RESUMO
OBJECTIVE: In highly aggressive malignant cancers including breast cancer, vasculogenic mimicry (VM) is the potential of tumor cells to generate a vascular channel network for delivering blood to tumor cells. Detection of genes involved in this process is critical to designing targeted therapy against breast cancer metastasis. In this study, we evaluated the roles of FAK and ILK in the progression of VM in metastatic breast tumor cells. RESULTS: Primary (4T1T), and highly metastatic (4T1B and 4T1L) breast tumor cells were isolated from cancerous mice. The potential of cancer cells to organize themselves into vascular-like structures (VM) has been evaluated with in vitro assessment. The expression of ILK and FAK were examined using real-time polymerase chain reaction. We confirmed the high ability of metastatic tumor cells in vascular-like structure formation. In molecular analysis, our data showed that ILK and FAK expression was significantly elevated in metastatic breast tumor cells. These results indicated that the higher potential of metastatic tumor cells in vascular-like structure formation may be related to higher expression of ILK and FAK. Analysis of molecular features of metastatic tumor cells could be utilized to create a targeted therapeutic strategy against metastasis in breast cancer.
Assuntos
Neoplasias da Mama , Metástase Neoplásica , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Feminino , Camundongos , Linhagem Celular Tumoral , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Camundongos Endogâmicos BALB C , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Severe viral pneumonia can induce rapid expansion of KRT5+ basal-like cells in small airways and alveoli; this forms a scar-like structure that persists in the injured alveoli and impedes normal alveolar epithelium regeneration. In this study, we investigated the mechanism by which viral infection induced this remodeling response. Through comparing different lung-injury models, we demonstrated that infection induced strong IFN-γ signal-stimulated dysplastic KRT5+ cell formation. Inactivation of interferon receptor 1 (Ifngr1) reduced dysplastic cell formation, ameliorated lung fibrosis, and improved lung-function recovery. Mechanistically, IFN-γ regulated dysplastic cell formation via the focal adhesion kinase (FAK)/Yes-associated protein 1 (YAP) pathway. Inhibiting FAK/Src diminished IFN-γ-induced YAP nuclear translocation and dysplastic cell formation. Inhibiting YAP during viral infection prevented dysplastic cell formation, whereas inhibiting YAP in persistent KRT5+ cells led to their conversion into distal club cells. Importantly, human dysplastic cells exhibited elevated FAK and YAP activity, and IFN-γ treatment promoted the transformation of human alveolar progenitor cells into dysplastic cells. These findings uncover the role of infection-induced inflammatory response in alveolar remodeling and may provide potential therapeutic avenues for the treatment of alveolar remodeling in patients with severe viral pneumonia.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quinase 1 de Adesão Focal , Interferon gama , Alvéolos Pulmonares , Proteínas de Sinalização YAP , Proteínas de Sinalização YAP/metabolismo , Animais , Camundongos , Humanos , Interferon gama/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/virologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transdução de Sinais , Camundongos Knockout , Inflamação/patologia , Inflamação/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
BACKGROUND: High-grade endometrial cancers (EAC) are aggressive tumors with a high risk of progression after treatment. As EAC may harbor mutations in the RAS/MAPK pathways, we evaluated the preclinical in vitro and in vivo efficacy of avutometinib, a RAF/MEK clamp, in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718, against multiple primary EAC cell lines and xenografts. METHODS: Whole-exome sequencing (WES) was used to evaluate the genetic landscape of five primary EAC cell lines. The in vitro activity of avutometinib and defactinib as single agents and in combination was evaluated using cell viability, cell cycle, and cytotoxicity assays. Mechanistic studies were performed using Western blot assays while in vivo experiments were completed in UTE10 engrafted mice treated with either vehicle, avutometinib, VS-4718, or their combination through oral gavage. RESULTS: WES results demonstrated multiple EAC cell lines to harbor genetic derangements in the RAS/MAPK pathway including KRAS/PTEN/PIK3CA/BRAF/ARID1A, potentially sensitizing to FAK and RAF/MEK inhibition. Five out of five of the EAC cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition. By Western blot assays, exposure of EAC cell lines to defactinib, avutometinib, and their combination demonstrated decreased phosphorylated FAK (p-FAK) as well as decreased p-MEK and p-ERK. In vivo the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition compared to single-agent treatment and controls starting at Day 9 (p < 0.02 and p < 0.04) in UTE10 xenografts. CONCLUSIONS: Avutometinib, defactinib, and to a larger extent their combinations, demonstrated promising in vitro and in vivo activity against EAC cell lines and xenografts. These preclinical data support the potential clinical evaluation of this combination in high-grade EAC patients.
Assuntos
Neoplasias do Endométrio , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Humanos , Animais , Camundongos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/genética , Linhagem Celular Tumoral , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/patologia , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/metabolismo , Sequenciamento do Exoma , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Gradação de Tumores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Oxazepinas , Sulfonamidas , Pirazinas , Benzamidas , ImidazóisRESUMO
SPHK1 (sphingosine kinase type 1) is characterized as a rate-limiting enzyme in sphingolipid metabolism to phosphorylate sphingosine into sphingosine-1-phosphate (S1P) that can bind to S1P receptors (S1PRs) to initiate several signal transductions leading to cell proliferation and survival of normal cell. Many studies have indicated that SPHK1 is involved in several types of cancer development, however, a little is known in bladder cancer. The TCGA database analysis was utilized for analyzing the clinical relevance of SPHK1 in bladder cancer. Through CRISPR/Cas9 knockout (KO) and constitutive activation (CA) strategies on SPHK1 in the bladder cancer cells, we demonstrated the potential downstream target could be programmed cell death 1 ligand 2 (PD-L2). On the other hand, we demonstrated that FDA-approved SPHK1 inhibitor Gilenya® (FTY720) can successfully suppress bladder cancer metastasis by in vitro and in vivo approaches. This finding indicated that SPHK1 as a potent therapeutic target for metastatic bladder cancer by dissecting the mechanism of action, SPHK1/S1P-elicited Akt/ß-catenin activation promoted the induction of PD-L2 that is a downstream effector in facilitating bladder cancer invasion and migration. Notably, PD-L2 interacted with c-Src that further activates FAK. Here, we unveil the clinical relevance of SPHK1 in bladder cancer progression and the driver role in bladder cancer metastasis. Moreover, we demonstrated the inhibitory effect of FDA-approved SPHK1 inhibitor FTY720 on bladder cancer metastasis from both in vitro and in vivo models.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool) , Transdução de Sinais , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/genética , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Metástase Neoplásica , Camundongos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Quinases da Família src/metabolismo , Movimento Celular , Camundongos Nus , Lisofosfolipídeos/metabolismo , Proteína Tirosina Quinase CSK/metabolismo , Cloridrato de Fingolimode/farmacologia , Proliferação de CélulasRESUMO
Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both in vitro and in vivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in in vivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of in situ tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.
Assuntos
Mecanotransdução Celular , Camundongos Knockout , Tendões , Animais , Masculino , Camundongos , Células Cultivadas , Matriz Extracelular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Adesões Focais/metabolismo , Mecanotransdução Celular/fisiologia , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Tendões/metabolismo , Tendões/fisiologia , Tendões/citologia , FemininoRESUMO
BACKGROUND: Fibrosis cataract occurs in patients receiving cataract extraction. Still, no medication that can cure the disease exists in clinical. This study aims to investigate the effects and mechanisms of Entrectinib on fibrotic cataract in vitro and in vivo. METHODS: The human lens cells line SRA 01/04 and C57BL/6J mice were applied in the study. Entrectinib was used in animals and cells. Cataract severity was assessed by slit lamp and Hematoxylin and Eosin staining. Expression of alpha-smooth muscle actin, fibronectin, and collagen I were examined by real-time quantitative PCR, western blotting, and immunofluorescence. Cell proliferation was evaluated by Cell Counting Kit-8. Cell migration was measured by wound healing and transwell assays. Molecular docking, Drug Affinity Responsive Target Stability, and Cellular Thermal Shift Assay were applied to seek and certify the target of Entrectinib treating fibrosis cataract. RESULTS: Entrectinib can ameliorate fibrotic cataract in vitro and in vivo. At the RNA and the protein levels, the expression of alpha-smooth muscle actin, collagen I, and fibronectin can be downgraded by Entrectinib, while E-cadherin can be upregulated. The migration and proliferation of cells were inhibited by Entrectinib. Mechanistically, Entrectinib obstructs TGFß2/Smad and TGFß2/non-Smad signaling pathways to hinder the fibrosis cataract by targeting PYK2 protein. CONCLUSIONS: Targeting with PYK2, Entrectinib can block TGF-ß2/Smad and TGF-ß2/non-Smad signaling pathways, lessen the activation of EMT, and alleviate fibrosis cataract. Entrectinib may be a potential treatment for fibrosis cataract in clinic.
Assuntos
Catarata , Quinase 2 de Adesão Focal , Transdução de Sinais , Fator de Crescimento Transformador beta2 , Animais , Camundongos , Transdução de Sinais/efeitos dos fármacos , Catarata/etiologia , Catarata/tratamento farmacológico , Catarata/metabolismo , Catarata/patologia , Humanos , Fator de Crescimento Transformador beta2/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Movimento Celular/efeitos dos fármacos , Linhagem Celular , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Indazóis/farmacologia , Indazóis/uso terapêutico , Masculino , Quinase 1 de Adesão FocalRESUMO
Based on the joint analysis of multi-omic data and the biological experiments, we demonstrate that FOXF1 inhibits invasion and metastasis of lung adenocarcinoma cells and enhances anti-tumor immunity via regulating MFAP4/FAK signal axis in this study. The levels of FOXF1 and MFAP4 are significantly down-regulated in LUAD, and the increased levels of two genes can improve the clinical prognosis of LUAD patients. Fluorescein reporter gene determination, chromatin immunoprecipitation and gene co-expression analysis indicate that MFAP4 level is positively regulated by transcription factor FOXF1. The function enrichment analysis shows that the levels of FOXF1 and MFAP4 are closely associated with an enrichment of tumor metastasis signatures. FOXF1 can inhibit the migration and invasion of LAUD cells by transcriptionally activating MFAP4 expression. And the overexpression of FOXF1/MFAP4 can reduce focal adhesion kinase (FAK) phosphorylation, while their knockdown result in the opposite effects. The increased levels of FOXF1/MFAP4 enhance the antitumor immunity by increasing the infiltration of dendritic cells and CD4+ T cells, and the interactions between LUAD cells and immune cells, and activating multiple anti-tumor immunity-related pathways. In conclusion, our study reveals the potential function of FOXF1/MFAP4/FAK signal axis in inhibiting metastasis of LUAD cells and modulating anti-tumor immunity of LUAD patients.
Assuntos
Adenocarcinoma de Pulmão , Fatores de Transcrição Forkhead , Neoplasias Pulmonares , Invasividade Neoplásica , Transdução de Sinais , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Humanos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Movimento Celular , Camundongos , Animais , Proteína-Tirosina Quinases de Adesão Focal/metabolismoRESUMO
Thrombosis is a key process that determines acute coronary syndrome and ischemic stroke and is the leading cause of morbidity and mortality in the world, together with cancer. Platelet adhesion and subsequent activation and aggregation are critical processes that cause thrombus formation after endothelial damage. To date, high hopes are associated with compounds of natural origin, which show anticoagulant action without undesirable effects and can be proposed as supportive therapies. We investigated the effect of the new combination of four natural compounds, escin-bromelain-ginkgo biloba-sage miltiorrhiza (EBGS), on the initial process of the coagulation cascade, which is the adhesion of platelets to activated vascular endothelium. Our results demonstrated that EBGS pretreatment of endothelial cells reduces platelet adhesion even in the presence of the monocyte-lymphocyte population. Our data indicate that EBGS exerts its effects by inhibiting the transcription of adhesion molecules, including P-selectin, platelet membrane glycoprotein GP1b, integrins αV and ß3, and reducing the secretion of the pro-inflammatory cytokines interleukin 6, interleukin 8, and the metalloproteinases MMP-2 and MMP-9. Furthermore, we demonstrated that EBGS inhibited the expression of focal adhesion kinase (FAK), strictly involved in platelet adhesion, and whose activity is correlated with that of integrin ß3. The results shown in this manuscript suggest a possible inhibitory role of the new combination EBGS in the reduction in platelet adhesion to activated endothelium, thus possibly preventing coagulation cascade initiation.
Assuntos
Endotélio Vascular , Adesividade Plaquetária , Transdução de Sinais , Fator de Necrose Tumoral alfa , Humanos , Adesividade Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Salvia miltiorrhiza/química , Quinase 1 de Adesão Focal/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Extratos Vegetais/farmacologiaRESUMO
High invasive capacity and acquired tyrosine kinase inhibitors (TKI) resistance of kidney renal clear cell carcinoma (KIRC) cells remain obstacles to prolonging the survival time of patients with advanced KIRC. In the present study, we reported that sine oculis homeobox 1 (SIX1) was upregulated in sunitinib-resistant KIRC cells and metastatic KIRC tissues. Subsequently, we found that SIX1 mediated metastasis and sunitinib resistance via Focal adhesion (FA) signaling, and knockdown of SIX1 enhanced the antitumor efficiency of sunitinib in KIRC. Mechanistically, Integrin subunit beta 1 (ITGB1), an upstream gene of FA signaling, was a direct transcriptional target of SIX1. In addition, we showed that DExH-box helicase 9 (DHX9) was an important mediator for SIX1-induced ITGB1 transcription, and silencing the subunits of SIX1/DHX9 complex significantly reduced transcription of ITGB1. Downregulation of SIX1 attenuated nuclear translocation of DHX9 and abrogated the binding of DHX9 to ITGB1 promoter. Collectively, our results unveiled a new signal axis SIX1/ITGB1/FAK in KIRC and identified a novel therapeutic strategy for metastatic KIRC patients.
Assuntos
Carcinoma de Células Renais , RNA Helicases DEAD-box , Resistencia a Medicamentos Antineoplásicos , Adesões Focais , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio , Integrina beta1 , Neoplasias Renais , Metástase Neoplásica , Transdução de Sinais , Sunitinibe , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Linhagem Celular Tumoral , Integrina beta1/genética , Integrina beta1/metabolismo , Animais , Adesões Focais/genética , Adesões Focais/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Camundongos , Transcrição Gênica , Integrinas/metabolismo , Integrinas/genética , Quinase 1 de Adesão FocalRESUMO
Immune cells survey their microenvironment by forming dynamic cellular protrusions that enable chemotaxis, contacts with other cells, and phagocytosis. Podosomes are a unique type of protrusion structured by an adhesive ring of active integrins that surround an F-actin-rich core harboring degradative proteases. Although the features of podosomes, once-established, have been well defined, the steps that lead to podosome formation remain poorly understood by comparison. In this study, we report that spleen tyrosine kinase (Syk) is a critical regulator of podosome formation. Deletion of Syk or targeting its kinase activity eliminated the ability for murine macrophages to form podosomes. We found that the kinase activity of Syk was important for the phosphorylation of its substrates, HS1 and Pyk2, both of which regulate podosome formation. Additionally, before podosomes form, we report that the tandem Src homology 2 domains of Syk afforded multivalent clustering of ITAM-containing adaptors that associated with integrins to structure platforms that initiate podosomes. We therefore propose that Syk has a dual role in regulating podosomes: first, by facilitating the assembly of multivalent signaling hubs that nucleate their formation and second, by sustaining tyrosine kinase activity of the podosomes once they form against their substrates. In cells expressing recently identified gain-of-function variants of SYK, podosomes were dysregulated. These results implicate SYK in the (patho)physiological functions of podosomes in macrophages.
Assuntos
Macrófagos , Podossomos , Quinase Syk , Quinase Syk/metabolismo , Animais , Camundongos , Podossomos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Fosforilação , Quinase 2 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/genética , Camundongos Knockout , Integrinas/metabolismo , Transdução de Sinais , Humanos , Camundongos Endogâmicos C57BL , Quinase 1 de Adesão FocalRESUMO
Neurofibromatosis type 2 (NF2) is a rare disorder that causes vestibular schwannomas (VS), meningiomas and ependymomas. To date, there is no FDA approved drug-based treatment for NF2. We have previously identified that BET inhibition can selectively reduce growth of the NF2-null schwannoma and Schwann cells in vitro and tumorigenesis in vivo and, separately, reported that inhibition of Focal Adhesion Kinase 1 (FAK1) via crizotinib has antiproliferative effects in NF2-null Schwann cells. The current study was aimed at determining whether combined BET and FAK inhibition can synergize and to identify the mechanisms of action. A panel of normal and NF2-null Schwann and schwannoma cell lines were used to characterize the effects of combined BET and FAK inhibition in vitro and in vivo using pharmacological and genetic approaches. The mechanism of action was explored by chromatin immunoprecipitation, ChIP-PCR, western blotting, and functional approaches. We find that combined BET and FAK inhibition are synergistic and inhibit the proliferation of NF2-null schwannoma and Schwann cell lines in vitro and in vivo, by arresting cells in the G1/S and G2/M phases of the cell cycle. Further, we identify the mechanism of action through the downregulation of FAK1 transcription by BET inhibition, which potentiates inhibition of FAK by 100-fold. Our findings suggest that combined targeting of BET and FAK1 may offer a potential therapeutic option for the treatment of NF2-related schwannomas.
Assuntos
Proliferação de Células , Quinase 1 de Adesão Focal , Neurilemoma , Neurofibromina 2 , Neuroma Acústico , Animais , Humanos , Camundongos , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Neuroma Acústico/patologia , Neuroma Acústico/genética , Neuroma Acústico/tratamento farmacológico , Neuroma Acústico/metabolismo , Neurilemoma/patologia , Neurilemoma/genética , Neurilemoma/tratamento farmacológico , Neurilemoma/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Neurofibromatoses/tratamento farmacológico , Neurofibromatoses/genética , Neurofibromatoses/patologia , Neurofibromatose 2/genética , Neurofibromatose 2/tratamento farmacológico , Neurofibromatose 2/patologia , Neurofibromatose 2/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/patologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Irreversible pulmonary fibrosis induced by paraquat is the most prevalent cause of death in patients with paraquat poisoning. Pulmonary fibrosis is characterized by abnormal deposition of extracellular matrix (ECM). Currently, the role of fibrotic ECM microenvironment in paraquat-induced pulmonary fibrosis has not been established. METHODS: Rat pulmonary fibrosis model was induced by paraquat, ATN-161 (an integrin-ß1 antagonist) was given to investigate their effect on Rat survival and pulmonary fibrosis. Lungs were decellularized to generate normal and fibrotic acellular ECM scaffolds using Triton and SDS. Fibroblasts were cocultured with ECM scaffolds to established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. Then we explored the effect of fibrotic ECM microenvironment systematically promoting on integrin-ß1/FAK/ERK1/2 pathway and established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. RESULTS: Antagonism of integrin-ß1 could alleviate paraquat-induced pulmonary fibrosis and ameliorate survival status of rats. Compared to normal ECM, fibrotic extracellular microenvironment promoted the differentiation of fibroblasts to myofibroblasts. Antagonism of integrin-ß1 could also ameliorate the promotion of fibrotic extracellular microenvironment on differentiation of fibroblasts to myofibroblasts. Fibrotic ECM microenvironment promotes fibroblasts transforming into myofibroblasts through integrin-ß1/FAK/ERK1/2 signaling pathway. Moreover, this phenomenon holds independent on exogenous integrin-ß1. CONCLUSIONS: Activation of integrin-ß1/FAK/ERK1/2 pathway aggravates paraquat-induced pulmonary fibrosis depend on fibrotic ECM and integrin-ß1 may be a prospective therapeutic target for paraquat-induced pulmonary fibrosis in the future.
Assuntos
Matriz Extracelular , Fibroblastos , Integrina beta1 , Sistema de Sinalização das MAP Quinases , Paraquat , Fibrose Pulmonar , Ratos Sprague-Dawley , Animais , Paraquat/toxicidade , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Matriz Extracelular/metabolismo , Masculino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/metabolismo , Ratos , Integrina beta1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Miofibroblastos/efeitos dos fármacos , Células Cultivadas , Modelos Animais de DoençasRESUMO
The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.
Assuntos
Substância Própria , Fibroblastos , Adesões Focais , Mecanotransdução Celular , Humanos , Adesões Focais/metabolismo , Fibroblastos/metabolismo , Substância Própria/citologia , Substância Própria/metabolismo , Fosforilação , Matriz Extracelular/metabolismo , Células Cultivadas , Proteínas de Sinalização YAP/metabolismo , Colágenos Fibrilares/metabolismo , Âmnio/citologia , Âmnio/metabolismo , Quinase 1 de Adesão Focal/metabolismoRESUMO
M64HCl, which has drug-like properties, is a water-soluble Focal Adhesion Kinase (FAK) activator that promotes murine mucosal healing after ischemic or NSAID-induced injury. Since M64HCl has a short plasma half-life in vivo (less than two hours), it has been administered as a continuous infusion with osmotic minipumps in previous animal studies. However, the effects of more transient exposure to M64HCl on monolayer wound closure remained unclear. Herein, we compared the effects of shorter M64HCl treatment in vitro to continuous treatment for 24 hours on monolayer wound closure. We then investigated how long FAK activation and downstream ERK1/2 activation persist after two hours of M64HCl treatment in Caco-2 cells. M64HCl concentrations immediately after washing measured by mass spectrometry confirmed that M64HCl had been completely removed from the medium while intracellular concentrations had been reduced by 95%. Three-hour and four-hour M64HCl (100 nM) treatment promoted epithelial sheet migration over 24 hours similar to continuous 24-hour exposure. 100nM M64HCl did not increase cell number. Exposing cells twice with 2-hr exposures of M64HCl during a 24-hour period had a similar effect. Both FAK inhibitor PF-573228 (10 µM) and ERK kinase (MEK) inhibitor PD98059 (20 µM) reduced basal wound closure in the absence of M64HCl, and each completely prevented any stimulation of wound closure by M64HCl. Rho kinase inhibitor Y-27632 (20 µM) stimulated Caco-2 monolayer wound closure but no further increase was seen with M64HCl in the presence of Y-27632. M64HCl (100 nM) treatment for 3 hours stimulated Rho kinase activity. M64HCl decreased F-actin in Caco-2 cells. Furthermore, a two-hour treatment with M64HCl (100 nM) stimulated sustained FAK activation and ERK1/2 activation for up to 16 and hours 24 hours, respectively. These results suggest that transient M64HCl treatment promotes prolonged intestinal epithelial monolayer wound closure by stimulating sustained activation of the FAK/ERK1/2 pathway. Such molecules may be useful to promote gastrointestinal mucosal repair even with a relatively short half-life.
Assuntos
Mucosa Intestinal , Cicatrização , Humanos , Cicatrização/efeitos dos fármacos , Células CACO-2 , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Movimento Celular/efeitos dos fármacos , Piridinas/farmacologia , Animais , Amidas/farmacologiaRESUMO
The destruction of the blood-brain barrier and damage to the gastrointestinal mucosa after intracerebral hemorrhage (ICH) are important reasons for its high disability and mortality rates. However, the exact etiology is not yet clear. In addition, there are currently no effective treatments for improving cerebral edema and gastric mucosal damage after ICH. Trefoil factor 1 (TFF1) is a secretory protein that plays a crucial role in maintaining the integrity and barrier function of the gastric mucosa, and it has been reported to have a protective effect on brain damage induced by various causes. This study utilized a rat model of ICH induced by type IV collagenase was utilized, and intervened with recombinant TFF1 protein from an external institute to investigate the protective mechanisms of TFF1 against brain edema and gastric mucosal damage after ICH. The results demonstrated that TFF1 alleviated the neurological function and gastric mucosal damage in the rat model of ICH induced by type IV collagenase. TFF1 may ensure the integrity of the blood-brain and gastric mucosal barriers by regulating the EGFR (epidermal growth factor receptor)/Src (non-receptor tyrosine kinase)/FAK (focal adhesion kinase) pathway. Clearly, the disruption of the blood-brain barrier and the destruction of the gastric mucosal barrier are key pathological features of ICH, and TFF1 can improve the progression of blood-brain barrier and gastric mucosal barrier disruption in ICH by regulating the EGFR/Src/FAK pathway. Therefore, TFF1 may be a potential target for the treatment of ICH.
Assuntos
Edema Encefálico , Hemorragia Cerebral , Modelos Animais de Doenças , Receptores ErbB , Mucosa Gástrica , Fator Trefoil-1 , Quinases da Família src , Animais , Masculino , Ratos , Barreira Hematoencefálica/metabolismo , Edema Encefálico/metabolismo , Hemorragia Cerebral/metabolismo , Receptores ErbB/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/lesões , Ratos Sprague-Dawley , Transdução de Sinais , Quinases da Família src/metabolismo , Fator Trefoil-1/metabolismoRESUMO
BACKGROUND: Recycling of integrin via endosomal vesicles is critical for the migration of cancer cells, which leads to the metastasis of pancreatic cancer and devastating cancer-related death. So, new diagnostic and therapeutic molecules which target the recycling of endosomal vesicles need to be developed. METHODS: Public databases including TCGA, ICGC, GSE21501, GSE28735, and GENT are analyzed to derive diagnostic and therapeutic targets. To reveal biological roles and underlying mechanisms of molecular targets, various molecular biological experiments were conducted. RESULTS: First, we identified UNC13D's overexpression in patients with pancreatic cancer (n = 824) and its prognostic significance and high hazard ratio (HR) in four independent pancreatic cancer cohorts (TCGA, n = 178, p = 0.014, HR = 3.629; ICGC, n = 91, p = 0.000, HR = 4.362; GSE21501, n = 102, p = 0.002, HR = 2.339; GSE28735, n = 45, p = 0.022, HR = 2.681). Additionally, its expression is associated with the clinicopathological progression of pancreatic cancer. Further biological studies have shown that UNC13D regulates the migration of pancreatic cancer cells by coupling the exocytosis of recycling endosomes with focal adhesion turnover via the regulation of FAK phosphorylation. Immunoprecipitation and immunocytochemistry showed the formation of the RAB11-UNC13D-FAK axis in endosomes during integrin recycling. We observed that UNC13D directly interacted with the FERM domain of FAK and regulated FAK phosphorylation in a calcium-dependent manner. Finally, we found co-expression of UNC13D and FAK showed the poorest survival (TCGA, p = 0.000; ICGC, p = 0.036; GSE28735, p = 0.006). CONCLUSIONS: We highlight that UNC13D, a novel prognostic factor, promotes pancreatic cancer progression by coupling integrin recycling with focal adhesion turnover via the RAB11-UNC13D-FAK axis for the migration of pancreatic cancer cells.
Assuntos
Movimento Celular , Adesões Focais , Integrinas , Neoplasias Pancreáticas , Proteínas rab de Ligação ao GTP , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Integrinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Feminino , Masculino , Transdução de Sinais , Pessoa de Meia-Idade , Prognóstico , Regulação Neoplásica da Expressão Gênica , Endossomos/metabolismo , Progressão da DoençaRESUMO
Senescence plays a key role in various physiological and pathological processes. We reported that injury-induced transient senescence correlates with heart regeneration, yet the multi-omics profile and molecular underpinnings of regenerative senescence remain obscure. Using proteomics and single-cell RNA sequencing, here we report the regenerative senescence multi-omic signature in the adult mouse heart and establish its role in neonatal heart regeneration and agrin-mediated cardiac repair in adult mice. We identified early growth response protein 1 (Egr1) as a regulator of regenerative senescence in both models. In the neonatal heart, Egr1 facilitates angiogenesis and cardiomyocyte proliferation. In adult hearts, agrin-induced senescence and repair require Egr1, activated by the integrin-FAK-ERK-Akt1 axis in cardiac fibroblasts. We also identified cathepsins as injury-induced senescence-associated secretory phenotype components that promote extracellular matrix degradation and potentially assist in reducing fibrosis. Altogether, we uncovered the molecular signature and functional benefits of regenerative senescence during heart regeneration, with Egr1 orchestrating the process.
Assuntos
Proliferação de Células , Senescência Celular , Proteína 1 de Resposta de Crescimento Precoce , Miócitos Cardíacos , Regeneração , Animais , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Regeneração/fisiologia , Senescência Celular/fisiologia , Miócitos Cardíacos/metabolismo , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Transdução de Sinais , Fibroblastos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Cultivadas , Animais Recém-Nascidos , Modelos Animais de Doenças , Fenótipo Secretor Associado à Senescência , Proteômica , Análise de Célula Única , Masculino , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Knockout , Quinase 1 de Adesão FocalRESUMO
Cancer cells mechanically interact with the tumor microenvironment during cancer development. Mechano-reciprocity has emerged as a crucial factor affecting anti-cancer drug resistance during adjuvant therapy. Here, we investigated the focal adhesion kinase (FAK)/Yes-associated protein (YAP) signaling axis as a prospective strategy for circumventing cisplatin resistance in ovarian cancer (OC). The Cancer Genome Atlas (TCGA) data analysis revealed that FAK overexpression significantly correlated with unfavorable clinical outcomes in patients with ovarian cancer. AFM indentation experiments showed that cell elasticity depends on FAK activity. Notably, the combination of FAK inhibition and cisplatin treatment led to a 69â¯% reduction in the IC50 of cisplatin. This combined treatment also increased apoptosis compared to the individual treatments, along with the upregulation of the pro-apoptotic factor BAX and cleaved PARP. Suppressing FAK expression sequestered YAP in the cytosol, potentially reducing cellular proliferation and promoting apoptosis. Moreover, reduced FAK expression sensitized drug-resistant OC cells to cisplatin treatment owing to a decrease in nuclear tension, allowing the relocation of YAP to the cytosol. In a mouse model, the co-administration of an FAK inhibitor and cisplatin significantly suppressed tumor growth and increased apoptotic events and DNA fragmentation. Our findings suggest that drug resistance can be attributed to the perturbation of mechanosensing signaling pathways, which drive the mechanical reinforcement of cancer cells. OC cells can restore their sensitivity to cisplatin treatment by strategically reducing YAP localization in the nucleus through FAK downregulation.
Assuntos
Apoptose , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Quinase 1 de Adesão Focal , Camundongos Nus , Neoplasias Ovarianas , Fatores de Transcrição , Proteínas de Sinalização YAP , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Feminino , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Animais , Cisplatino/farmacologia , Linhagem Celular Tumoral , Proteínas de Sinalização YAP/metabolismo , Apoptose/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , Mecanotransdução Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The potential therapeutic benefits of human dental pulp stem cells (HDPSCs) in dental regenerative medicine have been demonstrated. However, little is known about the molecular mechanisms regulating the biological characteristics of HDPSCs. The experiment aims to explore whether VEGF activates signaling pathways such as FAK, PI3K, Akt, and p38 in HDPSCs, and to investigate the molecular mechanisms by which VEGF influences proliferation and migration of HDPSCs. Normal and inflamed human dental pulp (HDP) samples were collected, and the levels of VEGF in HDP were assessed. HDPSCs were cultured and purified. HDPSCs were stimulated with lipopolysaccharide (LPS) at gradient concentrations, and real-time quantitative polymerase chain reaction (qPCR) was used to assess changes in VEGF mRNA. Gradient concentrations of VEGF were used to stimulate HDPSCs, and cell migration ability was evaluated through scratch assays and Transwell chamber experiments. Phosphorylation levels of FAK, AKT, and P38 were assessed using Western blotting. Inhibitors of VEGFR2, FAK, AKT, P38, and VEGF were separately applied to HDPSCs, and cell migration ability and phosphorylation levels of FAK, AKT, and P38 were determined. The results indicated significant differences in VEGF levels between normal and inflamed HDP tissues, with levels in the inflamed state reaching 435% of normal levels (normal: 87.91 ng/mL, inflamed: 382.76 ng/mL, P < 0.05). LPS stimulation of HDPSCs showed a significant increase in VEGF mRNA expression with increasing LPS concentrations (LPS concentrations of 0.01, 0.1, 1, and 10 µg/mL resulted in VEGF mRNA expressions of 181.2%, 274.2%, 345.8%, and 460.9%, respectively, P < 0.05). VEGF treatment significantly enhanced the migration ability of HDPSCs in Transwell chamber experiments, with migration rates increasing with VEGF concentrations (VEGF concentrations of 0, 1, 10, 20, 50, and 100 ng/mL resulted in migration rates of 8.41%, 9.34%, 21.33%, 28.41%, 42.87%, and 63.15%, respectively, P < 0.05). Inhibitors of VEGFR2, FAK, AKT, P38, and combined VEGF stimulation demonstrated significant migration inhibition, with migration rates decreasing to 8.31%, 12.64%, 13.43%, 18.32%, and 74.17%, respectively. The migration rate with combined VEGF stimulation showed a significant difference (P < 0.05). The analysis of phosphorylation levels revealed that VEGF stimulation significantly activated phosphorylation of FAK, AKT, and P38, with phosphorylation levels increasing with VEGF concentrations (P < 0.05). The VEGF/VEGFR2 signaling axis regulated the migration ability of HDPSCs through the FAK/PI3K/AKT and P38MAPK pathways. This finding highlighted not only the crucial role of VEGF in injury repair of HDPSCs but also provided important clues for a comprehensive understanding of the potential applications of this signaling axis in dental regenerative medicine.
Assuntos
Movimento Celular , Proliferação de Células , Polpa Dentária , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Células-Tronco , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Humanos , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Movimento Celular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Células Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Fosforilação/efeitos dos fármacos , Adulto JovemRESUMO
OBJECTIVE: In this study, we investigated the mechanism of action of LIMK1 in cervical cancer progression. METHODS: The biological role of LIMK1 in regulating the growth, invasion, and metastasis of cervical cancer was studied in SiHa, CaSki cells and nude mice tumor models. The role of LIMK1 in the growth of cervical cancer was evaluated by HE staining. The role of LIMK1 in the invasion, metastasis, and proliferation of cervical cancer was evaluated by cell scratch, Transwell, and monoclonal experiments. The interaction among LIMK1, ROS, and Src was evaluated by Western blotting. The effects of regulating ROS and p-Src expression on LIMK1 in the migration/invasion and proliferation of cervical cancer cells were evaluated through cellular functional assays. RESULTS: Overexpression of LIMK1 promoted tumor growth in nude mice. Cell scratch, Transwell, and monoclonal experiments suggested that LIMK1 promoted the invasion, metastasis, and proliferation of cervical cancer cells. Western blotting suggested that LIMK1 can promote the expression of ROS-related proteins NOX2, NOX4, p-Src, and downstream proteins p-FAK, p-ROCK1/2, p-Cofilin-1, F-actin and inhibit the expression of p-SHP2 protein. Correction experiments showed that LIMK1 regulated the expression of p-FAK and p-Cofilin-1 proteins by regulating ROS and p-Src. Through the detection of cervical cancer cell functions, it was found that the activation of ROS and p-Src induced by LIMK1 is an early event that promotes the migration, proliferation, and invasion of cervical cancer cells. CONCLUSIONS: LIMK1 promotes the expression of F-actin and promotes the development of cervical cancer by regulating the oxidative stress/Src-mediated p-FAK/p-ROCK1/2/p-Cofilin-1 pathway.