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The enigmatic role of cyclin D1 in multiple myeloma

2005; Wiley; Volume: 115; Issue: 2 Linguagem: Inglês

10.1002/ijc.20907

1097-0215

Denis Lesage, Xavier Troussard, Brigitte Sola,

Microtubule and mitosis dynamics

Multiple myeloma (MM) reflects the proliferation of terminally differentiated plasma cells with low proliferative capacities, synthesizing monoclonal immunoglobulin (Ig). Tumoral cells are post-germinal centre cells that have been subjected to rearrangement of variable/diversity/joining (VDJ) sequences of Ig heavy chain (IgH) genes, isotype switching and somatic hypermutation.1, 2 As the result of illegitimate switch recombinations, reciprocal translocations involving the IgH locus located on chromosome 14q32 arise in MM cells with a frequency of 50–70%.3, 3, 3, 6 Chromosomal translocation results in the cis-activation of candidate oncogenes located on chromosomal partners by enhancer elements within IgH genes. Several recurrent translocations, likely relevant to pathogenesis, have been identified: t(11;14)(q13;q32), t(4;14)(p16;q32), t(14;16)(q32;q23), t(6;14)(p25;q32), t(6;14)(p21;q32) targeting cyclin D1/PRAD1/BCL-1, FGFR3/MMSET, c-Maf, IRF4, cyclin D3, respectively.7, 8, 9, 10, 11, 12, 13 Analyzing the data of gene expression profiling from 2 different laboratories,14, 15 Bergsagel and Kuehl reported that MM tumors deregulate at least 1 of the 3 cyclin D genes.16 They established a classification for subtypes of MM (TC classification, presented in Table I) based both on the presence of a translocation and on the type of expressed cyclin. Indeed, cyclin D1 is present in almost 40% of MM cells lacking the t(11;14), and cyclin D2 exhibited an increased expression in the remaining tumors. This classification could help in predicting prognosis and response to treatments.17 They proposed that the deregulation of one cyclin D-type renders cells more susceptible to proliferative stimuli and stated that this mechanism is a unifying oncogenic event in MM. All cyclin D-types, acting as sensors of external stimuli, control the G1 and S phases of cell cycle and, in turn, cell proliferation. All cyclin Ds have been recognized as proto-oncogenes, and overexpression of cyclin D1 is commonly observed in human mature B-cell hemopathies.18, 19 Indeed, in addition to MM, cyclin D1 is overexpressed in more than 90% of mantle cell lymphomas (MCL), all harboring the t(11;14)(q13;q32) activating translocation and in a large subset of hairy cell leukemias (HCL, 50–65%) for which the mechanism of cyclin D1 gene activation is unknown.20 This review focuses on the role of cyclin D1 in the pathogenesis of MM and underlines some unexpected facts. We present evidence arguing that the presence of cyclin D1 does not give proliferative advantage to tumor cells. As the role of cyclin D1 as a transcriptional regulator is being more documented, we speculate on its involvement in MM pathogenesis. The t(11;14)(q13;q32) is the most frequent translocation occurring in MM,3, 5, 21, 22 i.e., in 15–25% of the cases. In MM cell lines, as well as in MM primary tumors, the breakpoints on 14q13 are coincident with switch region or JH region, indicating that errors occurred either during switch recombination or somatic hypermutation.4, 7 Chromosomal breakpoints on 14q32 are dispersed over a region of 350 kb centromeric to cyclin D1 encoding CCND1 gene (Fig. 1). In all cases, CCND1 coding sequences are placed under the control of a strong enhancer on der(14).5, 7, 23 Cyclin D1, normally not expressed in B cells nor in plasma cells,24 is then overexpressed in t(11;14)-bearing cells, although the precise mechanism of such an upregulation is unknown. Cyclin D1 is an integrator of mitogenic or oncogenic factors. In response to appropriate signals, cyclin D1 accumulates, associates with and activates its kinase partner, the cyclin-dependent kinase (CDK)-4 or -6 early in the cell cycle. CDK/cyclin D1 complexes initiate the phosphorylation and then the inactivation of pRb tumor suppressor protein, allowing the progression through the G1 phase and the transition G1 to S (Fig. 2). Cyclin D1 is regulated at several levels including transcriptional activation, protein accumulation, protein degradation and nuclear export. Any perturbation of those regulation steps as well as any alteration upstream of the pRb pathway is thought to give a proliferative advantage to modified cells by comparison with normal cells and, in turn, to participate in the tumorigenic process.25, 26 However, clinical observations, in vitro studies on MM cell lines or primary cells and finally mouse models argue against the statement that overexpression of cyclin D1 contributes to MM pathogenesis through uncontrolled proliferation. Schematic representation of chromosomes 11 and 14 breakpoints in multiple myeloma (MM). The IgH locus on chromosome 14q32 contains 3 different enhancers (closed circles), the 5′Eμ intronic enhancer located in the intron between the IgH joining (J) and switch μ (Sμ) sequences and 2 3′Eα1 and Eα2 enhancers downstream of the constant region. In MM tumoral cells, breakpoints (heavy arrows) occur in the switch region and juxtapose the CCND1 gene and the 3′Eα1 and/or Eα2 enhancers. Occasionally, breakpoints are in the VDJ region. In that case, CCND1 is placed under the control of the 5′Eμ enhancer. Within the BCL-1 region, the MTC breakpoint locus is restricted to 360 kb flanked by the CCND1 and MYEOV genes. MYEOV, identified as an oncogene in a gastric carcinoma, can be overexpressed in MM cells57 but its expression is lost in most cases, arguing against a prominent role.46 Schematic representation of the G1 to S phase transition. Mitogenic factors exert their effects during the G1 phase. Progression trough G1 phase and G1 to S transition are controlled by the phosphorylarion of pRb family members (pRb, p107, p130). Hypophosphorylated pRb proteins sequester E2F family members and thus prevent E2F-dependent gene activation. Upon appropriate signals, cyclin D1 associates with its kinase partners CDK4 or CDK6, phosphorylates pRb and thereby allows the transcription of genes required for the progression G1 to S, notably cyclin E. The complete inactivation of pRb is achieved by its hyperphosphorylation, which is mediated by the cyclin E/CDK2 complexes. The restriction point R defines the point after which cells are committed to undergo DNA synthesis and are no longer sensitive to external stimuli. Activity of cyclin/CDK complexes are regulated by inhibiting/activating phosphorylation and by association/dissociation of CDK inhibitors (CKI) belonging to the INK4 (p15INK4b, p16INK4a, p17INK4c, p18INK4d) and CIP/KIP (p21CIP1, p27KIP1, p57KIP2) families. For a more detailed review on cyclin D1 cascade during the cell cycle, see Sherr and McCormick.58 In sharp contrast with initial reports in which the amplification of cyclin D1 gene or the presence of t(11;14) were unfavorable prognostic factors27, 28, 29, 30 or in which the t(11;14) displays no association with the patients' survival or therapeutic response or other known parameters,4, 5, 31, 32 the presence of cyclin D1 was more recently found a favorable parameter. Indeed, MM patients with the t(11;14) or a chromosome 11 trisomy, i.e., all expressing cyclin D1, show better overall survival, better response to intensive chemotherapy and longer duration of remission after autologous transplantations (Table II).33, 34, 35, 36, 37 Those observations are in good correlation with a lower proliferative index of cyclin D1-expressing MM cells. Indeed, the proliferative activity measurement, assessed using BrdU labeling index, indicates that patients with t(11;14), i.e., expressing cyclin D1, have a lower proliferative index than the others.37, 38 The speculation that overexpression of cyclin D1 triggers MM cell proliferation is not supported by those clinical data. This apparent discrepancy between the supposed role of cyclin D1 in favoring cell proliferation and the nondividing state of cyclin D1-expressing MM cells is further reinforced by studies on monoclonal gammopathy of undetermined significance (MGUS) and systemic light chain amyloidosis (AL). Although MM cells are low proliferating cells, the S-phase fraction is higher than for normal plasma cells.39 By contrast, in MGUS and AL, the proportion of abnormal plasma cells within the S fraction is comparable to the one of normal plasma cells, and translocations involving the IgH locus are frequent, notably the t(11;14)(q13;q32).40, 41, 42 The t(11;14)(q13;q32) occurs in 15–20% of MM cells; however, cyclin D1 is detected in 40–50% of cases with various techniques including the highly sensitive real-time quantitative RT-PCR (Table III).31, 32, 33, 43, 44, 45 In the absence of t(11;14)(q13;q32), the only chromosomal abnormality associated with cyclin D1 expression is a polysomy of chromosome 11.33, 43, 44, 46 The use of real-time quantitative PCR demonstrates that the level of cyclin D1 expression was of 3 types depending on the chromosome 11 abnormality: high expression with t(11;14)(q13;q32), intermediate expression with trisomy 11 and very weak expression with no abnormality.33, 47 Interestingly, the presence of cyclin D1 whatever the associated chromosome 11 abnormality or in the absence of overt chromosome 11 abnormality gives the same prognostic advantage.33 On a clinical point of view, the characterization of cyclin D1-expressing patients is particularly important but not formally the detection of t(11;14). Alterations of the cyclin D1/p16INK4A/pRb pathway are frequent in MM.48 RB1 deletions occur in 30% of the patients and up to 70% in MM cell lines.49 In the remaining cases, it has been reported that pRb is predominantly phosphorylated.49 We have analyzed the expression of cyclin D1 and of cyclin D1 partners in MM cell lines with a well-defined cytogenetic status.43 Karpas 620 possesses a t(11;14)(q13;q32), U266 has a switch insertion 10 kb upstream of the CCND1 promoter, RPMI 8226 and NCI-H929 have a trisomy 11 and LP-1 and OPM-2 display no chromosome 11 abnormalities. As depicted in Figure 3, the cyclin D1 level is in good correlation with cytogenetic data. But pRb is expressed in all out of one cell line in agreement with a previous report,50 although at various levels. The Ser807/811 phosphorylated form of pRb, specific of CDK4/6/cyclin D1 activity, is also variously expressed and without any correlation with cyclin D1 level. This lack of pRb hyperphosphorylation is probably the result of the absence of upregulation of CDK4/6, which becomes limiting for the catalytic activity and explains the low proliferative capacity of MM cells. The presence of high levels of cyclin D1 in cell lines derived from MM does not correlate with CDK4/6 activity on pRB in MM cell lines. Lack of correlation between cyclin D1 level and CDK4/6 activity in multiple myeloma (MM) cell lines. MM cell lines were Karpas 620, U266, RPMI 8226, NCI-H929, LP-1 and OPM-2. As control of cyclin D1 expression, we used GRANTA-519, a mantle cell lymphoma cell line, with the t(11;14)(q13;q32). Ramos is a Burkitt lymphoma cell line, which responds to serum (FCS) re-stimulation after starvation in proliferating. Whole-cell extracts were prepared as previously described.59 Proteins were submitted to SDS-PAGE (8–12%); then gels were transferred into nitrocellulose membranes and immunoblotted with the indicated antibodies (Abs). Abs were purchased from Cell Signalling (Beverly, MA) (anti-phospho-pRb, #9308), BD Pharmingen (Palo Alto, CA) (anti-pRb, #554136), Santa Cruz Biotechnology (Santa Cruz, CA) (anti-cyclin D1, sc-718; anti-CDK4, sc-260; anti-CDK6, sc-177, anti-b-tubulin, sc-9104) and Oncogene Research Products (San Diego, CA) (anti-cyclin D3, no. CC13). Blots were revealed using the Super Signal West Pico chemiluminescent substrate (Pierce, Erembodegem, Belgium) and data acquired with the FluorSImager (Bio-Rad, Marnes la Coquette, France). In another experimental model, we have found that the ectopic expression of cyclin D1 in BaF3 lymphoid cells blocks the cell cycle at the G1 phase and potentiates the apoptosis triggered either by p53-independent (interleukin withdrawal) or by p53-dependent (anticancer drugs) signals.51 Interestingly in this model, G1-arrest and apoptosis amplification are promoted through the activation of cyclin D1/CDK4 complexes. Although cyclin D1 is regarded as a proto-oncogene,52 cyclin D1 is not oncogenic per se in vivo in B cells. Cyclin D1-transgenic mice (Eμ-cyclin D1) show almost no alterations of proliferation capacity of bone marrow B cells and do not develop lymphoid tumors. However, cyclin D1 can cooperate with Myc to provoke the development of clonal pre-B or B lymphomas.53, 54 This indicates that cyclin D1 is not a potent oncogene unless acting with strong oncogenes such as Myc or Ras and has little effect on B-cell proliferation. Therefore, the fact that cyclin D1 enhances proliferative capacity of expressing B cells is still not experimentally demonstrated. Altogether the previous data indicate that the pathologic function of cyclin D1 in tumoral cells is not simply to promote proliferation. Moreover, the oncogenic activity of cyclin D1 must be exerted through a pathway different from the CDK-dependent regulation of cell cycle. Cyclin D1 is also known as a transcriptional regulator affecting the activity of several transcription factors such as estrogen receptor, androgen receptor, cyclin D1-interacting myb-like protein or DMP1, signal transducer and activator of transcription-3 or STAT-3, NeuroD, MyoD, thyroid receptors (Fig. 4).55 Moreover, the modulation of transcriptional activity is CDK-independent. Recently, using gene expression profiling in solid epithelial tumors, Lamb et al. have identified the transcription factor C/EBPβ as a constitutive repressor of cyclin D1 target genes and that cyclin D1 acts by antagonizing this repression function in tumors.56 They speculate that cyclin D1 perturbs the differentiation-specific transcriptional program controlled by C/EBPβ and in turn contributes to its oncogenic function. Such a global analysis of gene expression has been done for MM cells as well as MGUS and normal plasma cells.14, 15 Interestingly, novel candidate genes have been identified that discriminate between normal and tumoral cells, many of them being involved in adhesion, apoptosis, cell cycle and growth arrest but also transcription and signaling. Among them, CEPBD is downregulated in MM cells vs. normal bone marrow plasma cells.14 In fine, we propose that CDK-independent and transcriptional functions of cyclin D1 are required for the development of malignant plasma cells but also more generally in B-cell tumorigenesis. Such cyclin D1 functions should be studied in detail to build new therapeutic strategies.5 Examples of mechanisms of cyclin D1 action. (a) Hypophosphorylated pRb sequesters E2F/DP1 complexes and in turn represses genes whose products are necessary for cell cycle progression. Cyclin D1 associates with CDK4, phosphorylates pRb, liberating E2F transcription factors and leading to the activation of genes controlling G1 progression and S transition. (b) Dimers of activated androgen receptors (AR) bound on ARE (androgen-responsive elements) on DNA are able to drive the transcription of androgen-responsive genes. Cyclin D1 is able to form directly specific complexes with AR and to inhibit the transcription of target genes.60 Cyclin D1 activities potentially involved in tumorigenesis. The authors thank the members of the laboratory: Ms. A. Barbaras, Ms. J. Gauduchon, Ms. S. Krieger and Mr. M. Roussel. Laboratory studies on multiple myeloma have been financially supported by ARERS-Verre Espoir (no. 236.01 to B.S.), Fondation de France (no. 2002004328 to B.S.) and Ligue Nationale contre le Cancer—Comité de la Manche et Comité du Calvados (to B.S.).