RESUMO
Background: Fibroblast activation protein (FAP), a cell surface serine protease, plays roles in tumor invasion and immune regulation. However, there is currently no pan-cancer analysis of FAP. Objective: We aimed to assess the pan-cancer expression profile of FAP, its molecular function, and its potential role in head and neck squamous cell carcinoma (HNSC). Methods: We analyzed gene expression, survival status, immune infiltration, and molecular functional pathways of FAP in The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) tumors. Furthermore, to elucidate the role of FAP in HNSC, we performed proliferation, migration, and invasion assays post-FAP overexpression or knock-down. Results: FAP expression was elevated in nine tumor types and was associated with poor survival in eight of them. In the context of immune infiltration, FAP expression negatively correlated with CD8+ T-cell infiltration in five tumor types and positively with regulatory T-cell infiltration in four tumor types. Our enrichment analysis highlighted FAP's involvement in the PI3K-Akt signaling pathway. In HNSC cells, FAP overexpression activated the PI3K-Akt pathway, promoting tumor proliferation, migration, and invasion. Conversely, FAP knockdown showed inhibitory effects. Conclusion: Our study unveils the association of FAP with poor tumor prognosis across multiple cancers and highlights its potential as a therapeutic target in HNSC.
Assuntos
Biomarcadores Tumorais , Movimento Celular , Proliferação de Células , Endopeptidases , Neoplasias de Cabeça e Pescoço , Proteínas de Membrana , Serina Endopeptidases , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Prognóstico , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Endopeptidases/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Gelatinases/metabolismo , Gelatinases/genética , Linhagem Celular Tumoral , Transdução de Sinais , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismoRESUMO
Introduction: This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Methods: Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response. Results: Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. Conclusions: These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development.
Assuntos
Fibroblastos Associados a Câncer , Imunoterapia , Serina Endopeptidases , Microambiente Tumoral , Microambiente Tumoral/imunologia , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/imunologia , Imunoterapia/métodos , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Citocinas/metabolismo , Endopeptidases , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Gelatinases/metabolismo , Gelatinases/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Neoplasias Renais/metabolismo , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/terapia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismoRESUMO
Head and neck squamous cell carcinoma (HNSCC) poses significant challenges with poor survival rates and limited therapeutic strategies. Our study, using The Cancer Genome Atlas (TCGA) data, assesses cancer-associated fibroblast (CAF) gene signatures' clinical relevance. In our analysis across TCGA tumor types, differential gene expression analysis revealed that fibroblast activation protein (FAP) is upregulated in tumor tissues and associated with poorer survival rates in HNSCC. Furthermore, mechanistic studies employing gene-silencing techniques substantiated that FAP knockout led to a significant decrease in cellular proliferation, invasion, and migration in HNSCC cell lines. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we established that high FAP expression correlates with vital biological processes such as extracellular matrix organization, angiogenesis, and cellular motility. Importantly, FAP was found to regulate these processes by promoting the expression of key proteins involved in epithelial-mesenchymal transition-related pathways. Additionally, our analysis revealed a significant correlation between FAP expression and the expression profiles of immune checkpoint molecules, underscoring its potential role in immune modulation. Collectively, our findings illuminate FAP's pivotal role in HNSCC pathogenesis and its potential as a prognostic biomarker and therapeutic target. This research lays the groundwork for understanding the multifaceted roles and regulatory mechanisms of CAFs in HNSCC, thereby offering valuable perspectives for the development of targeted therapeutic strategies aimed at improving patient outcomes.
Assuntos
Biomarcadores Tumorais , Endopeptidases , Gelatinases , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço , Proteínas de Membrana , Serina Endopeptidases , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Endopeptidases/metabolismo , Endopeptidases/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Gelatinases/metabolismo , Gelatinases/genética , Transição Epitelial-Mesenquimal/genética , Proliferação de Células/genética , Movimento Celular/genéticaRESUMO
Radiotherapy combined with immune checkpoint blockade holds great promise for synergistic antitumor efficacy. Targeted radionuclide therapy delivers radiation directly to tumor sites. LNC1004 is a fibroblast activation protein (FAP)-targeting radiopharmaceutical, conjugated with the albumin binder Evans Blue, which has demonstrated enhanced tumor uptake and retention in previous preclinical and clinical studies. Herein, we demonstrate that 68Ga/177Lu-labeled LNC1004 exhibits increased uptake and prolonged retention in MC38/NIH3T3-FAP and CT26/NIH3T3-FAP tumor xenografts. Radionuclide therapy with 177Lu-LNC1004 induced a transient upregulation of PD-L1 expression in tumor cells. The combination of 177Lu-LNC1004 and anti-PD-L1 immunotherapy led to complete eradication of all tumors in MC38/NIH3T3-FAP tumor-bearing mice, with mice showing 100% tumor rejection upon rechallenge. Immunohistochemistry, single-cell RNA sequencing (scRNA-seq), and TCR sequencing revealed that combination therapy reprogrammed the tumor microenvironment in mice to foster antitumor immunity by suppressing malignant progression and increasing cell-to-cell communication, CD8+ T-cell activation and expansion, M1 macrophage counts, antitumor activity of neutrophils, and T-cell receptor diversity. A preliminary clinical study demonstrated that 177Lu-LNC1004 was well-tolerated and effective in patients with refractory cancers. Further, scRNA-seq of peripheral blood mononuclear cells underscored the importance of addressing immune evasion through immune checkpoint blockade treatment. This was emphasized by the observed increase in antigen processing and presentation juxtaposed with T cell inactivation. In conclusion, our data supported the efficacy of immunotherapy combined with 177Lu-LNC1004 for cancer patients with FAP-positive tumors.
Assuntos
Inibidores de Checkpoint Imunológico , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Endopeptidases/genética , Células NIH 3T3 , Compostos Radiofarmacêuticos/uso terapêutico , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Imunoterapia , Gelatinases/genética , Gelatinases/imunologia , Lutécio/farmacologia , Linhagem Celular TumoralRESUMO
Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell coculture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the proinflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of proinflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies is through effects on the immune microenvironment and antitumor immune response. SIGNIFICANCE: The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.
Assuntos
Fibroblastos Associados a Câncer , Endopeptidases , Imunoconjugados , Microambiente Tumoral , Humanos , Animais , Imunoconjugados/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Fibroblastos Associados a Câncer/imunologia , Camundongos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Endopeptidases/genética , Endopeptidases/metabolismo , Linhagem Celular Tumoral , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Gelatinases/metabolismo , Gelatinases/genética , Oligopeptídeos/farmacologia , FemininoRESUMO
PURPOSE: In this study, we report the results from the esophageal squamous cell carcinoma (SCC) cohort of a phase II, noncomparative, basket study evaluating the antitumor activity and safety of fibroblast activation protein-IL2 variant (FAP-IL2v) plus atezolizumab in patients with advanced/metastatic solid tumors (NCT03386721). PATIENTS AND METHODS: Eligible patients had an Eastern Cooperative Oncology Group performance status of 0 to 1; measurable metastatic, persistent, or recurrent esophageal SCC; progression on ≥1 prior therapy; and were checkpoint inhibitor-naïve. Patients received FAP-IL2v 10 mg plus atezolizumab 1,200 mg intravenously every 3 weeks, or FAP-IL2v weekly for 4 weeks and then every 2 weeks plus atezolizumab 840 mg intravenously every 2 weeks. The primary endpoint was investigator-assessed objective response rate (ORR). RESULTS: In the response-evaluable population (N = 34), the best confirmed ORR was 20.6% [95% confidence interval (CI), 10.4-36.8], with a complete response seen in 1 patient and partial responses in 6 patients. The disease control rate was 44.1% (complete response = 2.9%; partial response = 17.6%; stable disease = 23.5%), and the median duration of response was 10.1 mon/ths (95% CI, 5.6-26.7). The median progression-free survival was 1.9 months (95% CI, 1.8-3.7). Analysis of response by PDL1 expression (Ventana SP263) resulted in an ORR of 26.7% for patients with PDL1-positive tumors (tumor area positivity cutoff ≥1%; n = 15) and 7.1% for patients with PDL1-negative tumors (tumor area positivity cutoff <1%; n = 14). Overall, the treatment combination was tolerable, and adverse events were consistent with the known safety profiles of each drug. CONCLUSIONS: FAP-IL2v plus atezolizumab demonstrated clinical activity and was tolerable in patients with previously treated esophageal SCC.
Assuntos
Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Esofágicas , Humanos , Feminino , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Masculino , Pessoa de Meia-Idade , Idoso , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Adulto , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Endopeptidases/genética , Proteínas de Membrana/genética , Gelatinases/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversosRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. METHODS: Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. RESULTS: We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. CONCLUSION: This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Glicoproteínas de Membrana , Proteínas de Membrana , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Prognóstico , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Feminino , Masculino , Endopeptidases , Gelatinases/metabolismo , Gelatinases/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Idoso , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Microambiente TumoralRESUMO
This study reports the whole-genome sequence of Lactiplantibacillus plantarum cqf-43 isolated from healthy sow feces. Based on genomic analysis, we performed a comprehensive safety assessment of strain cqf-43, using both in vitro and in vivo experiments, and explored its probiotic potential. The total genome length measures 3,169,201 bp, boasting a GC content of 44.59%. Through phylogenetic analyses, leveraging both 16S rRNA gene and whole-genome sequences, we confidently categorize strain cqf-43 as a member of Lactiplantibacillus. Genome annotation using Prokka unveiled a total of 3141 genes, encompassing 2990 protein-coding sequences, 71 tRNAs, 16 rRNAs, and 1 tmRNA. Functional annotations derived from COG and KEGG databases highlighted a significant abundance of genes related to metabolism, with a notable emphasis on carbohydrate utilization. The genome also revealed the presence of prophage regions and CRISPR-Cas regions while lacking virulence and toxin genes. Screening for antibiotic resistance genes via the CARD database yielded no detectable transferable resistance genes, effectively eliminating the potential for harmful gene transfer. It is worth highlighting that the virulence factors identified via the VFDB database primarily contribute to bolstering pathogen resilience in hostile environments. This characteristic is particularly advantageous for probiotics. Furthermore, the genome is devoid of menacing genes such as hemolysin, gelatinase, and biogenic amine-producing genes. Our investigation also unveiled the presence of three unannotated secondary metabolite biosynthetic gene clusters, as detected by the online tool antiSMASH, suggesting a great deal of unknown potential for this strain. Rigorous in vitro experiments confirmed tolerance of strain cqf-43 in the intestinal environment, its antimicrobial efficacy, sensitivity to antibiotics, absence of hemolysis and gelatinase activity, and its inability to produce biogenic amines. In addition, a 28-day oral toxicity test showed that the strain cqf-43 did not pose a health hazard in mice, further establishing it as a safe strain.
Assuntos
Genoma Bacteriano , Probióticos , Animais , Feminino , Suínos , Camundongos , RNA Ribossômico 16S , Filogenia , Antibacterianos , Gelatinases/genética , Análise de SequênciaRESUMO
Fibroblast activation protein (FAP) is frequently expressed in the tumor stroma, whereas expression by normal organs is highly restricted. Despite these promising features, FAP-targeted therapies have shown limited success so far. FAP imaging offers new opportunities to select patients for FAP-targeted therapies and monitor tumor response. See related article by Lee et al., p. 5330.
Assuntos
Gelatinases , Receptores de Antígenos Quiméricos , Humanos , Gelatinases/genética , Serina Endopeptidases , Receptores de Antígenos Quiméricos/metabolismo , Medicina de Precisão , Endopeptidases , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Terapia Baseada em Transplante de Células e TecidosRESUMO
Legionella-like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32-37 °C (with an optimum at 32 °C) on buffered charcoal-yeast extract agar with l-cysteine, glycine-vancomycin-polymyxin B-cycloheximide agar and Wadowsky-Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l-1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5â%, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32â% L. quateirensis NCTC 12376T, 91.45â% L. quateirensis ATCC 49507T and 91.45â% L. quateirensis ATCC 49507T, respectively. The digital DNA-DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species (L. quateirensis ATCC 49507T, ≤40.10â% DNA-DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T=DSM 112526T) as the type strain.
Assuntos
Legionella , Vancomicina , Ágar , Técnicas de Tipagem Bacteriana , Composição de Bases , Catalase/genética , Carvão Vegetal , Ciprofloxacina , Cicloeximida , Cisteína/genética , DNA Bacteriano/genética , Ácidos Graxos/química , Gelatinases/genética , Glicina/genética , Hipuratos , Nucleotídeos , Filogenia , Polimixina B/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , ÁguaRESUMO
Matrix metalloproteinase-9 (MMP-9) is a gelatinase, which is a member of the MMPs family. We know that MMP-9 is not only an important gelatinase to regulate the extracellular matrix balance, but also one of the most closely related proteases to the pathogenesis of intracranial aneurysm. The purpose of this study is to investigate the relationship between MMP-9 gene polymorphism and intracranial aneurysm. In this paper, 98 patients who were admitted to a hospital from 2018 to 2019 were selected as the experimental group and the control group according to the relevant standards of intracranial aneurysms. The MMP-9 positive and MMP-9 absorbance values between the two groups were compared, so as to determine the concentration of MMP-9 between the two groups. In addition, the gene distribution and gene frequency analysis of the C-1562T promoter region of MMP-9 were carried out. The results showed that in the control group, the gene distribution frequency of CC type was 67% that of CT type was 31% that of TT type was 2%, that of the experimental group was 52%, and that of CT type was 44%. The results showed that there was a correlation between MMP-9 gene polymorphism and intracranial aneurysm.
Assuntos
Aneurisma Intracraniano , Metaloproteinase 9 da Matriz , Gelatinases/genética , Frequência do Gene/genética , Humanos , Aneurisma Intracraniano/genética , Metaloproteinase 9 da Matriz/genética , Polimorfismo GenéticoRESUMO
Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities, such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis. IMPORTANCE In Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.
Assuntos
Enterococcus faecalis , Gelatinases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Enterococcus faecalis/metabolismo , Gelatinases/genética , Gelatinases/metabolismo , Peptidilprolil Isomerase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Quorum sensing (QS) is a cell-to-cell communication process that regulates major pathogenic attributes in bacteria including biofilm formation, secretion of virulence factors, and antimicrobial resistance. The two-component Fsr-QS system of the nosocomial pathogen Enterococcus faecalis controls the production of extracellular gelatinase that contributes to biofilm development by enhancing the release of nucleic acids into the biofilm matrix. However, the contribution of this system to the deposition of other biofilm matrix components such as polysaccharides and proteins remains unknown. Using wild type and mutant strains, we discovered that biofilm formation was attenuated by inactivation of the Fsr system or its downstream gelatinase production. Inactivation of the Fsr system caused a modest, yet significant reduction in biofilm metabolic activity without affecting cell counts. Inactivation of the QS-signal sensor FsrC and response regulator FsrA resulted in decreased extracellular polysaccharides and proteins in biofilms in a temporal manner. Irrespective of biofilm age, eDNA levels were reduced in the gelatinase mutant strain. Our results collectively suggest that the Fsr system contributes to the temporal deposition of polysaccharides and proteins into the extracellular polymeric matrix (EPS) of E. faecalis biofilm, without affecting bacterial viability. This understanding of the role of the Fsr-QS system in biofilm development may reveal a novel target to develop effective antibiofilm agents to tackle E. faecalis-mediated infections such as in dental root canals, heart valves, and surgical sites.
Assuntos
Enterococcus faecalis , Percepção de Quorum , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Enterococcus faecalis/genética , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Gelatinases/genética , Gelatinases/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Regiões Promotoras Genéticas , Transgenes , Microambiente Tumoral/genética , Animais , Linhagem Celular Tumoral , Quimiocina CXCL12/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Endopeptidases , Gelatinases/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína Jagged-1/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Osteonectina/genética , Serina Endopeptidases/genética , Fatores de Transcrição da Família Snail/genética , Ativação TranscricionalRESUMO
In order to invent a porcine gelatine detection device using microbial resources, bacterial enzymes with a preference towards porcine gelatine and their candidate genes were evaluated. Five (n = 5) bacterial strains isolated from hot spring water and wet clay, Malaysia were screened for their gelatinase activity. The gelatinase enzyme was extracted and purified using ammonium sulphate precipitation prior to performing gelatinase assay on porcine, bovine and fish gelatine medium substrates. The G2 strain or Enterobacter aerogenes (Strain EA1) was selected for whole genome sequenced after showing a consistent trend of preference towards porcine gelatine. The gelatinase candidate gene gelEA1_9 was cloned and expressed. Based on one-way analysis of variance (ANOVA) with POST-HOC Duncan test (α = 0.05), the final product of gelEA1_9 was identified as a novel gelatinase. This gelatinase presented no significant difference in activity towards porcine gelatine. Hence, the present study demonstrated an enzyme-substrate interaction for porcine gelatine identification.
Assuntos
Proteínas de Bactérias/metabolismo , Gelatina/metabolismo , Gelatinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bovinos , Enterobacter aerogenes/enzimologia , Enterobacter aerogenes/genética , Peixes/metabolismo , Gelatinases/química , Gelatinases/genética , Expressão Gênica , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato , SuínosRESUMO
BACKGROUND: High-grade serous ovarian cancer (HGSOC) is a fatal form of ovarian cancer. Previous studies indicated some potential biomarkers for clinical evaluation of HGSOC prognosis. However, there is a lack of systematic analysis of different expression genes (DEGs) to screen and detect significant biomarkers of HGSOC. METHODS: TCGA database was conducted to analyze relevant genes expression in HGSOC. Outcomes of candidate genes expression, including overall survival (OS) and progression-free survival (PFS), were calculated by Cox regression analysis for hazard rates (HR). Histopathological investigation of the identified genes was carried out in 151 Chinese HGSOC patients to validate gene expression in different stages of HGSOC. RESULTS: Of all 57,331 genes that were analyzed, FAP was identified as the only novel gene that significantly contributed to both OS and PFS of HGSOC. In addition, FAP had a consistent expression profile between carcinoma-paracarcinoma and early-advanced stages of HGSOC. Immunological tests in paraffin section also confirmed that up-regulation of FAP was present in advanced stage HGSOC patients. Prediction of FAP network association suggested that FN1 could be a potential downstream gene which further influenced HGSOC survival. CONCLUSIONS: High-level expression of FAP was associated with poor prognosis of HGSOC via FN1 pathway.
Assuntos
Cistadenocarcinoma Seroso/patologia , Gelatinases/genética , Proteínas de Membrana/genética , Neoplasias Ovarianas/patologia , Serina Endopeptidases/genética , Regulação para Cima , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Bases de Dados Genéticas , Endopeptidases , Feminino , Fibronectinas/metabolismo , Gelatinases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Prognóstico , Serina Endopeptidases/metabolismo , Transdução de Sinais , Análise de SobrevidaRESUMO
Fibroblast activation protein-α (FAP) is a type-II transmembrane serine protease expressed almost exclusively to pathological conditions including fibrosis, arthritis, and cancer. Across most cancer types, elevated FAP is associated with worse clinical outcomes. Despite the clear association between FAP and disease severity, the biological reasons underlying these clinical observations remain unclear. Here we review basic FAP biology and FAP's role in non-oncologic and oncologic disease. We further explore how FAP may worsen clinical outcomes via its effects on extracellular matrix remodeling, intracellular signaling regulation, angiogenesis, epithelial-to-mesenchymal transition, and immunosuppression. Lastly, we discuss the potential to exploit FAP biology to improve clinical outcomes.
Assuntos
Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Serina Endopeptidases/metabolismo , Animais , Endopeptidases , Gelatinases/química , Gelatinases/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Neoplasias/genética , Neoplasias/patologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Targeting Fibroblast Activation Protein (FAP) is a new approach for glioblastoma imaging. In a recent pilot study glioblastomas showed elevated tracer uptake with high intratumoral heterogeneity in projection on the corresponding T2w/FLAIR and contrast enhanced MRI lesions. In this study, we correlated FAP-specific signaling with apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) signals in MRI to further characterize the significance of FAP uptake. METHODS: Clinical PET/CT scans of 13 glioblastoma patients were performed post i. v. administration of 68Ga-labelled-FAP-specific tracer molecules. PET- and corresponding MRI-scans were co-registrated. 3d volumetric segmentations were performed of T2w/FLAIR lesions and contrast enhancing lesions within co-registrated MRI slides. Signal intensity values of FAP-specific PET signaling, ADC and rCBV were analyzed for their pixel wise correlation in each patient. Pooled estimates of the correlation coefficients were calculated by using the Fisher z-transformation. RESULTS: FAP-specific PET signals showed a moderately positive correlation with rCBV values which is more pronounced within the T2w/FLAIR lesion (pooled correlation 0,229) than in the contrast enhancing tumor region (pooled correlation 0.09). FAP-specific PET signals showed no correlation with ADC values. CONCLUSIONS: The moderately positive correlation of FAP-specific signals with rCBV values in MRI indicates that FAP-signaling is not independent from perfusion, but also does not only reflect intratumoral perfusion differences. The missing correlation of FAP-specific signals with ADC indicates that FAP-specific imaging does not reflect cell density, but the spot-like expression of FAP in glioblastomas. The clinical value of FAP-specific imaging needs further investigation.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Volume Sanguíneo Cerebral/fisiologia , Gelatinases/genética , Glioblastoma/diagnóstico por imagem , Glioblastoma/genética , Proteínas de Membrana/genética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Serina Endopeptidases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/patologia , Endopeptidases , Feminino , Radioisótopos de Gálio , Glioblastoma/patologia , Humanos , Imageamento Tridimensional , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
BACKGROUND: Gastric cancer is one of the leading causes of death worldwide. MicroRNA-30a (miR-30a) has been demonstrated to be involved in several types of cancer development. OBJECTIVE: We aimed to identify the molecular mechanism of miR-30a in gastric cancer. METHODS: We investigated the expression of miR-30a in gastric cancer tissues by qRT-PCR. The role of miR-30a on the metastasis and proliferation of gastric cancer was evaluated by cell migration assay, CCK-8 assay and tumor peritoneal dissemination model. The target of miR-30a in gastric cancer was identified. RESULTS: We discovered that miR-30a was significantly downregulated in gastric cancer tissues compared with adjacent nonmalignant tissues. The expression of miR-30a was inversely correlated with progression of gastric cancer. Gain- and loss-of function revealed that miR-30a acted as a potent tumor suppressor in gastric cancer. Re-expressed miR-30a inhibited gastric cancer cells migration, knock down miR-30a have the opposite effects. Furthermore, overexpression of miR-30a suppressed tumor peritoneal dissemination in vivo. We identified that fibroblast activation protein α (FAPα) was a direct target of miR-30a. The relative expression of FAPα was significantly higher in gastric cancer tissues compared with adjacent nonmalignant tissues. Inhibition of FAPα could recapitulate the effects of miR-30a, and overexpression of FAPα could abrogate the effect of miR-30a. CONCLUSION: MiR-30a inhibited gastric cancer metastasis by targeting FAPα, suggesting that miR-30a may function as a novel tumor suppressor in gastric cancer.
Assuntos
Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Serina Endopeptidases/metabolismo , Neoplasias Gástricas/patologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Endopeptidases , Transição Epitelial-Mesenquimal , Feminino , Gelatinases/genética , Genes Supressores de Tumor , Humanos , Metástase Linfática , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Serina Endopeptidases/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Esophageal squamous cancer cell (ESCC), with late diagnosis and poor rate of survival, is a significant cause of mortality in the developing countries. The hypothesis of rare high penetrance with mutations in new genes may explain the underlying predisposition in some of these familial cases. METHODS: Exome sequencing was performed in the patients with ESCC with strong disease aggregation, two sisters with ESCC cancer, and one with breast cancer. Data analysis selected only very rare variants (0-0.1%) located in genes with a role compatible with cancer. In addition, the homology modeling of the novel mutation (A459D) discovered in FAP gene was performed by using the online Swiss-Prot server for automated modeling and the resulted structure has been modified and analyzed by using bioinformatics software to thoroughly study the structural deficiencies caused by the novel mutation. RESULTS: Ten final candidate variants were selected and six genes validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in FAP, BOD1L, RAD51, Gasdermin D, LGR5, and CERS4. A novel, human mutation C1367A encoding Ala459 Asp (accession number: KT988039), occurring in the blade of the ß propeller domain, was identified in two sisters with ESCC. CONCLUSIONS: We identified novel mutations in three drug delivery genes, a tumor suppressor and also a stem cell marker of esophageal that may have a role in cancer treatment and are involved in cellular pathways, which supports their putative involvement in germ-line predisposition to this neoplasm.