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Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
Assuntos
Microscopia Crioeletrônica , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos , Proteína Fosfatase 1 , Proteínas ras , Motivos de Aminoácidos , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação de Sentido Incorreto , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/ultraestrutura , Estabilidade Proteica , Quinases raf , Proteínas ras/química , Proteínas ras/metabolismo , Proteínas ras/ultraestruturaRESUMO
As words can have multiple meanings that depend on sentence context, genes can have various functions that depend on the surrounding biological system. This pleiotropic nature of gene function is limited by ontologies, which annotate gene functions without considering biological contexts. We contend that the gene function problem in genetics may be informed by recent technological leaps in natural language processing, in which representations of word semantics can be automatically learned from diverse language contexts. In contrast to efforts to model semantics as "is-a" relationships in the 1990s, modern distributional semantics represents words as vectors in a learned semantic space and fuels current advances in transformer-based models such as large language models and generative pre-trained transformers. A similar shift in thinking of gene functions as distributions over cellular contexts may enable a similar breakthrough in data-driven learning from large biological datasets to inform gene function.
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Processamento de Linguagem Natural , Semântica , Humanos , Genes/genética , Ontologia Genética , Biologia Computacional/métodos , AnimaisRESUMO
Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.
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Diferenciação Celular , Movimento Celular , Disferlina , Matriz Extracelular , Mioblastos , Sarcoglicanas , Animais , Mioblastos/metabolismo , Mioblastos/citologia , Matriz Extracelular/metabolismo , Camundongos , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Disferlina/genética , Disferlina/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Distrofina/genética , Distrofina/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Decorina/genética , Decorina/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Músculo Esquelético/metabolismoRESUMO
The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.
Assuntos
Distrofia Muscular do Cíngulo dos Membros , Distrofias Musculares , Camundongos , Animais , Camundongos Endogâmicos DBA , Distrofias Musculares/genética , Músculos , Matriz Extracelular , Camundongos KnockoutRESUMO
High-throughput screening (HTS) methods enable the empirical evaluation of a large scale of compounds and can be augmented by virtual screening (VS) techniques to save time and money by using potential active compounds for experimental testing. Structure-based and ligand-based virtual screening approaches have been extensively studied and applied in drug discovery practice with proven outcomes in advancing candidate molecules. However, the experimental data required for VS are expensive, and hit identification in an effective and efficient manner is particularly challenging during early-stage drug discovery for novel protein targets. Herein, we present our TArget-driven Machine learning-Enabled VS (TAME-VS) platform, which leverages existing chemical databases of bioactive molecules to modularly facilitate hit finding. Our methodology enables bespoke hit identification campaigns through a user-defined protein target. The input target ID is used to perform a homology-based target expansion, followed by compound retrieval from a large compilation of molecules with experimentally validated activity. Compounds are subsequently vectorized and adopted for machine learning (ML) model training. These machine learning models are deployed to perform model-based inferential virtual screening, and compounds are nominated based on predicted activity. Our platform was retrospectively validated across ten diverse protein targets and demonstrated clear predictive power. The implemented methodology provides a flexible and efficient approach that is accessible to a wide range of users. The TAME-VS platform is publicly available at https://github.com/bymgood/Target-driven-ML-enabled-VS to facilitate early-stage hit identification.
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Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphy's Roth Large (MRL) strain has "super-healing" properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized "myoscaffolds". Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy.
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In high-throughput functional genomic screens, each gene product is commonly assumed to exhibit a singular biological function within a defined protein complex or pathway. In practice, a single gene perturbation may induce multiple cascading functional outcomes, a genetic principle known as pleiotropy. Here, we model pleiotropy in fitness screen collections by representing each gene perturbation as the sum of multiple perturbations of biological functions, each harboring independent fitness effects inferred empirically from the data. Our approach (Webster) recovered pleiotropic functions for DNA damage proteins from genotoxic fitness screens, untangled distinct signaling pathways upstream of shared effector proteins from cancer cell fitness screens, and predicted the stoichiometry of an unknown protein complex subunit from fitness data alone. Modeling compound sensitivity profiles in terms of genetic functions recovered compound mechanisms of action. Our approach establishes a sparse approximation mechanism for unraveling complex genetic architectures underlying high-dimensional gene perturbation readouts.
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Genômica , Genômica/métodos , HumanosRESUMO
Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.
Assuntos
Proteólise , CinéticaRESUMO
Collateral lethality occurs when loss of a gene/protein renders cancer cells dependent on its remaining paralog. Combining genome-scale CRISPR/Cas9 loss-of-function screens with RNA sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase vaccinia-related kinase 1 (VRK1) for their survival in vivo. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout sensitized cells to VRK1 loss, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 loss. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced barrier-to-autointegration factor phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas.
Assuntos
Glioma , Neuroblastoma , Vacínia , Criança , Glioma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema Nervoso , Neuroblastoma/genética , Proteínas Serina-Treonina Quinases/genética , Vaccinia virusRESUMO
Metastatic castration-resistant prostate cancers (mCRPCs) are treated with therapies that antagonize the androgen receptor (AR). Nearly all patients develop resistance to AR-targeted therapies (ARTs). Our previous work identified CREB5 as an upregulated target gene in human mCRPC that promoted resistance to all clinically approved ART. The mechanisms by which CREB5 promotes progression of mCRPC or other cancers remains elusive. Integrating ChIP-seq and rapid immunoprecipitation and mass spectroscopy of endogenous proteins, we report that cells overexpressing CREB5 demonstrate extensive reprogramming of nuclear protein-protein interactions in response to the ART agent enzalutamide. Specifically, CREB5 physically interacts with AR, the pioneering actor FOXA1, and other known co-factors of AR and FOXA1 at transcription regulatory elements recently found to be active in mCRPC patients. We identified a subset of CREB5/FOXA1 co-interacting nuclear factors that have critical functions for AR transcription (GRHL2, HOXB13) while others (TBX3, NFIC) regulated cell viability and ART resistance and were amplified or overexpressed in mCRPC. Upon examining the nuclear protein interactions and the impact of CREB5 expression on the mCRPC patient transcriptome, we found that CREB5 was associated with Wnt signaling and epithelial to mesenchymal transitions, implicating these pathways in CREB5/FOXA1-mediated ART resistance. Overall, these observations define the molecular interactions among CREB5, FOXA1, and pathways that promote ART resistance.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
SHOC2 is a prototypical leucine-rich repeat protein that promotes downstream receptor tyrosine kinase (RTK)/RAS signaling and plays important roles in several cellular and developmental processes. Gain-of-function germ line mutations of SHOC2 drive the RASopathy Noonan-like syndrome, and SHOC2 mediates adaptive resistance to mitogen-activated protein kinase (MAPK) inhibitors. Similar to many scaffolding proteins, SHOC2 facilitates signal transduction by enabling proximal protein interactions and regulating the subcellular localization of its binding partners. Here, we review the structural features of SHOC2 that mediate its known functions, discuss these elements in the context of various binding partners and signaling pathways, and highlight areas of SHOC2 biology where a consensus view has not yet emerged.
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Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/metabolismo , Proteínas ras/metabolismo , Animais , Humanos , Proteínas de Repetições Ricas em Leucina , Sistema de Sinalização das MAP Quinases , Transdução de Sinais/fisiologiaRESUMO
MicroRNA-29 (miR-29) is a critical regulator of fibroinflammatory processes in human diseases. In this study, we found a decrease in miR-29a in experimental and human chronic pancreatitis, leading us to investigate the regulatory role of the miR-29a/b1 cluster in acute pancreatitis (AP) utilizing a conditional miR-29a/b1-KO mouse model. miR-29a/b1-sufficient (WT) and -deficient (KO) mice were administered supramaximal caerulein to induce AP and characterized at different time points, utilizing an array of IHC and biochemical analyses for AP parameters. In caerulein-induced WT mice, miR-29a remained dramatically downregulated at injury. Despite high-inflammatory milieu, fibrosis, and parenchymal disarray in the WT mice during early AP, the pancreata fully restored during recovery. miR-29a/b1-KO mice showed significantly greater inflammation, lymphocyte infiltration, macrophage polarization, and ECM deposition, continuing until late recovery with persistent parenchymal disorganization. The increased pancreatic fibrosis was accompanied by enhanced TGFß1 coupled with persistent αSMA+ PSC activation. Additionally, these mice exhibited higher circulating IL-6 and inflammation in lung parenchyma. Together, this collection of studies indicates that depletion of miR-29a/b1 cluster impacts the fibroinflammatory mechanisms of AP, resulting in (a) aggravated pathogenesis and (b) delayed recovery from the disease, suggesting a protective role of the molecule against AP.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Pancreatite Crônica/metabolismo , Pancreatite/genética , Animais , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Pancreatite/patologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer with a dismal prognosis. miR-29a is commonly downregulated in PDAC; however, mechanisms for its loss and role still remain unclear. Here, we show that in PDAC, repression of miR-29a is directly mediated by MYC via promoter activity. RNA sequencing analysis, integrated with miRNA target prediction, identified global miR-29a downstream targets in PDAC. Target enrichment coupled with gene ontology and survival correlation analyses identified the top five miR-29a-downregulated target genes (LOXL2, MYBL2, CLDN1, HGK, and NRAS) that are known to promote tumorigenic mechanisms. Functional validation confirmed that upregulation of miR-29a is sufficient to ablate translational expression of these five genes in PDAC. We show that the most promising target among the identified genes, LOXL2, is repressed by miR-29a via 3'-untranslated region binding. Pancreatic tissues from a PDAC murine model and patient biopsies showed overall high LOXL2 expression with inverse correlations with miR-29a levels. Collectively, our data delineate an antitumorigenic, regulatory role of miR-29a and a novel MYC-miR-29a-LOXL2 regulatory axis in PDAC pathogenesis, indicating the potential of the molecule in therapeutic opportunities. IMPLICATIONS: This study unravels a novel functional role of miR-29a in PDAC pathogenesis and identifies an MYC-miR-29a-LOXL2 axis in regulation of the disease progression, implicating miR-29a as a potential therapeutic target for PDAC. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/2/311/F1.large.jpg.
Assuntos
Aminoácido Oxirredutases/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Aminoácido Oxirredutases/metabolismo , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , TransfecçãoRESUMO
MicroRNAs (miRNA) are small non-coding RNAs (â¼22 nt in length) that are known as potent master regulators of eukaryotic gene expression. miRNAs have been shown to play a critical role in cancer pathogenesis, and the misregulation of miRNAs is a well-known feature of cancer. In recent years, miR-29 has emerged as a critical miRNA in various cancers, and it has been shown to regulate multiple oncogenic processes, including epigenetics, proteostasis, metabolism, proliferation, apoptosis, metastasis, fibrosis, angiogenesis, and immunomodulation. Although miR-29 has been thoroughly documented as a tumor suppressor in the majority of studies, some controversy remains with conflicting reports of miR-29 as an oncogene. In this review, we provide a systematic overview of miR-29's functional role in various mechanisms of cancer and introspection on the contradictory roles of miR-29.
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The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Proteínas ras/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Pelados , Camundongos SCID , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, â¼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.
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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies with a 5-year survival rate of 8%. Dense, fibrotic stroma associated with pancreatic tumors is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Targeting stroma is considered as a potential therapeutic strategy to improve anti-cancer drug efficacy and patient survival. Although numerous stromal depletion therapies have reached the clinic, they add little to overall survival and are often associated with toxicity. Furthermore, increasing evidence suggests the anti-tumor properties of stroma. Its complete ablation enhanced tumor progression and reduced survival. Consequently, efforts are now focused on developing stromal-targeted therapies that normalize the reactive stroma and avoid the extremes: stromal abundance vs. complete depletion. In this review, we summarized the state of current and emerging anti-stromal targeted therapies, with major emphasis on the role of miRNAs in PDAC stroma and their potential use as novel therapeutic agents to modulate PDAC tumor-stromal interactions.
Assuntos
Adenocarcinoma/terapia , Carcinoma Ductal Pancreático/terapia , MicroRNAs/metabolismo , Células Estromais/patologia , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Humanos , Microambiente TumoralRESUMO
Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal malignancy that responds poorly to current therapeutic modalities. In an effort to develop novel therapeutic strategies, we found downregulation of miR-29 in pancreatic cancer cells, and overexpression of miR-29a sensitized chemotherapeutic resistant pancreatic cancer cells to gemcitabine, reduced cancer cell viability, and increased cytotoxicity. Furthermore, miR-29a blocked autophagy flux, as evidenced by an accumulation of autophagosomes and autophagy markers, LC3B and p62, and a decrease in autophagosome-lysosome fusion. In addition, miR-29a decreased the expression of autophagy proteins, TFEB and ATG9A, which are critical for lysosomal function and autophagosome trafficking respectively. Knockdown of TFEB or ATG9A inhibited autophagy similar to miR-29a overexpression. Finally, miR-29a reduced cancer cell migration, invasion, and anchorage independent growth. Collectively, our findings indicate that miR-29a functions as a potent autophagy inhibitor, sensitizes cancer cells to gemcitabine, and decreases their invasive potential. Our data provides evidence for the use of miR-29a as a novel therapeutic agent to target PDAC.
Assuntos
Autofagia , Carcinoma Ductal Pancreático/patologia , MicroRNAs/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Transição Epitelial-Mesenquimal , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Invasividade Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/genética , GencitabinaRESUMO
Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression, and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability, and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-ß1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.