RESUMO
A major question in hematopoiesis is how the system maintains long-term homeostasis whereby the generation of large numbers of differentiated cells is balanced with the requirement for maintenance of progenitor pools, while remaining sufficiently flexible to respond to periods of perturbed cellular output during infection or stress. We focused on the development of the myeloid lineage and present evidence that promyelocytic leukemia zinc finger (PLZF) provides a novel function that is critical for both normal and stress-induced myelopoiesis. During homeostasis, PLZF restricts proliferation and differentiation of human cord blood-derived myeloid progenitors to maintain a balance between the progenitor and mature cell compartments. Analysis of PLZF promoter-binding sites revealed that it represses transcription factors involved in normal myeloid differentiation, including GFI-1, C/EBPalpha, and LEF-1, and induces negative regulators DUSP6 and ID2. Loss of ID2 relieves PLZF-mediated repression of differentiation identifying it as a functional target of PLZF in myelopoiesis. Furthermore, induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells.
Assuntos
Diferenciação Celular , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Homeostase/fisiologia , Mielopoese/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Sangue Fetal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: The landscape of extracellular matrix (ECM) alterations in soft tissue sarcomas (STS) remains poorly characterized. We aimed to investigate the tumor ECM and adhesion signaling networks present in STS and their clinical implications. EXPERIMENTAL DESIGN: Proteomic and clinical data from 321 patients across 11 histological subtypes were analyzed to define ECM and integrin adhesion networks. Subgroup analysis was performed in leiomyosarcomas (LMS), dedifferentiated liposarcomas (DDLPS), and undifferentiated pleomorphic sarcomas (UPS). RESULTS: This analysis defined subtype-specific ECM profiles including enrichment of basement membrane proteins in LMS and ECM proteases in UPS. Across the cohort, we identified three distinct coregulated ECM networks which are associated with tumor malignancy grade and histological subtype. Comparative analysis of LMS cell line and patient proteomic data identified the lymphocyte cytosolic protein 1 cytoskeletal protein as a prognostic factor in LMS. Characterization of ECM network events in DDLPS revealed three subtypes with distinct oncogenic signaling pathways and survival outcomes. Evaluation of the DDLPS subtype with the poorest prognosis nominates ECM remodeling proteins as candidate antistromal therapeutic targets. Finally, we define a proteoglycan signature that is an independent prognostic factor for overall survival in DDLPS and UPS. CONCLUSIONS: STS comprise heterogeneous ECM signaling networks and matrix-specific features that have utility for risk stratification and therapy selection, which could in future guide precision medicine in these rare cancers.
Assuntos
Matriz Extracelular , Proteômica , Sarcoma , Humanos , Matriz Extracelular/metabolismo , Sarcoma/patologia , Sarcoma/genética , Sarcoma/metabolismo , Proteômica/métodos , Prognóstico , Feminino , Masculino , Transdução de Sinais , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , IdosoRESUMO
PURPOSE: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. EXPERIMENTAL DESIGN: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. RESULTS: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. CONCLUSIONS: Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.
Assuntos
Biomarcadores Tumorais , MicroRNA Circulante , Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos , Compostos de Fenilureia , Piridinas , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/sangue , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Piridinas/uso terapêutico , Piridinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Animais , Feminino , Estudos Prospectivos , Masculino , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Idoso , Biópsia Líquida/métodos , Pessoa de Meia-Idade , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/sangueRESUMO
Deregulated polyamine biosynthesis is emerging as a common feature of neuroblastoma and drugs targeting this metabolic pathway such as DFMO are in clinical and preclinical development. The polyamine analog verlindamycin inhibits the polyamine biosynthesis pathway enzymes SMOX and PAOX, as well as the histone demethylase LSD1. Based on our previous research in acute myeloid leukemia (AML), we reasoned verlindamycin may also unblock neuroblastoma differentiation when combined with all-trans-retinoic acid (ATRA). Indeed, co-treatment with verlindamycin and ATRA strongly induced differentiation regardless of MYCN status, but in MYCN-expressing cells, protein levels were strongly diminished. This process was not transcriptionally regulated but was due to increased degradation of MYCN protein, at least in part via ubiquitin-independent, proteasome-dependent destruction. Here we report that verlindamycin effectively induces the expression of functional tumor suppressor-antizyme via ribosomal frameshifting. Consistent with previous results describing the function of antizyme, we found that verlindamycin treatment led to the selective targeting of ornithine decarboxylase (the rate-limiting enzyme for polyamine biosynthesis) as well as key oncoproteins, such as cyclin D and Aurora A kinase. Retinoid-based multimodal differentiation therapy is one of the few interventions that extends relapse-free survival in MYCN-associated high-risk neuroblastoma and these results point toward the potential use of verlindamycin in this regimen.
Assuntos
Biguanidas , Neuroblastoma , Biguanidas/uso terapêutico , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Ornitina Descarboxilase/metabolismo , Ornitina Descarboxilase/uso terapêutico , Poliaminas/metabolismo , Poliaminas/uso terapêuticoRESUMO
Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
Assuntos
Antineoplásicos/farmacologia , Autofagossomos/enzimologia , Autofagossomos/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Furanos/farmacologia , Macroautofagia , Fosfatidilcolinas/biossíntese , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Células CHO , Linhagem Celular Tumoral , Colina/metabolismo , Colina-Fosfato Citidililtransferase/genética , Cricetulus , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Macroautofagia/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metabolômica , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an "intrinsic" spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. SIGNIFICANCE: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion-positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890.
Assuntos
Fusão Gênica/genética , Glioma/genética , Humanos , Lactente , Gradação de Tumores , Prognóstico , Resultado do TratamentoRESUMO
Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetilação , Acetiltransferases/análise , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fluoresceína-5-Isotiocianato , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Regulação Neoplásica da Expressão Gênica , Células HeLa , Histona Acetiltransferases , Humanos , Fatores de Transcrição Kruppel-Like , Leucemia Promielocítica Aguda/genética , Microscopia Confocal , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transativadores/química , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Histone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eµ). Here, we report that the Eµ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eµ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.
Assuntos
Linfócitos B/enzimologia , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/genética , Linfoma de Células B/enzimologia , Linfoma de Células B/genética , Transtornos Linfoproliferativos/enzimologia , Transtornos Linfoproliferativos/genética , Proteínas Repressoras/genética , Acetilação , Animais , Linfócitos B/patologia , Ciclo Celular/genética , Perfilação da Expressão Gênica , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Células HeLa , Histona Desacetilases/metabolismo , Humanos , Linfoma de Células B/patologia , Transtornos Linfoproliferativos/patologia , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The PLZF gene is translocated in a subset of all-trans-retinoic acid resistant acute promyelocytic leukaemia (APL) cases, encodes a DNA binding transcription factor and is expressed highly in haematopoietic progenitor cells as well-developing central nervous system (CNS). The spatially restricted and temporally dynamic pattern of PLZF expression in the developing CNS suggested that it might play a role in the circuitry regulating hindbrain segmentation. We have now identified a PLZF binding site (PLZF-RE) in an enhancer region of Hoxb2 that itself is required for directing high-level expression in rhombomers 3 and 5 of the developing hindbrain. The wild-type r3/r5 enhancer linked to a heterologous promoter was responsive to regulation by PLZF, and this activity was lost in variants containing a mutated PLZF-RE. Compared with the wild-type protein, the binding of the APL-associated reciprocal RARalpha-PLZF fusion to PLZF-RE was much stronger, suggesting that the N-terminal PLZF sequences missing from the fusion may play a role in the regulation of DNA binding. Consistent with this, the N-terminal POZ domain was required for cooperative binding of PLZF to a multimerized PLZF-RE. In the context of the r3/r5 enhancer, the PLZF-RE cooperated for PLZF binding with an additional A/T-rich motif positioned downstream of the PLZF-RE. This A/T motif was previously shown to be essential for the regulation of Hoxb2 expression in r3 and r5 in cooperation with another Krüppel-like zinc finger protein Krox 20. The presence of both the PLZF-RE and the A/T-rich motif was required for a maximal effect of PLZF on a heterologous promoter and was essential in vivo to direct the expression of a lacZ reporter in the chick neural tube. Hence, both PLZF and Krox20 cooperate with a common A/T motif in mediating in vivo activity of the Hoxb2 enhancer. Our findings indicate that Hoxb2 is a direct target for regulation by PLZF in the developing CNS and suggest that deregulation of Hox gene expression may contribute to APL pathogenesis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação , Embrião de Galinha , DNA/metabolismo , Elementos Facilitadores Genéticos , Hematopoese , Humanos , Fatores de Transcrição Kruppel-Like , Leucemia Promielocítica Aguda/etiologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores do Ácido Retinoico/fisiologia , Proteínas Repressoras/fisiologia , Receptor alfa de Ácido Retinoico , Rombencéfalo/embriologiaRESUMO
Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.
Assuntos
Neoplasias Cerebelares/patologia , Cerebelo/patologia , Meduloblastoma/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/patologia , Proteínas Proto-Oncogênicas/genética , Aloenxertos , Animais , Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Azepinas/farmacologia , Linhagem Celular Tumoral , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/mortalidade , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Feminino , Expressão Gênica , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/mortalidade , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Análise de Componente Principal , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de SobrevidaRESUMO
Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Feminino , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Estrutura Terciária de Proteína , Complexo Correpressor Histona Desacetilase e Sin3 , Esferoides Celulares , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Triple-negative breast cancers (TNBC) lacking estrogen, progesterone, and HER2 receptors account for 10% to 20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide, we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3-PAH2 interaction with MAD, induce expression of CDH1 and ESR1, and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes.
Assuntos
Ivermectina/análogos & derivados , Proteínas Repressoras/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antígenos CD , Antiparasitários/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ivermectina/química , Ivermectina/farmacologia , Camundongos , Modelos Moleculares , Conformação Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.
Assuntos
Neoplasias Cerebelares/genética , Meduloblastoma/genética , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Animais , Antineoplásicos/uso terapêutico , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Criança , Pré-Escolar , Feminino , Amplificação de Genes , Humanos , Lactente , Masculino , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Dados de Sequência Molecular , Mutação , Proteína Proto-Oncogênica N-Myc , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Experimentais , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Adulto JovemRESUMO
UNLABELLED: Although the ETV6-RUNX1 fusion is a frequent initiating event in childhood leukemia, its role in leukemogenesis is only partly understood. The main impact of the fusion itself is to generate and sustain a clone of clinically silent preleukemic B-cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, are required for overt disease. The understanding of the features and interactions of ETV6-RUNX1-positive cells during this "latency" period may explain how these silent cells can persist and whether they could be prone to additional genetic changes. In this study, two in vitro murine models were used to investigate whether ETV6-RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6-RUNX1-expressing cells showed a significant defect in the chemotactic response to CXCL12, caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-associated calcium flux and lack of ERK phosphorylation. Moreover, the induction of ETV6-RUNX1 caused changes in the expression of cell-surface adhesion molecules. The expression of genes regulating the cytoskeleton was also affected, resulting in a block of CDC42 signaling. The abnormalities described here could alter the interaction of ETV6-RUNX1 preleukemic BCP with the microenvironment and contribute to the pathogenesis of the disease. IMPLICATIONS: Alterations in the expression of cytoskeletal regulatory genes and migration properties of BCP represent early events in the evolution of the disease, from the preleukemic phase to the clinical onset, and suggest new strategies for effective eradication of leukemia.
Assuntos
Quimiocina CXCL12/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Células Precursoras de Linfócitos B/citologia , Receptores CXCR4/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Camundongos , Modelos Biológicos , Proteínas de Fusão Oncogênica/genética , Células Precursoras de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4(me2)) across the genome, but it did increase H3K4(me2) and expression of myeloid-differentiation-associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Tretinoína/uso terapêutico , Animais , Antígenos CD34/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Antígeno CD11b/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Interações Medicamentosas , Inibidores Enzimáticos/uso terapêutico , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Interferente Pequeno/metabolismo , Fator de Células-Tronco/metabolismo , Fatores de Tempo , Transplantes , Tranilcipromina/uso terapêutico , Tretinoína/farmacologiaRESUMO
Histone deacetylases (HDACs) perform an important function in transcriptional regulation by modifying the core histones of the nucleosome. We have now fully characterized a new member of the Class II HDAC family, HDAC9. The enzyme contains a conserved deacetylase domain, represses reporter activity when recruited to a promoter, and utilizes histones H3 and H4 as substrates in vitro and in vivo. HDAC9 is expressed in a tissue-specific pattern that partially overlaps that of HDAC4. Within the human hematopoietic system, expression of HDAC9 is biased toward cells of monocytic and lymphoid lineages. The HDAC9 gene encodes multiple protein isoforms, some of which display distinct cellular localization patterns. For example, full-length HDAC9 is localized in the nucleus, but the isoform lacking the region encoded by exon 7 is in the cytoplasm. HDAC9 interacts and co-localizes in vivo with a number of transcriptional repressors and co-repressors, including TEL and N-CoR, whose functions have been implicated in the pathogenesis of hematological malignancies. These results suggest that HDAC9 plays a role in hematopoiesis; its deregulated expression may be associated with some human cancers.