RESUMEN
Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.
Asunto(s)
Movimiento Celular , Ciclina D1/metabolismo , Citoplasma/metabolismo , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/patología , Transporte Activo de Núcleo Celular , Adulto , Anciano , Anciano de 80 o más Años , Animales , Núcleo Celular/metabolismo , Transformación Celular Neoplásica , Citosol/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , ProteómicaRESUMEN
The interactions of multiple myeloma (MM) cells with their microenvironment are crucial for pathogenesis. MM cells could interact differentially with their microenvironment depending on the type of cyclin D they express. We established several clones that constitutively express cyclin D1 from the parental RPMI8226 MM cell line and analyzed the impact of cyclin D1 expression on cell behavior. We performed a gene expression profiling study on cyclin D1-expressing vs. control cells and validated the results by semi-quantitative RT-PCR. The expression of cyclin D1 altered the transcription of genes that control adhesion and migration. We confirmed that cyclin D1 increases cell adhesion to stromal cells and fibronectin, stabilizes F-actin fibers, and enhances chemotaxis and inflammatory chemokine secretion. Both control and cyclin D1-expressing cells were more resistant to acute carfilzomib treatment when cultured on stromal cells than in suspension. However, this resistance was specifically reduced in cyclin D1-expressing cells after pomalidomide pre-treatment that modifies tumor cell/microenvironment interactions. Transcriptomic analysis revealed that cyclin D1 expression was also associated with changes in the expression of genes controlling metabolism. We also found that cyclin D1 expression disrupted the redox balance by producing reactive oxygen species. The resulting oxidative stress activated the p44/42 mitogen-activated protein kinase (or ERK1/2) signaling pathway, increased cell adhesion to fibronectin or stromal cells, and controlled drug sensitivity.Our results have uncovered a new function for cyclin D1 in the control of redox metabolism and interactions of cyclin D1-expressing MM cells with their bone marrow microenvironment.
Asunto(s)
Ciclina D1/fisiología , Mieloma Múltiple/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Quimiocinas/metabolismo , Ciclina D1/genética , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacología , Microambiente TumoralRESUMEN
BACKGROUND: Cyclin D1 and its kinase partners control cell cycle progression. Cyclin D1 is frequently deregulated in various cancers, including malignant hemopathies, and tumor cells display uncontrolled cell proliferation. Cyclin D1 is not expressed in the B-cell lineage but is found in multiple myeloma (MM) cells in almost 50% of patients with this condition. Paradoxically, cyclin D1 expression is associated with a good prognosis and longer overall survival in MM patients. METHODS: We used two independent MM cell lines (RPMI 8226 and LP1) to generate several clones stably expressing either the green fluorescent protein (GFP) or a GFP-cyclin D1 fusion protein, and we analyzed the properties acquired following cyclin D1 expression. RESULTS: Whole-genome expression analysis in the cell clones indicated that cyclin D1 profoundly modified several cellular functions, including the regulation of apoptotic cell death. We studied the apoptotic response of GFP- and GFP-cyclin D1-expressing clones to bortezomib-treatment. We found that the apoptotic response occurred faster and was of a greater amplitude in cyclin D1-expressing cells. Cyclin D1 expression enhanced the caspase-dependent apoptosis mediated by the intrinsic mitochondrial pathway. More importantly, cyclin D1 also activated the unfolded protein response (UPR) and induced endoplasmic reticulum (ER) stress-mediated apoptosis. CONCLUSION: The ER is well known to be a crucial regulator of plasma cell death and it plays the same role in their malignant counterparts, myeloma cells. This role involves activation of the UPR controlled at least in part by cyclin D1.
Asunto(s)
Ciclina D1/biosíntesis , Mieloma Múltiple/genética , Proteínas de Neoplasias/biosíntesis , Respuesta de Proteína Desplegada/genética , Apoptosis/genética , Línea Celular Tumoral , Linaje de la Célula/genética , Proliferación Celular , Ciclina D1/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Proteínas de Neoplasias/genética , Pronóstico , Transducción de Señal/efectos de los fármacosRESUMEN
Deregulated expression of heat shock proteins (HSPs) encoding genes is frequent in multiple myeloma. HSPs, which are molecular chaperones involved in protein homeostasis pathways, have emerged recently as promising therapeutic targets. Using human myeloma cell lines and primary myeloma cells belonging to various molecular groups, we tested the efficacy of HSP90, HSP70, and heat shock factor 1 (HSF1) inhibitors alone or associated with current antimyeloma drugs. We report here that KNK-437 (an inhibitor of HSF1) and bortezomib have additive effects on apoptosis induction in cells belonging to groups with bad prognosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN/biosíntesis , Mieloma Múltiple/patología , Factores de Transcripción/biosíntesis , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Compuestos de Bencidrilo/administración & dosificación , Western Blotting , Bortezomib/administración & dosificación , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Factores de Transcripción del Choque Térmico , Humanos , Mieloma Múltiple/metabolismo , Pirrolidinonas/administración & dosificación , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
PURPOSE: Clinical responses to the immmunomodulatory drug lenalidomide have been observed in patients with relapsed/refractory mantle cell lymphoma (MCL), although its mechanism of action remains partially unknown. We investigated whether the expression and subcellular localization of cyclin D1, a major cell-cycle regulator overexpressed in MCL, and the cyclin-dependent kinase inhibitor p27(KIP1), could identify MCL cases sensitive to lenalidomide, and whether the compound could modulate cyclin D1/p27(KIP1) complexes in MCL cells. EXPERIMENTAL DESIGN: MCL primary samples and cell lines were analyzed for subcellular levels of cyclin D1/p27(KIP1) complexes by Western blot, immunohistochemistry, immunoprecipitation, and flow cytometry. Activity of lenalidomide in vitro and its effect on cyclin D1/p27(KIP1) complexes were evaluated by real-time PCR, immunoprecipitation, immunofluorescence, and Western blot. In vivo validation was carried out in a mouse xenograft model of human MCL. RESULTS: We found cyclin D1 and p27(KIP1) to be coordinately expressed in all the MCL samples tested. Immunoprecipitation analyses and siRNA assays suggested a direct role of cyclin D1 in the regulation of p27(KIP1) levels. The nuclear accumulation of both proteins correlated with MCL cell tumorigenicity in vivo, and sensitivity to lenalidomide activity in vitro and in vivo. Lenalidomide mechanism of action relied on cyclin D1 downregulation and disruption of cyclin D1/p27(KIP1) complexes, followed by cytosolic accumulation of p27(KIP1), cell proliferation arrest, apoptosis, and angiogenesis inhibition. CONCLUSIONS: These results highlight a mechanism of action of lenalidomide in MCL cases with increased tumorigenicity in vivo, which is mediated by the dissociation of cyclin D1/p27(KIP1) complexes, and subsequent proliferation blockade and apoptosis induction.
Asunto(s)
Antineoplásicos/farmacología , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Linfoma de Células del Manto/metabolismo , Talidomida/análogos & derivados , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Xenoinjertos , Humanos , Lenalidomida , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Masculino , Ratones , Persona de Mediana Edad , Unión Proteica/efectos de los fármacos , Estabilidad Proteica , Talidomida/farmacología , Carga Tumoral/efectos de los fármacosAsunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/patología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Linfoma de Células del Manto/genética , Transporte de ProteínasRESUMEN
Multiple myeloma (MM) is a malignancy characterized by the accumulation of clonal plasma cells in the bone marrow. Despite extensive efforts to design drugs targeting tumoral cells and their microenvironment, MM remains an incurable disease for which new therapeutic strategies are needed. We demonstrated here that antiestrogens (AEs) belonging to selective estrogen receptor modulators family induce a caspase-dependent apoptosis and trigger a protective autophagy. Autophagy was recognized by monodansylcadaverin staining, detection of autophagosomes by electronic microscopy, and detection of the cleaved form of the microtubule-associated protein light chain 3. Moreover, autophagy was inhibited by drugs such as bafilomycin A1 and 3-methyladenosine. Autophagy was mediated by the binding of AEs to a class of receptors called the antiestrogen binding site (AEBS) different from the classical estrogen nuclear receptors. The binding of specific ligands to the AEBS was accompanied by alteration of cholesterol metabolism and in particular accumulation of sterols: zymostenol or desmosterol depending on the ligand. This was due to the inhibition of the cholesterol-5,6-epoxide hydrolase activity borne by the AEBS. We further showed that the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathway mediated autophagy signaling. Moreover, AEBS ligands restored sensitivity to dexamethasone in resistant MM cells. Since we showed previously that AEs arrest MM tumor growth in xenografted mice, we propose that AEBS ligands may have a potent antimyeloma activity alone or in combination with drugs used in clinic.
Asunto(s)
Colesterol/metabolismo , Moduladores de los Receptores de Estrógeno/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Humanos , Ligandos , Mieloma Múltiple/patologíaRESUMEN
p27(Kip1) is a cyclin-dependent kinase inhibitor that plays a critical role in regulating G(1)/S transition, and whose activity is, in part, regulated through interactions with D-type cyclins. We have generated the BD1-9 cell line, a BaF3 pro-B cells derivative in which cyclin D1 can be induced rapidly and reversibly by ponasterone A. The induction of cyclin D1 expression leads to a targeted p27(Kip1) accumulation in both cytoplasmic and nuclear compartments. But, only the p27(Kip1) form phosphorylated on serine 10 (pSer10-p27(Kip1)) accumulates in BD1-9 cells. We found that the binding of cyclin D1 and pSer10-p27(Kip1) prevents p27(Kip1) degradation by the cytoplasmic Kip1 ubiquitylation-promoting complex (KPC) proteosomic pathway. Importantly, the nuclear CDK2 activity which is crucial for G(1)/S transition is not altered by p27(Kip1) increase. Using siRNA techniques, we revealed that p27(Kip1) inhibition does not affect the distribution of BD1-9 cells in the different phases of the cell cycle. Our study demonstrates that aberrant cyclin D1 expression acts as a p27(Kip1) trap in B lymphocytes but does not induce p27(Kip1) relocation from the nucleus to the cytoplasm and does not modulate the G(1)/S transition. Since our cellular model mimics what observed in aggressive lymphomas, our data bring new insights into the understanding of their physiopathology.