RESUMEN
Intrabdominal dissemination of malignant mesothelioma (MM) and pseudomyxoma peritonei (PMP) is poorly characterized with respect to the stemness window which malignant cells activate during their reshaping on the epithelial-mesenchymal (E/M) axis. To gain insights into stemness properties and their prognostic significance in these rarer forms of peritoneal metastases (PM), primary tumor cultures from 55 patients selected for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy were analyzed for cancer stem cells (CSC) by aldehyde dehydrogenase 1 (ALDH1) and spheroid formation assays, and for expression of a set of plasticity-related genes to measure E/M transition (EMT) score. Intratumor heterogeneity was also analyzed. Samples from PM of colorectal cancer were included for comparison. Molecular data were confirmed using principal component and cluster analyses. Associations with survival were evaluated using Kaplan-Meier and Cox regression models. The activity of acetylsalicylic acid (ASA), a stemness modifier, was tested in five cultures. Significantly increased amounts of ALDH1bright-cells identified high-grade PMP, and discriminated solid masses from ascitic/mucin-embedded tumor cells in both forms of PM. Epithelial/early hybrid EMT scores and an early hybrid expression pattern correlated with pluripotency factors were significantly associated with early peritoneal progression (p = .0343 and p = .0339, respectively, log-rank test) in multivariable models. ASA impaired spheroid formation and increased cisplatin sensitivity in all five cultures. These data suggest that CSC subpopulations and hybrid E/M states may guide peritoneal spread of MM and PMP. Stemness could be exploited as targetable vulnerability to increase chemosensitivity and improve patient outcomes. Additional research is needed to confirm these preliminary data.
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Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.
Asunto(s)
Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Linfoma/genética , Células Madre Neoplásicas/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Células Jurkat , Linfoma/tratamiento farmacológico , Linfoma/virología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/virología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Idiopathic pulmonary fibrosis (IPF) is mainly characterized by aberrant extracellular matrix deposition, consequent to epithelial lung injury and myofibroblast activation, and inflammatory response. Glycogen synthase kinase 3 (GSK-3) is a serine-threonine kinase involved in several pathways, and its inhibition has been already suggested as a therapeutic strategy for IPF patients. There is evidence that GSK-3 is able to induce matrix metalloproteinase (MMP) expression and that its inhibition modulates MMP expression in the tissues. The aim of our study was to investigate the role of GSK-3 and its inhibition in the modulation of MMP-9 and -2 in an in vivo mouse model of lung fibrosis and in vitro using different cell lines exposed to pro-inflammatory or pro-fibrotic stimuli. We found that GSK-3 inhibition down-modulates gene expression and protein levels of MMP-9, MMP-2, and their inhibitors TIMP-1 and TIMP-2 in inflammatory cells harvested from bronchoalveolar lavage fluid (BALF) of mice treated with bleomycin as well as in interstitial alveolar macrophages and cuboidalized epithelial alveolar cells. To the same extent, GSK-3 inhibition blunted the increased MMP-9 and MMP-2 activity induced by pro-fibrotic stimuli in a human lung fibroblast cell line. Moreover, the αSMA protein level, a marker of fibroblast-to-myofibroblast transition involved in fibrosis, was decreased in primary fibroblasts treated with TGFß following GSK-3 inhibition. Our results confirm the implication of GSK-3 in lung inflammation and fibrosis, suggesting that it might play its role by modulating MMP expression and activity but also pushing fibroblasts toward a myofibroblast phenotype and therefore enhancing extracellular matrix deposition. Thus, its inhibition could represent a possible therapeutic strategy.
RESUMEN
Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.
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Proliferación Celular , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/metabolismo , Transcriptoma , Canales de Calcio Tipo L/metabolismo , Dihidropiridinas/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Células Madre Mesenquimatosas/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
PURPOSE: Interpatient clinical variability in soft-tissue sarcomas (STS) highlights the need for novel prognostic markers supporting patient risk stratification. As sarcomas might exhibit a more mesenchymal or a more epithelial state, we focused on epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT/MET) for prognostic clues, and selected three histotypes with variable aggressiveness. EXPERIMENTAL DESIGN: The expression of EMT/MET-related factors was measured by qRT-PCR in 55 tumor samples from patients with leiomyosarcoma, myxofibrosarcoma, or undifferentiated pleomorphic sarcoma. The identified marker was further evaluated by IHC in 31 leiomyosarcomas and by measuring its circulating levels in 67 patients. The prognostic value of a sarcoma-tailored EMT score was analyzed. Epirubicin chemosensitivity and migration were studied in primary STS cultures. Associations with overall survival (OS) were assessed using Kaplan-Meier and Cox regression methods. RESULTS: High expression of periostin, a mesenchymal matricellular protein, in sarcoma tissues (P = 0.0024), its high stromal accumulation in leiomyosarcomas (P = 0.0075), and increased circulation (>20 ng/mL, P = 0.0008) were associated with reduced OS. High periostin expression [HR 2.9; 95% confidence interval (CI), 1.3-6.9; P = 0.0134] and circulation (HR 2.6; 95% CI, 1.3-5.1; P = 0.0086), and a mesenchymal EMT score (mesenchymal vs. transitioning; HR, 5.2; 95% CI, 2.1-13.0, P = 0.0005) were associated with increased risk in multivariable models. An intrinsic or induced mesenchymal state enhanced chemoresistance and migration in sarcoma cell lines. CONCLUSIONS: Although limited to a pilot study, these findings suggest that periostin might contribute prognostic information in the three studied STS histotypes. Moreover, a transitioning EMT score measured in the tumor might predict a less active and a more chemosensitive disease.
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Biomarcadores de Tumor/análisis , Moléculas de Adhesión Celular/metabolismo , Transición Epitelial-Mesenquimal , Fibrosarcoma/patología , Leiomiosarcoma/patología , Neoplasias de los Tejidos Blandos/patología , Anciano , Femenino , Fibrosarcoma/metabolismo , Fibrosarcoma/cirugía , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Leiomiosarcoma/metabolismo , Leiomiosarcoma/cirugía , Masculino , Proyectos Piloto , Pronóstico , Estudios Prospectivos , Estudios Retrospectivos , Neoplasias de los Tejidos Blandos/metabolismo , Neoplasias de los Tejidos Blandos/cirugía , Tasa de Supervivencia , Análisis de Matrices TisularesRESUMEN
Adult T cell Leukemia/Lymphoma (ATLL) is a mature T cell malignancy associated with Human T cell Leukemia Virus type 1 (HTLV-1) infection. Among its four main clinical subtypes, the prognosis of acute and lymphoma variants remains poor. The long latency (3-6 decades) and low incidence (3-5%) of ATLL imply the involvement of viral and host factors in full-blown malignancy. Despite multiple preclinical and clinical studies, the contribution of the stromal microenvironment in ATLL development is not yet completely unraveled. The aims of this study were to investigate the role of the host microenvironment, and specifically fibroblasts, in ATLL pathogenesis and to propose a murine model for the lymphoma subtype. Here we present evidence that the oncogenic capacity of HTLV-1-immortalized C91/PL cells is enhanced when they are xenotransplanted together with human foreskin fibroblasts (HFF) in immunocompromised BALB/c Rag2-/-γc-/- mice. Moreover, cell lines derived from a developed lymphoma and their subsequent in vivo passages acquired the stable property to induce aggressive T cell lymphomas. In particular, one of these cell lines, C91/III cells, consistently induced aggressive lymphomas also in NOD/SCID/IL2Rγc KO (NSG) mice. To dissect the mechanisms linked to this enhanced tumorigenic ability, we quantified 45 soluble factors released by these cell lines and found that 21 of them, mainly pro-inflammatory cytokines and chemokines, were significantly increased in C91/III cells compared to the parental C91/PL cells. Moreover, many of the increased factors were also released by human fibroblasts and belonged to the known secretory pattern of ATLL cells. C91/PL cells co-cultured with HFF showed features reminiscent of those observed in C91/III cells, including a similar secretory pattern and a more aggressive behavior in vivo. On the whole, our data provide evidence that fibroblasts, one of the major stromal components, might enhance tumorigenesis of HTLV-1-infected and immortalized T cells, thus throwing light on the role of microenvironment contribution in ATLL pathogenesis. We also propose that the lymphoma induced in NSG mice by injection with C91/III cells represents a new murine preclinical ATLL model that could be adopted to test novel therapeutic interventions for the aggressive lymphoma subtype.
RESUMEN
BACKGROUND: Tubulointerstitial fibrosis, the final outcome of most kidney diseases, involves activation of epithelial mesenchymal transition (EMT). Endothelin-1 (ET-1) activates EMT in cancer cells, but it is not known whether it drives EMT in the kidney. We therefore tested the hypothesis that tubulointerstitial fibrosis involves EMT driven by ET-1. METHODS AND RESULTS: Transgenic TG[mRen2]27 (TGRen2) rats developing fulminant angiotensin II-dependent hypertension with prominent cardiovascular and renal damage were submitted to drug treatments targeted to ET-1 and/or angiotensin II receptor or left untreated (controls). Expressional changes of E-cadherin and α-smooth muscle actin (αSMA) were examined as markers of renal EMT. In human kidney HK-2 proximal tubular cells expressing the ETB receptor subtype, the effects of ET-1 with or without ET-1 antagonists were also investigated. The occurrence of renal fibrosis was associated with EMT in control TGRen2 rats, as evidenced by decreased E-cadherin and increased αSMA expression. Irbesartan and the mixed ET-1 receptor antagonist bosentan prevented these changes in a blood pressure-independent fashion (P < 0.001 for both versus controls). In HK-2 cells ET-1 blunted E-cadherin expression, increased αSMA expression (both P < 0.01), collagen synthesis, and metalloproteinase activity (P < 0.005, all versus untreated cells). All changes were prevented by the selective ETB receptor antagonist BQ-788. Evidence for involvement of the Rho-kinase signaling pathway and dephosphorylation of Yes-associated protein in EMT was also found. CONCLUSIONS: In angiotensin II-dependent hypertension, ET-1 acting via ETB receptors and the Rho-kinase and Yes-associated protein induces EMT and thereby renal fibrosis.
Asunto(s)
Actinas/efectos de los fármacos , Antagonistas de Receptores de Angiotensina/farmacología , Cadherinas/efectos de los fármacos , Endotelina-1/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Hipertensión/metabolismo , Enfermedades Renales/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Compuestos de Bifenilo/farmacología , Bosentán , Cadherinas/metabolismo , Antagonistas de los Receptores de la Endotelina B/farmacología , Endotelina-1/antagonistas & inhibidores , Fibrosis , Humanos , Hipertensión/complicaciones , Irbesartán , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/patología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Oligopéptidos/farmacología , Piperidinas/farmacología , Ratas , Receptor de Endotelina B/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , Tetrazoles/farmacología , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: Glycogen Synthase Kinase-3 (GSK-3) α and ß are two serine-threonine kinases controlling insulin, Wnt/ß-catenin, NF-κB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3α and GSK-3ß function in multiple myeloma (MM). METHODS: GSK-3 α and ß expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 α and ß isoforms. Survival signaling pathways were studied with WB analysis. RESULTS: GSK-3α and GSK-3ß were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3ß knock down decreased MM cell viability, while GSK-3α knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of ß-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3α knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. CONCLUSIONS: These data suggest that in MM cells GSK-3α and ß i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors.
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Ácidos Borónicos/farmacología , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Mieloma Múltiple/metabolismo , Pirazinas/farmacología , Transporte Activo de Núcleo Celular , Antineoplásicos/farmacología , Apoptosis , Bortezomib , Muerte Celular , Núcleo Celular/metabolismo , Proliferación Celular , Silenciador del Gen , Glucógeno Sintasa Quinasa 3 beta , Humanos , Potenciales de la Membrana , Fosforilación , Interferencia de ARN , Transducción de SeñalRESUMEN
Glycogen synthase kinase (GSK)-3 modulates the production of inflammatory cytokines. Because bleomycin (BLM) causes lung injury, which is characterized by an inflammatory response followed by a fibrotic degeneration, we postulated that blocking GSK-3 activity with a specific inhibitor could affect the inflammatory and profibrotic cytokine network generated in the BLM-induced process of pulmonary inflammation and fibrosis. Thus, here we investigated the effects of the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) on a BLM-induced lung fibrosis model in mice. SB216763 prevented lung inflammation and the subsequent fibrosis when coadministered with BLM. Bronchoalveolar lavage fluid analysis of mice treated with BLM plus SB216763 revealed a significant reduction in BLM-induced alveolitis. Furthermore, SB216763 treatment was associated with a significantly lower production of inflammatory cytokines by macrophages. BLM-treated mice that received SB216763 developed alveolar epithelial cell damage and pulmonary fibrosis to a significantly lower extent compared with BLM-treated controls. These findings suggest that GSK-3 inhibition has a protective effect on lung fibrosis induced by BLM and candidate GSK-3 as a potential therapeutic target for preventing pulmonary fibrosis.
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Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Indoles/uso terapéutico , Pulmón/efectos de los fármacos , Maleimidas/uso terapéutico , Neumonía/tratamiento farmacológico , Mucosa Respiratoria/efectos de los fármacos , Animales , Bleomicina , Quimiocina CCL2/biosíntesis , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/inmunología , Fibrosis Pulmonar Idiopática/patología , Pulmón/inmunología , Pulmón/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neumonía/inducido químicamente , Neumonía/inmunología , Neumonía/patología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/patología , Mucosa Respiratoria/patología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Casein kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase that regulates relevant biologic processes, many of which are dysregulated in malignant plasma cells. Here we investigated its role in multiple myeloma (MM). Analysis of MM cell lines and highly purified malignant plasma cells in patients with MM revealed higher protein and CK2 activity levels than in controls (normal in vitro-generated polyclonal plasma cells and B lymphocytes). The inhibition of CK2 with specific synthetic compounds or by means of RNA interference caused a cytotoxic effect on MM plasma cells that could not be overcome by IL-6 or IGF-I and that was associated with the activation of extrinsic and intrinsic caspase cascades. CK2 blockage lowered the sensitivity threshold of MM plasma cells to the cytotoxic effect of melphalan. CK2 inhibition also resulted in impaired IL-6-dependent STAT3 activation and in decreased basal and TNF-alpha-dependent I kappaB alpha degradation and NF-kappaB-driven transcription. Our data show that CK2 was involved in the pathophysiology of MM, suggesting that it might play a crucial role in controlling survival and sensitivity to chemotherapeutics of malignant plasma cells.