RESUMEN

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.

Asunto(s)

Mecanotransducción Celular , Ratones Noqueados , Tendones , Animales , Masculino , Ratones , Células Cultivadas , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Adhesiones Focales/metabolismo , Mecanotransducción Celular/fisiología , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Tendones/metabolismo , Tendones/fisiología , Tendones/citología , Femenino

RESUMEN

The mechanical environment generated through the adhesive interaction of endothelial cells (ECs) with the matrix controls nuclear tension, preventing aberrant gene synthesis and the transition from restrictive to leaky endothelium, a hallmark of acute lung injury (ALI). However, the mechanisms controlling tension transmission to the nucleus and EC-restrictive fate remain elusive. Here, we demonstrate that, in a kinase-independent manner, focal adhesion kinase (FAK) safeguards tension transmission to the nucleus to maintain EC-restrictive fate. In FAK-depleted ECs, robust activation of the RhoA-Rho-kinase pathway increased EC tension and phosphorylation of the nuclear envelope protein, emerin, activating DNMT3a. Activated DNMT3a methylates the KLF2 promoter, impairing the synthesis of KLF2 and its target S1PR1 to induce the leaky EC transcriptome. Repleting FAK (wild type or kinase dead) or inhibiting RhoA-emerin-DNMT3a activities in damaged lung ECs restored KLF2 transcription of the restrictive EC transcriptome. Thus, FAK sensing and control of tension transmission to the nucleus govern restrictive endothelium to maintain lung homeostasis.

Asunto(s)

Núcleo Celular , Células Endoteliales , Factores de Transcripción de Tipo Kruppel , Transcriptoma , Proteína de Unión al GTP rhoA , Animales , Humanos , Ratones , Núcleo Celular/metabolismo , ADN Metiltransferasa 3A , Células Endoteliales/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fosforilación , Regiones Promotoras Genéticas/genética , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Transcriptoma/genética , Masculino , Femenino

RESUMEN

Podocyte apoptosis or loss is the pivotal pathological characteristic of diabetic kidney disease (DKD). Insulin-like growth factor-binding protein 2 (IGFBP2) have a proinflammatory and proapoptotic effect on diseases. Previous studies have shown that serum IGFBP2 level significantly increased in DKD patients, but the precise mechanisms remain unclear. Here, we found that IGFBP2 levels obviously increased under a diabetic state and high glucose stimuli. Deficiency of IGFBP2 attenuated the urine protein, renal pathological injury and glomeruli hypertrophy of DKD mice induced by STZ, and knockdown or deletion of IGFBP2 alleviated podocytes apoptosis induced by high concentration of glucose or in DKD mouse. Furthermore, IGFBP2 facilitated apoptosis, which was characterized by increase in inflammation and oxidative stress, by binding with integrin α5 (ITGA5) of podocytes, and then activating the phosphorylation of focal adhesion kinase (FAK)-mediated mitochondrial injury, including membrane potential decreasing, ROS production increasing. Moreover, ITGA5 knockdown or FAK inhibition attenuated the podocyte apoptosis caused by high glucose or IGFBP2 overexpression. Taken together, these findings unveiled the insight mechanism that IGFBP2 increased podocyte apoptosis by mitochondrial injury via ITGA5/FAK phosphorylation pathway in DKD progression, and provided the potential therapeutic strategies for diabetic kidney disease.

Asunto(s)

Apoptosis , Diabetes Mellitus Experimental , Nefropatías Diabéticas , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Mitocondrias , Podocitos , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/genética , Podocitos/metabolismo , Podocitos/patología , Animales , Ratones , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/genética , Masculino , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Estrés Oxidativo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Ratones Endogámicos C57BL , Transducción de Señal , Fosforilación , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Ratones Noqueados , Integrinas

RESUMEN

Glioblastoma (GBM), an aggressive form of brain cancer, has a higher incidence in non-Hispanics when compared to the US Hispanic population. Using data from RT-PCR analysis of 21 GBM tissue from Hispanic patients in Puerto Rico, we identified significant correlations in the gene expression of focal adhesion kinase and proline-rich tyrosine kinase (PTK2 and PTK2B) with NGFR (nerve growth factor receptor), PDGFRB (platelet-derived growth factor receptor B), EGFR (epithelial growth factor receptor), and CXCR1 (C-X-C motif chemokine receptor 1). This study further explores these correlations found in gene expression while accounting for sex and ethnicity. Statistically significant (p < 0.05) correlations with an r value > ±0.7 were subsequently contrasted with mRNA expression data acquired from cBioPortal for 323 GBM specimens. Significant correlations in Puerto Rican male patients were found between PTK2 and PTK2B, NGFR, PDGFRB, EGFR, and CXCR1, which did not arise in non-Hispanic male patient data. The data for Puerto Rican female patients showed correlations in PTK2 with PTK2B, NGFR, PDGFRB, and EGFR, all of which did not appear in the data for non-Hispanic female patients. The data acquired from cBioPortal for non-Puerto Rican Hispanic patients supported the correlations found in the Puerto Rican population for both sexes. Our findings reveal distinct correlations in gene expression patterns, particularly involving PTK2, PTK2B, NGFR, PDGFRB, and EGFR among Puerto Rican Hispanic patients when compared to non-Hispanic counterparts.

Asunto(s)

Neoplasias Encefálicas , Regulación Neoplásica de la Expresión Génica , Glioblastoma , Hispánicos o Latinos , Transducción de Señal , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/etnología , Receptores ErbB/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Glioblastoma/genética , Glioblastoma/etnología , Hispánicos o Latinos/genética , Puerto Rico , Transducción de Señal/genética

RESUMEN

OBJECTIVE: Genomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading to the fusion of the adhesion protein CLDN18 and RHO regulator ARHGAP26. It remains unclear how these fusion constructs impact the activity of the RHO pathway and what is their broader impact on gastric cancer development. Herein, we developed a model to allow us to study the function of this fusion protein in the pathogenesis of DGC and to identify potential therapeutic targets for DGC tumours with these alterations. DESIGN: We built a transgenic mouse model with LSL-CLDN18-ARHGAP26 fusion engineered into the Col1A1 locus where its expression can be induced by Cre recombinase. Using organoids generated from this model, we evaluated its oncogenic activity and the biochemical effects of the fusion protein on the RHOA pathway and its downstream cell biological effects in the pathogenesis of DGC. RESULTS: We demonstrated that induction of CLDN18-ARHGAP26 expression in gastric organoids induced the formation of signet ring cells, characteristic features of DGC and was able to cooperatively transform gastric cells when combined with the loss of the tumour suppressor geneTrp53. CLDN18-ARHGAP26 promotes the activation of RHOA and downstream effector signalling. Molecularly, the fusion promotes activation of the focal adhesion kinase (FAK) and induction of the YAP pathway. A combination of FAK and YAP/TEAD inhibition can significantly block tumour growth. CONCLUSION: These results indicate that the CLDN18-ARHGAP26 fusion is a gain-of-function DGC oncogene that leads to activation of RHOA and activation of FAK and YAP signalling. These results argue for further evaluation of emerging FAK and YAP-TEAD inhibitors for these deadly cancers.

Asunto(s)

Claudinas , Proteínas Activadoras de GTPasa , Ratones Transgénicos , Transducción de Señal , Neoplasias Gástricas , Factores de Transcripción , Proteínas Señalizadoras YAP , Proteína de Unión al GTP rhoA , Animales , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Ratones , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Claudinas/genética , Claudinas/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Factores de Transcripción de Dominio TEA , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Organoides/metabolismo , Organoides/patología

RESUMEN

BACKGROUND: Ribosomal RNA processing protein 15 (RRP15) has been found to regulate the progression of hepatocellular carcinoma (HCC). Nevertheless, the extent to which it contributes to the spread of HCC cells remains uncertain. Thus, the objective of this research was to assess the biological function of RRP15 in the migration of HCC. METHODS: The expression of RRP15 in HCC tissue microarray (TMA), tumor tissues and cell lines were determined. In vitro, the effects of RRP15 knockdown on the migration, invasion and adhesion ability of HCC cells were assessed by wound healing assay, transwell and adhesion assay, respectively. The effect of RRP15 knockdown on HCC migration was also evaluated in vivo in a mouse model. RESULTS: Bioinformatics analysis showed that high expression of RRP15 was significantly associated with low survival rate of HCC. The expression level of RRP15 was strikingly upregulated in HCC tissues and cell lines compared with the corresponding controls, and TMA data also indicated that RRP15 was a pivotal prognostic factor for HCC. RRP15 knockdown in HCC cells reduced epithelial-to-mesenchymal transition (EMT) and inhibited migration in vitro and in vivo, independent of P53 expression. Mechanistically, blockade of RRP15 reduced the protein level of the transcription factor POZ/BTB and AT hook containing zinc finger 1 (PATZ1), resulting in decreased expression of the downstream genes encoding laminin 5 subunits, LAMC2 and LAMB3, eventually suppressing the integrin ß4 (ITGB4)/focal adhesion kinase (FAK)/nuclear factor κB kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. CONCLUSIONS: RRP15 promotes HCC migration by activating the LAMC2/ITGB4/FAK pathway, providing a new target for future HCC treatment.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Procesamiento Postranscripcional del ARN , Proteínas Ribosómicas , Animales , Ratones , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , FN-kappa B/metabolismo , Ribosomas/metabolismo , Ribosomas/patología , Factores de Transcripción/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo

RESUMEN

BACKGROUND: Circular RNAs are highly stable regulatory RNAs that have been increasingly associated with tumorigenesis and progression. However, the role of many circRNAs in triple-negative breast cancer (TNBC) and the related mechanisms have not been elucidated. METHODS: In this study, we screened circRNAs with significant expression differences in the RNA sequencing datasets of TNBC and normal breast tissues and then detected the expression level of circRPPH1 by qRT‒PCR. The biological role of circRPPH1 in TNBC was then verified by in vivo and in vitro experiments. Mechanistically, we verified the regulatory effects between circRPPH1 and ZNF460 and between circRPPH1 and miR-326 by chromatin immunoprecipitation (ChIP), fluorescence in situ hybridization assay, dual luciferase reporter gene assay and RNA pull-down assay. In addition, to determine the expression of associated proteins, we performed immunohistochemistry, immunofluorescence, and western blotting. RESULTS: The upregulation of circRPPH1 in TNBC was positively linked with a poor prognosis. Additionally, both in vivo and in vitro, circRPPH1 promoted the biologically malignant behavior of TNBC cells. Additionally, circRPPH1 may function as a molecular sponge for miR-326 to control integrin subunit alpha 5 (ITGA5) expression and activate the focal adhesion kinase (FAK)/PI3K/AKT pathway. CONCLUSION: Our research showed that ZNF460 could promote circRPPH1 expression and that the circRPPH1/miR-326/ITGA5 axis could activate the FAK/PI3K/AKT pathway to promote the progression of TNBC. Therefore, circRPPH1 can be used as a therapeutic or diagnostic target for TNBC.

Asunto(s)

MicroARNs , Factores de Transcripción , Neoplasias de la Mama Triple Negativas , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Endógeno Competitivo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Circular/genética , Hibridación Fluorescente in Situ , Línea Celular Tumoral , Integrinas/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Proteínas de Unión al ADN/metabolismo

RESUMEN

Abnormal metabolism is regarded as an oncogenic hallmark related to tumor progression and therapeutic resistance. Present study employed multi-omics, including phosphoproteomics, untargeted metabolomics and lipidomics, to demonstrate that the pAKT2 Ser128 and pCCTα Ser315/319/323-positive cancer-associated fibroblasts (CAFs) substantially release phosphatidylcholines (PCs), contributing to the resistance of focal adhesion kinase (FAK) inhibitors in esophageal squamous cell carcinoma (ESCC) treatment. Additionally, we observed extremely low levels of FAK Tyr397 expression in CAFs, potentially offering no available target for FAK inhibitors playing their anti-growth role in CAFs. Consequently, FAK inhibitor increased the intracellular concentration of Ca2+ in CAFs, promoting the formation of AKT2/CCTα complex, leading to phosphorylation of CCTα Ser315/319/323 sites and eventually enhancing stromal PC production. This activation could stimulate the intratumoral Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway, triggering resistance to FAK inhibition. Analysis of clinical samples demonstrated that stromal pAKT2 Ser128 and pCCTα Ser315/319/323 are related to the tumor malignancy and reduced patient survival. Pseudo-targeted lipidomics and further validation cohort quantitatively showed that plasma PCs enable to distinguish the malignant extent of ESCC patients. In conclusion, inhibition of stroma-derived PCs and related pathway could be possible therapeutic strategies for tumor therapy.

Asunto(s)

Fibroblastos Asociados al Cáncer , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Transducción de Señal , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo

RESUMEN

The incidence of diabetic patients with non-alcoholic fatty liver disease (NAFLD) is continuously increasing worldwide. However, the specific mechanisms of NAFLD patients with diabetes are still not clear. Recent studies have indicated that integrins play an important role in NAFLD. In this study, we considered the relationship between integrin αv (IGTAV)/FAK pathway and sinusoidal capillarization. We investigated the difference between the expression of IGTAV, laminin (LN), focal adhesion kinase (FAK), and phosphor-FAK protein in human liver sinusoidal endothelial cells (HLSECs) to explore the specific mechanisms of the diseases of NAFLD with diabetes under high glucose. We cultured and identified the HLSECs and constructed the recombinant lentivirus vector with IGTAV shRNA by quantitative real-time PCR (qRT-PCR) to silence the IGTAV gene. Cells were divided into groups of 25 mmol/L glucose and 25 mmol/L mannitol. We measured the protein levels of IGTAV, LN, FAK, and phosphor-FAK by western blot at 2 h, 6 h, and 12 h before and after IGTAV gene silencing. The lentivirus vector was successfully constructed with IGTAV shRNA. The HLSECs under high glucose were observed by scanning electron microscope. SPSS19.0 was used for statistical analysis. High glucose significantly increased the expression of IGTAV, LN, and phosphor-FAK protein in HLSECs; the shRNA IGTAV could effectively inhibit the expression of phosphor-FAK and LN at 2 h and 6 h. Inhibition of the phosphor-FAK could effectively decrease the expression of LN in HLSECs at 2 h and 6 h under high glucose. Inhibition of IGTAV gene of HLSECs under high glucose could improve hepatic sinus capillarization. Inhibition of IGTAV and phosphor-FAK decreased the expression of LN. High glucose led to hepatic sinus capillarization via IGTAV/ FAK pathway.

Asunto(s)

Diabetes Mellitus , Enfermedad del Hígado Graso no Alcohólico , Humanos , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrina alfaV/metabolismo , Células Endoteliales , Capilares/metabolismo , Glucosa/metabolismo , ARN Interferente Pequeño

RESUMEN

Lenvatinib is a clinically effective multikinase inhibitor approved for first-line therapy of advanced hepatocellular carcinoma (HCC). Although resistance against lenvatinib often emerges and limits its antitumor activity, the underlying molecular mechanisms involved in endogenous and acquired resistance remain elusive. In this study, we identified focal adhesion kinase (FAK) as a critical contributor to lenvatinib resistance in HCC. The elevated expression and phosphorylation of FAK were observed in both acquired and endogenous lenvatinib-resistant (LR) HCC cells. Furthermore, inhibition of FAK reversed lenvatinib resistance in vitro and in vivo. Mechanistically, FAK promoted lenvatinib resistance through regulating lysine-deficient kinase 1 (WNK1). Phosphorylation of WNK1 was significantly increased in LR-HCC cells. Further, WNK1 inhibitor WNK463 resensitized either established or endogenous LR-HCC cells to lenvatinib treatment. In addition, overexpression of WNK1 desensitized parental HCC cells to lenvatinib treatment. Conclusively, our results establish a crucial role and novel mechanism of FAK in lenvatinib resistance and suggest that targeting the FAK/WNK1 axis is a promising therapeutic strategy in HCC patients showing lenvatinib resistance.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Lisina/uso terapéutico , Línea Celular Tumoral

RESUMEN

The characteristic genetic abnormality of neuroendocrine neoplasms (NENs), a heterogeneous group of tumors found in various organs, remains to be identified. Here, based on the analysis of the splicing variants of an oncogene Focal Adhesion Kinase (FAK) in The Cancer Genome Atlas datasets that contain 9193 patients of 33 cancer subtypes, we found that Box 6/Box 7-containing FAK variants (FAK6/7) were observed in 7 (87.5%) of 8 pancreatic neuroendocrine carcinomas and 20 (11.76%) of 170 pancreatic ductal adenocarcinomas (PDACs). We tested FAK variants in 157 tumor samples collected from Chinese patients with pancreatic tumors, and found that FAK6/7 was positive in 34 (75.6%) of 45 pancreatic NENs, 19 (47.5%) of 40 pancreatic solid pseudopapillary neoplasms, and 2 (2.9%) of 69 PDACs. We further tested FAK splicing variants in breast neuroendocrine carcinoma (BrNECs), and found that FAK6/7 was positive in 14 (93.3%) of 15 BrNECs but 0 in 23 non-NEC breast cancers. We explored the underlying mechanisms and found that a splicing factor serine/arginine repetitive matrix protein 4 (SRRM4) was overexpressed in FAK6/7-positive pancreatic tumors and breast tumors, which promoted the formation of FAK6/7 in cells. These results suggested that FAK6/7 could be a biomarker of NENs and represent a potential therapeutic target for these orphan diseases.

Asunto(s)

Empalme Alternativo , Neoplasias de la Mama , Carcinoma Ductal Pancreático , Tumores Neuroendocrinos , Neoplasias Pancreáticas , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/uso terapéutico , Proteínas del Tejido Nervioso/genética , Tumores Neuroendocrinos/genética , Oncogenes , Neoplasias Pancreáticas/metabolismo

RESUMEN

BACKGROUND: Colorectal cancer (CRC) can be classified into four molecular subtypes (CMS) among which CMS1 is associated with the best prognosis, while CMS4, the mesenchymal subtype, has the worst outcome. Although mitochondria are considered to be hubs of numerous signaling pathways, the study of mitochondrial metabolism has been neglected for many years. Mitochondrial Complex I (CI) plays a dual role, both in energy and reactive oxygen species (ROS) production. However, the possible contribution of CI to tumorigenesis in cancer remains unclear. The purpose of this study was to investigate the CI under the prism of the CMS classification of CRC in ex vivo models. METHODS: Biochemical dosages, bioenergetics analysis and western-blot were used to characterize CI expression, function and redox balance in LoVo and MDST8 cell lines, belonging to CMS1 and CMS4 subgroups, respectively. Cell proliferation and migration were assessed by xCELLigence technology. Overproduction or scavenging of mitochondrial ROS (mtROS) were performed to analyze the effect of mtROS on proliferation, migration, and mesenchymal markers. Focal adhesion kinase (FAK) and its activation were analyzed by immunofluorescence. We assessed the distribution of two CI scores in CRC cohorts according to CMS classification and their relevance for patient survival. RESULTS: We found that CI is downregulated in CMS4 cells and is associated with elevated mtROS. We establish for the first time that in these migrating cells, mtROS production is maintained at optimal levels not only through changes in CI activity but also by inactivation/acetylation of superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. We show that promoting or scavenging mtROS both mitigate CMS4 cells' migration. Our results also point to a mtROS-mediated focal adhesion kinase (FAK) activation, which likely sustains their migratory phenotype. Using cohorts of CRC patients, we document that the expression of CI is downregulated in the CMS4 subgroup, and that low CI expression is associated with poor prognosis. Patients' datasets reveal an inverse correlation between CI and the epithelial-mesenchymal transition (EMT) pathway. CONCLUSION: We showed that inhibition of CI contributes to heighten mtROS, which likely foster MDST8 migration and might account for the specific EMT signature of CMS4 tumors. These data reveal a novel role of mitochondrial CI in CRC, with biological consequences that may be targeted with anti- or pro-oxidant drugs in clinical practice.

Asunto(s)

Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/genética , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Abajo , Transducción de Señal , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo

RESUMEN

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

Asunto(s)

Adhesiones Focales , Animales , Adhesiones Focales/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Fosforilación , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Movimiento Celular/fisiología , Adhesión Celular/fisiología

RESUMEN

Gastric cancer (GC) is one of the most common malignancies in China and is associated with high mortality. The occurrence and development of gastric cancer are related to genetic and environmental factors. Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor protein tyrosine kinase that is activated by the extracellular matrix and growth factors. FAK is highly expressed in cancer and promotes its development by regulating cancer cell proliferation, migration, and angiogenesis. The expression of IL-8 is increased in many types of malignant tumor cells and is linked to their proliferation, migration, invasion, angiogenesis, and EMT. In this study, we found FAK to be essential for the proliferation, migration, and peritoneal metastasis of gastric cancer cells. To examine the molecular regulatory mechanisms of FAK in the peritoneal dissemination of gastric cancer, we performed RNA-seq analysis of MKN-45-FAK-/- and MKN45 cells and demonstrated that IL-8 was downregulated in FAK-deficient cells. Conversely, we confirmed that IL-8 activates FAK activity. We established that IL-8 promotes the proliferation, colony formation, and migration of gastric cancer cells that are partially mediated by FAK. Thus, we propose that an IL-8-FAK-IL-8 positive feedback loop effects the proliferation and migration of gastric cancer cells.

Asunto(s)

Neoplasias Gástricas , Humanos , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Neoplasias Gástricas/patología , Interleucina-8/genética , Proliferación Celular , Movimiento Celular/genética , Línea Celular Tumoral

RESUMEN

Non-small-cell lung cancer (NSCLC) predominates lung cancer with a striking percentage of 85%. Eupafolin is documented to possess anti-tumor efficacy, which prompts efforts to uncover its impacts on the pathology of diseases including cancers. Focal adhesion kinase (FAK)-mediated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) has been found to be associated with several carcinomas. Nevertheless, how eupafolin exerts its effects in NSCLC and whether FAK/PI3K/AKT is related to the corresponding mechanism remain unclear. Thus, the relevant experiments were carried out with NSCLC cells treated with eupafolin and/or LY294002 at first. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing, and transwell assays were used to assess cell viability, proliferation, migration, and invasion, respectively. Western blot assay was performed to measure the relative protein expressions of phosphorylated (p)-FAK/FAK, p-PI3K/ PI3K, p-AKT/AKT, matrix metalloproteinase 9 (MMP9), and ras homolog gene family member A (RhoA), and to determine transfection efficiency. From experimental results, it was found that eupafolin inhibited the viability, proliferation, migration, and invasion of NSCLC cells, and inactivated the FAK/PI3K/AKT pathway by downregulating the ratios of p-FAK/FAK, p-PI3K/PI3K, and p-AKT/AKTand the expressions of MMP9 and RhoA. On the contrary, overexpressed FAK upregulated the expressions of FAK, MMP9, and RhoA and the ratios of p-PI3K/ PI3K and p-AKT/AKT, and promoted cell proliferation, migration, and invasion. LY294002, conversely, could partly reverse the effects of FAK on the aforementioned aspects of NSCLC cells. Collectively, it was verified in our study that eupafolin regulates the proliferation, migration, and invasion of NSCLC cells by downregulating MMP9 and RhoA expressions via the FAK/PI3K/AKT axis, which may provide a promising avenue for cancer therapy.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasa/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Transducción de Señal , Proliferación Celular/genética , Movimiento Celular/genética , Línea Celular Tumoral , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/farmacología

RESUMEN

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.

Asunto(s)

Carcinogénesis , Proteína-Tirosina Quinasas de Adhesión Focal , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Humanos , Carcinogénesis/genética

Asunto(s)

Neoplasias Meníngeas , Meningioma , Neurofibromatosis 2 , Humanos , Meningioma/tratamiento farmacológico , Meningioma/genética , Neurofibromatosis 2/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Mutación , Neoplasias Meníngeas/genética

RESUMEN

PURPOSE: Patients with progressive or recurrent meningiomas have limited systemic therapy options. Focal adhesion kinase (FAK) inhibition has a synthetic lethal relationship with NF2 loss. Given the predominance of NF2 mutations in meningiomas, we evaluated the efficacy of GSK2256098, a FAK inhibitor, as part of the first genomically driven phase II study in recurrent or progressive grade 1-3 meningiomas. PATIENTS AND METHODS: Eligible patients whose tumors screened positively for NF2 mutations were treated with GSK2256098, 750 mg orally twice daily, until progressive disease. Efficacy was evaluated using two coprimary end points: progression-free survival at 6 months (PFS6) and response rate by Macdonald criteria, where PFS6 was evaluated separately within grade-based subgroups: grade 1 versus 2/3 meningiomas. Per study design, the FAK inhibitor would be considered promising in this patient population if either end point met the corresponding decision criteria for efficacy. RESULTS: Of 322 patients screened for all mutation cohorts of the study, 36 eligible and evaluable patients with NF2 mutations were enrolled and treated: 12 grade 1 and 24 grade 2/3 patients. Across all grades, one patient had a partial response and 24 had stable disease as their best response to treatment. In grade 1 patients, the observed PFS6 rate was 83% (10/12 patients; 95% CI, 52 to 98). In grade 2/3 patients, the observed PFS6 rate was 33% (8/24 patients; 95% CI, 16 to 55). The study met the PFS6 efficacy end point both for the grade 1 and the grade 2/3 cohorts. Treatment was well tolerated; seven patients had a maximum grade 3 adverse event that was at least possibly related to treatment with no grade 4 or 5 events. CONCLUSION: GSK2256098 was well tolerated and resulted in an improved PFS6 rate in patients with recurrent or progressive NF2-mutated meningiomas, compared with historical controls. The criteria for promising activity were met, and FAK inhibition warrants further evaluation for this patient population.

Asunto(s)

Neoplasias Meníngeas , Meningioma , Humanos , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/uso terapéutico , Neoplasias Meníngeas/tratamiento farmacológico , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patología , Meningioma/tratamiento farmacológico , Meningioma/genética , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico

RESUMEN

Integrins are cellular adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Integrins can stimulate various signaling pathways by binding to different ligands, thereby exerting immunological functions. While integrins have been found to primarily play a role in bacterial agglutination, phagocytosis, and inhibition of apoptosis in invertebrates, the specific signaling pathway and mechanism of action remain unclear. In vertebrates, ß1 integrin and extracellular matrix interactions can associate with focal adhesion kinase (FAK) to initiate MAPK/ERK signaling and regulate cell survival; however, in invertebrates (e.g., Chinese mitten crab), the mechanisms of integrins are poorly understood. The purpose of this study was to investigate whether integrinß1/FAK activation of the MAPK/ERK pathway regulates hemocyte survival and the associated mechanism. Treatment with an integrinß1 inhibitor RGD (a conserved tripeptide Arg-Gly-Asp), decreased the levels of FAK and ERK expression and phosphorylation, followed by an intensification of apoptosis. Similar results were obtained following siRNA knockdown of integrinß1 expression. We further found that the attenuation of ERK phosphorylation enhanced the level of Caspase-3 expression. Together, these findings suggest that integrinß1 activates the FAK/ERK signaling cascade and is involved in the survival of Chinese mitten crab hemocytes.

Asunto(s)

Hemocitos , Sistema de Señalización de MAP Quinasas , Animales , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Sistema de Señalización de MAP Quinasas/fisiología , Transducción de Señal/fisiología , Integrinas

RESUMEN

Nonalcoholic steatohepatitis (NASH), a common clinical disease, is becoming a leading cause of hepatocellular carcinoma (HCC). Dual specificity phosphatase 22 (DUSP22, also known as JKAP or JSP-1) expressed in numerous tissues plays essential biological functions in immune responses and tumor growth. However, the effects of DUSP22 on NASH still remain unknown. Here, we find a significant decrease of DUSP22 expression in human and murine fatty liver, which is mediated by reactive oxygen species (ROS) generation. Hepatic-specific DUSP22 deletion particularly exacerbates lipid deposition, inflammatory response and fibrosis in liver, facilitating NASH and non-alcoholic fatty liver disease (NAFLD)-associated HCC progression. In contrast, transgenic over-expression, lentivirus or adeno-associated virus (AAV)-mediated DUSP22 gene therapy substantially inhibit NASH-related phenotypes and HCC development in mice. We provide mechanistic evidence that DUSP22 directly interacts with focal adhesion kinase (FAK) and restrains its phosphorylation at Tyr397 (Y397) and Y576 + Y577 residues, subsequently prohibiting downstream activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) cascades. The binding of DUSP22 to FAK and the dephosphorylation of FAK are indispensable for DUSP22-meliorated NASH progression. Collectively, our findings identify DUSP22 as a key suppressor of NASH-HCC, and underscore the DUSP22-FAK axis as a promising therapeutic target for treatment of the disease.

Asunto(s)

Carcinoma Hepatocelular , Fosfatasas de Especificidad Dual/metabolismo , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Animales , Carcinoma Hepatocelular/metabolismo , Progresión de la Enfermedad , Fosfatasas de Especificidad Dual/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Hepatocitos/metabolismo , Humanos , Lípidos , Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Especies Reactivas de Oxígeno/metabolismo