identical twin samples. Blood. 2004, 103: 1799-1806. 10.1182/blood-2003-02-0402View ArticlePubMedGoogle Scholar
- Heller G, Schmidt WM, Ziegler B, Holzer S, Müllauer L, Bilban M, Zielinski CC, Drach J, Zöchbauer-Müller S: Genome-wide transcriptional response to 5-aza-2'-deoxycytidine and trichostatin in multiple myeloma cells. Cancer Res. 2008, 68: 44-54. 10.1158/0008-5472.CAN-07-2531View ArticlePubMedGoogle Scholar
- Batsche E, Yaniv M, Muchardt C: The human SWI/SNF subunit Brm is a regulator of alternative splicing. Nat Struct Mol Biol. 2006, 13: 22-29. 10.1038/nsmb1030View ArticlePubMedGoogle Scholar
- Sanchez G, Bittencourt D, Laud K, Barbier J, Delattre O, Auboeuf D, Dutertre M: Alteration of cyclin D1 transcript elongation by a mutated transcripion factor up-regulates the oncogenic D1b splice isoform in cancer. Proc Natl Acad Sci USA. 2008, 105: 6004-6009. 10.1073/pnas.0710748105PubMed CentralView ArticlePubMedGoogle Scholar
- Wang Y, Dean JL, Millar EKA, Tran TH, McNeil CM, Burd CJ, Henshall SM, Utama FE, Witkiewicz A, Rui H, Sutherland RL, Knudsen KE, Knudsen ES: Cyclin D1b is aberrantly regulated in response to therapeutic challenge and promotes resistance to estrogen antagonists. Cancer Res. 2008, 68: 5628-5638. 10.1158/0008-5472.CAN-07-3170View ArticlePubMedGoogle Scholar
- Millar EK, Dean JL, McNeil CM, O'Toole SA, Henshall SM, Tran T, Lin J, Quong A, Comstock CE, Witkiewicz A, Musgrove EA, Rui H, Lemarchand L, Setiawan VW, Haiman CA, Knudsen KE, Sutherland RL, Knudsen ES: Cyclin D1b is aberrantly regulated in response to therapeutic challenge and promotes resistance to estrogen antagonists. Oncogene. 2009, 28: 1812-1820. 10.1038/onc.2009.13PubMed CentralView ArticlePubMedGoogle Scholar
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