Focal Adhesion Pathways Research Articles

An integrated analysis of mRNAs and lncRNAs in goat's hypothalamus to explore the onset of puberty.

The time of puberty onset is crucial for female animal, as it can affect the generation interval, feeding costs and the utilization of animals. However, little is known about the mechanism of hypothalamic lncRNAs (long noncoding RNAs) in regulatory goat puberty onset. Therefore, genome-wide transcriptome analysis was performed in goats to clarify the roles of hypothalamic lncRNAs and mRNAs in the onset of puberty. In the present study, the co-expression network of differentially expressed (DE) mRNAs in goat hypothalamus identified FN1 as the hub gene, and ECM-receptor interaction, Focal adhesion and PI3K-Akt signalling pathways are involved in puberty. We also observed the crucial hub transcription factors (TCF12, STAT1, STAT2, GATA3 and TEAD4) associated with reproduction and puberty. Then, genetic correlation analysis of DE mRNAs and DE lncRNAs identified the key lncRNAs involved in puberty. This research supplies a resource for transcriptome studies in goat puberty and indicated DE lncRNAs in the ECM-receptor interaction pathway were novel candidate regulators for genetic studies on female reproduction.

Inhibiting redox-mediated endothelial migration by gadofullerenes for inducing tumor vascular normalization and improving chemotherapy

Tumor vascular normalization (TVN) reverses abnormal tumor vasculatures, which could boost anti-cancer efficiency and especially increase drug intratumoral delivery. Endothelial cells play a vital role in angiogenesis, yet continuous modulating endothelial cell migration to improve TVN is ingenious but challenging. Here we propose a potential strategy for TVN based on inhibiting endothelial migration using antioxidative fullerene nanoparticles (FNPs). We demonstrate that FNPs inhibit cell migration upon their anti-oxidation effects in vitro. The optimized alanine-modified gadofullerene (GFA) exhibits superior TVN ability and inhibits tumor growth in vivo. Mechanically, facilitated with the protein microarray, we confirm that GFA could suppress the focal adhesion pathway to restrain endothelial migration. Subsequently, remarkable anti-tumor efficacy of chemotherapy synergy was obtained, which benefited from a more normalized vascular network by GFA. Together, our study introduces the potential of FNPs as promising TVN boosters to consider in cancer nanomedicine design.

P-325 Evidence for dysregulation of oncogene pathways at the gene expression level in the epithelium of deep infiltrating endometriosis compared to adenomyosis

Abstract Study question How does the gene expression profile of epithelium cells differ between deep infiltrating endometriosis (DIE) and adenomyosis (FA)? Summary answer The gene expression of epithelial cells from DIE reveals a significant upregulation of the PI3K-pathway and downregulation of the RAS-pathway compared to epithelium from FA. What is known already Both DIE and FA, are histologically considered benign but have features of malignant diseases, such as a tendency for local tissue invasion. Recent next generation sequencing studies have detected driver mutations in cancer-associated genes such as PIK3CA, ARID1A, PPP2R1A and KRAS in the epithelium of DIE, as well as in KRAS in epithelium of FA. Furthermore, identical mutations in the KRAS gene were detected in coexisting adenomyotic and endometriotic lesions, supporting the theory of a common molecular mechanism. The gene expression differences between these two entities were therefore investigated in the present study. Study design, size, duration This is an experimental analysis of 7 adenomyosis and 19 DIE samples (histologically confirmed), that were formalin-fixed and paraffin-embedded (FFPE) collected between 2003 and 2018. These were provided by the tissue bank of the National Centre for Tumour Diseases (Heidelberg, Germany), in accordance with the Ethics Committee of the University of Heidelberg (approval number S-362/2017). In addition, the medical and epidemiological data of the corresponding patients were retrospectively analyzed based on the clinical data collected. Participants/materials, setting, methods For this study, the epithelia of FFPE samples were microdissected in a laser-guided fashion. After RNA extraction, the expression of 770 genes was analyzed using the nCounter Technology with the Human PanCancer Pathways Panel (Nanostring). All genes with an adjusted false discovery rate of p < 0.05 and a fold change of < 0.66 or > 1.5 were considered differentially expressed and subjected to functional annotation and clustering using DAVID bioinformatics resources. Main results and the role of chance Our analysis revealed a total of 162 differentially expressed genes, that were either significantly increased (n = 116) or decreased (n = 46) in epithelium of DIE compared to that of FA (with log2 fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05). Gene ontology and KEGG pathway analysis for genes with increased expression in DIE compared with FA revealed significant upregulation of genes belonging to the PI3K-pathway and the focal adhesion pathway, as well as pathways related to viral infection, endocrine resistance, and malignancy. The upregulated pathways in adenomyosis mainly included the RAS-pathway. Limitations, reasons for caution The average age of DIE patients was lower, but many of them were hormonally inactive due to GnRH analogues, which mitigates any age-related differences. Furthermore, only relative expression was studied between the groups without comparison to normal endometrium. Overall, the sample size is relatively small and further studies are needed. Wider implications of the findings The expression of different signaling pathways in the two entities could, for example, explain the different sensitivity of the two diseases to certain therapeutic approaches. While DIE responds well to therapy with the progestogen dienogest, progesterone resistance is often described in adenomyosis patients. Trial registration number not applicable

Overexpression of Pasteurella multocida OmpA induces transcriptional changes and its possible implications for the macrophage polarization

Pasteurella multocida (P. multocida) is a highly infectious, zoonotic pathogen. Outer membrane protein A (OmpA) is an important virulence component of the outer membrane of P. multocida. OmpA mediates bacterial biofilm formation, eukaryotic cell infection, and immunomodulation. It is unclear how OmpA affects the host immune response. We estimated the role of OmpA in the pathogenesis of P. multocida by investigating the effect of OmpA on the immune cell transcriptome. Changes in the transcriptome of rat alveolar macrophages (NR8383) upon overexpression of P. multocida OmpA were demonstrated. A model cell line for stable transcription of OmpA was constructed by infecting NR8383 cells with OmpA-expressing lentivirus. RNA was extracted from cells and sequenced on an Illumina HiSeq platform. Key gene analysis of genes in the RNA-seq dataset were performed using various bioinformatics methods, such as gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, Gene Set Enrichment Analysis, and Protein–Protein Interaction Analysis. Our findings revealed 1340 differentially expressed genes. Immune-related pathways that were significantly altered in rat alveolar macrophages under the effect of OmpA included focal adhesion, extracellular matrix and vascular endothelial growth factor signaling pathways, antigen processing and presentation, nucleotide oligomerization domain-like receptor and Toll-like receptor signaling pathways, and cytokine-cytokine receptor interaction. The key genes screened were Vegfa, Igf2r, Fabp5, P2rx1, C5ar1, Nedd4l, Gas6, Cxcl1, Pf4, Pdgfb, Thbs1, Col7a1, Vwf, Ccl9, and Arg1. Data of associated pathways and altered gene expression indicated that OmpA might cause the conversion of rat alveolar macrophages to M2-like. The related pathways and key genes can serve as a reference for OmpA of P. multitocida and host interaction mechanism studies.

MDB-09. IMPACT OF PRDM6 ON CHROMATIN ACCESSIBILITY, GENE EXPRESSION, AND MEDULLOBLASTOMA FORMATION

Abstract Group 4 medulloblastoma (MB) is the most common medulloblastoma subgroup and shows high incidence of metastasis and late-onset relapse cases. These tumors lack a unifying oncogenic driver and treatment targets, despite extensive genomic characterization. Group 4 MBs are characterized by recurrent genetic alterations in chromatin modifiers and amplification of stemness genes. A substantial fraction of Group 4 MBs are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. Some PRDM6-overexpressing medulloblastoma cases show additional mutations in chromatin regulators and high MYCN expression. We set out to elucidate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and patient-derived medulloblastoma cells. We find that PRDM6 expression in human iPSC-derived neuroepithelial stem cells (NESCs) results in tumor growth in mice at low penetrance. Moreover, we find that PRDM6 expression in MYCN-overexpressing NESCs does not further alter tumor aggressiveness or survival in vivo. Notably, PRDM6 overexpression in the patient-derived Group 3 MYC-amplified D283-Med cell line causes significantly increased aggressiveness of tumor growth and shorter survival in mice. At the cellular level, PRDM6 localizes to the nucleus, suggesting a role in gene expression regulation. Consistent with this notion, ATAC-seq and RNA-seq analysis of PRDM6-NESCs and PRDM6-D283-Med cells reveals major changes in chromatin accessibility and gene expression, specifically upregulation of integrin family members and gene family members of focal adhesion and extracellular matrix receptor interaction pathways. We conclude that PRDM6 promotes tumor growth in NESCs and may play a role in tumor maintenance through interactions with the extracellular matrix and tumor microenvironment.

Establishment of a novel cytokine-related 8-gene signature for distinguishing and predicting the prognosis of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a common carcinoma in women, and the prognosis of TNBC is the worst. Using data from The Cancer Genome Atlas (TCGA) database, we analyzed the functional roles of cytokine-related genes in TNBC. The clinical and transcriptome data of TNBC patients were downloaded from TCGA database. A systematical analyses of the data from TCGA database were conducted to screen the prognostic genes and identify the main cytokine-related pathways related to TNBC. We identified 499 prognostic genes in TNBC patients from TCGA database and the cytokine-related pathways closely related to TNBC. TCGA-TNBC patients were divided into the high-risk cluster (C1) group and the low-risk cluster (C2) group based on the cytokine-related genes. The C1 group patients exhibited tumor metastasis and an advanced tumor stage. The functional analysis revealed that the upregulated differentially expressed genes (DEGs) in the C1 group were mainly associated with the extracellular matrix (ECM)-receptor interaction, stem cell proliferation, focal adhesion, and cyclic adenosine monophosphate (cAMP) signaling pathway, while the downregulated DEGs in the C1 group were mainly associated with cytokine and cytokine receptors, T-helper 17 (Th17) cell differentiation, and primary immunodeficiency. The immune activity of C1 group was lower than that of C2 group, and the identified half-maximal inhibitory concentration scores of 3 chemotherapy drugs (i.e., doxorubicin, methotrexate, and paclitaxel) were lower in C2 group than C1 group. More importantly, we constructed a novel prognostic signature and identified the following 8 genes: CCL25, CXCL13, IL12RB2, IL21, TNFRSF13C, TNFRSF8, CCL7 and GDF5. The status of the cytokine-related pathway was closely related to tumor classification and immune activity in the TNBC patients. The gene signature of the cytokine-related genes showed an good performance in predicting the prognosis of TNBC patients, and could predict the prognosis of TNBC patients.

Single-cell and spatial transcriptomics to analyze radiation therapy-induced tumor microenvironment reshaping in HPV- cervical cancer.

e17528 Background: Radiotherapy (RT) is the most widely used conventional first-line treatment in cervical cancer (CC). However, the treatment response rate was only 10%-40%, and even lower in HPV- negative CC (HPV- CC). So far, previous knowledge of radiotherapy on tumor and tumor immune microenvironment (TME) remodeling is still insufficient. In this study, we aimed to comprehensively understand the dynamic change of tumor and TME of HPV- CC at the single-cell level. Methods: The single-cell and spatial transcriptome sequencing (STRNAseq) were used to analyze the TME of newly diagnosed HPV- CC tumor tissues, including pre-radiotherapy and 3 weeks after radiotherapy. Tissue samples were split into two parts, the freshly lysed single-cell suspension for 10X single-cell sequencing, and another part that was immediately processed into frozen and embedded for STRNAseq. Cell clustering, annotation and Single-cell sequencing and spatial transcriptome integration were analyzed by Seurat 4.0. The CNV of epithelial cells (ECs) was estimated by InferCNV. Results: A total of 14000 cells were obtained from paired HPV-CC patient samples (pre-RT and 3 weeks after RT) and clustered into 21 cell subsets. Six EC subsets, four fibroblast subsets, three macrophage subsets, two T cell subsets, two B cell subsets, one mast cell cluster, and an endothelial cell cluster were discovered. CNV analysis revealed that all ECs were malignantly proliferating tumor cells. Tumor cells accounted for 90% of the total tumor tissue prior to RT, but only less than 1% of tumor cells remain after RT. Fibroblasts and myeloid cells, which make up 60% of tumor tissue, increase significantly after radiotherapy, with myeloid cells accounting for nearly 50% of the total. Furthermore, we found a population of cells in the Post-RT sample that were both highly expressed CD14 and CD68 (macrophage biomarkers) and highly expressed COL1A2 (fibroblast biomarker). Differential analysis showed that this group of cells was down-regulated in Focal adhesion and ECM-receptor interaction pathways, while up-regulated in cytokine secretion and phagosome pathways, indicating that this group of cells might remodel the intercellular matrix and interacted with other cells to regulate the immune microenvironment. Integrating single-cell and spatial transcriptome data, fibroblasts and myeloid cells formed a tight co-distribution in tissues after radiotherapy, implying that their interaction is critical for the formation of the tumor immune microenvironment. Conclusions: This study comprehensively analyzed the dynamic change of tumor and TME of HPV- CC treated with RT at the single-cell transcriptional scale. After radiotherapy, fibroblasts and myeloid cells significantly increased and were highly heterogeneous, and the interaction between them is critical in forming a tumor ecosystem for radiotherapy.

A novel Chr1-miR-200 driven whole transcriptome signature shapes tumor immune microenvironment and predicts relapse in early-stage lung adenocarcinoma

BackgroundIn Lung adenocarcinoma (LUAD), targeted therapies and immunotherapies have moved from metastatic to early stage and stratification of the relapse risk becomes mandatory. Here we identified a miR-200 based RNA signature that delineates Epithelial-to-mesenchymal transition (EMT) heterogeneity and predicts survival beyond current classification systems.MethodsA miR-200 signature was identified using RNA sequencing. We scored the miR-200 signature by WISP (Weighted In Silico Pathology), used GSEA to identify pathway enrichments and MCP-counter to characterize immune cell infiltrates. We evaluate the clinical value of this signature in our series of LUAD and using TCGA and 7 published datasets.ResultsWe identified 3 clusters based on supervised classification: I is miR-200-sign-down and enriched in TP53 mutations IIA and IIB are miR-200-sign-up: IIA is enriched in EGFR (p < 0.001), IIB is enriched in KRAS mutation (p < 0.001). WISP stratified patients into miR-200-sign-down (n = 65) and miR-200-sign-up (n = 42). Several biological processes were enriched in MiR-200-sign-down tumors, focal adhesion, actin cytoskeleton, cytokine/receptor interaction, TP53 signaling and cell cycle pathways. Fibroblast, immune cell infiltration and PDL1 expression were also significantly higher suggesting immune exhaustion. This signature stratified patients into high-vs low-risk groups, miR-200-sign-up had higher DFS, median not reached at 60 vs 41 months and within subpopulations with stage I, IA, IB, or II. Results were validated on TCGA data on 7 public datasets.ConclusionThis EMT and miR-200-related prognostic signature refines prognosis evaluation independently of tumor stage and paves the way towards assessing the predictive value of this LUAD clustering to optimize perioperative treatment.

Study on the Mechanism of MC5R Participating in Energy Metabolism of Goose Liver.

Nutrition and energy levels have an important impact on animal growth, production performance, disease occurrence and health recovery. Previous studies indicate that melanocortin 5 receptor (MC5R) is mainly involved in the regulations of exocrine gland function, lipid metabolism and immune response in animals. However, it is not clear how MC5R participates in the nutrition and energy metabolism of animals. To address this, the widely used animal models, including the overfeeding model and the fasting/refeeding model, could provide an effective tool. In this study, the expression of MC5R in goose liver was first determined in these models. Goose primary hepatocytes were then treated with nutrition/energy metabolism-related factors (glucose, oleic acid and thyroxine), which is followed by determination of MC5R gene expression. Moreover, MC5R was overexpressed in goose primary hepatocytes, followed by identification of differentially expressed genes (DEGs) and pathways subjected to MC5R regulation by transcriptome analysis. At last, some of the genes potentially regulated by MC5R were also identified in the in vivo and in vitro models, and were used to predict possible regulatory networks with PPI (protein-protein interaction networks) program. The data showed that both overfeeding and refeeding inhibited the expression of MC5R in goose liver, while fasting induced the expression of MC5R. Glucose and oleic acid could induce the expression of MC5R in goose primary hepatocytes, whereas thyroxine could inhibit it. The overexpression of MC5R significantly affected the expression of 1381 genes, and the pathways enriched with the DEGs mainly include oxidative phosphorylation, focal adhesion, ECM-receptor interaction, glutathione metabolism and MAPK signaling pathway. Interestingly, some pathways are related to glycolipid metabolism, including oxidative phosphorylation, pyruvate metabolism, citrate cycle, etc. Using the in vivo and in vitro models, it was demonstrated that the expression of some DEGs, including ACSL1, PSPH, HMGCS1, CPT1A, PACSIN2, IGFBP3, NMRK1, GYS2, ECI2, NDRG1, CDK9, FBXO25, SLC25A25, USP25 and AHCY, was associated with the expression of MC5R, suggesting these genes may mediate the biological role of MC5R in these models. In addition, PPI analysis suggests that the selected downstream genes, including GYS2, ECI2, PSPH, CPT1A, ACSL1, HMGCS1, USP25 and NDRG1, participate in the protein-protein interaction network regulated by MC5R. In conclusion, MC5R may mediate the biological effects caused by changes in nutrition and energy levels in goose hepatocytes through multiple pathways, including glycolipid-metabolism-related pathways.

Weighted gene co-expression network analysis identifies the prognosis-related models of left- and right-sided colon cancer.

Left-sided colon cancer (LC) and right-sided colon cancer (RC) are 2 essentially different diseases, and the potential mechanisms regulating them remain unidentified. In this study, we applied weighted gene co-expression network analysis (WGCNA) to confirm a yellow module, mainly enriched in metabolism-related signaling pathways related to LC and RC. Based on the RNA-seq data of colon cancer in The Cancer Genome Atlas (TCGA) and GSE41258 dataset with their corresponding clinical information, a training set (TCGA: LC: n = 171; RC: n = 260) and a validation set (GSE41258: LC: n = 94; RC: n = 77) were divided. Least absolute shrinkage and selection operator (LASSO) penalized COX regression analysis identified 20 prognosis-related genes (PRGs) and helped constructed 2 risk (LC-R and RC-R) models in LC and RC, respectively. The model-based risk scores accurately performed in risk stratification for colon cancer patients. The high-risk group of the LC-R model showed associations with ECM-receptor interaction, focal adhesion, and PI3K-AKT signaling pathway. Interestingly, the low-risk group of the LC-R model showed associations with immune-related signaling pathways like antigen processing and presentation. On the other hand, the high-risk group of the RC-R model showed enrichment for cell adhesion molecules and axon guidance signaling pathways. Furthermore, we identified 20 differentially expressed PRGs between LC and RC. Our findings provide new insights into the difference between LC and RC, and uncover the potential biomarkers for the treatment of LC and RC.

SPTBN2 regulates endometroid ovarian cancer cell proliferation, invasion and migration via ITGB4‑mediated focal adhesion and ECM receptor signalling pathway.

Ovarian cancer is as a major contributor to gynaecologic death globally. The present study aimed to investigate the regulatory role of spectrin β non-erythrocytic 2 gene (SPTBN2) in endometroid ovarian cancer and its mechanism of action. According to the Gene Expression Profiling Interactive Analysis (GEPIA) database, SPTBN2 expression is elevated in ovarian cancer tissues and higher SPTBN2 expression indicated a worse prognosis. The present study assessed SPTBN2 mRNA and protein expression levels by reverse transcription-quantitative PCR and western blotting, respectively. Cell viability, proliferation, migration and invasion were assessed with Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, wound healing and Transwell assays, respectively. SPTBN2 expression was notably enhanced in ovarian cancer cell lines, especially in A2780 cells compared with HOSEPiC cells (P<0.001). Following transfection with small interfering (si)RNA targeting SPTBN2, the viability, proliferation, migration and invasion of A2780 cells were decreased compared with those of A2780 cells transfected with siRNA-NC (P<0.001). Gene Set Enrichment Analysis database revealed that SPTBN2 was primarily enriched in 'focal adhesion' and 'extracellular matrix (ECM)-receptor interaction', whereas SPTBN2 was significantly associated with integrin β4 (ITGB4) in the GEPIA database. In addition, rescue experiments were performed to determine the mechanism of SPTBN2 in endometroid ovarian cancer. ITGB4 overexpression reversed the inhibitory effects of the SPTBN2 knockdown on viability, proliferation, migration and invasion of A2780 cells (P<0.05). The impacts of SPTBN2 on the expression of focal adhesion and downstream ECM receptor signalling-related proteins, including Src and p-FAK/FAK, were significantly reversed by ITGB4 overexpression (P<0.01). Collectively, SPTBN2 may regulate endometroid ovarian cancer cell proliferation, invasion and migration through the ITGB4-mediated focal adhesion and ECM receptor signalling pathway.

Identification of Differentially Expressed Genes and Molecular Pathways Involved in Osteoclastogenesis Using RNA-seq.

Osteoporosis is a disease that is characterised by reduced bone mineral density (BMD) and can be exacerbated by the excessive bone resorption of osteoclasts (OCs). Bioinformatic methods, including functional enrichment and network analysis, can provide information about the underlying molecular mechanisms that participate in the progression of osteoporosis. In this study, we harvested human OC-like cells differentiated in culture and their precursor peripheral blood mononuclear cells (PBMCs) and characterised the transcriptome of the two cell types using RNA-sequencing in order to identify differentially expressed genes. Differential gene expression analysis was performed in RStudio using the edgeR package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify enriched GO terms and signalling pathways, with inter-connected regions characterised using protein-protein interaction analysis. In this study, we identified 3201 differentially expressed genes using a 5% false discovery rate; 1834 genes were upregulated, whereas 1367 genes were downregulated. We confirmed a significant upregulation of several well-established OC genes including CTSK, DCSTAMP, ACP5, MMP9, ITGB3, and ATP6V0D2. The GO analysis suggested that upregulated genes are involved in cell division, cell migration, and cell adhesion, while the KEGG pathway analysis highlighted oxidative phosphorylation, glycolysis and gluconeogenesis, lysosome, and focal adhesion pathways. This study provides new information about changes in gene expression and highlights key biological pathways involved in osteoclastogenesis.

Investigation of shared genes and regulatory mechanisms associated with coronavirus disease 2019 and ischemic stroke.

Clinical associations between coronavirus disease (COVID-19) and ischemic stroke (IS) have been reported. This study aimed to investigate the shared genes between COVID-19 and IS and explore their regulatory mechanisms. Published datasets for COVID-19 and IS were downloaded. Common differentially expressed genes (DEGs) in the two diseases were identified, followed by protein-protein interaction (PPI) network analysis. Moreover, overlapping module genes associated with the two diseases were investigated using weighted correlation network analysis (WGCNA). Through intersection analysis of PPI cluster genes and overlapping module genes, hub-shared genes associated with the two diseases were obtained, followed by functional enrichment analysis and external dataset validation. Moreover, the upstream miRNAs and transcription factors (TFs) of the hub-shared genes were predicted. A total of 91 common DEGs were identified from the clusters of the PPI network, and 129 overlapping module genes were screened using WGCNA. Based on further intersection analysis, four hub-shared genes in IS and COVID-19 were identified, including PDE5A, ITGB3, CEACAM8, and BPI. These hub-shared genes were remarkably enriched in pathways such as ECM-receptor interaction and focal adhesion pathways. Moreover, ITGB3, PDE5A, and CEACAM8 were targeted by 53, 32, and 3 miRNAs, respectively, and these miRNAs were also enriched in the aforementioned pathways. Furthermore, TFs, such as lactoferrin, demonstrated a stronger predicted correlation with the hub-shared genes. The four identified hub-shared genes may participate in crucial mechanisms underlying both COVID-19 and IS and may exhibit the potential to be biomarkers or therapeutic targets for the two diseases.

Abstract 300: Mechanisms of growth hormone action in the aging tissue microenvironment

Abstract The aging tissue microenvironment undergoes progressive changes enabling age-related pathologies. Senescent cells increase with age, and the secretome released from senescence cells (SASP) affects neighboring cells, often promoting proliferation, tissue inflammation, angiogenesis, and, depending on tissue type, creating a milieu favoring neoplasia. We showed that local non-pituitary growth hormone (GH) accumulates with age in human colon tissue, is induced in response to DNA damage, and suppresses DNA repair. We now show that GH is a SASP component. To examine SASP-derived GH actions on the tissue microenvironment, we co-cultured intact human 3D intestinal organoids with organoids infected with lentivirus expressing either hGH (lentiGH) or vector (lentiV) for 5 weeks. Using whole-exome sequencing to evaluate somatic mutations and somatic copy number alterations, we found that local paracrine GH emanating from lentiGH organoids increases chromosomal instability in intact organoid cells, inducing somatic mutations including deletions, breakends, duplications, and insertions, as compared to organoids co-cultured with lentiV organoids. Mechanisms for chromosomal instability observed in these organoids likely occur as a consequence of GH-induced suppression of DNA damage repair proteins including pATM, pATR, and pBRCA2, leading to DNA damage accumulation, and, in the long run, favoring cell transformation. KEGG pathway analysis of intact organoids subjected to paracrine GH exposure identified changes in focal adhesion pathways (p=0.0016), consistent with observed increased migration, invasion, and anchorage-independent growth of normal human colon cells (hNCC) exposed to GH. Furthermore, IPA analysis revealed EMT pathway changes (p=0.0018),,also confirmed by showing increased EMT transcription factors Twist2 and Snai1 expression.Although senescence has been defined as irreversible proliferative arrest, recent studies now show that senescence can be reversed. Treating hNCC with etoposide to induce senescence and culturing senescent cells in the presence of GH for 10 days resulted in p53 suppression, enabling cells to re-enter the cell cycle, as evidenced by induced Ki67, a marker of proliferation. These post-senescent cells also acquired the stem cell marker SOX2 and exhibited increased Twist2, consistent with anchorage-independent growth and increased migration as compared to senescence cells not treated with GH. These results identify local paracrine GH as an important determinant of the local tissue microenvironment promoting age-associated changes consistent with a pro-neoplastic milieu. Citation Format: Vera Chesnokova, Svetlana Zonis, Tugce Apaydin, Christian Wang, Robert Barrett, Shlomo Melmed. Mechanisms of growth hormone action in the aging tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 300.

TMEM30A is essential for hair cell polarity maintenance in postnatal mouse cochlea

BackgroundPhosphatidylserine is translocated to the inner leaflet of the phospholipid bilayer membrane by the flippase function of type IV P-tape ATPase (P4-ATPase), which is critical to maintain cellular stability and homeostasis. Transmembrane protein 30A (TMEM30A) is the β-subunit of P4-ATPase. Loss of P4-ATPase function causes sensorineural hearing loss and visual dysfunction in human. However, the function of TMEM30A in the auditory system is unclear.MethodsP4-ATPase subtype expression in the cochlea was detected by immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) at different developmental stages. Hair cell specific TMEM30A knockout mice and wild-type littermates were used for the following functional and morphological analysis. Auditory function was evaluated by auditory brainstem response. We investigated hair cell and stereocilia morphological changes by immunofluorescence staining. Scanning electron microscopy was applied to observe the stereocilia ultrastructure. Differentially expressed transcriptomes were analyzed based on RNA-sequencing data from knockout and wild-type mouse cochleae. Differentially expressed genes were verified by qRT-PCR.ResultsTMEM30A and subtypes of P4-ATPase are expressed in the mouse cochlea in a temporal-dependent pattern. Deletion of TMEM30A in hair cells impaired hearing onset due to progressive hair cell loss. The disrupted kinocilia placement and irregular distribution of spectrin-α in cuticular plate indicated the hair cell planar polarity disruption in TMEM30A deletion hair cells. Hair cell degeneration begins at P7 and finishes around P14. Transcriptional analysis indicates that the focal adhesion pathway and stereocilium tip-related genes changed dramatically. Without the TMEM30A chaperone, excessive ATP8A2 accumulated in the cytoplasm, leading to overwhelming endoplasmic reticulum stress, which eventually contributed to hair cell death.ConclusionsDeletion of TMEM30A led to disrupted planar polarity and stereocilia bundles, and finally led to hair cell loss and auditory dysfunction. TMEM30A is essential for hair cell polarity maintenance and membrane homeostasis. Our study highlights a pivotal role of TMEM30A in the postnatal development of hair cells and reveals the possible mechanisms underlying P4-ATPase-related genetic hearing loss.

Use of a gene expression signature to identify trimetazidine for repurposing to treat bipolar depression.

The aim of this study was to repurpose a drug for the treatment of bipolar depression. A gene expression signature representing the overall transcriptomic effects of a cocktail of drugs widely prescribed to treat bipolar disorder was generated using human neuronal-like (NT2-N) cells. A compound library of 960 approved, off-patent drugs were then screened to identify those drugs that affect transcription most similar to the effects of the bipolar depression drug cocktail. For mechanistic studies, peripheral blood mononuclear cells were obtained from a healthy subject and reprogrammed into induced pluripotent stem cells, which were then differentiated into co-cultured neurons and astrocytes. Efficacy studies were conducted in two animal models of depressive-like behaviours (Flinders Sensitive Line rats and social isolation with chronic restraint stress rats). The screen identified trimetazidine as a potential drug for repurposing. Trimetazidine alters metabolic processes to increase ATP production, which is thought to be deficient in bipolar depression. We showed that trimetazidine increased mitochondrial respiration in cultured human neuronal-like cells. Transcriptomic analysis in induced pluripotent stem cell-derived neuron/astrocyte co-cultures suggested additional mechanisms of action via the focal adhesion and MAPK signalling pathways. In two different rodent models of depressive-like behaviours, trimetazidine exhibited antidepressant-like activity with reduced anhedonia and reduced immobility in the forced swim test. Collectively our data support the repurposing of trimetazidine for the treatment of bipolar depression.

Cancer cell-derived CD69 induced under lipid and oxygen starvation promotes ovarian cancer progression through fibronectin.

Cancer tissues generally have molecular oxygen and serum component deficiencies because of poor vascularization. Recently, we revealed that ICAM1 is strongly activated through lipophagy in ovarian clear cell carcinoma (CCC) cells in response to starvation of long-chain fatty acids and oxygen and confers resistance to apoptosis caused by these harsh conditions. CD69 is a glycoprotein that is synthesized in immune cells and is associated with their activation through cellular signaling pathways. However, the expression and function of CD69 in nonhematological cells is unclear. Here, we report that CD69 is induced in CCC cells as in ICAM1. Mass spectrometry analysis of phosphorylated peptides followed by pathway analysis revealed that CD69 augments CCC cell binding to fibronectin (FN) in association with the phosphorylation of multiple cellular signaling molecules including the focal adhesion pathway. Furthermore, CD69 synthesized in CCC cells could facilitate cell survival because the CD69-FN axis can induce epithelial-mesenchymal transition. Experiments with surgically removed tumor samples revealed that CD69 is predominantly expressed in CCC tumor cells compared with other histological subtypes of epithelial ovarian cancer. Overall, our data suggest that cancer cell-derived CD69 can contribute to CCC progression through FN.

Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus.

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log2 fold change [|log2FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis. IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

Tissue and plasma proteomic profiling indicates AHSG as a potential biomarker for ascending thoracic aortic aneurysms

BackgroundThoracic Aortic Aneurysms (TAAs) develop asymptomatically and are characterized by dilatation of the aorta. This is considered a life-threatening vascular disorder due to the risk of aortic dissection and rupture. There is an urgent need to identify blood-borne biomarkers for the early detection of TAA. The goal of the present study was to identify potential protein biomarkers associated with TAAs, using proteomic analysis of aortic tissue and plasma samples.MethodsExtracted proteins from 14 aneurysmal and 12 non-aneurysmal thoracic aortic tissue specimens as well as plasma samples from six TAA patients collected pre-and postoperatively and six healthy controls (HC), were analyzed by liquid chromatography-tandem mass spectrometry. Proteomic data were further processed and following filtering criteria, one protein was selected for verification and validation in a larger cohort of patients and controls using a targeted quantitative proteomic approach and enzyme-linked immunosorbent assay, respectively.ResultsA total of 1593 and 363 differentially expressed proteins were identified in tissue and plasma samples, respectively. Pathway enrichment analysis on the differentially expressed proteins revealed a number of dysregulated molecular pathways that might be implicated in aneurysm pathology including complement and coagulation cascades, focal adhesion, and extracellular matrix receptor interaction pathways. Alpha-2-HS glycoprotein (AHSG) was selected for further verification in 36 TAA and 21 HC plasma samples using targeted quantitative proteomic approach. The results showed a significantly decreased concentration of AHSG (p = 0.0002) in the preoperative plasma samples compared with HC samples. Further analyses using a larger validation dataset revealed that AHSG protein levels were significantly lower (p = 0.03) compared with HC. Logistic regression analysis on the validation dataset revealed males, advanced age, hypertension and hyperlipidaemia as significant risk factors for TAA.ConclusionAHSG concentrations distinguish plasma samples derived from TAA patients and controls. The findings of this study suggest that AHSG may be a potential biomarker for TAA that could lead to better diagnostic capabilities.

A comprehensive investigation regarding the clinical significance of ITGB4 in oral squamous cell carcinoma combining immunohistochemistry, RNA-seq, and microarray data

ObjectiveIntegrin subunit beta 4 (ITGB4), a receptor for laminins, was an oncoprotein in several malignancies. However, its clinical role in oral squamous cell carcinoma (OSCC) remains unclear. Materials and methodsFirstly, 99 OSCC and 13 normal oral epithelium samples were employed for immunohistochemistry (IHC) for detecting the expression level of ITGB4 protein in OSCC. Subsequently, 971 OSCC and 281 non-cancerous specimens from RNA-seq and 18 microarrays were applied for investigating the expression of ITGB4 mRNA. Furthermore, to explore the potential mechanism of ITGB4 in OSCC, the co-expressed genes of ITGB4 were initially screened using all available datasets, and were further utilized for the gene enrichment analysis. ResultsFirst, IHC showed a distinctively higher expression level of the ITGB4 protein in the OSCC group than that in the normal controls. Second, expression profile from RNA-seq and microarrays reflected that ITGB4 mRNA was dramatically overexpressed in OSCC tissues compared with non-tumor tissues. Third, standardized mean difference (SMD) with the area under the summary receiver operating characteristic (sROC) curve combining all incorporated data revealed that ITGB4 was consistently significantly upregulated in OSCC tissues, with the SMD value being 1.31 and the area under the sROC curve being 0.82. Lastly, 184 upregulated and 179 downregulated co-expressed genes of ITGB4 were utilized for enrichment analysis, which demonstrated that ITGB4 might influence the pathogenesis of OSCC through cell cycle, ECM-receptor interaction and focal adhesion pathways. ConclusionsITGB4 might play a pivotal role in the tumorigenesis and progression of OSCC, making it a promising biomarker of OSCC.