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Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I â KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.
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Neoplasias da Mama , Epigênese Genética , Histona Desmetilases , Interferon Tipo I , Antraciclinas/metabolismo , Antraciclinas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Histona Desmetilases/metabolismo , Humanos , Interferon Tipo I/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
BACKGROUND: Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. METHODS: In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. RESULTS: We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. CONCLUSION: In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.
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Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.
Assuntos
Imunoprecipitação/métodos , Complexos Multiproteicos/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Apoptose , Biotinilação , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Humanos , Carioferinas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteína Fosfatase 2C/metabolismo , Proteômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Reprodutibilidade dos Testes , Receptor fas/metabolismoRESUMO
MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting.
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Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas , OligonucleotídeosRESUMO
Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Adulto , Animais , Neoplasias Encefálicas/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Células HEK293 , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células Tumorais CultivadasRESUMO
Cutaneous melanoma is the fastest increasing cancer worldwide. Although several molecular abnormalities have been associated with melanoma progression, the underlying mechanisms are still largely unknown and few targeted therapies are under evaluation. Here we show that the HOXB7/PBX2 dimer acts as a positive transcriptional regulator of the oncogenic microRNA-221 and -222. In addition, demonstrating c-FOS as a direct target of miR-221&222, we identify a HOXB7/PBX2âmiR-221&222 âc-FOS regulatory link, whereby the abrogation of functional HOXB7/PBX2 dimers leads to reduced miR-221&222 transcription and elevated c-FOS expression with consequent cell death. Taking advantage of the treatment with the peptide HXR9, an antagonist of HOX/PBX dimerization, we recognize miR-221&222 as effectors of its action, in turn confirming the HXR9 efficacy in the treatment of human melanoma malignancy, whilst sparing normal human melanocytes. Our findings, besides suggesting the potential therapeutic of HXR9 or its derivatives in malignant melanoma, suggest the disruption of the HOXB7/PBX2 complexes, miR-221&222 inhibition or even better their combination, as innovative therapeutic approaches.
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Apoptose/fisiologia , Proteínas de Homeodomínio/genética , Melanoma/patologia , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas/genética , Neoplasias Cutâneas/patologia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Dimerização , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/fisiologia , Humanos , MicroRNAs/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , RNA Interferente Pequeno , Transcrição GênicaRESUMO
BACKGROUND: Homeobox (HOX) genes deregulation has been largely implicated in the development of human leukemia. Among the HOXB cluster, HOXB1 was silent in a number of analyzed acute myeloid leukemia (AML) primary cells and cell lines, whereas it was expressed in normal terminally differentiated peripheral blood cells. METHODS: We evaluated the biological effects and the transcriptome changes determined by the retroviral transduction of HOXB1 in the human promyelocytic cell line HL60. RESULTS: Our results suggest that the enforced expression of HOXB1 reduces cell growth proliferation, inducing apoptosis and cell differentiation along the monocytic and granulocytic lineages. Accordingly, gene expression analysis showed the HOXB1-dependent down-regulation of some tumor promoting genes, paralleled by the up-regulation of apoptosis- and differentiation-related genes, thus supporting a tumor suppressor role for HOXB1 in AML. Finally, we indicated HOXB1 promoter hypermethylation as a mechanism responsible for HOXB1 silencing. CONCLUSIONS: We propose HOXB1 as an additional member of the HOX family with tumour suppressor properties suggesting a HOXB1/ATRA combination as a possible future therapeutic strategy in AML.
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Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.
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Biomarcadores/metabolismo , Diferenciação Celular , Colo/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Miofibroblastos , Adipogenia/fisiologia , Biópsia , Células da Medula Óssea/citologia , Antígeno CD146/imunologia , Antígeno CD146/metabolismo , Condrogênese/fisiologia , Colo/citologia , Hematopoese/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia Confocal , Osteogênese/fisiologiaRESUMO
MiR-378a-3p plays a critical role in carcinogenesis acting as a tumor suppressor, promoting apoptosis and cell cycle arrest and reducing invasion and drug resistance in several human cancers, including colorectal cancer (CRC), where its expression is significantly associated with histological classification and prognosis. In this study, we investigated the biological and cellular processes affected by miR-378a-3p in the context of CRC carcinogenesis. In agreement with the literature, miR-378a-3p is downregulated in our cohort of CRC patients as well as, in 15 patient-derived colorectal cancer stem-like cell (CRC-SC) lines and 8 CRC cell lines, compared to normal mucosae. Restoration of miR-378a-3p restrains tumorigenic properties of CRC and CRC-SC lines, as well as, significantly reduces tumor growth in two CRC-SC xenograft mouse models. We reported that miR-378a-3p modulates the expression of the lncRNAs MALAT1 and NEAT1. Their expression is inversely correlated with that of miR-378a-3p in patient-derived CRC-SC lines. Silencing of miR-378a-3p targets, MALAT1 and NEAT1, significantly impairs tumorigenic properties of CRC-SCs, supporting the critical role of miR-378a-3p in CRC carcinogenesis as a tumor-suppressor factor by establishing a finely tuned crosstalk with lncRNAs MALAT1 and NEAT1.
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Chemotherapy is the mainstay for the treatment of non-small cell lung cancer (NSCLC). However, NSCLC cells are either intrinsically chemoresistant or rapidly develop therapy resistance. Cancer stem cells (CSCs) are widely recognized as the cell population responsible for resistance to systemic therapies, but the molecular responses of CSCs to chemotherapeutic agents are largely unknown. We identified the embryonic protein CRIPTO in stem cell-enriched spheroid cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC) derived from NSCLC surgical specimens. The CRIPTO-positive population had increased clonogenic capacity and expression of stem cell-related factors. Stemness-related properties were also obtained with forced CRIPTO expression, whereas CRIPTO downregulation resulted in cell cycle blockade and CSCs death. Cell populations positive and negative for CRIPTO expression were interconvertible, and interfering with their reciprocal equilibrium resulted in altered homeostasis of cell expansion both in spheroid cultures and in tumor xenografts. Chemotherapy treatment of NSCLC cells resulted in reduction of cell number followed by increased CRIPTO expression and selective survival of CRIPTO-positive cells. In NSCLC tumor xenografts, chemotherapeutic agents induced partial cell death and tumor stabilization followed by CRIPTO overexpression and tumor progression. Altogether, these findings indicate CRIPTO as a marker of lung CSCs possibly implicated in cancer cell plasticity and post-chemotherapy tumor progression.
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BACKGROUND: Circulating tumor cells (CTCs) are responsible for the metastatic dissemination of colorectal cancer (CRC) to the liver, lungs and lymph nodes. CTCs rarity and heterogeneity strongly limit the elucidation of their biological features, as well as preclinical drug sensitivity studies aimed at metastasis prevention. METHODS: We generated organoids from CTCs isolated from an orthotopic CRC xenograft model. CTCs-derived organoids (CTCDOs) were characterized through proteome profiling, immunohistochemistry, immunofluorescence, flow cytometry, tumor-forming capacity and drug screening assays. The expression of intra- and extracellular markers found in CTCDOs was validated on CTCs isolated from the peripheral blood of CRC patients. RESULTS: CTCDOs exhibited a hybrid epithelial-mesenchymal transition (EMT) state and an increased expression of stemness-associated markers including the two homeobox transcription factors Goosecoid and Pancreatic Duodenal Homeobox Gene-1 (PDX1), which were also detected in CTCs from CRC patients. Functionally, CTCDOs showed a higher migratory/invasive ability and a different response to pathway-targeted drugs as compared to xenograft-derived organoids (XDOs). Specifically, CTCDOs were more sensitive than XDOs to drugs affecting the Survivin pathway, which decreased the levels of Survivin and X-Linked Inhibitor of Apoptosis Protein (XIAP) inducing CTCDOs death. CONCLUSIONS: These results indicate that CTCDOs recapitulate several features of colorectal CTCs and may be used to investigate the features of metastatic CRC cells, to identify new prognostic biomarkers and to devise new potential strategies for metastasis prevention.
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Neoplasias Colorretais , Células Neoplásicas Circulantes , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Células Neoplásicas Circulantes/metabolismo , Organoides/metabolismo , Células-Tronco/metabolismoRESUMO
Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.
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BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by a poor prognosis mainly due to recurrence and therapeutic resistance. It has been widely demonstrated that glioblastoma stem-like cells (GSCs), a subpopulation of tumor cells endowed with stem-like properties is responsible for tumor maintenance and progression. Moreover, it has been demonstrated that GSCs contribute to GBM-associated neovascularization processes, through different mechanisms including the transdifferentiation into GSC-derived endothelial cells (GdECs). METHODS: In order to identify druggable cancer-related pathways in GBM, we assessed the effect of a selection of 349 compounds on both GSCs and GdECs and we selected elesclomol (STA-4783) as the most effective agent in inducing cell death on both GSC and GdEC lines tested. RESULTS: Elesclomol has been already described to be a potent oxidative stress inducer. In depth investigation of the molecular mechanisms underlying GSC and GdEC response to elesclomol, confirmed that this compound induces a strong increase in mitochondrial reactive oxygen species (ROS) in both GSCs and GdECs ultimately leading to a non-apoptotic copper-dependent cell death. Moreover, combined in vitro treatment with elesclomol and the alkylating agent temozolomide (TMZ) enhanced the cytotoxicity compared to TMZ alone. Finally, we used our experimental model of mouse brain xenografts to test the combination of elesclomol and TMZ and confirmed their efficacy in vivo. CONCLUSIONS: Our results support further evaluation of therapeutics targeting oxidative stress such as elesclomol with the aim of satisfying the high unmet medical need in the management of GBM.
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Neoplasias Encefálicas/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Hidrazinas/uso terapêutico , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Hidrazinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
BACKGROUND: The NCAM or CD56 antigen is a cell surface glycoprotein belonging to the immunoglobulin super-family involved in cell-cell and cell-matrix adhesion. NCAM is also over-expressed in many tumour types and is considered a tumour associated antigen, even if its role and biological mechanisms implicated in tumour progression and metastasis have not yet to be elucidated. In particular, it is quite well documented the role of the interaction between the NCAM protein and the fibroblast growth factor receptor-1 in metastasis and invasion, especially in the ovarian cancer progression. OBJECTIVE: Here we describe the isolation and preliminary characterization of a novel human anti-NCAM single chain Fragment variable antibody able to specifically bind NCAM-expressing cells, including epithelial ovarian cancer cells. METHODS: The antibody was isolate by phage display selection and was characterized by ELISA, FACS analysis and SPR experiments. Interference in EOC migration was analyzed by scratch test. RESULTS: It binds a partially linear epitope lying in the membrane proximal region of two fibronectin-like domains with a dissociation constant of 3.43 × 10-8 M. Interestingly, it was shown to interfere with the NCAM-FGFR1 binding and to partially decrease migration of EOC cells. CONCLUSIONS: According to our knowledge, this is the first completely human antibody able to interfere with this newly individuated cancer mechanism.
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Bacteriófagos , Transdução de Sinais , Bacteriófagos/metabolismo , Humanos , Imunoglobulinas , Moléculas de Adesão de Célula Nervosa/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Ets-1 is a widely expressed transcription factor implicated in several biological processes including hematopoiesis, where it contributes to the regulation of cellular differentiation. The functions of Ets-1 are regulated by transcription factors as well as by phosphorylation events: phosphorylation of threonine 38 activates Ets-1, whereas phosphorylation of a cluster of serines within exon VII reduces DNA binding activity. This study focuses on the role of Ets-1 during granulocytic differentiation of NB4 promyelocytic and HL60 myeloblastic leukemia cell lines induced by all-trans retinoic acid. DESIGN AND METHODS: Ets-1 expression was measured by real-time reverse transcriptase polymerase chain reaction and western blotting. The role of Ets-1 during all-trans retinoic acid-induced differentiation was analyzed by using a transdominant negative molecule or small interfering RNA. RESULTS: NB4 and HL60 cell lines expressed high levels of p51 Ets-1, while the splice variant isoform that lacks exon VII (p42) was almost undetectable. The addition of all-trans retinoic acid reduced p51 Ets-1 levels and induced inhibitory phosphorylation of the remaining protein. Expression of Ets-1 was also reduced during dimethylsulfoxide-induced differentiation and during granulocytic differentiation of human CD34(+) hematopoietic progenitor cells but not in NB4.R2 and HL60R cells resistant to all-trans retinoic acid. In line with these observations, transduction of a transdominant negative molecule of Ets-1, which inhibited DNA binding and transcriptional activity of the wild-type Ets-1, significantly increased chemical-induced differentiation. Consistently, Ets-1 knockdown by small interfering RNA increased the number of mature neutrophils upon addition of all-trans retinoic acid. Interestingly, p51 Ets-1 over-expression was frequently observed in CD34(+) hematopoietic progenitor cells derived from patients with acute myeloid leukemia, as compared to its expression in normal CD34(+) cells. CONCLUSIONS: Our results indicated that a decreased expression of Ets-1 protein generalizes to granulocytic differentiation and may represent a crucial event for granulocytic maturation.
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Diferenciação Celular/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Granulócitos/efeitos dos fármacos , Proteína Proto-Oncogênica c-ets-1/genética , Linhagem Celular Tumoral , Granulócitos/citologia , Células HL-60 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucemia/patologia , Leucemia Promielocítica Aguda , Proteína Proto-Oncogênica c-ets-1/fisiologia , RNA Interferente Pequeno/farmacologiaRESUMO
BACKGROUND: Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 geneâtype III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis. METHODS: Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3). RESULTS: Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan-Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT). CONCLUSION: In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.
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Glioblastoma , RNA Longo não Codificante , Adulto , Proteínas de Ligação ao Cálcio , Proliferação de Células , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Impressão Genômica , Glioblastoma/genética , Humanos , Proteínas de Membrana/genética , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. METHODS: A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. RESULTS: Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. CONCLUSIONS: These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.
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Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Neoplásicas/metabolismo , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Oxaliplatina/farmacologia , PrognósticoRESUMO
Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of long-lived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options. SIGNIFICANCE: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.
Assuntos
Antineoplásicos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Compostos de Pirvínio/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colesterol/metabolismo , Reposicionamento de Medicamentos , Feminino , Glucose/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The common beta chain subunit (beta(c)), also known as CDw131, shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, is required for high-affinity ligand binding and signal transduction. The present study explored the expression of CDw131 in 105 de novo cases of acute myeloid leukaemia (AML). The levels of CDw131 expression were used to identify two AML subgroups characterized by low (75/105) and high (30/105) expression of this receptor chain. It was observed that (i) the level of CDw131 expression strictly correlated with the level of CD116 (GM-CSFalpha receptor chain) and CD123 (IL-3Ralpha chain); (ii) AMLs with high CDw131 expression were characterized by low CD34 expression and usually high CD11b, CD14 expression; (iii) AMLs with high CDw131 expression frequently co-expressed receptors for angiogenic growth factors (vascular endothelial growth factor R2, Tie-2); (iv) AMLs with high CDw131 expression were more cycling than those with low CDw131 expression; (v) AMLs with high CDw131 frequently displayed Feline Murine Sarcoma (FMS-related) tyrosine kinase 3 (FLT3) internal tandem duplication and constitutively activated Signal Transducer and Activator of Transcription-5 (STAT5). In conclusion, the analysis of the level of CDw131 expression enabled the identification of a subset of AMLs characterized by a high cycling status, the expression of myelo-monocytic markers, mutated FLT3 and the co-expression of receptors for angiogenic growth factors. These findings are of value for the development of new therapeutic strategies for the treatment of these AMLs.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/análise , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/imunologia , Mutação , Tirosina Quinase 3 Semelhante a fms/genética , Biomarcadores/análise , Western Blotting/métodos , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Citometria de Fluxo , Humanos , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-3/análise , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Contagem de Leucócitos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Translocação Genética , Células Tumorais Cultivadas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
Emerging data support the rationale of combined therapies in advanced melanoma. Specifically, the combined use of drugs with different mechanisms of action can reduce the probability of selecting resistant clones. To identify agents active against melanoma cells, we screened a library of 349 anti-cancer compounds, currently in clinical use or trials, and selected PIK-75, an inhibitor of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, as the 'top active' drug. PIK-75 was then used alone or in combination with vemurafenib, the first BRAF inhibitor approved for patients with melanoma harboring BRAF mutations. We identified a combined dose of PIK-75 and vemurafenib that inhibited both the PI3K/AKT and mitogen-activated protein kinase pathways, thereby overcoming any compensatory activation. In view of the important tumor suppressor function induced by restoring expression of microRNA (miR)-126 in metastatic melanoma cells, we examined whether miR-126 has a synergistic role when included in a triple combination alongside PIK-75 and vemurafenib. We found that enforced expression of miR-126 (which alone can reduce tumorigenicity) significantly increased PIK-75 activity when used as either a single agent or in combination with vemurafenib. Interestingly, PIK-75 proved to be effective against early passage cell lines derived from patients' biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, thus suggesting that it potentially has the capability to overcome drug resistance. Finally, the synergistic role played by miR-126 in combination with vemurafenib and/or PIK-75 was demonstrated in vivo in mouse xenograft models, in which tumor growth inhibition was associated with increased apoptosis. These results not only show the efficacy of PIK-75 and vemurafenib co-treatment but also indicate that restoration of miR-126 expression in advanced melanoma can enhance their antitumor activity, which may possibly allow dose reduction to decrease adverse events without reducing the therapeutic benefits.