Reactome Analysis Research Articles

Exploring MPC1 as a potential ferroptosis-linked biomarker in the cervical cancer tumor microenvironment: a comprehensive analysis

BackgroundThe increasing problems of drug and radiotherapy resistance in cervical cancer underscores the need for novel methods for its management. Reports indicate that the expression of MPC1 may be associated with the tumor microenvironment and the occurrence of ferroptosis in cervical cancer. The objective of this study was to visually illustrate the prognostic significance and immunological characterization of MPC1 in cervical cancer.MethodsThe expression profile and prognostic significance of MPC1 were analyzed using various databases, including UALCAN, TIMER2, GEPIA2, and Kaplan–Meier Plotter. TISIDB, TIMER2, and immunohistochemical analysis were used to investigate the correlation between MPC1 expression and immune infiltration. GO enrichment analysis, KEGG analysis, Reactome analysis, ConsensusPathDB, and GeneMANIA were used to visualize the functional enrichment of MPC1 and signaling pathways related to MPC1. The correlation analysis was carried out to examine the relationship between MPC1 and Ferroptosis gene in TIMER 2.0, ncFO, GEPIA Database and Kaplan–Meier Plotter.ResultsWe demonstrated that the expression levels of MPC1 in cervical cancer tissues were lower than those in normal cervical tissues. Kaplan–Meier survival curves showed shorter overall survival in cervical cancer patients with low levels of MPC1 expression. The expression of MPC1 was related to the infiltrating levels of tumor-infiltrating immune cells in cervical cancer. Moreover, MPC1 expression was associated with the iron-mediated cell death pathway, and several important ferroptosis genes were upregulated in cervical cancer cells. Furthermore, after knocking down MPC1 in HeLa cells, the expression of these genes decreased.ConclusionThese findings indicate that MPC1 functions as a prognostic indicator and plays a role in the regulation of the ferroptosis pathway in cervical cancer.

Biology of Healthy Aging: Biological Hallmarks of Stress Resistance Related and Unrelated to Longevity in Humans

Stress resistance is highly associated with longer and healthier lifespans in various model organisms, including nematodes, fruit flies, and mice. However, we lack a complete understanding of stress resistance in humans; therefore, we investigated how stress resistance and longevity are interlinked in humans. Using more than 180 databases, we identified 541 human genes associated with stress resistance. The curated gene set is highly enriched with genes involved in the cellular response to stress. The Reactome analysis identified 398 biological pathways, narrowed down to 172 pathways using a medium threshold (p-value < 1 × 10−4). We further summarized these pathways into 14 pathway categories, e.g., cellular response to stimuli/stress, DNA repair, gene expression, and immune system. There were overlapping categories between stress resistance and longevity, including gene expression, signal transduction, immune system, and cellular responses to stimuli/stress. The categories include the PIP3-AKT-FOXO and mTOR pathways, known to specify lifespans in the model systems. They also include the accelerated aging syndrome genes (WRN and HGPS/LMNA), while the genes were also involved in non-overlapped categories. Notably, nuclear pore proteins are enriched among the stress-resistance pathways and overlap with diverse metabolic pathways. This study fills the knowledge gap in humans, suggesting that stress resistance is closely linked to longevity pathways but not entirely identical. While most longevity categories intersect with stress-resistance categories, some do not, particularly those related to cell proliferation and beta-cell development. We also note inconsistencies in pathway terminologies with aging hallmarks reported previously, and propose them to be more unified and integral.

Investigating long noncoding RNA HA117 and its possible regulatory network in osteosarcoma

Osteosarcoma, a primary malignant bone tumor, is the third most common cancer in children and adolescents under 20 years old. In recent decades, many osteosarcomar elated molecular targets, including long noncoding RNA (lncRNA), have been discovered or confirmed. This study aims to elucidate the gene expression and possible regulatory mechanisms of lncRNA HA117 in osteosarcoma. To achieve this, we downloaded 51 whole-transcriptome osteosarcoma sequencing samples from the NCBI Sequence Read Archive database and performed bioinformatics analysis. Geneexpression analysis of HA117 revealed no significant difference between the tumor and adjacent tissues. However, HA117 exhibited significant down-regulation in bothfresh and formalin‐fixed paraffin-embedded (FFPE) samples after chemotherapy. Bycombining two target gene prediction methods, we identified 11 and 83 target genes for HA117 in fresh and FFPE samples, respectively. Functional analysis indicated that these target genes are mainly located in the cytoplasm and nucleus, with most related to protein binding. In addition, Reactome analysis demonstrated that these target genes participate in the regulation of multiple metabolic pathways, predominantly in cellular responses to stress. Our findings suggest that chemotherapy may regulate downstream target genes by altering the expression of HA117, thereby inducing cellular stress response. Overall, our results indicate that HA117 may not function as an oncogene but could serve as a therapeutic target in osteosarcoma.

Abstract We062: Characterization of Transcriptional Responses to Streptococcus pneumoniae Infection in the Heart versus Sterile Cardiac Injury

Background: Streptococcus pneumoniae ( Spn ) remains the leading cause of community-acquired pneumonia (CAP). As many as 30% of patients hospitalized for CAP experience a major adverse cardiac event (MACE). Importantly, the elevated risk for MACE is not only limited to the time of acute infection but also extends several years post-hospitalization. Research Question: Is the host response to Spn- mediated injury in the heart different from the response to sterile injury? Goals: To identify defining features of the host response to Spn in the heart via differences in gene expression. Methods: We performed a bioinformatic analysis of bulk RNAseq data collected from hearts of mice acutely infected with Spn and compared this to publicly available data sets of mice that received experimental myocardial infarction, i.e. ischemia reperfusion and permanent ligation. Using DeSeq2, we identified 2,971 genes with differential expression between the infected and infarcted conditions. To identify gene expression differences that might be relevant to human patients, we subsequently used the Reactome GSA software to map our differential expression results onto predicted human pathways. Results: Categorization of the gene roles identified those in the classical antibody-mediated complement activation as the dominant pathway with differential expression. Reactome analysis of the top 5 differentially expressed genes (p < 0.05) at the individual level identified BAIAP2L1 , CXCL9 , IRF7 , IL18BP , RPL12 , and PSMB10 as significantly upregulated in Spn infected hearts versus infarcted hearts. As one feature of Spn mediated damage is the recruitment of immune cells to the affected sites and cardiac remodeling, we validated upregulation of CXCL9 as well as CXCL10, potent recruiters of T-cells, as enhanced during Spn infection. Conclusions: Our analyses provide information on how the host response to disseminated pneumococcal infection in the heart is distinct from sterile injury and identified mediators that may be contributing to adverse cardiac remodeling.

Transcriptomic analysis of primary nasal epithelial cells reveals altered interferon signalling in preterm birth survivors at one year of age.

Introduction: Many survivors of preterm birth (<37weeks gestation) have lifelong respiratory deficits, the drivers of which remain unknown. Influencers of pathophysiological outcomes are often detectable at the gene level and pinpointing these differences can help guide targeted research and interventions. This study provides the first transcriptomic analysis of primary nasal airway epithelial cells in survivors of preterm birth at approximately 1year of age. Methods: Nasal airway epithelial brushings were collected, and primary cell cultures established from term (>37 weeks gestation) and very preterm participants (≤32 weeks gestation). Ex vivo RNA was collected from brushings with sufficient cell numbers and in vitro RNA was extracted from cultured cells, with bulk RNA sequencing performed on both the sample types. Differential gene expression was assessed using the limma-trend pipeline and pathway enrichment identified using Reactome and GO analysis. To corroborate gene expression data, cytokine concentrations were measured in cell culture supernatant. Results: Transcriptomic analysis to compare term and preterm cells revealed 2,321 genes differentially expressed in ex vivo samples and 865 genes differentially expressed in cultured basal cell samples. Over one third of differentially expressed genes were related to host immunity, with interferon signalling pathways dominating the pathway enrichment analysis and IRF1 identified as a hub gene. Corroboration of disrupted interferon release showed that concentrations of IFN-α2 were below measurable limits in term samples but elevated in preterm samples [19.4 (76.7) pg/ml/µg protein, p = 0.03]. IFN-γ production was significantly higher in preterm samples [3.3 (1.5) vs. 9.4 (17.7) pg/ml/µg protein; p = 0.01] as was IFN-β [7.8 (2.5) vs. 13.6 (19.5) pg/ml/µg protein, p = 0.01]. Conclusion: Host immunity may be compromised in the preterm nasal airway epithelium in early life. Altered immune responses may lead to cycles of repeated infections, causing persistent inflammation and tissue damage which can have significant impacts on long-term respiratory function.

Bradykinin promotes immune responses in differentiated embryonic neurospheres carrying APPswe and PS1dE9 mutations

BackgroundNeural progenitor cells (NPCs) can be cultivated from developing brains, reproducing many of the processes that occur during neural development. They can be isolated from a variety of animal models, such as transgenic mice carrying mutations in amyloid precursor protein (APP) and presenilin 1 and 2 (PSEN 1 and 2), characteristic of familial Alzheimer’s disease (fAD). Modulating the development of these cells with inflammation-related peptides, such as bradykinin (BK) and its antagonist HOE-140, enables the understanding of the impact of such molecules in a relevant AD model.ResultsWe performed a global gene expression analysis on transgenic neurospheres treated with BK and HOE-140. To validate the microarray data, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on 8 important genes related to the immune response in AD such as CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF alpha and Iba-1. Furthermore, comparative analysis of the transcriptional profiles was performed between treatments, including gene ontology and reactome enrichment, construction and analysis of protein-protein interaction networks and, finally, comparison of our data with human dataset from AD patients. The treatments affected the expression levels of genes mainly related to microglia-mediated neuroinflammatory responses, with BK promoting an increase in the expression of genes that enrich processes, biological pathways, and cellular components related to immune dysfunction, neurodegeneration and cell cycle. B2 receptor inhibition by HOE-140 resulted in the reduction of AD-related anomalies caused in this system.ConclusionsBK is an important immunomodulatory agent and enhances the immunological changes identified in transgenic neurospheres carrying the genetic load of AD. Bradykinin treatments modulate the expression rates of genes related to microglia-mediated neuroinflammation. Inhibiting bradykinin activity in Alzheimer’s disease may slow disease progression.

Identification of Potential Breast Cancer Stem Cell Biomarkers in the Secretome Using a Network Interaction Approach Analysis.

Breast cancer stem cells (BCSCs) play a role in the high rates of resistance, recurrence, and metastasis. The precise biomarkers of BCSCs can assist effectively in identifying cancer, assessing prognosis, diagnosing, and monitoring therapy. The aim of this study was to give a complete analysis for predicting specific biomarkers of BCSCs. We aggregated profile datasets in this work to shed light on the underlying critical genes and pathways of BCSCs. We obtained two expression profiling by array datasets (GSE7513 and GSE7515) from the Gene Expression Omnibus (GEO) database to identify biomarkers in BCSCs. Enrichr was used to do functional analysis, including gene ontology (GO) and reactome pathway. Furthermore, the protein-protein interaction (PPI) of these differential expression genes (DEGs) was visualized using Cytoscape with the search tool for the retrieval of interacting genes (STRING). The hub genes in the PPI network were chosen for further investigation. We identified 65 up-regulated and 190 down- regulated DEGs and the GO enrichment analysis revealed that these DEGs were enriched in biological process related to tumorigenesis and stemness, including alter the extracellular matrix's physicochemical properties, cytoskeletal reorganisation, adhesion, motility, migration, growth, and survival. The Reactome analysis indicated that these DEGs were also involved in modulating function of ECM, regulation cancer metabolism and angiogenesis, tumor growth, proliferation, and metastasis. Our bioinformatic study revealed that FYN, INADL, OCLN, F11R, and TOP2A were potential biomarker panel of BCSCs from secretome.

Clinical Significance of Upregulation of EZH1 Expression in Hepatocellular Carcinoma Tissues.

The incidence and mortality of hepatocellular carcinoma (HCC) are increasing. It is urgent to develop more effective HCC biomarkers for diagnosis and treatment. This project intends to verify the expression of enhancer of zeste 1 polycomb repressive complex 2 subunit (EZH1) and its mechanism in HCC. This study integrates global microarray and high-throughput sequencing datasets, combined with internal immunohistochemistry, to analyze the expression and prognostic value of EZH1 in HCC. Functional enrichment analysis was conducted to investigate transcriptional targets, which were achieved by intersecting HCC over-expressed genes, EZH1 co-expressed genes and putative transcriptional targets. The relationship between EZH1 and anticancer drugs was detected by drug sensitivity analysis. In this study, 84 datasets from 40 platforms (3,926 HCC samples and 3,428 non-cancerous liver tissues) were included to show the high expression of EZH1 in HCC. Immunohistochemistry with 159 HCC samples and 62 non-HCC samples confirmed the high expression level. HCC patients with high EZH1 expression had worse survival prognoses. Gene ontology and Reactome analysis revealed that metabolism-related pathways, including autophagy, are critical for HCC. Interestingly, as one of the EZH1 potential transcriptional targets, autophagy-related 7 (ATG7) appeared in the above pathways. ATG7 was positively correlated with EZH1, upregulated in HCC, and mediated poor prognosis. Upregulation of EZH1 was found to be in contact with HCC anti-tumor drug resistance. The upregulation of EZH1 expression can promote the occurrence of HCC and lead to poor clinical progression and drug resistance; these effects may be mediated by regulating ATG7.

Abstract 3766: Utilizing whole-transcriptome digital spatial profiling for glioblastoma clinical biomarker discovery

Abstract Objective: IDH-wildtype glioblastoma (GBM) is the most aggressive brain tumor in adults. Notable for substantial inter- and intra-tumoral heterogeneity, GBM is highly resistant to standard-of-care treatment. Emerging spatial omics technologies, such as GeoMx Digital Spatial Profiling (DSP), offer novel approaches to interrogate the molecular features of regionally defined cellular ecosystems in cancer. Here, we leverage DSP to study whole-transcriptome gene expression signatures in a spatially defined manner across five IDH-wildtype GBMs. Methods: Formalin-fixed paraffin-embedded tumors were assessed histologically and three spatially distinct areas from each tumor were used to construct a custom “spatial” tissue microarray. From each set of three areas, we identified nine spatially distinct regions of interest (ROIs) for bulk transcriptomic analysis according to two histopathological features: distribution (cellular tumor core or tumor periphery) and vascular proximity (near or far). We evaluated normalized target expression data statistically using linear mixed modeling (LMM) with Benjamini-Hochberg correction. Results: 44 profiled ROIs and 8531 transcriptomic targets were analyzed following quality control trimming. 3D principal component analysis and unsupervised hierarchical clustering analysis showed clustering according to tumor specimen as well as distribution (core versus periphery), thereby substantiating the presence of inter- and intra-tumoral transcriptional heterogeneity. LMM-based differential gene expression analysis identified distinct molecular signatures when comparing core and periphery. Specifically, we observed elevated log2-transformed expression of migration and proliferation markers including LAMC1 (p=0.00297) and FABP7 (p=0.00434) in core ROIs and neurodevelopmental markers including NTRK2 (p=0.00734) and DAAM2 (p=0.00263) in periphery ROIs. Additionally, reactome analysis demonstrated considerable enrichment for the aberrant programmed cell death pathway (R-HSA-9645723, coverage=71.84%) in core ROIs and the MHC class II antigen presentation pathway (R-HSA-2132295, coverage=69.11%) in periphery ROIs. Interestingly, differential gene expression analysis did not reveal any biologically significant targets when subsetting for vascular proximity within cellular tumor core ROIs. However, reactome analysis of this regional subset demonstrated upregulation of various cellular proliferation pathways (G2/M transition, APC/C activity, DNA synthesis, etc.) within vessel-near ROIs when compared with vessel-far ROIs. Conclusions: We preliminarily characterize the molecular heterogeneity of GBM using a spatially guided approach. Ongoing analyses are focused on dataset validation, mixed cell deconvolution, further regional genotyping, and potential biomarkers. Citation Format: Adam Luo, Kelly Casella, Shivang Sharma, Ezra Baraban, Calixto-Hope Lucas, Eugene Shenderov. Utilizing whole-transcriptome digital spatial profiling for glioblastoma clinical biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3766.

Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

Experimental and pharmacoinformatic approaches unveil the neuropharmacological and analgesic potential of chloroform fraction of Roktoshirinchi (Achyranthes ferruginea Roxb.)

Ethnopharmacological relevanceAchyranthes ferruginea (A. ferruginea) Roxb. is a common plant used in traditional medicine in Asia and Africa. It has a variety of local names, including “Gulmanci” in Nigeria, “Dangar” in Pakistan, “Thola” in Ethiopia, and “Roktoshirinchi” in Bangladesh. It is edible and has several ethnomedical uses for a wide range of illnesses, including hysteria, dropsy, constipation, piles, boils, asthma, and shigellosis. However, the neuropharmacological and analgesic potential of A. ferruginea remains uninvestigated. Aim of the studyTo assess the neuropharmacological and analgesic potential of A. ferruginea through a multifaceted approach encompassing both experimental and computational models. Materials and methodsMethanol was used to extract the leaves of A. ferruginea. It was then fractionated with low to high polar solvents (n-hexane, chloroform, ethyl acetate, and water) to get different fractions, including chloroform fraction (CLF). The study selected CLF at different doses and conducted advanced chemical element and proximate analyses, as well as phytochemical profiling using GC-MS. Toxicological studies were done at 300 μg per rat per day for 14 days. Cholinesterase inhibitory potential was checked using an in-vitro colorimetric assay. Acetic acid-induced writhing (AAWT) and formalin-induced licking tests (FILT) were used to assess anti-nociceptive effects. The forced swim test (FST), tail suspension test (TST), elevated plus maze (EPM), hole board test (HBT), and light and dark box test (LDB) were among the behavioral tests used to assess depression and anxiolytic activity. Network pharmacology-based analysis was performed on selected compounds using the search tool for interacting chemicals-5 (STITCH 5), Swiss target prediction tool, and search tool for the retrieval of interacting genes and proteins (STRING) database to link their role with genes involved in neurological disorders through gene ontology and reactome analysis. ResultsQualitative chemical element analysis revealed the presence of 15 elements, including Na, K, Ca, Mg, P, and Zn. The moisture content, ash value, and organic matter were found to be 11.12, 11.03, and 88.97%, respectively. GC-MS data revealed that the CLF possesses 25 phytoconstituents. Toxicological studies suggested the CLF has no effects on normal growth, hematological and biochemical parameters, or cellular organs after 14 days at 300 μg per rat. The CLF markedly reduced the activity of both acetylcholinesterase and butyrylcholinesterase (IC50: 56.22 and 13.22 μg/mL, respectively). Promising dose-dependent analgesic activity (p < 0.05) was observed in chemically-induced pain models. The TST and FST showed a dose-dependent substantial reduction in immobility time due to the CLF. Treatment with CLF notably increased the number of open arm entries and time spent in the EPM test at doses of 200 and 400 mg/kg b.w. The CLF showed significant anxiolytic activity at 200 mg/kg b.w. in the HBT test, whereas a similar activity was observed at 400 mg/kg b.w. in the EPM test. A notable increase in the amount of time spent in the light compartment was observed in the LDB test by mice treated with CLF, suggesting an anxiolytic effect. A network pharmacology study demonstrated the relationship between the phytochemicals and a number of targets, such as PPARA, PPARG, CHRM1, and HTR2, which are connected to the shown bioactivities. ConclusionsThis study demonstrated the safety of A. ferruginea and its efficacy in attenuating cholinesterase inhibitory activity, central and peripheral pain, anxiety, and depression, warranting further exploration of its therapeutic potential.

Enhancing the anticancer potential of metformin: fabrication of efficient nanospanlastics, in vitro cytotoxic studies on HEP-2 cells and reactome enhanced pathway analysis

Metformin (MET), an oral antidiabetic drug, was reported to possess promising anticancer effects. We hypothesized that MET encapsulation in unique nanospanlastics would enhance its anticancer potential against HEP-2 cells. Our results showed the successful fabrication of Nano-MET spanlastics (d = 232.10 ± 0.20 nm; PDI = 0.25 ± 0.11; zeta potential = (−) 44.50 ± 0.96; drug content = 99.90 ± 0.11 and entrapment efficiency = 88.01 ± 2.50%). MTT assay revealed the enhanced Nano-MET cytotoxicity over MET with a calculated IC50 of 50 μg/mL and > 500 μg/mL, respectively. Annexin V/PI apoptosis assay showed that Nano-MET significantly decreased the percentage of live cells from 95.49 to 93.70 compared to MET and increased the percentage of cells arrested in the G0/G1 phase by 8.38%. Moreover, Nano-MET downregulated BCL-2 and upregulated BAX protein levels by 1.57 and 1.88 folds, respectively. RT-qPCR revealed that Nano-MET caused a significant 13.75, 4.15, and 2.23-fold increase in caspase-3, −8, and − 9 levels as well as a 100 and 43.47-fold decrease in cyclin D1 and mTOR levels, respectively. The proliferation marker Ki67 immunofluorescent staining revealed a 3-fold decrease in positive cells in Nano-MET compared to the control. Utilizing the combined Pathway-Enrichment Analysis (PEA) and Reactome analysis indicated high enrichment of certain pathways including nucleotides metabolism, Nudix-type hydrolase enzymes, carbon dioxide hydration, hemostasis, and the innate immune system. In summary, our results confirm MET cytotoxicity enhancement by its encapsulation in nanospanlastics. We also highlight, using PEA, that MET can modulate multiple pathways implicated in carcinogenesis.

Ectopic pregnancy: search for biomarker in salivary proteome

Ectopic pregnancy (EP) is associated with high maternal morbidity and mortality. Ultrasonography is the only dependable diagnostic tool for confirming an ectopic pregnancy. In view of inadequate early detection methods, women suffer from a high-life risk due to the severity of EP. Early detection of EP using pathological/molecular markers will possibly improve clinical diagnosis and patient management. Salivary proteins contain potential biomarkers for diagnosing and detecting various physiological and/or pathological conditions. Therefore, the present investigation was designed to explore the salivary proteome with special reference to EP. Gel-based protein separation was performed on saliva, followed by identification of proteins using Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS). Totally, 326 proteins were identified in the salivary samples, among which 101 were found to be specific for ruptured ectopic pregnancy (EPR). Reactome analysis revealed innate immune system, neutrophil degranulation, cell surface interactions at the vascular wall, and FCERI-mediated NF-kB activation as the major pathways to which the salivary proteins identified during EPR are associated. Glutathione-S-transferase omega-1 (GSTO1) is specific for EPR and has been reported as a candidate biomarker in the serum of EPR patients. Therefore, saliva would be a potential source of diagnostic non-invasive protein biomarker(s) for EP. Intensive investigation on the salivary proteins specific to EP can potentially lead to setting up of a panel of candidate biomarkers and developing a non-invasive protein-based diagnostic kit.

Weighted gene coexpression network analysis and machine learning reveal oncogenome associated microbiome plays an important role in tumor immunity and prognosis in pan-cancer

BackgroundFor many years, the role of the microbiome in tumor progression, particularly the tumor microbiome, was largely overlooked. The connection between the tumor microbiome and the tumor genome still requires further investigation.MethodsThe TCGA microbiome and genome data were obtained from Haziza et al.’s article and UCSC Xena database, respectively. Separate WGCNA networks were constructed for the tumor microbiome and genomic data after filtering the datasets. Correlation analysis between the microbial and mRNA modules was conducted to identify oncogenome associated microbiome module (OAM) modules, with three microbial modules selected for each tumor type. Reactome analysis was used to enrich biological processes. Machine learning techniques were implemented to explore the tumor type-specific enrichment and prognostic value of OAM, as well as the ability of the tumor microbiome to differentiate TP53 mutations.ResultsWe constructed a total of 182 tumor microbiome and 570 mRNA WGCNA modules. Our results show that there is a correlation between tumor microbiome and tumor genome. Gene enrichment analysis results suggest that the genes in the mRNA module with the highest correlation with the tumor microbiome group are mainly enriched in infection, transcriptional regulation by TP53 and antigen presentation. The correlation analysis of OAM with CD8+ T cells or TAM1 cells suggests the existence of many microbiota that may be involved in tumor immune suppression or promotion, such as Williamsia in breast cancer, Biostraticola in stomach cancer, Megasphaera in cervical cancer and Lottiidibacillus in ovarian cancer. In addition, the results show that the microbiome-genome prognostic model has good predictive value for short-term prognosis. The analysis of tumor TP53 mutations shows that tumor microbiota has a certain ability to distinguish TP53 mutations, with an AUROC value of 0.755. The tumor microbiota with high importance scores are Corallococcus, Bacillus and Saezia. Finally, we identified a potential anti-cancer microbiota, Tissierella, which has been shown to be associated with improved prognosis in tumors including breast cancer, lung adenocarcinoma and gastric cancer.ConclusionThere is an association between the tumor microbiome and the tumor genome, and the existence of this association is not accidental and could change the landscape of tumor research.

Abstract P2110: Fibroblast-specific Smad6 Restrains Adverse Remodeling And Dysfunction Following Myocardial Infarction

Signaling induced by the Transforming Growth Factor (TGF)-β superfamily members in fibroblasts plays a central role in repair and remodeling after myocardial infarction (MI). However, TGF-b signals need to be tightly regulated, as excessive or sustained fibroblast activation after MI can accentuate fibrosis, precipitating heart failure. The inhibitory Smads, Smad6 and Smad7, function as endogenous negative regulators that restrain TGF-β and BMP signaling. We have recently demonstrated that fibroblast-specific Smad7 restrains fibrosis after MI acting through TGF-dependent and -independent pathways. However, the role of Smad6 in tissue repair and fibrosis following injury is not known. We hypothesized that Smad6 expression in infarct fibroblasts may play an important role in repair and remodeling of the heart, regulating TGF-b and BMP-mediated responses. In a mouse model of non-reperfused MI, Smad6 expression was increased in infarct fibroblasts and myofibroblasts at 7d post-infarction. To investigate the role of Smad6 in injury and repair, we used CRISPR/Cas9 to generate Smad6 loxp mice. These animals were bred with Col1a2CreER mice to generate fibroblast-specific conditional Smad6 KOs (FS6KO). 7d after MI, FS6KO mice had accentuated systolic and diastolic dysfunction (p&lt;0.01, N=10) associated with scar expansion, accentuated fibrosis, and higher myofibroblast density in the infarct area and border zone. Fibroblast-specific Smad6 loss increased collagen denaturation in the infarct and border zone. In vitro, siRNA knockdown experiments showed that Smad6 suppresses collagen and fibronectin expression, and modulates protease and matricellular gene synthesis. Reactome analysis of RNA-Seq data suggested effects of Smad6 on pathways involved in assembly and crosslinking of collagen fibrils, collagen biosynthesis and degradation, and VEGF-dependent angiogenesis. Furthermore, Ingenuity Pathway Analysis identified signaling by Integrins, Ephrin receptors and Receptor Tyrosine Kinases as key regulators of the effects of Smad6 loss on the fibroblast transcriptome. In conclusion, fibroblast-specific Smad6 restrains adverse remodeling after MI. Ongoing studies explore the molecular mechanisms for the Smad6 effects.

Alteration of Cognitive Function in Aging and Alzheimer's Disease Mice Is Related to Dysfunction of the Neuroimmune System.

Both Alzheimer's disease (AD) and aging have aging-related cognitive dysfunction with a high incidence. These neurological diseases cause serious cognitive problems in patients' daily life. But the cognitive dysfunction mechanism in-depth of aging is far less known than that of AD. To reveal the different mechanisms of AD and aging-related cognitive dysfunction, we compared the mechanisms of aging and AD through analysis of differentially expressed genes. Mice were divided into four groups (3-month C57BL, 16-month C57BL, 3-month 3xTg AD mice, and 16-month 3xTg AD mice) according to genotype and age. The Morris water maze was employed to investigate the spatial cognition of mice. Differential expressions of genes of AD and aging were analyzed through RNA sequencing and GO, KEGG, Reactome analysis, and the dynamic change trend analysis. Microglia was stained with immunofluorescence and its numbers were counted for analysis. The cognitive function of elderly mice were worse through testing with the Morris water maze. The cognitive function of 16-month 3xTg AD mice were worse than 16-month C57BL mice. The alteration tendencies of DE genes were uncovered, and microglia numbers increased during aging and AD progression through immunofluorescence. These results suggest that immune-related pathways might play a critical role in aging and AD-related cognitive dysfunction. Our research will help to provide some new potential targets for treating cognitive dysfunction in aging and AD.

CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions.

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors

PurposePatient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs).Methods94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively.ResultsExome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways.ConclusionThe availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Pan-cancer analysis identifies PD-L2 as a tumor promotor in the tumor microenvironment.

Programmed cell death protein 1 (PD-1) receptor has two ligands,programmed death-ligand 1 (PD-L1) and PD-L2. When compared with PD-L1, PD-L2 has not received much attention, and its role remains unclear. The expression profiles of pdcd1lg2 (PD-L2-encoding gene) mRNA and PD-L2 protein were analyzed using TCGA, ICGC, and HPA databases. Kaplan-Meier and Cox regression analyses were used to assess the prognostic significance of PD-L2. We used GSEA, Spearman's correlation analysis and PPI network to explore the biological functions of PD-L2. PD-L2-associated immune cell infiltration was evaluated using the ESTIMATE algorithm and TIMER 2.0. The expressions of PD-L2 in tumor-associated macrophages (TAMs) in human colon cancer samples, and in mice in an immunocompetent syngeneic setting were verified using scRNA-seq datasets, multiplex immunofluorescence staining, and flow cytometry. After fluorescence-activated cell sorting, flow cytometry and qRT-PCR and transwell and colony formation assays were used to evaluate the phenotype and functions of PD-L2+TAMs. Immune checkpoint inhibitors (ICIs) therapy prediction analysis was performed using TIDE and TISMO. Last, a series of targeted small-molecule drugs with promising therapeutic effects were predicted using the GSCA platform. PD-L2 was expressed in all the common human cancer types and deteriorated outcomes in multiple cancers. PPI network and Spearman's correlation analysis revealed that PD-L2 was closely associated with many immune molecules. Moreover, both GSEA results of KEGG pathways and GSEA results for Reactome analysis indicated that PD-L2 expression played an important role in cancer immune response. Further analysis showed that PD-L2 expression was strongly associated with the infiltration of immune cells in tumor tissue in almost all cancer types, among which macrophages were the most positively associated with PD-L2 in colon cancer. According to the results mentioned above, we verified the expression of PD-L2 in TAMs in colon cancer and found that PD-L2+TAMs population was not static. Additionally, PD-L2+TAMs exhibited protumor M2 phenotype and increased the migration, invasion, and proliferative capacity of colon cancer cells. Furthermore, PD-L2 had a substantial predictive value for ICIs therapy cohorts. PD-L2 in the TME, especially expressed on TAMs, could be applied as a potential therapeutic target.

Comprehensive Kinase Activity Profiling Revealed the Kinase Activity Patterns Associated with the Effects of EGFR Tyrosine Kinase Inhibitor Therapy in Advanced Non-Small-Cell Lung Cancer Patients with Sensitizing EGFR Mutations

EGFR mutations are strong predictive markers for EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy in patients with non-small-cell lung cancer (NSCLC). Although NSCLC patients with sensitizing EGFR mutations have better prognoses, some patients exhibit worse prognoses. We hypothesized that various activities of kinases could be potential predictive biomarkers for EGFR-TKI treatment among NSCLC patients with sensitizing EGFR mutations. In 18 patients with stage IV NSCLC, EGFR mutations were detected and comprehensive kinase activity profiling was performed using the peptide array PamStation12 for 100 tyrosine kinases. Prognoses were observed prospectively after the administration of EGFR-TKIs. Finally, the kinase profiles were analyzed in combination with the prognoses of the patients. Comprehensive kinase activity analysis identified specific kinase features, consisting of 102 peptides and 35 kinases, in NSCLC patients with sensitizing EGFR mutations. Network analysis revealed seven highly phosphorylated kinases: CTNNB1, CRK, EGFR, ERBB2, PIK3R1, PLCG1, and PTPN11. Pathway analysis and Reactome analysis revealed that the PI3K-AKT and RAF/ MAPK pathways were significantly enriched in the poor prognosis group, being consistent with the outcome of the network analysis. Patients with poor prognoses exhibited high activation of EGFR, PIK3R1, and ERBB2. Comprehensive kinase activity profiles may provide predictive biomarker candidates for screening patients with advanced NSCLC harboring sensitizing EGFR mutations.